Apparatus and method for detecting multiple diseases using multiple electrodes

Abstract

This application is directed to biosensors and methods of forming and using such biosensors. Each biosensor includes different nanowells on multiple working electrodes for detecting multiple diseases. One exemplary embodiment of a biosensor includes a plurality of first nanowells formed on a first working electrode and a plurality of second nanowells formed on a second working electrode. The biosensor includes a first biomarker for detecting a first disease and a second biomarker for detecting a second disease. The first biomarker is immobilized in at least two of the first nanowells. The second biomarker is immobilized in at least two of the second nanowells. A shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells on the first or second working electrode. Each of the two adjacent nanowells contains the first or second biomarker.

Description

Attorney Docket No.132414-5006-WO APPARATUS AND METHOD FOR DETECTING MULTIPLE DISEASES USING MULTIPLE ELECTRODES TECHNICAL FIELD

[0001] The present disclosure relates generally to biosensors and methods of forming and using such biosensors, and more particularly, to such biosensors each of which includes different nanowells on multiple working electrodes for detecting multiple diseases. BACKGROUND

[0002] Biosensors are used to detect the presence of biological molecules such as proteins, amino acids (e.g., DNA and / or RNA containing specific base sequences), or other organic molecules. Some of the examples of biosensors include pregnancy tests and glucose monitoring sensors. These biosensors can detect biomolecules such as human chronic gonadotropin (hCG) or glucose that are present in bodily fluids such as blood or urine. Examples of different types of biosensors include electrochemical biosensors, nano-cantilever biosensors, and micro- or nano-electromechanical systems (MEMS / NEMS). Electrochemical biosensors are highly desirable because they can conveniently interface with an electronic device for further analysis of detected signals without user intervention. However, each electrochemical biosensor is oftentimes targeted at only one disease or antigen and is limited to diagnosing a particular disease. Many electrochemical biosensors require special sample treatment (e.g., incubation and separation), before samples are injected into the biosensors. The electrochemical biosensors have a resolution or measurement limit (e.g., in ng / mL), which has limited a concentration of a target material that can be reliably detected. As such, it oftentimes takes an extended time to use special sample treatment to prepare samples that must reach the required concentration, while the electrochemical biosensors can be used only once to detect only one disease or antigen. It would be beneficial to have more effective and efficient biosensors to detect diseases compared with many existing electrochemical biosensors.Attorney Docket No.132414-5006-WO SUMMARY

[0003] The embodiments described herein are directed to biosensors each including different nanowells on multiple working electrodes for detecting multiple diseases concurrently, and methods of forming and using such biosensors.

[0004] In one aspect of this application, a biosensor includes a first working electrode, a second working electrode, a plurality of first nanowells formed on the first working electrode, and a plurality of second nanowells formed on the second working electrode. In some embodiments, the biosensor further includes a first biomarker for detecting a first disease, and a second biomarker for detecting a second disease. The first biomarker is immobilized in at least two of the first nanowells. The second biomarker is immobilized in at least two of the second nanowells.

[0005] In some embodiments, a shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells on the first or second working electrode, each of the two adjacent nanowells containing the first or second biomarker. In some embodiments, the shortest distance between the first working electrode and the second working electrode is longer than a longest distance between the two adjacent nanowells on the first or second working electrode.

[0006] In another aspect, a method is implemented to form a biosensor. The method includes forming, on a substrate, a first working electrode and a second working electrode. The method further includes forming an insulation layer on the first working electrode and the second working electrode. The method further includes forming, in the insulating layer based on a first photolithography process, (1) a plurality of first nanowells to expose a partial area of the first working electrode and (2) a plurality of second nanowells to expose a partial area of the second working electrode. In some embodiments, a shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells formed in the insulating layer.

[0007] In yet another aspect, a method is implemented for detecting a plurality of diseases includes: placing a sample on a biosensor. The biosensor includes a first working electrode, a second working electrode, a plurality of first nanowells formed on the first working electrode, and a plurality of second nanowells formed on the second working electrode. In some embodiments, the biosensor further includes a first biomarker for detecting a first disease, and aAttorney Docket No.132414-5006-WO second biomarker for detecting a second disease. The first biomarker is immobilized in at least two of the first nanowells. The second biomarker is immobilized in at least two of the second nanowells. The method further includes detecting one or more of the plurality of diseases based on one or more corresponding biomarkers in the nanowells on the biosensor.

[0008] These illustrative embodiments and implementations are mentioned not to limit or define the disclosure, but to provide examples to aid understanding thereof. Additional embodiments are discussed in the Description of Embodiments, and further description is provided there. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The features and advantages of the present invention will be more fully disclosed in, or rendered obvious by the following detailed description of the preferred embodiments, which are to be considered together with the accompanying drawings wherein like numbers refer to like parts and further wherein:

[0010] FIG.1A is a top view of an example biosensor including multiple working electrodes, in accordance with some embodiments.

[0011] FIG.1B is a cross sectional view of the example biosensor shown in FIG.1A, in accordance with some embodiments.

[0012] FIG.1C is another cross sectional view of the biosensor shown in FIG.1A, in accordance with some embodiments.

[0013] FIG.2 is a top view of a biosensor including multiple working electrodes, a counter electrode, and a reference electrode, in accordance with some embodiments.

[0014] FIG.3 is a top view of another biosensor including multiple working electrodes, a counter electrode, and a reference electrode, in accordance with some embodiments.

[0015] FIG.4 is a flow diagram of an exemplary method for forming a biosensor, in accordance with some embodiments.

[0016] FIGS.5A-5G are cross-sectional views of an exemplary biosensor at a sequence of fabrication stages, in accordance with some embodiments. DETAILED DESCRIPTION

[0017] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerousAttorney Docket No.132414-5006-WO specific details are set forth in order to provide a thorough understanding of the various described embodiments. However, it will be apparent to one of ordinary skill in the art that the various described embodiments may be practiced without these specific details. In other instances, well-known methods, procedures, components, mechanical structures, circuits, and networks have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.

[0018] Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, and use of the devices disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present disclosure is defined solely by the claims. The features illustrated or described in collection with one 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.

[0019] Additionally, the figures 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 can be used in conjunction with such devices and methods. A person skilled in the art will recognize that an equivalent to such linear and circular dimensions can easily be determined for any geometric shape. Still further, sizes and shapes of the devices, and the components thereof, can depend at least on the anatomy of the subject in which the device will be used, the size and shape of components with which the device will be used, and the methods and procedures in which the device will be used.

