Methods for making biosensor nanopores

Abstract

An improved method for making a highly sensitive and selective electrochemical biosensor is provided. The method may include washing the nanopore array electrode of the biosensor with ferrocyanide, preferably potassium ferrocyanide. In addition to or without the ferrocyanide and / or H2SO4 washing steps, the method may also include washing the electrode of the biosensor with methylene blue (i.e., methylene blue).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 647,280, filed on March 23, 2018, and Korean Patent Application No. 10-2018-0033974, filed on March 23, 2018, the contents of each of which are incorporated herein by reference. Field of the Invention

[0003] The present disclosure generally relates to methods for making highly sensitive and highly selective biosensors. More particularly, the present disclosure relates to methods for making highly sensitive and highly selective biosensors that reduce or eliminate impurities from electrochemical biosensors. Background Art

[0004] Biosensors are used to detect the presence of biomolecules, such as proteins, amino acids (e.g., DNA and / or RNA containing specific base sequences), or other organic molecules. Some examples of biosensors include pregnancy tests and glucose monitoring sensors. These biosensors can detect biomolecules such as human chronic gonadotropin (hCG) or glucose present in body fluids such as blood or urine.

[0005] To detect a specific analyte (e.g., a biomolecule), a biosensor may comprise an analyte-binding surface to which a probe specific for the analyte (e.g., a single-stranded DNA or antibody specific for a target molecule) is immobilized. Different types of biosensors have been developed that can detect the presence of specific biomolecules using different scientific principles.

[0006] Examples of different types of biosensors include electrochemical biosensors, nano-cantilever biosensors, and micro-electromechanical systems or nano-electromechanical systems (MEMS / NEMS). Like other types of biosensors, electrochemical biosensors include an analyte-binding surface that can interact and / or bind to specific biomolecules (e.g., specific proteins or specific sequences of DNA). In particular, electrochemical biosensors use the principles of electrochemical analysis to detect specific analytes, wherein the chemical response to electrical excitation applied to the system is measured and analyzed to detect whether the analyte is bound to the electrode surface. Unlike nano-cantilever biosensors and MEMS / NEMS, the signal of the electrochemical biosensor can be directly detected by electronic means for analysis, thereby allowing rapid diagnosis.

[0007] Potential future applications for electrochemical biosensors include diagnostics in traditional medical and healthcare settings (e.g., testing of blood and / or urine samples for specific biomolecules); medical diagnostics in non-hospital settings (e.g., military use in combat zones and / or self-administered consumer diagnostics), non-medical testing for biological and / or small molecule detection (e.g., water quality testing, environmental testing, quality control and / or quality assurance testing in the food industry); companion diagnostics for drug therapy; research applications in which detection of small molecules is required; and / or other environments or conditions in which detection of biomolecules is required. Those skilled in the art will understand that although the present disclosure is referred to as a "biosensor," its application is not limited to the detection of biomolecules. In other words, the present disclosure can be used to detect other small non-biological (e.g., inorganic, metallic, solute, electrolyte and / or elemental) molecules. In addition, although the embodiments provided herein consist of detection in a fluid and / or aqueous environment, those skilled in the art will understand that the present disclosure can be used to detect small molecules in other fluid environments, such as oils, solvents, gases and / or colloidal solutions.

[0008] In order for electrochemical biosensors to be widely adapted for a wide range of applications, the biosensors must be highly sensitive and highly selective, and the manufacturing cost of such sensors must be competitive. Electrochemical biosensors with significantly improved sensitivity and selectivity can enable miniaturization of such devices, which in turn can reduce production costs and further facilitate the adoption of electrochemical biosensors for a wide range of applications.

[0009] Specific types of highly sensitive and highly selective biosensors have been described in published U.S. Patent Application No. US2017 / 0219554A1, the contents of which are incorporated herein by reference. These biosensors include a perforated insulation layer (or insulating layer) laid on the electrodes of the electrochemical biosensor to form a nanowell (or nano-well). Such electrochemical biosensors can highly selectively detect analytes present in the fM range in biological samples.

[0010] However, the manufacture of such biosensors is technically challenging. In particular, contamination of the biosensor with impurities during the manufacturing process can render the biosensor defective or adversely affect the sensitivity and selectivity of the biosensor.

[0011] Therefore, there is a need for improved methods of making highly sensitive and highly selective biosensors having a perforated insulating layer laid over electrodes, wherein contamination of the biosensor by impurities is reduced or eliminated. Summary of the invention

[0012] Provided herein is an improved method for making highly sensitive and highly selective electrochemical biosensors. An exemplary embodiment of the method includes washing the nanopore (or nano-pore) array electrode of the biosensor with ferrocyanide, preferably potassium ferrocyanide. In another exemplary embodiment, the method includes washing the electrode of the biosensor with H2SO4 and additionally washing the electrode with ferrocyanide. In another exemplary embodiment, in addition to the ferrocyanide and / or H2SO4 washing steps, or in the absence of the ferrocyanide and / or H2SO4 washing steps, the method includes washing the electrode of the biosensor with methylene blue (i.e., methylthioninium chloride).

[0013] The methods disclosed herein result in the removal of impurities from the biosensor.In some embodiments, impurities are removed from the surface of the electrode (eg, a gold electrode).

[0014] In an exemplary embodiment, the present invention is directed to improving the production of electrochemical biosensor devices. An example of such an electrochemical biosensor device includes a plurality of electrodes, the electrodes being made of a buffer layer laid on a substrate layer, an electrode layer laid on the buffer layer, and a perforated insulator layer laid on the electrode layer, such that a plurality of nanopores are formed on the electrode layer and the size of the nanopores is defined by the size of the perforations, the walls of the nanopores are defined by the insulator layer, and the bottom layer of the nanopores is defined by the upper surface of the electrode layer. In some cases, the nanopores of the biosensor have a spacing ratio of 1:1. In other cases, the biosensor can detect analytes present in the fM concentration range.

[0015] In some embodiments, the electrochemical biosensor may include a glass substrate layer, a silicon substrate layer, a silicon dioxide insulator layer, a titanium buffer layer, a chromium buffer layer, and / or a gold electrode layer.

[0016] In yet other embodiments, the electrochemical biosensor can have a perforated insulator layer, wherein the perforations (e.g., holes and / or holes) can define the size of the nanopore so that the nanopore is cylindrical in shape. In yet other embodiments, the nanopore has a circular opening with a diameter of about 230nm, 100nm, and / or 50nm. In yet other embodiments, the nanopore has a spacing ratio of about 1:5, about 1:3, and / or about 1:1.

[0017] In some embodiments, the electrochemical biosensor can be operated in conjunction with an electronic device, wherein the electrochemical biosensor is capable of sending a signal to the electronic device, so that the signal can be used by the electronic device to detect one or more electrochemical reaction parameters between an electrode containing a reference sample and an electrode containing a test sample to determine whether an analyte is present in the test sample. In an exemplary embodiment, the electrochemical reaction includes an oxidation reaction and a reduction reaction. In another exemplary embodiment, the parameter includes a change in a redox current.

