System and method for a digital, multiplexed, extracellular vesicle-derived biomarker diagnostic lab-on-a-chip

Abstract

In one embodiment, a method for isolating and detecting one or more target biomarkers on a dielectrophoresis device comprises: receiving a first biological sample containing one or more target biomarkers on a dielectrophoresis (DEP) electrode array; applying a DEP force through a specific electrode of the DEP electrode array, wherein the intensity, direction, and duration of the DEP force are specific to one or more target biomarkers of the first biological sample; and determining the amount of one or more target biomarkers of the first biological sample via a digital sensor.

Description

Technical Field

[0001] The present disclosure generally relates to a lab-on-a-chip diagnostic platform, and more particularly to the detection of extracellular vesicle biomarkers using lab-on-a-chip diagnostics.

Background Art

[0002] There is an increasing need for diagnostic markers for early disease detection that can increase survival rates. Early disease detection requires highly sensitive diagnostic tools for detecting small amounts of biomarkers indicative of disease. There are few methods with the sensitivity and specificity required to detect diseases such as cancer and neurodegenerative diseases before they progress significantly. Therefore, it may be useful to develop very sensitive lab-on-a-chip tests for the detection of early diseases.

[0003] Extracellular vesicles (EVs) are membranous nanoparticles that facilitate the intercellular communication system through their biomolecular components (e.g., proteins, lipids, carbohydrates, and nucleic acids). EVs are dense information compartments that are continuously released from the original cells and contain biomarkers similar to the original cells. EVs are present in biological fluids (e.g., blood, urine, cerebrospinal fluid, etc.), and the markers associated with EVs can exhibit a longer half-life and increased stability compared to freely circulating biomarkers. Therefore, EVs provide an available source of biomarkers that are continuously released from living cells in the body.

[0004] Methods for detecting cancer and neurodegenerative diseases often use costly, time-consuming, and invasive methods (e.g., tissue biopsies, computed tomography, magnetic resonance imaging, endoscopies, etc.), and there are no tests available for early detection of many of these diseases. Currently, liquid biopsy tests utilize freely circulating markers released during tumor cell death, rather than continuous and ongoing cellular processes such as EV secretion. Thus, EVs represent a useful biocompartment for detecting disease-related biomarkers from parental cells (e.g., tumor cells, neurons affected by neurodegeneration, inflammatory cells, etc.) early and with very low invasiveness. Existing methods for detecting EVs either use dirty "brute force" concentration methods such as centrifugation and size exclusion, or utilize dilution circulation concentration. Many existing EV biomarker test methods use fluorescence-based detection, which has limited sensitivity and specificity. Therefore, it may be useful to improve the prior art through efficient biomarker concentration and highly sensitive and specific biomarker detection using electronic methods. Summary of Specific Embodiments

[0005] In certain embodiments, a biological sample can be loaded onto a solid-state detection device, where EV102 can be concentrated and / or isolated using DEP. The detection device (e.g., a lab-on-a-chip device) can utilize built-in microfluidics (either conventional / pneumatic and / or digital) to divide the concentrated EV volume and automate multimarker antibody-based capture. By way of example and not limitation, each biomarker concentration can be directly quantified using a sensor, where data from the sensor can be processed through one or more algorithms to yield a composite result.

[0006] In certain embodiments, a method for isolating and detecting one or more target biomarkers on a dielectrophoresis (DEP) device may comprise receiving, on a DEP electrode array of the DEP device, a first biological sample comprising one or more target biomarkers 104. In certain embodiments, one or more processors of the DEP device may instruct the DEP device to apply a DEP force through one or more electrodes of the DEP electrode array. By way of example and not limitation, one or more processors of the DEP device may instruct the DEP device to apply a DEP force of a particular intensity, direction, duration, or other configurable setting specific to one or more target biomarker(s) of a biological sample (e.g., EV). In certain embodiments, one or more sensors of the DEP device may determine the amount of one or more target biomarkers of the biological sample.

[0007] In certain embodiments, a method for detecting a target biomarker on a DEP device may comprise receiving, on one or more arrays of the DEP device, a first biological sample comprising one or more target biomarkers. In certain embodiments, a current may be applied through one or more electrode arrays to separate one or more biomarkers from a plurality of biomarkers in the biological sample. In certain embodiments, the DEP device may filter a particular target biomarker from a plurality of biomarkers in the biological sample into one or more chambers of the DEP device via one or more microfluidics. In certain embodiments, one or more particular biomarkers may be tagged with one or more markers, which may then be detected via one or more sensors, where the amount of each biomarker may be determined and displayed.

[0008] In certain embodiments, a method for detecting at least one target biomarker on a device may comprise receiving a first biological sample and transferring the first biological sample through a microfluidic channel to a first chamber. By way of example, and not limitation, a first process may be performed on the first biological sample in the first chamber. In certain embodiments, the first biological sample may be passed from the first chamber through a microfluidic channel to a second chamber. By way of example, and not limitation, a second process may be performed on the first biological sample in the second chamber. In certain embodiments, the first biological sample may be passed from the second chamber through a microfluidic channel to a third chamber. By way of example, and not limitation, a third process may be performed on the first biological sample in the third chamber.

[0009] In certain embodiments, a method for isolating at least one target biomarker on a solid state device may comprise receiving a first biological sample on the solid state device. The solid state device may then transfer the first biological sample to a DEP chamber. By way of example, and not limitation, one or more processes may be performed on the first biological sample in the DEP chamber. In certain embodiments, the solid state device may pass the first biological sample through a microfluidics (MF) channel to a tagging chamber, where one or more target biomarkers of the first biological sample may be tagged based on one or more characteristics.

[0010] In certain embodiments, the solid state device may pass one or more target biomarkers from the tagging chamber to a sensor chamber. In certain embodiments, the solid state device may digitally determine the amount of one or more target biomarkers based on the amount of tags in the first biological sample within the sensor chamber.

[0011] In certain embodiments, a method 1800 for isolating at least one target biomarker on a solid-state device. In stage 1810, one or more processors of the solid-state device may provide instructions for receiving a first biological sample within the solid-state device. By way of example, but not limitation, the first biological sample may include one or more target biomarkers. As another example, but not limitation, the first biological sample may be input via a microfluidics (MF) channel.

[0012] In certain embodiments, one or more processors of the solid-state device may provide instructions for transferring the first biological sample via the MF to a DEP chamber and applying an alternating current (AC) through one or more electrodes of the DEP chamber to the first biological sample.

[0013] In certain embodiments, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to transfer the first biological sample from the MF to a tagging chamber from the DEP chamber.

[0014] In certain embodiments, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to assign a tag to each of one or more target biomarkers of the first biological sample. By way of example, but not limitation, each tag may define a particular label among a plurality of labels. In certain embodiments, the first target biomarker of the first biological sample may be assigned a first tag based on one or more particular characteristics. In certain embodiments, the second target biomarker of the first biological sample may be assigned a second tag based on one or more particular characteristics.

[0015] In certain embodiments, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to transfer a first biological sample to one or more detector chambers via MF, where each detector chamber is programmed for a particular target biomarker.

[0016] In certain embodiments, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to digitally determine the amount of one or more target biomarkers based on assigned tags by one or more detectors within one or more detector chambers.

[0017] For early cancer detection, certain technical challenges exist. One technical challenge may involve utilizing freely circulating markers released during tumor cell death. To address this challenge, the solution presented by the embodiments disclosed herein may be a solid-state device for isolating specific biomarker concentrations. Another technical challenge may involve detecting biomarker concentrations. To address this challenge, the solution presented by the embodiments disclosed herein may be a solid-state device that isolates specific biomarkers but also quantifies biomarker concentrations in a digital manner.

[0018] Certain embodiments disclosed herein may provide one or more technical advantages. The technical advantages of the embodiments may include utilizing dielectrophoresis (DEP) on the solid-state device. Another technical advantage of the embodiments may include utilizing multiple chambers on the solid-state device, where each chamber may perform a process specific to the biological sample. Certain embodiments disclosed herein may not provide the above technical advantages, or may provide some or all of the above technical advantages. One or more other technical advantages may become readily apparent to those skilled in the art in view of the figures, description, and claims of the present disclosure.

[0019] The embodiments disclosed in this specification are merely examples and the scope of the present disclosure is not limited thereto. A particular embodiment may or may not include all, some, or none of the components, elements, features, functions, operations, or steps of the embodiments disclosed herein. The embodiments described in the present invention are disclosed in particular in the appended claims relating to a method, apparatus or system, where any feature mentioned in one claim category, for example a method, may also be claimed in another claim category, for example a system. The dependencies or references in the appended claims are selected only for formal reasons. However, any subject matter resulting from an intentional backward reference to any previous claim (in particular multiple dependencies) may equally be claimed, so that any combination of claims and their features may be disclosed and claimed, regardless of the dependencies selected in the appended claims. The subject matter that may be claimed includes not only combinations of features set forth in the appended claims, but also any other combination of features in the claims, and each feature described in the claims may be combined with any other feature or combination of other features in the claims. Furthermore, any of the embodiments and features described or illustrated herein may be claimed in an individual claim and / or in any combination with any of the embodiments or features described or illustrated herein or features of the appended claims.

Brief Description of the Drawings

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[0044] FIG. 1A shows an exemplary diagram 100 of exemplary surface chemistries and capture techniques. As used herein, "extracellular vesicles (EVs)" can refer to membranous nanoparticles secreted from cells that can facilitate intercellular communication through their biomolecular components. In certain embodiments, one or more EVs 102 can include a biomarker 104 on the surface of the EV 102. The previously described properties of the EV 102 can provide an opportunity for the early detection of biomarkers corresponding to early stage diseases. Thus, the combined detection of the presence of multiple cancer-related biomarkers from EVs 102, in conjunction with analysis using advanced machine learning (ML) algorithms, can be useful for the sensitive and specific diagnosis of early stage cancer and other diseases from biological fluids. Disclosed herein are compositions, methods, and exosome detection devices for biomarker detection.

[0045] In certain embodiments, a biological sample can be loaded onto an electrode array, where the EVs 102 can be concentrated and / or isolated using dielectrophoresis (DEP). A detection device (e.g., a lab-on-chip device) can utilize built-in microfluidics (either conventional / pneumatic and / or digital) to divide the concentrated EV volume and automate multimarker antibody-based capture. By way of example and not limitation, the concentration of each biomarker can be directly quantified using an electrochemical sensor, where the data from the sensor can be processed through one or more algorithms to yield a synthetic result.

[0046] In certain embodiments, the biological fluid can be obtained via standard point-of-care procedures including, but not limited to, blood collection by a vacuum blood collection tube (e.g., in the case of blood), spinal puncture (e.g., in the case of cerebrospinal fluid), and urine collection devices (e.g., in the case of urine). In certain embodiments, the biological fluid can be processed to remove interfering cells. By way of example, and not limitation, processing of the biological fluid can involve centrifugation, filtration through a membrane, and / or other standard preparation procedures for preparing a biological sample for testing. The resulting sample can be plasma, serum, CSF, and / or urine.

[0047] In certain embodiments, the prepared biological sample can be flowed over an array of energized electrodes that apply dielectrophoretic (DEP) forces to the particles in the biological sample. The strength and direction of the DEP forces can be specific to multiple biological particles in the sample, enabling the isolation and further concentration of EV102 from the biological sample. As used herein, “dielectrophoresis (DEP)” can refer to the phenomenon in which a force is applied to dielectric particles, molecules, or macromolecular structures in an aqueous or organic solution when exposed to a non-uniform electric field. In certain embodiments, the period during which the DEP force is applied can be specific to a particular target biomarker in the biological sample.

