CROSS-REFERENCE TO RELATED APPLICATION

[01] This application claims priority to Provisional Application No. 10202260046Q, filed in Singapore on November 10, 2022, which is herein incorporated by reference in its entirety.

FIELD

[02] The present disclosure relates to bio-sensors for use in disease diagnostic and particularly to nanopore sensors.

BACKGROUND

[03] Proteomics, genomics, and bio-sensing studies have revolutionized early disease diagnosis and treatment. Proteins are the major bio macromolecule of life whose structure and sequence contain crucial pathological and physiological information. The proteins that are expressed in the sera of a diseased person are different than those in a healthy person. These proteins serve as disease biomarkers which are used to diagnose infectious, acute, and autoimmune illnesses in immunodiagnostic tests.

[04] Techniques such as PCR (Polymerase chain reaction), ELISA (enzyme-linked immunosorbent assay), Gel electrophoresis, and blotting have proven accurate for assessing infectious disease outbreaks. They use molecular probes, fluorescent, and luminescent protein stains to identify proteins. However, fluorescence or luminescencebased detection involves labor-intensive staining procedures for combining the target protein with fluorophore in solution, gels, blots, or arrays which are expensive, do not provide real-time results and most importantly, cannot be reused after detection.

[05] To address some of the shortcomings mentioned above, Field Effect Transistors (FETs) sensors emerged as an alternative to traditional protein detection sensors. These sensors use silicon nanowires or graphene as conducting layer placed in a liquid electrode. The principle of their operation is that the current flowing through the layer is measured as a function of gate voltage. The receptors or antibodies are immobilized on the surface and when the target molecules approach the surface, there is a change in surface potential on the surface which in turn changes the electrical conductivity (i.e.) amount of current that flows through the source and drain when electrostatic gating is applied. While FET sensors have several advantages including high sensitivity and high selectivity, their fabrication is complicated and involves lengthy data gathering and analysis times which makes it unfeasible for commercialization.

[06] Nanopore sensors are considered as an alternative to FETs. This sensors at their core comprise a membrane with one or more nano-sized pores, also recited herein interchangeably as nanopores, drilled therethrough. By applying a voltage across the membrane, an ionic current is established across the membrane and through the nanopore. This method will be better explained herein after. While such nanopore sensor may be able to detect a group of analytes due to a temporarily shift in current, it lacks selectivity for a specific target analyte or multiple target analytes among a larger group of analytes. Moreover, this method lacks the ability to determine the amount of a specific target analyte.

[07] Therefore, there is a need for a new type of bio-sensor and particularly a nanopore sensor which is not only easy-to-implement with inexpensive fabrication process but also provides fast detection capability, high selectivity and high sensitivity to one or more target analytes in a sample.

SUMMARY

[08] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key aspects or essential aspects of the claimed subject matter.

[09] According to a first aspect, the invention is directed to a nanopore sensor for detecting at least one target analyte. The nanopore sensor comprises a compartment containing an electrolyte solution; a membrane located in the compartment for dividing the compartment into a cis chamber and a trans chamber, the membrane comprising a substrate and at least one nanopore therethrough, each of the at least one nanopore defining an inner wall extending from a lumen; a pair of electrodes for applying a bias voltage across the membrane establishing an ionic current of the electrolyte solution across the at least one nanopore from the cis chamber to the trans chamber; and a gold coating covering, at least in part, the inner wall of each of the at least one nanopore. Each of the at least one nanopore is functionalized with probes covering, at least in part, the gold coating, the probes being adapted for selectively binding with the at least one target analyte for detecting the same.

[010] According to a preferred embodiment, the probes or the at least one target analyte may comprise at least one of: antigens, antibodies, aptamers, RNA, DNA, XNA, PNA, proteins or viruses. Preferably, the probes comprises apatmers for detecting proteins as the at least one target analyte.

[Oil] According to a preferred embodiment, the substrate comprise silicon nitride, silicon oxide, metal oxides, graphene, M0S2, borophene, gold, platinum and aluminum, preferably silicon nitride.

[012] According to a preferred embodiment, the membrane has a thickness from 0.5 nm to 1 pm.

[013] According to a preferred embodiment, each of the at least one nanopore has a conical or cylindrical shape, with a diameter of the lumen being from 5 nm to 1 pm.

[014] According to a preferred embodiment, the nanopore sensor may further comprise a layer of titanium underneath the gold coating for improving adhesion of the gold coating to the substrate.

[015] According to a preferred embodiment, the nanopore sensor may further comprising a passivation coating covering a remaining portion of the gold coating, the passivation layer being adapted to reduce or inhibit binding of non-target analytes to the at least one nanopores. Preferably, the passivation coating comprises: compounds having -COOH or -OH group, such as 6-Mercaptohexaol or 6-Mercaptohexanoic acid; bovine serum albumin (BSA), or blocking buffers.

[016] According to a preferred embodiment, each of the at least one nanopore is functionalized with same type of probes for detecting one single target analyte. Alternatively, each of the at least one nanopore is functionalized with different types of probes for detecting different target analytes.

[017] According to a preferred embodiment, the nanopore sensor is configured for regeneration upon reversing the bias voltage. [018] According to a first aspect, the invention is directed to process for manufacturing a nanopore sensor for detecting at least one target analyte, the method comprising:

- drilling at least one nanopore through a substrate for making a membrane, each of the at least one nanopore defining an inner wall extending from a lumen;

- depositing a gold coating on the substrate for covering, at least in part, the inner wall of each of the at least one nanopore;

- incorporating the membrane in a compartment filled with an electrolyte solution for dividing the compartment into a cis chamber and a trans chamber, and

- functionalizing each of the at least one nanopore by covering at least a portion of the gold coating with probes, the probes being adapted for selectively binding with the at least one target analyte for detecting the same.

[019] According to a preferred embodiment, the drilling step of the process is performed using transmission electron microscopy, helium ion microscope, controlled breakdown dielectric, focused ion beam or electron beam lithography, preferably helium ion microscope.

[020] According to a preferred embodiment, the depositing step of the process is performed using thermal evaporation, e-beam evaporation, sputtering, electroplating or electroless plating.

[021] According to a preferred embodiment, the functionalizing step of the process is performed using vapor phase deposition, layer-by-layer self-assembly, silanization and coating of fluid lipids.

[022] According to a preferred embodiment, the process further comprises covering a remaining portion of the gold coating with a passivation layer coating, the passivation layer coating being adapted to reduce or inhibit binding of non-target analytes to the at least one nanopore. Preferably, the passivation coating comprises: compounds having -COOH or -OH group, such as 6-Mercaptohexaol or 6-Mercaptohexanoic acid; bovine serum albumin (BSA), or blocking buffers. [023] According to a preferred embodiment, the functionalization step of the process comprises: functionalizing each of the at least one nanopore with same type of probes for the making of a nanopore sensor detecting one single target analyte, or functionalizing each of the at least one nanopore with different types of probes for the making of a nanopore sensor detecting different target analytes.

