Microfluidic devices comprising micro-immunobead based electromagnetic manipulation systems and methods of use thereof

Abstract

This invention provides a microfluidic device comprising an electromagnetic operating system based on microimmunobeads and a method of using the same. The microfluidic device includes a biochip and an electromagnetic (EM) chip with a programmable navigation matrix. The biochip and the EM chip are aligned parallel to each other, with the EM chip directly beneath the biochip. During immunoassay, droplets containing immunomagnetic beads are transported to various chambers of the biochip. The EM chip generates localized magnetic fields by activating specific coils directly beneath the droplet path. These magnetic fields attract and move the magnetic beads within the droplets, facilitating targeted interactions with binders and traps, mixing with wash buffer, and precise positioning for detection. Because the EM chip is aligned directly beneath the biochip, the generated magnetic fields are tightly coupled to the spatial layout of the fluid path, enabling accurate, contactless, and programmable manipulation of the assay workflow within the microfluidic system.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 786,606, filed April 10, 2025, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention belongs to the field of microfluidic devices. Specifically, this invention relates to a microfluidic device comprising an electromagnetic operating system based on micro-immune beads and a method of using the same. Background Technology

[0004] The growing aging population has presented new challenges to global health and social systems. This demographic shift necessitates public health preparedness, particularly in addressing age-related and chronic diseases requiring timely diagnosis and management, such as neurological disorders, cardiovascular diseases, and metabolic syndromes. 1,2 These symptoms typically develop quietly over time and can significantly impair quality of life if not detected early. 3-5 With the continued expansion of the elderly population, the incidence and prevalence of such diseases are expected to rise significantly, placing an increasing burden on the healthcare system, caregivers, and society as a whole. 2,3,6-8 Therefore, there is an increasing need for early, accessible, and automated diagnostic tools to facilitate timely intervention and large-scale screening procedures. 9-12 .

[0005] Currently, the diagnostic criteria for many chronic symptoms rely on clinical evaluation. 13-15 and advanced imaging technology 16-18 Its assessment may lack objectivity. 19-21 Furthermore, there is the problem of limited accessibility to a wider range of implementation methods. 16,18,22 Invasive molecular tests, such as those involving cerebrospinal fluid sampling or tissue biopsy, are generally unsuitable for routine or early screening due to the associated risks and discomfort. 23,24 To address these limitations, blood-based testing has emerged as a potential direction in diagnostics, offering a minimally invasive and scalable solution for detecting a variety of analytes associated with chronic and age-related symptoms. 3,25-28 .

[0006] Despite existing efforts, challenges remain in developing effective blood assays for disease-related biomarkers. For example, the use of specific proteins, exosome contents, or molecular markers as analytes in plasma or serum is still under evaluation, and previous findings are inconsistent. 29,30 In some cases, the lack of a clear correlation between circulating biomarker levels and clinical or imaging findings limits the application of these biomarkers in diagnosis.31-33 Furthermore, there is a lack of automated, user-friendly platforms capable of efficiently and on a large scale performing blood-based tests. 34,35 Conventional tools such as enzyme-linked immunosorbent assays (ELISA), although widely used, are typically time-intensive, labor-dependent, and require skilled personnel due to their multi-step manual procedures. 36 Alternative technologies include electrochemical sensors (e.g., field-effect transistors). 37 And optical methods (e.g., surface-enhanced Raman spectroscopy). 38 The need for specialized equipment and strictly controlled conditions reduces their feasibility for use in distributed or point-of-care detection applications. 39 .

[0007] Therefore, there is a need for systems and devices that can rapidly and accurately detect clinically relevant analytes, while also supporting automation, scalability, and ease of use in a variety of healthcare and point-of-care testing environments. Summary of the Invention

[0008] One object of the present invention is to provide an automated microfluidic device for detecting analytes in biological samples. This device exhibits high sensitivity and specificity, significantly improving the accuracy and reliability of detection, and achieves fully automated operation from sample loading to result readout.

[0009] Another object of the present invention is to provide a microfluidic system for automated analyte detection.

[0010] Another object of the present invention is to provide a method for detecting analytes in biological samples using a microfluidic device. This method is simple to operate, requires no professional training, and users can complete the detection with just a few simple steps.

[0011] In a first aspect, the present invention provides a microfluidic device and system thereof for automated analyte detection. The microfluidic device includes (a) a biochip and (b) an electromagnetic chip including a programmable navigation matrix. The biochip and the electromagnetic chip are oriented parallel to each other, and the electromagnetic chip is directly below the biochip.

[0012] This invention aligns a biochip and an electromagnetic chip containing a programmable navigation matrix parallel to each other, with the electromagnetic chip positioned directly beneath the biochip. This allows the local magnetic field generated by the electromagnetic coil to directly and efficiently act on the magnetic beads and droplets at corresponding positions within the biochip, avoiding magnetic field attenuation or shift and ensuring driving efficiency and control precision. Simultaneously, the programmable navigation matrix in the electromagnetic chip precisely guides the magnetic motor along a preset path, enabling non-contact, programmable manipulation of the magnetic beads and droplets. It also allows for flexible switching between "bead operation mode" and "droplet operation mode," meeting the operational requirements of multiple steps in immunoassay, such as mixing, washing, labeling, and reaction. Based on this structure, the microfluidic device of this invention achieves fully automated operation from sample loading to result reading. Users only need to load the sample and start the device; detection can be completed without manual intervention, significantly reducing the operational threshold and reliance on professional personnel.

[0013] According to the microfluidic device provided by the present invention, the biochip includes (i) a sample loading chamber, (ii) one or more washing chambers, (iii) a binding chamber, (iv) a detection chamber, and optionally (v) a termination buffer chamber. Optionally, the sample loading chamber, one or more washing chambers, the binding chamber, and the detection chamber are in fluid communication with each other.

[0014] According to the microfluidic device provided by the present invention, the microfluidic device further includes a plurality of immunomagnetic exosome beads; optionally, the plurality of immunomagnetic exosome beads are provided as separate components or pre-loaded in a sample loading chamber.

[0015] According to the microfluidic device provided by the present invention, the biochip includes a first washing chamber and a second washing chamber, wherein the first washing chamber is in direct fluid communication with the sample loading chamber and the binding chamber, respectively, and the second washing chamber is in direct fluid communication with the detection chamber and the binding chamber, respectively.

[0016] According to the microfluidic device provided by the present invention, the biochip further includes a termination buffer chamber, and the detection chamber is in direct fluid communication with the termination buffer chamber.

[0017] According to the microfluidic device provided by the present invention, wherein the electromagnetic chip includes a programmable navigation matrix, the programmable navigation matrix including: (1) a plurality of electromagnetic coils, (2) one or more switching integrated circuits electrically connected to the plurality of electromagnetic coils, (3) one or more magnetic motors, and (4) a microcontroller configured to control the activation of the plurality of electromagnetic coils to electromagnetically drive the magnetic motors.

[0018] According to the microfluidic device provided by the present invention, the plurality of electromagnetic coils are arranged such that they are aligned with the sample loading chamber, one or more washing chambers, binding chambers and detection chambers.

[0019] According to the microfluidic device provided by the present invention, the plurality of electromagnetic coils are arranged such that they are aligned with the termination buffer chamber.

[0020] According to the microfluidic device provided by the present invention, the microfluidic device further includes a material exchange region, wherein the material exchange region is in fluid communication with the sample loading chamber, one or more washing chambers, binding chambers and detection chambers, and optionally, wherein the material exchange region is centrally located on the biochip.

[0021] According to the microfluidic device provided by the present invention, each of the sample loading chamber, one or more washing chambers, binding chamber and detection chamber includes a mixing region and a flow region.

[0022] According to the microfluidic device provided by the present invention, the microfluidic device further includes: (c) an optical sensing module for detecting signals generated in a detection chamber; and (d) an optional operation module for processing the detected signals.

[0023] According to the microfluidic device provided by the present invention, the optical sensing module comprises: (i) a light source, which is aligned with the detection chamber for emitting light through a signal readout droplet; (ii) a photodetector configured to receive transmitted or absorbed light from the droplet in the detection chamber and generate an output signal corresponding to the level of the transmitted or absorbed light; and (iii) an optional support structure for fixing the optical sensing module aligned with the detection chamber.

[0024] According to the microfluidic device provided by the present invention, the light source of the optical sensing module is aligned below the detection chamber.

[0025] According to the microfluidic device provided by the present invention, the biochip includes: (a) a barrier layer comprising a top layer and a bottom layer; (b) one or more chamber layers; and (c) optionally one or more adhesive layers, wherein when the biochip is assembled, the one or more chamber layers form one or more chambers and one or more flow paths, and the one or more chamber layers and the one or more adhesive layers are located between the top layer and the bottom layer.

[0026] According to the microfluidic device provided by the present invention, the barrier layer is formed of one or more polymers selected from the following: polyethylene terephthalate, polycarbonate, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polyethylene naphthalate (PEN), transparent polypropylene, fluoropolymers such as Cytop, cellulose acetate membranes, and thermoplastic polyurethane.

[0027] According to the microfluidic device provided by the present invention, the one or more chamber layers are formed of one or more polymers selected from the following: polytetrafluoroethylene, DMS, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polycarbonate, transparent polypropylene, fluorinated ethylene propylene, polyethylene naphthalate (PEN), and TPU.

[0028] In a second aspect, the present invention provides a microfluidic system for automated analyte detection, comprising the microfluidic device described in the first aspect of the present invention.

[0029] According to the microfluidic system provided by the present invention, the microfluidic system further includes:

[0030] (1) A housing for enclosing internal components; and / or

[0031] (2) A user display, wherein the user display is used to display real-time system status, measurement progress, and user prompts; and / or

[0032] (3) One or more reagents used to detect analytes in biological samples.

[0033] Thirdly, the present invention also provides a method for detecting analytes in biological samples using the microfluidic device described in the first aspect of the present invention or the microfluidic system described in the second aspect of the present invention.

[0034] The detection method of this invention is simple to operate, requires no professional training, and users can complete the test with just a few simple steps. This method requires low amounts of biological samples and is applicable to various sample types such as blood, plasma, serum, and cerebrospinal fluid. Furthermore, this method demonstrates excellent diagnostic performance in distinguishing between Alzheimer's disease patients and healthy controls, providing a feasible technical solution for early screening and disease progression monitoring.

[0035] According to the method for detecting analytes in biological samples provided by the present invention, the exemplary method includes the following steps: (a) loading the biological sample into the sample loading chamber of a biochip in a microfluidic device; and (b) activating an electromagnetic chip to generate an electromagnetic field. In the present invention, when the user starts the device, the microcontroller selectively activates a specific electromagnetic coil to generate an electromagnetic field according to a preset measurement program or user instruction via a switching IC.

[0036] According to the method for detecting analytes in biological samples provided by the present invention, the method includes: functionalizing the plurality of immunomagnetic exosome beads with a binding agent, wherein the binding agent specifically binds to one or more exosome markers.

[0037] According to the method for detecting analytes in biological samples provided by the present invention, the exosome markers are selected from CD63, CD9, CD81, CD82, CD151, anti-HSP70, anti-HSP90, annexin V and Tim4 protein or combinations thereof.

[0038] According to the method for detecting analytes in biological samples provided by the present invention, the binding agent is selected from antibodies, aptamers, nanobodies, peptides, and ligands.

[0039] The method for detecting analytes in biological samples according to the present invention, wherein the biological samples are selected from tissue fluid, blood, plasma, serum, saliva, urine, cerebrospinal fluid (CSF), breast milk, and ascites.

[0040] The method for detecting analytes in biological samples, wherein the biological sample is a plasma sample or a serum sample, is provided by the present invention.

[0041] According to the method for detecting analytes in biological samples provided by the present invention, wherein the biological sample is a fluid sample ranging from 1 μL to 500 μL.

[0042] The method for detecting analytes in biological samples according to the present invention, wherein the biological samples are obtained from mammals.

[0043] The method for detecting analytes in biological samples according to the present invention, wherein the biological sample is obtained from humans, rodents, livestock, pets or wild animals.

[0044] According to the method for detecting analytes in biological samples provided by the present invention, wherein the sample loading chamber of the biochip includes a plurality of immunomagnetic exosome beads preloaded therein; or the plurality of immunomagnetic exosome beads are provided by a separate component, and the method further includes loading the plurality of immunomagnetic exosome beads into the biochip before or during step (a).

