Nanosensor chip with composite nanopores

Abstract

A nanosensor chip for detecting and / or quantifying target molecules in a liquid sample is provided. [Solution] A nanosensor chip for detecting and / or quantifying target molecules in a liquid sample includes a semiconductor or other substrate and one or more electrode structures. The substrate has one or more composite nanopores, called "compores." Each compore is an aperture formed in the substrate and contains multiple nanopores. Each nanopore is functionalized with immobilized probe molecules for detecting target molecules. For each compore, a corresponding electrode structure is disposed on the substrate. The electrode structure has a shape and position relative to the compore to apply an electric field to all nanopores within the compore and to provide a conductive path for the total current flowing through all nanopores within the compore. The total current changes as a function of the electric field in response to target molecules in the liquid sample binding to the probe molecules.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS)

[0001] This application claims priority to U.S. Patent Application No. 16 / 383,491, filed April 12, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002]

[0001] Nanopipettes have been developed to detect biomolecules in liquid samples. Current nanopipette sensors have a single funnel-shaped structure with probe molecules on the inner wall of the structure. The nanopipette sensor is immersed in a liquid sample and an electric field is applied to the sensor. When the electric field is applied at a certain voltage, target molecules bind to the probe molecules. This binding produces a detectable change in current in the nanopipette sensor. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0002] Current nanopipette sensors made from quartz glass capillaries have significant drawbacks. They are difficult to fabricate and manufacture on a large scale, and are fragile and easily damaged. In addition, there is a large variation in individual nanopipettes, so they must be individually calibrated to obtain accurate results. Also, after repeated use, nanopipette tips undergo chemical and electrical degradation, which significantly impairs their performance and limits their reuse. [Means for solving the problem]

[0004]

[0003] Disclosed herein is a nanosensor chip for detecting and / or quantifying target molecules in a liquid sample. The nanosensor chip includes a semiconductor substrate having one or more compound nanopores formed therein. Each compound nanopore is an aperture that includes a plurality of nanopores, each of which is functionalized with an immobilized probe molecule. The probe molecules are used to detect the target molecules in the liquid sample. The compound nanopores are referred to herein as "compores."

[0005]

[0004] Each compore has a corresponding electrode structure on a semiconductor substrate. The electrode structure has a shape and a position relative to the compore that allows an electric field to be applied to all nanopores in the compore. The electrode structure also provides a conductive path for detecting a total current flowing through all nanopores in the compore. The detected total current changes in response to target molecules in the liquid sample binding to the probe molecules. This binding is a function of the applied electric field.

[0006]

[0005] For example, if a liquid sample contains some target molecule (e.g., a specific viral protein in a biological fluid sample), the target molecule will bind to the probe molecule in the nanopore when a specific electric field (or voltage) is applied to the compore. The probe molecule functionalizing the compore is selected to bind to a specific target molecule when a specific electric field is applied. When the target molecule binds to the probe molecule, the electrical characteristics of the nanopore opening change, which causes a change in the total current through the compore. A given pairing of probe molecule and target molecule binds in the presence of a specific electric field strength or field strength range. If the liquid sample does not contain the target molecule and an electric field is applied, no change in the total current occurs. Furthermore, if an electric field other than the specific electric field strength or field strength range is applied, the target molecule will not bind to the probe molecule and no change in the total current occurs.

[0007]

[0006] The Compore structure provides a higher level of reliability than conventional nanopipette sensors. For example, if one of the nanopores of the Compore becomes blocked or clogged, a change in the total current is detected based on the binding of the probe molecules and the target molecules in the other unblocked nanopores. Furthermore, if the nanopores of a given Compore are not uniformly functionalized with the probe molecules, for example, if some nanopores have a high concentration of the probe molecules and other nanopores have a low concentration of the probe molecules, the change in the total current through all the nanopores will average out this concentration variation when detecting the presence of the target molecule in the liquid sample. Due to this increased reliability, the Compore sensor is more accurate and reliable than conventional nanopipette sensors.

[0008]

[0007] Nanosensor chips may also have other advantages over nanopipette technology. Specifically, nanosensor chips can be efficiently and inexpensively manufactured at scale. An entire wafer containing multiple chips can be functionalized with probe molecules simultaneously, whereas conventional nanopipettes are functionalized individually. With improved consistency, a single chip can be used to calibrate an entire wafer containing multiple chips, rather than calibrating the nanopipettes individually.