[0020] In some embodiments, the disclosure provides a system. In some embodiments, the system includes a sensor. As used herein with reference to the present disclosure, “sensor,” “biosensor,” “nanowell sensor,” and “nanowell biosensor” are used interchangeably. In some embodiments, the system includes a processor. The processor of the system may be a hardware device implemented by various electronic circuits (e.g., computer, microprocessor, CPU, ASIC, circuitry, logic circuits, etc.). The processor may be implemented by a non-transitory memory storing, e.g., a program(s), software instructions reproducing algorithms, etc., which, when executed, performs various functions described hereinafter, and a processor configured toAttorney Docket No.132414-5006-WO execute the program(s), software instructions reproducing algorithms, etc. Herein, the memory and the processor may be implemented as separate semiconductor circuits. Alternatively, the memory and the processor may be implemented as a single integrated semiconductor circuit. The processor may embody one or more processor(s). In some embodiments, the processor is configured to perform one or more methods disclosure herein. In some embodiments, the system includes a display. The display may be configured to display visualized data. In some embodiments, the visualized data represents the presence of one or more diseases in a sample.

[0021] Furthermore, while the exemplary embodiments provided herein describe use of the device in detecting biomolecules (e.g., proteins and / or nucleic acid molecules), a person skilled in the art will recognize that the device may be adopted to be used to detect presence of non-biological molecules and / or samples that are not biological samples. As an example, presence of inorganic material may be detected using the present invention for water quality testing, environmental testing and / or quality control / quality assurance testing in other industrial settings).

[0022] In order for an electrochemical sensor to be adopted in a wide range of applications such as diagnosis in traditional medical, pharmaceutical, and / or healthcare settings (e.g., blood and / or urine sample testing for specific biological molecules), medical diagnosis in non-hospital setting (e.g., military use in combat zone, self-administered consumer diagnostics such as pregnancy test or blood glucose monitoring), non-medical detection of biological and / or small molecule detection (e.g., water quality testing, environmental testing, quality control and / or quality assurance testing in food industry), companion diagnostics for pharmaceutical therapeutics; research applications where detection of small molecules are required, and / or other settings or circumstances where detection of biological molecules is needed, the electrochemical sensor must be sensitive (i.e., being able to detect low concentrations of analyte), selective (i.e., being able to distinguish and differentiate target analytes in the presence of other components), easy to use (i.e., simple to operate, requires small amounts of test samples), and readily available to users (i.e., able to manufacture scalably, in large quantities, and / or at a low cost).

[0023] The present disclosure is directed to sensors each including different nanowells on multiple working electrodes for detecting multiple diseases, and methods of forming and using such sensors. In some embodiments, the sensors may be highly sensitive and highly selective electrochemical biosensors made using components that are more resilient and stable comparedAttorney Docket No.132414-5006-WO to past electrochemical biosensors. In some embodiments, one or more electrochemical biosensors may be produced on a single substrate layer, such as a glass or silicon substrate layer (e.g., a wafer). Each electrochemical biosensor may include multiple working electrodes (also called sensing electrodes). A working electrode may include a plurality of nanowells.

[0024] In some embodiments, in order to detect specific analytes (e.g., biological molecules), biosensors may contain an analyte-binding surface where probes specific for an analyte (e.g., single-strand DNA or antibody specific for the target molecule) are immobilized to the analyte binding surface. Different types of biosensors using distinct scientific principles have been developed that can detect the presence of specific biological molecules.

[0025] Examples of different types of biosensors include electrochemical biosensors, nano-cantilever biosensors, and micro- or nano-electromechanical systems (MEMS / NEMS). Like other types of biosensors, electrochemical biosensors include an analyte-binding surface that is capable of interacting with and / or binding to specific biomolecules (e.g., a specific protein or a specific sequence of DNA). In particular, electrochemical biosensors use the principle of electrochemical analysis to detect specific analytes, where chemical response to an electrical excitation applied to a system is measured and analyzed to detect whether an analyte is bound to the surface of an electrode.

[0026] In some embodiments, applications for electrochemical biosensors include diagnosis in traditional medical and healthcare settings (e.g., blood and / or urine sample testing for specific biological molecules); medical diagnosis non-hospital settings (e.g., military use in combat zone and / or self-administered consumer diagnostics), non-medical detection of biological and / or small molecule detection (e.g., water quality testing, environmental testing, quality control and / or quality assurance testing in food industry); companion diagnostics for pharmaceutical therapeutics; research applications where detection of small molecules are required; and / or other settings or circumstances where detection of biological molecules is needed. A person skilled in the art will appreciate that, although the present disclosure is called “biosensors,” its application is not limited to detection of biological molecules. In other words, the present disclosure may be used for detection of other small non-biological (e.g., inorganic, metallic, solute, electrolyte, and / or elemental) molecules. In addition, although examples provided here consist of detection in fluidic and / or aqueous milieu, one skilled in the art willAttorney Docket No.132414-5006-WO appreciate that the present disclosure may be used to detect small molecules in other fluidic milieu such as in oil, solvents, gas, and / or colloidal solutions.

[0027] In some embodiments, electrochemical analysis methods for in vitro diagnostics measure the amount of change in current and impedance relative to the amount of binding of the antigen-antibody based on protein aggregation response, and various types of biosensors have been developed to perform these electrochemical analysis methods. In order for electrochemical biosensors to be adapted widely for a broad range of applications, the biosensors must be highly sensitive and selective, and cost of manufacturing of such sensor must be competitive. Electrochemical biosensors with significantly improved sensitivity and selectivity may enable miniaturization of such devices, which in turn may reduce the production cost and further contribute to adoption of electrochemical biosensors for a wide range of applications. In some embodiments, an electrochemical biosensor includes a perforated insulation layer (or insulating layer) laid on an electrode of an electrochemical biosensor to form nanowells (or nano-wells), and is configured to detect analytes that are present in fM-range in biological samples with high selectivity.

[0028] FIG.1A is a top view of a biosensor 100-1 including multiple working electrodes, in accordance with some embodiments. While FIG. 1A shows an electrode portion including two working electrodes of the biosensor 100-1, the biosensor 100-1 may include any number of working electrodes and any number of electrode portions, according to various embodiments of the present disclosure.

[0029] As shown in FIG.1A, the biosensor 100-1 includes a first working electrode 110 and a second working electrode 120. A plurality of first nanowells 111 are formed on the first working electrode 110. A plurality of second nanowells 121 are formed on the second working electrode 120. In some examples (as shown in FIG.1A), the first working electrode 110 and the second working electrode 120 have a same number of nanowells. In some examples not shown, the first working electrode 110 and the second working electrode 120 have different numbers of nanowells.

[0030] In some embodiments, the biosensor 100-1 further includes a first biomarker for detecting a first disease. The first biomarker may include a first analyte probe used to detect specific analytes (e.g., biological molecules) for the first disease. For example, the first analyte probe may be a single-strand DNA or antibody specific for the target molecule related to the firstAttorney Docket No.132414-5006-WO disease. The first biomarker may be immobilized in any of the first nanowells 111. In some embodiments, the first biomarker is immobilized in at least two of the first nanowells 111. In some embodiments, the term “immobilized” means binding a biomarker (e.g. a specific analyte probe) to a bottom surface of a nanowell (i.e. a top surface of an exposed working electrode), for example, by covalent bonding, hydrogen bonding, ionic bonding, and / or Van der Walls forces.