[0018] In some other embodiments, the electrochemical biosensor can be used to detect an analyte in a sample solution by: (1) applying the test sample to a sensing electrode of the electrochemical biosensor to allow binding of any analyte that may be present in the test sample; (2) flushing the sensing electrode with an appropriate buffer to wash away any unbound and / or non-specifically bound analyte and / or non-analyte from the sensing electrode; (3) applying an electric current to the sensing electrode in a manner to cause a chemical change in the sensing electrode; (4) measuring the electrochemical properties of the sensing electrode using an electronic device; and (5) analyzing the difference in the electrochemical properties between the test sample and the reference sample to determine the presence of the analyte at the sensing electrode. In an exemplary embodiment, the electrochemical properties of the sensing electrode are measured using cyclic voltammetry analysis.

[0019] In another exemplary embodiment, the present invention provides a method for manufacturing a biosensor, the method comprising the following steps: forming a metal layer; forming an electrode layer by patterning the metal layer using a first photolithography process; forming an inorganic insulator layer on the electrode layer; forming a plurality of nanopores on the inorganic insulator layer, wherein a portion of the electrode layer is exposed by using a second photolithography process; and washing the plurality of nanopores by using H2SO4 to remove impurities from the exposed electrode layer.

[0020] In some embodiments, the method further comprises washing the nanopore by using ferrocyanide etching (or washing) after the H2SO4 washing step. The ferrocyanide etching washing step is performed by immersing the biosensor in a mixed solution of K3Fe(CN)6 and KCl solution and applying a voltage of 1.0-1.5 V. For example, the ferrocyanide etching can be performed at a temperature in the range of 15-25°C for 1 to 10 seconds.

[0021] In some embodiments, the electrode comprises gold (Au), and the ferrocyanide etching flattens the surface of the electrode. The inorganic insulator layer comprises SiO2 or Si3N4.

[0022] In some embodiments, the H2SO4 washing step is performed by immersing the biosensor in a H2SO4 solution and applying a voltage of 1.0-1.5 V for 1-5 minutes.

[0023] In some embodiments, the pitch ratio of the nanopores is defined as the ratio of the size of each nano-pore to the shortest distance between adjacent nano-pores, and may be 1:3 or less, preferably 1:1.

[0024] In other embodiments, the present invention provides a manufacturing method, which includes the following steps: forming an electrode on a base layer; forming an inorganic insulator layer on the electrode; forming a plurality of nanopores on the insulator layer using a photolithography process so that a portion of the electrode is exposed; performing a first washing step by immersing the nanopore in a H2SO4 solution and applying a voltage of 1.5-2.0V; performing a second washing step by immersing the nanopore in a mixed solution of K3Fe(CN)6 and KCl solution and applying a voltage of 1.0-1.5V.

[0025] The present disclosure also relates to methods for manufacturing highly sensitive and highly selective electrochemical biosensors that minimize contamination of the biosensor by impurities. In some embodiments, the washing step using a sulfuric acid solution after forming the nanopore prevents adverse effects on the sensitivity and selectivity of the biosensor, which are caused by contamination of the biosensor, for example, contamination of the electrode on the surface of the nanopore by impurities during the manufacturing process. In some embodiments, in addition to the washing process using the sulfuric acid solution, a ferrocyanide etching (or washing) process is used to more effectively remove contaminants and improve the flatness of the electrode surface. Therefore, these processes improve the sensitivity and selectivity of the biosensor.

[0026] The electrochemical biosensor manufactured according to the manufacturing method of the present invention can detect analytes present in a fM concentration range in a biological sample with high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A-1D Photographic and microscopic images of embodiments of the present disclosure are presented.

[0028] Figure 2 A schematic cross-sectional view of an embodiment of a sensing electrode is presented.

[0029] Figure 3 A perspective schematic diagram of an embodiment of the present disclosure is presented, showing various sensing electrodes configured to detect different analytes.

[0030] Figure 4A schematic diagram is presented of how a biosensor can be used in conjunction with a potentiostat and electronics to detect analytes in a sample.

[0031] Figures 5A-5C Schematic diagrams showing exemplary pitch ratios of nanopores with respect to biosensor sensitivity are presented.

[0032] Figures 6A-6C Representative cyclic voltammetry (CV) analysis data demonstrating the effect of varying spacing ratios on biosensor sensitivity are presented.

[0033] Figure 7 Representative data demonstrating the detection of varying concentrations of DNA analytes in solution using embodiments of the present disclosure are presented.

[0034] Figure 8 A flow chart showing the method of fabrication of the biosensor is presented.

[0035] Figures 9A-9H A schematic diagram showing the main steps of the biosensor fabrication method is presented.

[0036] Figures 10A-10B Representative scanning electron micrographs (SEM) and cyclic voltammetry (CV) analysis data of a biosensor electrode including a Si3N4 insulating layer are presented, wherein the biosensor was produced without a H2SO4 wash or ferrocyanide etch (or wash) step.

[0037] Figures 11A-11B Representative SEM and CV analysis data of a biosensor electrode including a Si3N4 insulating layer are presented, where the biosensor was produced using a method including a H2SO4 washing step.

[0038] Figures 12A-12B Representative SEM and CV analysis data of a biosensor electrode comprising a Si3N4 insulating layer are presented, wherein the biosensor was produced using a method comprising a H2SO4 washing step and a ferrocyanide etching (or washing) step.

[0039] Figures 13A-13B Representative SEM and CV analysis data of a biosensor electrode including a SiO 2 insulating layer are presented, where the biosensor was produced without a H 2 SO 4 wash or a ferrocyanide etch (or wash) step.

[0040] Figures 14A-14B Representative SEM and CV analysis data of a biosensor electrode including a SiO2 insulating layer are presented, where the biosensor was produced using a method including a H2SO4 washing step.

[0041] Figures 15A-15BRepresentative SEM and CV analysis data of a biosensor electrode comprising a SiO2 insulating layer are presented, wherein the biosensor was produced using a method comprising a H2SO4 washing step and a ferrocyanide etching (or washing) step.

[0042] Figures 16A-16B Representative SEM and CV analysis data of biosensor electrodes fabricated using photolithography are presented, where the biosensor was produced without H2SO4 wash or ferrocyanide etching (or washing) steps.

[0043] Figures 17A-17B Representative SEM and CV analysis data of biosensor electrodes fabricated using photolithography are presented, where the biosensor was produced using a method that included a H2SO4 wash step.

[0044] Figures 18A-18B Representative SEM and CV analysis data of biosensor electrodes fabricated using photolithography are presented, where the biosensor was produced using a method that included a H2SO4 wash step and a ferrocyanide etch (or wash) step.

[0045] While the invention encompasses many different forms of embodiment, several specific embodiments are shown in the drawings and will be described in detail herein, it being understood that this disclosure is considered an exemplification of the principles of the technology and is not intended to limit the invention to the embodiments shown. DETAILED DESCRIPTION

[0046] In order to provide a comprehensive understanding of the principles of the methods disclosed herein, certain exemplary embodiments will now be described. One or more examples in these embodiments are shown in the accompanying drawings. It will be appreciated by those skilled in the art that the methods particularly described herein and shown in the accompanying drawings are non-limiting exemplary embodiments, and the scope of the present disclosure is limited only by the claims. The features shown or described in the collection with an exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present application.

[0047] Additionally, the drawings are not necessarily to scale, and to the extent that linear or circular dimensions are used in the description of the disclosed devices and methods, such dimensions are not intended to limit the types of shapes and sizes that may be used in conjunction with such devices and methods. One skilled in the art will recognize that equivalents to such linear and circular dimensions may be readily determined for any geometric shape. Still further, the size and shape of the device and its components may depend at least on the anatomy of the subject in which the device will be used, the size and shape of the components with which the device will be used, and the methods and procedures in which the device will be used.