[0009]

[0008] Other aspects include components, devices, systems, improvements, methods, processes, applications, and other techniques related to any of the above. [Brief description of the drawings]

[0010] [Figure 1]

[0009] FIG. 1 is a top view of a nanosensor chip with multiple composites. [Diagram 2]

[0010] A perspective view of a single compoa. [Figure 3A]

[0011] FIG. 1 is a top view of a single compore. [Figure 3B]

[0012] FIG. 3B is a cross-sectional view of the Compore shown in FIG. 3A. [Figure 4]

[0013] FIG. 1 shows a cross-sectional view of a single nanopore within a Composite. [Figure 5A]

[0014] 1 is a cross-sectional view showing the operation of a nanopore. [Figure 5B]

[0014] FIG. 1 is a cross-sectional view showing the operation of a nanopore. [Figure 6A]

[0015] 1 is a graph showing the operation of Compore. [Figure 6B]

[0015] A graph showing the operation of Compoa. [Figure 7]

[0016] FIG. 1 is a top view showing a first alternative arrangement of nanopores within a composite. [Figure 8]

[0017] FIG. 13 is a top view showing a second alternative arrangement of nanopores within a composite. [Figure 9]

[0018] FIG. 1 is a cross-sectional view of a Compore fabricated using semiconductor technology. [Figure 10]

[0019] FIG. 1 is a block diagram of a detection system including a nanosensor chip. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011]

[0020] The drawings and the following description relate to preferred embodiments by way of example only. It should be noted that from the following discussion, alternative embodiments of the structures and methods disclosed herein will be readily recognized as viable alternatives that may be employed without departing from the principles of what is claimed.

[0012]

[0021] Reference will now be made in detail to several embodiments, examples of which are illustrated in the accompanying drawings. It should be noted that wherever practicable, similar or similar reference numbers are used in the drawings to indicate similar or similar functionality. The drawings illustrate, for purposes of illustration only, embodiments of the disclosed nanosensor chip. From the following description, those skilled in the art will readily appreciate that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles described herein.

[0013]

[0022] FIG. 1 is a top view of a nanosensor chip 100 with multiple conical composite nanopores 120, also referred to as Compores 120. The nanosensor chip 100 is formed from a semiconductor substrate 110, such as a silicon or gallium arsenide (GaAs) substrate. Alternative substrates, such as glass, plastic, films, and other materials, can be used instead. One or more Compores 120 are formed in the semiconductor substrate 110. The Compores 120 are apertures formed in the semiconductor substrate 110 that contain multiple nanopores 130. The nanopores 130 form holes through the semiconductor substrate 110. The nanopores 130 have openings at the top of the Compores 120 (as shown in FIG. 1) and at the bottom of the Compores 120 on the back side of the nanosensor chip 100 (not visible in FIG. 1). The nanopores 130 of the Compores 120 are functionalized with immobilized probe molecules and collectively form an aperture through the semiconductor substrate 110. The pore 120 may have a maximum width of 1 micron to 1000 microns. The pore 120 may be placed in contact with a liquid sample, for example positioned vertically between the sample and a buffer tank. This may be used to detect the presence or absence of one or more target molecules in a liquid sample and / or to quantify the concentration of the target molecules in the liquid sample based on the binding of the target molecules to the probe molecules.

[0014]

[0023] The Compore 120 can be used to assay any liquid sample in which a target molecule of interest may be present, such as blood, saliva, spinal fluid, urine, food, beverages, water, etc. Different nanosensor chips 100, and different Compore 120 within a single nanosensor chip 100, can be configured to assay different types of liquid samples and detect one or more types of target molecules within the liquid sample. For example, one nanosensor chip 100 can be configured to test for a set of antibodies in a blood sample, and another nanosensor chip 100 can be configured to test for a set of contaminants in a water sample.

[0015]

[0024] Each of the nanopores 130 in the compore 120 is functionalized with an immobilized probe molecule. Each nanopore 130 has a sidewall extending between two openings, and the probe molecule is attached to this sidewall of the nanopore 130. Exemplary arrangements of the probe molecules in the nanopore 130 are shown in Figures 5A and 5B. The probe molecules can be selected to detect a specific type or set of target molecules in a liquid sample, for example to detect a specific antibody or set of antibodies in a blood sample or to detect a specific contaminant in water. The probe molecules have a binding affinity for the target molecules such that in the presence of a specific electric field, the target molecules in the sample bind to the probe molecules. The target molecules can be reversibly bound to the probe molecules, so that when the electric field is removed, the target molecules are released from the probe molecules. Examples of probe molecules include antibodies, antibody analogs, proteins, aptamers, polymers, oligonucleotides, and nanobodies.