[0045] The present invention has at least the following beneficial effects:

[0046] 1. This invention provides a highly sensitive and specific automated microfluidic detection device. The microfluidic device of this invention integrates a biochip and a programmable electromagnetic chip to achieve targeted identification of dual biomarkers of analytes (such as exosome membrane proteins and disease-related proteins), with detection sensitivity reaching the picogram / mL level. The device employs a vertically stacked integrated structure, with the biochip and electromagnetic chip aligned parallel to each other, and the electromagnetic chip positioned directly below the biochip. This enables non-contact, programmable manipulation of magnetic beads and droplets, significantly improving the accuracy and reliability of detection.

[0047] 2. The microfluidic detection device of this invention achieves fully automated operation from sample loading to result reading. Users only need to load the sample onto the biochip, and the device automatically completes steps such as magnetic bead mixing, washing, labeling, signal response, and optical detection, eliminating reliance on professional operators. The device is compact, portable, and suitable for point-of-care testing scenarios, and can be used in hospitals, community clinics, and even home environments.

[0048] 3. This invention also provides a simple and efficient detection method. The detection method of this invention is easy to operate, requires no professional training, and users can complete the test with just a few simple steps. This method requires low amounts of biological samples and is suitable for various sample types such as blood, plasma, serum, and cerebrospinal fluid. Clinical validation results show that this method achieves a sensitivity of up to 95% and a specificity of up to 90% in distinguishing between Alzheimer's disease patients and healthy controls, demonstrating excellent diagnostic performance and providing a feasible technical solution for early screening and disease progression monitoring. Attached Figure Description

[0049] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0050] Figure 1 A schematic diagram of MEMOS is shown. Figure 1 Figure a shows a schematic diagram of using MEMOS to detect Exo-Aβ42 in plasma for the diagnosis of Alzheimer's disease (AD). Figure 1 Figure b shows an exploded view of the main components of the MEMOS, which include a microfluidic exosome detection biochip for sample processing (μ-Exo Biochip), a microbead-driven electromagnetic chip (μ-Bead EMchip), and an optical signal detection module. Figure 1 Image c is an optical image of a representative MEMS operating under sample loading conditions. Scale bar: one Hong Kong dollar coin. Figure 1 Figure d is a schematic diagram showing the μ-Exo Biochip combined with the iME bead-based Exo-Aβ42 identification assay (iME beads and reagents), which is designed for the detection of Exo-Aβ42. Figure 1 The figure shows the mechanism of Exo-Aβ42 identification assay based on iME beads on μ-Exo Biochip, which includes first identifying the endogenous protein CD63 of the exosome membrane, then recognizing the exogenous protein Aβ42 bound to the exosome membrane, and finally generating a signal. Figure 1Figure f shows that the μ-Bead EMchip in MEMOS facilitates automated sample handling, including droplet and bead manipulation. Illustration: Stepping motion mechanism of a magnetic motor, which begins by applying a programmed current to a specific coil, followed by the induction of a magnetic field, and ultimately drives the motor in the direction of the induced magnetic field. Figure 1 The g-plot shows the overall workflow of the μ-Bead EMchip used for automated control. The yellow array represents the programmed navigation paths of the magnetic motors that execute various bead and droplet motion commands. Figure 1 The h-plot is a cross-sectional view showing the interface between the integrated μ-Exo Biochip and μ-Bead EMchip. A magnetic motor, controlled by the μ-Bead EMchip, triggers the movement of beads and droplets within the μ-ExoBiochip via a programmed magnetic field induction to complete the Exo-Aβ42 identification assay based on iME beads. Figure 1 The i-figure is a general illustration of an integrated system capable of performing different functions, including bead mixing, bead extraction, droplet merging, and readout. Figure 1 Figure j shows the optical readings of the Exo-Aβ42 signal detected by MEMOS, which is used to differentiate between Alzheimer's disease (AD), mild cognitive impairment (MCI), and healthy controls (CON).

[0051] Figure 2 The capture efficiency and sensing performance of Exo-Aβ42 based on iME beads are characterized. Figure 2 Figure a is a schematic diagram of functionalized immunomagnetic exosome beads (iME beads). Anti-CD63 primary antibody (pink) is coated on the surface of the beads. Figure 2 Figure b shows a flow cytometry scatter plot analysis of the control beads (left) and iME beads (right). APC dye was used to target antibody signal transduction. Figure 2 Figure c is a histogram of the APC signal strength of the blank bead and the iME bead. Figure 2 The d-plot shows the elemental spectra of the iME beads obtained by SEM-EDS analysis (Fe - green, O - red, Au - blue). Gold nanoparticles (AuNPs) were conjugated with antibodies for visualization. Scale bar = 1 μm. Figure 2 Figure e shows the process of identifying and capturing the exosomal membrane endogenous protein CD63 in the iME bead-based Exo-Aβ42 identification assay. Figure 2 The f-plot is a flow cytometry scatter plot analysis of a single blank iME bead (left) and beads captured with exosomes (right). FITC dye was used to label the exosomes. Figure 2 The g plot is a histogram of the FITC signal intensity of blank iME beads and beads captured by exosomes. Error bars represent SD (n = 3). Figure 2The h-image is a fluorescence microscopy image of iME beads captured by FITC-labeled exosomes (after extraction). Scale bar = 10 μm. Figure 2 The i-figure shows the process of identifying the exosome membrane-bound exogenous protein Aβ42 and signal transduction in the Exo-Aβ42 identification assay based on iME beads. Figure 2 The j-plot shows the improvement in iME bead exosome capture as a function of incubation time. Error bars represent SD (n = 3). Figure 2 The k-plot shows the improvement in iME bead exosome capture as a function of primary antibody concentration. Error bars represent SD (n = 3). Figure 2 The figure shows the specificity of the Exo-Aβ42 identification assay based on iME beads, displaying the sensing signals of the target Exo-Aβ42 protein and three non-target proteins (Aβ42, CD63, and BSA). The baseline represents the background signal of the optical detection sensor. Figure 2 The m-plot uses 0-50 pg ml -1 Calibration plot of the response of Exo-Aβ42 based on iME beads for Exo-Aβ42 identification assay (four-parameter logical fit, R) 2 = 0.987). The error bars represent SD (n = 3). The baseline represents the background signal of the optical detection sensor.

[0052] Figure 3A This demonstrates the layer structure design of the μ-Exo Biochip and the automated operation process for identifying and measuring Exo-Aβ42 based on iME beads on the chip. Figure 3A Figure a is a schematic diagram of the layer design of an exemplary μ-Exo Biochip. Figure 3A Figure b shows the simulated (line) and experimental (point) operating areas of the bead and droplet based on the adjusted bead loading mass and the programmed magnetic motor speed. Figure 3A Figure c is a series of optical images depicting the bead operation (bead extraction, ∆t = 150 ms, top) and the droplet operation (droplet transport, ∆t = 500 ms, bottom). Scale bar = 2 mm. Figure 3A Image d is a series of automated, sequential optical images showing the identification of Exo-Aβ42 based on iME beads on the chip. The bottom section features a magnified view of the driving details, while the upper right corner depicts the corresponding measurement steps.

[0053] Figure 3B The results show the performance of MEMOS in detecting Exo-Aβ42 levels, validated using Alzheimer's disease mouse models and wild-type mouse models. Figure 3BThe figure shows animal samples tested on the chip (Alzheimer's disease mouse models: 5xFAD and wild-type mice, control: C57BL / 6J). The 5xFAD Alzheimer's disease mouse model is a transgenic model of five gene mutations associated with AD. Figure 3B Image f shows the immunohistochemical (IHC) staining of Aβ42 aggregates in the hippocampus of C57BL / 6J (right) and 5xFAD (left) mice. Dark patches indicate Aβ42 deposition. Figure 3B The g and h plots are graphs showing the performance of MEMOS in detecting Exo-Aβ42 levels in plasma from AD mice (n = 10) and wild-type mice (n = 10). Box plots ( Figure 3B (g-chart) and waterfall chart ( Figure 3B The h-plot shows the optical signal readout via MEMOS. Statistical analysis was performed using the unpaired Mann-Whitney nonparametric test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0054] Figure 4A This paper compares the automated MEMOS detection workflow with conventional manual methods, and shows the signal distribution of both methods in clinical samples from Alzheimer's disease, mild cognitive impairment, and healthy controls. Figure 4A Figure a is a schematic diagram illustrating an automated MEMOS inspection workflow. Figure 4A Figure b shows the workflow of a typical manual method. Figure 4A Figure c shows the corresponding distribution of response signals for 60 clinical samples, as tested by an automated MEMOS detection system (AD, Alzheimer's disease patients; MCI, mild cognitive impairment patients; CON, healthy controls), highlighting the trends of change associated with different stages of AD (n = 20), MCI (n = 10), and CON (n = 30). Figure 4A The d-plot shows the corresponding distribution of response signals for the same 60 clinical samples, illustrating the trends of AD (n = 20), MCI (n = 10), and CON (n = 30) at various stages as tested by conventional manual methods.

[0055] Figure 4B The pie chart shows the distribution of MEMOS sensor signal values ​​in 60 clinical samples, the results of statistical significance analysis between groups, and the receiver operating characteristic (ROC) curves used to assess diagnostic performance. Figure 4B The e-plot is a circular plot showing the MEMOS sensor signal values ​​of 60 clinical samples. Figure 4BThe f-plot shows the statistical significance of MEMOS sensor signals in clinical plasma samples from AD, MCI, and CON. Statistical analysis was performed using the unpaired Mann-Whitney nonparametric test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 4B The g-plot shows the receiver operating characteristic curve analysis of 60 clinical samples with plasma Exo-Aβ42 levels based on the MEMOS test.

[0056] Figure 5 This section showcases the development and evaluation of the iME bead. Figure 5 Figure a shows the functionalization process of immunomagnetic exosome beads (iME beads). Figure 5 Figures b through g are flow cytometry characteristics of the functionalization process of iME beads. Blank beads ( Figure 5 (b image), iME beads ( Figure 5 Figure c), blank beads with APC antibody dye ( Figure 5 (d image) and iME beads with APC antibody dye ( Figure 5 Flow cytometry scatter plot analysis (Figure e) showed no cross-reaction between the blank beads and the APC antibody dye. Histograms of APC signal intensity for (Figures b, c, d, and e, n=3) Figure 5 (f-plot) and average ( Figure 5 (g-graph).

[0057] Figure 6 Characterization of extracellular vesicles derived from SH-SY5Y neurons. Figure 6 Figure a is a transmission electron micrograph of extracellular vesicles deposited in SH-SH5Y cells. Scale bar: 100 nm. Figure 6 Figure b is a graph showing the size distribution of isolated extracellular vesicles as determined by nanoparticle tracking analysis, showing an average value of approximately 150 nm. Figure 6 Figure c is a Western blot image of isolated extracellular vesicles targeting exosome markers (CD63 and TSG-101).

[0058] Figure 7 Flow cytometry characterization of the exosome capture performance of iME beads. Figure 7 Figures a through c are blank iME beads ( Figure 7 Figure a), iME beads with exosome FITC dye ( Figure 7 Figure b) and iME beads captured by exosomes labeled with FITC ( Figure 7 Flow cytometry scatter plot analysis (Figure c) showed no cross-reaction between iME beads and exosome FITC dye. Figure 7The d-plot and e-plot, and histograms of APC signal strength for plots a, b, and c (n = 3). Figure 7 (d graph) and average value ( Figure 7 (e-graph).

[0059] Figure 8 Characterization of the prepared Exo-Aβ42 protein is shown. Figure 8 Figure a is a schematic diagram of the preparation method. Figure 8 Figure b shows transmission electron microscopy images of the assembled Aβ42 aggregates (left) and exosome-Aβ42 protein conjugates (right). Figure 8 Figure c shows scanning transmission electron microscopy images of AuNP (10 nm) immunolabeled Aβ42 aggregates (left) and exosome-Aβ42 protein conjugates (right). Exo-Aβ42 was labeled with AuNP (10 nm) via an Aβ42-specific antibody. The nanoparticles appear as bulk dots.

[0060] Figure 9 This is a quantitative standard curve for exosomes (n = 3). Four-parameter logistic fitting, R0 2 = 0.996.

[0061] Figure 10 This demonstrates an improvement in on-chip iME bead-based Exo-Aβ42 identification assays. Blocking buffer ( Figure 10 Figure a), washing buffer ( Figure 10 (Figure b), number of washes () Figure 10 (Figure c), concentration of HRP-conjugated anti-Aβ42 antibody ( Figure 10 (d-plot) and added signal enhancer ( Figure 10 The signal-to-noise ratio of the e-graph.

[0062] Figure 11 This displays the corresponding calibration process for MEMOS. Figure 11 Figure a is a schematic diagram of the corresponding calibration process for MEMOS. Figure 11 Graph b is the standard curve (n = 3) from a commercially available Aβ42 protein kit. Four-parameter logistic fitting was performed, R0. 2 = 0.999. Figure 11 Figure c shows the reference signal for the intermediate process. The Exo-Aβ42 marker captured by iME beads was lysed and quantified using a commercially available Aβ42 kit to characterize the level of Aβ42 bound to exosomes (n = 3).