[0048] In addition, although the exemplary embodiments provided herein describe methods for producing devices capable of detecting biomolecules (e.g., proteins and / or nucleic acid molecules), those skilled in the art will recognize that the devices can be used to detect the presence of non-biological molecules and / or samples that are not biological samples. As an example, biosensors made using the present invention can be used to detect the presence of inorganic materials for water quality testing, environmental testing, and / or quality control / quality assurance testing in other industrial environments.

[0049] In order to employ electrochemical sensors in a wide range of applications, such as diagnostics in traditional medical, pharmaceutical and / or healthcare settings (e.g., testing of blood and / or urine samples for specific biomolecules), medical diagnostics in non-hospital settings (e.g., military use in combat zones, self-administered consumer diagnostics such as pregnancy tests or blood glucose monitoring), non-medical testing for biological and / or small molecule detection (e.g., water quality testing, environmental testing, quality control and / or quality assurance testing in the food industry), companion diagnostics to drug therapies; research applications in which detection of small molecules is required; and / or other environments or conditions in which detection of biomolecules is desired, the electrochemical sensor must be sensitive (i.e., capable of detecting low concentrations of the analyte), selective (i.e., capable of discriminating and distinguishing the target analyte in the presence of other components), easy to use (i.e., simple to operate, requiring small amounts of test sample), and readily available to users (i.e., capable of being manufactured scalably, in large quantities, and / or at low cost).

[0050] The present disclosure relates to methods of making highly sensitive and highly selective electrochemical biosensors that minimize contamination of the biosensor by impurities.

[0051] Detailed description of electrochemical biosensors

[0052] Various embodiments of electrochemical biosensors are described. The fabrication methods described herein can be used to improve the production of electrochemical biosensors such as those described below and published in U.S. Patent Application No. US2017 / 0219554A1.

[0053] Figures 1A-1D Photographic and microscopic images of one embodiment of the electrochemical biosensor 100 are shown. Figure 1A As shown, a plurality of 100 of one embodiment of the present disclosure may be produced on a single substrate layer 110 such as a glass or silicon substrate layer (eg, a wafer). Figure 1B is a photographic image of a single electrochemical biosensor 100 including a plurality of sensing electrodes 120 . Figure 1C yes Figure 1B1 is a scanning electron microscope image of a sensing electrode of , the sensing electrode comprising a plurality of nanopores 130 having a pitch ratio (ratio between the diameter of the nanopore opening and the shortest distance between adjacent nanopores) of about 1:1 and a nanopore opening diameter of about 230 nm. In other embodiments, the nanopore pitch ratio and / or the nanopore opening diameter may be different values, as disclosed in the later part of the present disclosure. Figure 1D is a perspective atomic force microscope image of a single nanopore 130, showing the cylindrical dimensions of the nanopore 130 with a floor 140 defined by the top surface of the electrode layer.

[0054] Figure 2 Shown Figure 1B The sensing electrode Figure 2 yes Figure 1D In one embodiment, the sensing electrode may include a glass or silicon substrate layer 210, a buffer layer 220 laid on the substrate layer, an electrode layer 230 laid on the substrate layer, and an insulator layer 240 laid on the electrode layer.

[0055] In one embodiment, the substrate layer 210 can be made of glass. The substrate layer can also include silicon, silicon dioxide (e.g., quartz), borosilicate, and / or other glass compositions used in semiconductor manufacturing. In other embodiments, the glass or silicon substrate layer 210 can be a circular wafer. In yet other embodiments, the glass or silicon substrate layer 210 can be configured to accommodate multiple electrochemical biosensors, such as Figure 1A shown.

[0056] The buffer layer 220 of the sensing electrode 120 can provide an enhanced bond between the electrode layer 230 and the substrate layer 210, thereby minimizing the risk of the electrode layer 230 separating from the substrate layer 210. In other words, the buffer layer 220 allows the electrode layer 230 and the substrate layer 210 to form a tighter seal. Such an enhanced bond or seal between the electrode layer 230 and the substrate layer 210 can make the electrochemical biosensor easier and more reliable to manufacture and / or reduce manufacturing costs. In a preferred embodiment, the buffer layer may include titanium, chromium and / or an alloy of titanium or chromium.

[0057] The insulator layer 240 of the sensing electrode 120 may be perforated so that the insulator layer 240 includes a plurality of holes 241 (ie, holes). Figure 1C Also shown are a plurality of holes that form nanopores 130. In a preferred embodiment, hole 241 defines the internal dimensions of a plurality of nanopores 242. In some embodiments, hole 241 is cylindrical in shape, such as Figure 1DAs shown, the nanopore 242 whose size is defined by the hole 241 has a circular opening, and the insulator layer forms the wall 243 of the cylindrical nanopore 242. Because the perforated insulator layer 240 is laid on the electrode layer 230, due to the relative position between the two layers, the top surface 231 of the electrode layer not covered by the insulator layer 240 can form the bottom surface of the nanopore 231a. In a preferred embodiment, the insulator layer 240 of the sensing electrode may include silicon nitride (Si3N4) because silicon nitride is more resilient and more stable than certain alternatives (such as organic or inorganic polymers). In another preferred embodiment, the insulator layer 240 of the sensing electrode may include silicon dioxide (SiO2). Features such as the resilience and stability of silicon nitride can enable more reliable and more consistent manufacturing of the sensing electrode portion of the present disclosure, resulting in reduced occurrence of defective products and reduced manufacturing costs.

[0058] In some embodiments, where the opening of the nanopore is circular, the diameter of the circular opening of the nanopore 242 may be less than 1000nm. In other embodiments, the diameter of the circular opening of the nanopore 242 may be less than 300nm. In yet other embodiments, the diameter of the circular opening of the nanopore 242 may be about 230nm, 100nm, and / or 50nm. Although the above-described embodiments have a cylindrical nanopore 242 with a circular opening, those skilled in the art will recognize that the nanopore 242 may have various other opening shapes, such as rectangular, elliptical, and / or polygonal shapes. In these embodiments with nanopores 242 with various other opening shapes, the size of the opening may be less than 1000nm or 300nm, or may be about 230nm, 100nm, and / or 50nm. In addition, those skilled in the art will also recognize that the present invention is not limited to the above-described composition and structure, but may also include compositions and structures having similar features or improved features.

[0059] In other embodiments, the bottom surface 231a of the nanopore 242 (e.g., the top surface of the electrode layer not covered by the insulator layer) may include a probe molecule 245 that is capable of binding to a specific analyte. Figure 2As shown, a biotinylated antibody 245 specific for the analyte can be fixed to the bottom surface 231a of the nanopore by using an intermediate binding molecule 244 such as avidin or streptavidin. It will be appreciated by those skilled in the art that other well-known methods of fixing the analyte probe 245 can be incorporated into the present disclosure and fall within the scope of the present invention, as discussed below. In yet another preferred embodiment, an insulator layer 240 having a plurality of holes 241 can limit the binding of the analyte with its probe 245 to the bottom surface 231a of the nanopore 242, while preventing the binding and / or aggregation of the analyte to the insulator layer 240.