[0016]

[0025] Each compore 120 has a corresponding electrode structure 140 disposed on the semiconductor substrate 110. The electrode structure 140 has a shape and position relative to the compore 120 suitable for applying an electric field to all the nanopores 130 of the compore 120. The electrode structure 140 also provides a conductive path for conducting the total current through the nanopores 130 of the compore 120. For example, the electrode structure 140 may include one or more electrodes on the top of the compore 120 (shown in FIG. 1) and one or more electrodes on the back side of the compore 120 (not visible in FIG. 1). Exemplary electrode structures are shown in FIGS. 3 and 9. The electrode structure 140 may be connected to a circuit disposed on the nanosensor chip 100 for providing a voltage source of the electric field and / or for detecting the total current. Alternatively, the electrode structure 140 may be connected to an off-chip source and / or detector.

[0017]

[0026] If the liquid sample contains a target molecule and the correct electric field is applied to the Compore 120, the target molecule will bind to the immobilized probe molecule of the nanopore 130. This binding will change the total current flowing through the Compore 120. The total current will flow via the electrode structure 140 to a current detector on or connected to the nanosensor chip 100. A change in the total current flowing through all the nanopores 130 of the Compore 120 indicates the presence of the target molecule in the liquid sample. The amount by which the total current changes can be used to determine the concentration or amount of the target molecule in the liquid sample. In contrast, if a particular electric field is applied to the Compore 120 but no change in the total current is detected, this indicates the absence of the target molecule in the liquid sample.

[0018]

[0027] Compore 120 is more reliable than previous nanosensors because each Compore 120 contains multiple nanopores 130 subjected to the same electric field and the total current through all the nanopores is detected. For example, even if one or a few of the nanopores 130 are blocked or clogged, the total current through the set of nanopores 130 in Compore 120 changes in response to the binding of target molecules in the liquid sample to probe molecules in the presence of an electric field. Similarly, even if the nanopores 130 are not uniformly functionalized, for example, some nanopores have more immobilized probe molecules than others, a change in the total current through the set of nanopores 130 can be detected. Furthermore, the use of multiple nanopores 130 increases the number of types of target molecules that can be detected using a single Compore 120, since the nanopores 130 can be functionalized with multiple different types of probe molecules. Configurations with multiple types of probe molecules are described in connection with Figures 5A and 5B.

[0019]

[0028] The nanosensor chip 100 shown in FIG. 1 includes four compores 120. The nanosensor chip 100 in other configurations may include any number of compores, such as 1, 4, 16, or hundreds or thousands of compores. The two compores 120 on the nanosensor chip 100 may test the same liquid sample or different liquid samples. The compores on the nanosensor chip 100 may be identical, or some or all of the compores may be different from each other. For example, the two compores 120 on a single nanosensor chip 100 may have different sizes, different shapes, different numbers of nanopores, nanopores of different sizes or shapes, or nanopores with different probe molecules. The inclusion of different compores on a single nanosensor chip 100 allows the single nanosensor chip 100 to perform multiple different tests, for example, to test multiple different target molecules, to perform tests of different sensitivity, or to include controls to verify accuracy and to authenticate the nanosensor chip 100. The inclusion of multiple identical compores on a single nanosensor chip 100 can also be used to improve the accuracy and reliability of a single nanosensor chip 100 .

[0020]

[0029] In FIG. 1, each compore 120 on the nanosensor chip 100 can be individually addressed using a respective electrode structure 140. Since each compore 120 has a separate electrode structure 140, the total current flowing through each compore 120 can be measured individually by a current detector connected to the electrode structure 140. In some embodiments, each electrode structure 140 is also used to apply an electric field to each compore 120 individually. In other embodiments, the electrode structure for applying an electric field to the compore is separate from the electrode structure used to measure the total current flowing through the compore. In such embodiments, the electrode structure for applying an electric field may be connected to two or more compores so that the same electric field or voltage can be applied to multiple compores simultaneously.

[0021]

[0030] FIG. 2 is a perspective view of a single Compoor 120, but does not show the electrode structure. FIG. 2 shows the structure of the Compoor 120 in detail. The Compoor 120 has a central region 210 that is thinner than the semiconductor substrate 110. The nanopores 130 are formed in this thin central region. For example, the central region 210 can be thinned to a thickness of less than 1 micron. The thickness of the central region 210 is also the height of the nanopores 130 formed in the Compoor 120. In a nanosensor chip with multiple Compoors, such as the nanosensor chip 100 shown in FIG. 1, the thin regions of the Compoor are separated from each other by non-thin regions. This can be used to maintain separation between the liquid samples of the different Compoors, as well as between the voltages and currents of the different Compoors, if desired.