[0031] In some embodiments, the biosensor 100-1 further includes a second biomarker for detecting a second disease. The second biomarker may include a second analyte probe used to detect specific analytes (e.g., biological molecules) for the second disease. For example, the second analyte probe is a single-strand DNA or antibody specific for the target molecule related to the second disease. The second biomarker is immobilized in any of the second nanowells 121. In some embodiments, the second biomarker is immobilized in at least two of the second nanowells 121.

[0032] The first disease is different from the second disease. The first analyte probe is different from the second analyte probe. Accordingly, the first biomarker immobilized in the first nanowells 111 is different from the second biomarker immobilized in the second nanowells 121.

[0033] In the example shown in FIG.1A, the first working electrode 110 and the second working electrode 120 are separated from each other by a distance L2, which is a shortest distance between the first working electrode 110 and the second working electrode 120. In the example shown in FIG.1A, the first nanowells 111 form a 4 * 4 nanowell array, and the second nanowells 121 form another 4 * 4 nanowell array. In other embodiments, the first nanowells 111 and the second nanowells 121 forms nanowell arrays with other dimensions. In the example shown in FIG.1A, a distance L1 is between any two adjacent (or neighboring) nanowells on the first working electrode 110 and any two adjacent nanowells on the second working electrode 120. In this example, L2 is longer than L1.

[0034] In some embodiments, L2 is longer than a shortest distance between two adjacent nanowells on the first working electrode 110 or the second working electrode 120, where the two adjacent nanowells contains a same biomarker, which may be the first biomarker or the second biomarker. The shortest distance between the two adjacent nanowells is larger than L1 when some nanowells on the first working electrode 110 and the second working electrode 120 do not contain any biomarker.Attorney Docket No.132414-5006-WO

[0035] In some embodiments, L2 is longer than a longest distance between two adjacent nanowells on the first working electrode 110 or the second working electrode 120, where the two adjacent nanowells contains a same biomarker, which may be the first biomarker or the second biomarker. The longest distance between the two adjacent nanowells is larger than L1 when some nanowells on the first working electrode 110 and the second working electrode 120 do not contain any biomarker.

[0036] In some embodiments, different distances are between different pairs of adjacent nanowells on the first working electrode 110 and the second working electrode 120. For example, some of the distances is larger than L1, while some of the distances is smaller than L1. In some embodiments, the shortest distance between the first working electrode 110 and the second working electrode 120 is longer than a shortest distance between all possible two adjacent nanowells on the first working electrode 110 and the second working electrode 120, where each of the two adjacent nanowells contains a same biomarker, which may be the first biomarker or the second biomarker. In other embodiments, the shortest distance between the first working electrode and the second working electrode is longer than a longest distance between all possible two adjacent nanowells on the first working electrode 110 and the second working electrode 120, where each of the two adjacent nanowells contains a same biomarker, which may be the first biomarker or the second biomarker.

[0037] As shown in FIG.1A, the biosensor 100-1 further includes a first connection electrode 101 and a second connection electrode 102. While the first connection electrode 101 is physically coupled to the first working electrode 110; the second connection electrode 102 is physically coupled to the second working electrode 120. In some embodiments, a shortest distance between the first connection electrode 101 and the second connection electrode 102 is larger than zero. In some embodiments, the first connection electrode 101 and the second connection electrode 102 are electrically isolated from each other, and the first working electrode 110 and the second working electrode 120 are also electrically isolated from each other.

[0038] In some embodiments, the biosensor 100-1 may further include one or more microfluidic lane(s) configured to move a sample to the first nanowells 111 and / or the second nanowells 121. In some embodiments, the one or more microfluidic lane(s) is configured toAttorney Docket No.132414-5006-WO process the sample as the sample flows through the one or more microfluidic lane(s) to the first nanowells 111 and / or the second nanowells 121.

[0039] FIG.1B shows a cross sectional view of the biosensor in FIG.1A, in accordance with some embodiments. The cross sectional view of the biosensor 100-2 in FIG.1B is generated by cutting the biosensor 100-1 along the line 1B-1B in FIG. 1A. In some embodiments, the biosensor 100-2 may include a glass or silicon substrate layer 130, a buffer layer 140 laid on the substrate layer 130, a metal layer or electrode layer 150 laid on the buffer layer 140, and an insulator layer 160 laid on the electrode layer 150.

[0040] In some embodiments, the substrate layer 130 is made of glass. The substrate layer may 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 130 is a circular wafer. In yet other embodiments, the glass or silicon substrate layer 130 is configured to accommodate a plurality of electrochemical biosensors.

[0041] The buffer layer 140 of the biosensor 100-2 may provide enhanced bonding of the electrode layer 150 to the substrate layer 130, thereby minimizing risk of the electrode layer 150 detaching from the substrate layer 130. In other words, the buffer layer 140 allows the electrode layer 150 and the substrate layer 130 to form a tighter seal. Such enhanced bonding or formation of seal between the electrode layer 150 and substrate layer 130 may enable easier and more reliable manufacturing of the electrochemical biosensor and / or reduce cost of manufacture. In some embodiments, the buffer layer 140 may include titanium, chromium, and / or alloys of titanium or chromium.

[0042] As shown in FIG.1B, the electrode layer 150 includes the first connection electrode 101 and the first working electrode 110 coupled to each other. In some embodiments, the electrode layer 150 may include graphite or metal material, e.g. Au, Pt, Cu, Pd.

[0043] In some embodiments, the insulator layer 160 of the biosensor 100-2 is perforated, such that the insulator layer 160 includes a plurality of bores (i.e., holes) defining the internal dimensions of the first nanowells 111. In some embodiments, the bores are cylindrical in shape. The first nanowells 111 whose dimensions are defined by the bores have a circular opening, and the insulator layer 160 form the walls of the cylindrical nanowells 111. As the perforated insulator layer 160 is laid on the electrode layer 150, by virtue of the relative positions between the two layers, a portion of the top surface of the electrode layer 150 is not covered byAttorney Docket No.132414-5006-WO the insulator layer 160 and forms the bottom surfaces of the nanowells 111. In some embodiments, the insulator layer 160 includes silicon nitride (Si3N4), and silicon nitride is more resilient and stable compared to certain alternatives, such as organic or inorganic polymers. In another embodiment, the insulator layer 160 may include silicon dioxide (SiO2). Resilience and stability of silicon nitride allow the sensing electrode portion of the present disclosure to be manufactured in a reliable and consistent manner, reducing both occurrences of defective products and cost of manufacture.

