Simultaneous detection of multiple alzheimer biomarkers

Abstract

This application is directed to detecting presence or quantity of at least two different molecules. A sensor includes a plurality of chambers, a nanowell array electrode, and a circuit board platform. The sensor separates, cleaves, filters, and detects the at least two different molecules selected from the group consisting of Aβ peptides and tau-protein from a sample in the plurality of nanowells. In some embodiments, the presence or quantity of at least three different Aβ peptides is detected, and an effective amount of a therapeutic compound is administrated to treat Alzheimer in a subject in need thereof.

Description

Attorney Docket No.132414-5009-WO Simultaneous Detection Of Multiple Alzheimer Biomarkers TECHNICAL FIELD

[0001] The disclosed embodiments relate generally to an electronic device, and more particularly, to methods and systems for simultaneous detection of multiple biological molecules. 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. However, biosensors must be highly sensitive and selective in performance and competitive in cost of manufacturing before they can be adapted widely for a broad range of applications. A conventional biosensor can diagnose a particular disease by targeting at only one disease or antigen, requiring use of different biosensors to diagnose multiple diseases. A sample undergoes processing, such as incubation and separation prior to being injected into the biosensor. While it takes an extended duration time to use a biosensor, the biosensors are for only one-time use and reuse of the biosensors is impossible. Additionally, the resolution or measurement limit of the conventional biosensors are at a ng / mL level, which requires that a concentration of a particular target material must exceed this level for proper measurement. Accordingly, there is a need for high-performance and low-cost devices, and methods and systems thereof, for detecting the presence or quantity of biological molecules. SUMMARY

[0003] Improved devices, and methods and systems thereof, for detecting the presence or quantity of at least two different molecules are provided herein. In yet another aspect, a sensor is applied for detecting presence or quantity of at least two different molecules in a sample. The sensor includes a plurality of chambers including a first chamber configured to separate and / or cleave, a second chamber configured to filter, and a third chamber configured to detect. TheAttorney Docket No.132414-5009-WO sensor further includes a nanowell array electrode and a circuit board platform. The at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein. By these means, this application provides a partially or entirely integrated medical device that collects, separates, purifies, and detects the sample, thereby reducing cost of manufacturing and providing high performance (e.g., providing a high sensitivity at a fast rate using a smaller amount of sample).

[0004] Potential future applications for such an electrochemical biosensor 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 will 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.

[0005] In one aspect, a method is implemented to detect presence or quantity of at least two different molecules. The method includes separating, cleaving, filtering, and detecting the at least two different molecules selected from the group consisting of Aȕ peptides and tau-protein from a sample in a plurality of nanowells on a sensor.

[0006] In another aspect, a method is implemented to treat Alzheimer in a subject in need thereof. The method includes detecting the presence or quantity of at least three different Aȕ peptides, and administering an effective amount of a therapeutic compound to the subject. Detecting the presence or quantity of at least three different Aȕ peptides further includes separating, cleaving, filtering, and detecting the at least three different molecules selected fromAttorney Docket No.132414-5009-WO the group consisting of Aȕ peptides and tau-protein from a sample in a plurality of nanowells on a sensor.

[0007] In one aspect, a system includes a sensor, a processor, and a display. The sensor includes a plurality of chambers configured to provide a plurality of functions to detect presence or quantity of at least two different molecules in a sample. The plurality of chambers includes a first chamber configured to provide separating and / or cleaving functions, a second chamber configured to provide filtering function, and a third chamber configured provide detecting function. The processor is configured to individually control each of the plurality of chambers. The display is configured to display visualized data including data representing the presence or quantity of the at least two different molecules in the sample. The at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein.

[0008] In yet another aspect, an electronic device includes a substrate, a working electrode (e.g., included in a nanowell array electrode), a plurality of nanowells, and a detection circuit. The working electrode includes a sensor portion and a detection portion that extends from the sensor portion on the substrate. The plurality of nanowells is coupled to the sensor portion of the working electrode, and is configured to hold a host sample and receive a microfluidic sample including at least two different molecules selected from the group consisting of Aȕ peptides and tau-protein. The detection circuit is coupled to the detection portion of the working electrode, and configured to detect an output signal that indicates a presence or a quantity of the at least two different molecules.

[0009] In some embodiments, the working electrode includes a first working electrode, and the plurality of nanowells includes first nanowells, the electronic device further includes a second working electrode, a plurality of second nanowells, a second host sample, and a second microfluidic sample separated from the microfluid sample received by the first nanowells. Two different molecules from the at least two different molecules are detected from the first and second working electrodes, respectively. Further, in some embodiments, the detection circuit is configured to detect the two different molecules from the first and second working electrodes in a time-multiplexed manner.

[0010] In some embodiments, the electronic device further includes an insulating structure formed on the working electrode. The plurality of nanowells are opened through the insulating structure to expose a subset of a surface of the working electrode.Attorney Docket No.132414-5009-WO

[0011] In some embodiments, the electronic device further includes a reference electrode and a counter electrode. The electronic device is configured to detect the output signal between the reference electrode and the working electrode and determine the presence or quantity of at least two different molecules based on antibody binding to the at least two different molecules.

[0012] In some embodiments, the host sample is immobilized in the plurality of nanowells and includes an antibody. Further, in some embodiments, at least a subset of the plurality of nanowells has a first structure configured to facilitate binding with the antibody, causing the antibody to be bound with a first orientation.

[0013] In some embodiments, the electronic device further includes one or more of: a sample inlet, a sample preparation module, a sample outlet, and a sample detection module mechanically coupled to the sample outlet. The sample detection module further includes the working electrode and the plurality of nanowells. Further, in some embodiments, the electronic device further includes one or more of: a first inlet, a second inlet, a normalization module, a first outlet, and a second outlet. The sample preparation module further includes a sample separation module and a filtration module.

[0014] 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

[0015] For a better understanding of the various described embodiments, reference should be made to the Description of Embodiments below, in conjunction with the follow drawings in which like reference numerals refer to corresponding parts throughout the figures.

[0016] Figure 1A illustrates an example self monitoring biosensor system including a nanowell array, in accordance with some embodiments.

[0017] Figure 1B illustrates an example data processing system for simultaneous and continuous detection of electrochemical signals using an electrode array including six nanowell biosensors, in accordance with some embodiments.Attorney Docket No.132414-5009-WO

[0018] Figure 2 is a schematic diagram of an example nanowell sensor-based microfluidic device that implements sample pretreatment (e.g., anchoring, normalization, cleavage, and filtration) and electrochemical detection, in accordance with some embodiments.

[0019] Figure 3 is a structural diagram of an example 5anowell sensor-based microfluidic device for managing sample flows and chamber compositions, in accordance with some embodiments.

[0020] Figure 4A is a list of parameters of example nanowell array electrodes having different well sizes, in accordance with some embodiments.

[0021] Figure 4B is an example quantitative scoring table for determining quantitative scores of AD biomarkers based on cut-off ranges of AD biomarkers, in accordance with some embodiments.

[0022] Figure 5 is a flow diagram of an example method for detecting presence or quantity of at least two different molecules, in accordance with some embodiments. DESCRIPTION OF EMBODIMENTS

[0023] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerous 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.

[0024] Examples of different types of biosensors include electrochemical biosensors, nano-cantilever biosensors, and micro- or nano-electromechanical systems (MEMS / NEMS). In various embodiments of this application, 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 isAttorney Docket No.132414-5009-WO bound to the surface of an electrode. Electrochemical biosensors’ signals can be directly detected by an electronic device for analysis, allowing for fast diagnosis.

[0025] The disclosure provides, in some embodiments, an engineered multiplex nanowell device that applies urinary cleared nanoprobes for sensitive Aȕ detection. In some embodiments, the multiplex nanowell device includes a microfluidic device having a nanowell array, and integrates at least three electrochemical nanowell sensors used for simultaneous and specific detection of at least three different Aȕ peptides (e.g., Aȕ-38, Aȕ-40, and Aȕ-42). In some embodiments, the multiplex nanowell device includes at least 4 functional chambers applied for normalization, separation and cleavage, filtration, and detection, respectively. In some embodiments, each chamber is individually controlled for performing a distinct task without interference from any other chambers. In some embodiments, the microfluidic device is fully automated, e.g., programmed to direct flows at pre-set time points. In some embodiments, each of the nanowell sensors integrated in the multiplex nanowell device is customized with different sizes and densities to improve sensitivity, signal-to-noise ratio (SNR), and specificity of the biosensors. In some embodiments, the multiplex nanowell device includes a nanowell array electrode that is configured to provide a reliable platform for monitoring AD biomarkers. In some embodiments, the sensitivity is amplified by simultaneous investigation of multiplex biomarkers. In some embodiments, the multiplex nanowell device corresponds to a quantitative scoring system applied for clinical validations.

[0026] In some embodiments, the disclosure provides the design of a multifunctional electrochemical nanowell-based microfluidic device. In some embodiments, such a nanowell- based microfluidic device facilitates sample (e.g., urine) analysis by integrating sample treatment (e.g., anchoring, normalization, cleavage, filtration, and detection) with immunoassays, thereby enabling quick measurement of Aȕ peptide concentrations. Specifically, in some embodiments, the microfluidic device includes 3 inlets for sequential injections of different liquids for the sample (e.g., urine), a buffer solution, and a pH solution (inlets 1-3, respectively), 4 chambers for electrode integration (chambers 4-7), and 2 outlets for directing the sampling volume back to the bioreactor (outlets 8-10), and valves 1-8 control liquid flows solely in each microfluidic channel.

[0027] In some embodiments, the disclosure provides a nanowell sensor-based point of care (POC) diagnostic device that operates in a portable analytical instrument and enables rapid quantification of a target biomarker (i.e., an AD disease biomarker) in a sample (e.g., urineAttorney Docket No.132414-5009-WO sample) . The POC diagnostic device enables highly sensitive, specific, multiplex, rapid, real- time, and cost-effective detection of AD, for early AD diagnostic assessments. In some embodiments, the disclosure provides simultaneous detection of multiple AD biomarkers by way of a quantitative scoring platform. In some embodiments, the sensitivity and specificity of nanowell biosensors is improved to achieve digital monitoring for molecular diagnostics by optimizing: 1) immobilization of antibodies on the specific position, 2) targetability by minimizing nonspecific adsorption, and 3) quantitative scoring of meaningful signals.