[0022]

[0031] The Compore 120 has Compore sidewalls 220 between the top of the central region 210 and the top of the semiconductor substrate 110. The Compore sidewalls 220 are shown as slanted, but may be straight, curved, or have other configurations. In some embodiments, a liquid sample may be placed in the depression formed by the central region 210 and the Compore sidewalls 220, and a buffer solution may be placed on the other side of the Compore 120. In other embodiments, the nanosensor chip 100 may be positioned vertically between two reservoirs, one containing a buffer solution and the other containing a liquid sample.

[0023]

[0032] Various other configurations for providing a liquid sample to the pore 120 may also be used. For example, the opposite side of the pore 120 may be exposed to the liquid sample. In such an embodiment, the portion of the pore 120 surrounded by the pore sidewall 220 may receive a buffer solution.

[0024]

[0033] FIG. 3A shows a top view of a single Composite 120. FIG. 3A shows an exemplary arrangement of nanopores 130 within Composite 120. Adjacent nanopores 130 are separated by a gap 230. The gap distance 230 between two adjacent nanopores within a single Composite 120 may be, for example, 1 nanometer to 100 nanometers. In the example shown in FIG. 3A, Composite 120 has 10 nanopores 130. In other embodiments, Composite 120 may have a different number of nanopores 130, for example, from 2 to hundreds of nanopores. Although the nanopores 130 in FIG. 3A show the nanopores 130 arranged in three rows, in other embodiments, the nanopores 130 may have different arrangements.

[0025]

[0034] FIG. 3B is a cross-sectional view of the Compore 120 shown in FIG. 3A along line A-A'. The Compore cross-section includes three nanopores 130a, 130b, and 130c that penetrate the semiconductor substrate 110. In this embodiment, one side of the nanopore structure is characterized by a depression formed by the top of a thin central region and the Compore sidewalls. For convenience, this side is referred to as the top side, and the back side is referred to as the bottom side. Either the top side or the bottom side can be exposed to the liquid sample, while the other side is typically exposed to a buffer. In FIG. 3B, the Compore 120 has an upper electrode 330 formed on the top surface and a lower electrode 340 formed on the bottom surface. However, in alternative embodiments, one or both of these electrodes may be mounted external to the chip, for example in a chip receptacle.

[0026]

[0035] The upper electrode 330 and the lower electrode 340 form an electrode structure for applying an electric field to the nanopore 120 and conducting the total current through the nanopore 130. Since all the nanopores 130 of the nanopore 120 are in close proximity to each other, an electrode structure including the upper electrode 330 and the lower electrode 340 disposed on the sides of the nanopores 130 may be sufficient to apply an electric field to all the nanopores 130 and detect the total current through all the nanopores 130. In alternative embodiments, the electrode structure may be more complex. For example, the upper electrode 330 may have a portion to the left of the nanopore 130a or a portion surrounding all the nanopores (as shown in FIG. 1). The upper electrode 330 may also extend to the area between the nanopores 130a-c. Similarly, bottom electrode 340 may have an additional portion formed to the left of nanopore 130a, an additional portion surrounding the nanopore, and / or an additional portion extending into the area between nanopores 130a-130c.

[0027]

[0036] The upper electrode 330 and the lower electrode 340 are connected to a voltage source 350 and a current detector 360. The voltage source 350 supplies a selected voltage to the upper and lower electrodes 340, thereby generating an electric field across all of the nanopores 130 in the Compore 120. The current detector 360 detects the total current flowing through all of the nanopores 130 in the Compore 120. The voltage source 350 and the current detector 360 may use different electrodes.

[0028]

[0037] In some embodiments, the voltage source 350 and / or the current detector 360 are integrated into the nanosensor chip 100. In such embodiments, the nanosensor chip 100 may further include a controller for controlling the voltage source 350 and the current detector 360. For example, the controller may control the voltage source 350 to vary the magnitude or frequency of the applied voltage. Different patterns of applied voltage (i.e., electric field) may be used to interrogate different target molecules. From the current detector 360, the controller may determine whether the measured total current through the compore 120 is changing as a function of the applied voltage. For example, the controller may command the voltage source 350 to apply a series of different voltages to the compore 120 and detect the level of change in the measured total current at each voltage. The controller may generate a signal indicative of the current detected by the current detector 360 or indicative of the determined change in the detected current and send the signal to an off-chip processor for further processing. The controller may be implemented on the nanosensor chip and / or as part of an external device or component.

