System for reducing the translocation velocity of molecules through a substrate for detecting biological reactions

Abstract

A device for fluorescence-based detection of at least one polymer, including at least one container, at least one light source, at least one detector and at least two electrodes, the container including a nanopore membrane, a first layer and a physical barrier, the nanopore membrane including at least one nanopore, the first layer including at least one opening respectively positioned in line over the nanopore, the physical barrier covering the opening, the light source for illuminating the polymer with light beams, the detector for detecting emissions emitted from the illuminated polymer, the polymer being placed within an ionic solution within the container, the electrodes generating a current in the ionic solution, wherein the physical barrier increases a viscosity of the ionic solution in a region adjacent to the opening and the nanopore for slowing down a translocation velocity of the polymer as it passes through the opening and the nanopore.

Description

[0001] SYSTEM FOR REDUCING THE TRANSLOCATION VELOCITY OF MOLECULES THROUGH A SUBSTRATE FOR DETECTING BIOLOGICAL REACTIONS

[0002] KORMAN, Maayan, WILNER, Ofer Israel, LEIBA, Yigal, BAUM, David, MANSOUR, Oren and NIMRI, Shai

[0003] FIELD OF THE DISCLOSED TECHNIQUE

[0004] The disclosed technique relates to fluorescence-based detection and analysis of biological molecules and polymers which are labeled via biochemical reactions, in general, and to methods and systems for controlled reduction of the translocation velocity of molecules through nanopores in a membrane during optical detection, in particular.

[0005] BACKGROUND OF THE DISCLOSED TECHNIQUE

[0006] Fluorescence is a well-known physical phenomenon characterized by an emission of light in reaction to either absorbed light or other electromagnetic radiation when impinging upon certain substances. Fluorescence can be used for detection purposes, such as the detection of a physical phenomenon, the detection of chemical reactions as well as the detection of biological molecules and polymers (herein referred to as biomolecules). Examples in the field of detecting biomolecules and biochemical reactions include protein quantification at low concentration, water quality analysis, time- resolved fluorescence for monitoring food composition, studying heart disease and the like. Fluorescence can also be used to analyze and determine at least a portion of the constituents of biomolecules such as DNA, RNA and proteins (which are all examples of polymers) as different fluorescent monomers within the polymer may emit fluorescent radiation at different wavelengths. In general, the detection of biomolecules is made possible by labeling the biomolecules via a biochemical reaction or by measuring them directly via absorbance and / or electric current.

[0007] Fluorescence-based detection and analysis systems of biomolecules using a membrane having nanopores are known in the art. Such systems typically include a light source and a fluorescence detector as well as a container or reservoir filled with an ionic solution in which a polymer of interest (herein referred to as a biopolymer) is placed. The biopolymer is usually electrically charged by the bonding of at least one charged molecule to the polymer. The reservoir is usually divided into two by a membrane and has at least one nanopore, which is a nanometer-sized hole which is drilled and / or inserted into the membrane. The membrane can also have a plurality of nanopores. An electric field is generated in the ionic solution (by applying a voltage across the two sides of the reservoir), thereby encouraging migration of the charged biopolymer from one side of the membrane to the other via the nanopore or the plurality of nanopores. The light source is directed at the nanopore to cause fluorescence in the migrating biopolymers as they pass through the nanopore. The fluorescence detector detects fluorescence from the biopolymers (as they pass through the nanopore) which can then be used to analyze the constitution of the biopolymers.

[0008] Reference is now made to Figure 1 A, which is a schematic illustration of a fluorescence based detection and analysis system, generally referenced 10, as is known in the prior art. Detection system 10 includes a container 12, a membrane 14, a light source 18 and a detector 20. As shown, an ionic solution 13 is placed in container 12 which fills container 12 and is located on both sides of membrane 14. As shown as well, membrane 14 includes a nanopore 16 through which biopolymers and biomolecules, such as a biopolymer 22, can pass there through. Detection system 10 may also include an electric field generator (not shown), for generating an electric field in ionic solution 13, shown schematically as an arrow 32. When electric field 32 is generated, biopolymer 22 is encouraged to migrate through nanopore 16 from one side of membrane 14 to the other, shown schematically by an arrow 24.

[0009] An optical excitation zone 28 can be defined as a region adjacent to nanopore 16 in which light beams are directed to for causing excitation that will cause fluorescence in biopolymer 22. As shown, light source 18 emits a plurality of light beams 26 towards optical excitation zone 28 which is adjacent to nanopore 16. As biopolymer 22 migrates through nanopore 16, biopolymer 22 is impinged upon by plurality of light beams 26, thereby causing a plurality of fluorescent rays 30 to be emitted. Plurality of fluorescent rays 30 are detected by detector 20, which can then use the detected fluorescent rays to analyze biopolymer 22.

[0010] Detection system 10 can also be referred to generally as an optical nanopore sensor and uses the following general detection principles: a) Insert a membrane in a reservoir containing an ionic liquid solution thereby splitting it into two. The membrane may be non-biological or biological. In the case of a non-biological membrane, such as a solid- state membrane, the membrane is typically silicon-based having a general chemical composition of SiNx. In the case of a biological membrane, the membrane may be a lipid membrane made from phospholipids, such as diphethanolphosphatidylcholine (DphPC), a lipid bilayer as well as using cells such as Xenopus ooctyes which can be formed into a membrane. b) Insert a biopolymer sample (such as a DNA, RNA and / or protein sample) to be analyzed in one side of the reservoir. c) The membrane can have any amount of nanometer-sized holes, as known as nanopores that are the only path for the charged biopolymer sample to pass from one side of the reservoir to the other. d) Generate an electric field in the ionic solution across the membrane. The electric field can be generated by applying an external voltage gradient to the two sides of the membrane and / or by using different ionic solution concentrations in the two sides of the membrane. e) In the case the biopolymer sample is a protein, the protein molecules may be fully or partly denatured by the presence of a denaturing agent in the ionic solution or by the application of an external denaturing force, such as heat. The denaturing agent at least partially unfolds the protein molecules for detection of the constituent molecules of the protein and also serves to provide the protein molecules with an electrical charge (for example by using sodium dodecyl sulfate (SDS)). It is noted that in the case of a protein molecule, the molecules forming the protein can be described as forming a chain. f) The biomolecules electrophoretically translocate (i.e., thread) through the membrane via the nanopores due to their charge and the presence of the electric field. g) As the biomolecules translocate through the membrane via the nanopores, their passage leads them via a spatial zone, immediately adjacent to the nanopores, in which the fluorescence generated by the optical excitation will reach a detector. Optical excitation can be in the form of free-space laser radiation or by using solid state waveguides adjacent to the nanopores. Either form of excitation may be potentially enhanced and better confined spatially using plasmonic effects. h) The excited biomolecules emit fluorescence as photons which are detected by a detector, positioned in the vicinity of the reservoir, which serves to gather information about the constitution of the biomolecules.

[0011] In the case of proteins, which are large biomolecules that are folded into various 3D shapes and usually include long chains of amino acids, the desire of detection system 10 is to determine not only the constituent amino acids of a protein of interest but also the order of the amino acids forming the protein. To this end, proteins of interest are typically treated, for example by labeling specific amino acids in the protein with fluorescent dyes, and are also at least partially denatured, to simplify the collection of information on the proteins as they translocate through the nanopores. Time-stamped detection of fluorescent emissions from the proteins as they translocate through the nanopores enable detection of the constituent amino acids as well as their order in the protein chain. In general, there is a desire that the optical excitation zone, which is adjacent to the nanopores, be very narrow in the direction in which the protein molecules travel (i.e. , perpendicular to the plane of the thickness of membrane 14), for example on the order of the length of a few amino acids at most, in order to detect individual amino acids in the protein. If the optical excitation zone is not sufficiently narrow, then it is likely that multiple amino acids will be excited simultaneously as the protein molecules pass through nanopore 16. Such a scenario makes it harder to distinguish the precise order of amino acids that cross through the optical excitation zone.

[0012] Systems similar to detection system 10 are known in the art. A first example of such can be found in US patent application publication no. 2021 / 0003547 A1 , to Meller et al., entitled “Light Enhancing Plasmonic Nanowell Nanopore Biosensor and Use Thereof”. The system of Meller is directed to a biosensor for detecting fluorescence from a molecule and comprises an ion impermeable film comprising at least one ion conducting nanopore. An upper liquid reservoir and a lower liquid reservoir are separated by the film. Electrodes coupled with the upper and lower liquid reservoirs are used as a means to induce movement of the molecule from the upper reservoir to the lower reservoir via the nanopore. The system of Meller includes a light source capable of exciting the molecule to emit fluorescence, wherein the light source shines into the lower reservoir. A metallic layer adhered to the film by an adhesion layer is also included and comprises a nanowell structure located adjacent to the nanopore. The system of Meller further includes a detector for detecting the fluorescence emitted by the molecule.

[0013] A second example of such can be found in US patent application publication no. 2017 / 0003227 A1 , to Peumans et al., entitled “Integrated waveguide structure for fluorescence analysis”. The system of Peumans is directed to an integrated waveguide structure for characterizing one or more fluorescent particles. The integrated waveguide structure includes a substrate as well as a waveguide layer arranged on top of the substrate. The waveguide layer includes one or more excitation waveguides, one or more emission waveguides, and a particle radiation coupler, which includes a resonator element. In addition, the integrated waveguide structure includes one or more sensing sites configured with respect to the one or more excitation waveguides and the one or more emission waveguides such that a fluorescent particle at one of the sensing sites is activated by an excitation radiation transmitted via the one or more excitation waveguides. The radiation emitted by the fluorescent particle is coupled into at least one of the emission waveguides by the particle radiation coupler.

[0014] A third example of such can be found in PCT international application publication no. WO 2023 / 139586 A2, to Genopore Ltd., entitled “System for detecting biological reactions on a substrate using waveguides and nanopores”. The system of Genopore is directed to a device for fluorescence-based detection of at least one polymer. The device includes at least one container, at least one light source, at least one detector and at least one optical element. The container includes a nanopore membrane and a waveguide layer, with the nanopore membrane including at least one nanopore and the waveguide layer including at least one opening. The light source is optically coupled with the waveguide layer and is for generating at least one light beam for illuminating the polymer and the detector is for detecting at least one emission emitted from the illuminated polymer. The optical element is for entering the light beam into the waveguide layer, with the opening being positioned substantially in line with the nanopore. The polymer is illuminated by the light beam propagating through the waveguide layer through the opening and the illuminated polymer emitting the emission as it passes through the nanopore and the opening.

[0015] Whereas the above described detection systems and general detection principles are known, the prior art suffers from a fundamental drawback which limits its use. Considering the optical excitation zone is very narrow and thin, biopolymers translocating through nanopores into the optical excitation zone must cross through the optical excitation zone slowly enough such that a sufficient amount of fluorescence (i.e., photons) are emitted to ensure reliable detection of the constituents of the biopolymer by the detector. However, as explained below, prior art detection systems lead to polymers passing through the optical excitation zone too rapidly for sufficient fluorescent photons to be detected for each constituent amino acid in the biopolymer to be identified. The velocity at which biopolymers translocate or cross through nanopores in the membrane can be referred to as the translocation velocity. Thus biopolymers must have a sufficiently slow translocation velocity while migrating through the nanopores in order for sufficient photons to be emitted from the biopolymers and detected by the detector to allow for reliable detection and analysis. The translocation velocity v of a biopolymer in an ionic solution depends on the strength of the generated electric field E as well as the electrophoretic mobility of the biopolymer, defined as p and can be written as:

[0016] The electrophoretic mobility p of a biopolymer itself depends on the viscosity, the ionic strength and temperature of the ionic solution, the size of the biopolymer as well as other factors. Whereas the temperature of the ionic solution can be easily controlled, significant changes in temperature can alter the chemical structure of the biopolymer. The viscosity of the ionic solution as well as its ionic strength is more difficult to control and the size of the biopolymer is not an adjustable parameter. Thus if the translocation velocity of a biopolymer is to be slowed down, a simple solution based on Equation (1 ) would be to decrease the strength of the generated electric field E. Thus the translocation velocity can be controlled by controlling by strength of the generated electric field E. Such a simple solution however is not an effective and practical solution for controlling the translocation velocity of a biopolymer, the reasons for which are explained with the aid of Figure 1 B.

[0017] Reference is now made to Figure 1 B, which is a schematic illustration of a detection system showing a generated electric field, generally referenced 50, as is known in the prior art. Detection system 50 is similar to detection system 10 (Figure 1 A) although some elements have been excluded for the sake of brevity. Detection system 50 includes a container 52 in which an ionic solution 54 is placed. Container 52 forms a reservoir which is separated into two sections via a membrane 55 having a nanopore 56 through which a biopolymer (not shown) can migrate through. Shown as well is a generated electric field 58 with a plurality of electric field lines 60. When an electric field is generated across membrane 55, ionic solution 54 is subject to Ohm’s Law which dictates the relation between an applied voltage and the resulting current through a conductor. In its classic form, Ohm’s Law is stated as: wherein V is the applied voltage, R is the resistance of the conductor and / is the current. According to Ohm’s Law, the resistance itself R of a conductor having a volume defined by a length and a cross-section (i.e., an area) is proportional to the length of the volume while being inversely proportion to the cross-section of the volume. The resistance R can thus be defined as: wherein / is the length of the volume, a is the cross-sectional area of the volume and p is a constant known as the resistivity. Given the structure of detection system 50, it can be seen that ionic solution 54 acting as a conductor presents a space-varying electric field which changes drastically from nanopore 56 to other areas in container 52. The cross-section of nanopore 56 is tiny as compared to the cross-section of ionic solution 54 in the two sections of the reservoir. Therefore even though the length of nanopore 56 is short (i.e., the thickness of membrane 55 which may be on the order of a few nanometers to hundreds of nanometers), nanopore 56 constitutes the majority of the resistance in ionic solution 54. The majority of the voltage potential drop occurs in nanopore 56, with only a small residue occurring immediately outside it. Since the generated electric field is the gradient of the voltage potential, this means that the strength of the generated electric field quickly diminishes outside nanopore 56. Thus as shown, a plurality of electric field lines 62 are highly concentrated in nanopore 56 whereas at a distance from nanopore 56, a lone and weak electric field line 64 is shown.

[0018] Based on Figure 1 B, if the voltage potential in ionic solution 54 is reduced in order to reduce the generated electric field, the generated electric field in nanopore 56 will be reduced, thereby reducing the translocation velocity of polymers crossing through nanopore 56. However such a solution also causes a voltage potential reduction which proportionally reduces the generated electric field outside nanopore 56 which is used to attract biopolymers to nanopore 56. Thus the reduced generated electric field will significantly reduce the chances of attracting biopolymers to nanopore 56 such that they can thread through nanopore 56. Said otherwise, reducing the generated electric field may enable a suitable translocation velocity for biopolymers reaching nanopore 56 however practically speaking, biopolymers will not migrate to nanopore 56 because the generated electric field in ionic solution 54 is too weak. It is noted as well that from a pragmatic standpoint, a detection system like detection system 50 is to be used to detect and analyze biopolymers at a relatively quick rate, for example on the order of minutes or possibly hours. Whereas reducing the generated electric field in ionic solution 54 may bring the translocation velocity of biopolymers that migrate eventually to nanopore 56 to a reasonable value, it may take days for biopolymers in ionic solution 54 to actually migrate to nanopore 56. Such a solution is thus not practical for a detection system. Therefore, as mentioned above, simply controlling the generated electric field in an ionic solution is not sufficient to control the translocation velocity of biopolymers through a nanopore in order for sufficient photons to be emitted from the biopolymers and detected by the detector to allow for reliable detection and analysis.

[0019] What is needed therefore is a solution to the aforementioned fundamental drawback of optical nanopore sensors and biochemical detection systems based on fluorescence detection wherein biopolymers and biochemicals are simultaneously attracted to a nanopore yet can have their translocation velocity controlled and slowed down while migrating through the nanopore to enable sufficient photons to be emitted from the biopolymers to allow for reliable detection and analysis.

[0020] SUMMARY OF THE DISCLOSED TECHNIQUE

[0021] The disclosed technique overcomes the disadvantages of the prior art by providing a novel fluorescence-based detection system for slowing down the translocation velocity of polymers passing through an excitation zone. According to a first aspect of the disclosed technique, there is thus provided a device for fluorescence-based detection of at least one polymer, including at least one container, at least one light source, at least one detector and at least two electrodes. The container includes a nanopore membrane, a first layer and a physical barrier. The nanopore membrane includes at least one nanopore and the first layer includes at least one opening respectively positioned substantially in line over the nanopore. The physical barrier covers the opening. The light source is for illuminating the polymer with at least one light beam and the detector is for detecting at least one emission emitted from the illuminated polymer. The polymer is placed within an ionic solution within the container and the two electrodes generate a current in the ionic solution. The physical barrier increases a viscosity of the ionic solution in a region adjacent to the opening and the nanopore for slowing down a translocation velocity of the polymer as it passes through the opening and the nanopore.

[0022] According to another aspect of the disclosed technique, the physical barrier is a gel coating, coating the first layer.

[0023] According to a further aspect of the disclosed technique, the physical barrier is at least one folded nucleic acid molecule, designed to substantially fit into the opening, with the folded nucleic acid molecule having a diameter larger than a diameter of the nanopore.

[0024] According to a second aspect of the disclosed technique, there is thus provided a device for fluorescence-based detection of at least one polymer, including at least one container, at least one light source, at least one detector and at least two electrodes. The container includes a nanopore membrane and an electric field strength altering layer. The nanopore membrane includes at least one nanopore and the electric field strength altering layer includes at least one opening respectively positioned substantially in line over the nanopore. The light source is for illuminating the polymer with at least one light beam and the detector is for detecting at least one emission emitted from the illuminated polymer. The polymer is placed within an ionic solution within the container and the two electrodes generate a current in the ionic solution. The electric field strength altering layer generates a microelectric field in a region adjacent to the opening and the nanopore for slowing down a translocation velocity of the polymer as it passes through the opening and the nanopore.

[0025] According to another aspect of the disclosed technique, the device further includes a lead wire, for coupling the electric field strength altering layer to a first voltage source.

[0026] According to a further aspect of the disclosed technique, the electric field strength altering layer includes at least one metal film and the lead wire couples the metal film to the first voltage source.

[0027] According to another aspect of the disclosed technique, the device further includes a dielectric isolation layer, deposited over the metal film.

[0028] According to a further aspect of the disclosed technique, the electric field strength altering layer includes a dielectric distancing layer and at least one metal film. The dielectric distancing layer is deposited on the nanopore membrane and distances an excitation zone, where the light source illuminates the polymer, from the nanopore.