[0028] In one aspect, the disclosure provides a system. In some embodiments, the system includes a sensor 101. As used herein with reference to the present disclosure, “nanowell sensor” and “nanowell biosensor” are used interchangeably.

[0029] Figure 1A illustrates an example self monitoring biosensor system 100 including a nanowell array, in accordance with some embodiments. In some embodiments, the system 100 includes: a sensor 101 including a plurality of chambers configured to provide a plurality of functions to detect presence or quantity of a target biomarker 108 (i.e., the at least two different molecules, e.g., Aȕ peptides and / or tau-protein) in a sample, the plurality of chambers includes: a first chamber configured to provide separating and / or cleaving functions, a second chamber configured to provide filtering function, and a third chamber configured provide detecting function; a processor configured to individually control each of the plurality of chambers; and a display configured to display visualized data including data representing the presence or quantity of the at least two different molecules in the sample; where the at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein.

[0030] In some embodiments, the at least two different molecules are bound to one or more particles in the sample. In some embodiments, the one or more particles in the sample are one or more nanoprobes. In some embodiments, the one or more particles in the sample are one or more antibodies. In some embodiments, the one or more antibodies include an anti-Tau antibody. In some embodiments, the one or more antibodies include an anti-Aȕ antibody, for example, an anti-Aȕ38, anti-Aȕ40, and / or anti-Aȕ42 monoclonal antibody.

[0031] A system 100 provided herein includes a nanowell sensor 101 including a plurality of chambers configured to provide a plurality of functions to detect the presence or quantity of a target biomarker 108 (i.e., the at least two different molecules, e.g., Aȕ peptides and / or tau-protein) in a sample.Attorney Docket No.132414-5009-WO

[0032] The nanowell sensor 101 may include a working electrode (WE) 102, a counter electrode (CE) 104, and / or a reference electrode (RE) 103, in accordance with some embodiments. In some embodiments, the nanowell sensor 101 includes at least 2 electrodes. In some embodiments, the nanowell sensor 101 includes 3 electrodes.

[0033] In some embodiments, the nanowell sensor 101 includes the reference electrode 103. In some embodiments, the reference electrode 103 includes a transition metal. The reference electrode 103 may include a transition metal. In some embodiments, the reference electrode 103 includes Ag.

[0034] In some embodiments, the nanowell sensor 101 includes the counter electrode 104. In some embodiments, the counter electrode 104 includes a transition metal including, but not limited to, Rh, Pt, Pd and / or Au. In some embodiments, the counter electrode 104 includes Pt.

[0035] In some embodiments, the nanowell sensor 101 includes a substrate 111. In some embodiments, the working electrode 102, the reference electrode 103, and the counter electrode 104 are on the substrate 111. In some embodiments, the nanowell array electrode 105 is formed on the substrate 111. The substrate 111 may include any substrate suitable for the application including, but not limited to, a polymer, glass, and silicon. In some embodiments, the substrate 111 includes one or more of polypropylene (PP), polystyrene (PS), polydimethylsiloxane (PDMS), polyethylene (PE), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyimide (PI), polyetheretherketone (PEEK), polyether sulfone (PES), polycarbonate (PC), polyester, and polyamide. In some embodiments, the substrate 111 includes polymethylmethacrylate (PMMA). In some embodiments, the substrate 111 is one of glass and silicon.

[0036] The nanowell sensor 101 includes a nanowell array electrode 105, in accordance with some embodiments. As used herein with reference to the present disclosure, “nanowell array” and “nanowell array electrode” are used interchangeably.

[0037] In some embodiments, the nanowell array electrode 105 includes the working electrode 102. In some embodiments, the working electrode 102 includes a transition metal. The working electrode 102 may include a transition metal including, but not limited to, Au, Ti, W, Pt, and / or Ir. In some embodiments, the working electrode 102 includes Au.Attorney Docket No.132414-5009-WO

[0038] In some embodiments, the nanowell array electrode 105 includes an insulating structure 106. In some embodiments, the insulating structure 106 includes a polymer layer. In some embodiments, the insulating structure 106 includes an insulation layer. In some embodiments, the insulating structure 106 includes a polymer layer and an insulation layer.

[0039] The nanowell array electrode 105 may include a polymer layer, in accordance with some embodiments. In some embodiments, the polymer layer is applied to one side of the working electrode. In some embodiments, the polymer layer has a thickness of from about 50 to about 300 nm, for example, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm. In some embodiments, the polymer layer has a thickness of from about 50 to about 300 nm, from about 60 to about 300 nm, from about 70 to about 300 nm, from about 80 to about 300 nm, from about 90 to about 300 nm, from about 100 to about 300 nm, from about 110 to about 300 nm, from about 120 to about 300 nm, from about 130 to about 300 nm, from about 140 to about 300 nm, from about 150 to about 300 nm, from about 50 to about 200 nm, from about 60 to about 200 nm, from about 70 to about 200 nm, from about 80 to about 200 nm, from about 90 to about 200 nm, from about 100 to about 200 nm, from about 110 to about 200 nm, from about 120 to about 200 nm, from about 130 to about 200 nm, from about 140 to about 200 nm, from about 150 to about 200 nm, from about 50 to about 250 nm, from about 50 to about 200 nm, from about 50 to about 190 nm, from about 50 to about 180 nm, from about 50 to about 170 nm, from about 50 to about 160 nm, from about 50 to about 150 nm, from about 150 to about 200 nm, from about 140 to about 210 nm, from about 130 to about 220 nm, from about 120 to about 230 nm, from about 110 to about 240 nm, or from about 100 to about 250 nm. In some embodiments, the polymer layer has a thickness of from about 150 to about 200 nm.

[0040] In some embodiments, the polymer layer is a negative polymer layer. In some embodiments, the polymer layer is a positive polymer layer. In some embodiments, the polymer layer includes poly(methyl methacrylate) (PMMA), acrylate / styrene copolymers, poly(hydroxystyrene), poly(methyl glutarimide) (PMGI), a blend of phenol formaldehyde resin (novolac resin) with diazonaphthoquinone resin (DNQ), partially protected phenols typified by tradename resists KRS™ and APEX™, acrylate / methacrylate copolymers, hybrid aliphatic / phenolic structures typified by trade-name ESCAP™, epoxy-based resins (e.g., SU-8), and / or a combination thereof. In some embodiments, the polymer layer includes anAttorney Docket No.132414-5009-WO acrylate / styrene copolymer, such as a copolymer of Į-chloromethacrylate and Į-methylstyrene (e.g., ZEP520). In some embodiments, the polymer layer includes ZEP520.

[0041] The nanowell array electrode 105 may include an insulation layer, in accordance with some embodiments. The insulation layer may be applied to one side of the working electrode. In some embodiments, the insulation layer has a thickness of from about 50 to about 300 nm, for example, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm. In some embodiments, the insulation layer has a thickness of from about 50 to about 300 nm, from about 60 to about 300 nm, from about 70 to about 300 nm, from about 80 to about 300 nm, from about 90 to about 300 nm, from about 100 to about 300 nm, from about 110 to about 300 nm, from about 120 to about 300 nm, from about 130 to about 300 nm, from about 140 to about 300 nm, from about 150 to about 300 nm, from about 50 to about 200 nm, from about 60 to about 200 nm, from about 70 to about 200 nm, from about 80 to about 200 nm, from about 90 to about 200 nm, from about 100 to about 200 nm, from about 110 to about 200 nm, from about 120 to about 200 nm, from about 130 to about 200 nm, from about 140 to about 200 nm, from about 150 to about 200 nm, from about 50 to about 250 nm, from about 50 to about 200 nm, from about 50 to about 190 nm, from about 50 to about 180 nm, from about 50 to about 170 nm, from about 50 to about 160 nm, from about 50 to about 150 nm, from about 150 to about 200 nm, from about 140 to about 210 nm, from about 130 to about 220 nm, from about 120 to about 230 nm, from about 110 to about 240 nm, or from about 100 to about 250 nm. In some embodiments, the insulation layer has a thickness of from about 150 to about 200 nm.

[0042] In some embodiments, the insulation layer includes an organic compound. In some embodiments, the insulation layer includes an inorganic compound. In some embodiments, the insulation layer includes silicon. In some embodiments, the insulation layer includes an oxide, for example, SiO2, Al2O3, Y2O3, Sc2O3, Ga2O3, In2O3, B2O3, TiO2, SnO2, ZrO2, GeO2, HfO2, Nb2O5, Ta2O5, V2O5, and / or WO3. In some embodiments, the insulation layer includes SiO2.

[0043] In some embodiments, the nanowell sensor 101 includes a plurality of nanowells 107. In some embodiments, the nanowell array electrode 105 includes a plurality of nanowells 107. In some embodiments, the polymer layer includes the plurality of nanowells 107. In someAttorney Docket No.132414-5009-WO embodiments, the insulation layer includes the plurality of nanowells 107. The plurality of nanowells 107 may be fabricated using lithography, for example, electron beam nanolithography.

[0044] In some embodiments, the plurality of nanowells 107 is from about 2 to 100,000 nanowells, for example, about 2, 10, 50, 100, 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 nanowells for each of the at least two different molecules. In some embodiments, the plurality of nanowells 107 is about 40,000 nanowells for each of the at least two different molecules. In some embodiments, the plurality of nanowells 107 is from about 10,000 to 100,000, from about 15,000 to 100,000, from about 20,000 to 100,000, from about 25,000 to 100,000, from about 30,000 to 100,000, from about 35,000 to 100,000, from about 40,000 to 100,000, from about 10,000 to 95,000, from about 10,000 to 90,000, from about 10,000 to 85,000, from about 10,000 to 80,000, from about 10,000 to 75,000, from about 10,000 to 70,000, from about 10,000 to 65,000, from about 10,000 to 60,000, from about 10,000 to 55,000, from about 10,000 to 50,000, from about 10,000 to 45,000, from about 10,000 to 40,000, from about 20,000 to 80,000, from about 20,000 to 60,000, from about 30,000 to 50,000, from about 35,000 to 55,000, or from about 30,000 to 60,000 nanowells for each of the at least two different molecules. In some embodiments, the plurality of nanowells includes from about 10,000 to 100,000 nanowells for each of the at least two different molecules.