[0063] Figure 12 This demonstrates the management and implementation of μ-EMChip manipulation at both the software and hardware levels. Figure 12 Figure a is a simplified diagram of an integrated programmable microcontroller, which is programmed through different predetermined motion commands. Figure 12(Figure b) to start the magnetic motor and achieve the desired operation within the defined area ( Figure 12 (Figure c).

[0064] Figure 13 This is a graph representing the optical sensor readings versus the readings of a standard microplate reader. The error bars represent the SD (n = 3).

[0065] Figure 14 This describes the characterization of total plasma Aβ42 protein levels in animal samples, including Alzheimer's disease mouse models: 5xFAD (n = 10) and wild-type mouse models: C57BL / 6J (n = 10). Figure 14 Figure a shows the standard curve (n = 3) of a commercially available Aβ42 protein kit. Four-parameter logistic fitting, Ra 2 = 0.979. Figure 14 Figures b and c show the box plot and waterfall plot of total Aβ42 levels in plasma measured by ELISA, respectively.

[0066] Figure 15 This is a receiver operating characteristic (ROC) curve analysis of plasma Exo-Aβ42 levels measured by MEMOS and plasma Aβ42 levels measured by ELISA in animal samples. Alzheimer's disease mouse model: 5xFAD (n = 10) and wild-type mouse model: C57BL / 6J (n = 10).

[0067] Figure 16 This demonstrates the characterization of CD63 protein levels in exosomes of human samples. Figure 16 Figure a is the standard curve of a commercially available CD63 protein kit (n = 3). Figure 16 Figure b shows the CD63 protein levels in clinical samples AD (n = 10), MCI (n = 10), and CON (n = 10) (AD, Alzheimer's disease patients; MCI, patients with mild cognitive impairment; CON, healthy controls).

[0068] Figure 17 This describes the characterization of total plasma Aβ42 protein levels in human samples, including AD (n = 20), MCI (n = 10), and healthy controls (n = 30). Figure 17 Figure a is a waterfall plot showing the total Aβ42 level in plasma as measured by ELISA. Figure 17 Figure b shows the statistical significance analysis of total plasma Aβ42 protein levels in clinical plasma samples from AD, MCI, and CON. Figure 17 Figure c is a receiver operating characteristic (ROC) curve analysis of total Aβ42 levels in plasma measured by ELISA in clinical samples.

[0069] Figure 18A This is an exploded view of an exemplary microfluidic device.

[0070] Figure 18B This is a top perspective view of an exemplary biochip, showing the chamber of the biochip.

[0071] Figure 18C This is an exploded perspective view of an exemplary biochip, showing the layers of the biochip.

[0072] Figure 18D and 18E These are the top and bottom views of an exemplary electromagnetic chip, respectively.

[0073] Figure 18F This is a side view of an exemplary electromagnetic chip.

[0074] Figure 18G This is a top perspective view of an exemplary optical sensing module.

[0075] Figure 19A and 19B This is a perspective view of an exemplary microfluidic device in its open state.

[0076] Figure 19C and 19D This is a perspective view of an exemplary microfluidic device in its off state. Detailed Implementation

[0077] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0078] I. Electromagnetic Operating System (MEMOS) Based on Microimmune Beads

[0079] This invention discloses a microfluidic device and its automated system (also referred to herein as a "microfluidic system"). The microfluidic device and its automated system are particularly suitable for detecting one or more analytes (e.g., Aβ42 protein) in biological samples (such as plasma / blood samples or cerebrospinal fluid samples) obtained from subjects.

[0080] While not intended to limit, microfluidic devices and their automated systems offer the following advantages over conventional microfluidic immunoassays. First, microfluidic systems integrate sample handling, biochemical analysis, and data transmission into a compact, portable form, making them highly suitable for point-of-care or distributed assay environments. Therefore, microfluidic devices and systems facilitate fully automated operation, enabling the entire assay process—from sample loading to result readout—without manual intervention, thereby reducing user error and increasing consistency. Second, microfluidic systems support dual-marker targeting, such as the simultaneous detection of surface proteins (e.g., on exosomes) and disease-specific biomarkers (e.g., Aβ42 protein), which can increase specificity and improve diagnostic accuracy. Microfluidic systems also incorporate programmable electromagnetic actuation, enabling precise and responsive control of both magnetic bead clusters and droplets during multiple assay steps. Furthermore, microfluidic systems are capable of detecting low concentrations of analytes from small sample volumes, such as in the picogram per milliliter (pg / mL) range.

[0081] Microfluidic systems typically comprise: (a) a biochip and (b) an electromagnetic chip (“EM chip”) including a programmable navigation matrix. The biochip and the electromagnetic chip are usually oriented parallel to each other, with the electromagnetic chip positioned directly beneath the biochip to facilitate seamless interaction with assay components within the biochip, i.e., droplets such as biological samples and buffer solutions, and immunomagnetic beads. This vertical alignment allows the electromagnetic chip to generate a precisely positioned magnetic field that can directly influence the position and movement of the assay components in real time.

[0082] For example, during immunoassays, droplets containing immunomagnetic beads are transported across the various chambers of a biochip. As the droplets arrive at each chamber, the EM chip activates a specific electromagnetic coil directly beneath the droplet's path within the biochip to generate a localized magnetic field. These fields attract and move the magnetic beads within the droplet, facilitating targeted interactions with binders and traps, mixing with the wash buffer, and precise localization for detection. Because the EM chip is aligned directly beneath the biochip, the generated magnetic field is tightly coupled to the spatial layout of the fluid path, enabling accurate, contactless, and programmable manipulation of the assay workflow within the microfluidic system.

[0083] A. Biochip

[0084] Microfluidic systems include biochips (e.g., μ-Exo Biochip). A biochip includes one or more chambers for performing immunoassays. In some embodiments, the biochip includes a sample loading chamber, one or more washing chambers, a binding chamber, and a detection chamber. The sample loading chamber, one or more washing chambers, the binding chamber, and the detection chamber are in fluid communication with each other, allowing droplets and / or immunomagnetic beads to move from / to different chambers and between different chambers.

[0085] Each chamber can have any suitable shape and size, as long as they can perform their respective design functions, such as mixing, antigen binding, chemical reactions, etc. In some embodiments, each chamber includes a mixing region and a flow region, wherein mixing occurs in the mixing region and the flow region provides pathways for droplets / fluids to flow into and out of the chamber.

[0086] Generally, biochips are stationary, meaning they do not require rotation to generate centrifugal force to drive droplets throughout the biochip. In contrast, droplets and immunomagnetic beads can move through the biochip via a magnetic mechanism generated by an electromagnetic chip, as described below.

[0087] Buffers and other reagents can be preloaded into the individual chambers of the biochip during manufacturing. In some embodiments, this can be achieved by forming multiple access holes in the top layer of the biochip, allowing reagents to be manually introduced into their respective chambers before final sealing. Alternatively, individual access holes can be located above the sample loading chamber to allow the user to manually load biological samples during use. In other forms, such as for large-scale manufacturing, automated dispensing techniques, such as inkjet or piezoelectric dispensing systems, or robotic displacement pipetting, can be used to perform reagent preloading, enabling precise and efficient deposition of reagents into designated chambers. After reagent loading, the biochip can be sealed, with the sample loading holes either remaining open or covered by a thin, flexible membrane that can be easily punctured or removed by the user upon sample introduction. This method is capable of producing fully or partially preloaded biochips.

[0088] The size of a biochip can be smaller or larger than that of a standard microtiter plate. The layout of the microfluidic patterns in the biochip can be configured to support a variety of assay protocols. For example, in some embodiments, the size of the biochip can be customized to perform immunomagnetic enrichment (iME) bead-based assays for identifying protein biomarkers. In some embodiments, the height of the biochip ranges from about 2 mm to about 6 mm, such as about 3 mm, about 3.5 mm, or about 4 mm. In some embodiments, the width of the biochip ranges from about 20 mm to about 60 mm, such as about 30 mm, about 43 mm, or about 50 mm. In some embodiments, the length of the biochip ranges from about 20 mm to about 60 mm, such as about 30 mm, about 43 mm, or about 55 mm. In one exemplary form, the size of the biochip is about 43 mm × about 43 mm × about 3.5 mm (length × width × height).

[0089] Biochips can be formed from various materials and in various configurations depending on the assay. In some embodiments, a biochip can be formed from one or more layers of material. In some embodiments, a biochip includes one or more barrier layers, one or more chamber layers, and optionally one or more adhesive layers. For example, a biochip includes a first barrier layer, a chamber layer, and a second barrier layer arranged in a stacked manner, wherein the first barrier layer is attached to a first surface of the chamber layer via a first adhesive layer, and the second barrier layer is attached to a second surface of the chamber layer via a second adhesive layer, wherein the first and second surfaces are opposite each other. When assembled, the one or more chamber layers form one or more chambers and one or more flow paths within which assay components and immunomagnetic beads can be transported.

[0090] One or more chamber layers may include a top layer and a bottom layer, which together enclose the internal fluid structure, thus providing structural integrity. One or more barrier layers may be formed from a variety of materials compatible with microfluidic devices and suitable for optical pathways within the detection chamber. For example, one or more barrier layers, such as the top and bottom layers, may be formed from transparent polymer films such as polyethylene terephthalate (PET). Other suitable materials for forming one or more barrier layers include, but are not limited to, polycarbonate, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polyethylene naphthalate (PEN), transparent polypropylene, thermoplastic polyurethane, fluoropolymers such as Cytop, and cellulose acetate membranes.

[0091] One or more chambers are situated between top and bottom barrier layers and generally define the internal three-dimensional structure of the fluidic network (e.g., fluid transport pathways) within the biochip. The one or more chambers may be formed from one or more substrates composed of materials suitable for microfluidic patterning and fluid control, such as polymers with low surface energy and high chemical resistance. For example, materials chosen to form the chambers can facilitate the smooth transport of immunomagnetic beads and droplets containing analytes, and minimize non-specific adsorption of assay reagents and biological samples. Suitable materials for forming one or more chambers include, but are not limited to, polytetrafluoroethylene, DMS, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polycarbonate, clear polypropylene, fluorinated ethylene propylene, polyethylene naphthalate (PEN), and TPU.

[0092] One or more chamber layers are typically patterned (e.g., via laser patterning or 3D printing) to have features such as cavities, channels, and openings, which, when closed by a barrier layer, form multiple fluid chambers and flow paths. The fluid chambers and flow paths within one or more chamber layers support a variety of functions of the biochip, including fluid loading, reagent handling, binding reactions, and signal detection.

[0093] Biochips can be manufactured using any suitable method, employing one or more suitable materials to form one or more layers. For example, biochips can be made from thermoplastic polymers, including polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), cyclic olefin polymers (COP), and cyclic olefin copolymers (COC), as well as other polymers suitable for large-scale production. Optionally, biochips can be manufactured by CNC machining of large plates (including those larger than the SBS standard plate), and blank plates used for CNC machining can be self-injection molded. Biochips can also be manufactured by injection molding of large plates (including those larger than the SBS standard plate). Optionally, biochips can be coated (before assembly, during an intermediate step in assembly, or after assembly) to improve performance. Various coatings and treatments can be applied. For example, exposing channels to 0.5% bovine serum albumin reduces the binding of proteins present in the sample to the channel walls. Optionally, hydrophobic products such as PFC 502A (Cytonix, Beltsville, MD) can be applied to channels and chambers to minimize bubble formation. Similarly, elastomeric or non-elastomeric membranes of pneumatic and mechanical valve structures can be coated or otherwise treated to provide improved sealing properties.

[0094] In some embodiments, the surfaces of the fluid chamber and / or flow path can be coated with one or more coatings to reduce nonspecific binding, to enable ligand binding, for biocompatibility, and for flow resistance. For example, polymeric hydrogels and gel pads can be coated on the surfaces of the fluid chamber and flow path to increase the adhesion of biomolecules to the fluid chamber surface. Polymeric hydrogels and gel pads that can be used as binding layers to adhere to the surface of biomolecules include, but are not limited to, proteins, peptides, oligonucleotides, polynucleotides, and larger nucleic acid fragments. For example, oligonucleotide probes can bind to the surface of a continuous layer of hydrogel or to an array of gel pads. Gel pads can be readily fabricated into sheets or plates, typically by depositing a solution of acrylamide monomer, a crosslinking agent such as methylenebisacrylamide, and a catalyst such as N,N,N',N'-tetramethylethylenediamine (TEMED) between two glass surfaces (e.g., glass plates or microscope slides) using spacers to obtain a polymeric gel of desired thickness. For example, biochips can be configured for assays involving the functionalization of solid substrate surfaces within microfluidic devices. In such configurations, specific regions of the solid substrate within the biochip can be coated with trapping ligands or other binding molecules. Magnetic beads can then be used not only as functional assay components but also as tools for driving and transporting sample droplets across the chip. The movement of the beads under an applied magnetic field can be used to drive the droplets toward a designated surface-coated area, thereby facilitating controlled interactions between the analyte in the droplet and the immobilized ligands on the substrate. Alternatively, as demonstrated in non-limiting embodiments, the surface of the magnetic beads is coated with ligands to minimize non-specific binding to the inner surface of the biochip, rather than immobilizing the inner surface of the biochip with ligands.