[0029] According to another aspect of the disclosed technique, the electric field strength altering layer includes a dielectric distancing layer and a waveguide. The dielectric distancing layer is deposited on the nanopore membrane and distances an excitation zone, where the light source illuminates the polymer, from the nanopore.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The disclosed technique will be understood and appreciated more fully from the following detailed description taken in conjunction with the drawings in which:

[0032] Figure 1A is a schematic illustration of a fluorescence-based detection and analysis system, as is known in the prior art;

[0033] Figure 1 B is a schematic illustration of a detection system showing a generated electric field, as is known in the prior art;

[0034] Figure 2A is a schematic illustration of a fluorescence-based detection system using plasmonic excitation, constructed and operative in accordance with an embodiment of the disclosed technique;

[0035] Figure 2B is a schematic illustration of a fluorescence-based detection system using waveguide excitation, constructed and operative in accordance with another embodiment of the disclosed technique;

[0036] Figure 3A is a schematic illustration of a first substrate of a fluorescence-based detection system using plasmonic excitation, for locally increasing the viscosity of an ionic solution adjacent to a nanopore, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0037] Figure 3B is a schematic illustration of a second substrate of a fluorescence-based detection system using waveguide excitation, for locally increasing the viscosity of an ionic solution adjacent to a nanopore, constructed and operative in accordance with another embodiment of the disclosed technique;

[0038] Figure 4A is a schematic illustration of a third substrate of a fluorescence-based detection system using plasmonic excitation, for locally increasing the viscosity of an ionic solution adjacent to a nanopore, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0039] Figure 4B is a schematic illustration of a fourth substrate of a fluorescence-based detection system using waveguide excitation, for locally increasing the viscosity of an ionic solution adjacent to a nanopore, constructed and operative in accordance with another embodiment of the disclosed technique;

[0040] Figure 40 is a schematic illustration of a substrate of a fluorescence-based detection system showing the bonding of a folded nucleic acid molecule with a functional group to the substrate, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0041] Figure 4D is a schematic illustration of a substrate of a fluorescence-based detection system showing the bonding of a folded nucleic acid molecule with staple strands to the substrate, constructed and operative in accordance with another embodiment of the disclosed technique;

[0042] Figure 5A is a schematic illustration of a membrane used in a detection system showing a generated electric field, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0043] Figure 5B is a schematic illustration of a fifth substrate of a fluorescence-based detection system using plasmonic excitation, for distancing an excitation zone from a nanopore, constructed and operative in accordance with another embodiment of the disclosed technique;

[0044] Figure 5C is a schematic illustration of a sixth substrate of a fluorescence-based detection system using waveguide excitation, for distancing an excitation zone from a nanopore, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0045] Figure 6A is a schematic illustration of a substrate of a fluorescence-based detection system wherein a non-isolated microelectrode is deposited around a nanopore, constructed and operative in accordance with another embodiment of the disclosed technique;

[0046] Figure 6B is a schematic illustration of a substrate of a fluorescence-based detection system wherein an isolated microelectrode is deposited around a nanopore, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0047] Figure 7A is a schematic illustration of a substrate of a fluorescence-based detection system showing the underlying electric principle of operation of non-isolated microelectrodes deposited around nanopores, constructed and operative in accordance with another embodiment of the disclosed technique;

[0048] Figure 7B is a schematic illustration of a substrate of a fluorescence-based detection system showing the underlying electric principle of operation of isolated microelectrodes deposited around nanopores, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0049] Figure 8A is a schematic illustration a seventh substrate of a fluorescence-based detection system using plasmonic excitation, for locally modifying the electric field adjacent to a nanopore using a non-isolated microelectrode, constructed and operative in accordance with another embodiment of the disclosed technique;

[0050] Figure 8B is a schematic illustration an eighth substrate of a fluorescence-based detection system using plasmonic excitation, for locally modifying the electric field adjacent to a nanopore using an isolated microelectrode, constructed and operative in accordance with a further embodiment of the disclosed technique;

[0051] Figure 8C is a schematic illustration a ninth substrate of a fluorescence-based detection system using waveguide excitation, for locally modifying the electric field adjacent to a nanopore using a non-isolated microelectrode, constructed and operative in accordance with another embodiment of the disclosed technique; and

[0052] Figure 8D is a schematic illustration a tenth substrate of a fluorescence-based detection system using waveguide excitation, for locally modifying the electric field adjacent to a nanopore using an isolated microelectrode, constructed and operative in accordance with a further embodiment of the disclosed technique.

[0053] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The disclosed technique overcomes the disadvantages of the prior art by providing novel structures, systems and methods for reducing and controlling the translocation velocity of biomolecules and biopolymers through membranes, either solid state or biological, having nanopores as used in fluorescence-based detection systems for analyzing and testing translocating biopolymers. The novel structures, systems and methods enable a sufficiently strong electric field to be generated (based on an applied voltage difference between the two sides of a membrane) which attracts biopolymers and biomolecules to the nanopores yet enables the rapidly moving biopolymers to slow down their translocation velocity as they migrate through the nanopores and their respective adjacent optical excitation zones. The controlled velocity of the biopolymers through the nanopores enables an increased signal-to-noise ratio (herein abbreviated SNR) of the photons emitted by the biopolymers thereby enabling reliable optical detection and analysis.

[0055] According to a first embodiment of the disclosed technique, the viscosity of the ionic solution adjacent to the nanopores is increased. The viscosity increase is localized to the nanopores and the region around the nanopores such that biopolymers in the ionic solution can still rapidly migrate towards the nanopores. However once the region adjacent to the nanopores is reached by the biopolymers, the increased viscosity reduces their translocation velocity as they migrate through the nanopores. As previously explained above, in biological reaction detection systems using a membrane with nanopores which use a generated electric field, the electric field has significant strength only in the immediate vicinity of the nanopores in the membrane separating the reservoir into two, however elsewhere in the ionic solution, the strength of the electric field is so weak it is practically speaking nonexistent. Therefore, according to the disclosed technique, the increase in viscosity of the ionic solution should be localized to the immediate vicinity of the nanopores where the generated electric field has significant strength. If this were not the case and the viscosity of the entire ionic solution were increased, the rate at which biopolymers approach the vicinity of the nanopores would be slowed down significantly and it may take hours or even days for a biopolymer to cross through a nanopore. According to the disclosed technique, biopolymers are enabled to move quickly and rapidly towards the region adjacent to the nanopores however then their translocation velocity is slowed down significantly due to the localized increase in viscosity of the ionic solution. In a first instance of this embodiment, the viscosity of the ionic solution adjacent to the nanopores is increased by bonding a hydrogel layer of controlled thickness to the membrane structure of the detection system in which the nanopores are located before the ionic solution is placed in the reservoir (i.e., the container) of the detection system. The hydrogel enables the ionic solution as well as most biopolymers to move through the nanopores however the translocation velocity of the biopolymers will be slowed down when they enter the hydrogel while nonetheless allowing for continual movement of the biopolymers through the nanopores. It is noted that the hydrogel used according to the disclosed technique should not affect the ions in the ionic solution thereby maintaining the strength of the generated electric field in the region adjacent to the nanopores. As described in greater detail below, this embodiment can be achieved through the use of a hydrogel layer which is applied to the membrane separating the reservoir into two. According to the disclosed technique, the hydrogel can be applied in the case of plasmonic optical excitation as well as waveguide optical excitation. The hydrogel essentially acts as a kind of physical impediment which biopolymers need to travel through to get to the nanopores.

[0056] In a second instance of this embodiment, folded nucleic acid molecules (such as DNA origami) can be used as a sieve adjacent to the nanopores to slow down the translocation velocity, thereby increasing the viscosity of the ionic solution locally. An electromagnetic (herein abbreviated EM) concentration zone, located above each nanopore and also slightly larger than each nanopore, can be used as a receptacle for holding the folded nucleic acid molecule. The folded nucleic acid molecules themselves may be charged, thereby also being attracted to the nanopores when the electric field is generated. The folded nucleic acid molecules however are designed to be larger than the nanopores, thus being attracted locally to the region adjacent to the nanopores but not actually being able to pass through the nanopores. When biopolymers approach the region adjacent to the nanopores, they will need to migrate through the folded nucleic acid molecules before being able to migrate through the nanopores. This will effectively slow down the translocation velocity of the biopolymers while nonetheless keeping this reduced velocity localized to the region adjacent to the nanopores.

[0057] According to a second embodiment of the disclosed technique, the excitation zone, where a biopolymer is illuminated by light beams for generating fluorescence, is moved to an area in the detection system where the generated electric field is weaker. As described above, one of the problems of the prior art is that fluorescence-based detection systems using a membrane with nanopores which use a generated electric field are subject to Ohm’s Law and that due to the very small size and area of the nanopores positioned in the membrane, the strength of the generated electric field increases significantly in the excitation zone near the nanopores which increases the translocation velocity of biopolymers crossing through the nanopores. In this embodiment, the excitation zone is distanced, or jacked, from its proximity to the nanopores, thereby leading to a positioning of the excitation zone where the generated electric field is weaker. The result is that even though biopolymers of interest may cross through the nanopores with a high translocation velocity, they will cross through the excitation zone with a slower translocation velocity that enables an increase in SNR of the fluorescence emitted, therein leading to reliable optical detection and analysis of the biopolymers. This embodiment is achieved by adding a distancing layer between the membrane and either the metal film or the waveguide layer such that the excitation zone provided by the metal film or the waveguide layer is moved to an area in the detection system which has a weaker electric field. The distancing layer can be made from a dielectric material.

[0058] According to a third embodiment of the disclosed technique, the translocation velocity of biopolymers and biochemicals through a nanopore is slowed down by locally varying the effective electric field in the region adjacent to the nanopore and the excitation zone. In this embodiment, a local electric field is generated in the region adjacent to the nanopore and the excitation zone which can be controlled independently. Thus even if there is a strong electric field in the region adjacent to the nanopore due to the geometry of the substrate and membrane, an opposite strength local electric field can be generated in the region adjacent to the nanopore to cause an effective electric field (i.e., the electric field experienced by the biopolymer) with reduced strength, thereby leading to a slowing down of the translocation velocity of the biopolymer. In this embodiment, a respective microelectric field can be generated around each nanopore which may have an opposite polarity to the macroelectric field generated in the ionic solution where the substrate and reservoir are located. The microelectric fields can be controlled independently of the macroelectric field and thus the translocation velocity of biopolymers through nanopores can be controlled accordingly. Therefore, as opposed to the second embodiment described above wherein the excitation zone is migrated away from the nanopore to thus expose the excitation zone to a lower strength electric field, in this embodiment, the strength of the effective electric field adjacent to the nanopore is modified directly.

[0059] Local control of the electric field adjacent to a nanopore can be exercised according to the disclosed technique by depositing microelectrodes adjacent to the nanopores in the substrate. Thus the substrate of the disclosed technique includes a membrane having nanopores, either a metal film or a waveguide positioned over the membrane and then microelectrodes deposited over each region adjacent to the nanopores. In one embodiment, the microelectrodes may be formed in the metal film positioned over the membrane when plasmonic excitation is used. According to the disclosed technique, two types of microelectrodes can be deposited for locally controlling the effective electric field adjacent to a nanopore. In a first instance of this embodiment, non-isolated microelectrodes are deposited adjacent to nanopores wherein the microelectrodes are exposed to the ionic solution placed in the container of the detection system. The current in the ionic solution thus may flow through the microelectrodes. In a second instance of this embodiment, isolated microelectrodes are deposited adjacent to nanopores. Isolation of the microelectrodes from the ionic solution can be performed by placing the microelectrodes between dielectric films.

[0060] In each instance of this embodiment, the microelectrodes serve the purpose of enabling local shaping of the effective electric field in the ionic solution adjacent to the nanopores. Local shaping of the effective electric field is achieved by controlling the local voltage in the vicinity of the nanopores with respect to the voltage difference generated in the ionic solution from the electrodes used to apply the macroelectric field to the ionic solution. According to the disclosed technique, control of the local electric field adjacent to the nanopore can be done in an open loop configuration or a closed loop configuration. In an open loop configuration, the generated local electric field is generated all the time regardless of whether biopolymers or biochemicals are passing through the nanopores or not by the constant application of a voltage differential in the ionic solution. In a closed loop configuration, the generated local electric field is only generated (i.e., through the application of a voltage differential in the ionic solution) when it can be identified that biopolymers or biochemicals are indeed passing through the nanopores. The passage of biopolymers through the nanopores can be identified, for example when the detector of the detection system detects fluorescent rays emanating from the ionic solution, thereby signaling a capture event of emitted fluorescent rays. Thus in this configuration, only when a capture event is identified is current provided to the microelectrodes for modifying the local electric field, thereby slowing down the translocation velocity of biopolymers only when they are indeed crossing through the nanopores. If a capture event is not identified, then no current is provided to the microelectrodes and the overall electric field of the ionic solution is maintained, even in the area adjacent to the nanopores, to encourage the migration of biopolymers and biochemicals to the nanopores.

[0061] It is noted that the disclosed technique is described in relation to the detection and analysis of biopolymers however the disclosed technique can relate to any kind of polymer whether organic or inorganic, biological or non-biological. As mentioned above, biopolymers can be proteins, DNA and RNA as well as other chains of molecules bonded together.

[0062] As the disclosed technique relates to fluorescence-based detection systems in general, the disclosed technique can be used with different configurations of such detection systems regarding how light beams are entered into the container and ionic solution and how fluorescent rays from the biopolymer are detected. Whereas the focus of the disclosed technique is on controlling the translocation velocity of biopolymers through nanopores in such detection systems, for the purposes of clarity, two generic configurations of detection systems are now presented which can be used with the disclosed technique. Reference is now made to Figure 2A, which is a schematic illustration of a fluorescence-based detection system using plasmonic excitation, generally referenced 100, constructed and operative in accordance with an embodiment of the disclosed technique. Detection system 100 includes a container 102, a substrate 105, a light source (not shown) and a detector (not shown). Container 102 effectively forms a reservoir which is separated into two separate sections via substrate 105. An ionic solution 104 is placed in container 102. Substrate 105 includes a membrane 106 in which at least one nanopore 114 is positioned in and also includes a metal film 108, deposited directly on membrane 106 in which an opening 1 16 is positioned. Membrane 106 can be positioned above or below metal film 108. As shown, opening 1 16 is larger than nanopore 114 and is positioned in line with nanopore 1 14. Figure 2A is schematic therefore it is clear to the worker skilled in the art that membrane 106 may include a plurality of nanopores and openings even though only a single nanopore and opening are shown. In general, membrane 106 and metal film 108 may be deposited and / or grown on a silicon wafer (not shown) which is then placed in container 102, as is known to the person skilled in the art.

[0063] A biopolymer 112 will migrate through nanopore 1 14 when a generated electric field 110 is activated, as biopolymer 1 12 may be pretreated to include at least one charged ion. Generated electric field 110 therefore effectively creates an Ohmic current via nanopore 1 14. Metal film 108 and opening 116 effectively function as an EM concentration zone for influencing the shape of the generated electric field near nanopore 114 and may also improve the spatial confinement of the generated electromagnetic excitation field using plasmonic effects. As shown, the light source emits a light beam 118 towards opening 1 16 and nanopore 114. As biopolymer 1 12 translocates through nanopore 114, it absorbs light beam 1 18 and in turn emits fluorescent ray 120 which can be detected by the detector. As shown, this occurs in an optical (also referred to as spatial) excitation zone 117 adjacent to opening 116 and nanopore 114. Light beam 118 is shown approaching opening 116 at an angle suggesting that the light source is positioned at an angle to opening 116. However the light source may be placed in a variety of positions, including directly above opening 116 and also under opening 116, as is known to the person skilled in the art. Likewise fluorescent ray 120 is shown propagating from opening 116 at an angle suggesting that the detector is also positioned at an angle to opening 116. However the detector may be placed in a variety of positions with additional optical elements (not shown) for guiding fluorescent ray 120 to the detector. Detection system 100 can be referred to as exciting biopolymer 112 via plasmonic excitation. Light beam 1 18 is an excitation light beam which propagates through ionic solution 104 for interacting with biopolymer 1 12 in optical excitation zone 117 where the interaction between light beam 1 18 and biopolymer 112 generates fluorescence as fluorescent ray 120, which is directed towards a detector (not shown). The spatial confinement of the optical excitation zone is achieved by the presence of opening 1 16 in metal film 108 which ensures that only photons generated within optical excitation zone 117 reach the detector, for example, in the case where the detector is on the same side of the membrane as the direction of travel of biopolymer 1 12 and / or more generally, according to the depth of field of the focusing system of the detector being focused on the optical excitation zone. The plasmonic effect enables further confinement of the excitation field in the spatial excitation zone by plasmons (not shown) on the surface of metal film 108 surrounding the nanopore.

[0064] It is noted as well that regarding average dimensions, membrane 106 may have a thickness ranging from 5 to 100 nanometers (herein abbreviated nm), with nanopore 1 14 having a diameter between 1 to 40 nm. Metal film 108 may have a thickness of 20 to 300 nm, with opening 116 also having a diameter of between 20 to 150 nm. Membrane 106 may be made from a silicon-based composition having the general chemical formulation of SiNx where ‘X’ is the ratio of nitride to silicon and is chosen to achieve a low stress condition for membrane 106. Membrane 106 can also be made from SiO2, HfC and 2D materials such as graphene. Metal film 108 can be made from conductor metals such as gold (Au), silver (Ag), aluminum (Al), titanium (Ti), palladium (Pd), platinum (Pt) or copper (Cu).

[0065] Reference is now made to Figure 2B, which is a schematic illustration of a fluorescence-based detection system using waveguide excitation, generally referenced 150, constructed and operative in accordance with another embodiment of the disclosed technique. Detection system 150 includes a container 152, a substrate 155, a light source (not shown) and a detector (not shown). Container 152 is effectively a reservoir separated into two separate sections via substrate 155. An ionic solution 154 is placed in container 152. Substrate 155 includes a membrane 156 in which at least one nanopore 164 is positioned in and also includes a light waveguide 158, deposited in close proximity to membrane 156 in which an opening 166 is positioned. Membrane 156 can be positioned above or below light waveguide 158. As shown, opening 166 is larger than nanopore 164 and is positioned in line with nanopore 164. Figure 2B is also schematic therefore it is clear to the person skilled in the art that membrane 156 may include a plurality of nanopores and openings even though only a single nanopore and opening are shown. Light waveguide 158 can be constructed as a dielectric layer (or thin dielectric film) in which light beams can be propagated towards opening 166 and nanopore 164. Light waveguide 158 can also include a metallic section (not shown). As mentioned above, light waveguide 158 and membrane 156 may be deposited and / or grown on a silicon wafer (not shown) which is then placed within container 152, as is known to the person skilled in the art.