[0029]

[0038] FIG. 4 is a cross-sectional view of a single nanopore 130 of a Compore 120. The Compore 130 has an upper opening 410 at the top of the nanopore 130 and a lower opening 430 at the bottom of the nanopore 130. The upper opening 410 has an upper diameter 415 and the lower opening 430 has a lower diameter 435. The upper diameter 420 may be in the range of 1 nanometer to 300 nanometers. In this example, the lower diameter 440 is smaller than the upper diameter 420 and may be less than 300 nanometers. However, in other embodiments, the nanopore may be "flipped" so that the upper opening is smaller. The nanopore 130 has a height 450 that is the same as the height of the thin central region 210 of the nanopore. The height 450 of the nanopore may be 1 micron or less. The nanopore has sidewalls that extend between the upper opening 410 and the lower opening 430. The slope of the sidewall is defined by an angle θ which may be between 6 degrees and 60 degrees. In other embodiments, the sidewall may be curved or have some other shape rather than being straight as shown in FIG.

[0030]

[0039] Figures 5A and 5B are cross-sectional views illustrating the operation of a nanopore. Figure 5A shows a nanopore 130 with a probe molecule 520 attached to a sidewall. As shown in Figure 5A, the probe molecule is attached to the sidewall near the smaller opening, i.e., the bottom opening 430 shown in Figure 4. The probe molecule 520 may span the entire sidewall or may be concentrated on a portion of the sidewall, for example, the bottom part of the sidewall near the bottom opening 430. The probe molecule 520 may be attached to the sidewall of the nanopore 130 by covalent or non-covalent bonding or by physical adsorption.

[0031]

[0040] In the examples shown in Figures 5A and 5B, the nanopore 130 is exposed to a liquid sample containing target molecules 530. In the example shown in Figure 5A, the sample with the target molecules is positioned below the nanopore 130. As mentioned above, in other embodiments, the sample containing the target molecules may be positioned on the other side of the nanopore 130. Figure 5A shows the nanopore without an appropriate electric field applied. In this state, the target molecules 530 remain separate from the probe molecules 520.

[0032]

[0041] 5B shows the same nanopore after applying a specific electric field to the compore. In the presence of this electric field, target molecules 530 are attracted into the nanopore and each target molecule 530 binds to a corresponding probe molecule 520 forming a probe / target bond 540. This binding generates an ionic current change in the current flowing through the nanopore 130. As other nanopores in the compore are also functionalized with the same probe molecule 520 and exposed to the same sample and electric field, other target molecules 530 in the sample will flow into other nanopores forming additional probe / target bonds 540 throughout the compore. The probe / target bonds 540 in the nanopores of the compore generate a total ionic current change that can be measured by a current sensor.

[0033]

[0042] When the electric field is removed or changed, the target molecule 530 is released from the probe molecule 520. The target molecule 530 can flow out of the nanopore 130 and return to the configuration shown in FIG. 5A. The probe / target bond 540 is reversible, so that the target molecule 530 is continuously bound to and released from the probe molecule 520 when a varying voltage is applied to the pore. After use of the pore 120, for example after the target molecule 530 is bound and released and the pore 120 is reset with a buffer solution, the probe molecule 520 remains attached to the sidewall of the nanopore. Because the probe molecule 520 remains attached after use, the pore 120 can be reused for multiple samples.

[0034]

[0043] In some applications, the nanopores of a single Compore 120 are functionalized with two or more different probe molecules. The probe molecules can be molecules of the same category (e.g., two antibodies) or molecules of different categories (e.g., one antibody and one protein). This allows a single Compore 120 to be used to detect multiple different types of target molecules. In one exemplary application, a first probe molecule pairs with a first target molecule at a first field strength (e.g., +0.2 volts), a second probe molecule pairs with a second target molecule at a second field strength (e.g., +0.4 volts), and a third probe molecule pairs with a third target molecule at a third field strength (e.g., +0.6 volts). A series of different electric fields can be applied to the Compore 120 to determine whether any of the three target molecules are present in the sample. This allows the Compore 120 to be used to perform multiple tests simultaneously on a single sample with a single sensor with high efficiency.

[0035]

[0044] In other applications, the probe molecules can be selected such that multiple target probe pairs can bind at the same voltage range. This configuration can be used to detect the presence of any of a set of target molecules, such as, for example, a set of contaminants that may be present in a food product or a set of target antibodies in a blood sample. By using multiple target probe pairs that bind at the same voltage, the Compore 120 can effectively identify negative results in a sample. In some applications, if a positive result is obtained, further tests can be performed after the first positive result to determine which target molecules are present.