[0095] The biochip optionally includes one or more adhesive layers to bond and seal one or more barrier layers and one or more chamber layers together. The one or more adhesive layers may include double-sided adhesives and / or pressure-sensitive membranes. Suitable adhesives may be formed from acrylic, silicone, or epoxy resin formulations. Other adhesives that can be used to bond and seal one or more barrier layers and one or more chamber layers together include, but are not limited to, UV-curable adhesives such as Norland Optical Adhesive (NOA) series, Dymax 204-CTH-F, or Loctite 3491, and thermal adhesives such as 3M DP420 epoxy resin, Loctite Ablestik 566K, or Henkel Technomelt TC series.

[0096] 1. Sample loading chamber

[0097] The sample loading chamber of the biochip is configured to receive one or more droplets of a biological sample, and a plurality of immunomagnetic exosome beads. Optionally, the plurality of immunomagnetic exosome beads are preloaded into the sample loading chamber. The sample loading chamber may be fluidly connected to a binding chamber (directly or indirectly) via a flow path or microfluidic channel. For example, the sample loading chamber may be in direct fluid communication with the binding chamber, in which a second binder binds the target antigen, or the sample loading chamber may be in direct fluid communication with a first washing chamber and / or the binding chamber.

[0098] The sample loading chamber can have any size and shape suitable for containing biological samples and facilitating efficient mixing with immunomagnetic beads. In some embodiments, the chamber can be defined by patterned circular or elliptical recesses in the chamber layer of a biochip, such as through laser micromachining. In some embodiments, the size of the sample loading chamber can be comparable to the size of the orifices used in microtiter plates, achieving compatibility with standard sample volumes. For example, the sample loading chamber can have a cylindrical shape with a flat bottom surface and continuous sidewalls. The sidewalls of the sample loading chamber may be tapered slightly outward from bottom to top to facilitate sample loading. Additionally, the bottom surface of the sample loading chamber may include flow regions, such as small ports or openings, that enable fluid communication with flow paths (such as microfluidic channels) leading to downstream chambers, such as binding or washing chambers.

[0099] The size and volume of the sample loading chamber can be selected based on the desired sample volume. For example, the sample loading chamber can have a size ranging from approximately 5 mm. 2 to about 30 mm 2 The area. For example, the area of ​​the sample loading chamber could be approximately 18 mm². 2 The chamber can have a diameter of 2-5 mm and a depth of 1-3 mm.

[0100] The diameter and / or depth of the sample loading chamber can vary based on the desired sample volume, hydrodynamics, and compatibility with magnetic bead manipulation or optical detection features within the biochip. In some embodiments, the diameter of the sample loading chamber ranges from about 1 mm to about 8 mm. For example, the diameter of the sample loading chamber can range from about 1 mm to about 7 mm, from about 1 mm to about 5 mm, from about 1 mm to about 3 mm, such as about 2.5 mm, about 3 mm, about 4 mm, or about 5 mm. In some embodiments, the depth of the sample loading chamber ranges from about 1 mm to about 5 mm, such as about 1.5 mm, about 2 mm, about 3 mm, or about 4 mm. In one exemplary form, the sample loading chamber has a diameter of about 5 mm and a depth of about 3 mm.

[0101] In some embodiments, the sample loading chamber may include a removable or permeable seal or diaphragm on the top opening to maintain sterility prior to use. The sample loading chamber may also optionally include a hydrophobic coating on the inner surface to prevent adhesion of the sample and / or magnetic beads.

[0102] In some implementations, the sample loading chamber includes a sub-chamber for containing pre-loaded immunomagnetic beads. For example, as in... Figure 18B As illustrated in the diagram, the sample loading chamber 210 may include a bead chamber 202 configured to include pre-loaded immunomagnetic beads. The bead chamber 202 may be integrated within the sample loading chamber 210 to enable immediate interaction between the sample and the beads upon loading. The biochip 200 may also include an inlet port or inlet 204 above the sample loading chamber 210 through which a user can manually introduce a biological sample, such as whole blood or plasma. Once introduced, the sample enters the sample loading chamber 210 and comes into contact with the pre-loaded immunomagnetic beads contained within the bead chamber 202.

[0103] 2. Laundry room

[0104] Biochips include two or more washing chambers for sequential washing and reagent exchange steps during the assay workflow. For example, depending on the specific assay performed on the biochip, it may include two, three, four, five, six, seven, eight, or other washing chambers.

[0105] In some implementations, the biochip includes four washing chambers. Optionally, a first washing chamber is arranged in direct fluid communication with a sample loading chamber and a binding chamber to facilitate the removal of unbound exosomes from the immunomagnetic beads. Optionally, a second washing chamber may be in direct fluid communication with a binding chamber and a detection chamber to provide a washing buffer after the binding step and before the detection step for the removal of unbound binders.

[0106] The washing chamber can have the same or different dimensions and shape as the sample loading chamber described above. For example, each washing chamber can be circular or elliptical, such as in the form of a hole. Furthermore, each washing chamber can have a diameter ranging from approximately 5 mm. 2 Approximately 30 mm 2 The area, such as approximately 18 millimeters.

[0107] In some embodiments, the diameter of the washing chamber ranges from about 1 mm to about 8 mm. For example, the diameter of the washing chamber can range from about 1 mm to about 7 mm, from about 1 mm to about 5 mm, from about 1 mm to about 3 mm, such as about 2.5 mm, about 3 mm, about 4 mm, or about 5 mm. In some embodiments, the depth of the washing chamber ranges from about 1 mm to about 5 mm, such as about 1.5 mm, about 2 mm, about 3 mm, or about 4 mm. In one exemplary form, the washing chamber has a diameter of about 4 mm and a depth of about 3 mm.

[0108] In some implementations, the washing chamber is configured to contain a pre-loaded buffer solution for washing, rinsing, or dilution steps. For example, as in... Figure 18B As illustrated in the diagram, washing chambers 220, 240, 250, and / or 260 may include one or more buffer solutions preloaded during biochip assembly. Biochip 200 may include fluid pathways that guide droplets, such as those containing immunomagnetic beads with an analyte of interest, into the washing chambers, where they are mixed with the buffer solutions to facilitate the removal of unbound material or to condition the sample for subsequent processing steps. Alternatively, the buffer solutions may not be preloaded into the washing chambers but added in later stages, such as before or during the assay.

[0109] 3. Combination chamber

[0110] Biochips include binding chambers (e.g., antibody chambers). These chambers are designed to expose samples and / or immunomagnetic beads to a binding agent against a target antigen (e.g., an Aβ42 antibody). The binding chambers are typically positioned along a fluid path after sample loading and an optional initial washing phase, and before subsequent washing and detection steps. For example, the binding chamber may be positioned between a first and a second washing chamber.

[0111] The binding chamber can have the same size and shape as other chambers on the biochip. For example, the binding chamber can have a circular geometry and can be formed as a recess or cavity within the chamber layer of the biochip. Alternatively, the binding chamber can have a different size and shape than other chambers on the biochip.

[0112] The binding chamber can have any size, any size suitable for providing sufficient volume for storing antibody reagents and facilitating interaction with the bead-bound analyte. In some embodiments, the diameter of the binding chamber ranges from about 1 mm to about 7 mm, from about 1 mm to about 5 mm, from about 1 mm to about 3 mm, such as about 2.5 mm, about 3 mm, about 4 mm, or about 5 mm. Additionally, the depth of the binding chamber generally corresponds to the thickness of the chamber layer. In some embodiments, the depth of the binding chamber ranges from about 1 mm to about 5 mm, such as about 1.5 mm, about 2 mm, about 3 mm, or about 4 mm. In one exemplary form, the washing chamber has a diameter of about 4 mm and a depth of about 3 mm.

[0113] For example, similar to a sample loading chamber, a binding chamber can have a cylindrical shape with a flat bottom surface and continuous sidewalls. The sidewalls of the antibody chamber may be slightly outwardly sloped from bottom to top to facilitate the entry and exit of droplets and / or bead clusters. Additionally, the bottom surface of the antibody chamber may include flow regions, such as small ports or openings, that enable fluid communication with flow paths to adjacent chambers, such as microfluidic channels, including an upstream chamber (e.g., a first washing chamber) and a downstream chamber (e.g., a second washing chamber).

[0114] In some implementations, the binding chamber is configured to contain a pre-loaded capture agent (e.g., antibody) for use in a target binding or detection step. For example, as in... Figure 18B As illustrated in the diagram, the binding chamber 230 may contain a variety of capture agents (such as various antibodies) preloaded during the assembly of the biochip. The biochip 200 may include a fluid path that guides droplets, such as those containing analytes of interest or immunomagnetic beads, into the binding chamber, where they mix with the capture agents to facilitate binding. Alternatively, the capture agents may not be preloaded into the binding chamber but added in a later stage, such as before or during the assay, allowing for flexibility in the selection or concentration of capture agents based on specific assay requirements.

[0115] The appropriate binding agent for use in the binding chamber can be selected based on the analyte to be measured and detected. In some embodiments, the binding agent can be an antibody, peptide, aptamer, or nucleic acid aptamer. Typically, the binding agent in the binding chamber can bind to the target antigen, but binds to a different region compared to the binding agent immobilized on the surface of the immunomagnetic beads. In other words, the first binding agent immobilized on the surface of the immunomagnetic beads and the second binding agent provided in the binding chamber are typically an affinity pair that binds the same target analyte.

[0116] In some embodiments, one or more binding agents are specific to a variety of target analytes associated with exosomes. These binding agents may include, but are not limited to, antibodies, aptamers, peptide ligands, or nucleic acid probes, which are designed to selectively recognize and bind target proteins, peptides, or nucleic acids such as DNA and RNA. In some embodiments, binding agents may be selected to identify surface biomarker features of exosomes, facilitating detection and analysis based on cell origin or disease state. Other binding agents may be tailored to interact with membrane lipid components of exosomes, facilitating the identification of exosome subtypes or functions. Additionally, biochips may incorporate binding agents specific to antigens derived from pathogens that become associated with exosomes upon infection or immune activation. These may include ligands or antibodies targeting lipopolysaccharides (LPS), bacterial virulence factors, or viral proteins present in exosomes shed from infected host cells.

[0117] 4. Testing Room

[0118] Biochips include a detection chamber, which serves as a reaction site for signal generation and optical detection of signals from a sample. For example, a binder in the binding chamber, which binds to an antigen, contains a reactive label that reacts with a reactant provided in the detection chamber and produces light and / or color for detection. Optionally, the detection chamber is in direct fluid communication with a termination buffer chamber, which allows the termination buffer to be delivered to the detection chamber after the reaction between the reactive label on the binder and the reactant.

[0119] The detection chamber typically has the same size and shape as the sample loading chamber and / or washing chamber. For example, the detection chamber can be a cylindrical or circular microfluidic chamber located within the chamber layer of a biochip. In some embodiments, the detection chamber includes a flat bottom surface and continuous sidewalls that may slope slightly outward from bottom to top to support stable confinement of droplets and facilitate controlled droplet coalescence and mixing. Alternatively, the detection chamber may be larger than the other chambers to accommodate a larger volume of fluid, such as droplets from the sample and a stop buffer from the stop buffer chamber.

[0120] In some implementations, the detection chamber is structurally different from other chambers in the biochip to facilitate accurate readout of optical signals. For example, the detection chamber can be directly sealed between the top and bottom layers of the biochip, both of which can be formed of a transparent material that allows light to pass through without interference. In contrast, the intermediate layer of the biochip can be made of polytetrafluoroethylene (PTFE) or a similar opaque material and can be hollowed out in the area corresponding to the detection chamber. This configuration prevents the opaque intermediate layer from obstructing the light path, thereby facilitating reliable detection of light-based signals (e.g., fluorescence, absorbance) during measurement readings.