[0048] In certain embodiments, the isolated and concentrated EVs can be mixed with immunochemical reagents and incubated. After immunochemistry, each of the one or more samples can be loaded onto a sensor array. By way of example, and not limitation, the original volume of EV102 may or may not be separated into multiple droplets at any point during this process, depending on the required workflow. In certain embodiments, the sensor array can include one or more digital sensors, where each digital sensor can determine the amount of one or more target biomarkers (e.g., EV102).

[0049] In certain embodiments, one or more EV102s can be suspended in solution, where biomarker 104 can be found on the surface of EV102. By way of example, and not limitation, labels of biomarker 104 can include, but are not limited to, antibodies, antibodies conjugated to enzymes, antibodies conjugated to metal nanoparticles, antibodies conjugated to cleavable single-stranded DNA barcodes, antibodies conjugated to cleavable single-stranded DNA barcodes conjugated to metal nanoparticles, antibodies conjugated to cleavable single-stranded DNA barcodes conjugated to enzymes, antibodies conjugated to cleavable single-stranded DNA barcodes conjugated to enzymes, and / or binding of antibodies conjugated to cleavable single-stranded DNA barcodes conjugated to enzymes. Cleavable linkages to antibodies can include, but are not limited to, proteolytic, chemical, electrochemical, and photolytic labile compounds. By way of example, and not limitation, EV102 can include a specific target biomarker on the surface of EV102, where the detector surface can be functionalized to capture EV102 and biomarker 104, where a detectable event can be used to quantify the presence of the biomarker. Table 1 below outlines a plurality of configurations, EV labels, label release mechanisms, surface capture mechanisms, and detected events.

[0050] In certain embodiments, biomarker 104 can be tagged with one or more labels and / or markers. Table 1: Examples of configurations of EV labels, label release mechanisms, surface capture mechanisms, and detected events

Table 1

[0051] As shown in Table 1, "ssDNA" can refer to single-stranded DNA and "MNP" can refer to metal nanoparticles. As shown in FIG. 1A, diagram 100 represents configuration "A" shown in Table 1, where there is no EV label or label release mechanism, the surface capture mechanism is a biomarker-specific antibody, and the event detected by binding event 110 is antibody-biomarker binding.

[0052] In certain embodiments, binding event 110 can be the detection by a sensor of a biomarker 104 of EV102 and a capture molecule 120. By way of example and not limitation, capture molecule 120 can be captured by an antibody. As used herein, “binding event” 110 can refer to a detectable event used to quantify the presence of a particular biomarker 104. By way of example and not limitation, when binding event 110 occurs, the event observed can either change the capacitance of surface chemistry 122 or increase the number of charge carriers, and either event can be measured electronically. Since the sensitivity of a particular biomarker 104 can vary, the detection device can be adjusted to a particular range of the subject.

[0053] As shown in diagram 100 of FIG. 1A, capture molecule 120 can be an antibody. By way of example and not limitation, a method of capturing an EV-derived biomarker (e.g., biomarker 104) and / or an EV-derived biomarker label can include exposing an electrode surface 124 (e.g., capture molecule) linked to an antibody 120 directly to EV102 with biomarker 104 such that the biomarker 104 attached to EV102 binds directly to and is captured by the antibody 120.

[0054] In certain embodiments, a method of capturing an EV-derived biomarker and / or an EV-derived biomarker label can include covalently attaching an antibody to a surface by leveraging the chemical reactivity of surface chemistry 122, where antibody 120 can bind directly to its EV-derived biomarker 104.

[0055] In certain embodiments, one or more surfaces 124 of the field effect transistor may be functionalized using standard surface chemistry processes, whereby chemical functional groups may be physically and / or chemically bonded via covalent bonds to a metal, metal oxide, glassy carbon, graphene, graphene nanoribbon, carbon nanotube, semiconductor, and / or dielectric surface. As used herein, "surface chemistry" 122 may refer to physical and chemical phenomena occurring at the interface between two phases. Surface chemistry 122 may include molecules with multiple chemical and / or physical reactive sites, whereby one site may interact physically and / or chemically with the surface and the other sites may be used to directly conjugate to a nucleic acid, protein, and / or other molecule of interest. Examples of surface chemistry 122 may include, but are not limited to, molecules containing functional groups such as silanes, thiols, disulfides, phosphonates, phosphonic acids, diazonium, alkenes, carboxylic acids, alkynes, alkanes, amines, ketones, esters, aldehydes, alcohols, amides, imines, hydrazines, ethers, nitriles, aromatics, halides, and azides, nanoparticles, and / or biomolecules. By way of example, and not limitation, surface chemistry 122 may be directly conjugated to nucleic acids, proteins, glycans, lipids, and / or other molecules used for the capture and / or detection of a biomarker of interest. In certain embodiments, an insulated gate field effect transistor (IGFET), extended gate field effect transistor (EGFET), and / or ion sensitive field effect transistor (ISFET) structure may functionalize the gate oxide instead of the metal gate.

[0056] In certain embodiments, a method for fabricating one or more electrodes for detecting one or more EV-derived biomarkers may include functionalizing a metal and / or metal oxide surface (hereinafter, “surface”) with one or more chemical groups, which may be referred to herein as surface chemistry 122. By way of example, but not limitation, the surface reactivity may be utilized to covalently attach single-stranded DNA to the surface. As another example, but not limitation, the inherent surface reactivity may be utilized to directly attach functionalized single-stranded DNA to the surface.

[0057] In certain embodiments, the working electrode surface 124 can function as a detector of one or more biomarkers 104. As used herein, "working electrode surface" can refer to one or more electrodes in an electrochemical system where a reaction of interest occurs. In certain embodiments, one or more EVs 102 are then moved in close proximity to the metal gates of one or more working electrode surfaces 124 during the capture and / or detection of biomarkers 104 mediated by biomolecules and / or molecules conjugated to the gate surface (e.g., hybridization by DNA, antibody binding, and / or enzymatic reactions). By way of example, and not limitation, one or more EVs 102 can be concentrated by DEP and moved in close proximity to the metal gates of one or more working electrode surfaces 124 (e.g., capture molecules 120). In certain embodiments, one or more EVs 102 in a biological sample can be labeled via one or more methods, where the labeled EVs can adhere to the surface of the working electrode surface 124. By way of example, and not limitation, one or more EVs 102 and / or biomarkers 104 can be labeled with one or more tags corresponding to one or more specific features of the EVs 102 and / or biomarkers 104. As another example, and not limitation, one or more EVs 102 and / or biomarkers 104 can be tagged with one or more specific markers, each marker corresponding to a specific feature of the EVs 102 and / or biomarkers 104. It should be understood that the terms labeled, tagged, and marked can be used interchangeably to describe the process of identifying one or more specific features of one or more EVs 102 and / or biomarkers 104. Although this disclosure describes labeling, tagging, and / or marking one or more specific features of one or more EVs 102 and / or biomarkers 104, this disclosure contemplates any suitable method for identifying and characterizing one or more specific features. In certain embodiments, the label on one or more EVs 102 can be cleaved from their respective biomarkers 104, where the working electrode surface 124 can capture only the label without EVs 102 at the surface via surface chemistry 122.As an example, without limitation, a method for releasing a label specific to an EV-derived biomarker may include a linker that can be cleaved chemically, photolytically, and / or electrochemically. The biomarker 104 and the linker between the detected biomolecule and / or molecules that can be cleaved chemically may include, but are not limited to, esters, carbamates, dialkoxydiphenylsilanes, azo, diazo, acylhydrazones, nitrobenzenesulfonamides, acylsulfonamides, and / or disulfides. The biomarker 104 and the linker between the detected biomolecule and / or molecules that can be cleaved photolytically may include, but are not limited to, nitrophenylethyl ethers and / or phenacyl esters. The biomarker 104 and the linker between the detected biomolecule and / or molecules that can be cleaved electrochemically may include, but are not limited to, aryl esters and imines.

[0058] In certain embodiments, digital sensing can occur when the target EV biomarker 104 is detected by a change in the ion concentration near the gate surface of the working electrode surface 124, mediated, for example, by an antibody binding event, a nucleic acid hybridization event, and / or an enzyme reaction. As an example, without limitation, the interaction between the biomarker 104 and its capture antibody 120 on the conjugated gate surface changes the electrical properties of the gate of the field effect transistor, resulting in a change in either the voltage or current of the transistor, depending on the surrounding circuit configuration. This electrical signal can then be amplified and digitized using an analog / digital converter.

[0059] In certain embodiments, when a force (e.g., DEP force, AC current) is applied to a biological sample within one or more chambers of a solid-state DEP device, the applied force can result in a change in the ion concentration of the biological sample.

[0060] In certain embodiments, the digital output for one or more biomarkers 104 can be analyzed by one or more machine learning algorithms. By way of example and not limitation, the one or more machine learning algorithms can include supervised learning, unsupervised learning, semi-supervised learning, deep learning, and / or reinforcement learning algorithms. In certain embodiments, a deep learning algorithm can include any artificial neural network (ANN) that can be utilized to learn deep levels of representation and abstraction from large amounts of data. For example, deep learning algorithms can include multi-layer perceptrons (MLPs), autoencoders (AEs), convolutional neural networks (CNNs), recurrent neural networks (RNNs), long short-term memory (LSTMs), gated recurrent units (GRUs), restricted Boltzmann machines (RBMs), deep belief networks (DBNs), bidirectional recurrent deep neural networks (BRDNNs), generative adversarial networks (GANs), and deep Q-networks, neural autoregressive distribution estimators (NADEs), adversarial networks (ANs), attention models (AMs), and ANNs such as deep reinforcement learning.

[0061] In certain embodiments, the digital output for one or more biomarkers 104 can be analyzed by one or more classification machine learning algorithms or functions that can include any algorithm that can utilize a supervised learning model (e.g., logistic regression, naive Bayes, stochastic gradient descent (SGD), k-nearest neighbor, decision tree, random forest, and support vector machine (SVM), etc.) to learn from the data input into the supervised learning model and make new observations or classifications based thereon.

[0062] The present disclosure refers to the machine learning algorithms described above, but the present disclosure contemplates any suitable machine learning algorithm. In certain embodiments, the one or more machine learning algorithms can analyze a wide range of data and can enable the detection of early-stage cancer (e.g., stage I, stage II) and / or other target diseases.

[0063] Figure 1B shows an exemplary diagram 130 of surface chemistry 122 and capture techniques. In certain embodiments, EV102 may include one or more biomarkers 104 on the surface of EV102. In certain embodiments, the label 140 may include, for example, an antibody, where the label 140 may bind to the biomarker 104 on EV102, and where the label 140 may be captured by the antibody 120 on the surface. As another example, without limitation, the label 140 may include an antibody and an MNP, where the capture antibody 120 may bind to the labeled antibody and result in an MNP proximity event. In certain embodiments, as shown in Figure 1B, the diagram 130 may represent Configuration "B" of Table 1, where the label 140 may label the antibody, there is no label release mechanism, and the surface capture mechanism is a secondary (capture) antibody binding to the EV-labeled antibody, and the event detected at the binding event 110 is an antibody-antibody binding.