[024] According to a third aspect, the invention is directed to a method for detecting a target analyte using the nanopore sensor as defined herein, or obtained by the process as defined herein, the method comprising:

- injecting a sample to be analysed in the compartment containing the electrolyte solution;

- establishing an ionic current across the at least one nanopore from the cis chamber to the trans chamber by applying the bias voltage across the at least one nanopore via the electrodes;

- determining a complete binding between the target analyte and the probes by measuring a constant electric current during a duration At; and

- comparing the measured current with a reference current, wherein a difference between the current and the reference current is indicative of presence of the target analyte in the sample.

[025] According to a preferred embodiment, the detected target analyte has a concentration ranging from about 0.3 fM to about 27 nM.

[026] According to a preferred embodiment, the duration At is ranging from about 1 to 40 s.

[027] Advantageously, the nanopore sensors as described herein provide high specificity and sensitivity for target analytes due to the presence of the gold coating on the substrate and the functionalization with the probes on the surface of the gold coating. Simple process for fabrication compared to FET biosensors. For example, the functionalization could be performed in- situ which considerably simplifies the fabrication process. [028] Other advantages of the instant invention in view of preferred embodiments thereof, will be described herein later.

[029] All features of exemplary embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[030] A detailed description of specific exemplary embodiments is provided herein below with reference to the accompanying drawings in which:

[031] Figure 1 shows schematic views of conventional nanopore sensors known in the art for detecting a group of analytes passing through a nanopore (top views), and corresponding currents (nA) applied in function of time (s).

[032] Figures 2A is schematic view of a nanopore sensor in accordance with an embodiment of the present invention before the analytes go across the nanopore with a constant current (nA), and Figure 2B shows the same when the analytes are bound to the probes of the nanopore when a current is applied.

[033] Figure 3 is a schematic view of a nanopore of the nanopore sensor in accordance with one embodiment of the present invention.

[034] Figures 4A, 4B and 4C are schematic views of respectively: a gold coated nanopore (Fig. 4A), a functionalized gold coated nanopore (Fig. 4B), and the same once the current is applied and the probes bonding the analytes (Fig. 4C), in accordance with an embodiment of the present invention.

[035] Figure 5 is a schematic view of a nanopore in accordance with an embodiment of the present invention comprising a passivation layer over the gold cotaing. [036] Figure 6 is a schematic of different nanopores of a nanopore sensor functionalized with different types of probes for detecting different target analytes. in accordance with an embodiment of the present invention.

[037] Figure 7 is an example flowchart of a process for manufacturing a nanopore sensor in accordance with an embodiment of the present invention.

[038] Figure 8 are images of drilled nanopores obtained via different drilling techniques in accordance with Example 1.

[039] Figure 9A is an image of a titanium coated nanopore and Figure 9A is an image of a gold coated nanopore in accordance with Example 2.

[040] Figures 10A and 10B are XPS characterization curves for the nanopores in accordance with Example 4.

[041] Figure 11 is a graph depicting the 1/f noise analysis results for a nanopore in accordance with Example 6.

[042] Figures 12A and 12B shows complementary noise analysis results for pores coated with gold and with aptamer in accordance with Example 7.

[043] Figures 13A and 13B shows IV characterization results in accordance with Example 8.

[044] Figures 14A and 14B are graphs showing respectively current vs. time and characteristic time vs. concentration, in accordance with Example 9.

[045] Figure 15 is a graph showing variations of nanopore conductance during multiple regeneration cycles and in accordance with Example 10.

[046] Figure 16 depicts effect of voltage on sensing time in accordance with Example 11.

[047] Figure 17 shows results of a control experiments with gold coated nanopore in accordance with Example 12.

[048] Figure 18 shows results for nanopore sensor specificity for non-specific proteins and in accordance with Example 13. [049] Figure 19 shows effect of nanopore diameter on sensitivity of the nanopore sensor and in accordance with Example 14.

[050] Figure 20 shows effect of nanopore thickness in the nanopore sensor and in accordance with Example 15.

[051] Figure 21 shows effect of nanopore diameter on sensitivity of the nanopore sensor and in accordance with Example 16.

[052] Figure 22 shows results for nanopore sensor performance in detecting Thrombin and in accordance with Example 17.

[053] Figure 23 shows results for a regenerative pre-thinned nanopore sensor in accordance with Example 18.

[054] Figure 24 shows results for regeneration of a functionalized nanopore sensor in accordance with Example 19.

[055] Figure 25 shows results for a functionalized nanopore sensor used for cancer detection in accordance with Example 20.

[056] Figure 26 shows a comparison between the nanopore sensor according to the present invention and other commercially available diagnostic tests (sensors) in term of limit of detection (LOD).

[057] Figure 27 shows a comparison between the nanopore sensor according to the present invention and other quick diagnostic tests in terms of testing time (i.e. detection time).

[058] Figure 28 is a qualitative comparison between the nanopore sensor according to the present invention and various standard protein detection methods.

[059] In the drawings, exemplary embodiments are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustrating certain embodiments and are an aid for understanding. They are not intended to be a definition of the limits of the invention. DETAILED DESCRIPTION

[060] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art considering the instant disclosure which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some embodiments of the technology, and not to exhaustively specify all permutations, combinations, and variations thereof.

Definitions

[061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods which will be described later are those well known and commonly employed in the art.

[062] The definition of main terms used in the detailed description of the invention is as follows.

[063] By "about", it is meant that the value of any data disclosed herein can vary within a certain range depending on the margin of error of the method or device used to measure or evaluate such data. A margin of + 10% encompassed by the term “about” is generally accepted.

[064] As used herein, the term “probe” refers to a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Examples of such molecules include, but are not limited to: Antigens, Antibodies, Aptamers, RNA, DNA, XNA, PNA, Protein, or Viruses. [065] As used herein, the term “sample” refers to a composition that might contain a target material of interest to be analyzed. It may be detected in a sample collected from one or more of liquids, soil, air, food, waste, animal and plant organs, and animal and plant tissues, but is not limited thereto. Herein, the liquids may be water, blood, urine, tears, sweat, salvia, lymph and cerebrospinal fluids. The scope of the instant invention cannot be limited to these examples of samples.

[066] As used herein, the term “analyte” means any solute in a solution which can be detected (or its concentration measured) using a sensor. Examples of such analytes include, but are not limited to: Antigens, Antibodies, Aptamers, RNA, DNA, XNA, PNA, Protein, Viruses.