[0121] Optionally, the bottom plate of the detection chamber can be formed of an optically transparent material and vertically aligned with one or more layers of the electromagnetic (EM) chip to enable unobstructed light transmission for signal readout. In some embodiments, the bottom surface of the detection chamber is precisely aligned with an optical sensor integrated into the bottom layer of the EM chip. To further facilitate optical detection, the substrate forming the bottom surface of the detection chamber may include cutouts or transparent areas to allow light to pass directly through the droplet and into the sensor without optical interference.

[0122] 5. Termination buffer chamber

[0123] The biochip optionally includes a termination buffer chamber to receive a quantity of termination buffer for transfer to the detection chamber to terminate the reaction between the reactive label on the binder and the reactant. For example, the termination buffer chamber may be located near the detection chamber to enable direct transfer of the termination buffer droplet into the detection chamber.

[0124] Similar to the other chambers described above, the termination buffer chamber can be a cylindrical or circular microfluidic chamber formed within the chamber layer of a biochip. For example, the termination buffer chamber may include a flat bottom surface and continuous sidewalls that may slope slightly outward from bottom to top to facilitate the entry and release of termination buffer droplets.

[0125] 6. Immunomagnetic exosome beads

[0126] The biochip may also include multiple immunomagnetic exosome beads, which are preloaded into the sample loading chamber or provided as a separate component from the microfluidic system for loading during assays.

[0127] In some implementations, immunomagnetic beads are preloaded into the sample loading chamber or a portion thereof. For example, immunomagnetic beads may be preloaded into the bead chamber 202 within sample loading chamber 210 (see [link to sample loading chamber 210]). Figure 18B In some implementations, the biochip includes an inlet port or inlet 204 on the top surface of the biochip 200, through which a user can manually introduce biological samples, such as whole blood or plasma (see [link to implementation]). Figure 18B After introduction, the sample enters directly into the sample loading chamber 210 and mixes with the preloaded immunomagnetic beads contained in the bead chamber 202.

[0128] In other embodiments, the immunomagnetic beads are not pre-loaded into the biochip, but are introduced by the user during sample preparation. In such a configuration, the user prepares a mixture of biological sample (e.g., whole blood or plasma) and immunomagnetic beads externally prior to loading. This prepared mixture can then be manually introduced into the biochip through an inlet or port above the sample loading chamber.

[0129] i. Magnetic beads

[0130] Biochips comprise multiple immunomagnetic beads. These beads are typically functionalized to bind a first binding agent to their surface, which is used to bind to a target analyte in a biological sample.

[0131] Magnetic beads can be any suitable bead formed from magnetic materials and can have any suitable shape. For example, magnetic beads are micron-sized spherical particles that include a magnetic core embedded within or coated with a functionalized outer layer. The core can be formed from superparamagnetic iron oxide nanoparticles (SPIONs), such as magnetite (Fe3O4) or maghemite (γ-Fe2O3).

[0132] Structurally, the beads are small enough to be manipulated through microfluidic channels without impeding flow, and large enough to be effectively driven by a localized magnetic field generated by the underlying electromagnetic chip. In some implementations, the diameter of the magnetic beads can range from about 5 nanometers to about 50 nanometers.

[0133] The outer surface of the beads is structurally modified or coated to contain a binding agent, such as an antibody, aptamer, nanobody, peptide, DNA / RNA, and / or other suitable ligands or chemical entities. The binding agent can be immobilized on the surface of the magnetic beads using any suitable coupling method, such as by binding to functional portions of the magnetic bead surface, such as streptavidin, antibodies, or other ligands. The binding agent on the surface of the magnetic beads can have any suitable density. For example, the binding agent can be immobilized on the surface of the magnetic beads at a high surface density—for example, in the range of 1–5 nanomoles of streptavidin per milligram bead—to facilitate interaction with the target analyte.

[0134] In some embodiments, magnetic beads may be incorporated into a polymer or inorganic matrix to form a bead structure with a total diameter ranging from 1 to 10 micrometers, for example, 2 to 5 micrometers. When a matrix material is used, the matrix material forming the bead body may include polymers such as polystyrene, silica, and / or agarose, providing mechanical stability and chemical compatibility with the assay reagents. In these forms, a binder may be fixed to the surface of the magnetic beads or the surface of the matrix, such as being fixed to the polymer and exposed on the surface of the matrix material.

[0135] In some embodiments, immunomagnetic beads include a functionalized outer layer containing a fixed binding agent for targeted separation of exosomes from a sample. Exemplary exosome markers that can be used as targets for the binding agent include, but are not limited to, CD63, CD9, CD81, CD82, CD151, or combinations thereof. Other exosome markers that can be used as targets for the binding agent include, but are not limited to, heat shock proteins such as anti-HSP70 and anti-HSP90 (capturing HSP70 and HSP90, respectively), and phospholipids such as annexin V and Tim4 (capturing phosphatidylserine). For example, the binding agent for exosome separation can be an antibody, such as a CD63-specific antibody, which can be immobilized on the surface of the magnetic beads via any suitable coupling reaction. For example, the antibody is a biotinylated antibody that can be immobilized on the surface of streptavidin-coated magnetic beads via a high-affinity biotin-streptavidin interaction. Once coated, the magnetic beads present binding sites that specifically bind to exosome membrane proteins, enabling selective capture of exosomes from the sample. The binder can be an antibody, aptamer, nanobody, or other peptide.

[0136] Magnetic beads can be manufactured using a multi-step process that may include co-precipitation of magnetic nanoparticles, emulsification to form uniform spherical particles, and surface functionalization for biocompatibility and assay of specific functions. The resulting beads should generally exhibit superparamagnetic behavior, with no residual magnetization in the absence of an external magnetic field, to enable precise control and repositioning within a biochip.

[0137] In some embodiments, the binder is immobilized on the surface of the magnetic beads via non-covalent protein-ligand binding, such as through streptavidin-biotin interactions. In this configuration, the magnetic beads can be functionalized with streptavidin, while the binder is biotinylated, and vice versa. Alternatively, several other immobilization strategies may be used, depending on the desired assay configuration or surface chemistry.

[0138] One such alternative is passive adsorption, where a binder, such as an antibody, adheres to the bead surface via non-covalent hydrophobic or electrostatic interactions. In some cases, the magnetic beads can be pretreated with proteins such as protein A or protein G, which promote more efficient and targeted binding of the antibody by interacting with the Fc region of the antibody. Another approach involves covalent coupling, where an antibody or other binder is chemically crosslinked to a functionalized bead surface with reactive groups such as carboxyl, amine, or epoxy moieties. Exemplary chemical methods for covalent coupling include EDC / NHS-mediated crosslinking and glutaraldehyde-based coupling.

[0139] Furthermore, click chemistry can be used to achieve highly specific and efficient covalent fixation under mild and biocompatible conditions via biorthogonal reactions such as azide-alkyne cycloaddition. Additionally, site-specific coupling techniques—including Sortase-mediated linkages or HaloTag technology—can be used to selectively control the orientation and position of binders on bead surfaces.

[0140] Alternatively, magnetic beads are commercially available. For example, uncoated or non-specifically coated polystyrene-based magnetic microparticles are available from Sigma-Aldrich (e.g., catalog numbers 49664, 39689, and 75597); streptavidin-coated magnetic beads, such as those from Sigma-Aldrich (catalog numbers 08014 and 49532); and Dynabeads, such as those from Invitrogen.

[0141] In some implementations, the magnetic beads are configured to be removed from the detection chamber before the analyte is detected. For example, such as... Figure 3A As shown in Figures b and c, magnetic beads can be extracted from a single droplet or used to transport the droplet, depending on the loading mass of the beads and the speed of the magnetic motor. By carefully controlling these parameters, the system allows for flexible manipulation of the droplet, enabling either bead-only extraction or bead-assisted droplet transport. In some embodiments, magnetic beads are first extracted from the sample loading chamber, where target molecules have already been captured, facilitating the separation of the analyte from the sample droplet. The beads are then sequentially extracted through various processing chambers, including washing chamber 1, antibody labeling chamber, washing chambers 2, 3, and 4, and finally enter the detection chamber.

[0142] 7. Other biochip components

[0143] i. Material exchange areas and / or passageways

[0144] Biochips may also include material exchange regions or microfluidic channels in the chamber layers to facilitate the flow of sample and bead mixtures between a sample loading chamber, one or more washing chambers, a binding chamber, and a detection chamber. Optionally, when a material exchange region is included, the material exchange region is located at the center of the biochip (e.g., see [link to biochip]). Figure 1 (See diagram d). Optionally, the material exchange region may include one or more microfluidic channels or conduits to facilitate droplet flow from one chamber to another (e.g., side-phase flow design).

[0145] In some embodiments, the material exchange region is configured as a central hub for the distribution and redirection of fluids within the biochip. For example, the material exchange region can be used to combine samples with reagents and facilitate the movement of these fluids to appropriate chambers for subsequent processing steps such as washing, binding, and detection. In some embodiments, the material exchange region is constructed with flow paths and droplet chambers arranged around a central oil zone. This configuration minimizes the water / oil interface, thereby reducing the risk of droplet contamination or mixing between adjacent reaction chambers. The central oil zone acts as a physical and chemical barrier, separating aqueous droplets loaded into different chambers and maintaining their integrity during handling and transport. Additionally, no coil pattern is provided in the corresponding central region of the electromagnetically driven chip (EMChip) described below.

[0146] 8. Exemplary biochip

[0147] exist Figure 18B and 18C The illustration in the middle section depicts an exemplary biochip, the μ-Exo Biochip. The μ-Exo Biochip 200 includes multiple functional chambers, including a sample loading chamber 210, washing chambers 220, 240, 250, and 260, a binding chamber 230, a detection chamber 270, and a termination buffer chamber 280, all of which are fluidly connected to each other via predetermined microfluidic flow paths. The μ-Exo Biochip can be configured as a stationary platform where the movement of droplets and clusters of magnetic beads is driven by a localized magnetic field generated by an underlying electromagnetic chip.

[0148] like Figure 18C As shown, the μ-Exo Biochip 200 is formed of multiple stacked layers, including two barrier layers 310 and 312, two chamber layers 322 and 324, and three adhesive layers 330a, 330b, and 332. The two barrier layers, namely the top barrier layer 310 and the bottom barrier layer 312, form the top and bottom surfaces of the biochip 200. The top and bottom barrier layers can be formed of a transparent material such as polyethylene terephthalate (PET) to facilitate optical detection through the top barrier layer of the μ-Exo Biochip 200.

[0149] The two chambers 322 and 324 of the μ-Exo Biochip 200 can be formed of polytetrafluoroethylene (PTFE) and include a first chamber and a second chamber. For example... Figure 18C As shown, the first chamber layer 322 can be patterned, such as by laser micromachining, to define fluid chambers and flow paths. The surface of the second chamber layer 324 can be used to form the chambers and the substrate of the biochip, on which assay reagents and samples will move during use.

[0150] Three adhesive layers are used to bond the two barrier layers 310, 312 and the two chamber layers 322, 324 of the μ-Exo Biochip together, sealing the fluid chambers and flow paths while maintaining flow passages between the chambers. Figure 18C As shown, a first adhesive layer 330a can be used to bond the top barrier layer 310 to the first chamber layer 322, a second adhesive layer 330b can be used to bond the first chamber layer 322 to the second chamber layer 324, and a third adhesive layer 332 can be used to bond the second chamber layer 324 to the bottom barrier layer 312. The first and second adhesive layers 330a and 330b can be patterned similarly to the first chamber layer 322 to define fluid chambers and flow paths, thereby facilitating the non-adhesiveness of the chamber floor formed by the second chamber layer 324.

[0151] like Figure 18B As shown, each chamber in the μ-Exo Biochip 200 can have a cylindrical geometry with a flat bottom surface and slightly sloping sidewalls. The sample loading chamber 210 is configured to receive a biological sample and immunomagnetic beads and includes a flow region connected to a downstream chamber. The sample loading chamber 210 can have a diameter of about 2 to 5 mm and a depth of 1 to 3 mm, sufficient to accommodate a sample volume of about 5 μL to about 200 μL. In some embodiments, the sample loading chamber has dimensions sufficient to accommodate sample volumes of about 5 μL to about 150 μL, about 5 μL to about 100 μL, about 5 μL to about 80 μL, about 5 μL to about 60 μL, about 5 μL to about 40 μL, about 5 μL to about 20 μL, or about 5 μL to about 10 μL.

[0152] The μ-Exo Biochip includes a binding chamber 230 located between a first washing chamber 220 and a second washing chamber 240.