[0060] Figure 3 A perspective schematic representation of one embodiment of an electrochemical biosensor 100 is shown, wherein each individual sensing electrode 310 is coated with a specific analyte probe 311-317 (e.g., an antibody) such that different analytes 321-327 (e.g., proteins) can bind to different probes 311-317 immobilized on the individual sensing electrodes 310 (e.g., via protein-protein interactions, DNA-DNA hybridization, and / or other intermolecular binding). The term "immobilization" means binding a specific analyte probe (e.g., 311) to the surface of the sensing electrode 310 by, for example, covalent bonding, hydrogen bonding, ionic bonding, and / or Van der Walls forces binding the probe to the electrode surface. In a preferred embodiment, the electrochemical biosensor includes a plurality of electrodes 310 capable of sensing very low amounts of analyte (e.g., less than 1000 fM in concentration, less than 500 fM in concentration, less than 100 fM in concentration, less than 10 fM in concentration, and / or less than 1 fM in concentration).

[0061] Figure 4 A schematic representation showing how a preferred embodiment of an electrochemical biosensor 410 can be used in conjunction with a potentiostat 440 and an electronic device 450 to detect an analyte in a sample. In one embodiment, a user (e.g., a consumer, laboratory personnel, nurse, physician, computer system, machine, or robotic device using the present disclosure as a component or step) can use the present disclosure to measure an analyte in a sample by performing the following steps: (1) applying a test sample to a sensing electrode to allow the analyte to bind to an analyte probe; (2) rinsing the sensing electrode to remove unbound and / or non-specifically bound molecules (analyte and / or non-analyte) from the sensing electrode, and (3) performing an electrochemical measurement and analysis on the sensing electrode.

[0062] Preferred embodiments of the present disclosure use cyclic voltammetry to measure the electrochemical properties of analytes in solution, such as Figure 4 Shown. Figure 4is a schematic diagram of how a biosensor can be used to detect an analyte in a sample.

[0063] In such an embodiment, the electrochemical biosensor (working electrode) 410 is used in conjunction with an electrochemical chamber 400, a potentiostat 440, and an electronic device 450 such as a computing device (e.g., a personal computer, a server, a laptop, a smartphone, a dedicated electronic device, and / or any other device that may be capable of receiving and analyzing electrical signals from the present disclosure). The electrochemical chamber 400 includes a reservoir 405, a reference electrode 420, a counter electrode 430, and a working electrode (the working electrode is a component of the electrochemical biosensor) 410. The reference electrode 420, the counter electrode 430, and the working electrode 410 may be immersed in a solution of an electrolyte 460 such that when an electrical excitation is applied to the system 400, the electrical excitation causes a chemical response (e.g., an oxidation and / or reduction reaction) that can be detected and analyzed by the electronic device 450. More specifically, when an electric current is applied to flow between the working electrode 410 and the counter electrode 430, the potential of the working electrode 410 relative to the reference electrode 420 may be controlled by the potentiostat 440. In this case, regardless of the current generated by the electrode reaction, the potential between the working electrode 410 and the reference electrode 420 can be accurately measured. Those skilled in the art will appreciate that other alternative electrochemical measurement methods may also be employed in the present disclosure, and these methods therefore fall within the scope of the present disclosure.

[0064] Figures 5A-5C Schematic cross-sectional representations of various embodiments of the present disclosure showing different distributions of nanopores 520 on a sensing electrode 500. The distribution of the nanopores 520 is shown in terms of a spacing ratio 525 between adjacent nanopores 520. The spacing ratio 525 is defined by the ratio between the opening diameter 530 of the nanopore 520 and the closest distance 535, 545, 555 between two adjacent nanopores. Figure 5A Another display of the closest distances 535, 545, 555 can be found in Figure 5A In , the closest distance between adjacent nanopores is shown to be 230 nm.

[0065] Figure 5A A preferred embodiment is shown in which the pitch ratio 525 of the nanopores 520 is 1: 1. In this embodiment, the nanopore 520 opening has a diameter 530 of 230 nm, and the shortest distance 535 between adjacent nanopores 520 is 230 nm. Therefore, the ratio between the nanopore opening diameter 530 and the shortest distance 535 between adjacent nanopores is 230 nm:230 nm or 1:1. Figure 5BYet another embodiment is shown in which the spacing ratio 525 is 1:3. In this embodiment, the nanopore 520 opening has a diameter 530 of 230 nm, and the shortest distance 545 between adjacent nanopores 520 is 690 nm. Therefore, the ratio between the nanopore opening diameter 530 and the shortest distance 545 between adjacent nanopores is 230 nm:690 nm or 1:3. Figure 5C Yet another embodiment is shown in which the spacing ratio 525 is 1:5. In this embodiment, the nanopore 520 opening has a diameter 530 of 230nm, and the shortest distance 550 between adjacent nanopores 520 is 1150nm. Therefore, the ratio between the nanopore opening diameter 530 and the shortest distance 550 between adjacent nanopores is 230nm:1150nm or 1:5. Another embodiment of the shortest distance (535, 545, 555) is Figure 5A , 5B and 5C. Figure 5B It shows that the shortest distance between adjacent nanopores is 230 nm.

[0066] Those skilled in the art will recognize that, as previously discussed in the present disclosure, these are exemplary embodiments, and other sizes and / or spacing ratios of nanopores are also within the scope of the present disclosure. For example, the nanopore opening diameter can be about 1000nm, about 500nm, about 100nm, about 50nm, about 20nm, or less than 20nm. Similarly, the spacing ratio range can be any ratio between 100:1 and 1:100, including 50:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:50. In addition, although the nanopores in the embodiments are distributed in a grid-like uniform pattern, other similar patterns or uneven distributions of nanopores and / or other similar, equivalent, and / or further improvements of the arrangement that can further improve the sensitivity and / or specificity of the present invention also fall within the scope of the present disclosure.

[0067] Figures 6A-6C The difference in sensitivity of electrochemical biosensors with different nanopore spacing ratios (i.e., the distance between adjacent nanopores) is demonstrated. In general, changing the spacing ratio between nanopores results in a change in detection sensitivity. In particular, cyclic voltammetry measurements of the present invention were performed, wherein Fig. 6A (4.4x10 -8 Cathodic peak of , biosensor with nanopore spacing ratio of 1:5) or Figure 6B (7.9x10 -8 The cathodic peak of the biosensor with a nanopore spacing ratio of 1:3 is compared to Figure 6CIt is shown that for the biosensor with a nanopore spacing ratio of 1:1, the highest sensitivity (1.75x10 -7 Those skilled in the art will appreciate that the nanopore 420 opening size, pitch ratio, and / or other dimensions, morphologies, and / or physical properties of the nanopores may both affect the sensitivity of the present disclosure, and that variable configurations of the nanopores fall within the scope of the present disclosure.

[0068] Figure 7 1 shows representative data showing the redox current ratios measured by embodiments of the present disclosure for detecting different concentrations of DNA analytes. Analytes are detected by measuring the change in redox current (expressed as a percentage) at concentrations ranging from mM (10 -4 M) range to fM(10 -15 This example illustrates that embodiments of the present disclosure can detect DNA analytes in the fM range by detecting statistically significant redox current ratios.