[0064] In certain embodiments, as shown in Figure 1B, the diagram 130 may represent Configuration "D" of Table 1. As an example, without limitation, the label 140 may label the antibody-MNP without a label release mechanism, the surface capture mechanism may be a secondary capture antibody binding to the EV-labeled antibody 140, and the event detected at the binding event 110 may be an MNP proximity event.

[0065] In certain embodiments, the event 110 may be detected by the working electrode surface 124 and the surface chemistry 122.

[0066] Figure 1C shows an exemplary diagram 150 of surface chemistry 122 and capture techniques. In certain embodiments, EV102 may include one or more biomarkers 104 and one or more labels 140 on the surface of EV102. In certain embodiments, as represented in Figure 1C, the diagram 150 may represent Configuration "C" of Table 1, where the label 140 may include an antibody-enzyme conjugate without a label release mechanism, the surface capture mechanism may be a secondary antibody 120 that binds to the EV-labeled antibody, and the event detected at the event 160 may be an enzyme activity.

[0067] In certain embodiments, event 160 can be detected by working electrode surface 124 and surface chemistry 122.

[0068] FIG. 1D shows an exemplary diagram 170 of exemplary surface chemistry 122 and capture techniques. In certain embodiments, EV102 can include one or more biomarkers 104 and one or more labels 140 on the surface of EV102. In certain embodiments, as represented in FIG. 1D, diagram 170 can represent configuration "E-1" of Table 1, where label 140 can be an antibody-ssDNA barcode with a chemical label release mechanism. By way of example, and not limitation, the label release mechanism can be represented by step 180, where the label can be cleaved from EV102. In this example, biomarker 104 and label 140 can be measured without EV102. By way of example, and not limitation, label 140 can represent a combination of antibody-DNA and / or antibody-DNA-MNP. In an example of configuration "E-1", the surface capture mechanism can be a DNA barcode that hybridizes to complementary ssDNA, where the detected binding event 110 can be DNA hybridization.

[0069] In certain embodiments, as represented in FIG. 1D, diagram 170 can represent configuration "E-2" of Table 1, where label 140 can be an antibody-ssDNA barcode with a photo label release mechanism. In an example of configuration "E-2", the surface capture mechanism can be a DNA barcode that hybridizes to complementary ssDNA, where the detected binding event 110 can be DNA hybridization.

[0070] In certain embodiments, as represented in FIG. 1D, diagram 170 may represent configuration "E-3" of Table 1, where label 140 may be an antibody-ssDNA barcode with an electrochemical label release mechanism. In an example of configuration "E-3", the surface capture mechanism may be a DNA barcode that hybridizes to a complementary ssDNA, where the detected binding event 110 may be DNA hybridization.

[0071] In certain embodiments, diagram 170 may represent configuration "G-1" of Table 1, where label 140 may be an antibody-ssDNA barcode-MNP with a chemical label release mechanism. In an example of configuration "G-1", the surface capture mechanism may be a DNA barcode that hybridizes to a complementary ssDNA, where the detected binding event 110 may be MNP proximity.

[0072] In certain embodiments, diagram 170 may represent configuration "G-2" of Table 1, where label 140 may be an antibody-ssDNA barcode-MNP with a photochemical label release mechanism. In an example of configuration "G-2", the surface capture mechanism may be a DNA barcode that hybridizes to a complementary ssDNA, where the detected binding event 110 may be MNP proximity.

[0073] In certain embodiments, diagram 170 may represent configuration "G-3" of Table 1, where label 140 may be an antibody-ssDNA barcode-MNP with an electrochemical label release mechanism. In an example of configuration "G-3", the surface capture mechanism may be a DNA barcode that hybridizes to a complementary ssDNA, where the detected binding event 110 may be MNP proximity.

[0074] In certain embodiments, event 110 may be detected by the working electrode surface 124 and surface chemistry 122.

[0075] FIG. 1E shows an exemplary diagram 190 of exemplary surface chemistry 122 and capture techniques. In certain embodiments, EV 102 may include one or more biomarkers 104 and one or more labels 140 on the surface of EV 102. In certain embodiments, as represented in FIG. 1E, diagram 190 may represent configuration "F-1" of Table 1, where the EV label may be an antibody-ssDNA barcode with a chemical label release mechanism. By way of example, and not limitation, the label release mechanism may be represented by step 180, where the label may be cleaved from EV 102. In this example, label 140 may be measured without EV 102. By way of example, and not limitation, label 140 may represent a combination of antibody-DNA and / or antibody-DNA-enzyme. In an example of configuration "F-1", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where the detected event 160 may be polymerase activity using DNA polymerase.

[0076] In certain embodiments, diagram 190 may represent configuration "F-2" of Table 1, where label 140 may be an antibody-ssDNA barcode with a photo label release mechanism. In an example of configuration "F-2", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where the detected event 160 may be polymerase activity using DNA polymerase.

[0077] In certain embodiments, diagram 190 may represent configuration "F-3" of Table 1, where label 140 may be an antibody-ssDNA barcode with an electrochemical label release. In an example of configuration "F-3", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where the detected event 160 may be polymerase activity using DNA polymerase.

[0078] In certain embodiments, diagram 190 may represent configuration "H-1" of Table 1, where label 140 may be an antibody-ssDNA barcode-enzyme with a chemical label release mechanism. In an example of configuration "H-1", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where detected event 160 may be polymerase activity using DNA polymerase.

[0079] In certain embodiments, diagram 190 may represent configuration "H-2" of Table 1, where label 140 may be an antibody-ssDNA barcode-enzyme with a photo label release mechanism. In an example of configuration "H-2", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where detected event 160 may be polymerase activity using DNA polymerase.

[0080] In certain embodiments, diagram 190 may represent configuration "H-3" of Table 1, where label 140 may be an antibody-ssDNA barcode-enzyme with an electrical label release mechanism. In an example of configuration "H-3", the surface capture mechanism may be a DNA barcode hybridized to complementary ssDNA, where detected event 160 may be polymerase activity using DNA polymerase.

[0081] In certain embodiments, event 160 may be detected by working electrode surface 124 and surface chemistry 122.

[0082] Figure 2 shows an exemplary flowchart 200 of a sample preparation and isolation and / or concentration chamber. In certain embodiments, flowchart 200 of FIG. 2 may illustrate a chamber and fluid channel architecture for a sample preparation and isolation / concentration chamber. In certain embodiments, flowchart 200 may begin at stage 202 where a biological sample is placed within the system and passed to sample preparation chamber 204. By way of example and not limitation, the sample preparation chamber may perform a plasma purification process. As another example and not limitation, the sample preparation chamber may perform a conductivity change. By way of example and not limitation, isolation and / or concentration may be achieved using DEP.

[0083] In certain embodiments, flowchart 200 may then pass the biological sample to DEP chamber 208, where DEP chamber waste 206 may be output from sample preparation chamber 204 and DEP reagent input 209 may be input to sample preparation chamber 204.

[0084] In certain embodiments, the biological sample may be passed from sample preparation chamber 204 to isolation / concentration chamber 210. In certain embodiments, a plurality of fluid reagents may be supplied to and from one or more fluid chambers using bulk and / or consumable reagent containers. In certain embodiments, a plurality of fluid reagents may be supplied to and from one or more fluid chambers using a fluid guiding manifold and a driving force that is either hydraulic, pneumatic, or electrostatic in nature. In an example where an electrostatic force is used, a method of electro-wetting the fluid using electrodes may be employed, where the electrodes may be disposed either in the fluid channel and either the top or bottom of the chamber, where the electrodes may be coated with a thin film selected to achieve a hydrophobic or hydrophilic nature to assist in electro-wetting.

[0085] In certain embodiments, the DEP chamber waste 206 can be output from the isolation / concentration chamber 210, and the DEP reagent input 209 can be input into the isolation / concentration chamber 210. In certain embodiments, the biological sample can be transferred from the isolation / concentration chamber 210 to the labeling chamber 214. By way of example, and not limitation, one or more processes (e.g., labeling of biomarkers) can occur within the labeling chamber 214. In certain embodiments, the DEP reagent input 209 can consist of one or more bulk reagents such as bulk reagent Z' 218, bulk reagent B' 220, and / or bulk reagent A' 222.

[0086] FIG. 3 shows an exemplary diagram 300 of a cross-sectional view of a dielectrophoresis concept. In certain embodiments, isolation of one or more EVs 102 can be achieved in a detection device by using DEP, where DEP is the net force acting on particles with an asymmetric polarizability in the presence of a radio frequency (RF) electric field. As shown by diagram 300, the plasma 320 of a biological sample can enter a DEP chamber (e.g., isolation and / or concentration chamber 210). By way of example, and not limitation, the plasma 320 can consist of EVs 102 and various other biological particles 312, 314, 316. In this example, the EVs 102 can be spherical objects with dimensions of about 30 to 10,000 nm. By way of example, and not limitation, for any given combination of voltage, frequency, electrode pitch, and / or electrode geometry, a partial range of this set of particles can be attracted to one or more electrodes net. This phenomenon can be called positive DEP. Hereinafter, particles that undergo positive DEP are called the induced band. As used herein, the "induced band" can refer to a partial range of EV102 sizes. For the sake of clarity to avoid misunderstanding, it is clear that the induced band is an adjustable range of EV102 sizes.

[0087] In certain embodiments, when an alternating current (AC) waveform is applied to the positive electrode 304 and the negative electrode 306, one or more EVs 102 can experience an attractive force that is a function of the RF frequency, voltage, plasma conductivity, EV particle size, and EV particle charge. For any given combination of voltages, it is understood that a sub-range of particle sizes can experience a net inducement to one or more of the electrodes.

[0088] In this example, the positive electrode 304 and / or the negative electrode 306 can be constructed of metal or any other suitable material. In certain embodiments, the positive electrode 304 and / or the negative electrode 306 can be separated by a dielectric material 302. In certain embodiments, EVs 102 in the induction band can be attracted to one or more of the positive electrode 304 and / or the negative electrode 306 on the surface of the dielectric material 302.

[0089] FIG. 4 shows an exemplary diagram 400 of a dielectrophoretic electrode array. In certain embodiments, one or more positive electrodes 304 and / or one or more negative electrodes 306 can be arranged in an interlocking configuration. By way of example, and not limitation, an interlocking configuration can create one or more regions with steep electric field gradients. In this example, the length 404 of the electrodes can be on the order of hundreds of microns to millimeters, where the width 402 of the electrodes can be less than 1 micron. Similarly, the pitch and thickness of the electrodes can be less than 1 micron. By way of example, and not limitation, the electrode material can be formed using sputtering. As another example, and not limitation, an alternating electrode deposition technique including, but not limited to, atomic layer deposition, chemical vapor deposition, physical vapor deposition, electroforming, or any other suitable method can be used. In certain embodiments, the metal material of the positive electrode 304 and / or the negative electrode 306 can be either a transition metal or a noble metal. In practice, noble metals such as silver, gold, platinum, tungsten, ruthenium, rhodium, palladium, osmium, iridium, and / or any other suitable material are desirable.

[0090] In certain embodiments, a polymer film is coated on the surface of one or more positive electrodes 304 and / or one or more negative electrodes 306 and the dielectric 302. This polymer film is a multi-component mixture spin-cast on the surface of a semiconductor wafer / chip. This material has a porosity adjusted for the band of induction, does not interfere with the electrical properties of one or more electrodes, and is adjusted to be non-adsorbent. After casting, the film can be cured using ultraviolet or thermal energy.