[0044] In some embodiments, the opening of each nanowell 111 is circular, and the diameter of the circular opening of the nanowells 111 is less than 1000 nm. In other embodiments, the diameter of the circular opening of the nanowells 111 is less than 300 nm. In yet other embodiments, the diameter of the circular opening of the nanowells 111 is approximately 230 nm, 100 nm, and / or 50 nm. Although the embodiments described above has nanowells 111 that are cylindrical in shape with a circular opening, a person skilled in the art will recognize that the nanowells 111 may have various other opening shapes, such as rectangular, oval, and / or polygonal shapes. In these embodiments having nanowells 111 with various other opening shapes, the dimension of the opening may be less than 1000 nm or 300 nm, or may be approximately 230 nm, 100 nm, and / or 50 nm. In addition, a person skilled in the art will also recognize that the present invention is not limited to the compositions and structure described above, but may also include compositions and structure with similar characteristics, or improved characteristics.

[0045] In some embodiments, the bottom surfaces of the first nanowells 111 may include the first biomarker, which may be analytes probes or probe molecules that are capable of binding with specific analytes. As an example, as shown in FIG.1B, analytes probes (e.g. biotinylated antibodies) 165 specific for an analyte is immobilized to the bottom surface of a first nanowell 111 by using an intermediary binding molecule 164 such as avidin or streptavidin. A person skilled in the art will appreciate that other well-known methods of immobilizing analytes probes 165 can be incorporated into present disclosure, and are within the scope of present invention. In some embodiments, the insulator layer 160 with a plurality of bores may restrict binding of an analyte to its probes 165 to the bottom surfaces of the first nanowells 111, while preventing binding and / or aggregation of the analyte to the insulator layer 160.Attorney Docket No.132414-5006-WO

[0046] FIG.1C shows another cross sectional view of the biosensor in FIG.1A, in accordance with some embodiments. The cross sectional view of the biosensor 100-3 in FIG.1C is generated by cutting the biosensor 100-1 along the line 1C-1C in FIG.1A. Same as the biosensor 100-2, the biosensor 100-3 includes the substrate layer 130, the buffer layer 140 laid on the substrate layer 130, the electrode layer 150 laid on the buffer layer 140, and the insulator layer 160 laid on the electrode layer 150.

[0047] In the cross sectional view shown in FIG.1C, the electrode layer 150 includes the first working electrode 110 and the second working electrode 120 separated from each other by a distance L2. In some embodiments, the first working electrode 110 and the second working electrode 120 may include a same material. In some embodiments, the first working electrode 110 and the second working electrode 120 include different materials.

[0048] Referring to FIG.1C, in some embodiments, the insulator layer 160 includes a first plurality of bores (i.e., holes) defining the internal dimensions of the first nanowells 111 on the first working electrode 110, and a second plurality of bores (i.e., holes) defining the internal dimensions of the second nanowells 121 on the second working electrode 120. In some embodiments, the bores are cylindrical in shape. Each of the first nanowells 111 and the second nanowells 121 has a circular opening, and the insulator layer 160 form the walls for the cylindrical nanowells 111, 121. The perforated insulator layer 160 is laid on the electrode layer 150, by virtue of the relative positions between the two layers, a portion of the top surface of the electrode layer 150 is not covered by the insulator layer 160 and may form the bottom surfaces of the first nanowells 111 and the second nanowells 121. As shown in FIG. 1C, the insulator layer 160 does not cover the gap between the first working electrode 110 and the second working electrode 120 in the electrode layer 150. Specifically, in an example, distance between two adjacent nanowells (e.g. two adjacent first nanowells 111 on the first working electrode 110, or two adjacent second nanowells 121 on the second working electrode 120) is equal to L1. In some embodiments, L2 is larger than L1.

[0049] In some embodiments, while the bottom surfaces of the first nanowells 111 may include the first biomarker, the bottom surfaces of the second nanowells 121 may include the second biomarker. The first and second biomarkers are different analytes probes (e.g. biotinylated antibodies) specific for different analytes. Each analytes probe is immobilized to theAttorney Docket No.132414-5006-WO bottom surface of a nanowell by using an intermediary binding molecule such as avidin or streptavidin, or based on other methods of immobilizing analytes probes.

[0050] In some embodiments, the nanowells 111, 121 in the biosensor 100-3 have a pitch ratio (ratio between the diameter of the nanowell openings and the shortest distance between neighboring nanowells) of approximately 1:1. In some embodiments, the diameter of the nanowell openings is approximately 230 nm. In some embodiments, the nanowell pitch ratio and / or the nanowell opening diameter are of different value. In some embodiments, the first nanowells 111 and the second nanowells 121 may have different pitch ratios and / or different nanowell opening diameters. In some embodiments, pitch ratios of the nanowells 111, 121 can range from any ratio between 100:1-1:100, including 50:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10 or 1:50.

[0051] FIG.2 is a top view of a biosensor 200, in accordance with some embodiments. While FIG.2 shows an electrode portion including three working electrodes of the biosensor 200, the biosensor 200 may include any number of working electrodes and any number of electrode portions, according to various embodiments of the present disclosure. As shown in FIG.2, the biosensor 200 includes a first working electrode 210, a second working electrode 220 and a third working electrode 230. A plurality of first nanowells 211 is formed on the first working electrode 210. A plurality of second nanowells 221 is formed on the second working electrode 220. A plurality of third nanowells 231 is formed on the third working electrode 230. In some examples (as shown in FIG.2), the working electrodes 210, 220, 230 have the same number of nanowells. Alternatively, in some examples, the working electrodes 210, 220, 230 have different numbers of nanowells.

[0052] In some embodiments, the biosensor 200 further includes a first biomarker (e.g. a first analyte probe), a second biomarker (e.g. a second analyte probe), and a third biomarker (e.g. a third analyte probe). The first biomarker is immobilized in the first nanowells 211 for detecting specific analytes (e.g., biological molecules) for a first disease. The second biomarker is immobilized in the second nanowells 221 for detecting specific analytes (e.g., biological molecules) for a second disease, and the second biomarker is immobilized in the third nanowells 231 for detecting specific analytes (e.g., biological molecules) for a third disease. In some embodiments, some nanowells of the biosensor 200 do not contain any biomarker. In some embodiments, some nanowells of the biosensor 200 do not contain any biomarker. In someAttorney Docket No.132414-5006-WO embodiments, at least two of the first nanowells 211 contain the first biomarker, at least two of the second nanowells 221 contain the second biomarker, and at least two of the third nanowells 231 contain the third biomarker. While the three diseases are different from each other, the three biomarkers are also different from each other.

[0053] In the example shown in FIG.2, the first working electrode 210 and the second working electrode 220 are separated from each other by a distance L2. The second working electrode 220 and the third working electrode 230 are separated from each other by a distance L2’. The L2 may or may not be equal to L2’. In the example shown in FIG.2, the first nanowells 211 form a first 4 * 4 nanowell array, the second nanowells 221 form a second 4 * 4 nanowell array, and the third nanowells 231 form a third 4 * 4 nanowell array. In other embodiments, the nanowells 211, 221, 231 may form nanowell arrays with other dimensions. In the example shown in FIG.2, a distance L1 is between any two adjacent (or neighboring) nanowells on the first working electrode 210, the second working electrode 220 and the third working electrode 230. In this example, both L2 and L2’ are longer than L1.