[0036]

[0045] By including multiple nanopores 130 within a single Compore 120, the Compore 120 can be functionalized with more types of probe molecules than conventional sensors. Specialized high-resolution printers can be used to place probe molecules simultaneously and with high precision across a wafer containing multiple nanosensor chips. This allows multiple tests and test panels to be produced at low cost. Additionally, the Compore 120 can be functionalized to detect positive and negative controls for validation and calibration, as well as markers to authenticate and verify the integrity of the nanosensor chips and reagents.

[0037]

[0046] 6A and 6B are graphs showing the operation of the Compore. FIG. 6A is a graph showing the behavior of the Compore in the absence of target molecules, and FIG. 6B shows the response by the target molecules. In this example, the voltage source 350 generates a square wave current, first at -400 millivolts (mV), then at -200 mV, 0 mV, and +200 mV. Each pair of probe and target molecules has a unique voltage at which they bind. This changes the electrical characteristics of the nanoprobe 130 aperture, causing the current to change as shown in FIG. 6B, where the unique voltage is -200 mV. The output current has changed compared to FIG. 6A. This indicates that there are target molecules in the sample that bind to the probe molecules at -200 mV, since the target molecules bind to the probe molecules in the presence of the -200 mV electric field. The magnitude of the current change can also indicate the concentration of the target molecules in the sample. When a variable voltage is used, the target molecules can bind to and release from the probe molecules. A particular target molecule may bind and remain bound rather than being bound and released.

[0038]

[0047] Although the Compore 120 shown in Figures 1 to 3 has a circular shape and a circular nanopore 130, in other embodiments, the Compore 120 and / or the nanopore 130 may have different shapes and the nanopores may be arranged differently within the Compore. Figure 7 shows a top view of a first alternative arrangement of nanopores within the Compore. Figure 7 shows a rectangular Compore 720 with a rectangular nanopore 730. In other embodiments, the Compore 720 may be elliptical, rectangular, another polygonal shape, or some other shape. Similarly, the nanopore 730 may be elliptical, rectangular, another polygonal shape, or some other shape. The shape of the nanopore 730 may be different from the shape of the Compore 720.

[0039]

[0048] FIG. 8 is a top view showing a second alternative arrangement of nanopores within a compore. While compore 120 and 720 had nanopores of consistent size, in other embodiments, nanopores within a single compore may have different sizes. FIG. 8 shows compore 820 with multiple different sized nanopores 830, including small nanopore 830A and large nanopore 830B. The different sized nanopores 830 may have slanted sidewalls at the same angle (e.g., each with a 10 degree sidewall angle) or at different angles. In some embodiments, using multiple different sized nanopores can improve the sensitivity and dynamic range of the nanosensor.

[0040]

[0049] FIG. 9 is a cross-sectional view of a Compore 900 made using semiconductor technology. The structure of the Compore 900 is similar to the Compore shown in FIG. 3. The Compore 900 includes a layer of silicon 910, which is an example of a semiconductor substrate 110. In other embodiments, other semiconductor materials may be used instead of the silicon 910. Alternatively, the nanosensor may be implemented on a substrate of glass, plastic, film, or other non-conductive or semiconducting material. Various etching processes, for example wet or dry etching, may be used to thin the substrate to form the Compore central region in the silicon 910. A separate process, such as ion beam lithography, may be used to form the nanopore. Although only one Compore 900 is shown in FIG. 9, multiple Compores may be formed and electrode structures may be placed as shown in FIG. 1.

[0041]

[0050] Two silicon nitride layers 920 and 930 are deposited on the top and bottom of the silicon 910, respectively. Various silicon nitride deposition processes can be used to deposit the two silicon nitride layers 920 and 930, such as chemical vapor deposition or plasma enhanced chemical vapor deposition. Although the silicon nitride layers 920 and 930 for only one compore 900 are shown in FIG. 9, the silicon nitride layers 920 and 930 may extend across the entire nanosensor chip for each compore included in the nanosensor chip.

[0042]

[0051] Two electrode layers 940 and 950 are deposited on the upper silicon nitride layer 920 and the lower silicon nitride layer 930, respectively. The electrode layers 940 and 950 can be formed of any conductive material, such as copper, silver, or platinum. To deposit the two electrode layers 940 and 950, various deposition processes for depositing conductive materials can be used, such as evaporation or chemical vapor deposition. In this embodiment, the electrode layers 940 and 950 are disposed on each side of the compore 900. In other embodiments, the electrodes 940 and 950 can be disposed differently on the compore 900, as described with respect to FIG. 3. For example, in other embodiments, the electrodes 940 can be formed on the silicon sidewalls and / or on the surface of the thinned silicon. Each compore included in the nanosensor chip can have a similar electrode structure. In other embodiments, any of the electrodes can be disposed off-chip, in a separate component, such as a chip receptacle in which the chip is mounted.