[0091] FIG. 5A shows an example of an isometric diagram 500 of a generalized fluid transport technique of a detection device. In certain embodiments, the detection device can be a consumable chip (e.g., a printed wiring board), where one or more biological samples (e.g., fluids) can be injected into the detection device by one or more syringes 510.

[0092] In certain embodiments, one or more syringes 510 of the detection device can be actuated by one or more motors 520, where the motor 520 can include one or more gears. In certain embodiments, the detection device can move fluid (e.g., a biological sample) through a manifold 530 to a plurality of solenoid-actuated valves 540 connected thereto. It should be understood that the solenoid-actuated valves can be arranged in parallel, in series, or in any other suitable configuration.

[0093] In certain embodiments, the detection device can move fluid through one or more reservoirs 550, where the one or more reservoirs 550 can be constructed of centrifuge tubes or any other suitable material. In certain embodiments, one or more syringes 510 of the detection device can be actuated by one or more motors 520, where the motors 520 can include one or more gears. In certain embodiments, the detection device can move fluid (e.g., a biological sample) through a manifold 530 to a plurality of solenoid-actuated valves 540 that are connected. It should be understood that the solenoid-actuated valves can be arranged in parallel, in series, or in any other suitable configuration. In certain embodiments, the detection device can move fluid through one or more reservoirs 550, where the one or more reservoirs 550 can be constructed of centrifuge tubes or any other suitable material.

[0094] In certain embodiments, the detection device can move fluid through one or more isolation and / or tagging chambers 560, where tagging of the fluid and / or isolation of the fluid can occur. By way of example and not limitation, the one or more isolation and / or tagging chambers 560 can tag specific chemical groups.

[0095] In certain embodiments, the detection device can move fluid through one or more sensor chambers 570. By way of example and not limitation, the detection device can receive one or more biological samples via one or more reservoirs 550, where the biological samples can be passed through one or more tagging and / or isolation chambers 560 and then through one or more sensor chambers 570. Although the present disclosure describes a particular order for processing biological samples within the detection device, the present disclosure contemplates any suitable order for processing biological samples within the detection device.

[0096] In certain embodiments, a particular bulk reagent can be dedicated to one or more specific chambers 560. By way of example, and not limitation, chamber 560 can include a detector chamber, where the detector chamber can receive one or more “detector” reagents. As another example, and not limitation, a particular detector reagent can be input into one or more specific chambers 560, or a particular detector reagent can be common to all of chambers 560. By way of example, and not limitation, each chamber of one or more chambers 560 can label a particular biomarker. For example, one chamber 560 can label one particular biomarker such as “biomarker 1,” where another chamber 560 can label “biomarker 2.” In this example, one or more chambers 560 can cleave a specific label, where the label is chemically bound to one or more sensors and can ultimately be quantified in a digital manner. In certain embodiments, one or more chambers 560 can receive fluid (e.g., biological sample) input and output waste from each particular chamber.

[0097] FIG. 5B shows an exemplary isometric diagram 580 of a generalized fluid transport technique. In certain embodiments, the assembly of the detection device described in diagram 500 of FIG. 5A can be encompassed within frame 590. By way of example, and not limitation, frame 590 can house one or more syringes 510, one or more motors 520, a manifold 530, one or more solenoid-actuated valves 540, one or more reservoirs 550, one or more chambers 560, and one or more sensors 570.

[0098] FIG. 6 shows an exemplary diagram 600 of a device architecture for examining a plurality of EV-derived biomarkers 104 from one or more biological samples using fluid multiplexing. In certain embodiments, isolation of the biological sample can be performed in a single chamber, where a sample sub-volume of the biological sample can be directed to biomarker-specific labeling and detection chambers (e.g., chamber 560). By way of example and not limitation, one or more reservoirs 550 containing the biological sample can be input into the device via a digital microfluidics sample input 610, where the one or more biological samples can be passed, flowed, or migrated through a dielectrophoresis (DEP) chamber 620. The DEP chamber 620 can be the only chamber within the device of diagram 600, or it should be understood that the DEP chamber 620 can be one of many DEP chambers 620 within the device of diagram 600.

[0099] In certain embodiments, the DEP chamber 620 can process one or more biological samples, where the biological samples can be passed through digital microfluidics (DMF) channels 630 until the biological samples reach one or more of the tagging chambers. It should be understood that the DMF channels 630 can also be microfluidics channels instead of digital microfluidics (DMF) channels. As shown in the diagram 600 of FIG. 6, one or more biological samples can be processed and tagged in a plurality of chambers such as tagging chamber "1" 640, tagging chamber "2" 642, and / or tagging chamber "N" 644. In certain embodiments, the biological samples can be passed from one or more of the tagging chambers 640, 642, 644, through the DMF channels 630, to one or more sensor chambers 650, 652, and / or 654. In certain embodiments, a single common detector chemistry can be used for each of the sensor chambers 650, 652, and / or 654. In this example, since the chemistry can be common, the specificity of what is detected can be determined by the label, where only the labeled exosomes of the biological samples can be detected after the one or more biological samples pass through the tagging chambers 640, 642, and / or 644. In this example, each of the N labeled chambers can label only one distinct specific biomarker. In certain embodiments, in one or more of the tagging chambers (e.g., tagging chambers 640, 642, 644), the label specific to the EV and / or biomarker can be cleaved from the EV102 and then can be chemically bound to one or more sensors (e.g., sensor 570), where the label can be quantified digitally or by suitable means.

[0100] FIG. 7 shows an exemplary diagram 700 of a device architecture for examining a plurality of EV-derived biomarkers 104 from one or more biological samples using fluid multiplexing. In certain embodiments, isolation of the one or more biological samples can be performed in a single chamber, where a sub-volume of the one or more biological samples can be directed to biomarker-specific labeling and detection chambers. By way of example, and not limitation, the one or more biological samples can be input to the device via a DMF sample input 610, where the one or more biological samples can be directed to a DEP chamber 620. The DEP chamber 620 can be the only chamber within the device of diagram 700, or it should be understood that the DEP chamber 620 can be one of many DEP chambers 620 within the device of diagram 700. Although the present disclosure describes directing the one or more biological samples to one or more chambers (e.g., DEP chamber 620), it should be understood that the present disclosure contemplates any suitable method for directing, transferring, and / or passing the one or more biological samples to one or more chambers and / or channels.

[0101] In certain embodiments, the DEP chamber 620 can process one or more biological samples, where the biological samples can be passed through the digital microfluidics (DMF) channels 630 until the biological samples reach one or more of the tagging chambers. As shown by the diagram 700 of FIG. 7, the one or more biological samples can be processed in a plurality of chambers, such as the tagging chamber "1" 640 and / or the tagging chamber "N" 644, and one or more tags can be assigned. In certain embodiments, the one or more biological samples can be passed from the one or more tagging chambers 640, 644, through the DMF channels 630, to an array of one or more detectors 710, 720, 730. In certain embodiments, each of the plurality of detector arrays can be programmed to detect a specific biomarker. By way of example, but not limitation, the detector array 710 can be specifically programmed for a specific biomarker, such as "biomarker 1". Similarly, the detector array 720 can be programmed for "biomarker 2", and the detector array 730 can be programmed for "biomarker N". In this example, each detector array can be programmed to operate with a different detector chemistry, where each detector chamber can detect the attachment of one distinct specific labeled biomarker. In certain embodiments, a label specific to an EV and / or biomarker can be cleaved from the EV 102 in the tagging chamber "1" 640, where the label can be chemically bound to one or more sensors (e.g., detector arrays 710, 720, 730) and quantified in a digital manner.

[0102] FIG. 8 shows an exemplary diagram 800 of a device architecture for examining a plurality of EV-derived biomarkers 104 from one or more biological samples using fluid multiplexing. In certain embodiments, isolation of the one or more biological samples can be performed in a single chamber, where a sub-volume of the one or more biological samples can be directed to biomarker-specific labeling and detection chambers. By way of example, and not limitation, the one or more biological samples can be input into the device via a DMF sample input 610, where the one or more biological samples can be directed to a DEP chamber 620. The DEP chamber 620 can be the only chamber within the device of diagram 800, or it should be understood that the DEP chamber 620 can be one of many DEP chambers 620 within the device of diagram 800.

[0103] In certain embodiments, the DEP chamber 620 can process one or more biological samples, where the biological samples can be passed through digital microfluidics (DMF) channels 630 until the biological samples reach one or more of the tagging chambers. By way of example, and not limitation, the DMF channels 630 can be microfluidics channels. As shown by the diagram 800 of FIG. 8, one or more biological samples can be processed and tagged by a single tagging chamber, such as tagging chamber “1” 640, where the biological samples can be tagged and labeled. By way of example, and not limitation, the labeled sample 810 can be passed from the tagging chamber “1” 640 through the DMF channels 630 or microfluidics channels to one or more detector arrays 710, 720, 730. As described with respect to FIG. 7, each of the plurality of detector arrays can be programmed to detect a particular biomarker. By way of example, and not limitation, the detector array 710 can be specifically programmed for a particular biomarker, such as “biomarker 1”. Similarly, the detector array 720 can be programmed for “biomarker 2”, and the detector array 730 can be programmed for “biomarker N”. In the example of the diagram 800 of FIG. 8, the volume of the labeled EVs (e.g., labeled sample 810) can be passed through all of the detection chambers (e.g., detector arrays 710, 720, 730), thereby increasing the efficiency of biological sample utilization. As described in FIGS. 6-7, labels specific to EVs and / or biomarkers can be cleaved from the EV 102 in the tagging chamber “1” 640 and then chemically bound to one or more sensors, where the labels can be quantified in a digital manner.

[0104] FIG. 9 shows an exemplary schematic diagram 900 that displays the open circuit potential of a working electrode. In certain embodiments, the circuit architecture of the device displayed by diagram 900 can be used to measure changes in the floating voltage of working electrode 940 compared to reference electrode 920, which is held at a potential by counter electrode 930. By way of example and not limitation, the circuit of diagram 900 can include voltage source 950, fluid cell 970, digital / analog converter (DAC) 952, analog / digital converters 960, 962, diodes 954, 956, 958, reference electrode 920, counter electrode 930, and working electrode 940. In certain embodiments, a method of providing an electronic circuit for detecting one or more events as previously described can use any of an open circuit potential configuration, an electrochemical impedance spectroscopy configuration, a cyclic voltammetry configuration, or an ammeter configuration to detect immunochemical potential changes, immunochemical impedance changes, and / or immunochemical current changes from an independent reference electrode (e.g., reference electrode 920), counter electrode 930, and working electrode 940 (displayed in diagram 900 of FIG. 9 and diagram 1000 of FIG. 10). By way of example and not limitation, reference electrode 920, counter electrode 930, and / or working electrode 940 can be made from any metal, conductive metal oxide, or suitable conductive material (e.g., graphene) including but not limited to Pt, Au, Ag, Ag / AgCl, Zn, Ti, W, Pd, Ru, Pb, Cu, In, InxOy, ITO, AZO, ICO, graphene, etc.

[0105] In certain embodiments, each of the reference electrode 920, counter electrode 930, and / or working electrode 940 can be made of a separate material. In certain embodiments, any one of the reference electrode 920, counter electrode 930, and / or working electrode 940 can be functionalized, while the remaining electrodes are not functionalized. By way of example, but not limitation, the working electrode 940 can be functionalized, while the reference electrode 920 and counter electrode 930 may not be functionalized. In certain embodiments, the potential, impedance, and / or current at each of the reference electrode 920, counter electrode 930, and / or working electrode 940 can be monitored using one or more ADCs (e.g., ADCs 960, 962), where the current can be stored for determination of biomarker quantification.