[0153] The μ-Exo Biochip can include four washing chambers: 220, 240, 250, and 260. For example... Figure 18B As shown, the first washing chamber 220 is in direct fluid communication with the sample loading chamber 210 and the bonding chamber 230. For example, the first washing chamber 220 may be located between the sample loading chamber and the bonding chamber, and adjacent to the bonding chamber, to achieve sequential washing steps to remove unbonded material. Figure 18B As shown, the second, third, and fourth washing chambers 240, 250, and 260 are located between the bonding chamber 230 and the detection chamber 270. Although the three washing chambers are located between the bonding chamber and the detection chamber, any number of washing chambers, such as one or two washing chambers, may be included between the bonding chamber and the detection chamber.

[0154] like Figure 18BAs shown, the μ-Exo Biochip includes a detection chamber 270, such as a reaction chamber. The detection chamber 270 is aligned with the optical sensor 130 below (see Figure 130). Figure 18A It may include a transparent bottom surface to allow light to pass through the droplet for signal readout based on absorbance.

[0155] An exemplary μ-Exo Biochip also includes a termination buffer chamber 280 adjacent to the detection chamber 270, which stores droplets of a termination solution that merge with reaction droplets in the detection chamber 270 to terminate the enzyme reaction used for signal generation.

[0156] B. Electromagnetic chip

[0157] The microfluidic device also includes an electromagnetic chip, which includes a programmable navigation matrix.

[0158] The electromagnetic chip includes a solid substrate on which are a plurality of electromagnetic coils, one or more switching integrated circuits electrically connected to the plurality of electromagnetic coils, one or more magnetic motors, and a microcontroller configured to regulate the activation of the electromagnetic coils for electromagnetic actuation of the magnetic motors. In some embodiments, the magnetic motors used for driving in the biochip system are permanent magnets, such as neodymium magnets, which are formed of an alloy of neodymium, iron, and boron (NdFeB). Alternative permanent magnets may also be used, such as iron oxide-based magnets (e.g., Fe3O4 or γ-Fe2O3), cobalt (Co) magnets, nickel (Ni) magnets, and samarium cobalt (SmCo) magnets.

[0159] Typically, multiple electromagnetic coils in an electromagnetic chip are arranged such that they are aligned with the sample loading chamber, one or more washing chambers, binding chamber, detection chamber, and, if present, termination buffer chamber.

[0160] The solid substrate of the electromagnetic (EM) chip can be formed from any suitable material capable of supporting the formation of circuits and components. In some embodiments, the solid substrate comprises a four-layer printed circuit board (PCB) structure that facilitates the compact and functional integration of electromagnetic coils, magnetic motors, control circuitry, and electrical interconnects. The multilayer PCB structure may include at least two conductive layers and insulating layers, such as a first layer and a second layer. The first layer may be configured to house the magnetic motor, providing structural support and alignment for its movement across the chip. The second layer may be configured to embed electromagnetic coil elements, enabling the generation of a localized magnetic field for driving the magnetic motor. Optionally, the PCB structure includes a third and fourth layer, which may be used to mount integrated circuits (ICs) and manage signal routing and power distribution.

[0161] Each layer of the EM chip can be formed from alternating conductive materials such as copper and dielectric insulating materials such as FR-4, a glass fiber reinforced epoxy laminate known for its mechanical strength, thermal stability, and electrical insulation. Depending on the application requirements, the EM chip can also be manufactured using other suitable substrate materials, including polyimide, PTFE-based laminates, or high-frequency compatible composites, all of which offer improved electrical performance and environmental durability.

[0162] The solid substrate can have any suitable thickness, for example, between about 0.1 mm and about 0.5 mm, between about 0.5 mm and about 1.0 mm, between about 1.0 mm and about 2.0 mm, between about 2.0 mm and about 3.0 mm, between about 3.0 mm and about 5.0 mm, such as between about 0.7 mm and about 1.0 mm. In some embodiments, the EM chip is constructed using a 4-layer PCB with a thickness ranging from about 0.6 mm to about 6 mm. Since magnetic flux density decreases with distance, a thinner PCB is preferred to improve magnetic drive capability and increase heat dissipation.

[0163] 1. Electromagnetic coil

[0164] An electromagnetic chip includes one or more structurally integrated electromagnetic coils, such as those mounted on or embedded in a solid substrate. The electromagnetic coils can be arranged in a defined array or matrix configuration, aligned with specific regions or chambers of the biochip, including but not limited to sample loading chambers, washing chambers, binding chambers, detection chambers, and, if present, termination buffer chambers. Such spatial alignment enables the application of targeted magnetic fields at predetermined locations within the biochip.

[0165] Each electromagnetic coil can be formed from wires such as copper wound around a core, which may include a magnetic material (e.g., ferrite) to increase the magnetic field strength. The coil dimensions—including inner diameter, number of turns, and spacing—are chosen to achieve the desired magnetic properties suitable for different microfluidic applications. The following are non-limiting considerations for coil arrangement when fabricating EM chips.

[0166] Coil shape: Coils can be designed as square rather than round because wires perpendicular to the direction of magnet movement have the highest driving efficiency. The geometry of a square coil allows for more segments to be perpendicular to the magnetic field direction during magnet movement, thereby improving the efficiency of interaction with the magnetic field.

[0167] Coil size: To facilitate efficient driving of the magnet and bead, the coil size should be smaller than the magnet's diameter but larger than its radius. In some implementations, the coil size can be approximately 2 / 3 of the magnet's diameter.

[0168] Core material: In PCB (printed circuit board) technology, coils are generally planar and, due to manufacturing limitations, usually do not include a magnetic core.

[0169] Coil fill factor: Increasing the coil fill factor helps improve the driving capability of the coil, which can be approximately 50%. To maximize the fill factor, the spacing between turns should be kept within manufacturable limits, meaning it should be as small as possible (e.g., approximately 0.1 mm) to increase the proportion of conductive material. Additionally, maximizing the number of coil turns within a limited space can also contribute to a higher fill factor and a stronger magnetic field.

[0170] Coil Inner Diameter: The coil inner diameter can be designed to accommodate a blind via of the minimum required size, which is used to connect coils between adjacent layers in series. This design aims to keep the coil structure compact while achieving effective electrical connection between layers.

[0171] Electromagnetic coil arrays comprise 150 or more coils, such as from about 150 to about 300 active coil elements. The number of coil elements required in an electromagnetic coil array depends on the specific application and the range of motion of the magnet. For continuous and efficient magnetic actuation, the array should include enough coil elements to completely cover the movement path of the magnet, achieving uninterrupted magnetic interaction throughout operation. Adding additional coils provides the advantage of backward compatibility. That is, coil arrays with more elements can be selectively configured or operated to match systems requiring fewer coils, providing improved flexibility and adaptability in a variety of biochip designs and use cases.

[0172] Figure 18D and 18E The images show top and bottom views of an exemplary EM chip 120, illustrating the arrangement of electromagnetic coils 122 and circuitry 124 on a solid substrate 126. The electromagnetic coils 122 are structurally integrated with the solid substrate 126, such as being mounted on or embedded within its surface. The coils 122 are arranged in a defined array or matrix configuration and spatially aligned with chambers of the biochip above, such as sample loading chambers, washing chambers, and detection chambers, to facilitate targeted actuation of magnetic beads via the biochip. Each coil 122 may be formed of wire, such as copper, wound around a core, which optionally includes a magnetic material (e.g., ferrite) to enhance the magnetic field strength. In some embodiments, each coil element comprises a four-turn coil (e.g., 2 mm × 2 mm). Figure 18E As shown, the bottom side of the EMchip may include passive components 121 for power regulation and signal filtering, such as resistors and capacitors.

[0173] 2. Magnetic motor

[0174] An EM chip includes at least one magnetic motor configured as a movable magnetic element, such as a permanent magnet or a disk. The size and shape of the magnetic motor generally correspond to the layout of the coil array, facilitating precise driving along the surface of the chip via a localized magnetic field generated by the electromagnetic coils.

[0175] A magnetic motor is positioned above an array of electromagnetic coils on the surface of the EM chip. In some embodiments, the magnetic motor can be housed or suspended within the interface region, preventing direct contact with the coil layer while maintaining sufficient proximity to respond to magnetic field gradients generated by the electromagnetic coils. Since each coil is independently activated via programmable control circuitry, the magnetic motor is guided across the EM chip by a series of controlled magnetic pulses, facilitating precise navigation beneath the biochip. Spatial alignment between the magnetic motor and the superimposed chambers (such as sample loading chambers, binding chambers, and detection chambers) on top of the biochip allows the magnetic motor to transfer mechanical forces to the overlying magnetic beads or droplets, facilitating targeted actuation within the microfluidic device.

[0176] For example, as in Figure 18F As illustrated in the figure, the EM chip 120 includes at least one magnetic motor 128 configured as a movable magnetic element, such as a permanent magnet or a disk. The size and shape of the magnetic motor 128 are chosen to correspond to the layout of the electromagnetic coils 122 to facilitate precise actuation along the surface of the chip via a localized magnetic field generated by the coils. The magnetic motor 128 is located above an array of electromagnetic coils 122 on the surface of the EM chip 120. In some embodiments, the magnetic motor 128 is suspended or contained within an interface region so that it does not directly contact the coil layer while remaining sufficiently close to the electromagnetic field gradient generated by the coils. When each coil in the array is independently activated via programmable control circuitry 124, the magnetic motor 128 is guided across the EM chip with a controlled sequence of magnetic pulses.

[0177] 3. Integrated Circuits

[0178] An EM chip comprises multiple programmable switching integrated circuits (ICs) mounted on a lower layer of a multilayer printed circuit board (PCB), e.g., between the second and third layers. The multiple ICs can be arranged to provide electrical connectivity and achieve compact integration within the chip architecture. Each IC may include one or more output ports. Each port may be electrically connected to a corresponding electromagnetic coil element located on one or more upper layers (e.g., the second layer) of the PCB to facilitate the activation of a specific coil. Optionally, the ICs are electrically connected to the coil element and other components via conductive traces and vertical interconnects (vias) within the PCB structure. In some embodiments, for example, the chip may include 20 or more ICs, such as 21 programmable switching ICs (e.g., the MAX14662, Maxim Integrated), arranged in a configuration supporting serial communication protocols such as the Serial Peripheral Interface (SPI).

[0179] 4. Exemplary electromagnetic chip

[0180] exist Figure 18D and 18E The illustration depicts an exemplary electromagnetic chip, the μ-Bead EMchip. The μ-Bead EMchip is configured to regulate the activation of electromagnetic coils for electromagnetically driving a magnetic motor. The μ-Bead EMchip includes a solid substrate, twenty-one switching integrated circuits electrically connected to an array of electromagnetic coils, a magnetic motor, and a microcontroller. The solid substrate for the μ-Bead EMchip is formed from a four-layer printed circuit board (PCB) structure. The PCB substrate includes four conductive layers and insulating layers. A first PCB layer can be configured to house the magnetic motor, providing structural support and alignment for its movement across the μ-Bead EMchip. A second PCB layer can be configured to embed electromagnetic coil elements, enabling the generation of a localized magnetic field for driving the magnetic motor. The PCB substrate includes third and fourth layers for mounting integrated circuits (ICs) and managing signal routing and power distribution.

[0181] The μ-Bead EMchip includes a magnetic motor configured as a movable magnetic element, such as a permanent magnet or a disk. The magnetic motor is configured to connect to a μ-Exo Biochip located above the μ-Bead EMchip. The size and shape of the magnetic motor correspond to the layout of the coil array on the EMchip, facilitating precise actuation across the chip's surface via a localized magnetic field generated by the electromagnetic coils. The magnetic motor of the μ-Bead EMchip is positioned above the coil array and is housed or suspended within an interface region that prevents direct contact with the coil layer while maintaining close proximity to the coils to effectively respond to the generated magnetic field gradient. Because each coil in the array is independently activated via programmable control circuitry, the magnetic motor can be guided across the chip in a stepwise manner using a series of controlled magnetic pulses.

[0182] The movement of the magnetic motor in the μ-Bead EMchip is spatially aligned with the layout of the sample loading chamber, binding chamber, and detection chamber in the μ-Exo Biochip. This alignment allows the magnetic motor to transmit mechanical forces through the biochip to manipulate clusters of magnetic beads or droplets, facilitating targeted actuation and controlled transport of assay components throughout the microfluidic system.

[0183] The μ-Bead EMchip includes an array of electromagnetic coil elements, each configured to generate a localized magnetic field for precisely driving a magnetic motor. For example, the EMchip includes 165 coil elements, each formed as a multi-turn (e.g., four-turn) coil with a size of approximately 2 mm × 2 mm, and arranged with a narrow spacing (e.g., 0.1 mm gap) between adjacent coils. The coils can be distributed across multiple upper PCB layers. The electromagnetic coil array in the μ-Bead EMchip is arranged such that it is aligned with the sample loading chamber, washing chamber, binding chamber, and detection chamber of the μ-Exo Biochip.