[0066] A biopolymer 162 will migrate through nanopore 164 when a generated electric field 160 is activated, as biopolymer 162 may be pre-treated to include at least one charged ion. Opening 166 in light waveguide 158 effectively allows biopolymer 162 to interact in free space with a light beam 168 propagating along light waveguide 158. As shown, the light source propagates light beam 168 through light waveguide 158 towards opening 166 and nanopore 164 in an optical excitation zone 165 which is adjacent to opening 166 and nanopore 164. As biopolymer 162 threads through nanopore 164, it absorbs light beam 168 and in turn emits fluorescent ray 170 which can be detected by the detector. The position of the light source is not shown as light beam 168 can be entered into light waveguide 158 in a variety of ways. For example, the light source may be embedded in the container, may be located above the ionic solution or may be positioned in line with light waveguide 158, as is known to the person skilled in the art. Likewise fluorescent ray 170 is shown propagating from opening 166 at an angle suggesting that the detector is also positioned at an angle to opening 166. However the detector may be placed in a variety of positions with additional optical elements (not shown) for guiding fluorescent ray 170 to the detector. Detection system 150 can be referred to as exciting biopolymer 162 via waveguide excitation as the light beams for causing fluorescence in biopolymer 162 are propagated through a dielectric layer acting as a light waveguide towards opening 166 and nanopore 164. Regarding average dimensions, membrane 156 may have a thickness ranging from 5 to 100 nm, with nanopore 164 having a diameter between 1 to 40 nm. Light waveguide 158 may have a thickness of 50 to 800 nm and a width of 50 to 800 nm, with opening 166 having a gap length of between 30 to 300 nm. Membrane 156 may be made from a silicon-based composition having the general chemical formulation of SiNx where ‘X’ is the ratio of nitride to silicon and is chosen to achieve a low stress condition for membrane 156. Membrane 156 can also be made from SiO2, HfO2and 2D materials such as graphene. Light waveguide 158 can be made out of any of the following materials: SiO2, TiO2, Ta2O5, Nb2O5, Si3N4, AI2O3, SiNx (where ‘X’ represents a ratio of silicon to nitrogen which is typically determined by the stress requirements of the waveguide in a given arrangement), PMMA (poly(methyl methacrylate)) or PDMS (polydimethylsiloxane).

[0067] As the disclosed technique relates to controlling the translocation velocity in the area and region adjacent to nanopores 114 (Figure 2A) and 164 (Figure 2B) and openings 116 (Figure 2A) and 166 (Figure 2B), for the sake of clarity, the continued description of the disclosed technique will not specifically refer to common elements of the detection systems mentioned above, such as the container, the light source, the detector and a generator for applying an electric field to the ionic solution, as these elements will be assumed to be present in the structures, configuration, systems and methods presented below. This includes the silicon wafer (in some embodiments) upon which the membrane which divides the reservoir into two is deposited and / or grown upon.

[0068] Reference is now made to Figure 3A, which is a schematic illustration of a first substrate of a fluorescence-based detection system using plasmonic excitation for locally increasing the viscosity of an ionic solution adjacent to a nanopore, generally referenced 200, constructed and operative in accordance with a further embodiment of the disclosed technique. As mentioned above, not all the elements of detection system 200 are shown in Figure 3A for the sake of clarity. Figure 3A substantially shows the substrate of detection system 200 which separates an ionic solution 204 in a reservoir formed in a container into two. In Figure 3A, detection system 200 is shown from a top view 202A and a cross-sectional view 202B, wherein cross-sectional view 202B is shown along the line A-A in top view 202A, shown by a line 216. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 206, a metal film 208 and a hydrogel coating 210. As shown, membrane 206 includes a nanopore 212 and metal film 208 includes an opening 214, which is substantially centered over nanopore 212. Both nanopore 212 and opening 214 are shown in top view 202A as being circular in shape however other shapes are possible. Opening 214 is larger in diameter than nanopore 212 and serves the function with metal film 208 as an EM concentration zone for influencing the shape of the generated electromagnetic field near nanopore 212 and may also improve the spatial confinement of the generated electromagnetic field using plasmonic effects. The electric field is spatially shaped primarily by nanopore 212 and encourages polymers (not shown) in ionic solution 204 to migrate towards opening 214 and then through nanopore 212.

[0069] As shown, metal film 208 is deposited over membrane 206 and hydrogel coating 210 is deposited over metal film 208. Even though hydrogel coating 210 is shown as only covering metal film 208 and filling opening 214, hydrogel coating 210 may also fill (not shown) nanopore 212 and may even coat a portion of the underside of membrane 206. Hydrogel coating 210 may chemically bind to membrane 206. It should be noted as well that if metal film 208 is not made from a noble metal (such as Au, Pt and / or Pd), then typically a dielectric layer (not shown) should be deposited over metal film 208 to protect it from ionic solution 204. Such a dielectric layer could be made, for example, from SiO2, TiC>2, HfO2 and / or AI2O3. In such a case, the hydrogel coating would bind to this dielectric layer thereby covering the entire upper surface of the substrate. It should be additionally noted that lithography methods may be employed over either the metal film or the dielectric layer to define where the hydrogel coating is bonded and thus where it is present and where it is not. The use of a dielectric layer in this scenario does not affect the reduction of the translocation velocity of the biopolymer by the hydrogel coating. In general, the deposition of the hydrogel layer comprises several stages. In a first stage, a layer to chemically bond the gel to is created. In a second stage, the gel is physically deposited. This usually includes physically depositing, for example, a liquid acrylamide and then setting the deposited gel to a desired thickness (for example by using spinning, spraying, gravity and the like). In a third stage and final stage, gel cross-linking is performed, in which the deposited liquid is polymerized to form a gel (for example, by a cross-linking agent). The final deposition stage used in the disclosed technique is similar to specific deposition techniques used in the semiconductor industry for depositing thin layers, such as the deposition of soft layers (for example, the deposition of a photoresist). The bottom left hand corner of top view 202A shows a cutaway of the layers of the substrate whereas cross-sectional view 202B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 202B, hydrogel coating 210 covers metal film 208 and also fills opening 214. Hydrogel coating 210 is permeable and thus a biopolymer 222 can traverse through hydrogel coating 210 towards opening 214 and then through nanopore 212. Shown as well in cross-sectional view 202B is a light beam 218 which is directed towards opening 214 and in general towards an optical excitation zone 223. Light beam 218 originates from a light source (not shown). As light beam 218 interacts with biopolymer 222 in optical excitation zone 223, biopolymer 222 emits a fluorescent ray 220 which can be detected by a detector (not shown). Due to the presence of hydrogel coating 210, the translocation velocity of biopolymer 222 through opening 214 and nanopore 212 (or vice-versa, through nanopore 212 and then through opening 214) will be slowed down significantly such that a first fluorescent ray (not labeled) emitted from biopolymer 222 can be distinguished from a second fluorescent ray (not labeled) in the detector. As mentioned in the prior art, without the presence of a mechanism for slowing down the translocation velocity, such as hydrogel coating 210, the first and second fluorescent rays will substantially arrive at the detector at the same time, thus making it very difficult to distinguish between the two fluorescent rays. In addition, without a reduction in the translocation velocity, the SNR of the detected fluorescent rays won’t be high enough to enable reliable optical detection and analysis of the biopolymer. As is clear to the person skilled in the art, the example given above is merely schematic. A biopolymer may give off tens of fluorescent rays simultaneously according to the prior art whereas the disclosed technique allows for sufficiently slow movement of the biopolymer through the opening in the metal film where the light beams are directed towards such that each fluorescent ray emitted can be resolved and distinguished from a subsequently emitted fluorescent ray with a high SNR for reliable detection and analysis.

[0070] Hydrogel coating 210 can be any gel which is typically used in DNA or protein electrophoresis. Thus examples of hydrogel coating 210 can include starch, agarose and polyacrylamide, however any material which is both soluble in water and also able to be polymerized to form a gel could also be used (such as polyethyleneglycol (also known as PEG) which is described below). Hydrogel coating 210 may thus also be embodied as a gel or as a nanoporous material. Whereas the specific process of depositing hydrogel coating 210 over metal film 208 depends on the type of gel used, in general, deposition of a liquid to be gelated is performed before the process of gelation. In general, once metal film 208 has been deposited on membrane 206, liquid deposition techniques as used in the semiconductor industry, such as spin-coating, spray-coating, dip-coating, roller-coating and the like, can be used to deposit a liquid to be gelated over metal film. The aforementioned techniques allow for control of the thickness of the liquid deposited and thus also the thickness of the hydrogel coating formed once the liquid goes through a process of gelation. In general, during liquid deposition for gelation, there is no strict control of the gelation process in the XY plane, thus when the liquid is deposited, it will substantially cover all of metal film 208. As mentioned above, lithographic masking can optionally be used during the processes of gel deposition to specify specific portions of the metal film where the hydrogel coating is to be deposited (usually adjacent to openings (i.e., nanopores) in the membrane). According to the disclosed technique, the thickness of hydrogel coating 210, shown by a marker 221 , should be as thin as possible since hydrogel coating 210 will reduce the velocity of any biopolymer entering into hydrogel coating 210 and the desire of the disclosed technique is to only reduce the velocity of biopolymer 222 when it enters optical excitation zone 223 and not elsewhere in ionic solution 204. Optical excitation zone 223 may be smaller in thickness than the thickness of opening 214. In general, the thickness of the optical excitation zone is defined by the plasmonic effect of the specific aperture used (in this case, the shape and size of opening 214 in metal film 208), including the thickness of metal film 208, the metal which metal film 208 is made from (as well as the material from which any dielectric layers are made from if dielectric layers are included in the substrate structure) and the wavelength of the excitation in the biopolymer. It is noted however that the thickness of hydrogel coating 210 should not be thinner than the optical excitation zone where the translocation velocity of a biopolymer is to be slowed down. In theory, the hydrogel coating would only be present in the region adjacent to opening 214, however it is not possible to strictly control the gelation process in the XY plane (unless lithographic masking techniques are used). However the Z-plane of the gelation process is controllable and thus according to the disclosed technique, the gel coating is as thin a layer as possible over metal film 208 however the thickness of the gel coating is increased in the region adjacent to opening 214 (to at least match the thickness of optical excitation zone 223).

[0071] Different gelation processes can be used to deposit a gel precursor which then becomes hydrogel coating 210. For example, in the case of temperature-based gelation, deposition of the gel precursor is performed over metal film 208 at a temperature at which the gel precursor is a liquid. The substrate (including membrane 206, metal film 208 and the gel precursor) is then cooled which results in the gel precursor becoming hydrogel coating 210. This is the case when hydrogel coating 210 is made from starch or agarose, both of which undergo gelation through changes in temperature. In the case of gelation based on a chemical polymerization reaction, which is one method for forming hydrogel coating 210 out of polyacrylamide, deposition of the gel precursor is done when the gel precursor is liquid which then leads to a polymerization reaction (which results in radical polymerization) that results in the formation of hydrogel coating 210. Polymerization reactions are not instantaneous and thus require time. Gelation can also occur based on photochemical polymerization, which is another method for forming hydrogel coating 210 out of polyacrylamide. Deposition of the gel precursor is also performed when the gel precursor is a liquid after which polymerization is initiated using optical curing (which also results in radical polymerization). Sufficient optical curing thus results in the formation of hydrogel coating 210.

[0072] As a specific example of the deposition of hydrogel coating 210, a 30% polyacrylamide gel can be prepared via a gel precursor having a ratio of acrylamide to bisacrylamide of 19:1 . As another example, polyacrylamide gels can be prepared have a gel range of 10% to 40% wherein the ratio of acrylamide to bisacrylamide can range from 9:1 to 39:1 respectively. The relatively high percentage of the polyacrylamide in the gel and the low ratio of acrylamide to bisacrylamide increases the generation of small gel pores in and around opening 214. Typical gel coatings used in DNA or protein electrophoresis are used for molecular separation wherein a slowing down of the velocity of the DNA and / or protein through the gel coating is proportional to the molecular length of the molecule. Thus, in the prior art, a plurality of different DNA molecules can be separated into smaller molecules based on size and / or weight, with smaller / lighter molecules congregating in one area of the gel coating whereas larger / heavier molecules will congregate in another area of the gel coating. The DNA molecules in each area are not separated and stuck in one place, as smaller / lighter molecules will move and migrate faster than bigger / heavier molecules which will move and migrate slower. In the case of the disclosed technique, hydrogel coating 210 is targeted as much as possible (for example, in its thickness) to the regions adjacent to where biopolymer excitation occurs (i.e., optical excitation zone 223) for the purposes of slowing down the translocation velocity of the biopolymer rather than the typical use of a gel coating for molecular separation which requires a slowing down of the velocity of a moving molecule which is proportional to the molecule’s length.

[0073] Hydrogel coating 210 as a polyacrylamide gel coating can also be prepared in the presence of ammonium persulphate (herein abbreviated APS) and TEMED (1 ,2bis(dimethylamino)ethane or tetramethylethylenediamine) which is used as a catalyst for chemical polymerization. In such a case, the chemical polymerization reaction is initiated by APS, which can take between 10--20 minutes to complete. In the case of photochemical polymerization, a photo-sensitive radical reaction initiator, such as riboflavin is added to polyacrylamide mixture and optical curing is initiated by light induction, which takes place over the course of 30-60 minutes, with gel casting being done at 23-25 °C. After polymerization, the gel coating is rinsed, typically by a water-based buffer, at which point the prepared substrate with hydrogel coating 210 can be placed in a container (not shown) and filled with ionic solution 204.

[0074] It is noted that hydrogel coating 210 embodied as acrylamide can actually serve two purposes regarding the structure of the substrate. First, a monolayer of cross-linked acrylamide may contribute to the overall force of the bonds in membrane 206 thus resulting in a more robust and long-lasting nanopore 212 (i.e., a nanopore which retains its initial dimensions over time). Second, an acrylamide hydrogel film (i.e., an example of hydrogel coating 210) on membrane 206 and metal film 208, on top of nanopore 212, can serve as a sieve structure that will slow down the translocation velocity and help in the separation of biopolymers (such as proteins) as they migrate through nanopore 212 (similar to what occurs in acrylamide gel electrophoresis).

[0075] Hydrogel coating 210 as a polyacrylamide gel coating can also be prepared by first depositing an adhesive monolayer formed from functionalized siloxanes (such as aminosilanes, carboxysilanes and the like) that bind to a dielectric (which can also be referred to as a chip passivation) layer (typically SiO2, TiOs, SiNx or HfOz) on the surface of the metal film and the membrane, through silicon-oxygen bonds. Different deposition schemes for a polyacrylamide gel coating are possible depending on the base surface upon which the gel coating is to be deposited. In the case of deposition on a dielectric layer being silicon-based, the acryl groups are linked to the dielectric layer by first forming an acrylic monolayer through siloxane chemistry. After that, the gel coating is grown by a common radical polymerization reaction. In the case of deposition on a metal film (assuming it is made from a noble metal such as Au, Pt and / or Pd), the acryl groups are linked to the metal film by first forming an acrylic monolayer through thiol-metal chemistry. After that, the gel coating is grown by a common radical polymerization reaction. In the case of deposition on the membrane (where covalent linking to the membrane is not utilized), a pre-prepared gel coating can be deposited on the membrane and remain attached to it due to weak forces (for example, the electrostatic force, the Van der Waals force and the like). This kind of formation can leave active and functional groups (such as amine, thiol, carboxyl and other groups) protruding towards the ionic solution. In a second step, functionalized acrylate and / or methacrylate groups can be bonded to the active silane groups in the functionalized siloxane monolayer that will result in a composite silane-acrylate monolayer. After washing, the protruding acrylate groups can be polymerized by the addition of acrylamide molecules, resulting in a covalently bonded 3D hydrogel layer by radical polymerization. Following radical polymerization, a polyacrylamide hydrogel layer as hydrogel coating 210 may then be formed on the metal film and membrane surfaces.

[0076] Besides the examples given above for hydrogel coating 210, such as starch, agarose and polyacrylamide, polyethylene glycol (herein abbreviated PEG) can also be used for forming a coating serving a similar function to hydrogel coating 210. PEG is a polymer which is available at various molecular weights and can be deposited over metal film 208 and / or membrane 206 in an aqueous solution which is then cured via the application of ultraviolet light. PEG is not a gel per se yet similar to microvilli in the nose regarding its structure, resembling long strands of molecules that elevate the viscosity which the biopolymer travels through, thereby slowing down the movement of biopolymers. PEG is a hydrophilic polymer which differs in its length according to how many repeats it has of a monomer unit and can also possess functional groups. A molecule of PEG can serve to elevate the viscosity of the path in which biopolymers pass through, also causing steric interference in that path and by these means reduce the translocation velocity of the biopolymers. In such an embodiment, the strands of PEG will bond with metal film 208 in opening 214 if metal film 208 is made from a noble metal as well as with membrane 206 in nanopore 212. If metal film 208 is not made from a noble metal, then the strands of PEG will bond to a dielectric layer (not shown yet mentioned above as a chip passivation layer) protecting metal film 208 from the ionic solution. In such a scenario, the strands of PEG can bond with membrane 206 as well as to the inside of nanopore 212. Besides PEG, other carbon-based chain molecules can be used as well to form a coating serving a similar function to hydrogel coating 210. As mentioned above, metal film 208 can be covered by a dielectric layer (for example, made from SiO2, TiO2, SiNx and / or HfO2) deposited over the metal film to which PEG can be bonded to using siloxane chemistry.

[0077] As another embodiment of the disclosed technique, hydrogel coating 210 can be embodied as a thin polymer coating using either natural polysaccharides or synthetic polymers for slowing down the translocation velocity of biological molecules passing through a nanopore. Thus hydrogel coating 210 can be embodied using polysaccharides such as dextran, agarose, alginate, chitosan or hyaluronic acid or using synthetic polymers such as polyacrylamide, polyethylene glycol (also known as PEG) or polyvinyl alcohol (also known as PVA). According to the disclosed technique as described herein, hydrogel coating 210 can be further modified to improve and fine-tune its functioning. For example, modifications to hydrogel coating 210 can be used to fine-tune how much the translocation velocity of biological molecules through a nanopore are slowed down, thus attaining an ideal translocation velocity of biological molecules for optimizing optical detection of the biological molecules as they travel through a nanopore. Modifications to hydrogel coating 210 can also be used to add functional groups to the hydrogel coating to achieve other desired features such as preventing the biological molecules from adhering to the substrate surface, maintaining the hydrophilicity and stability of the outer surface of the substrate especially under dry conditions for long term storage and maintaining the stability of the nanopores in the substrate under various conditions including changes in the electric field. It is noted as well that the modifications made to hydrogel coating 210 may be designed and applied differently for different types of biomolecules. For example, different modifications to hydrogel coating 210 can be made for proteins and biomolecules having different ranges of molecular weights.

[0078] As described in greater detail below, hydrogel coating 210 can be modified by adding cross-linking reagents. The addition of cross-linking reagents can create an additional mesh in the hydrogel coating, thus effectively enabling a slowing down of the translocation velocity of a biomolecule. As mentioned above, the amount of cross-linking can be fine-tuned because too much cross-linking can cause the hydrogel coating to be impassable by a biomolecule. Likewise, the hydrophilicity, hydrophobicity or electric charge of the hydrogel coating can be modified accordingly by an appropriate quantity of functional groups added to the hydrogel coating. In general, according to the disclosed technique, the modification of hydrogel coating 210 can be done under mild conditions in high yield, to ensure enduring of the hydrogel coating functioning. It is noted as well that functional groups added to hydrogel coating 210 can also function to bind or increase the binding strength of hydrogel coating 210 to metal film 208 and / or membrane 206.