[067] As used herein, the term “membrane” means a polymer film, plug of hydrogel, liquid-infused film, tiny pore, or other suitable material which is permi selective to transport of a solute through the membrane by solute parameters such as size, charge state, hydrophobicity, physical structure, or other solute parameters than can enable permiselectivity. For example, a dialysis membrane is permiselective by passing small solutes but not large solutes such as proteins. Membranes as understood herein comprise a substrate which is coated by one or more coating layers.

Nanopore sensor

[068] AS aforesaid, nanopore sensors are considered as an alternative to FETs. This sensors at their core comprise a membrane with one or more nano- sized pores (also recited interchangeably as nanopores) drilled therethrough. A simplified schematic of one nanopore drilled through a membrane of a typical nanopore sensor is shown in Figure 1. The membrane 2 divides a compartment 1 , filled with an electrolyte solution (ES), into two chambers: a cis chamber and a trans chamber. By applying a voltage across the membrane 2 and via electrodes 10, an ionic current is established across the membrane 2 and through the nanopore 3 as shown by the current vs. time plot (A) on the left side of Figure 1. Once a group of analytes 7 get to proximity of the nanopore 2 and pass through it, the nanopore becomes temporarily constricted (because of the passage of the group of analytes) creating a resistance which in turn temporarily reduces the current as shown by the current vs. time plot in the middle of Figure 1, (B). Upon complete passage of the group of analytes 7 through the nanopore, the previously created resistance due to the constriction of the nanopore ceases to exist and therefore, the current increases again and returns to its initial level as shown by the current vs. time plot on the right side of Figure 1 (C). While such nanopore sensor may be able to detect a group of analytes due to a temporarily shift in current, it lacks selectivity for a specific target analyte or multiple target analytes among a larger group of analytes. Moreover, since the shift in current is caused by passage of a group of analytes, the method lacks the ability to determine the amount of a specific target analyte.

[069] The above drawbacks of the know technique are resolved by the invention which is now described in the following detailed description in reference to Figures 2 to 7.

[070] Figures 2A and 2B are schematics of a nanopore 3 drilled through a membrane 2 of the nanopore sensor in accordance with an embodiment of the present invention. For sake of simplicity, the compartment that contains the electrolyte solution and is separated by the membrane into a cis chamber and a trans chamber have not been shown and only the cis side and trans side of the nanopore have been illustrated. Furthermore, only one nanopore is depicted while more than one nanopore can be drilled to the membrane 2 as shown in Figure 6.

[071] The membrane itself could be thought as a substrate which may be coated. The substrate can be made of different materials such as silicon nitride; silicon oxide; metal oxides; 2D materials such as Graphene, MoS2 or Borophene; Gold; Platinum and aluminum. In a preferred embodiment, the substrate is made of silicon nitride also recited interchangeably as silicon in the present disclosure.

[072] Returning to Figures 2 A and 2B, the nanopore 3 is functionalized with probes 4 at least on the inner walls of the pore 2. It is noteworthy to mention that the probes 4 (also known as ligands) once dispersed over the nanopore upon functionalization, they only cover a portion of the surface of the nanopore 3 and yet there is still a remaining portion of the nanopore 3 surface that is not covered by probes.

[073] By applying a bias voltage through the electrodes 10 (such as Ag/AgCl) immersed in the compartment containing the electrolyte solution (ES) and the membrane 2, a constant ionic current (current vs. time plot in Figure 2A) is established across the membrane 2 and through the nanopore 3. If a sample containing a group of analytes 7 is brought to the proximity of the nanopore as shown in Figure 2A, the group of analytes would pass through the nanopore along the direction of the ionic current. However, there is only one target analyte 5 (shown here by triangular shapes) among the group of analytes 7 that needs to be detected by the nanopore sensor and it is only the target analyte 5 that can interact with the functionalized nanopore 3. In other words, it is only the target analyte 5 that can bind to the probes 4 while the non-target analyte(s) 6 (also known as non-specific analytes) make no binding with the probes 4. Throughout the present disclosure, the terms non-target analyte and non-specific analyte may be used interchangeably.

[074] In a preferred embodiment, the binding between the probe 4 and the target analyte 5 is via adsorption.

[075] Since the nanopore ionic current is sensitive to the nanopore geometry, the current starts to gradually decrease upon formation of initial bindings between the target analyte 5 and the probes 4, as shown by the current vs. time plot in Figure 2B. As can be appreciated by the person skilled in the art, the current in this case would decrease toward a plateau which indicates that no further binding between the probes and the target analyte would occur and therefore the geometry of the nanopore would not be changed any further. This plateau is also referred to as “saturation state”.

[076] In some embodiments, the probes may be antigens, antibodies, aptamers, RNA, DNA, XNA, PNA, proteins, viruses, etc. In a preferred embodiment, the probes are aptamers.

[077] In some embodiments, the target analyte may be antigens, antibodies, aptamers, RNA, DNA, XNA, PNA, proteins, viruses, etc. In a preferred embodiment, the target analytes are proteins.

[078] Figure 3 is a schematic of the nanopore 3 which is coated with a layer of gold 8, at least on the inner wall of the nanopore 3, in accordance with the present invention (the compartment and the electrodes across the membrane are not shown for simplicity). Also, prior to gold deposition, a layer of titanium (not shown) may be preferably deposited at least on the inner wall of the nanopore. This layer is sandwiched between the surface of the nanopore 3 and the gold coating 8 and serves to improve adhesion between the gold coating 8 and the membrane 2. [079] As can be seen, upon coating the nanopore 3 with a layer of gold 8, functionalization with probes 4 is performed over the gold coating 8. The inventors have surprisingly found out through experiments that deposition of gold on the nanopore 3 will increase sensitivity of the nanopore sensor in detecting the target analyte 5. Details of the gold coating process will be described later in the present disclosure.

[080] Upon functionalization, if a sample containing the target analyte 5 and nontarget or non-specific analytes 6, the probes 4 will only bind with the target analyte, shown by round shapes in Figure 3, with an increased sensitivity conferred by the gold coating 8.

[081] Due to nano-metric size of the pore 4, both functionalization and binding would affect the size of the nanopore 3. To further elaborate, upon functionalization of the nanopore 3, probes 4 are adhered at least to the inner wall of the nanopore. Therefore, the pore becomes constricted and creates an additional resistance which in turn decreases the current. The pore becomes even more constricted when the target analyte 5 binds with the probe resulting in increased resistance and therefore further decrease in current. This is illustratively shown in Figures 4A-C as well.

[082] As can be seen in Figure 4A, a nanopore 3 has been drilled in a membrane 2 and the nanopore 3 (including its inner wall) is coated with the layer of gold 8. The purpose of the gold coating and related details will be described later in the text. In such arrangement, the ionic current established at the nanopore 3 is denoted by Ii. At the next stage, as shown by Figure 4B, when the surface of the coated membrane (i.e. the gold coating) is functionalized with probes 4, the geometry of the nanopore 3 is affected causing a reduction in conductance of the nanopore 3 and consequently a drop in the ionic current (I2 < Ii).