[0045] In some embodiments, each nanowell of the plurality of nanowells includes a nanowell depth of from about 10 to 200 nm, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell depth of 50 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell depth of 100 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell depth of from about 10 to 200 nm, from about 20 to 200 nm, from about 30 to 200 nm, from about 40 to 200 nm, from about 50 to 200 nm, from about 10 to 150 nm, from about 20 to 150 nm, from about 30 to 150 nm, from about 40 to 150 nm, from about 50 to 150 nm, from about 10 to 190 nm, from about 10 to 180 nm, from about 10 to 170 nm, from about 10 to 160 nm, from about 10 to 150 nm, from about 50 to 150 nm, from about 10 to 100 nm, from about 10 to 90 nm, from about 10 to 80 nm, from about 10 to 70 nm, from about 10 to 60 nm, from about 10 to 50 nm, from about 20 to 100 nm, from about 30 to 100 nm, from about 40 to 100 nm, from about 50 toAttorney Docket No.132414-5009-WO 100 nm, from about 20 to 80 nm, from about 30 to 70 nm, from about 40 to 60 nm, from about 50 to 200 nm, from about 60 to 200 nm, from about 70 to 200 nm, from about 80 to 200 nm, from about 90 to 200 nm, from about 100 to 200 nm, from about 50 to 190 nm, from about 50 to 180 nm, from about 50 to 170 nm, from about 50 to 160 nm, from about 50 to 150 nm, from about 50 to 140 nm, from about 50 to 130 nm, from about 50 to 120 nm, from about 50 to 110 nm, from about 50 to 100 nm, from about 70 to 130 nm, from about 80 to 120 nm, or from about 90 to 110 nm.

[0046] In some embodiments, each nanowell of the plurality of nanowells includes a nanowell diameter of from about 10 to 200 nm, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell diameter of 50 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell diameter of 90 nm. In some embodiments, each nanowell of the plurality of nanowells includes a nanowell diameter of from about 10 to 200 nm, from about 20 to 200 nm, from about 30 to 200 nm, from about 40 to 200 nm, from about 50 to 200 nm, from about 10 to 150 nm, from about 20 to 150 nm, from about 30 to 150 nm, from about 40 to 150 nm, from about 50 to 150 nm, from about 10 to 190 nm, from about 10 to 180 nm, from about 10 to 170 nm, from about 10 to 160 nm, from about 10 to 150 nm, from about 50 to 150 nm, from about 10 to 100 nm, from about 10 to 90 nm, from about 10 to 80 nm, from about 10 to 70 nm, from about 10 to 60 nm, from about 10 to 50 nm, from about 20 to 100 nm, from about 30 to 100 nm, from about 40 to 100 nm, from about 50 to 100 nm, from about 20 to 80 nm, from about 30 to 70 nm, from about 40 to 60 nm, from about 50 to 150 nm, from about 60 to 150 nm, from about 70 to 150 nm, from about 80 to 150 nm, from about 90 to 150 nm, from about 50 to 190 nm, from about 50 to 180 nm, from about 50 to 170 nm, from about 50 to 160 nm, from about 50 to 150 nm, from about 50 to 140 nm, from about 50 to 130 nm, from about 50 to 120 nm, from about 50 to 110 nm, from about 50 to 100 nm, from about 60 to 120 nm, from about 70 to 110 nm, or from about 80 to 100 nm.

[0047] In some embodiments, the nanowell array electrode 105 includes a total active area of from about 10 to 400 μm2, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 μm2. In some embodiments, the nanowell array electrode 105 includes a total active area of about 80 μm2. In some embodiments, theAttorney Docket No.132414-5009-WO nanowell array electrode 105 includes a total active area of about 250 μm2. In some embodiments, the nanowell array electrode 105 includes a total active area of about 260 μm2. In some embodiments, the nanowell array electrode 105 includes a total active area of about 254 μm2. In some embodiments, the nanowell array electrode 105 includes a total active area of from about 10 to 400 μm2, from about 50 to 400 μm2, from about 75 to 400 μm2, from about 100 to 400 μm2, about 10 to 100 μm2, from about 10 to 150 μm2, from about 10 to 200 μm2, from about 10 to 250 μm2, from about 10 to 300 μm2, from about 50 to 300 μm2, or from about 75 to 300 μm2, or from about 100 to 300 μm2. In some embodiments, the nanowell array electrode 105 includes a total active area of from about 10 to 300 μm2.

[0048] In some embodiments, each nanowell of the plurality of nanowells includes a biomolecule. In some embodiments, each nanowell of the plurality of nanowells includes one or more of an amino acid, a peptide, a polypeptide, and a protein. In some embodiments, the at least two different molecules interact with (e.g., bind to) the one or more of the amino acid, the peptide, the polypeptide, and the protein. In some embodiments, each nanowell of the plurality of nanowells includes an antibody 109. In some embodiments, the at least two different molecules interact with (e.g., bind to) the antibody 109. In some embodiments, the at least two different molecules bind to the antibody 109. In some embodiments, the antibody 109 includes an anti-Tau antibody. In some embodiments, the antibody 109 includes an anti-Aȕ antibody, for example, an anti-Aȕ38, anti-Aȕ40, and / or anti-Aȕ42 monoclonal antibody. In some embodiments, the antibody 109 is immobilized. In some embodiments, the orientation of biomolecules (e.g., antibody 109) is an important factor to achieve high sensitivity and specificity on chip-based sensors. In some embodiments, by using the well-oriented nanowell structure, site-specific immobilization of single or only a few biomolecules (e.g., antibody 109) is achieved. In some embodiments, nonspecific adsorption of peptides is minimized by surface chemical modification of nanowells. In some embodiments, for efficient binding of the target biomarker 108, the immobilized probes (i.e., nanoprobes, e.g., antibody 109) display free target binding regions in the nanowell. In some embodiments, such a well-controlled antibody orientation, therefore, improves binding of biomarkers and sensitivity. In some embodiments, the system 100 and / or the nanowell sensor 101 has improved sensitivity, which is determined by comparing a 50-100 nm nanowell array electrode 105 described herein to conventional microwell array systems. In some embodiments, molecular orientation relative to the targetAttorney Docket No.132414-5009-WO biomarkers can be controlled, which increases the overall sensitivity by diminishing background signals (i.e., high SNR). In some embodiments, the sensitivity and / or SNR of an electrode may vary depending on the sample volume applied to the electrode, the size and / or volume of the electrode chamber may be varied to select the best design after sequential sensitivity tests. In some embodiments, the magnitude of the signal is by amplified by creating a nanowell array (e.g., ca.40,000 wells per biomarker). In some embodiments, the system 100 and / or the nanowell sensor 101 has improved specificity, which is determined by comparing the performance of immobilized Aȕ-specific antibodies and structurally similar molecules (e.g., IgG) in nanowell array electrodes. In some embodiments, electrochemical impedance spectroscopy (EIS) is performed at a potential of about 111 mV and an alternating potential with amplitude of 10 mV at a frequency range from 2 KHz to 50 MHz using a wide range of biomarker concentrations. In some embodiments, Nyquist plots (íZre vs. Zim) are drawn to analyze the impedance results. In some embodiments, the impedance spectra of the antibody-grafted WE is measured to assess the effectiveness of the surface treatment and the difference of the electron- transfer resistance (dRet) is analyzed.

[0049] 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 to 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 individually control each of the plurality of chambers. In some embodiments, the processor is configured to individually control each of the plurality of nanowells.

[0050] In some embodiments, the processor is mounted on a circuit board. In some embodiments, the circuit board platform integrates the plurality of chambers and the nanowell array electrode.Attorney Docket No.132414-5009-WO

[0051] In some embodiments, the sensor and the processor are communicably connected to each other. In some embodiments, the sensor and the processor are implemented as a single device. In some embodiments, the sensor and the processor are implemented as separate devices. In some embodiments, the sensor and the processor may be implemented as a system on a chip (SoC). In some embodiments, the processor is further configured to process detecting result from the sensor to output the visualized data.

[0052] In some embodiments, the system includes a display. The display may be configured to display the visualized data. In some embodiments, the visualized data represents the presence or quantity of the at least two different molecules. In some embodiments, electrochemical detection 110 is related to the visualized data.

[0053] Figure 1B illustrates an example electronic device 150 for simultaneous and continuous detection of electrochemical signals using an electrode array including six nanowell biosensors, in accordance with some embodiments. In some embodiments, the electronic device 150 includes: a substrate 111 and a nanowell array electrode 105 including a sensor portion 152 and a detection portion 153 that extends from the sensor portion 152 on the substrate 111. The nanowell array electrode 105 is also called a working electrode 102 (Figure 1A). A plurality of nanowells 107 is coupled to the sensor portion 152 (Figure 1B) of the working electrode 102, and is configured to hold a host sample and receive a microfluidic sample including a at least two different molecules selected from the group consisting of Aȕ peptides and tau-protein; and a detection circuit 151 coupled to the detection portion 153 of the nanowell array electrode 105, where the detection circuit 151 is configured to detect an output signal that indicates a presence or a quantity of the at least two different molecules.

[0054] In some embodiments of the electronic device 150, the nanowell array electrode 105 includes a first working electrode 102A, and the plurality of nanowells 107 including first nanowells. The electronic device 150 further includes a second nanowell array electrode including a second working electrode 102B, a plurality of second nanowells, a second host sample, a second microfluidic sample separated from the microfluid sample received by the first nanowells. Two molecules (e.g., two different molecules) are detected from the first and second working electrodes 102A and 102B, respectively. In some embodiments, the electronic device 150 further includes a third nanowell array electrode including a third working electrode 102C, a plurality of third nanowells, a third host sample, and a third microfluidic sample separated fromAttorney Docket No.132414-5009-WO the microfluid sample received by the first and second nanowells. Three molecules (e.g., three different molecules) are detected from the first, second, and third working electrodes 102A, 102B, and 102C, respectively. In some embodiments, the electronic device 150 further includes a fourth nanowell array electrode including a fourth working electrode 102D, a plurality of fourth nanowells, a fourth host sample, and a fourth microfluidic sample separated from the microfluid sample received by the first, second, and third nanowells. Four molecules (e.g., four different molecules) are detected from the first, second, third, and fourth working electrodes 102A, 102B, 102C, and 102D, respectively. In some embodiments, the electronic device 150 further includes: a fifth nanowell array electrode including a fifth working electrode 102E, a plurality of fifth nanowells, a fifth host sample, and a fifth microfluidic sample separated from the microfluid sample received by the first, second, third, and fourth nanowells. Five molecules (e.g., five different molecules) are detected from the first, second, third, fourth, and fifth working electrodes 102A, 102B, 102C, 102D, and 102E, respectively. In some embodiments, the electronic device 150 further includes: a sixth nanowell array electrode including a sixth working electrode 102F, a plurality of sixth nanowells, a sixth host sample, and a sixth microfluidic sample separated from the microfluid sample received by the first, second, third, fourth, and fifth nanowells. Xix molecules (e.g., six different molecules) are detected from the first, second, third, fourth, fifth, and sixth working electrodes 102A, 102B, 102C, 102D, 102E, and 102F,, respectively.