[0069] Detailed description of the biosensor fabrication method

[0070] Generally, the present disclosure relates to methods for producing electrochemical biosensors, the methods comprising steps for removing and / or reducing impurities from the electrochemical biosensor. In certain embodiments, the present disclosure comprises steps for removing impurities from the surface of an electrode of the electrochemical biosensor. For example, Figure 8 and Figures 9A-9H Some embodiments of the present invention illustrate methods of fabricating electrochemical sensors that minimize contamination of the biosensor (eg, electrode surface) by impurities. Figure 8 is a flow chart showing one embodiment of a method for manufacturing a biosensor, and Figures 9A-9H is a schematic diagram showing some embodiments of the main steps of the method for manufacturing a biosensor.

[0071] In one embodiment of the present invention, the method for manufacturing a biosensor includes forming a buffer layer on a substrate layer (S610) and forming a metal layer on the buffer layer (S620). Fig.9A As described, the buffer layer 220 and the metal layer 230 are sequentially formed on the base layer 210 .

[0072] The base layer 210 may be made of glass, silicone, silicon dioxide (e.g., quartz), and / or borosilicate used in semiconductor manufacturing. The buffer layer 220 provides an enhanced bond between the metal layer 230' and the base layer 210. For example, the buffer layer 220 includes titanium (Ti), chromium (Cr), and / or an alloy thereof. In some embodiments, Cr / Ti may be used. The metal layer 230' is provided for an electrode and may include, for example, gold (Au). As a deposition process, various deposition processes such as sputtering and electron beam deposition may be used.

[0073] The method of the present invention further includes forming a desired patterned electrode 230 by patterning the metal layer 230' using a first photolithography process. Fig. 9B and 9C The above process is carried out by the process shown.

[0074] Reference Fig. 9B , a photoresist material is coated on the metal layer 230', and selective exposure is performed using an exposure device such as a KrF photolithography machine with a desired pattern and a mask M1, thereby forming a desired first photoresist pattern PR1. Fig. 9C As shown, the electrode 230 having a desired pattern may be formed by selectively removing the metal layer 230 ′ using the first photoresist pattern PR1 as a mask.

[0075] like Fig. 9B As shown, an insulator layer 240 is formed on the electrode 230 (S640). The insulator layer 240 formed in this step is provided as a structure for forming a nanopore. In this embodiment, an inorganic insulator layer 240 having restorability and stability is used. For example, silicon nitride (Si3N4) or silicon oxide (SiO2) can be used as the inorganic insulating layer 240. In particular, silicon nitride (Si3N4) or silicon oxide (SiO2) can be preferably used instead of a polymer material such as a photoresist because a washing process such as sulfuric acid washing and / or ferrocyanide etching is applied in a subsequent process.

[0076] Next, a plurality of holes may be formed in the insulator layer using a second photolithography process (S650). The plurality of holes formed in this process are provided as nanopores, and the electrodes exposed on the bottom surfaces of the nanopores may be used as sensing electrodes. In particular, the sensing electrodes may be formed by Fig.9E and 9F The process is carried out by the process shown.

[0077] Reference Fig.9E, a photoresist material is coated on the insulator layer 240, and similar to the previous step, a selective exposure is performed using an exposure device such as a mask M2 having a nanometer-scale hole pattern, thereby forming a desired second photoresist pattern PR2. Fig.9F As shown, the desired hole h1 is formed by selectively removing the insulator layer 240 using the second photoresist pattern PR2 as a mask, wherein the hole h1 can be provided as a nanopore structure 241. However, impurities (CP) are generated in the process of forming the nanopores (241), and these nanopores expose a portion of the electrode (230) in the insulator layer (240). The CP may remain on the electrode surface and greatly reduce the reliability of the biosensor.

[0078] To address this problem, various effective washing processes can be introduced after the formation of nanopores, e.g. Figure 9G As shown. In addition to the washing process using a sulfuric acid solution, a ferrocyanide etching process can also be used. Each process can be performed separately, but can be performed in parallel. In particular, by performing ferrocyanide etching (S670) after the sulfuric acid washing step, the most preferred effect can be expected. In particular, a sulfuric acid (H2SO4) solution can be used to clean multiple nanopores to remove impurities from electrodes exposed by multiple nanopores (S660). Sulfuric acid washing is performed by immersing the biosensor including the nanopores in sulfuric acid and applying a voltage for a predetermined time. Although not limited to this, the applied voltage is in the range of 1.5 to 2.0V and can be performed for 1 to 5 minutes.

[0079] After the sulfuric acid wash, ferrocyanide etching can be used to clean the multiple nanopores. Ferrocyanide etching is performed by immersing the biosensor in a mixed solution of K3Fe(CN)6 and KCl and applying a voltage of 1.0 to 1.5V.

[0080] Generally, it is known that electrodes made of gold are disadvantageous because of the reaction when a voltage of 1V or more is applied in a ferrocyanide solution. However, in the present invention, by intentionally applying a voltage of 1.0-1.5V, the electrode surface exposed on the bottom surface of the nanopore can be quickly processed to effectively remove residual impurities and greatly improve the flatness of the electrode surface. Although not limited thereto, ferrocyanide etching is performed at a temperature in the range of 15 to 25°C for 1 to 10 seconds.

[0081] according to Figure 9H , the washing process removes impurities (CP) from the electrode located on the bottom surface of the nanopore 241 and flattens the electrode region, so that high selectivity can be ensured.

[0082] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are set forth to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0083] Example

[0084] Example 1. Effect of washing an electrode having a Si3N4 or insulating layer with H2SO4 and / or K3Fe(CN)6.

[0085] Prepare a silicone base layer, and a Cr layer with a thickness of 300nm and a Ti layer with a thickness of 10nm are deposited on the silicone base layer as a buffer layer. Sputtering equipment and electron beam deposition equipment are used to form the Cr layer and the Ti layer, respectively. Sequentially, an Au layer with a thickness of 200nm is formed using an electron beam deposition equipment as a metal layer for an electrode. Finally, a photoresist material is applied to the Au layer, and an exposure device, a KrF photolithography machine and a mask are used to form a photoresist pattern corresponding to the desired patterned electrode. Using the photoresist pattern as a mask, Au is selectively removed in an inductively coupled plasma (ICP) etching device, and the Ti layer is selectively removed with the same pattern. The photoresist pattern is removed by immersing it in an ultrasonic beaker for 3-4 minutes using acetone.

[0086] Next, to form an insulating layer for the nanohole array, a Si3N4 layer having a thickness of 150 nm was deposited using plasma enhanced chemical vapor deposition (PECVD), and similar to the aforementioned photolithography process, the Si3N4 layer was selectively removed so that the holes had an opening diameter of 230 nm, with a pitch ratio of 1: 1. However, the biosensor manufactured according to Example 1 was subjected to different washing processes as described in Table 1 below:

[0087] Table 1

[0088] Example <![CDATA[Washing with sulfuric acid (H2SO4)]]> Ferrocyanide etching Example 1A X X Example 1B O X Example 1C O O

[0089] Sulfuric acid washing was performed by immersing the biosensor in a H2SO4 solution and pre-treating at +1.8V for 3 minutes before CV analysis. Ferrocyanide etching was performed by immersing the biosensor in a ferrocyanide solution and treating K3Fe(CN)6 at a scan rate of 100mV / s at room temperature. A mixed solution of 5mM K3Fe(CN)6 and 0.1M KCl solution was used as the ferrocyanide solution. In addition, a solid Ag / AgCl rod (with 3M KCl) and a platinum wire were used as a reference electrode and a counter electrode. In particular, CV analysis, sulfuric acid washing, and ferrocyanide etching were performed under the conditions described in Table 2 below.