[083] Finally, and as shown in Figure 4C, once probes 4 on the functionalized nanopore 3 bind with the target analyte 5, the geometry of the nanopore 3 is further affected causing another drop in the ionic current (I3 < I2). Again, and as illustrated in Figure 4C, once all the probes had their binding with the target analyte, the current reaches a plateau indicating the saturation state for the functionalized nanopore 3. [084] It would be apparent to the person skilled in the art that a change in geometry of the nanopore 3 may mean a linear or non-linear decrease in diameter of the nanopore 3 or any other changes in the dimensions of the nanopore 3 either at the lumen of the pore (pore entrance) or in its inner wall.

[085] Figure 5, is a schematic of a nanopore 3 in accordance with another embodiment of the present invention. As discussed above, the functionalization of the nanopore 3 is done such that only a portion of the nanopore 3 is covered by probes and there is still a remaining portion of bare gold coating. This remaining portion may have interactions with non-target analytes and therefore, in some embodiments, the surface of the nanopore 3, after functionalization, is further coated by a passivation layer 9 to reduce or inhibit non-specific interactions with the nanopore 3.

[086] It is noteworthy to mention that a membrane 2 may have more than one nanopore 3 in which at least one nanopore becomes being functionalized with a probe different from other probes that have been used for functionalizing other pores. The purpose of such arrangement is to have multiple pores for detecting multiple target analytes 5. A non-limiting example of such arrangement is schematically shown in Figure 6 and in accordance with another embodiment of the present invention. As can be seen, a snapshot of the membrane 2 is illustrated along with four nanopores 3A-3D drilled through the membrane. While the nanopores 3A-3D are all drilled in the membrane, they can be drilled with different methods which will be described later. Also, the nanopores 3A-3D may have different diameter while still being in the order of nanometers. Such arrangement may be used for detecting a group of analytes 7 at the same time. Each of the nanopore 3A-3D are functionalized with a different probe (4A- 4D) that interacts with a specific analyte (i.e., each probe interacts/binds its own target analyte which may be non-specific to other probes). The corresponding current vs. time plots for each nanopore is shown underneath each pore. As can be seen, pores 4A, 4C and 4D are functionalized with probes 4A, 4C and 4D and the variations in current over time for each probe is indicative of the detection of the target analyte. It is possible that a pore such as 3B that is functionalized with probe 4B does not interact with any of the analytes 7 shown in Figure 6. Instead, such pore may be used, in a specific experiment, as a control or may be used, in other experiments, for other types of analytes that are not included in Figure 6. Kinetics of the binding

[087] The binding between the probe and the target analyte as well as the detachment between the two is governed by a kinetic reaction. In a specific model for interaction between a protein (as the target analyte) and an aptamer (as the probe), the following reversible reaction takes place: fci

A + P -> AP fc ₂

AP A + P wherein “A” represents an aptamer, “P” represents a protein, and “AP” represents an aptamer-protein complex.

[088] The following is the equation for the chemical reaction, including the concentrations: d (Eq. 1)

- [A] = -k ₁ [A] - [P] + k ₂ [AP] dt

[089] Wherein [A] is the concentration of unbound aptamers at the pore's periphery at a particular time, [AP] is the concentration of aptamer-protein complexes at the pore surface, and [P] is the concentration of proteins inside the pore.

[090] Considering that the overall number of aptamers, whether free or bound to protein, remains unchanged:

[A] + [AP] = A _o = const. (Eq. 2)

[091] Wherein Ao denotes the initial concentration of aptamers at the pore's entrance. Since the supply of proteins is substantially more than the number of aptamers, the concentration of proteins does not vary over time and therefore: [P] = c _p = const.

[092] The bulk protein concentration co and the local protein concentration in the pore c _p are related by:

[093] Wherein is the pore enhancement factor. [094] When a negative voltage is applied, the proteins are electrophoretically concentrated at the pore entrance which means When a positive voltage is applied, the proteins are pushed away from the pore, resulting in a generation depletion zone in the pore's mouth where In general, is influenced by voltage and nanopore geometry.

[095] By combining equations 1-3, the following is obtained:

[096] Once Equation 4 is integrated with a boundary condition [A ] (t = 0) = 4 _o meaning that all aptamers are unreacted with proteins at the beginning, the following is obtained: wherein:

[097] Replacing equation 5 into equation 1 for {A] the following is obtained for m

[098] Further, in order to determine how the conductivity of the nanopore 3 varies over time as a result of adsorbing the proteins (as the target analyte), following equation is used: wherein G _o is the initial conductance of the pore and at any given time. The conductance is defined by the concentration of associated aptamers [AP]. In the case of very small pores, when access resistance is the primary factor in determining conductance, the relation is calculated as following: wherein D represents the nanopore's diameter after aptamer functionalization and G is the conductivity of the electrolyte solution. In this circumstance, there are two extreme conditions:

• when the nanopore is open at the beginning (no adsorption) a , in which Do is the initial diameter of the aptamer-functionalized pore without protein attachment; and

• when aptamers are fully saturated with proteins, where AD is the change in diameter due to the adsorption of proteins

[099] At every given time during adsorption, the overall conductance could be thought as the weighted average of the two limits above and therefore:

[0100] By replacing [A] and [AP] from equations 5 and 7, the following is obtained:

[0101] wherein 8 — D /D is the sensitivity of the pore on the protein adsorption (larger for smaller pores and bigger proteins).

[0102] Assuming that G(t) = G _s at t -> co (i.e. after sufficient time, the pore conductivity reaches a value corresponding to saturation) and that conductivity and current are proportionally related (such that - = — and — = — ), Equation 11 could

Go Io G _o Io be rewritten as:

[0103] Therefore, the saturation current (I _s ) is as follows:

Regeneration of the nanopore sensor

[0104] Attachment and removal of protein molecules on the surface is a reversible process in the nanopore sensor according to a preffered embodiment of the present invention.

[0105] Prior to the introduction of a fresh concentration (of a sample), the pore is regenerated back to its original condition by reversing the applied bias voltage for a period of time, resulting in the unblocking of the nanopore(s) over time. The molecules in the nanopore lumen, upon regeneration by applying reverse bias voltage, are essentially pushed away which returns the nanopore to its initially open state.

[0106] In some embodiments, a fresh DPBS solution is pumped through the nanopore before measuring a new protein sample.

[0107] During normal operation of the nanopore sensor, when a negative voltage is applied to transport the molecules into the nanopore, the molecules from the bulk concentration (Co) travel towards the nanopore and generate a local concentration surrounding the nanopore (C _p ). The relationship between C _p and Co is mediated by 0, enhancement factor from Eq. 3 as discussed earlier. Under this condition, target analytes (such as proteins) are electrophoretically concentrated at the nanopore entrance which indicates that 0»1.