[0106] FIG. 10 shows an exemplary schematic diagram 1000 that displays an exemplary electrode configuration. In certain embodiments, the circuit architecture of the device depicted by diagram 1000 can be used to measure changes in the floating voltage of the working electrode 940 compared to the reference electrode 920 held at the potential by the counter electrode 930. By way of example, but not limitation, the circuit of diagram 1000 can include a voltage source 950, a digital / analog converter (DAC) 952, analog / digital converters 960, 962, 1020, operational amplifiers 1032, 1034, 1036, 1038, a reference electrode 920, a counter electrode 930, and a working electrode 940. In certain embodiments, a switch 1010 can be added to the open circuit potential, thereby enabling current measurement and / or electrochemical impedance spectroscopy (EIS).

[0107] FIG. 11 shows an exemplary diagram 1100 of an electrode configuration in which an embodiment can operate. In a particular embodiment, the lower printed circuit board (PCB) 1110 can consist of a gasket 1132 and a working electrode 940. By way of example, and not limitation, the length 1150 of the lower PCB 1110 can be a dimension of about 12.7 millimeters, and the width 1140 of the lower PCB 1110 can be a dimension of about 12.7 millimeters. In a particular embodiment, the upper PCB 1120 can consist of gaskets 1132, one or more vents 1130, a reference electrode 920, and a counter electrode 1130. By way of example, and not limitation, the length 1150 of the upper PCB 1120 can be a dimension of about 12.7 millimeters, and the width 1140 of the upper PCB 1120 can be a dimension of about 12.7 millimeters. Although the present disclosure describes a particular geometry, the present disclosure contemplates any suitable geometry, including elliptical, rectangular, and / or non-planar electrode configurations.

[0108] In a particular embodiment, a detection device (e.g., a device) for quantifying EV-derived biomarkers in a biological sample can include a device platform capable of holding a cartridge. In a particular embodiment, a detection device (e.g., a device) for quantifying EV-derived biomarkers in one or more biological samples (e.g., blood, plasma, serum, cerebrospinal fluid, lymph fluid, saliva, urine, feces, cell lysate, cell culture medium) can include a device platform capable of holding a cartridge and delivering a sample, reagent, light, and / or an electrical pulse to a plurality of fluidic and electrical channels on the cartridge. By way of example, and not limitation, the device platform can include an electronics substrate with logic and / or power circuitry for driving and sensing an electronic device, sensor, and / or electrode. As another example, and not limitation, the device platform can include a fluid reservoir, tubing, and a fluid control system consisting of any of pneumatic, hydraulic, or electrical fluid control, where the fluid control can be actuated manually and / or electronically.

[0109] In certain embodiments, the fluid cartridge can be constructed from a molded upper material and a molded lower material, where the space between the upper and lower molded materials can accommodate one or more printed wiring boards and / or microelectronics chips. By way of example and not limitation, the upper and lower molded materials can be constructed from plastic or any other suitable material. In certain embodiments, one or more adhesive layers can form a fluid seal between the chambers and / or channels of the fluid cartridge. In certain embodiments, a "spacer" layer, in conjunction with one or more printed wiring boards, can define the boundaries of the fluid chambers and / or channels. By way of example and not limitation, the spacer can be constructed from plastic or any other suitable material. In certain embodiments, the fluid cartridge can include ports dedicated to the inlet and outlet of fluid for each channel. In certain embodiments, components including the walls of one or more fluid chambers and / or channels can be coated with a non-biofouling film. In certain embodiments, one or more valves can be included to direct a number of samples and / or reagents between various inlet and outlet ports and / or reservoirs.

[0110] In certain embodiments, a detection device for quantifying EV-derived biomarkers in a biological sample can include a fluid cartridge with a plurality of chambers and / or channels. In certain embodiments, a detection device for quantifying EV-derived biomarkers in a biological sample can include a printed circuit and / or a microelectronics silicon-based integrated chip (e.g., a complementary metal oxide semiconductor (CMOS) or silicon-based integrated chip), where the chip can be constructed to form a DEP cavity (e.g., DEP chamber 620). By way of example and not limitation, the DEP cavity can isolate and capture particles of an adjustable size and / or charge range from one or more biological samples (e.g., plasma samples).

[0111] In certain embodiments, a device for quantifying EV-derived biomarker 104 in a plasma sample may include a printed wiring board with electrodes and / or a silicon-based integrated chip, where the printed wiring board and / or the silicon-based integrated chip can electrostatically control one or more fluid transfer systems. In certain embodiments, a device for quantifying an EV-derived biomarker in a plasma sample may include a fluid supply (e.g., sample input 610) and / or a waste management system. In certain embodiments, a device for quantifying an EV-derived biomarker in a biological sample may include one or more reaction chambers. By way of example, but not limitation, one or more reaction chambers may include a DEP structure, where the DEP structure may assist in the labeling and / or cleavage of an EV-derived biomarker label.

[0112] In certain embodiments, a device for quantifying an EV-derived biomarker in a biological sample may include one or more printed circuits and / or microelectronics chips that include electrodes for the electrical capture and detection of labeled EVs and / or cleaved EV biomarker labels. In certain embodiments, a device for quantifying EV-derived biomarker 104 in a biological sample may include a plurality of electronics and / or sensors, where the electronics and / or sensors can report a digital sensor response that quantifies the presence of biomarker 104 and / or a biomarker label.

[0113] In certain embodiments, the EV biosensor device may perform DEP particle filtering to separate EV102 from other biological components, where the EV biosensor device may be constructed by one or more silicon chips. By way of example, but not limitation, the top of one or more silicon chips may be constructed by a metal layer, where the metal layer may include an alternating polarity electrode configuration that meshes with each other. In certain embodiments, the EV biosensor device may include an off-chip RF-AC waveform generator, where the RF AC waveform generator may apply one or more of a particular waveform to one or more electrodes (e.g., reference electrode 920, counter electrode 930, working electrode 940). In certain embodiments, the EV biosensor device may include an anti-biofouling surface coating or other coating on the electrodes within the DEP chamber 620 and / or on non-electrode surfaces.

[0114] FIG. 12 shows an exemplary schematic diagram 1200 displaying an electrode configuration example. In certain embodiments, the circuit architecture of the detection device displayed by diagram 1200, with the switch in the “open” position, can be used to measure the change in the open circuit potential of the working electrode 940 compared to the reference electrode 920 held at a potential by the counter electrode 930. Alternatively, the circuit architecture of the device displayed by diagram 1200, with the switch in the “closed” position, can be used to measure the current flowing through the working electrode 940 in response to a defined voltage on the reference electrode 920. By way of example, but not limitation, the circuit of diagram 1300 can include a digital / analog converter (DAC) 952, analog / digital converters (ADCs) 960, 962, 1020, operational amplifiers 1210, 1220, buffers 1230, 1240, a switch 1010, a reference electrode 920, a counter electrode 930, and a working electrode 940. In certain embodiments, a method of providing an electronic circuit for detecting one or more events as previously described can include an independent reference electrode (e.g., reference electrode 920), a counter electrode 930, and a working electrode 940 for detecting immunochemical potential changes, immunochemical impedance changes, and / or immunochemical current changes using either an open circuit potential configuration, an electrochemical impedance spectroscopy configuration, a cyclic voltammetry configuration, or an amperometry configuration. By way of example, but not limitation, the reference electrode 920, counter electrode 930, and / or working electrode 90 can be made of any metal, conductive metal oxide, or suitable conductive material.

[0115] In certain embodiments, each of the reference electrode 920, counter electrode 940, and / or working electrode 940 can be made of a distinct material. In certain embodiments, any one of the reference electrode 920, counter electrode 930, and / or working electrode 940 can be functionalized, while the remaining electrodes are not functionalized or are functionalized differently. In certain embodiments, the potential, impedance, and / or current at each of the reference electrode 920, counter electrode 930, and / or working electrode 940 can be monitored using one or more ADCs (e.g., ADCs 960, 962, 1020), where the current can be stored for determination of biomarker quantification.

[0116] FIG. 13 shows an exemplary diagram 1300 of an electrode configuration in which an embodiment can operate. In certain embodiments, the PCB 1330 can include a bottom surface 1380 that can consist of one or more working electrodes 940. By way of example and not limitation, the length 1312 of the bottom surface 1380 can be on the order of about 20 millimeters. As another example and not limitation, the width 1310 of the bottom surface 1380 can be on the order of about 10 millimeters. In certain embodiments, the top surface 1382 and the bottom surface 1380 can be separated by one or more layers of a transfer adhesive 1392 with one or more fluid ports 1340, 1350, and electrical via 1320.

[0117] In certain embodiments, the top surface 1382 can consist of electrical vias 1320, one or more reference electrodes 920, and one or more counter electrodes 930. By way of example and not limitation, the length 1312 of the top surface 1382 can be on the order of about 20 millimeters, and the width 1310 of the top surface 1382 can be on the order of about 10 millimeters. While the present disclosure describes approximate lengths 1312 and widths 1310 of the top surface 1382 and the bottom surface 1380, the present disclosure contemplates any suitable lengths 1312 and widths 1310. While the present disclosure describes a particular electrode configuration (e.g., the positions of the counter electrode 930, reference electrode 920, working electrode 940), the present disclosure contemplates any suitable configuration of the counter electrode 930, reference electrode 920, and / or working electrode 940.

[0118] FIG. 14 shows an exemplary method 1400 for isolating and detecting a target biomarker via a dielectrophoresis device. The method may begin at step 410 where the DEP device may receive a first biological sample containing one or more target biomarkers 104 on the DEP electrode array of the DEP device. At step 1420, one or more processors of the DEP device may instruct the DEP device to apply a DEP force through one or more electrodes of the DEP electrode array. By way of example, and not limitation, one or more processors of the DEP device may instruct the DEP device to apply a DEP force of a specific intensity, direction, duration, or other configurable setting specific to one or more target biomarker(s) 104 of the biological sample (e.g., EV 102). In certain embodiments, the DEP force may be an AC waveform. By way of example, and not limitation, the DEP force may be adjusted according to the size of the target biomarker 104.

[0119] In certain embodiments, the DEP electrode arrays may be arranged in an interlocking configuration with each other. In certain embodiments, the DEP electrode array may include a working electrode 940, a reference electrode 920, and / or a counter electrode 930. By way of example, and not limitation, the DEP electrode array may include one or more positive electrodes and one or more negative electrodes.

[0120] In certain embodiments, at step 1430, one or more sensors of the DEP device may determine the amount of one or more target biomarker(s) 104 of the biological sample. By way of example, and not limitation, the sensor may quantify the total number of tags, labels, and / or markers corresponding to a particular biomarker 104. In certain embodiments, the amount of one or more specific biomarker(s) 104 may be output and presented via the GUI of one or more computing devices.

[0121] Certain embodiments may repeat one or more steps of the method of FIG. 14 as needed. Although the present disclosure describes and illustrates certain steps of the method of FIG. 14 as occurring in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 14 may occur in any suitable order. The present disclosure describes and illustrates an exemplary method for isolating and detecting one or more target biomarkers on a DEP device that includes certain steps of the method of FIG. 14, but the present disclosure contemplates any suitable method for isolating and detecting one or more target biomarkers on a DEP device that may or may not include all or some of the steps of the method of FIG. 14 as needed. Further, the present disclosure describes and illustrates certain components, devices, or systems for performing certain steps of the method of FIG. 14, but the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 14.