[0054] In some embodiments, both L2 and L2’ are longer than a shortest distance between two adjacent nanowells containing a same biomarker on any one of the working electrodes 210, 220, 230. The shortest distance between the two adjacent nanowells is larger than L1 when some nanowells in the biosensor 200 do not contain any biomarker.

[0055] In some embodiments, both L2 and L2’ are longer than a longest distance between the two adjacent nanowells containing the same biomarker on any one of the working electrodes 210, 220, 230. The longest distance between the two adjacent nanowells is larger than L1 when some nanowells in the biosensor 200 do not contain any biomarker.

[0056] In some embodiments, different distances are between different pairs of adjacent nanowells on the working electrodes 210, 220, 230. For example, some of the distances are larger than L1, while some of the distances are smaller than L1. In some embodiments, a shortest distance between any two working electrodes in a sensor (e.g. a minimum of L2 and L2’ in the example of FIG. 2) is longer than a shortest distance between all possible two adjacent nanowells containing the same biomarker on any one of the working electrodes 210, 220, 230. In other embodiments, the shortest distance between any two working electrodes is longer than a longest distance between all possible two adjacent nanowells containing the same biomarker on any one of the working electrodes 210, 220, 230.Attorney Docket No.132414-5006-WO

[0057] As shown in FIG.2, the biosensor 200 further includes a first connection electrode 201 physically coupled to the first working electrode 210, a second connection electrode 202 physically coupled to the second working electrode 220, and a third connection electrode 203 physically coupled to the third working electrode 230. In some embodiments, a shortest distance between any two adjacent connection electrodes is larger than zero. For example, the distance L5 between the first connection electrode 201 and the second connection electrode 202 is larger than zero. In some embodiments, all of the connection electrodes 201, 202, 203 are electrically isolated from each other, and all of the working electrodes 210, 220, 230 are also electrically isolated from each other.

[0058] As shown in FIG.2, the biosensor 200 further includes a counter electrode 208 shared by the first working electrode 210, the second working electrode 220 and the third working electrode 230. In some embodiments, the first working electrode 210, the second working electrode 220 and the third working electrode 230 have a first constant distance L4 to the counter electrode 208. In some embodiments, L4 is larger than zero. In some embodiments, the counter electrode 208 is shared by all working electrodes in the biosensor 200, and all working electrodes in the biosensor 200 have the first constant distance to the counter electrode 208.

[0059] As shown in FIG.2, the biosensor 200 further includes a reference electrode 207 shared by the first working electrode 210, the second working electrode 220 and the third working electrode 230. In some embodiments, the first working electrode 210, the second working electrode 220 and the third working electrode 230 have a second constant distance L3 to the reference electrode 207. In some embodiments, L3 is larger than zero. In some embodiments, the reference electrode 207 is shared by all working electrodes in the biosensor 200, and all working electrodes in the biosensor 200 have the second constant distance to the reference electrode 207.

[0060] In some embodiments (as shown in FIG. 2), L3 is shorter than L4. In some embodiments, L3 is longer than L4. In some embodiments (as shown in FIG.2), the reference electrode 207 is disposed between the counter electrode 208 and the working electrodes 210, 220, 230. In some embodiments, the counter electrode 208 is disposed between the reference electrode 207 and the working electrodes 210, 220, 230.Attorney Docket No.132414-5006-WO

[0061] In some embodiments, the reference electrode 207, the counter electrode 208 and the working electrodes 210, 220, 230 are submerged in a solution of electrolyte such that when an electrical excitation is applied to the biosensor 200, the electrical excitation causes chemical responses (e.g., oxidation and / or reduction reactions) that can be detected and analyzed by an electronic device or processor. More specifically, when a current is applied to flow between a working electrode (e.g. one of the working electrodes 210, 220, 230) and the counter electrode 208, electric potential of the working electrode relative to the reference electrode 207 can be controlled by a potentiostat. In this instance, the electric potential between the working electrode and the reference electrode 207 can be measured accurately, irrespective of electric current resulting from electrode reaction. A person skilled in the art will appreciate that other alternative electrochemical measurement methods may also be adopted to the present disclosure, and thus are within the scope of the present disclosure.

[0062] FIG.3 shows a top view of another biosensor 300 including multiple working electrodes, a counter electrode, and a reference electrode, in accordance with some embodiments. While FIG.3 shows an electrode portion including three working electrodes of the biosensor 300, the biosensor 300 may include any number of working electrodes and any number of electrode portions, according to various embodiments of the present disclosure.

[0063] As shown in FIG.3, the biosensor 300 includes a first working electrode 310, a second working electrode 320, a third working electrode 330, a first connection electrode 301 physically coupled to the first working electrode 310, a second connection electrode 302 physically coupled to the second working electrode 320, and a third connection electrode 303 physically coupled to the third working electrode 330. Same as the biosensor 200 in FIG.2, each of the working electrodes 310, 320, 330 has nanowells containing different biomarkers, e.g. for detecting different diseases. As shown in FIG.3, the biosensor 300 further includes a counter electrode 308 shared by the first working electrode 310, the second working electrode 320 and the third working electrode 330, and a reference electrode 307 shared by the first working electrode 310, the second working electrode 320 and the third working electrode 330.

[0064] The dimension relationships between nanowells and electrodes in the biosensor 300 are similar to those relationships in the biosensor 200 in FIG.2, except that the reference electrode 307 and the counter electrode 308 have a zigzag shape, as shown in FIG. 3.Attorney Docket No.132414-5006-WO

[0065] In some embodiments, the first working electrode 310, the second working electrode 320 and the third working electrode 330 have a constant vertical distance L7 to the counter electrode 308, and have a constant horizontal distance L9 to the counter electrode 308. In some embodiments, L7 is equal to L9. In some embodiments, L7 is longer than L9. In some embodiments, L7 is shorter than L9.

[0066] In some embodiments, the first working electrode 310, the second working electrode 320 and the third working electrode 330 have a constant vertical distance L6 to the reference electrode 307, and have a constant horizontal distance L8 to the reference electrode 307. In some embodiments, L6 is equal to L8. In some embodiments, L6 is longer than L8. In some embodiments, L6 is shorter than L8.

[0067] In some embodiments (as shown in FIG.3), L6 is shorter than L7, and L8 is shorter than L9. In some embodiments, L6 is longer than L7, and L8 is longer than L9. In some embodiments (as shown in FIG.3), the reference electrode 307 is disposed between the counter electrode 308 and the working electrodes 310, 320, 330. In some embodiments, the counter electrode 308 is disposed between the reference electrode 307 and the working electrodes 310, 320, 330.

[0068] In some embodiments, any one of the biosensors 100, 200, 300 described above can be used for detecting a plurality of diseases simultaneously. For example, a system may include a sensor being one of the biosensors 100, 200, 300, and a sample to be placed on the sensor for detecting a plurality of diseases. In some embodiments, the system includes a plurality of samples to be placed into nanowells on different working electrodes on the sensor for detecting different diseases, respectively. In some embodiments, after a sample is placed on the sensor, the sensor can detect one or more of the plurality of diseases based on one or more corresponding biomarkers in the nanowells on the sensor.