[0079] As an example of a natural polysaccharide which can be used to embody hydrogel coating 210 which can be modified to include additional functional groups, dextran-alkyne can be used. Dextran is a polysaccharide from which hydrogels and thin hydrogel layers on surfaces can be formed and is commonly used in capillary gel electrophoresis of proteins. Dextran is a polymer made of glucose subunits to which functional groups can be coupled with. Alkynes can be coupled with dextran through a linker, such as an alkyl-ester group. According to the disclosed technique, in the case of using an alkyne group, other functional groups can be easily and simply bonded with the alkyne group. For example, it is known that alkyne groups can react and form covalent bonds with azide (N3) groups in high yield under mild conditions using the known CuAAC (Cu(l)-catalyzed azide-alkyne cycloaddition) click chemistry approach. Thus, hydrogel coating 210 as dextran-alkyne can bind surface azide groups and thus bind or increase the binding strength of hydrogel coating 210 to metal film 208 and / or membrane 206 and / or can be used to bind other functional groups to the dextran-alkyne hydrogel coating.

[0080] Some of the advantages of using a natural or pre-made polymer such as dextran as hydrogel coating 210 as opposed to grafting a synthetic polymer such as polyacrylamide (from its acrylamide monomers) include being both commercially and readily available as a gel coating that comes in various molecular weights. Thus unlike grafted polyacrylamide, dextran (and other natural or pre-made polymers) does not need to be grafted and can be deposited directly over metal film 208 and / or membrane 206. As a readily available gel coating, the thickness and uniformity of the hydrogel coating can be substantially guaranteed, thus ensuring that the thickness deposited on the substrate surface is controlled. In the case of a grafted polymer such as polyacrylamide, it is much more difficult to ensure uniformity of deposition as well as uniformity of thickness. As mentioned above and detailed below, numerous efficient and reliable chemical reactions exist for adding functional groups to polysaccharides such as dextran or its derivatives, thus easily enabling the addition of functional groups to the hydrogel coating without causing further complications in the hydrogel coating such as unanticipated chemical reactions. However, the option to chemically modify hydrogel coating 210 is not limited to natural or pre-made polymers, and can similarly be applied to grafted polymers having available functional groups, such as grafted polyacrylamide in which acrylic monomers with functional groups were incorporated during the grafting process. Example for such monomers may be acryl-linker-alkyne.

[0081] In the case of hydrogel coating 210 embodied as dextran-alkyne, the hydrogel coating as well as the substrate can be modified to have bonding properties between hydrogel coating 210 and metal film 208 and / or membrane 206. Membrane 206 may be prepared from silicon nitride to which hydroxide (OH) groups are added via a piranha solution treatment. The resulting membrane will include a plurality of hydroxide groups extending from the membrane surface. A silanization process can then be applied to the membrane surface which allows for azide functional groups to couple with the functionalized silicon (such as silicon nitride) that has available hydroxide groups. PEG can be used as a linker to couple the azide functional groups to the silicon nitride, namely azide-PEG-silane can be used to modify the surface by reaction of the silane groups with hydroxide groups on the surface. With the substrate (such as membrane 206) prepared as such, dextran-alkyne molecules of the hydrogel coating, having functional alkyne groups, can reliably and efficiently bond with the azide functional groups on membrane 206 through a CuAAC click chemistry reaction. Thus according to the disclosed technique, hydrogel coating 210 can be bonded to membrane 206.

[0082] Another use of hydrogel coating made of dextran-alkyne as described above for example, allows for cross-linking the dextran-alkyne hydrogel coating with itself, thus forming a mesh. Cross-linking of hydrogels is a known method for slowing down translocation velocities and can be used here to slow down the translocation velocity of biomolecules moving through a hydrogel. Dextran-alkyne can react and cross-link with reagents and molecules having a general azide-linker-azide structure. Cross-linking is formed under conditions in which both azide groups of one molecule react with alkyne groups in the hydrogel layer. According to another embodiment of the disclosed technique, dextran molecules can be cross-linked using borate complexes, which are commonly used to retard capillary electrophoresis of proteins. This may be done to a cross-link dextran-alkyne hydrogel coating or other types of dextran-containing hydrogel coatings. Even though borate does not form complete covalent bonding, cross-linking of the dextran-alkyne molecules via the borate complexes is still possible for forming a mesh for slowing down the translocation velocity of biomolecules. In general, any of the following cross-linking reagent structures are possible: functional group — linker — functional group (where the functional group can be any one of an alkyne group, an azide group, a dibenzocyclooctyne (DBCO) group, a carboxyl group, an amine group, a hydroxyl group, an aldehyde group, an epoxide group and / or an acrylic group), functional group — PEG — functional group, azide — linker — azide, azide — PEG — azide, acryl — linker — acryl, bisacrylamide, borate complexes, sodium tetraborate and boric acid.

[0083] As mentioned above, dextran-alkyne can also be used to couple functional groups to the alkyne for giving hydrogel coating 210 a specific functionality. Suppose that for given biomolecules (such as proteins) which are either hydrophobic or have hydrophobic regions, increasing the hydrophobicity in hydrogel coating 210 may result in an ideal reduction of their translocation velocity. Thus dextran-alkyne can be reacted with molecules having the general structure of an azide coupled with a hydrophobic group for increasing the hydrophobicity of hydrogel coating 210. Alternatively, dextran-alkyne can be reacted with molecules having the general structure of an azide coupled with an electrostatically charged group for adding electrostatic charge to hydrogel coating 210. Such charged groups may be for example carboxyl, sulfonate or amine groups, and may be bound to the azide group via a linker. According to the disclosed technique, the hydrogel coating layer can thus be functionalized to include electrostatic charge. Such electrostatic charge may reduce the translocation velocity of biomolecules by interaction with charged regions of the biomolecule having the opposite electrostatic charge.

[0084] As mentioned above, dextran-alkyne along with CuAAC click chemistry were used as an example of the disclosed technique to show how hydrogel coating 210 could be embodied along with an ability to be easily and reliably modified chemically. However other examples for embodying the hydrogel coating layer exist, including alternative synthesis approaches for easily and reliably bonding functional groups to the hydrogel coating. Alginate, carboxymethyl dextran and hyaluronic acid, which can all be used for hydrogel coating 210, all include carboxylic groups which can be reacted with amine groups to form amide bonds for adding functionality to hydrogel coating 210. Chitosan and aminoethyl dextran, which can also both be used for hydrogel coating 210, have amine groups which can be reacted with carboxylic acids, epoxides and aldehydes, also for adding functionality to hydrogel coating 210. Dextran, agarose, alginate, chitosan, hyaluronic acid and polyvinyl alcohol (PVA), which can all further be used for hydrogel coating 210, all include hydroxyl groups which can be reacted with acyl chlorides or anhydrides for adding functionality to hydrogel coating 210. And finally, dextran derivatives can further be used for hydrogel coating 210 which include functional groups that can easily be reacted with for adding functionality to hydrogel coating 210. The example of dextran-alkyne with CuAAC click chemistry was already mentioned above, however other dextran derivatives can be used for hydrogel coating 210 such as dextran-dibenzocyclooctyne (dextran-DBCO) which can react with azide groups via click chemistry, dextran-azide which can react with an alkyne or dibenzocyclooctyne (DBCO) via click chemistry as well as dextran-aldehyde which can react with amine groups.

[0085] In general, thickness 221 of hydrogel coating 210 is significantly thinner, by orders of magnitude, than the typical thickness of gel mediums used for molecular separation. Normally, gel thickness is measured as a distance between glass plates in which a gel is deposited. In the case of the disclosed technique, thickness 221 represents either the height and / or length of hydrogel coating 210 through which biopolymers travel through. For example, gel coatings used to separate DNA are usually at least a few centimeters thick and can be even thicker, whereas thickness 221 can be on the order of tens of nanometers. In addition, the desired thickness of hydrogel coating 210 may depend on the construction of the entire substrate (i.e., membrane, metal film and hydrogel coating) and thus may have different ranges depending on the construction of the substrate. Regardless, according to the disclosed technique, the desire is to have hydrogel coating 210 concentrated only in the region adjacent to opening 214 (i.e., optical excitation zone 223), for slowing down the translocation velocity of biopolymer 222 where polymer excitation occurs. Thus, as a further example, thickness 221 may range from around 20 nm to 1000 nm. Thus, the disclosed technique differs from the prior art not only in the thickness of the gel coating used but also in the function of the hydrogel coating as well as the chemical bonding used to bond the hydrogel to the substrate surface. In the prior art, gels used for DNA and protein separation are tailored to create a sieve for slowing down the velocity of molecules proportional to their length. In addition, very high voltages (i.e., 100-200 volts) are employed for the separation. In the disclosed technique, the hydrogel coating is used to slow down the translocation velocity of biopolymers, regardless of their size, in the region of the optical excitation zone where the biopolymers are excited by light beams. Thus, the gel coating is deposited such that it has the smallest thickness possible, only having an increased thickness which is localized to openings adjacent to nanopores. The chemical constitution as well as the method of deposition of hydrogel coating 210 is thus very different than what is typically in use for gel coatings of the prior art.

[0086] According to the disclosed technique, functional groups can be added to hydrogel coating 210 to enhance covalent bonding of hydrogel coating 210 with metal film 208 and / or enhancing the bonding of other structures and / or molecules to hydrogel coating 210. This also applies in the case of hydrogel coating 210 being embodied as PEG. The addition of functional groups to hydrogel coating 210 can strengthen the coupling of hydrogel coating 210 to metal film 208 (or the dielectric layer protecting it), as explained above. As mentioned above, the functional groups can include an alkyne group, an azide group, a dibenzocyclooctyne (DBCO) group, a carboxyl group, an amine group, a hydroxyl group, an aldehyde group, an epoxide group and / or an acrylic group. In one embodiment of the disclosed technique, a bonding layer (not shown), which is functionally equivalent to the dielectric layer mentioned above which can protect metal film 208, can be deposited on the upper layer of the substrate, for example metal film 208 in the example of Figure 3A, before gelation is performed for depositing gel coating 210. The bonding (i.e., equivalent dielectric) layer may be made from gold, platinum and / or silicon dioxide for covalently attaching a monolayer of hydrogel coating 210. Thus, when gelation is performed, hydrogel coating 210 will bond covalently with the bonding layer which is also coupled with metal film 208. The strength of the coupling of hydrogel coating 210 to metal film 208 is thus increased according to the disclosed technique. It is noted as well that the bonding of hydrogel coating 210 to the substrate also provides a consistent surface to nanopore 212 and membrane 206 for ionic solution 204 which acts as a liquid buffer and ensure consistent wetting of the surfaces of the substrate. Thus according to the disclosed technique, the hydrogel coating is not only used for slowing down the translocation velocity but also for stabilizing the chemical and physical structure of nanopore 212 and its immediate vicinity.

[0087] Reference is now made to Figure 3B, which is a schematic illustration of a second substrate of a fluorescence-based detection system using waveguide excitation for locally increasing the viscosity of an ionic solution adjacent to a nanopore, generally referenced 240, constructed and operative in accordance with another embodiment of the disclosed technique. Similar to Figure 3A, not all the elements of detection system 240 are shown in Figure 3B for the sake of clarity. As described below, Figure 3B is similar to Figure 3A, however, the excitation of polymers in the ionic solution is not via plasmonic excitation but rather via waveguide excitation. Figure 3B substantially shows the substrate of detection system 240 which separates an ionic solution 244 in a container (i.e., a reservoir) into two sections. In Figure 3B, detection system 240 is shown from a top view 242A and a cross-sectional view 242B, wherein cross-sectional view 242B is shown along the line B-B in top view 242A, shown by a line 256. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 246, a waveguide 248 and a gel coating 250. As shown, membrane 246 includes a nanopore 252 and waveguide 248 includes an opening 254, which is substantially centered over nanopore 252. In top view 242A, nanopore 252 is shown as being circular in shape and opening 254 is shown as being square in shape, however other shapes are possible. An optical excitation zone 263 is shown wherein biopolymers can interact with light beams and emit fluorescent rays adjacent to nanopore 252 and opening 254. Opening 254 is larger in size than nanopore 252 and serves the function of shaping the electric field strength in the immediate vicinity of nanopore 252. The size of nanopore 252 dictates the electric field strength in the immediate vicinity of nanopore 252 when an electric field is generated in ionic solution 244. The increased electric field generated in nanopore 252 encourages biopolymers (not shown) in ionic solution 244 to migrate towards opening 254 and then through nanopore 252.

[0088] As shown, waveguide 248 is positioned over membrane 246 and gel coating 250 is deposited over waveguide 248 and membrane 246. The deposition schemes for depositing gel coating 250 over waveguide 248 and membrane 246 are similar to what was described above regarding hydrogel coating 210. Thus in general, the deposition techniques used in the last stage of deposition in the disclosed technique are similar to the deposition techniques used in the semiconductor industry for depositing thin layers. The bottom left hand corner of top view 242A shows a cutaway of the layers of the substrate whereas cross-sectional view 242B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 242B, gel coating 250 covers waveguide 248 and also fills opening 254. Gel coating 250 does not need to cover the entire upper surface of waveguide 248 however gel coating should primarily be deposited in optical excitation zone 263 where it is needed for reducing the translocation velocity of biopolymers passing there through. Even though gel coating 250 is shown as only covering waveguide 248 and filling opening 254, gel coating 250 may also fill (not shown) nanopore 252 and may even coat a portion of the underside of membrane 246. Gel coating 250 is permeable and thus a biopolymer 262 can traverse through gel coating 250 towards opening 254 and then through nanopore 252. Shown as well in cross-sectional view 242B is a light beam 258 traversing through waveguide 248 which is directed towards opening 254. Light beam 258 originates from a light source (not shown) which is coupled with waveguide 248. Various techniques can be used to introduce light beam 258 into waveguide 248 from the light source. As light beam 258 interacts with biopolymer 262 in optical excitation zone 263, biopolymer 262 emits a fluorescent ray 260 which can be detected by a detector (not shown). Due to the presence of gel coating 250, the translocation velocity of biopolymer 262 through opening 254 and nanopore 252 (or vice-versa, through nanopore 252 and then through opening 254) will be slowed down significantly such that a first fluorescent ray (not labeled) emitted from biopolymer 262 can be distinguished from a second fluorescent ray (not labeled) in the detector. As mentioned in the prior art, without the presence of a mechanism for slowing down the translocation velocity, such as gel coating 250, the first and second fluorescent rays will substantially arrive at the detector at the same time, thus making it very hard to distinguish between the two fluorescent rays and also not leading to sufficient SNR for reliable optical detection and analysis. As mentioned above, the example given above is merely schematic and a biopolymer may give off tens of fluorescent rays simultaneously according to the prior art whereas the disclosed technique allows for sufficiently slow movement of the biopolymer through the opening in the waveguide where the light beams are directed towards such that each fluorescent ray emitted can be resolved and distinguished from a subsequently emitted fluorescent ray with a high SNR for reliable detection and analysis.

[0089] The deposition and composition of gel coating 250 is substantially equivalent to what was described above regarding gel coating 210 (Figure 3A), including the various natural and pre-made polysaccharides and synthetic polymers which can be used to embody a hydrogel coating and also to add functionality to the hydrogel coating. As shown in Figure 3B, a gel coating can be deposited not only over a metal film but also over a waveguide, wherein the gel coating can serve the same purpose of slowing down the translocation velocity of biopolymers crossing through an optical excitation zone adjacent to a nanopore where fluorescent excitation is to occur.

[0090] Reference is now made to Figure 4A, which is a schematic illustration of a third substrate of a fluorescence-based detection system using plasmonic excitation for locally increasing the viscosity of an ionic solution adjacent to a nanopore, generally referenced 280, constructed and operative in accordance with a further embodiment of the disclosed technique. Detection system 280 is similar in construction to detection 200 (Figure 3A) and will be briefly described before a disclosure of the structure of the substrate which allows for locally increasing the viscosity of the ionic solution adjacent to the nanopore is brought. As mentioned above, not all the elements of detection system 280 are shown in Figure 4A for the sake of clarity. Figure 4A substantially shows the substrate of detection system 280 which separates an ionic solution 284 in a container (i.e., a reservoir) into two sections. In Figure 4A, detection system 280 is shown from a top view 282A and a cross-sectional view 282B, wherein cross-sectional view 282B is shown along the line C-C in top view 282A, shown by a line 296. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 286 and a metal film 288. As shown, membrane 286 includes a nanopore 290 and metal film 288 includes an opening 292, which is substantially centered over nanopore 290. Both nanopore 290 and opening 292 are shown in top view 282A as being circular in shape however other shapes are possible. Opening 292 is larger in diameter than nanopore 290 and serves the function with metal film 288 as an EM concentration zone for influencing the shape of the generated electric field near nanopore 290 and may also improve the spatial confinement of the generated electromagnetic field using plasmonic effects. The electric field is spatially shaped primarily by nanopore 290 and encourages biopolymers (not shown) in ionic solution 284 to migrate towards opening 292 and then through nanopore 290. As described in greater detail below, a folded nucleic acid molecule 294 is positioned in opening 292, which acts as a sieve for slowing down the translocation velocity of a biopolymer 308. Folded nucleic acid molecule 294 is designed to be smaller than the diameter of opening 292 yet larger than the opening of nanopore 290, thus acting as a partial impediment to the movement of biopolymer 308. Folded nucleic acid molecule 294 thus does not block the movement of biopolymer 308 but merely slows down its translocation velocity as it passes through an optical excitation zone 303 adjacent to nanopore 290 where it will be excited by light beams for emitting fluorescence.

[0091] As shown, metal film 288 is deposited over membrane 286 in a manner similar to what was described in Figure 3A. The bottom left hand corner of top view 282A shows a cutaway of the layers of the substrate whereas cross-sectional view 282B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 282B, folded nucleic acid molecule 294 can enter the opening in metal film 288 due to its size but cannot cross into nanopore 290. In addition, since folded nucleic acid 294 is negatively charged, it will be attracted towards the positive electrode (not shown) in ionic solution 284 and thus towards nanopore 290. As mentioned above, folded nucleic acid molecule 294 acts as a sieve and is a partial impediment, thus a biopolymer 308 can traverse through folded nucleic acid 294 towards opening 292 and then through nanopore 290 albeit with a slower translocation velocity. Shown as well in cross-sectional view 282B is a light beam 298 which is directed towards opening 292. Light beam 298 originates from a light source (not shown). As light beam 298 interacts with biopolymer 308 in optical excitation zone 303, biopolymer 308 emits a fluorescent ray 300 which can be detected by a detector (not shown). As mentioned, due to the presence of folded nucleic acid molecule 294, the translocation velocity of biopolymer 308 through opening 292 and nanopore 290 (or vice-versa, through nanopore 290 and then through opening 292) will be slowed down significantly such that a first fluorescent ray (not labeled) emitted from biopolymer 308 can be distinguished from a second fluorescent ray (not labeled) in the detector. As mentioned in the prior art, without the presence of a mechanism for slowing down the translocation velocity, such as folded nucleic acid molecule 294, the first and second fluorescent rays will substantially arrive at the detector at the same time, thus making it very hard to distinguish between the two fluorescent rays. As is clear to the person skilled in the art, the example given above is merely schematic. A biopolymer may give off tens of fluorescent rays simultaneously according to the prior art whereas the disclosed technique allows for sufficiently slow movement of the biopolymer through the opening in the metal film where the light beams are directed towards such that each fluorescent ray emitted can be resolved and distinguished from a subsequently emitted fluorescent ray with a high SNR for reliable detection and analysis.