[0122] FIG. 15 shows an exemplary method 1500 for detecting a target biomarker on a dielectrophoresis device. The method may begin at step 1510 where the DEP device may receive a first biological sample that includes one or more target biomarkers 104 on one or more arrays of the DEP device. At step 1520, one or more processors of the DEP device may be instructed to apply a current through one or more electrode arrays. By way of example and not limitation, one or more processors of the DEP device may instruct the DEP device to apply a DEP force of a particular intensity, direction, duration, or other configurable setting that is specific to one or more target biomarker 104 of the biological sample (e.g., EV 102). In certain embodiments, the DEP force may be an AC waveform. By way of example and not limitation, the DEP force may be adjusted according to the size of the target biomarker 104.

[0123] In certain embodiments, the DEP electrode arrays can be arranged in an interlocking configuration. In certain embodiments, the DEP electrode arrays can include the working electrode 940, the reference electrode 920, and / or the counter electrode 930. By way of example, and not limitation, the DEP electrode arrays can include one or more positive electrodes and one or more negative electrodes.

[0124] In certain embodiments, one or more biomarkers 104 can be separated from the volume of the biological sample in response to applying a current of a particular configuration to one or more electrode arrays.

[0125] In stage 1530, one or more processors of the DEP device can instruct the DEP device to filter one or more biomarkers 104 from the plurality of biomarkers 104 of the biological sample via one or more microfluidics. By way of example, and not limitation, the biomarker 104 can be filtered into one or more chambers of the DEP device.

[0126] In certain embodiments, in stage 1540, one or more processors of the DEP device can instruct the DEP device to tag one or more biomarkers 104 of the biological sample using markers. By way of example, and not limitation, the first biomarker can be marked with the first marker and the second biomarker can be marked with the second marker. As another example, and not limitation, the marker can be a plurality of labels, tags, or other forms of identifiers. In certain embodiments, the first marker can include a particular one of the plurality of labels. By way of example, and not limitation, the plurality of labels can include antibody labels, metal nanoparticle (MNP) labels, single-stranded DNA (ssDNA) labels, or any other suitable label.

[0127] In certain embodiments, at step 1550, one or more sensors of one or more chambers of the DEP device may detect the amount of a particular biomarker 104. At step 1560, the DEP device may output and present a composite result of the amount of the particular biomarker via the GUI of a computing device. In certain embodiments, one or more additional biological samples may be flowed onto the DEP device simultaneously or in parallel.

[0128] Certain embodiments may repeat one or more steps of the method of FIG. 15, as needed. Although the present disclosure describes and illustrates particular steps of the method of FIG. 15 as occurring in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 15 may occur in any suitable order. The present disclosure describes and illustrates an exemplary method for isolating and detecting one or more target biomarkers on a DEP device that includes particular steps of the method of FIG. 15, but the present disclosure contemplates any suitable method for isolating and detecting one or more target biomarkers on a DEP device that may or may not include all or some of the steps of the method of FIG. 15, as needed, and that includes any suitable steps. Further, the present disclosure describes and illustrates particular components, devices, or systems for performing particular steps of the method of FIG. 15, but the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 15.

[0129] Figure 16 shows an exemplary method 1600 for isolating at least one target biomarker on a device. The method may begin at step 1610 where the device may receive a first biological sample. By way of example and not limitation, the biological sample may include one or more biomarkers 104. At step 1620, one or more processors of the device may instruct the device to transfer the biological sample into a first chamber via a microfluidic channel. By way of example and not limitation, the first chamber may perform a specific process (e.g., a first process) on the first biological sample. In certain embodiments, the first chamber may include a sample preparation chamber where the first process may modify one or more characteristics of the biological sample. In certain embodiments, the first process may modify plasma purification of the first biological sample.

[0130] In certain embodiments, at step 1630, one or more processors of the DEP device may instruct the DEP device to transfer the first biological sample from the first chamber to a second chamber via one or more microfluidic channels. By way of example and not limitation, a specific process may be performed on the first biological sample in the second chamber. The specific process performed on the first biological sample in the second chamber may be the same or a different process than the process performed on the first biological sample in the first chamber. By way of example and not limitation, the second chamber may include a concentration chamber where an electric current applied to the first biological sample in the concentration chamber results in a change in ion concentration.

[0131] In certain embodiments, at stage 1640, one or more processors of the DEP device may instruct the DEP device to transfer a first biological sample from a second chamber to a third chamber via a microfluidic channel. By way of example, and not limitation, a third process may be performed on the first biological sample in the third chamber. In certain embodiments, the third chamber may include an isolation chamber, where an electric current applied to the first biological sample in the isolation chamber may result in the separation of specific target biomarkers of the first biological sample.

[0132] In certain embodiments, one or more reagents may be input into any one of the first, second, or third chambers. In certain embodiments, any one of the first, second, or third chambers may output waste including a portion of the first biological sample and a portion of one or more reagents resulting from the first process. It should be understood that the DEP device may repeat this process using specific biological samples (e.g., a second biological sample, a third biological sample, etc.). In certain embodiments, one or more biological samples may be processed simultaneously or in parallel.

[0133] Certain embodiments may repeat one or more steps of the method of FIG. 16 as needed. Although the present disclosure describes and illustrates the specific steps of the method of FIG. 16 as occurring in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 16 may occur in any suitable order. The present disclosure describes and illustrates an exemplary method for isolating and detecting one or more target biomarkers on a DEP device that includes specific steps of the method of FIG. 16, but the present disclosure contemplates any suitable method for isolating and detecting one or more target biomarkers on a DEP device that may or may not include all or some of the steps of the method of FIG. 16 as needed. Further, although the present disclosure describes and illustrates specific components, devices, or systems for performing specific steps of the method of FIG. 16, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 16.

[0134] FIG. 17 shows an exemplary method 1700 for isolating at least one target biomarker on a solid state device. In step 1710, one or more processors of the solid state device may provide instructions for receiving a first biological sample within the solid state device. By way of example and not limitation, the first biological sample may be received via one or more microfluidic channels.

[0135] In stage 1720, one or more processors of the solid-state device may instruct one or more components of the solid-state device to transfer a first biological sample to the DEP chamber. By way of example, and not limitation, one or more processes may be performed on the first biological sample in the DEP chamber. In certain embodiments, the DEP chamber may include one or more electrode arrays. By way of example, and not limitation, each of the one or more electrode arrays may be arranged in an interlocking configuration with each other. As another example, and not limitation, each of the one or more electrode arrays may include a working electrode 940, a reference electrode 920, and a counter electrode 930.

[0136] In certain embodiments, in stage 1730, one or more processors of the solid-state device may instruct one or more components of the solid-state device to pass the first biological sample through a microfluidics (MF) channel to a tagging chamber.

[0137] In certain embodiments, in stage 1740, one or more processors of the solid-state device may instruct one or more components of the solid-state device to process the first biological sample in the tagging chamber. By way of example, and not limitation, one or more target biomarkers of the first biological sample may be tagged based on one or more characteristics. In certain embodiments, one or more target biomarkers of the first biological sample may be tagged with a specific label of a plurality of labels. By way of example, and not limitation, the plurality of labels may include at least an antibody label, a metal nanoparticle (MNP) label, and / or a single-stranded DNA (ssDNA) label.

[0138] In certain embodiments, in stage 1750, one or more processors of the solid-state device may instruct one or more components of the solid-state device to pass one or more target biomarkers to a sensor chamber.

[0139] In certain embodiments, at stage 1760, one or more processors of the solid-state device may instruct one or more components of the solid-state device to digitally determine the amount of one or more target biomarkers based on the amount of tags in a first biological sample within the sensor chamber.

[0140] It should be understood that the solid-state device may repeat this process using a particular biological sample (e.g., a second biological sample, a third biological sample, etc.). In certain embodiments, one or more biological samples may be processed simultaneously or in parallel.

[0141] Certain embodiments may repeat one or more stages of the method of FIG. 17 as needed. Although the present disclosure has described and illustrated the particular stages of the method of FIG. 17 as occurring in a particular order, the present disclosure contemplates that any suitable stages of the method of FIG. 17 may occur in any suitable order. The present disclosure has described and illustrated an exemplary method for isolating and detecting one or more target biomarkers on a solid-state device, including the particular stages of the method of FIG. 17, but the present disclosure contemplates any suitable method for isolating and detecting one or more target biomarkers on a DEP device that may or may not include all or some of the stages of the method of FIG. 17 as needed, and that includes any suitable stages. Further, although the present disclosure has described and illustrated particular components, devices, or systems for performing the particular stages of the method of FIG. 17, the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable stage of the method of FIG. 17.

[0142] Figure 18 shows an exemplary method 1800 for isolating at least one target biomarker on a solid state device. At step 1810, one or more processors of the solid state device may provide instructions for receiving a first biological sample within the solid state device. By way of example, and not limitation, the first biological sample may include one or more target biomarkers. As another example, and not limitation, the first biological sample may be input via a microfluidics (MF) channel.

[0143] At step 1820, one or more processors of the solid state device may provide instructions for transferring the first biological sample to a DEP chamber via the MF and applying an alternating current (AC) through one or more electrodes of the DEP chamber to the first biological sample. In certain embodiments, one or more electrodes of the DEP chamber may include at least a working electrode 940, a reference electrode 920, and a counter electrode 930.

[0144] At step 1830, one or more processors of the solid state device may provide instructions to one or more components of the solid state device for transferring the first biological sample from the MF to a tagging chamber from the DEP chamber.

[0145] In stage 1840, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to assign tags to each of one or more target biomarkers of the first biological sample. By way of example and not limitation, each tag may define a particular label among a plurality of labels. As another example and not limitation, the plurality of labels may include, but are not limited to, antibody labels, metal nanoparticle (MNP) labels, or single-stranded DNA (ssDNA) labels. In certain embodiments, the first target biomarker of the first biological sample may be assigned a first tag based on one or more specific characteristics. In certain embodiments, the second target biomarker of the first biological sample may be assigned a second tag based on one or more specific characteristics.

[0146] In stage 1850, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to transfer the first biological sample via the MF to one or more detector chambers, where each detector chamber is programmed for a particular target biomarker.

[0147] In stage 1860, one or more processors of the solid-state device may provide instructions to one or more components of the solid-state device to digitally determine the amount of one or more target biomarkers based on the assigned tags by one or more detectors within one or more detector chambers.

[0148] It should be understood that the solid-state device may repeat this process using a particular biological sample (e.g., a second biological sample, a third biological sample, etc.). In certain embodiments, one or more biological samples may be processed simultaneously or in parallel.