[0069] FIG.4 is a flow diagram showing an exemplary method 400 for forming a biosensor, in accordance with some embodiments. In some embodiments, the method 400 can be carried out to form any of the biosensors 100, 200, 300 as described above. Beginning at operation 410, a first working electrode and a second working electrode are formed on a substrate. At operation 420, an insulation layer is formed on the first working electrode and the second working electrode. At operation 430, nanowells are formed in the insulating layer based on a first photolithography process. The nanowells include (1) a plurality of first nanowellsAttorney Docket No.132414-5006-WO formed to expose a partial area of the first working electrode and (2) a plurality of second nanowells formed to expose a partial area of the second working electrode. In some embodiments, the working electrodes and the nanowells are formed in a manner such that a shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells formed in the insulating layer.

[0070] Although the methods described above are with reference to the illustrated flowcharts, it will be appreciated that many other ways of performing the acts associated with the methods can be used. For example, the order of some operations can be changed, and some of the operations described may be optional.

[0071] FIGS.5A-5G illustrate cross-sectional views of an exemplary biosensor 500 during various fabrication stages, in accordance with some embodiments of the present disclosure. In some embodiments, the biosensor 500 may be any one of the biosensors 100, 200, 300 described above. In addition, FIGS. 5A through 5G are simplified for a better understanding of the concepts of the present disclosure. For example, although the figures illustrate two working electrodes, it is understood the biosensor may include more than two working electrodes, and may include a number of other components including reference electrode, counter electrode, connection electrodes, etc., which are not shown in FIGS. 5A through 5G, for purposes of clarity of illustration.

[0072] FIG.5A is a cross-sectional view of the biosensor 500-1 including a substrate 530, which is provided at one of the various stages of fabrication, according to some embodiments of the present disclosure. The substrate 530 may be formed of glass, silicon, or another semiconductor material, according to various embodiments.

[0073] FIG.5B is a cross-sectional view of the biosensor 500-2 including a buffer layer 540, which is formed on the substrate 530 at one of the various stages of fabrication, according to some embodiments of the present disclosure. The buffer layer 540 may be formed by epitaxial growth or deposition. According to various embodiments, the buffer layer 540 may include materials like titanium, chromium, and / or alloys of titanium or chromium, and may serve as a buffer to reduce the stress and / or provide enhanced bonding between the substrate 530 and the layer on top of the buffer layer 540.

[0074] FIG.5C is a cross-sectional view of the biosensor 500-3 including an electrode layer 550, which is formed on the buffer layer 540 at one of the various stages of fabrication,Attorney Docket No.132414-5006-WO according to some embodiments of the present disclosure. In various embodiments, the electrode layer 550 may include materials like metal (e.g. Au, Pt, Cu, Pd) or graphite. In some embodiments, the electrode layer 550 may be formed based on a deposition method like physical vapor deposition (PVD) or chemical vapor deposition (CVD).

[0075] FIG.5D is a cross-sectional view of the biosensor 500-4 including a trench 552, which is formed in and through the electrode layer 550 at one of the various stages of fabrication, according to some embodiments of the present disclosure. As shown in FIG.5D, the trench 552 divides the electrode layer 550 into two electrode portions a first working electrode 510 and a second working electrode 520 that are separated from each other.

[0076] That is, the electrode layer 550 is patterned to form the working electrodes 510, 520 shown in FIG.5D, based on a photolithography process. In some embodiments, the patterning of the electrode layer 550 includes, e.g., (i) forming a masking layer (e.g., photoresist, etc.) over the electrode layer 550, the masking layer including openings over the portions of the electrode layer 550 that are to be removed, e.g. the trench 552, (ii) removing the portions of the electrode layer 550 that are left exposed by the masking layer (e.g., via a wet or dry etch procedure), and (iii) removing the masking layer and cleaning up the patterned electrode layer 550.

[0077] FIG.5E is a cross-sectional view of the biosensor 500-5 including an insulator layer 560, which is formed on the patterned electrode layer 550 at one of the various stages of fabrication, according to some embodiments of the present disclosure. The insulator layer 560 may be formed by epitaxial growth or deposition. In various embodiments, the insulator layer 560 may include materials like silicon nitride (e.g. Si3N4), silicon dioxide (e.g. SiO2), or another inorganic material.

[0078] FIG.5F is a cross-sectional view of the biosensor 500-6 including a plurality of first nanowells 511 and a plurality of second nanowells 521, which are formed in and through the insulator layer 560 at one of the various stages of fabrication, according to some embodiments of the present disclosure. As shown in FIG.5F, the plurality of first nanowells 511 and the plurality of second nanowells 521 are separated from each other by a distance L2, which is also the distance between the first working electrode 510 and the second working electrode 520. Two adjacent or neighboring nanowells in the insulator layer 560 may be separated by a distance L1. In this example, L2 is longer than L1. In some embodiments, a shortest distance between theAttorney Docket No.132414-5006-WO first working electrode 510 and the second working electrode 520 is longer than a shortest distance between two adjacent nanowells formed on a same working electrode in the insulating layer 560. In some embodiments, the shortest distance between the first working electrode 510 and the second working electrode 520 is longer than a longest distance between two adjacent nanowells formed on a same working electrode in the insulating layer 560.

[0079] The insulator layer 560 may be patterned to form the first nanowells 511 and the second nanowells 521 shown in FIG.5F, based on a photolithography process. In some embodiments, the patterning of the insulator layer 560 includes, e.g., (i) forming a masking layer (e.g., photoresist, etc.) over the insulator layer 560, the masking layer including openings over the portions of the insulator layer 560 that are to be removed, e.g. the first nanowells 511, the second nanowells 521, and the portion over the trench 552, (ii) removing the portions of the insulator layer 560 that are left exposed by the masking layer (e.g., via a wet or dry etch procedure), and (iii) removing the masking layer and cleaning up the patterned insulator layer 560 and the exposed portions of the electrode layer 550.

[0080] In some embodiments, the first nanowells 511 are formed according to a first pattern on the first working electrode 510, and the second nanowells 521 are formed according to the first pattern on the second working electrode 520. In some embodiments, the first nanowells 511 are formed according to a first pattern on the first working electrode 510, the second nanowells 521 are formed according to a second pattern on the second working electrode 520, and the second pattern is different from the first pattern.

[0081] As shown in FIG.5F, after the first nanowells 511 and the second nanowells 521 are formed, partial areas of the upper surfaces of the first working electrode 510 and the second working electrode 520 are exposed. In some embodiments, the exposed partial areas of the first working electrode 510 and the second working electrode 520 are cleaned, e.g. using a sulfuric acid solution, to remove impurities. In some embodiments, the first nanowells 511 and the second nanowells 521 are cleaned by etching.