[0090] In an exemplary embodiment, the method for producing a highly sensitive and highly selective electrochemical biosensor includes a step of washing the electrode with a K3Fe(CN)6 solution. Preferably, the washing step is performed under the conditions described in Table 2.

[0091] In another exemplary embodiment, the method for producing a highly sensitive and highly selective electrochemical biosensor comprises a step of washing the electrode with a K3Fe(CN)6 solution in combination with a step of washing the electrode with H2SO4. Preferably, these washing steps are performed under the conditions described in Table 2.

[0092] Table 2: Exemplary conditions for cyclic voltammetry (CV) analysis, H2SO4 wash step, and Fe(CN)6 wash step.

[0093] CV <![CDATA[H2SO4]]> <![CDATA[Fe(CN)6]]> Initial E(V) -0.6 0 -1.2 High E(V) 0.4 1.8 1.2 Low E(V) -0.6 0 -1.2 Initial P / N P P P Scan rate (V / s) 0.1 0.1 0.1 part 5 4 3 Sample interval (V) 0.01 0.01 0.01 Static time (Sec) 2 2 2 Sensitivity (A / V) 1e-5 1e-4 0.001

[0094] In certain embodiments, the Fe(CN)6 washing step is performed using 5 mM K3Fe(CN)6 and 0.1 M KCl solution.

[0095] In certain embodiments, prior to CV analysis, the electrode was pretreated in a H2SO4 solution at +1.7-1.8 V for 3 minutes. The electrochemical data were then measured in a solution containing K3Fe(CN)6 at a scan rate of 100 mV / s at room temperature.

[0096] In certain embodiments, solid Ag / AgCl rod (with 3M KCl) and platinum wire were used as reference and counter electrodes.

[0097] In certain embodiments, the production methods disclosed herein result in electrodes for biosensors having improved electrochemical properties. In a preferred embodiment, cyclic voltammetry analysis is used to measure the electrochemical properties.

[0098] In certain embodiments, the scanning cycle of the Fe(CN)6 washing step includes one cycle or two cycles. In certain other embodiments, the scanning cycle of the Fe(CN)6 washing step includes more than two cycles.

[0099] It is well known in the art that Fe(CN)6 washing conditions comprising applying a current having a voltage greater than 1V may adversely affect the surface integrity of the electrode. However, in certain embodiments of the present disclosure, a current having a voltage greater than 1V is applied for a short duration to produce unexpectedly favorable results, such as removal of impurities and enhanced electrochemical properties of the electrode of the biosensor. In certain other embodiments, similar unexpectedly favorable results may occur with Fe(CN)6 washing conditions comprising applying a current having a voltage of 0.9-1.5V, 1.0-1.4V, 1.1-1.3V, or 1.15-1.25V.

[0100] As previously described, the electrochemical biosensor including an insulating layer with nanopores can be composed of materials that do not react during the Fe(CN)6 washing step, such as inorganic materials. Some embodiments of the present disclosure are particularly well suited for electrochemical biosensors including an insulating layer made of Si3N4 or SiO2. In some embodiments, the present disclosure is not well suited for photolithography of electrochemical biosensors including an insulating layer with nanopores (due to weak bonding between gold and the insulating layer).

[0101] In some embodiments, such as with respect to the ferrocyanide etching step, the present disclosure may be adapted for use with a phosphate buffered saline solution.

[0102] The resulting biosensors, for example, the nanopore arrays of the biosensors fabricated in Examples 1A to 1C, were photographed by SEM, and CV analysis was performed on each biosensor.

[0103] In some embodiments, the present disclosure can be adapted for use with methylene blue (ie, methyl blue) as a wash solution in addition to, or in the absence of, ferrocyanide and / or H2SO4 washes.

[0104] like Figures 10A-15B The effect of washing an electrode having an insulating layer of Si3O4 or SiO2 with H2SO4 and / or K3Fe(CN)6 is shown.

[0105] Fig. 10A SEM images of nanopore array surfaces formed by Si3N4 resist layers are shown. In this embodiment, no cleaning by H2SO4 or ferrocyanide etching is performed. A uniform array of nanopores is shown. As shown in the lower panel, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0106] Fig. 10B Shows Fig. 10A CV analysis data of the nanopore array electrode shown in FIG.

[0107] Fig.11A SEM images of nanopore array surfaces formed by Si3N4 resist layers are shown. In this embodiment, H2SO4 washing steps were performed. Uniform arrays of nanopores are shown. As shown in the panel below, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0108] Fig. 11B Shows Fig.11A CV analysis data of the nanopore array electrode shown in FIG.

[0109] Fig. 12A SEM images of nanopore array surfaces formed by Si3N4 resist layers are shown. In this embodiment, H2SO4 washing steps and ferrocyanide etching (or washing) steps were performed. A uniform array of nanopores is shown. As shown in the lower panel, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0110] Fig. 12B Shows Fig. 12A CV analysis data of the nanopore array electrode shown in FIG.

[0111] The CV analysis data revealed the lack of well-defined redox peaks, such as Fig. 10A and 16A As shown, significant impurities are present on the electrode surface.

[0112] exist Fig.13A The CV analysis data also revealed a normal oxidation current peak but an irregular reduction current peak, indicating the presence of some impurities on the electrode surface.

[0113] Fig. 16B , 17B The CV analysis data of 18A and 18B show ill-defined redox peaks, indicating that the electrode surface is contaminated with higher levels of impurities compared to the nanopore array electrodes formed with Si3N4 or SiO2 resist layers.

[0114] In some embodiments, such as with respect to the ferrocyanide etching step, the present disclosure may be adapted for use with a phosphate buffered saline solution.

[0115] For example, CV analysis was measured in a solution containing 100 mM phosphate buffered saline solution (pH 7.4) at a scan rate of 100 mV / s, with an initial E of -0.6 V and a high E of +0.4 V.

[0116] In some embodiments, the present disclosure can be adapted for use with methylene blue (ie, methyl blue) as a wash solution in addition to, or in the absence of, ferrocyanide and / or H2SO4 washes.

[0117] As shown in Example 1, a uniform and stable nanopore array surface is formed by using an insulating layer including silicon nitride (Si3N4). Signals from electrodes exposed to such nanopores can be accurately quantified, thereby greatly improving the sensitivity and selectivity of the biosensor.

[0118] At the same time, if Fig. 10B ,11B As shown in Figures 12 and 12B, in Example 1A, the redox peak is rarely exhibited, while in Example 1B, a relatively well-defined redox peak is exhibited. In particular, in Example 1C, which includes ferrocyanide etching after sulfuric acid washing, the most clearly defined redox peak is exhibited. Therefore, it seems that in the case of Example 1A that does not include a washing process, there are significant impurities on the electrode surface, so that the electrode surface is not well exposed. By comparison, it can be understood that in Examples 1B and 1C that include a washing process, impurities on the bottom layer of the nanopore are removed, and thus the electrode surface is normally exposed.

[0119] Example 2. Effect of washing an electrode having a SiO2 insulating layer with H2SO4 and / or K3Fe(CN)6.