[0043]

[0052] 10 is a block diagram of a detection system 1000 including a nanosensor chip. The detection system 1000 includes a nanosensor chip 1010, a chip receptacle 1020, a voltage source 1030, a current detector 1040, a controller 1050, a user interface 1060, a display 1070, and a communication interface 1080. Other components (not shown) may include a processor, memory, a digital-to-analog converter, and an analog-to-digital converter. In other embodiments, the detection system 1000 has additional, alternative, or fewer components than those shown in FIG. 10.

[0044]

[0053] The nanosensor chip 1010 as described with respect to Figures 1 to 9 has one or more pores. The chip receptacle 1020 is configured to receive and hold the nanosensor chip 1010 and to form electrical connections between the components of the nanosensor chip 1010 and other components of the detection system 1000. For example, the chip receptacle 1020 may include electrodes configured to connect to the electrode structure 140 shown in Figure 1. The chip receptacle 1020 may also have one or more fluid connections to the nanosensor chip 1010, for example, to transfer one or more liquid samples to the pores of the nanosensor chip 1010 or to transfer buffer solutions to the pores of the nanosensor chip 1010.

[0045]

[0054] The voltage source 1030 generates the electric field that the electrodes supply to the compores. The voltage source 1030 may be a variable voltage source that generates a variable current over a voltage range to one or more compores. In some embodiments, the voltage source 1030 is integrated into the nanosensor chip 1010. The detection system 1000 may have one voltage source 1030 or multiple voltage sources, for example, one voltage source for each compore included in the nanosensor chip 1010. If the detection system 1000 has fewer voltage sources than compores, the nanosensor chip 1010 may have a switching mechanism to apply voltages to one compore at a time, or the nanosensor chip 1010 may be configured to apply the same voltage to two or more compores simultaneously.

[0046]

[0055] The current detector 1040 detects the current flowing through the compore. In some embodiments, the current detector 1040 is integrated into the nanosensor chip 1010. The detection system 1000 may have one current detector 1040, for example one current detector for each compore included in the nanosensor chip 1010. Alternatively, it may have multiple current detectors. If the detection system 1000 has fewer current detectors than compores, the nanosensor chip 1010 may include a switching mechanism that allows the current detectors to individually address selected compores.

[0047]

[0056] The controller 1050 controls the voltage source 1030 and the current detector 1040. The controller 1050 may be similar to the controller described with respect to FIG. 3. The controller 1050 may be integrated within the nanosensor chip 1050 or may be part of a separate component or device. The detection system 1000 may have one controller 1050 for controlling all of the voltage sources, current detectors, and any switching mechanisms included in the detection system 1000. Alternatively, the detection system 1000 may have multiple controllers 1050, e.g., one for each component. In addition to controlling the voltage source 1030 and the current detector 1040, the controller 1050 is configured to interact with other components of the detection system, such as a user interface 1060, a display 1070, and a communication interface 1080.

[0048]

[0057] The user interface 1060 is configured to receive user input, such as a command from a user to start a sample analysis or parameters for analyzing a sample. For example, the user interface 1060 can receive parameters describing one or more voltages to be applied to the compore or instructions for a pre-programmed test procedure with a set of voltages to be executed on the compore. The user interface 1060 passes these commands or parameters to the controller 1050. The user interface 1060 can include buttons, a keyboard, a touch screen, a microphone and voice recognition software, or any other suitable mechanism for receiving input from a user. Alternatively, the detection system 1000 can receive commands and parameters from a mobile phone application, a tablet, a PC, or a web application, or from an automated external control system.

[0049]

[0058] The display 1070 provides a user with visual output regarding the tests performed by the detection system 1000. For example, the display 1070 can be used in conjunction with the user interface 1060 and the controller 1050 to display options selectable by the user to the user. The display 1070 can also output test results generated by the controller 1050, such as, for example, whether or not a given target molecule was detected in the sample, or the concentration of the target molecule detected in the sample.

[0050]

[0059] The communications interface 1080 allows the detection system 1000 to communicate with one or more other devices, for example over a network such as a local network or the Internet, or by a serial or parallel, wireless or wired interface, for example by Bluetooth, USB, or other communications protocol. For example, the communications interface 1080 can upload results of tests performed by the detection system 1000 to another device or component for further processing, or upload the test results to a database.