[0082] FIG.5G is a cross-sectional view of the biosensor 500-7 including a first biomarker 565 and a second biomarker 567, which are filled into the first nanowells 511 and the second nanowells 521, respectively, at one of the various stages of fabrication, according to some embodiments of the present disclosure. In some embodiments, the bottom surfaces of the first nanowells 511 may include the first biomarker 565, which may be analytes probes or probeAttorney Docket No.132414-5006-WO molecules that are capable of binding with specific analytes associated with a first disease. For example, as shown in FIG.5G, analytes probes (e.g. biotinylated antibodies) 565 specific for an analyte may be immobilized to the bottom surface of a first nanowell 511 by using an intermediary binding molecule 564 such as avidin or streptavidin, or another methods of immobilizing. Similarly, the bottom surfaces of the second nanowells 521 may include the second biomarker 567, which may be analytes probes or probe molecules that are capable of binding with specific analytes associated with a second disease. As shown in FIG.5G, analytes probes (e.g. biotinylated antibodies) 567 specific for an analyte may be immobilized to the bottom surface of a second nanowell 521 by using an intermediary binding molecule 566 such as avidin or streptavidin, or another methods of immobilizing. While the first disease is different from the second disease, the first biomarker 565 and the second biomarker 567 are different from each other. The binding molecule 564 and the binding molecule 566 may or may not be the same to each other.

[0083] In some embodiments, as shown in FIG.5G, not all nanowells are filled with biomarkers. In some embodiments, at least two of the first nanowells 511 are filled with the first biomarker 565 for detecting a first disease, and at least two of the second nanowells 521 are filled with the second biomarker 567 for detecting a second disease.

[0084] In some embodiments, the fabrication of the biosensor 500 may further include forming a first connection electrode physically coupled to the first working electrode 510 and forming a second connection electrode physically coupled to the second working electrode 520. In some embodiments, a shortest distance between the first connection electrode and the second connection electrode is larger than zero.

[0085] In some embodiments, the fabrication of the biosensor 500 may further include forming a counter electrode shared by the first working electrode 510 and the second working electrode 520. In some embodiments, the first working electrode 510 and the second working electrode 520 have a first constant distance to the counter electrode, where the first constant distance is larger than zero. In some embodiments, the fabrication of the biosensor 500 may further include forming a reference electrode shared by the first working electrode 510 and the second working electrode 520. In some embodiments, the first working electrode 510 and the second working electrode 520 have a second constant distance to the reference electrode, whereAttorney Docket No.132414-5006-WO the second constant distance is larger than zero. The second constant distance may be longer than or shorter than the first constant distance, according to various embodiments.

[0086] In some embodiments, the fabrication of the biosensor 500 may further include forming one or more microfluidic lane(s) for moving a sample to the first nanowells 511 and / or the second nanowells 521. In some embodiments, the one or more microfluidic lane(s) is configured to process the sample as the sample flows through the one or more microfluidic lane(s) to the first nanowells 511 and / or the second nanowells 521.

[0087] In some embodiments, the fabrication of the biosensor 500 may further include forming a third working electrode in the electrode layer 550, forming, in the insulating layer 560, a plurality of third nanowells to expose a partial area of the third working electrode, and filling at least two of the third nanowells with a third biomarker for detecting a third disease. In some embodiments, a shortest distance between any two working electrodes in the biosensor 500 is longer than a shortest distance between two adjacent nanowells formed on a same working electrode in the insulating layer 560. In some embodiments, the shortest distance between any two working electrodes in the biosensor 500 is longer than a longest distance between two adjacent nanowells formed on a same working electrode in the insulating layer 560. In some embodiments, all working electrodes in the biosensor 500 have a same number of nanowells. In some embodiments, at least two of the working electrodes in the biosensor 500 have different numbers of nanowells.

[0088] The terminology used in the description of the various described implementations herein is for the purpose of describing particular implementations only and is not intended to be limiting. As used in the description of the various described implementations and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “includes,” “including,” “comprises,” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. Additionally, it will be understood that, although the terms “first,” “second,” etc. mayAttorney Docket No.132414-5006-WO be used herein to describe various elements should not be limited by these terms. These terms are only used to distinguish one element from another.

[0089] As used herein, the term “if” is, optionally, construed to mean “when” or “upon” or “in response to determining” or “in response to detecting” or “in accordance with a determination that,” depending on the context. Similarly, the phrase “if it is determined” or “if [a stated condition or event] is detect” is, optionally, construed to mean “upon determining” or “in response to determining” or “upon detecting [the stated condition or event]” or “in response to detecting [the stated condition or event]” or “in accordance with a determination that [a stated condition or event] is detected,” depending on the context.

[0090] The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions are not intended to be exhaustive or to limit the claims to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain principles of operation and practical applications, to thereby enable others skilled in the art.

[0091] Although various drawings illustrate a number of logical stages in a particular order, stages that are not order dependent may be reordered and other stages may be combined or broken out. While some reordering or other groupings are specifically mentioned, others will be obvious to those of ordinary skill in the art, so the ordering and groupings presented herein are not an exhaustive list of alternatives. Moreover, it should be recognized that the stages can be implemented in hardware, firmware, software, or any combination thereof.

Claims

Attorney Docket No.132414-5006-WO What is claimed is:

1. A sensor, comprising: a plurality of first nanowells formed on a first working electrode; a plurality of second nanowells formed on a second working electrode; a first biomarker for detecting a first disease, the first biomarker being immobilized in at least two of the first nanowells; and a second biomarker for detecting a second disease, the second biomarker being immobilized in at least two of the second nanowells, wherein a shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells on the first or second working electrode, each of the two adjacent nanowells containing the first or second biomarker.

2. The sensor according to claim 1, wherein: the shortest distance between the first working electrode and the second working electrode is longer than a longest distance between two adjacent nanowells on the first or second working electrode.

3. The sensor according to claim 1 or 2, further comprising: a first connection electrode physically coupled to the first working electrode; and a second connection electrode physically coupled to the second working electrode.

4. The sensor according to claim 3, wherein: a shortest distance between the first connection electrode and the second connection electrode is larger than zero.

5. The sensor according to any of claims 1-4, further comprising: a counter electrode shared by said first and second working electrodes, wherein: said first and second working electrodes have a first constant distance to the counter electrode, and the first constant distance is larger than zero.Attorney Docket No.132414-5006-WO 6. The sensor according to claim 5, wherein: the counter electrode is shared by all working electrodes in the sensor; and all working electrodes in the sensor have the first constant distance to the counter electrode.

7. The sensor according to claim 5 or 6, further comprising: a reference electrode shared by said first and second working electrodes, wherein: said first and second working electrodes have a second constant distance to the reference electrode, and the second constant distance is larger than zero.

8. The sensor according to claim 7, wherein: the reference electrode is shared by all working electrodes in the sensor; and all working electrodes in the sensor have the second constant distance to the reference electrode.