[0149] Certain embodiments may repeat one or more steps of the method of FIG. 18 as needed. Although the present disclosure describes and illustrates certain steps of the method of FIG. 18 as occurring in a particular order, the present disclosure contemplates that any suitable steps of the method of FIG. 18 may occur in any suitable order. The present disclosure describes and illustrates an exemplary method for isolating and detecting one or more target biomarkers on a solid-state device that includes certain steps of the method of FIG. 18, but the present disclosure contemplates any suitable method for isolating and detecting one or more target biomarkers on a solid-state device that may or may not include all or some of the steps of the method of FIG. 18 as needed and that includes any suitable steps. Further, the present disclosure describes and illustrates certain components, devices, or systems for performing certain steps of the method of FIG. 18, but the present disclosure contemplates any suitable combination of any suitable components, devices, or systems for performing any suitable steps of the method of FIG. 18. Systems and Methods

[0150] FIG. 19 shows an exemplary computer system 1900 that may be utilized to perform digital, multiplexed, extracellular vesicle-derived biomarker lab-on-a-chip diagnostics in accordance with the disclosed embodiments. In certain embodiments, one or more computer systems 1900 perform one or more steps of one or more of the methods described or illustrated herein. In certain embodiments, one or more computer systems 1900 provide the functionality described or illustrated herein. In certain embodiments, software executing on one or more computer systems 1900 performs one or more steps of one or more of the methods described or illustrated herein or provides the functionality described or illustrated herein. Certain embodiments include one or more portions of one or more computer systems 1900. As used herein, references to a computer system may, as needed, include a computing device and vice versa. Also, references to a computer system may, as needed, include one or more computer systems.

[0151] This disclosure contemplates any suitable number of computer systems 1900. This disclosure contemplates a computer system 1900 in any suitable physical form. By way of example, and not limitation, computer system 1900 may be an embedded computer system, a system-on-chip (SOC), a single-board computer system (SBC) (e.g., a computer-on-module (COM) or system-on-module (SOM)), a desktop computer system, a laptop or notebook computer system, an interactive kiosk, a mainframe, a mesh of computer systems, a mobile phone, a personal digital assistant (PDA), a server, a tablet computer system, an augmented / virtual reality device, or any combination of two or more thereof. Optionally, computer system 1900 may include one or more computer systems 1900; may be monolithic or distributed; may span multiple locations; may span multiple machines; may span multiple data centers; or may exist in the cloud, including one or more cloud components in one or more networks.

[0152] Optionally, one or more computer systems 1900 can perform one or more steps of one or more of the methods described or illustrated herein without substantial spatial or temporal limitation. By way of example, and not limitation, one or more computer systems 1900 can execute one or more steps of one or more of the methods described or illustrated herein in real time or in batch mode. One or more computer systems 1900 can perform one or more steps of one or more of the methods described or illustrated herein at different times or at different locations, as needed.

[0153] In certain embodiments, computer system 1900 includes processor 1902, memory 1904, storage 1906, input / output (I / O) interface 1908, communication interface 1910, and bus 1912. Although the present disclosure describes and illustrates a particular computer system having a particular number of particular components in a particular arrangement, the present disclosure contemplates any suitable computer system having any suitable number of any suitable components in any suitable arrangement. In certain embodiments, processor 1902 includes hardware for executing instructions, such as those that make up a computer program. By way of example and not limitation, to execute instructions, processor 1902 fetches (or retrieves) instructions from internal registers, internal cache, memory 1904, or storage 1906; decodes and executes them; and then may write one or more results to internal registers, internal cache, memory 1904, or storage 1906. In certain embodiments, processor 1902 may include one or more internal caches for data, instructions, or addresses. The present disclosure contemplates processor 1902 including any suitable number of any suitable internal caches, as needed. By way of example and not limitation, processor 1902 may include one or more instruction caches, one or more data caches, and one or more translation lookaside buffers (TLBs). Instructions in the instruction cache may be copies of instructions in memory 1904 or storage 1906, and the instruction cache can speed up the retrieval of these instructions by processor 1902.

[0154] Data in the data cache can be a copy of data in memory 1904 or storage 1906 for manipulation by instructions executed by processor 1902; the result of instructions executed prior to those executed by processor 1902 for access by subsequent instructions executed by processor 1902 or for writing to memory 1904 or storage 1906; or other suitable data. The data cache can be used by processor 1902 to speed up read or write operations. The TLB can be used by processor 1902 to speed up virtual address translation. In certain embodiments, processor 1902 can include one or more internal registers for data, instructions, or addresses. The present disclosure contemplates processor 1902 including any suitable number of any suitable internal registers, as needed. Optionally, processor 1902 can include one or more arithmetic logic units (ALUs); can be a multi-core processor; or can include one or more processors 1902. Although the present disclosure describes and illustrates particular processors, the present disclosure contemplates any suitable processor.

[0155] In certain embodiments, memory 1904 includes main memory for storing instructions for execution by processor 1902 or data for manipulation by processor 1902. By way of example and not limitation, computer system 1900 may load instructions from storage 1906 or another source (e.g., another computer system 1900, etc.) into memory 1904. Processor 1902 may then load the instructions from memory 1904 into internal registers or an internal cache. To execute the instructions, processor 1902 may fetch the instructions from the internal registers or internal cache and decode them. During or after execution of the instructions, processor 1902 may write one or more results (which may be intermediate or final results) to the internal registers or internal cache. Processor 1902 may then write one or more of these results to memory 1904. In certain embodiments, processor 1902 executes only instructions in one or more internal registers or internal cache or in memory 1904 (and not in storage 1906 or elsewhere) and manipulates only data in one or more internal registers or internal cache or in memory 140 (and not in storage 1906 or elsewhere).

[0156] One or more memory buses (which may each include an address bus and a data bus) may couple the processor 1902 to the memory 1904. The bus 1912 may include one or more memory buses as described below. In certain embodiments, one or more memory management units (MMUs) exist between the processor 1902 and the memory 1904 and facilitate access to the memory 1904 requested by the processor 1902. In certain embodiments, the memory 1904 includes random access memory (RAM). This RAM may be volatile memory, if desired. Optionally, this RAM may be dynamic RAM (DRAM) or static RAM (SRAM). Further, optionally, this RAM may be single-port or multi-port RAM. The present disclosure contemplates any suitable RAM. The memory 1904 may optionally include one or more memory devices 104. Although the present disclosure describes and illustrates particular memories, the present disclosure contemplates any suitable memory.

[0157] In certain embodiments, storage 1906 includes mass storage for data or instructions. By way of example and not limitation, storage 1906 can include a hard disk drive (HDD), floppy disk drive, flash memory, optical disk, magneto-optical disk, magnetic tape, or universal serial bus (USB) drive, or a combination of two or more of these. Storage 1906 can include removable or non-removable (or fixed) media, as desired. Storage 1906 can be internal or external to computer system 1900, as desired. In certain embodiments, storage 1906 is non-volatile solid state memory. In certain embodiments, storage 1906 includes read only memory (ROM). Optionally, this ROM can be mask-programmed ROM, programmable ROM (PROM), erasable PROM (EPROM), electrically erasable PROM (EEPROM), electrically variable ROM (EAROM) or flash memory, or a combination of two or more of these. The present disclosure contemplates mass storage 1906 taking any suitable physical form. Storage 1906 can include one or more storage control units that facilitate communication between processor 1902 and storage 1906, as desired. Optionally, storage 1906 can include one or more storage 1906. Although the present disclosure describes and illustrates particular storage, the present disclosure contemplates any suitable storage.

[0158] In certain embodiments, I / O interface 1908 includes hardware, software, or both that provide one or more interfaces for communication between computer system 1900 and one or more I / O devices. Computer system 1900 may optionally include one or more of these I / O devices. One or more of these I / O devices may enable communication between a person and computer system 1900. By way of example, and not limitation, I / O devices can include a keyboard, keypad, microphone, monitor, mouse, printer, scanner, speaker, still camera, stylus, tablet, touch screen, trackball, video camera, another suitable I / O device, or a combination of two or more of these. An I / O device can include one or more sensors. The present disclosure contemplates any suitable I / O devices and any suitable I / O interface 1906 therefor. Optionally, I / O interface 1908 can include one or more devices or software drivers that enable processor 1902 to drive one or more of these I / O devices. I / O interface 1908 can optionally include one or more I / O interfaces 1906. Although the present disclosure describes and illustrates particular I / O interfaces, the present disclosure contemplates any suitable I / O interface.

[0159] In certain embodiments, communication interface 1910 includes hardware, software, or both to provide one or more interfaces for communication (e.g., packet-based communication) between computer system 1900 and one or more other computer systems 1900 or one or more networks. By way of example, and not limitation, communication interface 1910 can include a network interface controller (NIC) or network adapter for communicating with an Ethernet® or other wired network, or a wireless NIC (WNIC) or wireless adapter for communicating with a wireless network such as a Wi-Fi® network. The present disclosure contemplates any suitable network and any suitable communication interface 1910 therefor.

[0160] By way of example, and not limitation, computer system 1900 can communicate with an ad hoc network, a personal area network (PAN), a local area network (LAN), a wide area network (WAN), a metropolitan area network (MAN), or one or more portions of the Internet, or a combination of two or more thereof. One or more portions of these networks can be wired or wireless. By way of example, computer system 1900 can communicate with a wireless PAN (WPAN) (e.g., a BLUETOOTH® WPAN), a Wi-Fi network, a WI-MAX® network, a cellular telephone network (e.g., a Global System for Mobile Communications (GSM) network), or other suitable wireless network, or a combination of two or more thereof. Computer system 1900 can include, as needed, any suitable communication interface 1910 for any of these networks. Communication interface 1910 can include, as needed, one or more communication interfaces 1910. Although the present disclosure describes and illustrates particular communication interfaces, the present disclosure contemplates any suitable communication interface.

[0161] In certain embodiments, bus 1912 includes hardware, software, or both that couple the components of computer system 1900 to each other. By way of example and not limitation, bus 1912 can include an Accelerated Graphics Port (AGP) or other graphics bus, an Extended Industry Standard Architecture (EISA) bus, a Front Side Bus (FSB), a HyperTransport (HT) interconnect, an Industry Standard Architecture (ISA) bus, an InfiniBand interconnect, a Low Pin Count (LPC) bus, a memory bus, a MicroChannel Architecture (MCA) bus, a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCIe) bus, a Serial Advanced Technology Attachment (SATA) bus, a Video Electronics Standards Association Local (VLB) bus, or another suitable bus, or a combination of two or more of these. Bus 1912 can include one or more buses 1912 as needed. Although the present disclosure describes and illustrates particular buses, the present disclosure contemplates any suitable bus or interconnect. Other matters

[0162] As used herein, "or" is inclusive and not exclusive, unless expressly indicated otherwise or indicated otherwise by context. Accordingly, as used herein, unless expressly indicated otherwise or indicated otherwise by context, "A or B" means "A, B, or both." Further, unless expressly indicated otherwise or indicated otherwise by context, "and" is both joint and several. Thus, as used herein, unless expressly indicated otherwise or indicated otherwise by context, "A and B" means "A and B" jointly or severally.

[0163] As used herein, "automatically" and derivatives thereof mean "without human intervention" unless expressly indicated otherwise or indicated otherwise by context.

[0164] The embodiments disclosed in this specification are merely examples and the scope of the present disclosure is not limited thereto. The embodiments described in the invention are disclosed particularly in the appended claims regarding methods, storage media, systems and computer program products, where any feature mentioned in one claim category, for example a method, may also be claimed in another claim category, for example a system. The dependencies or references in the appended claims are selected only for formal reasons. However, any subject matter resulting from an intentional reference to any preceding claim (in particular multiple dependencies) may equally be claimed, such that the claims and any combination of their features are disclosed and may be claimed regardless of the dependencies selected in the appended claims. The claimable subject matter includes not only combinations of features set in the appended claims, but also any other combination of features in the claims, and each feature described in the claims may be combined with any other feature or combination of other features in the claims. Furthermore, any of the embodiments and features described or illustrated in this specification may be claimed in an individual claim and / or in any combination with any of the embodiments or features described or illustrated in this specification or features of the appended claims.