[0108] Upon regeneration, when a positive voltage is applied, the proteins are pushed away from the nanopore, resulting in a generation depletion zone at the lumen of the nanopore, where J3« 1—0. Generally speaking, nanopore geometry and voltage have an impact on the value of 0.

Fabrication of the nanoporc sensor

[0109] As stated above, the nanopore sensor according to the present invention is much less complex to fabricate compared to other sensors such as FETs. Figure 7 is a flowchart example of a process 700 for fabricating a nanopore sensor in accordance with an embodiment of the present invention.

[0110] The process starts at step 710 by drilling one or more nanopores through a membrane substrate. The suitable materials for membrane substrate were previously disclosed above. The membrane substrates for use in a nanopore sensor may have a thickness from about 0.5 nm to about 1 pm.

[0111] Several methods can be employed to drill a nanopore on a membrane substrate. These methods include but are not limited to Transmission Electron Microscopy (TEM), Helium Ion Microscope (HIM), Controlled Breakdown Dielectric (CBD), Focused Ion Beam (FIB) and Electron Beam Lithography for large scale nanopore drilling in a membrane. These methods may differ in terms of the consistency and reproducibility of nanopores they could drill in a substrate.

[0112] In some embodiments only one nanopore may be drilled into the membrane substrate. In some embodiments there may be multiple nanopores drilled into the membrane substrate and these nanopores may form an array of nanopores for simultaneous detection/sensing of same or different target analytes.

[0113] The size of the nanopores that are drilled into a membrane substrate may range from about 5 nm to about 1000 nm (1 pm) in diameter depending on the drilling method used. As for the shape, the nanopores may be cylindrical meaning that the drilling is performed such that the pore entry (or pore lumen) has the same diameter as the channel created through the membrane. Alternatively, the nanopores may be conical meaning that the drilling is performed such that the pore entry (or pore lumen) has the largest diameter which tapers to a smaller diameter as drilling progresses toward the other side of the membrane substrate. The shape may also be dependent on the method use for drilling.

[0114] At the next step 730 of Figure 7, the process comprises depositing a gold coating for covering, at least in part, the inner wall of each of the at least one nanopore. Optionally, the nanopores may be previously coated with a layer of titanium at step 720 which as discussed above serves to improve adhesion between the membrane substrate and the gold coating 8 that will be subsequently deposited on the nanopore surface at step 730. Various methods could be used for deposition of titanium and gold over the nanopore(s). These methods include but are not limited to thermal Evaporation, E-beam Evaporation, Sputtering, Electroplating and Electroless plating. As stated above, the nanopore may be coated on its surface (pore lumen and regions adjacent to pore lumen), on the inner wall or on both the nanopore surface and the inner wall.

[0115] At the next step 740, the membrane with nanopore(s) drilled therethrough is incorporated in a compartment filled with an electrolyte solution such that it divides the compartment into two chambers (cis and trans) that are only in communication through the one or more nanopores.

[0116] Finally, at step 750, the nanopore is functionalized in-situ by covering at least a portion of the gold coating 8 with the probes 4, the probes being adapted for selectively binding with the at least one target analyte for detecting the same.

[0117] In an alternative embodiment, functionalization may be done ex-situ (i.e. outside the compartment).

[0118] Several methods can be employed to functionalize a nanopore. These methods include but are not limited to vapor phase deposition such as chemical vapor deposition (CVD) and atomic layer deposition (ALD); layer by layer self-assembly; silanization; coating of liquid lipids; self-assembled monolayers using gold such as EDC NHS chemistry and Thiol chemistry.

[0119] It should be noted that the functionalization step 750 could be done in-situ (while the membrane being placed in the compartment) which is one of the advantages associated with the fabrication process.

[0120] Further, in a preferred embodiments, the membrane substrate is patterned with a method such as electron-beam lithography (EBL) to limit the area of the substrate that is supposed to be covered by the gold layer. Such patterning results in less consumption of gold (only on regions adjacent to the pores lumen and/or the pores’ inner walls) and functionalization material (i.e. the probe 4).

[0121] In some embodiments, the method may further comprise coating a remaining portion of the gold coating with a passivation layer 9 to reduce or inhibit non-specific interactions with the nanopore 3 (not shown in Figure 7). Examples

[0122] The following examples describe some exemplary modes of making and practicing certain aspects of the presently claimed invention that are described herein. These examples are for illustrative purposes only and are not meant to limit the scope of the compositions and methods described herein.

Example 1 : nanopore drilling

[0123] In this Example, nanopores are drilled on free standing silicon nitride (SiN) membranes using following methods:

• Transmission Electron Microscopy (TEM) -(e.g. JEOL JEM-2010F with field- effect source, operating at 200 keV electron beam energies,

• Focused Ion Beam (FIB), Ga+ Ion beam (e.g. AURIGA 60 FIB-SEM system (ZEISS Microscopy GmBH, Oberkochen, Germany), and

• Helium Ion Microscope (HIM) (Zeiss Orion Nanofab)

[0124] The HIM method was specifically used to fabricate pores in size that were inaccessible by two different regimes by TEM and FIB. A comparison of nanopore drilled by FIB, TEM, and HIM is presented in Table 1 while images of each nanopore are shown in Figure 8.

Table 1 Example 2: Titanium and Gold deposition

[0125] In this Example, the surface and inner walls of the nanopores drilled in a membrane substrate are coated with a sticking layer of titanium followed by a thin layer of gold using E-beam evaporation at 0.2 A/s for titanium and 0.3 A/s for gold. Furthermore, the membrane is patterned by electron-beam lithography (EBL) as discussed previously. The results are presented in Figure 9 showing a 6nm coating layer of Ti topped by a 20nm coating layer of Au deposited on a 10 nm thick membrane (left) and a 300nm thick membrane (right).

Example 3: Functionalization of the nanopore

[0126] In this example, the nanopore from Example 2 was functionalized with the following probes:

• Thrombin: 15-mer DNA aptamer o 5 ’ -GGT TGG TGT GGT TGG-3 ’

• SARS-Cov-2 N protein: 58nt DNA aptamer o 5’-

GCTGGATGTTCATGCTGGCAAAATTCCTTAGGGGCACCGTTA CTTTGACACATCCAGC-3 ’

[0127] The aptamer in this example ends with a thiol group that could interact with gold. Originally, the thiol group in the aptamer is covalently capped/bonded with an inert group to prevent unwanted binding of the thiol group. However, , the thiol group needs to be de-protected before beginning of functionalization for interacting with gold. As for the de -protection of the thiol group of aptamer, 10 nM tris(2- carboxyethyl)phosphine) also known as TCEP was mixed with 100 pM aptamer at room temperature and stayed for 1 hour to cleave the inert capping group and expose the thiol group which is now free to interact with gold. For the purpose of nanopore functionalization, the resulting solution was let to flow into the nanopore and coat it.