[0120] In Example 2, a biosensor was manufactured according to the same method described in Example 1, except that SiO2 was used instead of Si3N4 to form the insulating layer of the nanopore. In addition, the biosensor manufactured according to Example 2 was subjected to different washing processes as described in Table 3 below. Each washing process was performed under the same conditions as described in Example 1.

[0121] Table 3

[0122] Example <![CDATA[Washing with sulfuric acid (H2SO4)]]> Ferrocyanide etching Example 2A X X Example 2B O X Example 2C O O

[0123] The resulting biosensors, for example, the nanopore arrays of the biosensors fabricated in Examples 2A to 2C, were photographed by SEM, and CV analysis was performed on each biosensor.

[0124] Fig.13A , 14A 15A and 15B show SEM images of biosensors (Examples 2A-2C) fabricated by using different washing processes, and Fig. 13B , 14B and 15B are shown respectively Fig.13A , 14A and CV analysis data of the biosensor of 15A.

[0125] Fig.13A SEM images of nanopore array surfaces formed by SiO2 resist layers are shown. In this embodiment, no cleaning by H2SO4 or ferrocyanide etching is performed. A uniform array of nanopores is shown. As shown in the lower panel, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0126] Fig. 13B Shows Fig.13A CV analysis data of the nanopore array electrode shown in FIG.

[0127] Fig.14A SEM images of nanopore array surfaces formed by SiO2 resist layers are shown. In this embodiment, H2SO4 washing steps were performed. Uniform arrays of nanopores are shown. As shown in the panel below, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0128] Fig. 14B Shows Fig.14A CV analysis data of the nanopore array electrode shown in FIG.

[0129] Fig.15A SEM images of nanopore array surfaces formed by SiO2 resist layers are shown. In this embodiment, H2SO4 washing steps and ferrocyanide etching (or washing) steps were performed. As shown in the lower panel, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0130] Fig. 15B Shows Fig.15A CV analysis data of the nanopore array electrode shown in FIG.

[0131] first, Fig.13A , 14A 15A show that the biosensor according to all embodiments has a uniform array of nanopores. In particular, as shown in the lower panel, the diameter of each nanopore opening is approximately 230 nm, and the nanopores are spaced at a 1:1 pitch ratio (see SEM images, where the scale bar is 200 nm).

[0132] In this way, a uniform and stable nanopore array surface can be formed by using SiO2, similar to the Si3N4 insulating layer as described in Example 1. Signals from electrodes exposed to such nanopores can be accurately quantified, thereby greatly improving the sensitivity and selectivity of the biosensor.

[0133] like Fig. 13B , 14B As shown in Figures 15 and 15B, a relatively normal oxidation current peak is exhibited, but a reduction current peak is not exhibited in Example 2A. By comparison, in Example 2B including sulfuric acid washing, a relatively well-defined reduction current peak is also exhibited. In particular, in Example 2C including ferrocyanide etching after sulfuric acid washing, the most clearly defined redox peak is exhibited. Therefore, it can be understood that the impurities on the bottom layer of the nanopore are removed by the washing process, and thus the electrode surface is normally exposed.

[0134] Comparative Example. Effect of washing an electrode having an insulating layer formed by photolithography with H2SO4 and / or K3Fe(CN)6.

[0135] In a comparative example, a biosensor was manufactured according to the same method described in Examples 1 and 2, except that the insulating layer of the nanopores was formed by using a photoresist material instead of an inorganic insulator layer (such as SiO2 or Si3N4). In addition, the biosensor manufactured according to the comparative example was subjected to different washing processes as described in Table 4 below. Each washing process was performed under the same conditions as described in Examples 1 and 2.

[0136] Table 4

[0137] Example <![CDATA[Washing with sulfuric acid (H2SO4)]]> Ferrocyanide etching Comparative Example A X X Comparative Example B O X Comparative Example C O O

[0138] The resulting biosensor, for example, the nanopore array of the biosensor manufactured by Comparative Examples AC, was photographed by SEM, and CV analysis was performed on each biosensor.

[0139] Fig.16A , 17A 18A and 18B show SEM images of biosensors (Comparative Examples AC) fabricated by using different washing processes, and Fig. 16B , 17B and 18B are respectively shown Fig.16A , 17A and CV analysis data of the biosensor of 18A.

[0140] like Figures 14A-16B The effect of washing an electrode having an insulating layer formed by photolithography with H2SO4 and / or K3Fe(CN)6 is shown.

[0141] Fig.16A SEM images of nanopore array surfaces formed using photolithography are shown. In this embodiment, no cleaning by H2SO4 or ferrocyanide etching is performed. A uniform array of nanopores is shown. As shown in the lower panel, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0142] Fig. 16B Shows Fig.16A CV analysis data of the nanopore array electrode shown in FIG.

[0143] Fig.17ASEM images of nanopore array surfaces formed using photolithography are shown. In this embodiment, H2SO4 washing steps were performed. Uniform arrays of nanopores are shown. As shown in the panel below, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in the SEM photo: 200nm).

[0144] Fig. 17B Shows Fig.17A CV analysis data of the nanopore array electrode shown in FIG.

[0145] Fig.18A SEM images of nanopore array surfaces formed using photolithography are shown. In this embodiment, H2SO4 washing steps and ferrocyanide etching (or washing) steps were performed. A uniform array of nanopores is shown. As shown in the panel below, the diameter of each nanopore is approximately 230nm, and the nanopores are spaced at a 1:1 spacing ratio (scale bar in SEM photo: 200nm).

[0146] Fig.18B Shows Fig.18A CV analysis data of the nanopore array electrode shown in FIG.

[0147] First, similar to other embodiments, Fig.16A , 17A 18A shows that the biosensor according to Comparative Example 1 has a uniform array of nanopores, each nanopore opening has a diameter of about 230 nm, and the nanopores are spaced at a pitch ratio of 1: 1. However, in Examples B and C including a washing process, the nanopores of the photoresist insulating layer are collapsed or damaged. Thus, an organic insulator layer such as a photoresist is not suitable for use in introducing the washing process of the present invention because it causes a chemical reaction with a solution applied to the washing process.

[0148] exist Fig. 16B , 17B and 18B, no well-defined redox peaks were exhibited, indicating that the electrode surface was not well exposed due to significant impurities.

[0149] In certain embodiments, the production methods disclosed herein result in electrodes for biosensors having improved electrochemical properties. In a preferred embodiment, cyclic voltammetry analysis is used to measure the electrochemical properties.

[0150] In certain embodiments, the scanning cycle of the Fe(CN)6 washing step includes one cycle or two cycles. In certain other embodiments, the scanning cycle of the Fe(CN)6 washing step includes more than two cycles.

[0151] It is well known in the art that, typically, Fe(CN)6 washing conditions comprising applying an electric current having a voltage greater than 1V may adversely affect the surface integrity of the electrode. However, in certain embodiments of the present disclosure, applying an electric current having a voltage greater than 1V for a short duration produces unexpectedly favorable results, such as removal of impurities and enhanced electrochemical properties of the electrode of the biosensor. In certain other embodiments, similar unexpectedly favorable results may occur with Fe(CN)6 washing conditions comprising applying an electric current having a voltage of 0.9-1.5V, 1.0-1.4V, 1.1-1.3V, or 1.15-1.25V.