[0165] The scope of the present disclosure encompasses all changes, substitutions, variations, alternatives, and modifications to the exemplary embodiments described or illustrated herein that would be understood by those skilled in the art. The scope of the present disclosure is not limited to the exemplary embodiments described or illustrated herein. Further, although the present disclosure describes and illustrates each embodiment herein as including a particular component, element, feature, function, operation, or step, any of these embodiments may include any combination or permutation of any of the components, elements, features, functions, operations, or steps described or illustrated anywhere herein that would be understood by those skilled in the art. Additionally, references in the appended claims to an apparatus or system, or a component of an apparatus or system, that is adapted, arranged, capable, configured, enabled, operable, or operative to perform a particular function include the apparatus, system, or component whether or not that particular function is activated, initiated, or unlocked, so long as the apparatus, system, or component is so adapted, arranged, capable, configured, enabled, operable, or operative. Further, although the present disclosure describes and illustrates particular embodiments as providing particular advantages, the particular embodiments may provide none, some, or all of these advantages.

Claims

1. A method for isolating and detecting one or more target biomarkers on a dielectrophoresis device, comprising: receiving a first biological sample containing the one or more target biomarkers on a dielectrophoresis (DEP) electrode array; applying a DEP force through specific electrodes of the DEP electrode array, wherein the intensity, direction and duration of the DEP force are specific to the one or more target biomarkers of the first biological sample; and determining the amount of the one or more target biomarkers of the first biological sample via a digital sensor A method comprising the above steps.

2. The method according to claim 1, wherein the DEP electrode arrays are arranged in an interlocking configuration.

3. The method according to claim 1, wherein the DEP electrode array includes at least a working electrode, a reference electrode and a counter electrode.

4. The method according to claim 1, wherein the DEP electrode array includes one or more positive electrodes and one or more negative electrodes.

5. The method according to claim 1, wherein the DEP force is an alternating current (AC) waveform.

6. The method according to any one of claims 1 to 5, wherein one target biomarker is an extracellular vesicle (EV).

7. The method according to claim 6, wherein the DEP force is adjusted according to the size of the EV particles.

8. A dielectrophoresis device for isolating and detecting one or more target biomarkers, comprising: means for receiving a first biological sample containing the one or more target biomarkers on a dielectrophoresis (DEP) electrode array; means for applying a DEP force through specific electrodes of the DEP electrode array, wherein the intensity, direction and duration of the DEP force are specific to the one or more target biomarkers of the first biological sample; and means for determining the amount of the one or more target biomarkers of the first biological sample via a digital sensor A dielectrophoresis device comprising the above means.

9. A method for detecting at least one target biomarker on a dielectrophoresis (DEP) device, comprising: receiving a first biological sample on one or more electrode arrays of the DEP device, wherein the first biological sample contains one or more target biomarkers; Applying a current through the one or more electrode arrays to separate a first target biomarker from a plurality of target biomarkers from the first biological sample; Filtering the first target biomarker from the plurality of biomarkers of the first biological sample into one or more chambers of the DEP device via one or more microfluidics; Tagging the first target biomarker with a first marker; Detecting the amount of the first target biomarker via an electrochemical sensor; and Displaying the synthesis result A method comprising.

10. The method according to claim 9, wherein each of the one or more electrode arrays includes at least a working electrode, a reference electrode, and a counter electrode.

11. The method according to claim 9, wherein the first marker is a specific marker among a plurality of labels.

12. The method according to claim 11, wherein the plurality of labels includes at least an antibody label, a metal nanoparticle (MNP) label, or a single-stranded DNA (ssDNA) label.

13. The method according to claim 9, wherein the one or more electrode arrays are arranged in an interlocking configuration.

14. The method according to claim 9, wherein each of the one or more electrode arrays includes one or more positive electrodes and one or more negative electrodes.

15. The method according to any one of claims 9 to 14, wherein the first target biomarker is an extracellular vesicle (EV).

16. A dielectrophoresis device for detecting at least one target biomarker, comprising: Means for receiving a first biological sample on one or more electrode arrays of the dielectrophoresis device, wherein the first biological sample includes one or more target biomarkers; Means for applying a current through the one or more electrode arrays to separate a first target biomarker from a plurality of target biomarkers from the first biological sample; Means for filtering the first target biomarker from the plurality of biomarkers of the first biological sample into one or more chambers of the dielectrophoresis device via one or more microfluidics; Means for tagging the first target biomarker with a first marker; Means for detecting the amount of the first target biomarker via an electrochemical sensor; and Means for displaying the synthesis result A dielectrophoresis device comprising

17. A method for detecting at least one target biomarker on a device, comprising: Receiving a first biological sample; Transferring the first biological sample into a first chamber via a microfluidic channel, wherein a first process is performed on the first biological sample in the first chamber; Passing the first biological sample from the first chamber to a second chamber via the microfluidic channel, wherein a second process is performed on the first biological sample in the second chamber; and Passing the first biological sample from the second chamber to a third chamber via the microfluidic channel, wherein a third process is performed on the first biological sample in the third chamber, A method comprising

18. The method according to claim 17, wherein the first chamber includes a sample preparation chamber, and wherein the first process changes one or more characteristics of the first biological sample.

19. The method according to claim 18, wherein the first process changes the plasma purification of the first biological sample.

20. Inputting one or more reagents into the first chamber; and Outputting waste including a portion of the first biological sample and a portion of the one or more reagents resulting from the first process from the first chamber The method according to claim 17, further comprising

21. The method according to claim 17, wherein the second chamber includes a concentration chamber, and wherein the current applied to the first biological sample in the concentration chamber results in a change in ion concentration.

22. The method according to any one of claims 17 to 21, wherein the third chamber includes an isolation chamber, and wherein the current applied to the first biological sample in the isolation chamber results in the separation of a specific target biomarker of the first biological sample.

23. A device for detecting at least one target biomarker, comprising: Means for receiving a first biological sample; Means for transferring the first biological sample into the first chamber via a microfluidic channel, where a first process is performed on the first biological sample in the first chamber; Means for passing the first biological sample from the first chamber to a second chamber via the microfluidic channel, where a second process is performed on the first biological sample in the second chamber; and Means for passing the first biological sample from the second chamber to a third chamber via the microfluidic channel, where a third process is performed on the first biological sample in the third chamber, A device comprising the above.

24. A method for isolating at least one target biomarker on a solid state device, comprising: Receiving a first biological sample within the solid state device; Transferring the first biological sample to a dielectrophoresis (DEP) chamber, where one or more processes are performed on the first biological sample in the DEP chamber; Passing the first biological sample through a microfluidics (MF) channel to a tagging chamber; Processing the first biological sample in the tagging chamber, where one or more target biomarkers of the first biological sample are tagged based on one or more characteristics; Passing the one or more target biomarkers with their respective tags to a sensor chamber; and Determining digitally the amount of the one or more target biomarkers with their respective tags through one or more processes performed in the sensor chamber A method comprising the above.

25. The method according to claim 24, wherein the first biological sample is received via the microfluidic channel.

26. The method according to claim 24, wherein the one or more target biomarkers of the first biological sample are tagged with a specific label among a plurality of labels.

27. The method according to claim 26, wherein the plurality of labels includes at least an antibody label, a metal nanoparticle (MNP) label, or a single-stranded DNA (ssDNA) label.

28. The method according to any one of claims 24 to 27, wherein the DEP chamber includes one or more electrode arrays. **Claim 29** The method according to claim 28, wherein each of the one or more electrode arrays is arranged in an interlocking configuration with each other. **Claim 30** The method according to claim 29, wherein each of the one or more electrode arrays includes at least a working electrode, a reference electrode, and a counter electrode. **Claim 31** A solid-state device for isolating at least one target biomarker, comprising: means for receiving a first biological sample within the solid-state device; means for transferring the first biological sample to a dielectrophoresis (DEP) chamber, wherein one or more processes are performed on the first biological sample in the DEP chamber; means for passing the first biological sample through a microfluidics (MF) channel to a tagging chamber; means for processing the first biological sample in the tagging chamber, wherein one or more target biomarkers of the first biological sample are tagged based on one or more characteristics; means for passing the one or more target biomarkers with respective tags to a sensor chamber; and means for digitally determining the amount of the one or more target biomarkers with respective tags through one or more processes performed in the sensor chamber. A device comprising the above. **Claim 32** A method for isolating at least one target biomarker on a solid-state device, comprising: receiving a first biological sample within the solid-state device, wherein the first biological sample includes one or more target biomarkers and is input via a microfluidics channel (MF); transferring the first biological sample to a dielectrophoresis (DEP) chamber via the MF and applying an alternating current (AC) to the first biological sample through one or more electrodes of the DEP chamber; transferring the first biological sample from the DEP chamber to a tagging chamber via the MF; assigning a tag to each of the one or more target biomarkers of the first biological sample; Transferring the first biological sample to one or more detector chambers via the MF, where each detector chamber is programmed for a specific target biomarker; and Determining, in a digital manner, the amount of the one or more target biomarkers based on the assigned tags by one or more detectors in the one or more detector chambers A method comprising the steps of:

33. The method according to claim 32, wherein the tag defines a specific label among a plurality of labels.

34. The method according to claim 33, wherein the plurality of labels includes at least an antibody label, a metal nanoparticle (MNP) label, or a single-stranded DNA (ssDNA) label.

35. The method according to claim 32, wherein a first target biomarker of the first biological sample is assigned a first tag based on specific characteristics.

36. The method according to claim 32, wherein a second target biomarker of the first biological sample is assigned a second tag based on specific characteristics.

37. The method according to any one of claims 32 to 36, wherein the one or more electrodes of the DEP chamber include at least a working electrode, a reference electrode, and a counter electrode.

38. A solid-state device for isolating at least one target biomarker, comprising: Means for receiving a first biological sample within the solid-state device, wherein the first biological sample includes one or more target biomarkers and is input via a microfluidics (MF) channel; Means for transferring the first biological sample to a dielectrophoresis (DEP) chamber via the MF channel and applying an alternating current (AC) to the first biological sample through one or more electrodes of the DEP chamber; Means for transferring the first biological sample from the DEP chamber to a tagging chamber via the MF channel; Means for assigning a tag to each of the one or more target biomarkers of the first biological sample; Means for transferring the first biological sample to one or more detector chambers via the MF channel, where each detector chamber is programmed for a specific target biomarker; and means for digitally determining the amount of the one or more target biomarkers based on the assigned tags by one or more detectors within the one or more detector chambers A solid state device comprising the same. **Claim 39** A syringe; A microfluidic channel, wherein the microfluidic channel receives a biological sample; One or more motors, wherein each motor includes one or more gears; One or more actuating valves, wherein each of the one or more actuating valves connects a plurality of the microfluidic channels; One or more reservoirs, wherein the one or more reservoirs are connected to the microfluidic channels; One or more dielectrophoresis (DEP) chambers, wherein each DEP chamber includes at least one or more working electrodes, one or more reference electrodes, and one or more counter electrodes; One or more tagging chambers, wherein the biological sample is assigned one or more tags; One or more detector chambers, wherein the biological sample and the associated one or more tags are processed; and One or more detectors, wherein the one or more detectors are operable to quantify the one or more tags, A solid state device comprising the same.

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