Example 4: nanopore XPS characterization

[0128] In this Example, a nanopore which is gold coated and functionalized with aptamers is subjected to X-ray Photoelectron Spectroscopy (XPS) for surface characterization and particularly for analyzing the binding energies and hybridization states of carbon, nitrogen, oxygen, sulfur, and phosphate atoms on the surface of functionalized nanopore. The results are shown in Figures 10A and 10B demonstrating the (P2p) and (S2p) peaks of the XPS spectra of aptamer-coated samples, respectively. The P2p peak is derived from the phosphate backbone of the DNA, whereas the S2p peak is derived from the Sulfur-Gold bond formed when DNA aptamers adhere to the surface.

Example 5: SARS-CoV-2 N rotein detections using aptamers as probes

[0129] In this example, nanopores are drilled in 10 nm thick silicon nitride (SiN) membrane substrate using Helium Ion Beam (HIM) and in a 300nm thick SiN membrane using Focused Ion Beam (FIB).

[0130] The membranes are then patterned for a small area around the nanopore to limit the amount of gold deposited on the surface using Electron Beam Lithography (EBL) method.

[0131] Gold is deposited on these patterned areas using E-beam evaporator. 6nm Titanium is used as a sticking layer between silicon and gold. The parameters used for depositions have been explained in Example 2.

[0132] The 58-nt DNA aptamer used to bind the SARS-Cov-2 N protein has the sequence 5'- thiol-C6-12T-

GCTGGATGTTCATGCTGGCAAAATTCCTTAGGGGCACCGTTACTTTGACAC ATCCAGC-3'. This aptamer will fold into a hairpin structure to attach to the target N protein. Typically, aptamers possess two binding modes. One is to fold into a definite, unchanging configuration prior to binding. The second is that aptamers induce folding in the presence of a target. It is anticipated that the secondary structure of this aptamer will fold into a hairpin configuration. Prior to functionalization, the SARS-Cov-2 N aptamer is heated at 950°C for 5 min and cooled down to room temperature gradually for about 15-20 min.

[0133] 20pM aptamer solution is mixed with 2mM TCEP bond breaker solution diluted with DPBS and reacted for 1 hour to de -protect the thiol. This process is done in order to dissolve its secondary structure. The nanopore is then coated with this deprotected aptamer solution for 7 hrs. inside the cell. This functionalization process is done in-situ as described before (i.e. the process happens inside the cell and we don’t have to remove the chip for coating and insert back for measurement).

[0134] The electrolyte used is DPBS, NaCl 137 mM, KC1 2.7mM, KH2PO4 1.5mM, Na ₂ HPO ₄ 8mM, CaC12 ImM, MgCh 0.5mM, pH 7.4.

[0135] The recombinant SARS-CoV-2 N protein molecular weight is 46000KDa with pl value of 10.07. This protein is positively charged at the electrolyte pH of 7.4.

Example 6: 1/f noise analysis

[0136] In this Example, lOnm-thick silicon nitride membranes are drilled using a Helium ion beam. The measurement traces for both positive and negative voltages (OmV, ±100mV, ±200mV) are recorded so that the power spectral density can be plotted (as shown in Figure 11), and 1/f noise can be analyzed to determine how it varies after coating.

[0137] Figure 11 depicts the 1/f noise below 100 Hz for a gold-coated nanopore (line# 1) and an aptamer-coated pore (line# 2). The noise power referred to as H is calculated to be 1.65 e-6 nA2 before aptamer coating and 2.21 e-6 nA2 after coating.

Example 7: complementary noise analysis

[0138] This example quantifies the change in 1/f noise more precisely by determining the Hooge parameter (A or a) by dividing the noise power by the squared current value (H=AI ² or A=H/I ² ) at various voltages (±100mV, ±200mV). The value for Hooge parameter is 7.078e-8 for pores coated with gold and 4.6768e-8 for those coated with aptamer as given in Figure 12A and 12B, respectively. Changes in this noise parameter indicate that surface characteristics have changed, supporting the modification of pores after coating.

Example 8: IV characterization and change in nanopores diameter

[0139] Given the sensitivity of nanopores (in DNA and protein sensing applications) to changes in diameter, the change was monitored during functionalization (coating with DNA aptamers) and binding of the aptamers with the target analyte. The initial nanopore diameter upon gold coating is determined by TEM while changes on the nanopore diameter after aptamer coating and after N protein binding is determined on the basis of variation in ionic conductance. The results are presented in Figures 13 A and 13B showing a plot of IV curves prior to and following N aptamer coating and respective diameter reduction after coatings and protein attachment, respectively.

[0140] As shown in Figure 13B there has been an overall reduction of 7 nm in nanopore diameter after the coating. After binding of the N protein, the nanopore diameter further shrunk.

Example 9: Concentration measurement and characteristic time (r)

[0141] In this Example, a stock concentration 18.91 pM of N protein is diluted to 0.7nM, 2nM, 7nM, 22nM, 70nM and are inserted into an 80cnm pore in the fluidic cell for measurements and current traces are obtained as shown in Figure 14 A.

[0142] At a working electrolyte pH of 7.4, the SARS-CoV-2- Nucleocapsid protein has an isoelectric point of 10.07 and is positively charged. Therefore, the protein molecules are driven into the nanopore using a negative bias voltage of -200mV.

[0143] From lower to higher concentrations, proteins were introduced into the nanopore sensor chamber. The first concentration inserted was 0.7nM and a decrease was noticed in current as a result of the aptamer and protein binding. The adsorption curves for various concentrations starting from 0.7nM to 70nM are fitted by Eq. 12. Further, the characteristic time (r) is calculated from Eq. 5.12 that can be quantitatively used to describe the rate of adsorption of the aptamer binding sites.

[0144] The characteristic time r decreases with concentration increase, as seen in the Figure 14B suggesting that it takes less time for the current to reach saturation which means faster adsorption. The characteristic time (r) value, which depends on time, can be used to calculate protein concentration. The characteristic time (r) fitted with two rate constants pki and k with values (1.8+ 0.5) x IO ⁵ [M ^-1 s ^_1 ] and (1.2 + 0.7) x 10’ ³ [s’ respectively, based on Eq. 6.

Example 10: Regeneration of a nanopore sensor in lOnm thick pores

[0145] In this Example, a nanopore sensor used for N protein sensing with 10 nm thick pores is regenerated for multiple cycles and the conductance is determined as described above. The results of the regeneration are presented in Figure 15 showing multiple usability of the nanopore sensor by which the conductance nearly returns to its initial value after each regeneration sequence.