[0092] Folded nucleic acid molecule 294 is another method according to the disclosed technique wherein an increase in the viscosity in optical excitation zone 303 which is near the region of opening 292 can be localized, thereby enabling biopolymers to migrate quickly towards opening 292 when an electric field is generated in ionic solution 284 yet to have their translocation velocities slowed down once in optical excitation zone 303 wherein light beams are propagated towards the biopolymers. Similar to a gel coating, folded nucleic acid molecule 294 does not have any substantial effects on the ions in ionic solution 284 and merely serves the purposes of slowing down the translocation velocity of biopolymer 308. Folded nucleic acid molecule 294 can be made from a strand of DNA, RNA, PNA (peptide nucleic acid) and / or any other nucleic acid molecule, which is spatially folded into a sieve structure having a 2D or 3D shape exhibiting properties similar to a gel coating for reducing the translocation velocity of biopolymers in the ionic solution. Schematically shown is an open strand of nucleic acid molecules 302 (i.e., a scaffold strand) which is folded (schematically shown as an arrow 304) through hydrogen bonds of complementary nucleotides making up the scaffold nucleic acid strand to form a sieve structure 306, referred to above generally as a folded nucleic acid molecule. Folded sieve structures made from nucleic acids are generally known as DNA origami nanostructures, which in the prior art have primarily been used as a medium of art. A DNA origami nanostructure is typically composed of a long single-stranded DNA molecule, called a “scaffold strand’’. A set of short (e.g., 20-60 bases long) single-stranded DNA molecules (referred to as “staple strands”), are specifically designed and attached to specific locations on the scaffold strand by hydrogen-bonded base pairing complementarity. According to the disclosed technique, folded nucleic acid molecules, such as DNA origami nanostructures, are designed and engineered and used as sieve structures for slowing down the translocation velocity of polymers of interest.

[0093] Folded nucleic acid molecule 294, by its nature as a nucleic acid molecule, has a high negative electric charge. Thus, when an electric field is generated in ionic solution 284, not only will biopolymer 308 be attracted to opening 292 and nanopore 290, folded nucleic acid molecule 294 will also be attracted to opening 292 and nanopore 290. However due to its large dimension as compared to nanopore 290, folded nucleic acid molecule 294 will remain in opening 292 thereby acting as a sieve which biopolymer 308 will have to cross to enter or exit nanopore 290.

[0094] Open strand of nucleic acid molecules 302 can be prepared as folded nucleic acid molecule 294 in another solution (not shown) and once prepared, can then be placed in ionic solution 284. As mentioned above, folded nucleic acid molecule 294 is folded as a large mesh of nucleic acid molecules that includes nanometer-sized cavities between the folded strands of nucleic acids. Biopolymer 308 is small enough to pass through such nanometer-sized cavities however at a slower translocation velocity. A more specific reduced translocation velocity can be achieved by the density of the 3D structure composed of the folded strand of nucleic acid molecules, wherein an increase in the number of folds of the scaffold strand generates a denser mesh and hence a slower translocation velocity. Embodied as a DNA origami nanostructure, folded nucleic acid molecule 294 can be a long DNA molecule (i.e., the scaffold strand), such as the M13mp18 phage, additionally including around 200 staple strands of DNA, each including approximately 20 nucleotides. Each short strand can be considered like a staple which will bind to a complementary staple strand over the long scaffold strand of the M13mp18 phage through the base pairing of hydrogen bonds. The order of nucleotides in each staple strand is selected and designed to bind to specific nucleotides in the long scaffold strand through base-complementary bonding (i.e., hydrogen bonds). The selected order of nucleotides can thus be used to design any desired 2D or 3D nanostructure having sub-nanometer resolution, wherein nanometer precision can be achieved. Thus, folded nucleic acid molecule 294 can be embodied as a long DNA molecule which includes hundreds of short synthetic DNA molecules that couple and attach to the long DNA molecule to fold it into a sieve structure. Regarding example dimensions of folded nucleic acid molecule 294, once a DNA origami nanostructure has been designed, typical expected dimensions of the resulting sieve structure are a consequence of the length of the scaffold strand and the spacing between adjacent strands of DNA in the nanostructure. In a solution, DNA generally has a helical pitch of 35.7 angstroms (herein abbreviated A) and an inter-strand base stack distance of 3.4 A, which results in 10.5 base pairs per turn and a twist angle of 34.3 degrees. The spacing between two adjacent DNA helices in a DNA origami nanostructure is therefore typically 6-8 A. The spacing between adjacent DNA loops in a highly coiled DNA structure depends on the sequence but is usually about 3 A.

[0095] A sieve structure according to the disclosed technique fulfilling the same function as folded nucleic acid molecule 294 can also be constructed through the use of nanostructures such as a 2D sheet containing a central cavity or an assembled 3D mushroom nanostructure. According to the disclosed technique, staple stands of folded nucleic acids can be added to a 2D or 3D nanostructure as micro-protrusions from such a structure. The micro-protrusions can serve as anchors that can bind to complementary strands that are covalently bonded to membrane 286 and / or metal film 288, thus resulting in a hollowed DNA nanostructure which is positioned on top of nanopore 290. The hollowed DNA nanostructure substantially adds an additional nanopore having a controlled diameter (not shown), through which biopolymer 308 can pass. This additional nanopore would be located within nanopore 290, thus serving as an additional barrier through which biopolymer 308 has to pass through and thus results in a further slowing down of the translocation velocity of biopolymer 288.

[0096] As mentioned above with regards to a gel coating, complementary nucleic acid molecules and / or functional groups can be bonded to the ends of a nucleic acid strand (such as a DNA strand). Such complementary nucleic acid molecules and / or functional groups not only strengthen the bonding of the 2D / 3D nanostructures to opening 292 and nanopore 290 but also prepare the 2D / 3D nanostructures for other uses if need be, for example, for adding an additional nanopore within nanopore 290, for attaching additional structures to the 2D / 3D nanostructure already in opening 292 and for further slowing down the translocation velocity of a biopolymer through the optical excitation zone and the nanopore. An example of such additional nanostructures and the bonding of a folded nucleic acid molecule to the substrate according to the disclosed technique is shown below in Figures 4C and 4D.

[0097] In comparing the structure of Figure 3A which uses a gel coating to the structure of Figure 4A which uses a folded nucleic acid molecule, the following differences can be noted. As opposed to a gel coating that needs to be prepared in advance and used to coat the substrate of the container during fabrication, in the case of a folded nucleic acid molecule, each DNA, PNA and / or RNA molecule, after folding, constitutes an independent 2D and / or 3D structure that can be designed with a given structure, charge and even electrical dipole moment. Consequently, the folded nucleic acid molecules can therefore be added into the reservoir of the container in ionic solution 284, either prior to the introduction of biopolymers and biomolecules, at the same time as their introduction or after their introduction into ionic solution 284, wherein a generated electric field is used, based on the structure, designed dipole moment and electrical charge of each folded nucleic acid molecule, to enable each folded nucleic acid molecule to reach their effective position inside a given opening above a nanopore (either in the case of plasmonic excitation or wavelength excitation). Once positioned, the folded nucleic acid molecules can be held there by the generated electric field and can additionally bind to complementary strands that are covalently bonded to the edges of opening 292 and / or nanopore 290, thus bonding the folded nucleic acid molecule to the membrane. In addition, the use of folded nucleic acid molecules is highly localized as they will only slow down the translocation velocity of biopolymer 308 in the region of opening 292.

[0098] Reference is now made to Figure 4B, which is a schematic illustration of a fourth substrate of a fluorescence-based detection system using waveguide excitation for locally increasing the viscosity of an ionic solution adjacent to a nanopore, generally referenced 320, constructed and operative in accordance with another embodiment of the disclosed technique. Similar to Figure 4A, not all the elements of detection system 320 are shown in Figure 4B for the sake of clarity. As described below, Figure 4B is similar to Figure 4A however the excitation of biopolymers in the ionic solution is not via plasmonic excitation but rather via waveguide excitation. Figure 4B substantially shows the substrate of detection system 320 which separates an ionic solution 324 in a container (i.e., a reservoir) into two sections. In Figure 4B, detection system 320 is shown from a top view 322A and a cross-sectional view 322B, wherein cross-sectional view 322B is shown along the line D-D in top view 322A, shown by a line 336. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 326 and a waveguide 328. As shown, membrane 326 includes a nanopore 330 and waveguide 328 includes an opening 332, which is substantially centered over nanopore 330. An optical excitation zone 341 is shown in the vicinity of nanopore 330 and opening 332 wherein a biopolymer interacts with light beams and emits fluorescent rays. In top view 322A, nanopore 330 is shown as being circular in shape and opening 332 is shown as being square in shape, however other shapes are possible. Opening 332 is larger in size than nanopore 330 and serves the function of shaping the electric field strength in the immediate vicinity of nanopore 330. The size of nanopore 330 dictates the electric field strength in the immediate vicinity of nanopore 330 when an electric field is generated in ionic solution 324. The increased electric field generated in nanopore 330 encourages a biopolymer 342 in ionic solution 324 to migrate towards opening 332 and then through nanopore 330. Shown as well is a folded nucleic acid molecule 334, which is designed to fit and remain in opening 332 however is larger in diameter such that it cannot pass through nanopore 332.

[0099] As shown, waveguide 328 is positioned over membrane 326, with waveguide 328 being deposited in a matter similar to the deposition of the waveguide in Figure 3B. The bottom left hand corner of top view 322A shows a cutaway of the layers of the substrate whereas cross-sectional view 322B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 322B, folded nucleic acid molecule 334 fills opening 332. As mentioned above folded nucleic acid molecule 334 acts as a sieve structure and as a partial impediment to the movement of biopolymer 342. Thus, biopolymer 342 can traverse through folded nucleic acid molecule 334 towards opening 332 and then through nanopore 330 albeit at a slower translocation velocity. Shown as well in cross-sectional view 322B is a light beam 338 traversing through waveguide 328 which is directed towards opening 332. Light beam 338 originates from a light source (not shown) which is coupled with waveguide 328. Various techniques can be used to introduce light beam 338 into waveguide 328 from the light source. As light beam 338 interacts with biopolymer 342 in optical excitation zone 341 , biopolymer 342 emits a fluorescent ray 340 which can be detected by a detector (not shown). Due to the presence of folded nucleic acid molecule 334, the translocation velocity of biopolymer 342 through opening 332 and nanopore 330 (or vice-versa, through nanopore 330 and then through opening 332) will be slowed down significantly such that a first fluorescent ray (not labeled) emitted from biopolymer 342 can be distinguished from a second fluorescent ray (not labeled) in the detector. As mentioned in the prior art, without the presence of a mechanism for slowing down the translocation velocity, such as folded nucleic acid molecule 334, the first and second fluorescent rays will substantially arrive at the detector at the same time, thus making it very difficult to distinguish between the two fluorescent rays. As mentioned above, the example given above is merely schematic and a biopolymer may give off tens of fluorescent rays simultaneously according to the prior art, whereas the disclosed technique allows for sufficiently slow movement of the biopolymer through the opening in the waveguide where the light beams are directed such that each emitted fluorescent ray can be resolved and distinguished from a subsequently emitted fluorescent ray with high SNR for enabling reliable detection and analysis.

[0100] The composition and method of manufacture of folded nucleic acid molecule 334 is substantially equivalent to what was described above regarding folded nucleic acid molecule 294 (Figure 4A). As shown in Figure 4B, a folded nucleic acid molecule can also be used in a detection system employing a waveguide. As mentioned above folded nucleic acid molecule 334 may have functional groups (not shown) attached to it for increasing its bonding to opening 332. An example of such is given below in Figures 4C and 4D. Thus when an electric field is generated in ionic solution 324, folded nucleic acid molecule 334, which has a high negative electric charge due to its composition from nucleic acids, will be attracted to opening 332 where there is an increase in the strength of the electric field. Once in opening 332, additional functional groups on folded nucleic acid molecule 334 may be used to further bond folded nucleic acid molecule 334 to the sides of opening 332 and / or to the upper surface of membrane 326 adjacent to nanopore 330.

[0101] Reference is now made to Figure 4C, which is a schematic illustration of a substrate of a fluorescence-based detection system showing the bonding of a nucleic acid molecule with a functional group to the substrate, generally referenced 880, constructed and operative in accordance with a further embodiment of the disclosed technique. Many of the elements of the fluorescence-based detection system have been omitted from Figure 4C for the sake of clarity and the fluorescence-based detection system described herein can refer to any of the fluorescence-based detection system described herein. Shown is a container (not labeled) in which an ionic solution 886 is placed, wherein the container includes a membrane 888 which includes a nanopore 890. Membrane 888 may also have a metal film (not shown), a dielectric layer (not shown) and the like deposited over it at different locations, similar to the other detection systems described herein.

[0102] The container is shown in two views, a first view 882A and a second view 882B. First view 882A shows nucleic acid molecules with a functional group before bonding to a substrate surface whereas second view 882B shows nucleic acid molecules with the functional group after bonding to the substrate surface. The movement from first view 882A to second view 882B is shown via an arrow 884. In first view 882A, as shown, membrane 888 is made from silicon (Si) wherein an upper surface 889 of membrane 888 has an exposed silicon dioxide (SiC ) layer. In ionic solution 886, a plurality of single strand nucleic acid molecules 892 is shown floating around. Each single strand nucleic acid molecule 892 includes a functional group, which in the embodiment shown is a silane (SiHs) end 894. Functional groups are chemical moieties in a molecule which can be used for reacting with other molecules and / or functional groups in an environment. As shown in second view 882B, the functional group of each single strand nucleic acid molecule 892 can be linked with the upper surface of membrane 888 depending on the selection of the chemical composition of the membrane and the functional group of the nucleic acid molecule. As shown, silane end 894 can interact with the exposed silicon dioxide layer of membrane 888 through siloxane bonds, shown schematically as a siloxane bond 896. The left-hand side of second view 882B shows plurality of single strand nucleic acid molecules 892 linked to upper surface 889 whereas the right-hand side of second view 882B shows the linking of one single strand nucleic acid molecule 892 to upper surface 889 via siloxane bond 896. As described below, the linking of single strand nucleic acid molecules to the upper surface of a membrane can be used to bond other structures and molecules to the membrane.

[0103] Shown in Figure 4C is a second container (not labeled) in which an ionic solution 904 is placed, wherein the container includes a membrane 906 which includes a nanopore 908. Membrane 906 may also have a metal film (not shown), a dielectric layer (not shown) and the like deposited over it at different locations, similar to the other detection systems described herein. The second container is shown in two views, a first view 900A and a second view 900B. First view 900A shows another example of nucleic acid molecules with a functional group before bonding to a substrate surface, whereas second view 900B shows nucleic acid molecules with the functional group after bonding to the substrate surface. The movement from first view 900A to second view 900B is shown via an arrow 902. In first view 900A, as shown, membrane 906 is made from gold (Au) having an upper surface 907. In ionic solution 904, a plurality of single strand nucleic acid molecules 910 is shown floating around. Each single strand nucleic acid molecule 910 includes a functional group, which in the embodiment shown is a thiol (SH) end 912. As shown in second view 900B, the functional group of each single strand nucleic acid molecule 910 can be linked with the upper surface of membrane 906 depending on the selection of the chemical composition of the membrane and the functional group of the nucleic acid molecule. As shown, thiol end 912 can interact with the upper surface of membrane 906 also through thiol-metal bonds, shown schematically as a thiol-metal bond 914. The left-hand side of second view 900B shows plurality of single strand nucleic acid molecules 910 linked to upper surface 907 whereas the right-hand side of second view 900B shows the linking of one single strand nucleic acid molecule 910 to upper surface 907 via thiol-metal bond 914. As described below, the linking of single strand nucleic acid molecules to the upper surface of a membrane can be used to bond other structures and molecules to the membrane.

[0104] Reference is now made to Figure 4D, which is a schematic illustration of a substrate of a fluorescence-based detection system showing the bonding of a folded nucleic acid molecule with staple strands to the substrate, generally referenced 940, constructed and operative in accordance with another embodiment of the disclosed technique. Figure 4D shows how a substrate to which single strand nucleic acid molecules are bonded to can be used to bond large structures such a folded nucleic acid molecule (for example, a DNA origami nanostructure). The folded nucleic acid molecule, following its folding, is coupled using staple strands that attach the folded nucleic acid molecule to the surface of the substrate using hydrogen bonds of covalently linked DNA. In general, it is noted that staple strands partly hybridize a folded nucleic acid molecule and also partly bind a single strand folded nucleic acid molecule to a substrate surface. Similar to Figure 4C, many of the elements of the fluorescence-based detection system have been omitted from Figure 4D for the sake of clarity and the fluorescence-based detection system described herein can refer to any of the fluorescence-based detection system described herein. Shown is a membrane 942 which includes a nanopore 944. Membrane 942 may also have a metal film (not shown), a dielectric layer (not shown) and the like deposited over it at different locations, similar to the other detection systems described herein. Membrane 942 is located within a container (not shown) wherein an ionic solution (not shown) is placed.

[0105] Figure 4D shows two views, a first view 951 A and a second view 951 B. First view 951 A shows a folded nucleic acid molecule with a plurality of staple strands before bonding to a substrate surface whereas second view 951 B shows the folded nucleic acid molecule with the plurality of staple strands after bonding to the substrate surface. The plurality of staple strands may also be involved in the hybridization of the folded nucleic acid molecule. The movement from first view 951 A to second view 951 B is shown via an arrow 947. In first view 951 A, a plurality of single strand nucleic acid molecules 946 is shown, coupled with the upper surface (not labeled) of membrane 942 using any of the linking methods shown above in Figure 4C. Assuming plurality of single strand nucleic acid molecules 946 are single strand DNA molecules, sections of plurality of single strand nucleic acid molecules 946 may include any one of the four base nucleic acid molecules (adenine (A), cytosine (C), guanine (G) and thymine (T)) found in DNA which are not bonded, shown as a plurality of not bonded molecules 948. Floating in the ionic solution is also a folded nucleic acid molecule 950 to which a plurality of staple strands 952 have been coupled with. Plurality of staple strands 952 are substantially also single strand nucleic acid molecules and assuming they are embodied as single strand DNA molecules, can also have not bonded sections consisting of a sequence composed of any one of the four base nucleic acid molecules (A, C, G and T) found in DNA, shown as a plurality of not bonded molecules 954, which can be used for hybridization of folded nucleic acid molecule 950. It is noted that folded nucleic acid molecule 950 is shown coupled with plurality of single strand nucleic acid molecules 946 by their not bonded sections however this is not the only way how folded nucleic acid molecule 950 can couple with plurality of single strand nucleic acid molecules 946. In general, hybridization can be used to couple folded nucleic acid molecule 950 with plurality of single strand nucleic acid molecules 946 provided that the base nucleic acid molecule sequence of a portion of plurality of single strand nucleic acid molecules 946 complementary matches the base nucleic acid molecule sequence of a respective portion of folded nucleic acid molecule 950.