9. The sensor according to claim 7 or 8, wherein: the second constant distance is longer than the first constant distance.

10. The sensor according to claim 7 or 8, wherein: the second constant distance is shorter than the first constant distance.

11. The sensor according to claim 7 or 8, wherein: the reference electrode is disposed between the counter electrode and said first and second working electrodes.

12. The sensor according to claim 7 or 8, wherein: the counter electrode is disposed between the reference electrode and said first and second working electrodes.

13. The sensor according to any one of claims 1-12, further comprising:Attorney Docket No.132414-5006-WO one or more microfluidic lane(s) configured to move a sample to the first nanowells and / or the second nanowells.

14. The sensor according to claim 13, wherein the one or more microfluidic lane(s) is configured to process the sample as the sample flows through the one or more microfluidic lane(s) to the first nanowells and / or the second nanowells.

15. The sensor according to any of claims 1-14, further comprising: a plurality of third nanowells formed on a third working electrode; and a third biomarker for detecting a third disease, the third biomarker being immobilized in at least two of the third nanowells, wherein a shortest distance between any two working electrodes in the sensor is longer than a shortest distance between two adjacent nanowells containing a same biomarker on any one of the working electrodes in the sensor.

16. The sensor according to claim 15, wherein: the shortest distance between any two working electrodes in the sensor is longer than a longest distance between two adjacent nanowells containing the same biomarker on any one of the working electrodes in the sensor.

17. The sensor according to any one of claims 1-16, wherein: the first and second working electrodes have a same number of nanowells.

18. The sensor according to any one of claims 1-16, wherein: the first and second working electrodes have different numbers of nanowells.

19. A method of applying a sensor of any one of claims 1-18 for detecting a plurality of diseases.

20. A system comprising the sensor of any one of claims 1-18.Attorney Docket No.132414-5006-WO 21. The system according to claim 20, further comprising a sample to be placed on the sensor for detecting a plurality of diseases.

22. The system according to claim 20, further comprising a plurality of samples to be placed into nanowells on different working electrodes on the sensor for detecting different diseases, respectively.

23. A method of detecting a plurality of diseases, comprising: placing a sample on the sensor of any one of claims 1-16; and detecting one or more of the plurality of diseases based on one or more corresponding biomarkers in the nanowells on the sensor.

24. A method for forming a sensor, comprising: forming, on a substrate, a first working electrode and a second working electrode; forming an insulation layer on the first working electrode and the second working electrode; and forming, in the insulating layer based on a first photolithography process, (1) a plurality of first nanowells to expose a partial area of the first working electrode and (2) a plurality of second nanowells to expose a partial area of the second working electrode, wherein a shortest distance between the first working electrode and the second working electrode is longer than a shortest distance between two adjacent nanowells formed on a same working electrode in the insulating layer.

25. The method according to claim 24, further comprising: filling at least two of the first nanowells with a first biomarker for detecting a first disease; and filling at least two of the second nanowells with a second biomarker for detecting a second disease.

26. The method according to claim 24 or 25, further comprising: forming a buffer layer on the substrate; andAttorney Docket No.132414-5006-WO forming a metal layer on the buffer layer based on a second photolithography process, wherein the first working electrode and the second working electrode are formed in the metal layer.

27. The method according to any one of claims 24-26, further comprising: removing impurities from the exposed partial areas of the first working electrode and the second working electrode using a sulfuric acid solution; and etching the plurality of first nanowells and the plurality of second nanowells.

28. The method according to any one of claims 24-27, wherein: the insulation layer is formed with an inorganic material.

29. The method according to any one of claims 24-28, wherein: the plurality of first nanowells are formed according to a first pattern on the first working electrode; and the plurality of second nanowells are formed according to the first pattern on the second working electrode.

30. The method according to any one of claims 24-28, wherein: the plurality of first nanowells are formed according to a first pattern on the first working electrode; and the plurality of second nanowells are formed according to a second pattern on the second working electrode, wherein the second pattern is different from the first pattern.

31. The method according to any one of claims 24-30, wherein: the shortest distance between the first working electrode and the second working electrode is longer than a longest distance between two adjacent nanowells formed on a same working electrode in the insulating layer. The method according to any one of claims 24-31, further comprising:Attorney Docket No.132414-5006-WO forming a first connection electrode physically coupled to the first working electrode; and forming a second connection electrode physically coupled to the second working electrode.

33. The method according to claim 32, wherein: a shortest distance between the first connection electrode and the second connection electrode is larger than zero.

34. The method according to any one of claims 24-33, further comprising: forming a counter electrode shared by said first and second working electrodes, wherein: said first and second working electrodes have a first constant distance to the counter electrode, and the first constant distance is larger than zero.

35. The method according to claim 34, wherein: the counter electrode is shared by all working electrodes in the sensor; and all working electrodes in the sensor have the first constant distance to the counter electrode.

36. The method according to claim 34 or 35, further comprising: forming a reference electrode shared by said first and second working electrodes, wherein: said first and second working electrodes have a second constant distance to the reference electrode, and the second constant distance is larger than zero.

37. The method according to claim 36, wherein: the reference electrode is shared by all working electrodes in the sensor; and all working electrodes in the sensor have the second constant distance to the reference electrode.Attorney Docket No.132414-5006-WO 38. The method according to claim 36 or 37, wherein: the second constant distance is longer than the first constant distance.

39. The method according to claim 36 or 37, wherein: the second constant distance is shorter than the first constant distance.

40. The method according to claim 36 or 37, wherein: the reference electrode is formed between the counter electrode and said first and second working electrodes.

41. The method according to claim 36 or 37, wherein: the counter electrode is formed between the reference electrode and said first and second working electrodes.

42. The method according to any one of claims 24-41, further comprising: forming one or more microfluidic lane(s) for moving a sample to the first nanowells and / or the second nanowells.

43. The method according to claim 42, wherein: the one or more microfluidic lane(s) is configured to process the sample as the sample flows through the one or more microfluidic lane(s) to the first nanowells and / or the second nanowells.

44. The method according to any one of claims 24-43, further comprising: forming, on the substrate, a third working electrode; forming, in the insulating layer based on the first photolithography process, a plurality of third nanowells to expose a partial area of the third working electrode; and filling at least two of the third nanowells with a third biomarker for detecting a third disease,Attorney Docket No.132414-5006-WO wherein a shortest distance between any two working electrodes in the sensor is longer than a shortest distance between two adjacent nanowells formed on a same working electrode in the insulating layer.

45. The method according to claim 44, wherein: the shortest distance between any two working electrodes in the sensor is longer than a longest distance between two adjacent nanowells formed on a same working electrode in the insulating layer.

46. The method according to any one of claims 24-45, wherein: the first and second working electrodes have a same number of nanowells.

47. The method according to any one of claims 24-45, wherein: the first and second working electrodes have different numbers of nanowells.

48. A sensor formed according to a method of any one of claims 24-47.

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