[0184] Each coil element can be individually manipulated and powered by a direct current (DC) power supply, such as at 0.4 A, to generate sufficient magnetic force to drive a movable magnetic motor. Control of the coil elements can be achieved using a set of programmable switching integrated circuits (ICs) mounted on a lower PCB layer, with each output port electrically connected to the corresponding coil element. In one example, the EM chip includes 21 switching ICs (e.g., MAX14662, Maxim Integrated) configured in a daisy-chain arrangement using the Serial Peripheral Interface (SPI) protocol.

[0185] The IC can connect to a microcontroller such as the Arduino Nano, which can be programmed using a pathfinding algorithm. This algorithm processes commands received from an external user interface, determines the required coil activation sequence, and transmits the corresponding SPI commands to the switching IC for automated, addressable magnetic actuation.

[0186] C. Optical sensing module

[0187] The microfluidic device may also include an optical sensing module to measure the light absorption of the droplets read out through a signal in the detection chamber, thereby enabling quantitative analysis of the measurement results. The optical sensing module can be integrated into the lower portion of the device, directly below the bottom layer of the EM chip, and vertically aligned with the detection chamber. In some embodiments, the optical sensing module can be mounted on or embedded within the housing of the microfluidic device (see [link to documentation]). Figure 1 (Figure c).

[0188] Optical sensing modules typically include a light-emitting component and a light-detecting component. The light-emitting component may be a blue LED with a peak emission wavelength of approximately 450 nm, and the light-detecting component may be an ambient light sensor with a peak absorption at approximately 560 nm. The optical sensing module may also include optical sensors for detecting light transmitted through or emitted from droplets within the detection chamber, such as absorbance, fluorescence, or color changes indicating the presence or concentration of a target analyte.

[0189] For example, an optical sensing module may include a blue light-emitting diode (LED) (e.g., L450R-04, USHIO) and an optical sensor (e.g., BH1721, ROHM Semiconductor, 3.3 V) for capturing or absorbing light. The optical sensor can be transmitted via an internal integrated circuit (I) for data transfer and control. 2 C) Communication Protocol: Connects to microcontrollers such as the Arduino Nano. In some implementations, the 2 mm × 2 mm board notch can be aligned with the center of the detection area of ​​the EMCchip (such as the μ-BeadEMchip) to allow unobstructed light path through the droplet. During operation, light from the LED passes through the droplet located in the detection chamber and reaches the optical sensor through the notch, facilitating the measurement of optical properties such as absorbance or colorimetric changes (see...). Figure 18A ).

[0190] exist Figure 18GThe illustration depicts an exemplary optical sensor 130. The optical sensor 130 may include a substrate 131, which serves as the structural basis for one or more integrated components. An optical sensor chip 132 may be mounted on the surface of the substrate 131 and serves as a detection element for capturing and processing optical signals. One or more conductive traces 139 may extend from the optical sensor chip 132 to form circuitry facilitating signal transmission. These conductive traces 139 connect the optical sensor chip 132 to a connection interface 138, which includes multiple terminals for power and data communication. For example, the connection interface 138 may include a ground terminal (GND) 133 for electrical stability, a reset pin (RST) 134 for initializing the sensor, a serial clock line (SCL) 135, and a connection for communication via I... 2 The serial data line (SDA) 136 for C protocol communication and the power supply terminal (VCC) 137 for providing the operating voltage.

[0191] D. Optional components

[0192] Microfluidic systems may include one or more optional components, such as a user display, a housing, and / or reagents and / or buffers. In some embodiments, one or more reagents and / or buffers may be provided as separate components in the packaging of the microfluidic system.

[0193] 1. Reagents and buffer solutions

[0194] Microfluidic systems may include one or more reagents and / or buffer solutions. Reagents for the detection and analysis of analytes may be pre-positioned in the detection chamber of the biochip (e.g., capture agents immobilized on a substrate of the analytical region). For example, reagents may be introduced into the detection chamber during assay execution or pre-loaded into the detection chamber prior to assay execution. Reagents suitable for the detection chamber may react with reactive labels of binders for the binding chamber, and the reaction between them may produce readable signals, such as optional signals. Buffer solutions used in microfluidic systems are generally suitable for washing immunomagnetic beads to remove non-specifically bound non-target molecules and / or binders. Suitable buffer solutions for the washing chamber of a biochip include, but are not limited to, phosphate-buffered saline.

[0195] In some embodiments, the microfluidic system includes one or more imaging agents. An imaging agent is a compound or molecule that includes or generates a detectable label. A detectable label is a compound or molecule capable of emitting or generating a measurable signal. In some embodiments, the imaging agent may specifically bind to an analyte molecule, or to a complex or product formed during assay. This interaction facilitates the label's localization within a specific region of the biochip's detection chamber—such as the region where the analyte molecule binds or where reaction products accumulate—so that the emitted signal is spatially correlated with a specific analytical event.

[0196] Examples of suitable imaging agents include, but are not limited to, fluorescent markers such as fluorescein (FITC), 5,6-carboxymethylfluorescein, Texas Red, nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4',6-diamidinyl-2-phenylindole (DAPI), and anthocyanin dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7. Common fluorescent markers include, but are not limited to, 5-carboxyfluorescein-N-hydroxysuccinimide and 5,6-tetramethylrhodamine, while FITC and anthocyanin dyes are particularly suitable for combined multicolor coding. Their respective excitation and emission maximum values ​​are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm), and Cy7 (755 nm; 778 nm). In some implementations, the optical module is configured to measure light absorption within the biochip. However, if the microfluidic device is to be suitable for detecting fluorescence signals, simple filters or lenses can be integrated into the module to selectively capture emitted fluorescence while blocking excitation light.

[0197] 2. Shell

[0198] Microfluidic systems are optionally encapsulated in a housing, such as a portable housing. For example, the microfluidic system can be provided as a point-of-care testing device. The housing can be formed of any material that provides protection against mechanical shock, moisture, and environmental contaminants during handling and operation outside of a laboratory environment. For example, the housing can be formed of lightweight, impact-resistant materials such as polycarbonate, ABS plastic, or aluminum alloy. In one exemplary form, the housing is formed of a resin such as Tough 2000 Resin.

[0199] In some embodiments, the length of the housing ranges from about 100 mm to about 150 mm, such as about 110 mm, about 120 mm, or about 130 mm. In some embodiments, the width of the housing ranges from about 60 mm to about 80 mm, such as about 65 mm, about 70 mm, or about 75 mm. In some embodiments, the height of the housing ranges from about 30 mm to about 50 mm, such as about 35 mm, about 40 mm, or about 50 mm. In one exemplary form, the housing has a length, width, and height of about 127 mm, 71 mm, and 36 mm, respectively.

[0200] The housing may include a base configured to securely hold internal components of the system, such as biochips, electromagnetic chips, optical sensor modules, and control electronics, with integrated mounting features or internal compartments for alignment. The top cover may include an access point for sample loading and a transparent window or cutout aligned with the detection chamber for visual inspection or optical reading. The cover may be connected via a hinged or latching mechanism for quick opening and closing in field applications.

[0201] For usability in mobile environments, the housing can incorporate ergonomic design features such as a carrying handle, low profile shape factor, and non-slip base to ensure stable placement and easy manipulation. Ventilation may be included to dissipate heat generated by onboard components during operation. Portable housings enable reliable, user-friendly deployment of microfluidic devices in remote, clinical, or resource-constrained environments.

[0202] like Figures 19A-19D As shown, the exemplary housing 100 may include a user interface 160 integrated into the upper surface of the housing 100. The user interface 160 may be in the form of a touchscreen interface, configured to display real-time system status, measurement progress, and user prompts. Figures 19A-19D In some implementations, the screen displays the current date and time, Wi-Fi connection, battery status, and a progress indicator corresponding to the "sample loading" step (see [link]). Figure 19A and 19B The user interface 160 enables users to interact with the device via a graphical user interface (GUI) and can also support touch-based input for navigation in the measurement workflow.

[0203] The housing 100 may include a compartment 110 configured to receive and immobilize the biochip 200 (see [link]). Figure 19B The compartment 110 can be housed in a drawer-type or sliding compartment, allowing for manual insertion of the biochip 200 by the user. In the closed position, the compartment 110 can be aligned with the underlying electromagnetic chip and optical sensing module, such that relevant chambers within the biochip, such as the sample loading chamber, binding chamber, and detection chamber, are appropriately positioned above the corresponding functional components within the device. If desired, the compartment 110 may also include features for stable placement and electrical or optical connection, such as guide rails, positioning pins, or recessed structures.

[0204] 3. User display / interface

[0205] The microfluidic system optionally includes an operator-operable user interface / user display. Optionally, the microfluidic system includes a user interface for operating and controlling one or more device functions. In some embodiments, the upper surface of the device housing may incorporate a user interface panel configured to provide access to system controls, such as initiating or pausing fluid operations, starting measurements, or displaying real-time status indicators. The user interface may be connected to internal components of the device, such as electromagnetic chips, optical sensors, or fluid control modules, via wired or wireless connections, including Bluetooth, Wi-Fi, or serial communication protocols.

[0206] The user interface may include one or more interactive controls, such as buttons, touch-sensitive buttons, LED-backlit buttons, knobs, or capacitive touch sliders, enabling users to control various operating parameters without turning on the device. For example, buttons may be provided to start or stop measurement cycles, while touch-sensitive areas may be designated for specific commands, such as initiating sample loading or triggering a readout process. In some embodiments, LED-backlit buttons are used to indicate different operating states—such as measurement in progress, completed, or error—through changes in color or brightness. Knobs may optionally be included to allow users to navigate menu options or adjust settings such as measurement duration or sensor calibration parameters. Alternatively, a capacitive slider may be integrated, allowing users to adjust values ​​(e.g., motor speed or sensitivity thresholds) by sliding their finger across a designated interface area.

[0207] In some implementations, a user interface is provided via a touchscreen display, which can serve as an interactive control panel and a visual output module, or both. The touchscreen allows the user to initiate the measurement run via buttons on the screen and save or record test results upon completion. The interface can also guide the user through various stages of the measurement using interactive prompts, status messages, and progress indicators. Multiple interface screens can be displayed throughout the workflow, which includes sample loading, processing, and result visualization. For example, as... Figure 19D As shown, the results from the measurement can be immediately displayed on the user's monitor.

[0208] In other forms, the user interface can be omitted from the system, and the microfluidic system can instead be remotely controlled via an external interface, such as a wireless handheld remote control or a software application running on a smartphone, tablet, or computer. This remote interface can provide functions such as power on / off, protocol selection, progress monitoring, and data output, allowing users to easily interact with the device without direct physical contact.

[0209] II. Methods using MEMS

[0210] This invention also provides a method for detecting one or more analytes in a biological sample. Generally, detection methods using microfluidic systems are highly automated, and the user simply loads the biological sample into the sample loading chamber of the biochip and then starts the system by generating an electromagnetic field. Optionally, prior to sample loading, the user loads immunomagnetic beads, one or more washing buffers, one or more binding agents, one or more reactants, and / or termination buffers into the respective chambers of the biochip. Optionally, the user prepares the buffers and / or reactants for loading into the chambers of the biochip.

[0211] An electromagnetic field generated by an EMChip guides the flow of magnetic beads between different chambers on a biochip for assaying. For example, upon pressing a button integrated into or externally connected to the microfluidic system, the generated electromagnetic field guides the mixing of immunomagnetic exosome beads loaded in the sample with the sample, enabling the binding of one or more target analytes to a first binder on the surface of the magnetic beads to form a bead-analyte complex. The bead-analyte complex is guided to an adjacent washing chamber and mixed with a washing buffer to remove non-specifically bound molecules. The washed bead-analyte complex is then guided to a binding chamber containing a second binder labeled with a reactive tag to bind the second binder to one or more target analytes, forming a bead-analyte-labeled complex. The bead-analyte-labeled complex is guided to one or more adjacent washing chambers and mixed with a washing buffer to remove non-specifically bound binders. The washed bead-analyte-labeled complex is then guided into a detection chamber containing one or more reactants for signal generation. The reactants in the detection chamber can react with the reactive markers on the second binder and generate readable signals, such as optical signals, which can be detected and quantified by an optical module included in the microfluidic system.

[0212] Biological samples can be any suitable biological sample, such as tissues, blood, plasma, serum, saliva, urine, cerebrospinal fluid (CSF), breast milk, and ascites. Generally, biological samples are obtained from mammals such as humans, rodents, livestock, pets, or wild animals. In some embodiments, the biological sample is a plasma or serum sample from a human.