[0055] In some embodiments, the detection circuit 151 is configured to detect the two different molecules from the first and second working electrodes in a time-multiplexed manner.

[0056] In some embodiments, the nanowell array electrode 105 includes an insulating structure 106 formed on the working electrode 102, the plurality of nanowells 107 are opened through the insulating structure 106 to expose a subset of a surface of the working electrode 102. In some embodiments, the insulating structure 106 includes a polymer layer. In some embodiments, the insulating structure includes an insulation layer. In some embodiments, the insulating structure 106 includes a polymer layer and an insulation layer. The polymer layer and insulating layer are discussed in greater detail above with reference to Figure 1A.

[0057] In some embodiments, the electronic device 150 further includes a reference electrode 103. In some embodiments, the electronic device 150 further includes a counter electrode 104. In some embodiments, the electronic device 150 is configured to detect the outputAttorney Docket No.132414-5009-WO signal between the reference electrode 103 and the working electrode 102 and determine the presence or quantity of the at least two different molecules based on the antibody binding to the at least two different molecules. In some embodiments, the electronic device 150 further includes: a reference electrode 103, and a counter electrode 104, where the electronic device 150 is configured to detect the output signal between the reference electrode 103 and the working electrode 102 and determine the presence or quantity of the at least two different molecules based on the antibody binding to the at least two different molecules. In some embodiments, electrochemical measurement includes detecting the output signal between the reference electrode 103 and the working electrode 102 and determining the presence or quantity of the at least two different molecules based on the antibody binding to the at least two different molecules.

[0058] In some embodiments, the electronic device 150 includes an electrochemical measurement. Exemplary electrochemical measurements include, but are not limited to, Nyquist measurements, cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, potentiometry, and amperometry.

[0059] In some embodiments, the plurality of nanowells 107 includes an antibody 109. The antibody 109 is discussed in greater detail above with reference to Figure 1A. In some embodiments, at least a subset of the plurality of nanowells has a first structure configured to facilitate binding with the antibody, causing the antibody 109 to be bound with a first orientation.

[0060] In some embodiments, the electronic device 150 further includes: a sample inlet; a sample preparation module; a sample outlet; and a sample detection module mechanically coupled to the sample outlet, the sample detection module further including the nanowell array electrode 105.

[0061] In some embodiments, the electronic device 150 further includes one or more of: a first inlet; a second inlet; a normalization module; a first outlet; a second outlet; where the sample preparation module further includes a sample separation module and a filtration module.

[0062] Figure 2 is a schematic diagram of an example nanowell sensor-based microfluidic device 200 that implements sample pretreatment (e.g., anchoring, normalization, cleavage, and filtration) and electrochemical detection, in accordance with some embodiments, and Figure 3 is a structural diagram of an example nanowell sensor-based microfluidic device 200 for managing sample flows and chamber compositions, in accordance with someAttorney Docket No.132414-5009-WO embodiments. In one aspect, the disclosure provides a sensor. The sensor optionally includes a microfluidic film 220. In some embodiments, the disclosure provides a sensor for detecting presence or quantity of a target biomarker 108 (i.e., at least two different molecules, e.g., Aȕ peptides and / or tau-protein) in a sample, including: a) a plurality of chambers 204-207 including: a first chamber 205 configured to separate and / or cleave; a second chamber 206 configured to filter; and a third chamber 207 configured to detect; b) a nanowell array electrode 105; and c) a circuit board platform, where the at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein.

[0063] Specifically, in some embodiments, a system 100 (Figure 1) provided herein includes a nanowell sensor 101 including a plurality of chambers configured to provide a plurality of functions to detect the presence or quantity of a target biomarker 108 (i.e., the at least two different molecules, e.g., Aȕ peptides and / or tau-protein) in a sample. In some embodiments, the plurality of chambers 204-207 includes a first chamber 205 configured to provide separating and / or cleaving functions, a second chamber 206 configured to provide filtering function, and a third chamber 207 configured provide detecting function. In some embodiments, the plurality of chambers includes a fourth chamber 204 configured to provide normalization function. In some embodiments, the fourth chamber 204 includes an endogenous protein (e.g., albumin). In some embodiments, the first chamber 205 includes a buffer solution 213. In some embodiments, the buffer solution 213 is at a pH of about 7.4. In some embodiments, the buffer solution 213 includes phosphate-buffered saline (PBS). In some embodiments, the first chamber 205 is configured to provide the separating function to separate the at least two different molecules from the one or more particles. In some embodiments, the first chamber 205 includes a tetrazine molecule and a trans-cyclooctene molecule. In some embodiments, the first chamber 205 includes a pH solution 212. In some embodiments, the pH solution 212 is at a pH of from about 7 to 14 (e.g., 9 to 11). In some embodiments, the first chamber 205 is configured to provide the cleaving function to cleave different molecules from each other from the at least two different molecules. In some embodiments, the second chamber 206 is configured to provide the filtering function to isolate the at least two different molecules to be detected. In some embodiments, the second chamber 206 includes a membrane (e.g., a 1% methyl cellulose gel membrane). In some embodiments, the third chamber 207 is configured to provide the detecting function including an electrochemical measurement. In someAttorney Docket No.132414-5009-WO embodiments, the at least two different molecules consists of Aȕ peptides (e.g., Aȕ-38, Aȕ-40, and Aȕ-42). In some embodiments, the sample 211 includes urine and / or serum. In some embodiments, the plurality of chambers further includes a chamber configured to provide amplifying function. In some embodiments, the third chamber 207 is further configured to provide the amplifying function. In some embodiments, the plurality of chambers are connected by a microfluidic channel 240. In some embodiments, the plurality of chambers are located in a microfluidic film 220. Stated another way, the plurality of chambers are formed in a material layer of the microfluidic film 220 by patterning and etching.

[0064] In some embodiments, the working electrode 102 is formed under the third chamber 207, and nanowells are formed in the third chamber 207. A sample contained in the third chamber 207 contacts the working electrode 102 via a sensor portion 152 (Figure 1B) lying on the bottom of the third chamber 207. A detection portion 153 of the working electrode 102 extends from the sensor portion 152. A detection circuit 151 is coupled to the detection portion 153 of the working electrode 102, and configured to detect an output signal that indicates a presence or a quantity of the at least two different molecules.

[0065] In some embodiments, each of the plurality of chambers 204-207 includes a plurality of nanowells 107 (Figure 1A). In some embodiments, the plurality of nanowells 107 is from about 2 to 100,000 nanowells 107 for each of the at least two different molecules, for example, about 2, 10, 50, 100, 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 nanowells 107 for each of the at least two different molecules. In some embodiments, the plurality of nanowells 107 is about 40,000 nanowells 107 for each of the at least two different molecules. In some embodiments, the plurality of nanowells 107 is from about 10,000 to 100,000, from about 15,000 to 100,000, from about 20,000 to 100,000, from about 25,000 to 100,000, from about 30,000 to 100,000, from about 35,000 to 100,000, from about 40,000 to 100,000, from about 10,000 to 95,000, from about 10,000 to 90,000, from about 10,000 to 85,000, from about 10,000 to 80,000, from about 10,000 to 75,000, from about 10,000 to 70,000, from about 10,000 to 65,000, from about 10,000 to 60,000, from about 10,000 to 55,000, from about 10,000 to 50,000, from about 10,000 to 45,000, from about 10,000 to 40,000, from about 20,000 to 80,000, from about 20,000 to 60,000, from about 30,000 to 50,000, from about 35,000 to 55,000, or from about 30,000 to 60,000 nanowells 107 for each of the at leastAttorney Docket No.132414-5009-WO two different molecules. In some embodiments, the plurality of nanowells 107 includes from about 10,000 to 100,000 nanowells 107 for each of the at least two different molecules.

[0066] In some embodiments, each nanowell of the plurality of nanowells 107 is individually controlled. In some embodiments, the plurality of nanowells 107 is simultaneously controlled.

[0067] In some embodiments, the plurality of chambers is located in a microfluidic film 220. As used herein, the terms “microfluidic film” and “microfluidic device” are used interchangeably and generally refers to a chip, system, or device which can incorporate a plurality of interconnected channels and / or chambers, through which materials, and particularly fluid materials can be transported to effect one or more preparative or analytical manipulations on those materials. A microfluidic device can exist alone or may be a part of a microfluidic system which, for example and without limitation, can include: pumps for introducing fluids, e.g., samples, reagents, buffers, and the like, into the system; detection equipment or systems; data storage systems; and control systems for controlling fluid transport and / or direction within the device, monitoring and controlling environmental conditions to which fluids in the device are subjected, e.g., temperature, current, and the like.

[0068] In some embodiments, an additional substrate is used to prevent leakage of the fluid from the microfluidic film 220 and / or microfluidic channel 240 which may be caused by, e.g., the complexity of the system. In some embodiments, the microfluidic film 220 is sandwiched between two PMMA boards and clamped together using screw / bolt sets. In some embodiments, the positive pressure created by the PMMA boards mechanically secures the PDMS bonding and prevents leakage.

[0069] In some embodiments, the microfluidic film 220 where the plurality of chambers 204-207 is formed includes any suitable polymer including, but not limited to, polypropylene (PP), polystyrene (PS), polydimethylsiloxane (PDMS), polyethylene (PE), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyimide (PI), polyetheretherketone (PEEK), polyether sulfone (PES), polycarbonate (PC), polyester, cyclic olefin copolymer (COC), polyetherimide (PEI), polyvinyl chloride (PVC), and perfluoropolyether (PFPE). In some embodiments, the microfluidic film 220 includes polydimethylsiloxane (PDMS).Attorney Docket No.132414-5009-WO

[0070] In some embodiments, the plurality of chambers includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 chambers. In some embodiments, the plurality of chambers includes from 1 to 10 chambers, from 1 to 9 chambers, from 1 to 8 chambers, from 1 to 7 chambers, from 1 to 6 chambers, from 1 to 5 chambers, from 1 to 4 chambers, from 2 to 8 chambers, from 2 to 7 chambers, from 2 to 6 chambers, from 2 to 5 chambers, from 2 to 4 chambers, or from 3 to 5 chambers.