Example 11: Effect of voltage on sensing time (electrostatic focusing)

[0146] In this Example, 7nM N protein concentration is inserted into a nanopore with a diameter of 41nm and a thickness of 300nm. Different voltages such as -200mV, - 250mV, -300mV, -350mV are applied to drive the protein molecules as presented in Figure 16 (left). Observations indicate that an increase in applied voltage leads to faster sensing time. This is measured by the characteristic time (r) calculated from Eq. 5.10, whose value fell as the voltage increased, as seen in Figure 16 (middle). Further, and as shown in Figure 16 (right), a trend is observed indicating that an increase of the inverse of characteristic time (1/ r) which confirms the hypothesis of electrophoretic focusing meaning that voltage causes electrostatic focusing of protein molecules near to the pore entrance and as the voltage is increased, it modifies (i.e.) increases the local concentration of protein molecules more and more leading to faster detection times. Also, at no applied voltage, P<=1. As the voltage is increased, the proteins from bulk concentrations concentrate around the pore and P, enhancement factor increases (P»l) permitting the detection of more molecules at a faster rate, hence increasing the sensitivity of detection.

Example 12: control experiment with gold coated nanopore

[0147] In this Example, the interaction between the bare gold surface (nonfunctionalized) and the N protein as probe is tested. The result in terms of current measurement is shown in Figure 17 in which the current before and after introduction of the N protein to the nanopore sensor is the same (i.e. the two lines coincide). This means the drop which is observed in the presence of the aptamers (upon functionalizing) is due to interaction and binding dynamics between the aptamer and the N protein which is slowly blocking the pore and not due to nonspecific adsorption of the protein molecules on the bare gold surface.

Example 13: Nanopore sensor specificity for non-specific proteins (Elastase and Thrombin) [0148] In this Example, the specificity of the nanopore sensor is experimentally verified using Thrombin as the non-specific protein (or analyte) and SARS-CoV-2 Nucleocapsid protein as the specific protein (or analyte). The nanopore in the sensor is functionalized with N aptamer.

[0149] Thrombin is a protein with molecular weight of 36.7kDa and a pl value of 7. This protein is slightly negatively charged at pH 7.4 and therefore a positive voltage is used to drive the molecules through the nanopore. As shown in Figure 18, it is observed that N aptamer did not interact with thrombin at IpM concentration and no drop in current was observed before or after Thrombin introduction for testing.

[0150] In contrast, when aptamer-coated nanopores were used to detect considerably lower amounts of N protein, protein adsorption occurred as evidenced by a current reduction which proves that the nanopore sensor is selective towards SARS-CoV-2 Nucleocapsid protein (N protein).

Example 14: Effect of nanopore diameter on sensitivity of lOnm thick nanopores

[0151] In this Example, the effect of nanopore diameter on sensitivity was tested by plotting characteristic time (T) against concentration comparing values between I) a nanopore of 24nm in diameter at concentrations: 2.2pM, 0.7nM, 7nM and 70nM and II) a nanopore of 80nm in diameter at concentrations: 0.7nM, 2nM, 7nM, 22nM and 70nM. Both nanopores were lOnm thick and were both subjected to a -200mV applied voltage. The results are shown in Figure 19.

[0152] The characteristic time for both nanopore diameters was fitted using pkl and k2 values shown in Table 2 using Eq. 6.

Table 2 [0153] Based on Table 2, an enhancement was observed in both pki and k2. The most significant enhancement is in pki with an order of magnitude change from 10 ⁵ to 10 ⁶ . The slight change in ki could also be attributed to the curvature of the pore.

[0154] Overall, it was determined that the sensitivity of detection is faster in nanopores with a smaller diameter compared to those with a larger diameter.

Example 15: Effect of nanopore thickness

[0155] In this Example, the effect of nanopore thickness on sensitivity was tested by plotting characteristic time (r) against concentration comparing values between I) a nanopore of 24 nm in diameter and 10 nm in thickness at concentrations: 2.2pM, 0.7nM, 7nM and 70nM and II) a nanopore of 17 nm in diameter and 300 nm in thickness at concentrations: 0.7nM, 2nM, 7nM, 22nM and 70nM. Both nanopores were subjected to a 200 mV applied voltage. The results are shown in Figure 20.

[0156] The characteristic time for both nanopores was fitted using pki and k2 values shown in Table 3 using Eq. 6.

Table 3

[0157] Based on Figure 20, it was observed that the characteristic time for protein adsorption of in thick and thin holes falls in different regimes. Particularly, the adsorption of proteins is faster in thicker pores for the injected concentrations. This is due to the fact that binding sites are abundant in pores with a large surface area. Using FIB to drill pores in thick pores results in a membrane cross-section with 70-degree sidewalls. This slanted sidewall allowed more molecules to cling to the nanopores' walls, which dominated pore conductivity and led to faster detection rates.

[0158] Moreover, from Figure 20, a steep curve is observed at higher concentrations that reaches saturation. When in saturation regime, the change in concentration results in a little change in characteristic time. Also, pki values do not change significantly indicating that the sensitivity does not change much with thickness but rather with diameter.

[0159] However, thick pores are faster in detection and the small k changes can be attributed to the nanopore geometry. Overall, it can be concluded that pki does not depend on the thickness, yet it is strongly dependent on the nanopore diameter, where is the enhancement factor. The smaller the nanopore diameter, the greater the sensitivity enhancement. Also, I is dependent on the diameter and its dependence on thickness is unclear. While it was expected that k2 only depends on chemistry, nanoscale curvatures and confinements may influence I . Therefore, by increasing ki value and decreasing the dissociation Io value, the sensitivity of the nanopore sensor according to the present invention is significantly increased by optimizing nanopore chemistry, nanopore geometry, and voltage.

Example 16: Diameter dependence on sensitivity in 300nm thick nanopores and limit of detection (LOD)

[0160] In this Example, the sensitivity dependence on nanopores diameter was tested for nanopores of 300 nm thick by plotting characteristic time (r) against concentration comparing values between I) a nanopore of 71 nm in diameter at concentrations: 0.3fM, 0.3pM, 0.3nM and 0.3pM and II) a nanopore of 17 nm in diameter at concentrations: 0.7nM, 2nM, 7nM, 22nM and 70nM. Both nanopores were subjected to a 200 mV applied voltage. In particular, the range of concentrations from femto-molar to micromolar were introduced to the 71 nm diameter nanopore to evaluate the detection limit of the sensors. The results are shown in Figure 21.