[0106] Second view 951 B shows what happens when plurality of free ends 948 on the upper surface of membrane 942 are selected to complementarily match plurality of free ends 954 on folded nucleic acid molecule 950, wherein hydrogen bond base-pairing can occur between adenine and thymine / uracil (AT bonding in DNA or AU bonding in RNA) and cytosine and guanine (CG bonding). As shown, plurality of hydrogen bonds 956 based on the complementary base-pairing of DNA molecules can be formed between plurality of free ends 948 and plurality of free ends 954, thereby bonding folded nucleic acid molecule 950 to the upper surface of membrane 942. The example shown in Figure 4D can be used to bond a folded nucleic acid molecule, acting as a sieve for reducing the translocation velocity of a biopolymer, to an opening positioned within a metal film and / or a dielectric layer substantially centered over a nanopore, as shown above in Figures 4A and 4B.

[0107] As mentioned above, the translocation velocity of a biopolymer is influenced, amongst other things, by the generated electric field in the ionic solution. As the electric field increases in strength, so does the translocation velocity, and vice-versa. As discussed regarding the prior art, globally reducing the generated electric field in the ionic solution will globally reduce the velocity of the biopolymer, not only in the excitation region but in the two sections of the reservoir as well. Thus, a biopolymer may cross an optical excitation zone and a nanopore at a desired velocity such that an increase in SNR of emitted fluorescence is obtained however it may take hours or even days for the biopolymer to migrate from a given location in one of the sections of the reservoir to the optical excitation zone. According to the disclosed technique, the excitation zone of the substrate is repositioned such that it is in an area of reduced electric field strength, thus enabling biopolymers to be excited by light beams while nonetheless exhibiting a reduced translocation velocity. Therefore, while it is not possible to globally change the generated electric field without impacting the electric field in the optical excitation zone and the region adjacent to the nanopores, it is possible to construct a substrate such that the optical excitation zone is migrated to an area where the generated electric field is weaker.

[0108] Reference is now made to Figure 5A, which is a schematic illustration of a membrane used in a detection system showing a generated electric field, generally referenced 360, constructed and operative in accordance with a further embodiment of the disclosed technique. Figure 5A shows a membrane 362 in which a nanopore 363 is located. Membrane 362 is in an ionic solution 365 and is contained within a container (not shown). An electric field 372 is generated in ionic solution 365 across membrane 362 in order to encourage the migration of a biopolymer (not shown) from one side of nanopore 363 to the other. Equipotential voltage field lines 364 are schematically shown to represent the voltage potential in ionic solution 365. The density and closeness of voltage field lines 364 schematically represent the strength of the voltage field. The electric field in ionic solution 365 can be derived from the gradient of the voltage field. A schematic representation of the generated electric field is shown by a plurality of electric field lines 372, which are perpendicular to the equipotential voltage field lines 364. The magnitude of the electric field is inversely related to the distance between equipotential voltage field lines, thus the voltage field lines within nanopore 363 are very dense with little distance between them and therefore represent a strong generated electric field. The further voltage field lines 364 are located from nanopore 363, the greater the distance between them and thus a weaker electric field is generated. As mentioned before, the electric field strength increases by increasing the current generated by electrodes (not shown) which are used to apply the voltage across membrane 362. The increased voltage potential in turn increases the translocation velocity of biopolymers in ionic solution 365. As already explained, simply reducing the current in ionic solution 365 is not a viable and practical solution for reducing the translocation velocity of biopolymers. As explained above in Equation (2), the detection systems of the disclosed technique are subject to Ohm’s Law, where V is the voltage across membrane 362, I is the current flowing through nanopore 363 and R is the overall resistance between the electrodes applying the voltage and the electric field to ionic solution 365. Using the example of Figure 5A wherein the simplified case of a single nanopore is shown, substantially all the current I must pass through nanopore 363 (even though there may be some leakage through membrane 362) and in order for Ohm’s Law to remain true, the increase in current through nanopore 363 will result in a corresponding increase in voltage across membrane 362. Generalizing this notion, the application of Ohm’s Law can be described regarding an equipotential voltage field line 368 representing a 2D slice of a volume of ionic solution 365 having a thickness 370 and an area 366. Thus, the voltage drop over a volume of ionic solution at a given location (such as the 2D slice of volume represented by voltage field line 368) can be described mathematically as:

[0109] A7(n) = I - AR(n) (4) where (n) is an index representing an equipotential voltage field line, with AV(n) being the change in voltage over (n) and AR(n) being the change in resistance in the ionic solution over (n). As described in Equation (3) above, the change in resistance itself can be described in terms of the area and the thickness of a volume of solution representing an equipotential voltage field line, thereby leading to: where / schematically represents thickness 370 of equipotential voltage field line 368 and A(n) schematically represents area 366, with p representing the resistivity of ionic solution 365. Thickness 370 and area 366 represent the area of a body of revolution obtained by rotating equipotential voltage field line 368 around an axis of symmetry (not shown) which passes through the center of nanopore 363. The electric field strength can then be defined as:

[0110] As can be understood from Figure 5A, the area A(n) increases rapidly as the distance from nanopore 363 increases, thus R(n) and E(n) decrease proportionally. For the sake of explaining the disclosed technique, one can assume that nanopore 363 has an infinitely small dimension and therefore equipotential surfaces having hemispherical shapes can be defined. The area A(n) of a hemisphere at a distance r from the nanopore, shown schematically in Figure 5A as an arrow 374, can then be defined as:

[0111] If the change in voltage AV(n) is then integrated over a given hemisphere, it can be seen that the voltage is proportional to f / r and the electric field strength is proportional to 1 / r2. Thus, the electric field strength reduces rapidly (i.e., proportionally to 1 / r2) as the distance from nanopore 363 increases. According to the disclosed technique as shown in Figures 5B and 5C below, a dielectric distancing layer of controlled thickness is added to the substrate of the detection system, to thereby distance the optical excitation zone of biopolymers from the nanopore. As shown mathematically above, doubling the distance of the optical excitation zone from the nanopore will result in a four-fold reduction in electric field strength, whereas quadrupling the distance of the optical excitation zone from the nanopore will result in a sixteen-fold reduction in electric field strength. The significantly reduced electric field strength will result in a reduced translocation velocity of biopolymers in the optical excitation zone such that an increased SNR can occur and reliable detection and analysis of a biopolymer is thus possible.

[0112] Reference is now made to Figure 5B, which is a schematic illustration of a fifth substrate of a fluorescence-based detection system using plasmonic excitation for distancing an excitation zone from a nanopore, generally referenced 400, constructed and operative in accordance with another embodiment of the disclosed technique. Detection system 400 is similar in construction to detection systems 200 (Figure 3A) and 280 (Figure 4A) wherein plasmonic excitation is employed and likewise Figure 5B does not show all the elements of detection system 400 for the sake of clarity. Figure 5B substantially shows the substrate of detection system 400 which separates an ionic solution 404 in a container (i.e., a reservoir) into two sections. In Figure 5B, detection system 400 is shown from a top view 402A and a cross-sectional view 402B, wherein cross-sectional view 402B is shown along the line E-E in top view 402A, shown by a line 416. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 406, dielectric distancing layer 408 and a metal film 410. As shown, membrane 406 includes a nanopore 412 and together dielectric distancing layer 408 and metal film 410 include an opening 414, which is substantially centered over nanopore 412. As mentioned above, metal film 410 may include an additional dielectric layer (not shown) for protecting and isolating metal film 410 from ionic solution 404 in case metal film 410 is not made from a noble metal. Both nanopore 412 and opening 414 are shown in top view 402A as being circular in shape however other shapes are possible. Opening 414 is larger in diameter than nanopore 412 and serves the function with metal film 410 as an EM concentration zone for influencing the shape of the generated electromagnetic field near nanopore 412 and may also improve the spatial confinement of the generated electromagnetic field using plasmonic effects. The increased electric field strength caused by nanopore 412 and opening 414 encourages a biopolymer 422 in ionic solution 404 to migrate towards opening 414 and then through nanopore 412. As shown in both top view 402A and cross-section view 402B, dielectric distancing layer 408 distances metal film 410 which effectively distances an optical excitation zone 424 from nanopore 41 . The distancing of metal film 410 from nanopore 412 places it in an area with a weaker electric field while maintaining the electric field strength that serves to attract biopolymers through the optical excitation zone 424 on their route to nanopore 412, which exhibits a very strong electric field due to the small area of nanopore 412 as compared to the area of ionic solution 404.

[0113] As shown, dielectric distancing layer 408 is deposited over membrane 406 and metal film 410 is deposited over dielectric distancing layer 408. In general, the deposition techniques and schemes used in this embodiment are as the same described as with reference to Figure 3A. The bottom left hand corner of top view 402A shows a cutaway of the layers of the substrate whereas cross-sectional view 402B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 402B, metal film 410 is now distanced from nanopore 412 by a distance 426, which is the thickness of dielectric distancing layer 408. Whereas in detection systems without dielectric distancing layer 408 (such as detection system 100 (Figure 2A)), a biopolymer will increase its velocity as it approaches the metal film due to the proximity of the metal film to the nanopore, in detection system 400, a polymer will only slightly increase its velocity when approaching metal film 410 as metal film 410 is distanced from nanopore 412 where there is a significant increase in electric field strength and thus a significant increase in translocation velocity. Shown as well in cross-sectional view 402B is a light beam 418 which is directed towards opening 414 in metal film 410. The dotted ellipse representing optical excitation zone 424 is where light beam 418 will interact with biopolymer 422. Light beam 418 originates from a light source (not shown). As light beam 418 interacts with biopolymer 422, biopolymer 422 emits a fluorescent ray 420 which can be detected by a detector (not shown). Due to the distancing of optical excitation zone 424 from nanopore 412 by distance 426, the translocation velocity of biopolymer 422 through opening 414 will be slowed down sufficiently such that a first fluorescent ray (not labeled) emitted from biopolymer 422 can be distinguished from a second fluorescent ray (not labeled) in the detector. According to the disclosed technique, the thickness of dielectric distancing layer 408 can be tweaked and controlled such that a desired translocation velocity of biopolymer 422 through optical excitation zone 424 can be achieved.

[0114] As explained above, the small size and diameter of nanopore 412 causes the current in ionic solution 404 to speed up as biopolymer 422 approaches nanopore 412. However, by distancing optical excitation zone 424 from nanopore 412, the translocation velocity at which biopolymer 422 enters optical excitation zone 424 as it approaches nanopore 412 is slowed down. Dielectric distancing layer 408 can be made from any dielectric material which is compatible with techniques used in manufacturing semiconductors. Thus, dielectric distancing layer 408 can be made from materials such as SiO2, SiNx, TiO2, AI2O3, Ta2O5, HfO2, for example. In addition, dielectric distancing layer 408 may be composed of a single dielectric layer or a combination of layers of dielectric materials. Provided dielectric distancing layer 408 does not narrow the path to nanopore 412, dielectric distancing layer 408 should not have any substantial effect on the electric field strength in ionic solution 404.

[0115] As mentioned above, the actual strength of the electric field in ionic solution 404 can be determined by solving for the exact resistance of ionic solution 404 based on the actual geometry of membrane 406 and the substrate in general. Based on the example described in Figure 5A and using the detection system of Figure 5B, assume as an approximation that nanopore 412 has an infinitely small area, and assuming, for example, that the thickness of metal film 410 is 40 nm and that the optical excitation zone has substantially the same size as opening 414 such that the center of the optical excitation zone is in the middle of metal film 410 (i.e., at around 20 nm). If the optical excitation zone is about 20 nm from nanopore 412 (without a dielectric distancing layer as per the assumption above), then moving the optical excitation zone from 20 nm above nanopore 412 to 80 nm above nanopore 412 (i.e., by the use of dielectric distancing layer 408), will involve a four-fold increase in the distance of optical excitation zone 424 from nanopore 412. This in turn will reduce the electric field strength at optical excitation zone 424 sixteen-fold, due to the 1 / r2proportionality described above in Figure 5A. According to the disclosed technique, a relatively small increase in the distance between nanopore 412 and metal film 410 can result in a considerable reduction in electric field strength and thus the translocation velocity at which biopolymers move through optical excitation zone 424 can be significantly reduced. It is noted that the opening in dielectric distancing layer 408 as shown in Figure 5B is substantially the same size as the opening in metal film 410. According to the disclosed technique, the opening of those layers do not need to be the same, however the opening in dielectric distancing layer 408 must have an area which is large enough as compared to the area of nanopore 412 to allow for the effect of the weakening of the electrical field in opening 414. Thus, in general, the size of the opening in dielectric distancing layer 408 should be more similar to the opening in metal film 410 and not to the size of nanopore 412. As mentioned above, deposition of dielectric distancing layer 408 and metal film 410 over membrane 406 is similar to deposition techniques used in the semiconductor industry and thus any known deposition technology in that industry can be used for fabricating the substrate structure shown in Figure 5B.

[0116] Reference is now made to Figure 5C, which is a schematic illustration of a sixth substrate of a fluorescence-based detection system using waveguide excitation for distancing an excitation zone from a nanopore, generally referenced 440, constructed and operative in accordance with a further embodiment of the disclosed technique. Similar to Figure 5B, not all the elements of detection system 440 are shown in Figure 5C for the sake of clarity. As described below, Figure 5C is similar to Figure 5B however the excitation of biopolymers in the ionic solution is not via plasmonic excitation but rather via waveguide excitation. Thus, detection system 440 includes two dielectric layers, one acting as a waveguide layer and one acting as a distancing layer. Figure 5C substantially shows the substrate of detection system 440 which separates an ionic solution 444 in a container (i.e., a reservoir) into two sections. In Figure 5C, detection system 440 is shown from a top view 442A and a cross-sectional view 442B, wherein cross-sectional view 442B is shown along the line F-F in top view 442A, shown by a line 456. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 446, a dielectric distancing layer 448 and a waveguide 450. As shown, membrane 446 includes a nanopore 452 and dielectric distancing layer 448 and waveguide 450 together include an opening 454, which is substantially centered over nanopore 452. In top view 442A, nanopore 452 is shown as being circular in shape and opening 454 is shown as being square in shape, however other shapes are possible. Opening 454 is larger in size than nanopore 452 and should not have any substantial impact on the electric field strength in the immediate vicinity of nanopore 452 when an electric field is generated in ionic solution 444. The increased electric field generated in opening 454 and nanopore 452 encourages a biopolymer 464 in ionic solution 444 to migrate towards opening 454 and then through nanopore 452. However as explained above, the distancing of waveguide 450 from nanopore 452 reduces the electric field strength in the optical excitation zone generated in opening 454 due to its distance from nanopore 452. The reduced electric field strength in the optical excitation zone nonetheless attracts biopolymer 464 towards opening 454 albeit at a slower translocation velocity in the optical excitation zone as compared to a detection system wherein the waveguide is directly positioned over the membrane.

[0117] As shown, waveguide 450 is positioned over dielectric distancing layer 448 which itself is deposited over membrane 446, using deposition techniques and schemes as described above. The bottom left hand corner of top view 442A shows a cutaway of the layers of the substrate whereas cross-sectional view 442B shows the placement of the layers of the substrate and their relative positions. As can be seen in cross-sectional view 442B, dielectric distancing layer 448 distances waveguide 450 from membrane 446 by a thickness shown by a marker 462. Similar to detection system 400 (Figure 5B), dielectric distancing layer 448 migrates an optical excitation zone 466 of biopolymer 464 away from nanopore 452, thus enabling a slower translocation velocity of biopolymer 464 through optical excitation zone 466. Shown as well in cross-sectional view 442B is a light beam 458 traversing through waveguide 450 which is directed towards opening 454. Light beam 458 originates from a light source (not shown) which is coupled with waveguide 450. Various techniques can be used to introduce light beam 458 into waveguide 450 from the light source. As light beam 458 interacts with biopolymer 464, biopolymer 464 emits a fluorescent ray 460 which can be detected by a detector (not shown). Due to the distancing of waveguide 450 from nanopore 452 the translocation velocity of biopolymer 464 through optical excitation zone 466 will be slowed down significantly such that a first fluorescent ray (not labeled) emitted from biopolymer 464 can be distinguished from a second fluorescent ray (not labeled) in the detector and a high enough SNR can be obtained for detected fluorescent rays to enable reliable detection and analysis of biopolymer 464. The composition of dielectric distancing layer 448 is substantially equivalent to what was described above regarding dielectric distancing layer 408 (Figure 5B). As shown in Figure 5C in top view 442A, the dielectric distancing layer is deposited over the entire surface of the membrane whereas the waveguide is positioned in the middle of the membrane around the region of the nanopore. Since waveguide 450 and dielectric distancing layer 448 are both made from dielectric materials, the index of refraction of dielectric distancing layer 448 as well as the index of refraction of ionic solution 444 need to be lower than the index of refraction of waveguide 450 to ensure that light beam 458 remains in waveguide 450 due to total internal reflection.

[0118] It is noted as well regarding Figures 5B and 5C that merely increasing the thickness of the membrane to increase the thickness of the nanopore to reduce the increased electric field strength in the nanopore will not result in a significant reduction of the electric field strength in the nanopore. Increasing the thickness of the nanopore (while maintaining the same current) will generate an additional voltage drop substantially in the nanopore. Thus an increase in the thickness of the nanopore will substantially maintain the electric field distribution in the ionic solution, thus resulting in a high translocation velocity similar to the prior art. The dielectric distancing layer therefore serves the purpose of positioning the optical excitation zone of the waveguide far enough from the nanopore such that there is some reduction in the electric field strength in the optical excitation zone that leads to a reduction in translocation velocity of a biopolymer crossing the optical excitation zone. As explained in Figures 5B and 5C, both the waveguide and the metal film serve the same general purpose of defining an optical excitation zone and impact the electromagnetic field responsible for excitation of the biopolymer. Ideally, the waveguide and the metal film would have an infinitesimal thickness thereby defining a thin optical excitation zone which will result in significantly better spatial resolution. In practice, the waveguide and the metal film do have a thickness which defines the electric field strength in the optical excitation zone. The distancing of the waveguide and the metal film from the nanopore by the inclusion of the dielectric distancing layer enables control in defining the electric field strength in the optical excitation zone, and specifically, its reduction.