[0071] The microfluidic film 220 and / or microfluidic channel 240 may include one or more inlets and one or more outlets, in accordance with some embodiments. In some embodiments, the one or more inlets includes 1, 2, 3, 4, 5, 6, 7, or 8 inlets. In some embodiments, the one or more inlets includes from 1 to 8 inlets, from 1 to 6 inlets, from 1 to 4 inlets, from 1 to 3 inlets, or from 2 to 4 inlets. In some embodiments, the one or more outlets includes 1, 2, 3, 4, 5, 6, 7, or 8 outlets. In some embodiments, the one or more outlets includes from 1 to 8 outlets, from 1 to 6 outlets, from 1 to 4 outlets, from 1 to 3 outlets, or from 2 to 4 outlets. In some embodiments, the microfluidic film 220 and / or microfluidic channel 240 includes 3 inlets and 3 outlets.

[0072] In some embodiments, the plurality of chambers is connected by one or more microfluidic channels 240. As used herein, the terms “channel” and “microfluidic channel” are used interchangeably and can mean a recess or cavity formed in a material by imparting a pattern from a patterned substrate into a material or by any suitable material removing technique, or can mean a recess or cavity in combination with any suitable fluid-conducting structure mounted in the recess or cavity, such as a tube, capillary, or the like. In some embodiments, the microfluidic film 220 includes the microfluidic channel 240. In some embodiments, the microfluidic film 220 includes one or more microfluidic channels 240 (e.g., 1, 2, 3, 4, or 5 microfluidic channels). The microfluidic channel 240 is optionally linear or branched, in accordance with some embodiments. In some embodiments, increasing the number of channels 240 causes the formation of air bubbles, which may be eliminated by miniaturized bubble traps to eliminate all bubbles generated in the fluid system.

[0073] The microfluidic film 220 and / or microfluidic channel 240 may include one or more valves (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), in accordance with some embodiments. In some embodiments, the microfluidic film 220 and / or microfluidic channel 240 includes from 1 to 15 valves, from 1 to 12 valves, from 1 to 10 valves, from 4 to 10 valves, from 4 to 12 valves, from 6 to 10 valves, from 6 to 12 valves, from 7 to 9 valves, or from 5 to 11Attorney Docket No.132414-5009-WO valves. In some embodiments, the microfluidic film 220 and / or microfluidic channel 240 includes 8 valves. In some embodiments, the valve controls the flow of a fluid (e.g., a liquid).

[0074] An exemplary sensor including a microfluidic film 220 is provided in Figure 2, in accordance with some embodiments. In some embodiments, an inlet 201 is connected to a first portion of the microfluidic channel 240, which branches into two parallel portions of the microfluidic channel 240. In some embodiments, the sample 211 flows into the microfluidic channel 240 from the inlet 201. In some embodiments, a first of the two parallel portions of the microfluidic channel 240 is connected to a first chamber 205, optionally including a valve between the inlet 201 and the first chamber 205. In some embodiments, a second portion of the microfluidic channel 240 includes an inlet 202 connected to the first chamber 205, optionally including a valve between the inlet 202 and the first chamber 205. In some embodiments, an inlet 203 is connected to the second portion of the microfluidic channel 240 between the inlet 202 and the first chamber 205, optionally including a valve between the inlet 203 and the second portion of the microfluidic channel 240. In some embodiments, the first chamber 205 is further connected to an outlet 208, optionally including a valve between the first chamber 205 and the outlet 208. In some embodiments, the first chamber 205 is further connected to an outlet 210. In some embodiments, a second chamber 206 is connected between the first chamber 205 and the outlet 210, optionally including a valve between the first chamber 205 and the second chamber 206. In some embodiments, a third chamber 207 is connected between the first chamber 205 and the outlet 210, optionally including a valve between the third chamber 207 and the outlet 210. In some embodiments, a third chamber 207 is connected between the second chamber 206 and the outlet 210, optionally including a valve between the third chamber 207 and the outlet 210. In some embodiments, the first chamber 205 is connected to the second chamber 206, the second chamber 206 is connected to the third chamber 207, and the third chamber 207 is connected to the outlet 210, where a valve is between the first chamber 205 and the second chamber 206 and between the third chamber 207 and the outlet 210. In some embodiments, a second of the two parallel portions of the microfluidic channel 240 is connected to a fourth chamber 204. In some embodiments, the fourth chamber 204 is connected to an outlet 209, optionally including a valve between the fourth chamber 204 and the outlet 209.

[0075] In some embodiments, the first chamber 205, the second chamber 206, the third chamber 207, and / or the fourth chamber 204 include an electrode. In some embodiments, anAttorney Docket No.132414-5009-WO electrode is integrated with the first chamber 205, the second chamber 206, the third chamber 207, and / or the fourth chamber 204. For example, an insulating material covers the electrode that is formed on the substrate 111, and a third chamber 207 is opened on the insulating material to reach the working electrode 102, includes nanowells 107, and is configured to expose part of the working electrode via at least the nanowells 107. Stated another way, in some embodiments, the nanowells 107 are formed to expose the working electrode 102 in the chamber. In some embodiments, the antibody 109 is immobilized in and above the nanowells 107.

[0076] An exemplary sensor including a microfluidic film 220 is provided in Figure 3. In some embodiments, the inlet 201 includes the sample 211 (also referred to as “host sample”). In some embodiments, the sample 211 is a biological material including, but not limited to, urine, whole blood, serum, plasma, and saliva. In some embodiments, the sample 211 includes urine. In some embodiments, the sample 211 includes serum. In some embodiments, the sample is a non-mammalian sample (e.g., mouse, rat, rabbit, hamster, guinea pig, ferret, dog, cat, pig, or sheep). In some embodiments, the sample is a mammalian sample (e.g., human, non-human primate, monkey, or ape). In some embodiments, the sample is a human sample. In some embodiments, the sample 211 includes the target biomarker 108 (i.e., at least two different molecules, e.g., Aȕ peptides and / or tau-protein).

[0077] In some embodiments, the at least two different molecules include a peptide, a polypeptide, and / or a protein. In some embodiments, the at least two different molecules include tau-protein. In some embodiments, the at least two different molecules include and / or consist of Aȕ peptides. In some embodiments, the at least two different Aȕ peptides include Aȕ-37, Aȕ-38, Aȕ-39, Aȕ-40, Aȕ-41, Aȕ-42, Aȕ-43, Aȕ-44, Aȕ-45, Aȕ-46, Aȕ-47, Aȕ-48, and Aȕ-49. In some embodiments, the at least two different Aȕ peptides include Aȕ-38, Aȕ-40, and Aȕ-42. As discussed above with reference to Figure 1A, the at least two different molecules are bound to one or more particles in the sample, e.g., one or more antibodies. In some embodiments, the first chamber 205 configured to separate includes separating the at least two different molecules from the one or more particles.

[0078] In some embodiments, the sensor 101 includes a first chamber 205 configured to separate. In some embodiments, the first chamber 205 includes a buffer solution 213. In some embodiments, the buffer solution 213 is at a pH of greater than or equal to about 7, for example, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, theAttorney Docket No.132414-5009-WO buffer solution 213 is at a pH of from about 7 to about 10, from about 7 to about 9, or from about 7 to about 8. In some embodiments, the buffer solution 213 is at physiological pH. In some embodiments, the buffer solution 213 is at a pH of about 7.4. Any buffer suitable for the present invention may be used including, but not limited to, HEPES, MOPS, Tris, MES, PIPES, phosphate buffer, phosphate-buffered saline (PBS), bis-Tris, ADA, ACES, MOPSO, bis-Tris propane, BES, TES, citrate buffer, SSC, TAE, TBE, and Tris-EDTA. In some embodiments, the buffer solution 213 includes phosphate-buffered saline (PBS).

[0079] In some embodiments, the first chamber 205 includes a bioorthogonal anchor. In some embodiments, the first chamber 205 includes a tetrazine bioorthogonal anchor (e.g., Tz- TCO). In some embodiments, the first chamber 205 includes a tetrazine molecule. In some embodiments, the tetrazine molecule includes a 1,2,4,5-tetrazine molecule, such as 1,2,4,5- tetrazine, (4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine, 6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3- yl)pyridin-3-amine, 4-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)benzoic acid, 3,6-dimethyl- 1,2,4,5-tetrazine, 3,6-diphenyl-1,2,4,5-tetrazine, (4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)methanamine, (4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methanamine, (4-(6- (pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methanamine, 3,6-bis(trifluoromethyl)-1,2,4,5- tetrazine, 5-((4-(1,2,4,5-tetrazin-3-yl)phenyl)amino)-5-oxopentanoic acid, or 5-oxo-5-((6-(6- (pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)pentanoic acid. In some embodiments, the first chamber 205 includes an alkene molecule. In some embodiments, the first chamber 205 includes a cycloalkene molecule. In some embodiments, the first chamber 205 includes a cyclooctene molecule. In some embodiments, the first chamber 205 includes a trans-cyclooctene molecule. In some embodiments, the first chamber 205 includes a tetrazine molecule and a trans-cyclooctene molecule.

[0080] In some embodiments, the first chamber 205 configured to cleave includes cleaving different molecules from each other from the at least two different molecules.

[0081] In some embodiments, the first chamber 205 includes a pH solution 212. In some embodiments, the pH solution 212 is acidic. In some embodiments, the pH solution 212 is basic. In some embodiments, the pH solution 212 is at a pH of from about 7 to 14, for example, a pH of about 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the pH solution 212 is at a pH of about 10. In some embodiments, the pH solution 212 is at a pH of from about 7 to 14, from about 7 to 13, from about 7 to 12, from about 7 to 11, from about 7 to 10, from about 8 to 14,Attorney Docket No.132414-5009-WO from about 8 to 13, from about 8 to 12, from about 8 to 11, from about 8 to 10, or from about 9 to 11. In some embodiments, the pH solution 212 is at a pH of from about 9 to 11. In some embodiments, the pH solution includes a base, for example, NaOH, KOH, Ca(OH)2, Mg(OH)2, and NaHCO3. In some embodiments, the pH solution includes NaOH. In some embodiments, the pH solution has a concentration of from about 0.1 to about 100 mM, for example, about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the pH solution has a concentration of from about 0.1 to 100 mM, from about 0.1 to 100 mM, from about 0.1 to 50 mM, from about 0.1 to 20 mM, from about 1 to 100 mM, from about 1 to 100 mM, from about 1 to 20 mM, or from about 0.5 to 30 mM. In some embodiments, the pH solution has a concentration of from about 1 to 20 mM.