[0152] As previously described, the insulating layer providing the nanopores may be composed of a material, such as an inorganic material, that does not react during the washing step of the Fe(CN)6 washing step. In some embodiments, an inorganic material may be used instead of an organic material, such as a photoresist material to form the insulating layer. In particular, some embodiments of the present disclosure are particularly well suited for use in electrochemical biosensors including an insulating layer made of Si3N4 or SiO2. In some embodiments, due to the weak bonding between the electrode (e.g., gold) and the insulating layer, the present disclosure is not well suited for use in photolithography of an electrochemical biosensor including an insulating layer having nanopores.

[0153] In some embodiments, the present disclosure may be adapted for use with phosphate buffered saline solutions other than, for example, those described with respect to the ferrocyanide etching step.

[0154] Exemplary embodiments of biosensors and their uses

[0155] An electrochemical biosensor device for sensing the presence of molecules in a solution, the device comprising:

[0156] basal layer; and

[0157] A plurality of electrodes, the electrodes further comprising:

[0158] a buffer layer laid on the base layer, the buffer layer being configured to provide bonding between the plurality of electrodes and the base layer;

[0159] an electrode layer disposed on the buffer layer, the electrode layer being configured to provide a binding site for an analyte; and

[0160] an insulator layer having a plurality of holes, the insulator layer being laid on the electrode layer, and the insulator having a plurality of holes configured to form a plurality of nanopores having sidewalls defined by the insulator layer and having a bottom layer defined by a top surface of the electrode layer not covered by the insulator layer,

[0161] wherein an analyte probe is immobilized on the bottom layer of the nanopore, the analyte probe being configured to bind to an analyte, and

[0162] The insulator layer is configured to substantially restrict binding of the analyte to a top surface of the electrode layer, the top surface of the electrode layer defining a bottom layer of the plurality of nanopores.

[0163] The electrochemical biosensor device of claim 1, wherein the substrate layer comprises glass.

[0164] The electrochemical biosensor device of claim 1, wherein the substrate layer comprises silicon.

[0165] The electrochemical biosensor device of claim 1, wherein the insulator layer comprises silicon nitride.

[0166] The electrochemical biosensor device of claim 1, wherein the insulator layer comprises silicon dioxide.

[0167] The electrochemical biosensor device of claim 1, wherein the buffer layer comprises titanium.

[0168] The electrochemical biosensor device of claim 1, wherein the buffer layer comprises chromium.

[0169] The electrochemical biosensor device of claim 1, wherein the electrode layer comprises gold.

[0170] The electrochemical biosensor device of claim 1, wherein the nanopore is cylindrical in shape and has a circular nanopore opening with a diameter of about 230 nm.

[0171] The electrochemical biosensor device of claim 1, wherein the nanopore is cylindrical in shape and has a circular nanopore opening with a diameter of about 100 nm.

[0172] The electrochemical biosensor device of claim 1, wherein the nanopore is cylindrical in shape and has a nanopore opening with a diameter of about 50 nm.

[0173] The electrochemical biosensor device of claim 1, wherein a spacing ratio between the plurality of nanopores is less than 1:5.

[0174] The electrochemical biosensor device of claim 1, wherein a spacing ratio between the plurality of nanopores is less than 1:3.

[0175] The electrochemical biosensor device of claim 1, wherein a spacing ratio between the plurality of nanopores is about 1:1.

[0176] The electrochemical biosensor device of claim 1, wherein the device is capable of sending a signal to an electronic device so that the signal can be used by the electronic device to detect a difference in one or more electrochemical reaction parameters between an electrode containing a reference sample and an electrode containing a test sample to determine whether the analyte is present in the test sample.

[0177] The electrochemical biosensor device according to claim 9, wherein the electrochemical reaction comprises an oxidation reaction and a reduction reaction.

[0178] The electrochemical biosensor device of claim 9, wherein the parameter comprises a change in redox current.

[0179] A method for detecting an analyte in a test sample using the biosensor device according to claim 1, the method comprising the steps of:

[0180] applying the test sample to a sensing electrode of the electrochemical biosensor device in a manner such that an analyte that may be present in the test sample is able to bind to the analyte probe;

[0181] flushing the sensing electrode with a suitable buffer in a manner such that unbound and / or non-specifically bound analytes and / or non-analytes to be removed from the sensing electrode are washed away;

[0182] applying an electric current to the sensing electrode in such a manner as to induce a chemical change in the sensing electrode;

[0183] measuring an electrochemical property of the sensing electrode using an electronic device; and

[0184] Differences in electrochemical characteristics between the test sample and the reference sample are analyzed to determine the presence of an analyte on the sensing electrode.

[0185] The method of claim 12, wherein the electrochemical properties of the sensing electrode are measured using cyclic voltammetry.

[0186] Although the foregoing description has been directed to specific embodiments, it will be apparent that other variations and modifications may be made to the described embodiments, wherein some or all of the advantages thereof are obtained. Therefore, this description is by way of example only and does not otherwise limit the scope of the embodiments herein. It is therefore the object of the appended claims to cover all such variations and modifications as come within the true spirit and scope of the embodiments described herein. Finally, all publications and references cited herein are expressly incorporated by reference in their entirety.

[0187] Those skilled in the art will appreciate that, although the present disclosure is referred to as a "biosensor", its application is not limited to the detection of biomolecules. In other words, the present disclosure can be used to detect other small non-biological (e.g., inorganic, metallic, solute, electrolyte and / or element) molecules. In addition, although the embodiments provided herein consist of detection in a fluid and / or aqueous environment, those skilled in the art will appreciate that the present disclosure can be used to detect small molecules in other fluid environments, such as oils, solvents, gases and / or colloidal solutions.

Claims

1. A method for manufacturing a biosensor, the method comprising: forming a buffer layer on the base layer; forming a metal layer on the buffer layer; forming a desired patterned electrode by patterning the metal layer using a first photolithography process; forming an inorganic insulating layer on the desired patterned electrode; forming a plurality of nanopores on the inorganic insulating layer by exposing a region of the desired patterned electrode using a second photolithography process; as well as washing the plurality of nanopores with a sulfuric acid (H2SO4) solution to remove impurities from the desired patterned electrodes exposed through the plurality of nanopores; and After washing with the sulfuric acid (H2SO4) solution and during a time period of 1 to 10 seconds, the multiple nanopores are washed by ferrocyanide etching and applying a voltage of 1.25-1.5 V for one or two scanning cycles, thereby removing impurities on the multiple nanopores of the desired patterned electrode, wherein the ferrocyanide etching is performed by immersing the multiple nanopores in a mixed solution of K3Fe(CN)6 and KCl.

2. The method according to claim 1, wherein: The ferrocyanide etch is performed at a temperature in the range of 15-25°C for 1 to 10 seconds.

3. The method according to claim 1 or 2, wherein: The desired patterned electrode includes gold (Au).

4. The method according to claim 1, wherein: The inorganic insulating layer includes SiO2 or Si3N4.

5. The method according to claim 1, further comprising: washing the plurality of nanopores with the sulfuric acid (H2SO4) solution by immersing the plurality of nanopores in the sulfuric acid (H2SO4) solution and applying a voltage of 1.0-1.5 V for 1-5 minutes.

6. The method according to claim 1, wherein: The pitch ratio of the plurality of nanopores is defined as the ratio of the size of each nanopore to the shortest distance between adjacent nanopores, wherein the pitch ratio is 1:3 or less.

Images