[0119] Reference is now made to Figure 6A, which is a schematic illustration of a substrate of a fluorescence-based detection system wherein a non-isolated microelectrode is deposited near a nanopore, generally referenced 500, constructed and operative in accordance with another embodiment of the disclosed technique. Similar to the figures above (such as Figures 3A and 3B), not all the elements of the detection system are shown in Figure 6A for the sake of clarity. Shown in Figure 6A is a portion of a container (not labeled) in which an ionic solution 502 is placed. A membrane 504 divides the container (i.e., a reservoir) into two sections 503A and 503B wherein ionic solution 502 can pass from one section of the reservoir to the next via a nanopore 506. Movement of ionic solution 502 and biopolymers (not shown) through nanopore 506 is facilitated by a generated electric field, shown via an arrow 516. Electric field 516 can be referred to as a macroelectric field which is generated by a pair of macroelectrodes 508A and 508B which each apply a voltage. Each of macroelectrodes 508A and 508B is coupled via a lead wire 512 to a first voltage source (not shown) for generating macroelectric field 516. In this embodiment, membrane 504 has deposited on its surface a non-isolated microelectrode 510, which may have a disc-like shape surrounding nanopore 506. Only a single nanopore is shown in membrane 504 however membrane 504 may have a plurality of nanopores, wherein surrounding each nanopore, a respective non-isolated microelectrode is deposited. Shown as well is a lead wire 514 to a second voltage source (not shown) for generating a microelectric field 518. First and second voltage sources are independently controlled and thus macroelectric field 516 and microelectric field 518 can be controlled independently. In addition, as mentioned above, microelectric field 518 can be controlled in an open loop or closed loop configuration. Macroelectric field 516 permeates the entire volume of ionic solution 502 and is distributed between macroelectrodes 508A and 508B. Microelectric field 518 is localized to the vicinity near nanopore 506 and can be used to alter the effective electric field experienced by a biopolymer in an optical excitation region 517, which is adjacent to nanopore 506. As described below, by changing the effective electric field experienced by the biopolymer in optical excitation region 517, the translocation velocity of the biopolymer can be slowed down when it crosses through nanopore 506 and optical excitation zone 517 adjacent to nanopore 506.

[0120] Non-isolated microelectrode 510 is not isolated from ionic solution 502 and is thus exposed to ionic solution 502. Therefore, non-isolated microelectrode 510 and macroelectrodes 508A and 508B operate under identical physical conditions within ionic solution 502 wherein the electric fields which they generate depend on their applied respective voltages as well as the resistance in ionic solution 502 present between the various electrodes in accordance with Ohm’s Law. Non-isolated microelectrode 510 may be deposited on membrane 504 using common conducting materials used in the semiconductor industry (such as Al, Cu, Ag, Au, Pt, Cr, Ti and Pd) and common deposition techniques (such as physical vapor deposition (herein abbreviated PVD), chemical vapor deposition (herein abbreviated CVD) and atomic layer deposition (herein abbreviated ALD)). When fabricating membrane 504 and a plurality of non-isolated microelectrodes deposited thereon, leakage currents may flow between various non-isolated microelectrode pairs. According to the disclosed technique, if such flowing leakage currents are not desired, then the concentration of ionic solution 502 on reservoir side where the non-isolated microelectrodes are located (i.e., section 503A) can be reduced to a concentration level were flowing leakage currents are tolerable. In the case of a non-isolated microelectrode, the exact composition of the microelectrode material has significance since there is DC current flowing through ionic solution 502 which can present potential chemical interactions of the salt constituents in ionic solution 502 that may destroy the microelectrodes because they are not isolated. Depending on the constitution of ionic solution 502, non-isolated microelectrode 510 may need to be fabricated from noble metals (such as gold (Au), platinum (Pt) or palladium (Rd)) to help avoid any destructive interaction between the microelectrode and the salt constituents in ionic solution 502. In another embodiment, destructive interaction between the microelectrode and the salt constituents in ionic solution 502 can be prevented by either depositing a sufficiently thick layer of metal as the microelectrode and / or selecting the metal composition of the microelectrode such that it is compatible with the salt constituents of ionic solution 502. For example, the microelectrode can be made from silver when it is known that ionic solution 502 contains a chlorine-based salt, such as lithium chloride, sodium chloride or potassium chloride. In such a case, the silver microelectrode may react with the chlorine in ionic solution 502 to form silver chloride when the microelectrode applies a microelectric field, which will not result in destruction or corrosion to the microelectrode.

[0121] Reference is now made to Figure 6B, which is a schematic illustration of a substrate of a fluorescence-based detection system wherein an isolated microelectrode is deposited around a nanopore, generally referenced 530, constructed and operative in accordance with a further embodiment of the disclosed technique. Similar to Figure 6A, not all the elements of the detection system are shown in Figure 6B for the sake of clarity. Shown in Figure 6B is a portion of a container (not labeled) in which an ionic solution 532 is placed. A membrane 534 divides the container (i.e., a reservoir) into two sections, 533A and 533B, wherein ionic solution 532 can pass from one section of the reservoir to the next via a nanopore 536. Movement of ionic solution 532 and biopolymers (not shown) through nanopore 536 is facilitated by a generated electric field, shown via an arrow 546. Electric field 546 can be referred to as a macroelectric field which is generated by a pair of macroelectrodes 538A and 538B. Each one of macroelectrodes 538A and 538B is coupled via a lead wire 544 to a first voltage source (not shown) for generating macroelectric field 546. In this embodiment, membrane 534 has deposited on its surface an isolated microelectrode 540, which may have a disc-like shape surrounding nanopore 536. Isolated microelectrode 540 includes an insulating layer 542 which surrounds the microelectrode and insulates it from ionic solution 532. Only a single nanopore is shown in membrane 534 however membrane 534 may have a plurality of nanopores, wherein surrounding each nanopore, a respective isolated microelectrode is deposited. Shown as well is a lead wire 550 to a second voltage source (not shown) for generating a microelectric field 548. First and second voltage sources are independently controlled and thus macroelectric field 546 and microelectric field 548 can be controlled independently. Macroelectric field 546 permeates the entire volume of ionic solution 532 and is distributed between macroelectrodes 538A and 538B. Microelectric field 548 is localized to the vicinity near nanopore 536 and can be used to alter the effective electric field experienced by a biopolymer in an optical excitation zone 547, adjacent to nanopore 536. As described below, by changing the effective electric field experienced by the biopolymer in optical excitation zone 547, the translocation velocity of the biopolymer can be slowed down when it crosses through nanopore 536 and optical excitation zone 547 adjacent to nanopore 536.

[0122] Isolated microelectrode 540 is isolated from ionic solution 532 by insulating layer 542 and is thus not exposed to ionic solution 532. As such, the method of operation of isolated microelectrode 540 is different from non-isolated microelectrode 510 (Figure 6A). When voltage is applied via lead wire 550 to isolated microelectrode 540, since isolated microelectrode 540 is insulated from ionic solution 532, isolated microelectrode 540 functions like a capacitor plate, generating a microelectric field with respect to macroelectrodes 538A and 538B. Since ions in ionic solution 532 can move freely in the ionic solution and through nanopore 536, the moving ions build a spatial charge density to counter the generated microelectric field generated by isolated microelectrode 540. This phenomenon is generally known as the electrical double layer (herein abbreviated EDL) phenomenon wherein a surface charge is not controlled by electrodes but is rather the outcome of free charge carriers countering a forced electrical field. In the case of isolated microelectrode 540 the EDL phenomenon presents itself as an extension of the spatial charge distribution and the associated electric field generated by isolated microelectrode 540. A parameter known as the Debye length, in reference to an electrode, represents how far the electrostatic effect of a charge carrier's net electrostatic effect of the electrode persists in a solution. The Debye length depends on the applied voltage to isolated microelectrode 540, the type of ions in ionic solution 532 as well as the solution concentration of ionic solution 532. The Debye length may range from sub-nanometer values to tens of nanometers for ionic solution concentrations ranging from 1 mole to 0.1 millimole. According to the disclosed technique, the Debye length can be altered by changing the applied voltage to isolated microelectrode 540 which in turn changes the local microelectric field in optical excitation zone 547 adjacent to nanopore 536. The strength of the local microelectric field can be altered to slow down the translocation velocity of biopolymers through nanopore 536. As mentioned above and similar to the embodiment which included a non-isolated microelectrode, isolated microelectrodes may be deposited on membrane 534 using conducting materials commonly used in the semiconductor industry (such as Al, Cu, Ag, Au, Pt, Cr, Ti and Pd) and common deposition processes (such as PVD, CVD and ALD). As shown, isolated microelectrode 540 requires isolation and insulation from ionic solution 532 and can be isolated via the deposition of insulating layer 542 which may be embodied as a dielectric layer (not shown) made from compounds such as SiO2, TiOa, SiNx, HfC and the like. Other methods of isolating isolated microelectrode 540 can include depositing a first dielectric layer (such as insulating layer 542) which may be followed by the deposition of optional additional dielectric layers for providing additional isolation. Examples of a dielectric layer can include SiO2, TiO2, HfO2, SisN4, AI2O3, SiNx, Ta2Os and Nb20s) and can be deposited using common deposition processes such as PVD, CVD and ALD.

[0123] Reference is now made to Figure 7A, which is a schematic illustration of a substrate of a fluorescence-based detection system showing the underlying electric principle of operation of non-isolated microelectrodes deposited around nanopores, generally referenced 570, constructed and operative in accordance with another embodiment of the disclosed technique. A first view 571 A shows the electric principle of operation of non-isolated microelectrodes within a substrate in an ionic solution (i.e., a physical representation) whereas a second view 571 B shows an equivalent circuit diagram (i.e., an electrical representation) of the electric principle of operation shown in first view 571 A. With reference to first view 571 A, a membrane 574 including a plurality of nanopores 576 is shown placed within an ionic solution 572 which is placed within a container (not shown and not labeled). A plurality of non-isolated microelectrodes 578 are respectively deposited and positioned around each nanopore 576. A pair of macroelectrodes 580A and 580B generate a current in ionic solution 572 by generating a voltage difference between a ground voltage 596 and a macro voltage VM 594 in ionic solution 572. As shown, each one of plurality of non-isolated microelectrodes 578 is coupled with a respective lead wire 582i, 5822, 582s and 5824 for generating respective localized microelectric fields near respective ones of plurality of nanopores 576. As mentioned above, each one of plurality of non-isolated microelectrodes 578 is independently controlled, thus lead wire 582i can generate a local voltage of Vi, lead wire 5822 can generate a local voltage of V2, lead wire 5823 can generate a local voltage of V3 and lead wire 582 can generate a local voltage of V4.

[0124] As mentioned above, ionic solution 572 and membrane 574 are subject to Ohm’s Law and thus the resistance at different locations within ionic solution 572 can be graphically illustrated to understand the electric principle of operation of non-isolated microelectrodes. As shown in first view 571 A, the geometry of each nanopore 576 generates a resistance, shown graphically as a resistor 584. Resistance is also present between adjacent nanopores, shown as a resistor 586 (in the upper section of the reservoir (not labeled) with the non-isolated microelectrodes) and a resistor 592 (in the lower section of the reservoir (not labeled) which does not include the non-isolated microelectrodes). There is furthermore resistance across membrane 574 and macroelectrodes 580A and 580B, respectively via plurality of nanopores 576 and ionic solution 572, respectively shown as a resistor 588 and a resistor 590. As illustrated, the resistance shown in each location can be defined separately even though separate reference numbers are not used. Thus, each non-isolated microelectrode may experience a different resistance between itself and macroelectrodes 580A and 580B, which is shown in first view 571 A however only two resistors are labeled as such as resistors 588 and 590.

[0125] An arrow 594 points to second view 571 B which shows an equivalent circuit diagram of the electric principle of operation shown in first view 571 A. With reference to second view 571 B, as mentioned above, since the microelectrodes are not isolated, both the macroelectrodes and the microelectrodes are subject to Ohm’s Law and the volume of ionic solution 572 can be modeled as an Ohmic resistance. Second view 571 B shows the various resistors 584, 586, 588, 590 and 592 and their connections. As shown, the different microelectrodes can force specific voltages to specific nanopores at specific spatial locations. Thus, a voltage gradient can be applied across a specific nanopore adding an additional level of control of the effective electric field generated around the specific nanopore which in turn can slow down the translocation velocity of a biopolymer through a given nanopore. As an example, a local electric field 598 around the adjacent nanopore (not shown or labeled) can be modified based on the voltage applied by lead wire 582i to the non-isolated microelectrode it is coupled with.

[0126] As mentioned above, the local electric field (i.e., the microelectric field) can be modified statically or dynamically. In a static embodiment, the local electric field is kept constant such that Vi resembles the voltage of VM. In a dynamic embodiment, Vi is modified only when a translocation event is detected of a biopolymer crossing the respective nanopore lead wire 582i it is coupled with. For example, when a detector (not shown) detects fluorescent rays being emitted from a given nanopore, this is an indication that a translocation event is occurring. Once this is detected, a voltage source (not shown) coupled with lead wire 582i is turned on to reduce Vi such that it resembles the voltage of macroelectrode 580A which is VM 594. The reduction in voltage of Vi translates into a reduction of the translocation velocity of the biopolymer. Once the detector no longer detects fluorescent rays being emitted from the given nanopore, it is an indication that the translocation event is over. The voltage source can then stop supplying current via lead wire 5821 , thus allowing the Vi to return to its previous higher voltage level before the translocation event occurred. As mentioned above, the higher voltage level encourages biopolymers to migrate more rapidly towards a given nanopore.

[0127] Reference is now made to Figure 7B, which is a schematic illustration of a substrate of a fluorescence-based detection system showing the underlying electric principle of operation of isolated microelectrodes deposited around nanopores, generally referenced 620, constructed and operative in accordance with a further embodiment of the disclosed technique. A first view 621 A and a second view 621 B show the electric principle of operation of an isolated microelectrode within a substrate in an ionic solution. First view 621 A shows the Ohmic resistance in the ionic solution when the isolated microelectrode is not provided with a voltage and second view 621 B shows the Ohmic resistance in the ionic solution when the isolated microelectrode is provided with a voltage. An arrow 646 shows the movement from first view 621 A to second view 621 B. With reference to first view 621 A, a membrane 624 including a nanopore 625 is shown placed within an ionic solution 622 which is placed within a container (not shown and not labeled). An isolated microelectrode 626 is respectively deposited and positioned around nanopore 625. A pair of macroelectrodes 628A and 628B generate a current in ionic solution 622 by generating a voltage difference between a ground voltage 632 and a macrovoltage VM 630 in ionic solution 622. As shown, isolated microelectrode 626 is coupled with a lead wire 634 for generating a localized microelectric field near nanopores 625. Isolated microelectrode 626 is independently controlled and thus lead wire 634 can generate a local voltage of Vi. In the case of a plurality of nanopores (not shown) and respective isolated microelectrodes (not shown), each isolated microelectrode would be provided with its own lead wire.

[0128] As mentioned above, ionic solution 622 and membrane 624 are subject to Ohm’s Law and thus the resistance at different locations within ionic solution 622 can be graphically illustrated to understand the electric principle of operation of an isolated microelectrode. As shown by a plurality of resistors 638, there is a first voltage drop 636i between macroelectrode 628A and the upper surface of membrane 624. A second voltage drop 6362 occurs within nanopore 625 and a third voltage drop 6863 occurs between the lower surface of membrane 624 and macroelectrode 628B. Since the microelectrode is isolated, there is no voltage drop or direct electric interaction between isolated microelectrode 626 and ionic solution 622. As explained above in Figure 6B, isolated microelectrode 626 acts as a capacitor plate 640 in the region adjacent to nanopore 625.

[0129] Turning now to second view 621 B, current is provided to isolated microelectrode 626 via lead wire 634. As the current is provided, since isolated microelectrode 626 is isolated, negative charge, shown as a plurality of negative charge carriers 642, starts to build up in isolated microelectrode 626. Likewise, due to the EDL phenomenon and an appropriate Debye length, positive charge, shown as a plurality of positive charge carriers 644, starts to build up spatially around isolated microelectrode 626 within ionic solution 622 and around nanopore 625, thus countering the generated electrical field created when current is provided to isolated microelectrode 626. The spatial charge causes a fourth voltage drop 6364 to occur in the area adjacent to nanopore 625, which is substantially an optical excitation zone (not shown). Like the embodiment shown above in Figure 7A, by adjusting the voltage provided by lead wire 634 (determined as Vi) to levels similar to macrovoltage VM 630, the effective electric field generated around nanopore 625 can be modified thereby reducing the translocation velocity of a biopolymer (not shown) through nanopore 625.

[0130] As described above, isolated microelectrode 626, which is close to nanopore 625, can be set to a specific voltage Vi respective of macrovoltage VM 630. If Vi is different than the voltage level determined by second voltage drop 6362, an electric field will start to be generated, with spatial charges beginning to accumulate to counter the generated electric field. Thus, changes to voltage Vi influence the resistance in ionic solution 622 by means of this accumulated spatial charge. As mentioned above, this additional level of control of Vi can be applied to control the microelectric field adjacent to nanopore 625 in the optical excitation zone either statically or dynamically. In an example of a dynamic use, the observation of a translocation event (for example through the detection of fluorescent rays in a detector (not shown)) could trigger reducing Vi to the levels of VM. This reduction in voltage is transferred to ionic solution 622 by means of the spatial charge induced by isolated microelectrode 626 and translates into a reduction in translocation velocity of a biopolymer (not shown) crossing through nanopore 625. Once the translocation event is over, determined also by observation and a lack of fluorescent rays being detected by the detector, voltage Vi can then be restored to its previous level before the translocation event began. Since the control method shown in Figure 7B is based on a capacitive effect, the capacitance responsible for the spatial charge and the equivalent change in resistance in the ionic solution should be tailored to yield time constants compatible with the duration of a translocation event.

[0131] It is noted, as mentioned above, that the microelectrodes of Figures 7A and 7B can be deposited using the deposition techniques and schemes as described above. It is noted as well that whether the microelectrodes are non-isolated or isolated, according to the disclosed technique, each microelectrode is coupled with an independent lead wire and thus the current provided to each microelectrode can be controlled independently.

[0132] Reference is now made to Figure 8A, which is a schematic illustration a seventh substrate of a fluorescence-based detection system using plasmonic excitation for locally modifying the electric generated field adjacent to a nanopore using a non-isolated microelectrode, generally referenced 670, constructed and operative in accordance with another embodiment of the disclosed technique. As mentioned above, not all the elements of detection system 670 are shown in Figure 8A for the sake of clarity. Figure 8A substantially shows the substrate of detection system 670 which separates an ionic solution 674 in a container into two sections. In Figure 8A, detection system 670 is shown from a top view 672A and a cross-sectional view 672B, wherein cross-sectional view 672B is shown along the line G-G in top view 672A, shown by a line 686. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 676 and a metal film 678 which is electrically coupled via a lead wire 680. As shown, membrane 676 includes a nanopore 682 and metal film 678 includes an opening 684, which is substantially centered over nanopore 682. Opening 684 is larger in diameter than nanopore 682 and serves the function with metal film 678 of generating the plasmonic effect (and therefore shaping the electromagnetic field strength) which defines an optical excitation zone 694. Metal film 678 also functions as a non-isolated microelectrode due to its coupling with lead wire 680 and thus in this embodiment, metal film 678 also influences the generated electric field in optical excitation zone 694 which influences the translocation velocity of a biopolymer 692 crossing there through and its migration towards opening 684 and then through nanopore 682. It is noted that the two roles of metal film 678 could also be served via two distinct metal entities (not shown) and thus metal film 678 can be embodied as at least one metal film or a plurality of metal films. As described above in Figures 6A and 7A, current can be delivered to metal film 678 via lead wire 680 to locally modify the effective electric field generated around opening 684 and nanopore 682. For example, current may be provided to metal film 678 when biopolymer 692 is detected in the vicinity of opening 684 due to the detection of fluorescent rays being emitted and detected on a detector (not shown).