[0082] In some embodiments, the sensor 101 includes a second chamber 206 configured to filter. In some embodiments, the second chamber 206 configured to filter includes isolating the at least two different molecules to be detected.

[0083] In some embodiments, the second chamber 206 includes a membrane 214. In some embodiments, the microfluidic channel 240 includes the membrane 214 before and / or after the second chamber 206. In some embodiments, the membrane 214 includes a gel membrane. Suitable gel membranes may include, for example, dextrans, agarose, polyacrylamide, polyvinylethylcarbitol, polyvinylpyrrolidone, cellulose, methyl cellulose, hydroxyethyl cellulose, polyvinyl alcohol, pullulan, silica, allyl dextran-bis-acrylamide, or any mixtures thereof. In some embodiments, the membrane 214 includes a cellulose-based gel membrane. In some embodiments, the membrane 214 includes a methyl cellulose gel membrane. In some embodiments, the methyl cellulose gel membrane includes from about 0.1% to 10% of methyl cellulose, for example, about 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, or 10% of methyl cellulose. In some embodiments, the methyl cellulose gel membrane includes from about 0.1 to 10%, from about 0.1 to about 2%, from about 0.5 to about 5%, or from about 0.5 to about 2% of methyl cellulose. In some embodiments, the membrane 214 includes a 1% methyl cellulose gel membrane. In some embodiments, the 1% methyl cellulose gel is applied with the second chamber 206 (e.g., before the chamber 206, after the chamber 206, or both).Attorney Docket No.132414-5009-WO

[0084] In some embodiments, the second chamber 206 provides a microfluidic sample. In some embodiments, the microfluidic sample includes the at least two different molecules. In some embodiments, the at least two different molecules are purified (i.e., at least 50% pure). In some embodiments, the microfluidic sample is provided to the third chamber 207 configured to detect.

[0085] In some embodiments, the sensor 101 includes a third chamber 207 configured to detect. In some embodiments, the third chamber 207 configured to detect includes an electrochemical measurement. More details on electrochemical measurement are discussed above with reference to Figure 1B. In some embodiments, the third chamber 207 includes the nanowell sensor 101. In some embodiments, the third chamber 207 includes the nanowell array electrode 105.

[0086] In some embodiments, the sensor 101 further includes a fourth chamber 204 configured to normalize. In some embodiments, the fourth chamber 204 includes a protein (e.g., an endogenous protein, a globular protein, and / or an immunoglobulin). In some embodiments, the fourth chamber 204 includes an endogenous protein (e.g., IgG, IgM, IgD, IgA, IgE, albumin, or hemoglobin). In some embodiments, the endogenous protein is albumin.

[0087] In some embodiments, the fourth chamber 204 includes a nanowell sensor. In some embodiments, the nanowell sensor includes a nanowell array electrode. In some embodiments, the fourth chamber 204 includes an electrochemical measurement of the protein (e.g., albumin). In some embodiments, the electrochemical measurement of the at least two different molecules is provided relative to the electrochemical measurement of the protein (e.g., albumin).

[0088] In some embodiments, the sensor further includes a chamber configured to amplify. In some embodiments, the third chamber 207 is further configured to amplify. In some embodiments, third chamber 207 is further configured to amplify includes amplifying the electrochemical measurement.

[0089] Referring to Figure 1A, in some embodiments, the sensor includes a nanowell array electrode 105. In some embodiments, the nanowell array electrode 105 includes a nanowell depth of from about 10 to 200 nm. In some embodiments, the nanowell array electrode 105 includes a nanowell diameter of from about 10 to 150 nm. In some embodiments, the nanowell array electrode 105 includes from about 10,000 to 100,000 nanowells 107 for each ofAttorney Docket No.132414-5009-WO the at least two different molecules. In some embodiments, the nanowell array electrode 105 includes a working electrode 102 (e.g., a transition metal working electrode). In some embodiments, the working electrode 102 (e.g., a transition metal working electrode) is selected from the group consisting of Au, Ti, W, Pt, and Ir. In some embodiments, the working electrode 102 (e.g., a transition metal working electrode) includes Au. In some embodiments, the working electrode 102 (e.g., a transition metal working electrode) includes an insulating structure 106. In some embodiments, the working electrode 102 (e.g., a transition metal working electrode) includes a polymer layer. In some embodiments, the nanowell array electrode 105 further includes a counter electrode 104. In some embodiments, the counter electrode 104 includes Pt. In some embodiments, the nanowell array electrode 105 further includes a reference electrode 103. In some embodiments, the reference electrode 103 includes Ag. In some embodiments, the nanowell array electrode 105 further includes an insulation layer. In some embodiments, the insulation layer includes SiO2. In some embodiments, the nanowell array electrode 105 includes a total active area of from about 10 to 300 μm2.

[0090] Referring to Figure 3, in some embodiments, the nanowell sensor-based microfluidic device 200 includes a circuit board 230. The substrate 111 includes the circuit board 230 or is mounted on the circuit board 230.

[0091] In some embodiments, the chamber design for electrode integration are as follows: the fourth chamber 204 includes a normalization chamber using albumin as an endogenous internal standard; the first chamber 205 includes an anchor chamber to separate Aȕ peptide-nanoprobe complexes from urinary debris using Tz-TCO bioorthogonal anchors and to cleave Aȕ peptides from the nanoprobes by using high pH buffer (e.g., pH 10); the second chamber 206 includes a filtration chamber to transfer only cleaved Aȕ peptides into the detection chamber through 1% methyl cellulose gel membrane; and the third chamber 207 includes an amplifying detection chamber to sensitively detect 3 Aȕ peptides (e.g., Aȕ38, Aȕ40 and Aȕ42) by immobilization of multiple specific antibodies. In some embodiments, the sensor includes a single electrode-based microfluidic device. In some embodiments, the sensor is scaled up to simultaneously accommodate 3 individual electrodes in parallel, while retaining the portable miniaturized size. In some embodiments, in the first chamber 205, Aȕ peptides are separated from the anchored nanoprobes by incubating the saturated electrodes with 10 mM NaOH, pH = 10 (i.e., the pH solution 212) for 1 min, followed by PBS (i.e., the buffer solution 213) rinsing.Attorney Docket No.132414-5009-WO In some embodiments, the pH solution 212 (e.g., NaOH; 10 mM; and pH = 10) incubation interrupts the nanoprobe-peptide interactions so that the dissociated peptides can be efficiently removed from the surface of the electrode. In some embodiments, the efficiency of peptide cleavage using NaOH in the microfluidic chamber may differ compared to the result from nanowell biosensors placed off-chip, due to the volume confinement and the flow effect; in which case the concentration and / or incubation period of the NaOH solution and the flow rate of the washing buffer can be varied to select the optimal cleavage condition. In some embodiments, the nanowell biosensor 101 is functionalized with Aȕ-specific antibodies and Nyquist measurements are followed until peptide binding reaches saturation on the electrode surface. In some embodiments, a series of NaOH solutions (i.e., the pH solution 212) at 1, 5, 10, 15, and 20 mM are applied for a certain period of time (0.5, 1, 1.5, and 2 min), washed with PBS (i.e., the buffer solution 213), and the process is repeated 2-3 times. In some embodiments, the electrical signals are immediately recorded and the Nyquist plots are compared across multiple cycles. In some embodiments, to capture only Aȕ peptides, a 1% methyl cellulose gel membrane is introduced in the filtration chamber (i.e., the second chamber 206). In some embodiments, the size-exclusion membrane composition (e.g., 1% methyl cellulose gel membrane) yields pore sizes small enough to allow buffer ion transport and current flow through the membrane, yet exclude a wide range of proteins (> 10 kDa). In some embodiments, the capacity and functionality of the single-electrode microfluidic device is fully tested using dissolved biomarkers and compared with off-chip results. In some embodiments, the detection chamber (i.e., the third chamber 207) is integrated with 4 terminal biosensor units (i.e., 3 units for 3 Aȕ peptides and 1 for reference). In some embodiments, the size of this sensor is a small portable form containing of 3 electric parts: a) a circuit board platform, b) a nanowell array electrode 105, and c) a polymer film fluidic chamber (i.e., the microfluidic film 220).

[0092] Figure 4A is a list 400 of parameters of example nanowell array electrodes having different well sizes (e.g., 90 nm and 50 nm), in accordance with some embodiments. In some embodiments, the nanowell array electrode 105 is fabricated using electron beam nanolithography with constant depths of 50 and 100 nm, and well sizes of 50 and 90 nm to control number of biomolecules immobilized. In some embodiments, the surface of Au electrodes is covered with a standard positive polymer layer, ZEP520 Nippon Zeon, for blocking biomolecules and insulating. In some embodiments, a 200x200 nanowell array is fabricated inAttorney Docket No.132414-5009-WO the layer of each pad with a thickness of 150-200 nm. In some embodiments, the nanowell electrodes are characterized by employing electrochemical measurements such as cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy in an electrolyte containing K3Fe(CN)6 / K4Fe(CN)6. In some embodiments, the measurements are performed in a 3-electrode system in which a nanowell structure on Au functions as the working electrode (WE) 102. In some embodiments, this is combined with a platinum counter electrode (CE) 104 and an Ag reference electrode (RE) 103. In some embodiments, the electrochemical characteristics of electrodes with different densities, sizes, and numbers of nanowells 107 to determine optimal conditions are compared.

[0093] In some embodiments, the disclosure provides a quantitative scoring platform for the target biomarker 108. In some embodiments, the disclosure provides simultaneous detection of the at least two different molecules at a 1 pg / mL level in urine. In some embodiments, the interaction between biological materials in the sample and nanostructures at the molecular level can provide new bioelectrochemical information where the single biomolecule behavior can be investigated from a redox reaction, which represents an important advantage to the analytical use of nanowell diagnostic device.