[0161] According to Figure 21, the lowest detection limit achieved by the nanopore sensor according to the present invention corresponded to concentrations as low as 0.3fM. With an increase in concentration, the characteristic time decreases. Since molecules take longer time to bind to surfaces at very low femto-molar concentrations, they need to be pulled into the pore lumen by the applied voltage for detection. Finally, and similar to the result obtained for nanopores that were lOnm thick, Figure 21 indicates that the smaller diameter pores are more sensitive than larger diameter pores. Example 17: Thrombin detection using aptamer functionalized nanopores

[0162] In this Example, the nanopore sensor which is functionalized with aptamers is used to detect Thrombin. Nanopores are drilled through a 300 nm thick Silicon nitride membrane by focus ion beam (FIB). The nanopore is coated with a sticking layer of titanium followed by a thin layer of gold. The 15-mer DNA oligonucleotide (5’-GGT TGG TGT GGT TGG-3’) is coated onto the gold surface as the probe to form stable binding with the protein molecules. 15-mer TBA aptamer is mixed with TCEP for one hour at 5:1 volume ratio to de-protect the thiol. The 15-mer DNA oligonucleotide will form a stable intramolecular G-quadruplex structure in antiparallel orientation with thrombin molecule in a chair-like conformation. Electrolyte used is IM KC1 solution buffered at pH 9.

[0163] Negatively charged thrombin molecules at pH 9 are attracted towards the nanopore by external applied voltage and the cis chamber is grounded while applying bias voltage at the trans chamber’s electrode. When the thrombin is attached around the nanopore, the ions passing through the nanopore decreases resulting in a drop in current and conductance.

[0164] Positive bias voltage at the trans chamber’s electrode can also be applied to push away the protein molecule from the nanopore surface (regeneration) for subsequent sensing with different concentration or different proteins.

[0165] On the other end, the covalent bond between the 15-mer DNA oligonucleotide and the gold surface remains stable allowing the regeneration of the pore for multiple sensing. The thrombin protein molecular weight is 36.7KDa with pl value around 7. This protein is negatively charged at pH 9.

[0166] The results obtained from this Example are presented in Figure 22. The top left plot depicts the reduction of the effective diameter of the nanopore before and after gold coating, after functionalization, followed by thrombin attachment as well as TEM images of the pristine nanopore before and after the gold coating. The bottom left plot depicts the drop of nanopore current as the thrombin molecules attach to the functionalized surface. The right graph shows the exponential decay constant T value after fitting the aforementioned current reduction curve with exponential decay function. Example 18: Regenerative 10 nm thick nanopore sensor

[0167] In this Example, a pristine nanopore is gold coated and then functionalized to bind with Thrombin at different concentrations. The results are presented in Figure 23. The top left plot depicts the reduction in nanopore diameter due to gold coating, functionalization and thrombin attachment. The inset shows the TEM image of the nanopore before and after the gold coating. The bottom left plot depicts the drop of normalized nanopore current after different thrombin concentration is injected into the nanopore chamber. The pre-thinned membrane, combined with sputtering gold uniformly enables ultra-low concentration sensing, achieving 0.3fM of thrombin molecule sensing that is 40% better than high performance FET biosensors, and one order of magnitude better than majority of FET sensor. The right plot shows the exponential decay r value for different thrombin molecule concentration.

Example 19: Regeneration of functionalized nanopore sensor

[0168] In this Example, detachment of Thrombin from a functionalized nanopore (i.e. regeneration) is done and the results are presented in Figure 24. The left plot depicts the dynamics of the pore opening by applying positive bias voltage for detachment of thrombin molecules from the functionalized surface. The right plot depicts the conductance change during four cycle of detachment and reattachment of thrombin molecules onto the functionalized surface. This reusable feature is possible as the binding force between the thrombin molecules and the functionalized surface is not as strong as antigen-antibody binding force. The thrombin molecules will be subjected to suction force to detach from the surface near the nanopore as the positive bias is applied and the molecules are negatively charged.

Example 20: Functionalized nanopore for cancer detection

[0169] In this Example, the sensing of cancer related protein is achieved by first functionalizing the gold surface with DSP-dithiobis(succinimidylpropionate). DSP forms stable binding with gold surface via disulfide bonds. After gold surface functionalization, the CEACAM Pan Antibody (D14HD11) is attached onto the gold surface via the active NHS group on the DSP. The surface is then prepared to form stable bond with the specific cancer related antigen, the CEACAM-5 Recombinant Human Protein (His-Tag). The antigen and antibody binds via electrostatic interaction, hydrogen bonds, Van Der Vaals forces, and hydrophobic interactions at the interaction sites.

[0170] The results for this Example are presented in Figure 25. The top left plot shows the nanopore reduction graph. The bottom left plot shows the normalized current drop when the nanopore functionalized gold surface is attached to the aforementioned antigen. The right plot shows the exponential decay r value with respect to different antigen concentrations. In medical term, it is generally accepted that 0.68nM or above means that the patient maybe developing cancer while below 0.68nM means that the patient is healthy with sensitivity of 43% and specificity of 100%.

Comparison with commercially available diagnostic tests (sensors) and overall performance

[0171] The nanopore sensor according to the present invention has been compared with commercially available diagnostic tests (sensors) in term of limit of detection (LOD) and the result in presented in Figure 26 showing a significantly lower detection limit of the current nanopore sensor compared to other available rapid diagnostic tests.

[0172] Moreover, a comparison between the testing time (i.e. detection time) of the current nanopore sensor and other quick diagnostic tests are presented in Figure 27 which again shows a significant improvement (in the order of 5 minutes) in terms of detection time whereas other quick diagnostic tests have detection times of 6 hours (RT- PCR) and 15 minutes (Rapid diagnostics).

[0173] Finally, the nanopore sensor according to the present invention is qualitatively compared against various standard protein detection methods such as Liquid Chromatography-Mass spectrometry (LC-MS), Enzyme-Linked Immunosorbent Assay (ELISA), Immunoelectrophoresis, and Western Blot in terms of sensitivity and testing/detection time in Figure 28. As can be seen, the performance of the nanopore sensor according to the present invention is significantly improved compared to the other methods.

[0174] Advantageously, the nanopore sensors as described herein provide high specificity and sensitivity for target analytes due to the presence on the substrate of the gold coating and the functionalization with the probes on the surface of the gold coating. The invention also provides a simple process for fabrication compared to FET biosensors. For example, the functionalization could be performed in-situ which considerably simplifies the fabrication process.

[0175] Among other advantages of preferred embodiments of the instant invention, there are:

• Ability to concentrate the molecules of interest at the lumen (entry or opening) of the nanopore through voltage application rather than diffusion. This is due to electrostatic focusing near the opening of the pore;

• Option to add further protective (passivation) layer for increased specificity;

• Fast detection/sensing (in seconds) and ability to shorten the detection/sensing time by increasing the applied bias voltage; and/or

• Capability for regeneration (by applying a reverse bias voltage) and therefore being reusable for multiple tests. The nanopore sensor is also compatible with chemical regeneration.

[0176] It would be apparent to the person skilled in the art that while the drawings in the present disclosure have illustrated a packing list envelope with a rectangular geometry, other non-rectangular geometries could also be envisioned without departing from the spirit of the present invention.

[0177] While illustrative and presently preferred embodiments of the invention have been described in detail hereinabove, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.