[0051]

[0060] Upon reading this disclosure, those skilled in the art will recognize additional alternative structural and functional designs for the nanosensor chip and detection system. Thus, while specific embodiments and applications have been illustrated and described, it will be understood that the described subject matter is not limited to the precise configuration and components disclosed herein, and that various modifications, changes, and variations may be made in the configuration, operation, and details of the devices disclosed herein, as will be apparent to those skilled in the art.

[0052]

[0061] As used herein, a reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. The appearances of the phrase "one embodiment" in various places in the specification do not necessarily all refer to the same embodiment.

[0053]

[0062] As used herein, the words "comprise," "comprising," "include," "including," "has," "having," or any other variation thereof, are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Furthermore, unless otherwise indicated, "or" indicates an inclusive or and not an exclusive or. For example, a condition A or B is satisfied if A is true (or exists) and B is false (or does not exist), if A is false (or does not exist) and B is true (or exists), or if both A and B are true (or exist).

[0054]

[0063] Furthermore, the use of "a" or "an" is used to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of the disclosure. This description should be read to include one or at least one, and the singular also includes the plural unless it is clear that a different meaning is indicated.

Claims

1. A nanosensor chip for detecting or quantifying a target molecule in a liquid sample, comprising: a substrate containing one or more composite nanopores ("Compore"); Each of the composites includes an aperture formed in the substrate with a central region that is thinner than the surrounding substrate, and includes a plurality of nanopores; each of said nanopores having a large opening and a small opening; each of the nanopores is functionalized with a plurality of immobilized probe molecules attached to a sidewall of the nanopore; The nanosensor chip further comprises, for each compore, a corresponding electrode structure having a shape surrounding the plurality of nanopores and arranged to conduct a total current to the plurality of nanopores, whereby the total current changes in response to binding of target molecules in the liquid sample to the probe molecules.

2. The nanosensor chip of claim 1 , wherein the substrate comprises a plurality of composites.

3. the electrode structure for each component includes a first electrode disposed on one side of the substrate and a second electrode on an opposite side of the substrate; the first electrode surrounds the plurality of nanopores; The nanosensor chip of claim 1 , wherein the first and second electrodes are positioned to generate an electric field at the plurality of nanopores and to conduct an electric current through the plurality of nanopores.

4. The nanosensor chip according to claim 1 , 2 or 3 , wherein the probe molecules are selected from the group consisting of antibodies, antibody analogs, proteins, aptamers, polymers, oligonucleotides and nanobodies.

5. The nanosensor chip of claim 4 , wherein the substrate comprises a plurality of pores functionalized with different probe molecules.

6. The nanosensor chip of claim 4 , wherein the substrate comprises a plurality of pores functionalized with identical probe molecules.

7. The nanosensor chip of claim 1 , wherein the substrate comprises a semiconductor material.

8. The nanosensor chip of claim 1 , wherein the probe molecule is attached to the nanopore by covalent, non-covalent or physical adsorption.

9. 1. A method for detecting and / or quantifying a target molecule in a liquid sample, comprising: (a) providing a nanosensor chip comprising a substrate containing one or more composite nanopores ("compores"); Each of the composites includes an aperture formed in the substrate having a plurality of nanopores formed therethrough; the nanopore is functionalized with an immobilized probe molecule that specifically binds to a target molecule; The compore further comprises an electrode structure configured to apply a voltage to all of the nanopores within the compore; The method further comprises: (b) allowing target molecules in the liquid sample to bind to the immobilized probe molecules; (c) applying a range of voltages from a voltage source to all of the plurality of nanopores in the composite using the electrode structure such that specific binding between target molecules and probe molecules varies as a function of the electric field applied to the plurality of nanopores; (d) detecting a total current through all of the plurality of nanopores in the composite in response to the voltage using a current sensor connected to the electrode structure; Binding of a target molecule to a probe molecule generates a change in the total current, thereby indicating the presence and / or concentration of the target molecule in the liquid sample.

10. The method of claim 9, wherein in (c), applying a voltage in the range using the electrode structure includes applying a voltage to one or more of the compoa simultaneously.

11. removing any bound target molecules from the nanosensor chip; reusing said nanosensor chip; The method of claim 9 further comprising:

12. 10. The method of claim 9, further comprising the step of applying the liquid sample in the form of blood, saliva, spinal fluid, urine, food, drink, or water.

13. 10. The method of claim 9, further comprising the step of applying alternating positive and negative voltages to the plurality of nanopores, whereby target molecules dissociate from the probe molecules.

14. 10. The method of claim 9, further comprising measuring a change in the total current at different voltages to determine a concentration of a target molecule in the sample.

15. The method of claim 9, further comprising the step of detecting different targets in the sample by using compores containing different probe molecules.

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