[0094] Figure 4B is an example quantitative scoring table 450 for determining quantitative scores of AD biomarkers based on cut-off ranges of AD biomarkers, in accordance with some embodiments. For example, the quantitative scoring table 450 is applied to determine if the target biomarker 108 (i.e., at least two different molecules, e.g., of Aȕ peptides and / or tau- protein) is at a normal concentration, border concentration, or AD concentration, including the reported cut-off ranges of 3 exemplary AD biomarkers. In some embodiments, in order to summarize the output reading, the concentration of the biomarkers is categorized into 5 levels (i.e., Normal, level 1-2; Border, level 3; and AD, level 4-5). In some embodiments, a normal concentration level 1 risk is from 0–0.2. In some embodiments, a normal concentration level 2 risk is from 0.2–0.4. In some embodiments, a border concentration level 3 risk is from 0.4–0.6. In some embodiments, an AD concentration level 4 risk is from 0.6–0.8. In some embodiments, an AD concentration level 4 risk is from 0.8–1.0. In some embodiments, the overall index for AD risk is established using statistical analyses of the quantitative scoring platform. In some embodiments, although 3 urinary biomarkers for AD is exemplified, the number of biomarkers (i.e., terminals) can be easily increased. In general, as displayed in the following statisticalAttorney Docket No.132414-5009-WO analysis, the power of disease detection is proportional to the number of biomarkers. In some embodiments, the conditional probability of having AD, given biomarker A is positive, can be written as follows (denote AD Ł Alzheimer's disease and {A, B, C} Ł {biomarker A, biomarker B, biomarker C}): (1) Similarly, in somebiomarkers A & B are can as: (2) InC are positive, can be written as: (3) In(4) or Similarly, in some(2) and (3): (5) or

[0095] In^(^ / ^,^,^)^(^ / ^) ^(^ / ^,^)are 1 for any values of A and B, how much more likely one is to have the disease with biomarkers 1, 2, or 3 depending on the final value can be determined. In some embodiments, when another biomarker test is added, it is multiplied by another probability ratio as was done above when a second and third biomarker (B and C) were added; thus, P(D / A) < P (D / A,B) < P (D / A,B,C).

[0096] Figure 5 is a flow diagram of an example method 500 for detecting presence or quantity of at least two different molecules, in accordance with some embodiments.

[0097] In some embodiments, the at least two different molecules are from (operation 501) a sample. In some embodiments, the sample includes urine. In some embodiments, theAttorney Docket No.132414-5009-WO sample includes serum. In some embodiments, the at least two different molecules are selected (operation 502) from the group consisting of Aȕ peptides and tau-protein. In some embodiments, the at least two different molecules consist of Aȕ peptides. In some embodiments, the at least two different Aȕ peptides include Aȕ-38, Aȕ-40, and Aȕ-42. In some embodiments, the at least two different molecules are bound (operation 503) to one or more particles in the sample. In some embodiments, the one or more particles in the sample are one or more nanoprobes. In some embodiments, the one or more particles are one or more antibodies.

[0098] In some embodiments, the sample is (operation 504) in a plurality of nanowells on a sensor. In some embodiments, the sensor includes (i) a plurality of chambers, and (ii) a microfluidic channel 240 connecting the chambers 505. In some embodiments, the sensor includes (i) a plurality of chambers, at least one of which includes a subset of the plurality of nanowells, and (ii) a microfluidic channel 240 connecting the chambers. In some embodiments, each of the plurality of chambers includes (operation 506) a plurality of nanowells. In some embodiments, each nanowell of the plurality of nanowells is individually controlled. In some embodiments, the plurality of nanowells includes from about 10,000 to 100,000 nanowells for each of the at least two different molecules.

[0099] In some embodiments, the method further includes normalizing (operation 507). In some embodiments, normalizing includes adding (operation 508) an endogenous protein to the sample. In some embodiments, the endogenous protein is albumin. In some embodiments, the normalizing is performed only in a fourth chamber of the plurality of chambers.

[0100] In some embodiments, the method includes separating (operation 509). In some embodiments, separating includes adding (operation 510) a buffer solution to the sample. In some embodiments, the buffer solution is at a pH of about 7.4. In some embodiments, the buffer solution includes phosphate-buffered saline (PBS). In some embodiments, the separating includes separating the at least two different molecules from the one or more particles. In some embodiments, the separating is performed in a first chamber of the plurality of chambers.

[0101] In some embodiments, the method includes cleaving (operation 511). In some embodiments, separating includes adding a tetrazine molecule and a trans-cyclooctene molecule. In some embodiments, cleaving includes adding (operation 512) a pH solution to the sample. In some embodiments, the pH solution is at a pH of from about 7 to 14. In some embodiments, the pH solution is at a pH of from about 9 to 11. In some embodiments, the cleaving includesAttorney Docket No.132414-5009-WO cleaving different molecules from each other from the at least two different molecules. In some embodiments, the separating and cleaving are performed only in a first chamber of the plurality of chambers.

[0102] In some embodiments, the method includes filtering (operation 513). In some embodiments, filtering includes isolating the at least two different molecules to be detected. In some embodiments, the filtering includes passing (operation 514) the at least two different molecules to be detected through a membrane. In some embodiments, the membrane includes a 1% methyl cellulose gel membrane. In some embodiments, the filtering is performed only in a second chamber of the plurality of chambers.

[0103] In some embodiments, the method further includes amplifying (operation 515). In some embodiments, the amplifying is performed only in a third chamber of the plurality of chambers.

[0104] In some embodiments, the method includes detecting (operation 516). In some embodiments, detecting includes using (operation 517) electrochemical measurement. In some embodiments, the detecting is performed only in a third chamber of the plurality of chambers. In some embodiments, the detecting and amplifying are performed only in a third chamber of the plurality of chambers.

[0105] In one aspect, the disclosure provides a method of treating Alzheimer’s disease (AD). In some embodiments, the disclosure provides a method of treating AD in a subject in need thereof, including detecting the presence or quantity of at least three different Aȕ peptides according to the method 500 described herein, and administering an effective amount of a therapeutic compound to the subject.

[0106] 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 orAttorney Docket No.132414-5009-WO 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. may 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.

[0107] 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.

[0108] 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.

[0109] 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.132414-5009-WO What is claimed is:

1. A method of detecting presence or quantity of at least two different molecules, comprising: separating, cleaving, filtering, and detecting the at least two different molecules selected from the group consisting of Aȕ peptides and tau-protein from a sample in a plurality of nanowells on a sensor.

2. The method according to claim 1, wherein the at least two different molecules are bound to one or more particles in the sample.

3. The method according to claim 2, wherein the one or more particles in the sample are one or more antibodies.

4. The method according to claim 3, wherein the one or more antibodies comprise anti-Aȕ antibodies.

5. The method according to any one of the preceding claims, wherein the sensor comprises (i) a plurality of chambers, at least one of which comprises a subset of the plurality of nanowells, and (ii) a microfluidic channel connecting the chambers.

6. The method according to any one of the preceding claims, further comprising normalizing.

7. The method according to claim 6, wherein the normalizing comprises adding an endogenous protein to the sample.

8. The method according to claim 7, wherein the endogenous protein is albumin.

9. The method according to any one of the preceding claims, wherein separating comprises adding a buffer solution to the sample.

10. The method according to claim 9, wherein the buffer solution is at a pH of 7.

4.

11. The method according to claim 9 or 10, wherein the buffer solution comprises phosphate- buffered saline (PBS).

12. The method according to any one of the preceding claims, wherein the separating comprises separating the at least two different molecules from the one or more particles.

13. The method according to claim 12, wherein separating comprises adding a tetrazine molecule and a trans-cyclooctene molecule.

14. The method according to any one of the preceding claims, wherein cleaving comprises adding a pH solution to the sample.

15. The method according to claim 14, wherein the pH solution is at a pH of from 7 to 14.Attorney Docket No.132414-5009-WO 16. The method according to claim 14, wherein the pH solution is at a pH of from 9 to 11.

17. The method according to claim 14, wherein the cleaving comprises cleaving different molecules from each other from the at least two different molecules.

18. The method according to any one the preceding claims, wherein filtering comprises isolating the at least two different molecules to be detected.

19. The method according to claim 18, wherein the filtering comprises passing the at least two different molecules to be detected through a membrane.

20. The method according to claim 19, wherein the membrane comprises a 1% methyl cellulose gel membrane.

21. The method according to any one the preceding claims, wherein detecting comprises using electrochemical measurement.

22. The method according to any one of the preceding claims, wherein the at least two different molecules consist of at least two different Aȕ peptides.

23. The method according to any one of the preceding claims, wherein the at least two different Aȕ peptides comprise Aȕ-38, Aȕ-40, and Aȕ-42.

24. The method according to any one of claims 6-8, wherein the normalizing is performed only in a fourth chamber of the plurality of chambers.

25. The method according to any one of the preceding claims, wherein the separating and cleaving are performed only in a first chamber of the plurality of chambers.

26. The method according to any one of the preceding claims, wherein the filtering is performed only in a second chamber of the plurality of chambers.

27. The method according to any one of the preceding claims, wherein the detecting is performed only in a third chamber of the plurality of chambers.

28. The method according to any one of the preceding claims, wherein each nanowell of the plurality of nanowells is individually controlled.

29. The method according to any one of the preceding claims, wherein the plurality of nanowells comprises from 10,000 to 100,000 nanowells for each of the at least two different molecules.

30. The method according to any one of the preceding claims, wherein the sample comprises urine.Attorney Docket No.132414-5009-WO 31. The method according to any one of the preceding claims, wherein the sample comprises serum.

32. The method according to any one of the preceding claims, further comprising amplifying.

33. The method according to claim 32, wherein the detecting and amplifying are performed only in a third chamber of the plurality of chambers.

34. A method of treating Alzheimer’s disease in a subject in need thereof, comprising detecting the presence or quantity of at least three different Aȕ peptides according to the method of any one of the preceding claims, and administering an effective amount of a therapeutic compound to the subject.

35. A sensor for detecting presence or quantity of at least two different molecules in a sample, comprising: a) a plurality of chambers comprising: a first chamber configured to separate and / or cleave; a second chamber configured to filter; and a third chamber configured to detect; b) a nanowell array electrode comprising a plurality of nanowells; and c) a circuit board platform, wherein the at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein.