[0133] As shown, metal film 678 is deposited over membrane 676 using the deposition techniques and schemes described above for depositing thin layers. The bottom left hand corner of top view 672A shows a cutaway of the layers of the substrate whereas cross-sectional view 672B shows the placement of the layers of the substrate and their relative positions. Lead wire 680 is shown electrically coupling metal film 678 to a voltage source (not shown). Shown as well in cross-sectional view 672B is a light beam 688 which is directed towards opening 684. Light beam 688 originates from a light source (not shown). As light beam 688 interacts with biopolymer 692 in optical excitation zone 694, biopolymer 692 emits a fluorescent ray 690 which can be detected by a detector (not shown). When voltage is supplied to metal film 678, a local microelectric field can be generated in optical excitation zone 694 in the vicinity of opening 684 for slowing down the translocation velocity of biopolymer 692 to enable increased SNR of detected fluorescent ray 690.

[0134] Reference is now made to Figure 8B, which is a schematic illustration an eighth substrate of a fluorescence-based detection system using plasmonic excitation for locally modifying the electric field adjacent to a nanopore using an isolated microelectrode, generally referenced 720, constructed and operative in accordance with a further embodiment of the disclosed technique. As mentioned above, not all the elements of detection system 720 are shown in Figure 8B for the sake of clarity. Figure 8B substantially shows the substrate of detection system 720 which separates an ionic solution 724 in a container into two sections. In Figure 8B, detection system 720 is shown from a top view 722A and a cross-sectional view 722B, wherein cross-sectional view 722B is shown along the line H-H in top view 722A, shown by a line 736. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 726 and a metal film 728 which is electrically coupled via a lead wire 738. Metal film 728 is covered with a dielectric isolation layer 730 for isolating metal film 728. Thus metal film 728 can function as an isolated microelectrode. As shown, membrane 726 includes a nanopore 732 and metal film 728 includes an opening 734, which is substantially centered over nanopore 732. Opening 734 is larger in diameter than nanopore 732 and serves the function with metal film 728 of shaping the electromagnetic field strength and thus generating the plasmonic effect in an optical excitation zone 746. As mentioned, metal film 728 also functions as an isolated microelectrode due to its coupling with lead wire 738 and being covered by dielectric isolation layer 730 and thus in this embodiment, metal film 728 also influences the generated electric field in optical excitation zone 746 which influences the translocation velocity of a biopolymer 744 crossing there through and its migration towards opening 734 and then through nanopore 732. It is noted that the two roles of metal film 728 could also be served via two distinct metal entities (not shown) and thus metal film 728 can be embodied as at least one metal film or a plurality of metal films. As described above in Figures 6B and 7B, current can be delivered to metal film 728 via lead wire 738 to locally modify the effective electric field generated around opening 734 and nanopore 732. For example, voltage may be provided to metal film 728 when biopolymer 744 is detected in optical excitation zone 746 in the vicinity of opening 734 due to the detection of fluorescent rays being emitted and detected on a detector (not shown).

[0135] As shown, metal film 728 is deposited over membrane 726 and dielectric isolation layer 730 is deposited over metal film 728 using the deposition techniques and schemes described above. The bottom left hand corner of top view 722A shows a cutaway of the layers of the substrate whereas cross-sectional view 722B shows the placement of the layers of the substrate and their relative positions. Lead wire 738 is shown electrically coupling metal film 728 to a voltage source (not shown). Shown as well in cross-sectional view 722B is a light beam 740 which is directed towards opening 734. Light beam 740 originates from a light source (not shown). As light beam 740 interacts with biopolymer 744, biopolymer 744 emits a fluorescent ray 742 which can be detected by a detector (not shown). When voltage is supplied to metal film 728, a local microelectric field can be generated in the vicinity of opening 734 for slowing down the translocation velocity of biopolymer 744 to enable increased SNR of detected fluorescent ray 742.

[0136] Reference is now made to Figure 8C, which is a schematic illustration a ninth substrate of a fluorescence-based detection system using waveguide excitation for locally modifying the electric field adjacent to a nanopore using a non-isolated microelectrode, generally referenced 770, constructed and operative in accordance with another embodiment of the disclosed technique. Similar to Figure 8A, not all the elements of detection system 770 are shown in Figure 8C for the sake of clarity. As described below, Figure 8C is similar to Figure 8A, however, the excitation of biopolymers in the ionic solution is not via plasmonic excitation but rather via waveguide excitation. Figure 8C substantially shows the substrate of detection system 770 which separates an ionic solution 774 in a container into two sections. In Figure 8C, detection system 770 is shown from a top view 772A and a cross-sectional view 772B, wherein cross-sectional view 772B is shown along the line l-l in top view 772A, shown by a line 786. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 776, a waveguide 778 and a metal film 780. As shown, membrane 776 includes a nanopore 782 and waveguide 778 includes an opening 784, which is substantially centered over nanopore 782. Metal film 780 is also substantially centered over nanopore 782 and functions as a non-isolated microelectrode. As shown, a lead wire 781 couples metal film 780 to a voltage source (not shown) for generating a local electric field in the vicinity of opening 784 in an optical excitation zone 794. Opening 784 is larger in size than nanopore 782 and serves the function of shaping the electromagnetic field strength in optical excitation zone 794. Metal film 780 functions as a non-isolated microelectrode due to its coupling with lead wire 781 and thus in this embodiment, metal film 780 influences the generated electric field in optical excitation zone 794 which influences the translocation velocity of a biopolymer 792 crossing there through and its migration towards opening 784 and then through nanopore 782. As described above in Figures 6A and 7A, current can be delivered to metal film 780 via lead wire 781 to locally modify the effective electric field generated around opening 784 and nanopore 782 in optical excitation zone 794. For example, voltage may be provided to metal film 780 when biopolymer 792 is detected in optical excitation zone 794 in the vicinity of opening 784 due to the detection of fluorescent rays being emitted and detected on a detector (not shown).

[0137] As shown, waveguide 778 is deposited over membrane 776 and metal film 780 is also deposited over membrane 776 using the deposition techniques and schemes described above. As an aside it is noted that Figures 8A-8D are schematic and thus the exact shape of the metal film (such as metal film 780 in Figure 8C) which acts as either a non-isolated microelectrode or an isolated microelectrode is not necessarily as drawn, for example encircling a nanopore (Figures 8A and 8B) or finger-like shapes approaching from opposite sides (Figures 8C and 8D). What is important however is the proximity of the microelectrode to the nanopore for the purposes of generating a microelectric field to influence the translocation velocity of a biopolymer in the optical excitation zone. In Figure 8C, in the case of a waveguide, the metal film could also be deposited above the waveguide (not shown), rather than being perpendicular to it, or both on top of and also perpendicular to the waveguide provided the metal film does not interfere with the function of the waveguide of guide a light beam to the optical excitation zone. The bottom left hand corner of top view 772A shows a cutaway of the layers of the substrate whereas cross-sectional view 772B shows the placement of the layers of the substrate and their relative positions. Since cross-sectional view 772B is along line 786 metal film 780 is not visible in cross-sectional view 772B. Shown as well in cross-sectional view 772B is a light beam 788 traversing through waveguide 778 which is directed towards opening 784. Light beam 788 originates from a light source (not shown) which is coupled with waveguide 778. Various techniques can be used to introduce light beam 788 into waveguide 778 from the light source. As light beam 788 interacts with biopolymer 792 in optical excitation zone 794, biopolymer 792 emits a fluorescent ray 790 which can be detected by a detector (not shown). When voltage is supplied to metal film 780, a local microelectric field can be generated in optical excitation zone 794 in the vicinity of opening 784 for slowing down the translocation velocity of biopolymer 792 to enable increased SNR of detected fluorescent ray 790.

[0138] Reference is now made to Figure 8D, which is a schematic illustration a tenth substrate of a fluorescence-based detection system using waveguide excitation for locally modifying the electric field adjacent to a nanopore using an isolated microelectrode, generally referenced 820, constructed and operative in accordance with a further embodiment of the disclosed technique. Similar to Figure 8B, not all the elements of detection system 820 are shown in Figure 8D for the sake of clarity. As described below, Figure 8D is similar to Figure 8B however the excitation of polymers in the ionic solution is not via plasmonic excitation but rather via waveguide excitation. Figure 8D substantially shows the substrate of detection system 820 which separates an ionic solution 824 in a container into two sections. In Figure 8D, detection system 820 is shown from a top view 822A and a cross-sectional view 822B, wherein cross-sectional view 822B is shown along the line J-J in top view 822A, shown by a line 836. Identical elements in both views are labeled using the same reference numbers. The substrate includes a membrane 826, a waveguide 828 and a metal film 830. As shown, metal film 830 is covered and isolated by a dielectric isolation layer 833. As shown, membrane 826 includes a nanopore 832 and waveguide 828 includes an opening 834, which is substantially centered over nanopore 832. Metal film 830 is also substantially centered over nanopore 832 and functions as an isolated microelectrode. As shown, a lead wire 831 couples metal film 830 to a voltage source (not shown) for generating a local electric field in the vicinity of opening 834 in an optical excitation zone 844. Opening 834 is larger in size than nanopore 832 and serves the function of shaping the electromagnetic field strength in optical excitation zone 844. As mentioned, metal film 830 functions as an isolated microelectrode due to its coupling with lead wire 831 and being covered by dielectric isolation layer 833 and thus in this embodiment, metal film 830 influences the generated electric field in optical excitation zone 844 which influences the translocation velocity of a biopolymer 842 crossing there through and its migration towards opening 834 and then through nanopore 832. As described above in Figures 6B and 7B, voltage can be delivered to metal film 830 via lead wire 831 to locally modify the effective electric field around opening 834 and nanopore 832. For example, voltage may be provided to metal film 830 when biopolymer 842 is detected in optical excitation zone 844 in the vicinity of opening 834 due to the detection of fluorescent rays being emitted and detected on a detector (not shown).

[0139] As shown, waveguide 828 is deposited over membrane 826 and metal film 830 is also deposited over membrane 826 using the deposition techniques and schemes described above. The bottom left hand corner of top view 822A shows a cutaway of the layers of the substrate whereas cross-sectional view 822B shows the placement of the layers of the substrate and their relative positions. Since cross-sectional view 822B is along line 836 metal film 830 is not visible in cross-sectional view 822B. Shown as well in cross-sectional view 822B is a light beam 838 traversing through waveguide 828 which is directed towards opening 834. Light beam 838 originates from a light source (not shown) which is coupled with waveguide 828. Various techniques can be used to introduce light beam 838 into waveguide 828 from the light source. As light beam 838 interacts with biopolymer 842 in optical excitation zone 844, biopolymer 842 emits a fluorescent ray 840 which can be detected by a detector (not shown). When voltage is supplied to metal film 830, a local microelectric field can be generated in optical excitation zone 844 in the vicinity of opening 834 for slowing down the translocation velocity of biopolymer 842 to enable increased SNR of detected fluorescent ray 840.

[0140] It will be appreciated by persons skilled in the art that the disclosed technique is not limited to what has been particularly shown and described hereinabove. Rather the scope of the disclosed technique is defined only by the claims, which follow.

Claims

CLAIMS1. A device for fluorescence-based detection of at least one polymer, the device comprising: at least one container; said at least one container comprising: a nanopore membrane, comprising at least one nanopore; a first layer, comprising at least one opening respectively positioned substantially in line over said at least one nanopore; and a physical barrier, covering said at least one opening, at least one light source, for illuminating said at least one polymer with at least one light beam; at least one detector, for detecting at least one emission emitted from said illuminated at least one polymer; and at least two electrodes, wherein said at least one polymer is placed within an ionic solution within said at least one container; wherein said at least two electrodes generate a current in said ionic solution; and wherein said physical barrier increases a viscosity of said ionic solution in a region adjacent to said at least one opening and said at least one nanopore for slowing down a translocation velocity of said at least one polymer as it passes through said at least one opening and said at least one nanopore.

2. The device according to claim 1 , wherein said first layer is a metal film, functioning with said at least one opening as an electromagnetic concentration zone.

3. The device according to claim 2, wherein said metal film is made from a conductor metal selected from the list consisting of: gold (Au); silver (Ag) aluminum (Al); titanium (Ti); palladium (Pd); platinum (Pt); and copper (Cu).

4. The device according to claim 1 , wherein said nanopore membrane is made from a silicon-based composition having a chemical formulation of SiNx, wherein ‘X’ is a ratio of nitride to silicon.

5. The device according to claim 1 , wherein said nanopore membrane is made from a material selected from the list consisting of:SIO2;HfO2; graphene; and a 2D material.

6. The device according to claim 1 , wherein said first layer is a dielectric layer functioning as a waveguide.

7. The device according to claim 6, wherein said dielectric layer comprises a metallic section.

8. The device according to claim 6, wherein said dielectric layer is made from a material selected from the list consisting of:SiO2;TiOa;Ta2O5;Nb2O5;Si3N4;AI2O3;SiNx; poly(methyl methacrylate) (PMMA); and polydimethylsiloxane (PDMS).

9. The device according to claim 1 , wherein said physical barrier is a gel coating, coating said first layer.

10. The device according to claim 9, wherein said gel coating is a hydrogel selected from the list consisting of:a polysaccharide; a synthetic polymer; starch; agarose; dextran; alginate; chitosan; hyaluronic acid; polyacrylamide; polyethyleneglycol (PEG); and polyvinyl alcohol (PVA).11 . The device according to claim 9, wherein said gel coating is modified to comprise at least one functional group.

12. The device according to claim 1 1 , wherein said at least one functional group is selected from the list consisting of: an alkyne group; an azide group; a dibenzocyclooctyne (DBCO) group; a carboxyl group; an amine group; a hydroxyl group; an aldehyde group; and an epoxide group; and an acrylic group.

13. The device according to claim 9, wherein said gel coating is modified to comprise at least one cross-linking reagent.

14. The device according to claim 13, wherein said at least one cross-linking reagent is selected from the list consisting of: functional group — linker — functional group; functional group — PEG — functional group; azide — linker — azide;azide — PEG — azide; acryl — li nke r — acryl ; bisacrylamide; borate complexes; sodium tetraborate; and boric acid.

15. The device according to claim 1 , wherein said physical barrier is at least one folded nucleic acid molecule, designed to substantially fit into said at least one opening; and wherein said at least one folded nucleic acid molecule has a diameter larger than a diameter of said at least one nanopore.

16. The device according to claim 15, wherein said at least one folded nucleic acid molecule is selected from the list consisting of:DNA;PNA; andRNA.

17. The device according to claim 1 , wherein said at least one polymer is selected from the list consisting of: a biological polymer; an organic polymer; an inorganic polymer; a non-biological polymer; a protein;DNA;RNA; andPNA.

18. The device according to claim 1 , wherein a diameter of said at least one opening is larger than a diameter of said at least one nanopore.

19. The device according to claim 9, wherein a thickness of said gel coating is at least as thick as a thickness of an excitation zone where said at least one light source illuminates said at least one polymer.

20. The device according to claim 9, wherein a thickness of said gel coating ranges between 20 nanometers to 1000 nanometers.21 . A device for fluorescence-based detection of at least one polymer, the device comprising: at least one container; said at least one container comprising: a nanopore membrane, comprising at least one nanopore; and an electric field strength altering layer, comprising at least one opening respectively positioned substantially in line over said at least one nanopore, at least one light source, for illuminating said at least one polymer with at least one light beam; at least one detector, for detecting at least one emission emitted from said illuminated at least one polymer; and at least two electrodes, wherein said at least one polymer is placed within an ionic solution within said at least one container; wherein said at least two electrodes generate a current in said ionic solution; and wherein said electric field strength altering layer generates a microelectric field in a region adjacent to said at least one opening and said at least one nanopore for slowing down a translocation velocity of said at least one polymer as it passes through said at least one opening and said at least one nanopore.

22. The device according to claim 21 , further comprising a lead wire, for coupling said electric field strength altering layer to a first voltage source.

23. The device according to claim 22, wherein said electric field strength altering layer comprises at least one metal film; and wherein said lead wire couples said at least one metal film to said first voltage source.

24. The device according to claim 23, wherein said at least one metal film is made from a conductor metal selected from the list consisting of:

25. The device according to claim 21 , wherein said nanopore membrane is made from a silicon-based composition having a chemical formulation of SiNx, wherein ‘X’ is a ratio of nitride to silicon.

26. The device according to claim 21 , wherein said nanopore membrane is made from a material selected from the list consisting of:

27. The device according to claim 23, further comprising a dielectric layer, comprising at least one opening, also respectively positioned substantially in line over said at least one nanopore, said dielectric layer functioning as a waveguide.

28. The device according to claim 27, wherein said dielectric layer is made from a material selected from the list consisting of:SiNx; poly(methyl methacrylate) (PMMA); and polydimethylsiloxane (PDMS).

29. The device according to claim 23, further comprising a dielectric isolation layer, deposited over said at least one metal film.

30. The device according to claim 29, wherein said dielectric isolation layer is made from a material selected from the list consisting of:31 . The device according to claim 21 , wherein said electric field strength altering layer comprises a dielectric distancing layer and at least one metal film; wherein said dielectric distancing layer is deposited on said nanopore membrane; and wherein said dielectric distancing layer distances an excitation zone, where said at least one light source illuminates said at least one polymer, from said at least one nanopore.

32. The device according to claim 21 , wherein said electric field strength altering layer comprises a dielectric distancing layer and a waveguide; wherein said dielectric distancing layer is deposited on said nanopore membrane; and wherein said dielectric distancing layer distances an excitation zone, where said at least one light source illuminates said at least one polymer, from said at least one nanopore.

33. The device according to claim 21 , wherein said at least one polymer is selected from the list consisting of: a biological polymer; an organic polymer; an inorganic polymer; a non-biological polymer; a protein;DNA;RNA; and PNA.

34. The device according to claim 22, wherein said current in said ionic solution generated by said at least two electrodes generates a macroelectric field; and wherein a polarity of said macroelectric field is opposite to a polarity of said microelectric field.

35. The device according to claim 34, wherein said at least two electrodes are coupled with a second voltage source; and wherein said microelectric field can be controlled independently of said macroelectric field.

36. The device according to claim 22, wherein said microelectric field is controlled in a loop configuration selected from the list consisting of: an open loop configuration; and a closed loop configuration.

37. The device according to claim 23, wherein said at least one metal film is deposited using a deposition technique selected from the list consisting of: physical vapor deposition; chemical vapor deposition; and atomic layer deposition.

38. The device according to claim 27, wherein said dielectric layer and said at least one metal film are deposited on said nanopore membrane proximate to said at least one nanopore in a configuration selected from the list consisting of:said dielectric layer and said at least one metal film being perpendicular to one another; said at least one metal film being deposited above said dielectric layer; and said at least one metal film being deposited above said dielectric layer and also perpendicular to said dielectric layer.

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