36. The sensor according to claim 35, wherein each of the plurality of chambers comprises a plurality of nanowells.

37. The sensor according to claim 36, wherein each nanowell of the plurality of nanowells is individually controlled.

38. The sensor according to claim 36 or 37, wherein the plurality of nanowells comprises from 10,000 to 100,000 nanowells for each of the at least two different molecules.

39. The sensor according to any one of claims 35-38, wherein the plurality of chambers is connected by a microfluidic channel.

40. The sensor according to claim 39, wherein the plurality of chambers is located in a microfluidic film.Attorney Docket No.132414-5009-WO 41. The sensor according to any one of claims 35-40, wherein the at least two different molecules are bound to one or more particles in the sample.

42. The sensor according to claim 41, wherein the one or more particles in the sample are one or more antibodies.

43. The sensor according to claim 42, wherein the one or more antibodies comprise anti-Aȕ antibodies.

44. The sensor according to any one of claims 35-43, further comprising a fourth chamber configured to normalize.

45. The sensor according to claim 44, wherein the fourth chamber comprises an endogenous protein.

46. The sensor according to claim 45, wherein the endogenous protein is albumin.

47. The sensor according to any one of claims 35-46, wherein the first chamber comprises a buffer solution.

48. The sensor according to claim 47, wherein the buffer solution is at a pH of 7.

4.

49. The sensor according to claim 47 or 48, wherein the buffer solution comprises phosphate- buffered saline (PBS).

50. The sensor according to any one of claims 35-49, wherein the first chamber configured to separate comprises separating the at least two different molecules from the one or more particles.

51. The sensor according any one of claims 35-50, wherein the first chamber comprises a tetrazine molecule and a trans-cyclooctene molecule.

52. The sensor according to any one of claims 35-51, wherein the first chamber comprises a pH solution.

53. The sensor according to claim 52, wherein the pH solution is at a pH of from 7 to 14.

54. The sensor according to claim 52, wherein the pH solution is at a pH of from 9 to 11.

55. The sensor according to any one of claims 35-54, wherein the first chamber configured to cleave comprises cleaving different molecules from each other from the at least two different molecules.

56. The sensor according to any one of claims 35-55, wherein the second chamber configured to filter comprises isolating the at least two different molecules to be detected.

57. The sensor according to any one of claims 35-56, wherein the second chamber comprises a membrane.Attorney Docket No.132414-5009-WO 58. The sensor according to claim 57, wherein the membrane comprises a 1% methyl cellulose gel membrane.

59. The sensor according to any one of claims 35-58, wherein the third chamber configured to detect comprises an electrochemical measurement.

60. The sensor according to any one of claims 35-59, wherein the at least two different molecules consist of at least two different Aȕ peptides.

61. The sensor according to any one of claims 35-60, wherein the at least two different Aȕ peptides comprise Aȕ-38, Aȕ-40, and Aȕ-42.

62. The sensor according to any one of claims 35-61, wherein the sample comprises urine.

63. The sensor according to any one of claims 35-62, wherein the sample comprises serum.

64. The sensor according to any one of claims 35-63, further comprising a chamber configured to amplify.

65. The sensor according to any one of claims 35-63, wherein the third chamber is further configured to amplify.

66. The sensor according to any one of claims 35-65, wherein each nanowell of the plurality of nanowells comprises a nanowell depth of from 10 to 200 nm.

67. The sensor according to any one of claims 35-66, wherein each nanowell of the plurality of nanowells comprises a nanowell diameter of from 10 to 150 nm.

68. The sensor according to any one of claims 35-67, wherein the nanowell array electrode comprises from 10,000 to 100,000 nanowells.

69. The sensor according to any one of claims 35-68, wherein the nanowell array electrode comprises a working electrode.

70. The sensor according to claim 69, wherein the working electrode comprises a transition metal selected from the group consisting of Au, Ti, W, Pt, and Ir.

71. The sensor according to claim 69 or 70, wherein the working electrode comprises Au.

72. The sensor according to any one of claims 69-71, wherein the working electrode comprises a polymer layer.

73. The sensor according to any one of claims 35-72, wherein the sensor further comprises a counter electrode.

74. The sensor according to claim 73, wherein the counter electrode comprises Pt.Attorney Docket No.132414-5009-WO 75. The sensor according to any one of claims 35-74, wherein the sensor further comprises a reference electrode.

76. The sensor according to claim 75, wherein the reference electrode comprises Ag.

77. The sensor according to any one of claims 35-76, wherein the nanowell array electrode further comprises an insulation layer.

78. The sensor according to claim 77, wherein the insulation layer comprises SiO2.

79. The sensor according to any one of claims 35-78, wherein the nanowell array electrode comprises a total active area of from 10 to 300 μm2.

80. A system, comprising: a sensor including a plurality of chambers configured to provide a plurality of functions to detect presence or quantity of at least two different molecules in a sample, the plurality of chambers includes: a first chamber configured to provide separating and / or cleaving functions, a second chamber configured to provide filtering function and a third chamber configured provide detecting function; a processor configured to individually control each of the plurality of chambers; and a display configured to display visualized data including data representing the presence or quantity of the at least two different molecules in the sample; wherein the at least two different molecules are selected from the group consisting of Aȕ peptides and tau-protein.

81. The system according to claim 80, wherein the sensor includes: a nanowell array electrode comprising a plurality of nanowells; and a circuit board platform into which the plurality of chambers and the nanowell array electrode are integrated.

82. The system according to claim 80, wherein each of the plurality of chambers comprises a plurality of nanowells.

83. The system according to claim 82, wherein each nanowell of the plurality of nanowells is individually controlled by the processor.

84. The system according to claim 82 or 83, wherein the plurality of nanowells comprises from 10,000 to 100,000 nanowells for each of the at least two different molecules.Attorney Docket No.132414-5009-WO 85. The system according to any one of claims 80-84, wherein the plurality of chambers are connected by a microfluidic channel.

86. The system according to claim 85, wherein the plurality of chambers are located in a microfluidic film.

87. The system according to any one of claims 80-86, wherein the at least two different molecules are bound to one or more particles in the sample.

88. The system according to claim 87, wherein the one or more particles in the sample are one or more antibodies.

89. The system according to claim 88, wherein the one or more antibodies comprise anti-Aȕ antibodies.

90. The system according to any one of claims 80-89, wherein the plurality of chambers includes a fourth chamber configured to provide normalization function.

91. The system according to claim 90, wherein the fourth chamber comprises an endogenous protein.

92. The system according to claim 91, wherein the endogenous protein is albumin.

93. The system according to any one of claims 80-92, wherein the first chamber comprises a buffer solution.

94. The system according to claim 93, wherein the buffer solution is at a pH of 7.

4.

95. The system according to claim 93 or 94, wherein the buffer solution comprises phosphate-buffered saline (PBS).

96. The system according to any one of claims 80-95, wherein the first chamber is configured to provide the separating function to separate the at least two different molecules from the one or more particles.

97. The system according any one of claims 80-96, wherein the first chamber comprises a tetrazine molecule and a trans-cyclooctene molecule.

98. The system according to any one of claims 80-97, wherein the first chamber comprises a pH solution.

99. The system according to claim 98, wherein the pH solution is at a pH of from 7 to 14.

100. The system according to claim 98, wherein the pH solution is at a pH of from 9 to 11.Attorney Docket No.132414-5009-WO 101. The system according to any one of claims 80-100, wherein the first chamber is configured to provide the cleaving function to cleave different molecules from each other from the at least two different molecules.

102. The system according to any one of claims 80-101, wherein the second chamber is configured to provide the filtering function to isolate the at least two different molecules to be detected.

103. The system according to any one of claims 80-102, wherein the second chamber comprises a membrane.

104. The system according to claim 103, wherein the membrane comprises a 1% methyl cellulose gel membrane.

105. The system according to any one of claims 80-104, wherein the third chamber is configured to provide the detecting function including an electrochemical measurement.

106. The system according to any one of claims 80-105, wherein the at least two different molecules consist of at least two different Aȕ peptides.

107. The system according to any one of claims 80-106, wherein the at least two different Aȕ peptides comprise Aȕ-38, Aȕ-40, and Aȕ-42.

108. The system according to any one of claims 80-107, wherein the sample comprises urine.

109. The system according to any one of claims 80-108, wherein the sample comprises serum.

110. The system according to any one of claims 80-109, wherein the plurality of chambers further comprises: a chamber configured to provide amplifying function.

111. The system according to any one of claims 80-110, wherein the third chamber is further configured to provide the amplifying function.

112. The system according to claim 81, wherein each nanowell of the plurality of nanowells comprises a nanowell depth of from 10 to 200 nm.

113. The system according to claim 81 or 112, wherein each nanowell of the plurality of nanowells comprises a nanowell diameter of from 10 to 150 nm.

114. The system according to any one of claims 81 or 112-113, wherein the nanowell array electrode comprises from 10,000 to 100,000 nanowells.

115. The system according to any one of claims 81 or 112-114, wherein the nanowell array electrode comprises a working electrode.Attorney Docket No.132414-5009-WO 116. The system according to claim 115, wherein the working electrode comprises a transition metal selected from the group consisting of Au, Ti, W, Pt, and Ir.

117. The system according to claim 115 or 116, wherein the working electrode comprises Au.

118. The system according to any one of claims 81 or 112-117, wherein the working electrode comprises a polymer layer.

119. The system according to any one of claims 81 or 112-118, wherein the sensor further comprises a counter electrode.

120. The system according to claim 119, wherein the counter electrode comprises Pt.

121. The system according to any one of claims 81 or 112-120, wherein the sensor further comprises a reference electrode.

122. The system according to claim 121, wherein the reference electrode comprises Ag.

123. The system according to any one of claims 81 or 112-122, wherein the nanowell array electrode further comprises an insulation layer.

124. The system according to claim 123, wherein the insulation layer comprises SiO2.

125. The system according to any one of claims 81 or 112-124, wherein the nanowell array electrode comprises a total active area of from 10 to 300 μm2.

126. The system according to any one of claims 80-125, wherein the sensor and the processor are implemented as a system on a chip (SoC).

127. The system according to any one of claims 80-125, wherein the sensor and the processor are implemented as a single device.

128. The system according to any one of claims 80-125, wherein the sensor and the processor are implemented as separate devices.

129. The system according to any one of claims 80-128, wherein the sensor and the processor are communicably connected to each other.

130. The system according to any one of claims 80-129, wherein the processor is further configured to process detecting result from the sensor to output the visualized data.

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