[0213] Typically, microfluidic systems enable detection using very small amounts of biological samples, such as less than 200 μL, less than 100 μL, less than 20 μL, or less than 10 μL. In some embodiments, the biological sample is a liquid sample ranging from 1 μL to 200 μL. For example, in some embodiments, the biological sample is a fluid sample ranging from about 5 μL to about 150 μL, about 5 μL to about 100 μL, about 5 μL to about 80 μL, about 5 μL to about 60 μL, about 5 μL to about 40 μL, about 5 μL to about 20 μL, or 5 μL to about 10 μL.

[0214] A more detailed description of methods using microfluidic devices is provided in the following non-limiting embodiments.

[0215] Example 1

[0216] 1. Materials and Methods

[0217] (1) Materials

[0218] Plasma samples from healthy individuals, Alzheimer's disease patients, and patients with mild cognitive impairment were obtained from Innovative Research, Inc. (Novi, MI, USA), BioIVT LLC. (Westbury, NY, USA), and Molecular Depot LLC (San Diego, USA) to minimize bias in the plasma sample processing. Mild cognitive impairment represents an early stage of memory loss, characterized by symptoms less severe compared to Alzheimer's disease, and was confirmed by screening using the Mini-Mental State Examination (MMSE). Alzheimer's disease (AD) was confirmed based on a clinical diagnosis. All plasma samples were stored at -20°C. Human anti-CD63 primary antibody (MA5-30187), APC goat anti-rabbit IgG (H+L) secondary antibody (A-10931), and 10 nm AuNP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (A-31566) were obtained from ThermoFisher Scientific. Mouse anti-CD63 primary antibody (ab281516), mouse anti-β-amyloid 1-42 antibody (ab280993), and human anti-β-amyloid 1-42 antibody (ab240233) were obtained from Abcam Limited. Total exosome isolation reagent (from cell culture medium) (4478359) was obtained from ThermoFisher Scientific. Exosome FITC staining (EXOFLOW800A-1) and FluoroCet exosome quantitative kit (FCET96A-1) were obtained from System Biosciences.

[0219] (2) Cell culture

[0220] The human neuroblastoma cell line SH-SY5Y was obtained from the American Type Culture Collection (ATCC). It was cultured in basal essential media (MEM, Gibco) and media containing GlutaMAX. TM SH-SY5Y cells were cultured for passages 1-15 in a 1:1 medium supplemented with Ham's F-12 nutrient mix (F-12, Gibco). The medium was supplemented with 10% exosome-free fetal bovine serum (EXO-FBS-250A-1, SystemBiosciences) and 1% penicillin-streptomycin (15070063, Gibco). The cells were maintained at 37°C in a humid atmosphere containing 5% CO2.

[0221] (3) Exosome isolation

[0222] SH-SY5Y cells from passages 1-15 were cultured for 48 hours in exosome-free medium containing 10% exosome-free FBS (EXO-FBS-250A-1, SystemBiosciences). The cell culture medium was collected and filtered through a 0.2 μm membrane filter. The filtered medium was then centrifuged at 2000×g for 30 minutes to remove cell debris. The supernatant was transferred to a new tube, mixed with total exosome separation reagent (4478359, Invitrogen) according to the manufacturer's protocol, and incubated overnight at 4°C. After incubation, the sample was centrifuged at 10000×g for 1 hour at 4°C. The supernatant was discarded, and the exosomes were collected in a precipitate at the bottom of the tube. The exosome precipitate was resuspended in PBS for subsequent experiments.

[0223] (4) Preparation for iME bead-based exosome identification assay

[0224] The anti-CD63 primary antibody was first conjugated with biotin using a commercially available biotinylation kit (ab201796, Abcam) according to the protocol. After conjugation, the biotin-conjugated CD63 primary antibody (0.07 mg / ml) was incubated with streptavidin-coated magnetic beads (4 mg / ml, 11206D, Invitrogen) in 100 μl PBS at room temperature with constant rotation for 2 hours, protected from light. The beads were then washed three times in PBS (Bio-Rad) containing 0.4% Block Ace and blocked for 1 hour at room temperature with PBST buffer containing 4% Block Ace. After blocking, the coated beads were concentrated to 10 mg / ml in Buffer A SignalPlus Antibody Enhancer (GeneTex) to enhance binding affinity. The iME beads were then prepared for exosome capture. To generate a signal, horseradish peroxidase (HRP) was conjugated to anti-Aβ42 antibody using a commercially available HRP antibody conjugation kit (ab102890, Abcam) according to the protocol. The HRP-conjugated anti-Aβ42 antibody was diluted 1:1000 in Buffer B SignalPlus AntibodyEnhancer (GeneTex) before use. Wash buffer was prepared using PBST containing 0.4% Block Ace. TMB buffer (34028) and stop buffer (SS03) were purchased from ThermoFisher Scientific. For each assay, the sample was loaded and mixed with 8 μl iME beads on the chip for 1 hour. Afterward, the beads were transferred to a 50 μl drop of wash buffer and washed for 5 minutes. The beads were then mixed and incubated with a 50 μl drop of HRP-conjugated anti-Aβ42 antibody for 1 hour, followed by three washes. The beads were then extracted and reacted with a 50 μl drop of TMB for 15 minutes. Finally, a 50 μL drop of stop buffer was added to terminate the reaction before reading. Uncoated polystyrene magnetic microspheres (49664, Sigma-Aldrich) are used to adjust the bead loading mass, facilitating the handling of beads and droplets.

[0225] (5) Fabrication of a microfluidic exosome detection biochip (μ-Exo Biochip)

[0226] μ-ExoBiochips were fabricated by laser-engraving different patterns on two polytetrafluoroethylene (PTFE) substrates, which were then sandwiched between two transparent polyethylene terephthalate (PET) films. This structure encapsulates mineral oil (5904, Sigma-Aldrich) and assay reagents in a sealed environment. The substrates were assembled using double-sided adhesive tape to construct a chamber approximately 3 mm high.

[0227] (6) Design of μ-Bead EMchip

[0228] μ-Bead EMchip is based on a four-layer PCB. 57 The microcontroller is designed and consists of 165 active coil elements. Each element features a four-turn coil (2 mm × 2 mm) stacked on a top three-layer PCB with a 0.1 mm gap separating it from adjacent elements. Each coil element can be independently powered by a 0.4 A direct current (DC) to generate localized magnetic force to attract a magnetic motor. A total of 21 programmable switch ICs (MAX14662, Maxim Integrated) are mounted on the bottom layer of the PCB, with each output port connected to a single coil element to selectively activate it. The switch ICs are daisy-chained to an Arduino Nano via a serial peripheral interface, which communicates with the computer via serial communication. A simple pathfinding algorithm is embedded in the microcontroller. The target address or operation sent from the user interface is processed by the pathfinding algorithm, then translated by the Arduino into a serial peripheral interface command, and subsequently transmitted to the switch ICs for automated, addressable activation of the coil elements.

[0229] The light signal was measured using a blue LED (L450R-04, USHIO) with a peak wavelength of 450 nm and an ambient light sensor (3.3 V) (BH1721, ROHM Semiconductor) with a peak absorption of 560 nm. The ambient light sensor was connected to the Arduino Nano via an internal inter-integrated circuit (I2C) protocol. A 2 mm × 2 mm board cutout was centrally located within the detection area of ​​the μ-BeadEMchip. Light emitted by the LED passed through a droplet, then through the cutout to reach the ambient light sensor. To ensure measurement accuracy, the light intensity was continuously sampled and filtered over a duration of 10 seconds.

[0230] (7) Preparation of synthetic exosome-amyloid β-conjugate

[0231] Lyophilized Aβ42 protein (rPeptide) was resuspended in PBS buffer containing isolated exosome precipitate and incubated at 4°C for 20 hours to facilitate conjugate formation. After incubation, the initial binding buffer was diluted with PBS to achieve the desired total Aβ42 concentration and used immediately. 100 μl aliquots were used for on-chip assays.

[0232] (8) Flow cytometry analysis

[0233] To characterize the immunocoating efficiency of the magnetic beads, primary antibodies were labeled with APC-conjugated secondary antibodies. Briefly, beads coated with primary antibody (100 μg / 100 μl PBS) and secondary antibody (10 μg / ml) were incubated at room temperature with constant rotation for 1 hour, protected from light. Blank uncoated beads served as a control for nonspecific binding assays. The beads were then washed three times in PBS containing 0.4% BlockAce, and the precipitate was resuspended in 1 ml PBS for flow cytometry analysis using a BD FACSAria III cell sorter. To evaluate the exosome capture performance of the immunobeads, a commercially available exosome purification kit was used. Briefly, exosome-captured beads (100 μg) were resuspended in 250 μl of exosome staining buffer containing 10 μl of FITC exosome staining agent and incubated on ice for 3 hours, with gentle tapping every 30 minutes to ensure mild mixing. Blank primary antibody-coated beads served as a control for nonspecific FITC exosome dye binding assays. The beads were then washed three times in PBS containing 0.4% Block Ace, and the precipitate was resuspended in 1 ml PBS for flow cytometry (BD FACSAria III cell sorter). Analysis was performed using FlowJo V10 software, which gated the beads based on their forward and lateral scattering characteristics.

[0234] (9) Electron microscope

[0235] Energy-dispersive X-ray spectroscopy (EDXS) images of AuNP-conjugated secondary antibody-labeled iME beads were obtained using a FEG scanning electron microscope (Hitachi S-4800), with no Au sputtering on the samples. Ex-Aβ samples were fixed with 4% paraformaldehyde (I28800, ThermoFisher Scientific), washed three times, and transferred to a nickel grid. Aβ42 protein was then subjected to double immunolabeling using Aβ42 primary antibody (ab180956, Abcam) and 10 nm AuNP-conjugated secondary antibody (A-31566, ThermoFisher Scientific). The labeled samples were washed and contrast-stained. The dried samples were imaged using a scanning transmission electron microscope (FEI Tecnai G2 20). Exosomes and Aβ42 aggregates were fixed with 4% paraformaldehyde, transferred to a copper grid, washed, and contrast-stained. The dried samples were imaged using a transmission electron microscope (Philips CM100).

[0236] (10) Confocal microscope

[0237] Fluorescence images of the fluorescent beads were obtained using confocal and multiphoton microscopy (Nikon NEW AXR high-speed confocal microscope, Nikon Ti2-E motorized inverted microscope) by depositing the beads on a 0.17 mm confocal slide. Fluorescence images were recorded by collecting light from 500–600 nm under 488 nm laser excitation.

[0238] (11) Animal sample analysis

[0239] C57BL / 6J and 5xFAD mice aged 7 to 9 months were housed at the Centre for Comparative Medicine Research (CCMR) at the University of Hong Kong, China, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The use of animals was approved by the Hong Kong Department of Health and the Committee on Live Animals for Teaching and Research Use (CULATR) of the University of Hong Kong (#12-127). Mice were housed in a temperature-controlled chamber between 20 and 22°C and 50 ± 10% humidity under a 12 / 12-hour light / dark cycle. Food and water were provided freely, and all possible efforts were made to minimize the number of animals used and their suffering. 0.6 ml of blood was collected via cardiac puncture into Eppendorf tubes pre-coated with 0.5 M EDTA. The blood was then centrifuged at 1200 × g for 15 minutes at 4°C. Plasma was carefully collected and stored at -80°C until use. 100 μl aliquots of mouse plasma were thawed to room temperature and then loaded onto a chip for testing.

[0240] (12) Analysis of human plasma samples

[0241] Before use, plasma samples from healthy individuals, Alzheimer's patients, and patients with mild cognitive impairment were thawed to room temperature. 100 μl aliquots of human plasma were loaded onto the chip for testing.

[0242] (13) Statistical analysis

[0243] All statistical analyses were performed using GraphPad Prism software version 10.0. Unpaired Mann-Whitney nonparametric tests were used to analyze animal samples (n = 20) from 5×FAD transgenic mice and C57BL / 6J mice, as well as clinical samples (n = 60). For all analyses, p < 0.05 was considered statistically significant.

[0244] 2. Results Analysis

[0245] (1) Development and characterization of MEMOS measurement

[0246] To detect the target biomolecule Exo-Aβ42 with high sensitivity and automation, an iME bead-based Exo-Aβ42 identification assay with dual biomarker site targeting capability was developed to specifically identify exosomal biomarkers and amyloid-β biomarkers, respectively. Figure 1 Magnetic microspheres were selected as the solid substrate to provide measurements with magnetic field responsiveness, enabling subsequent downstream separation, extraction, and transport between multiphase interfaces. 44,45 Furthermore, due to the large surface area of ​​magnetic microspheres, their use also improves the availability of fixation sites for biomolecules. 46,47 A series of assay procedures have been carefully developed and systematically evaluated to achieve target molecule identification, including iME bead development, exosome capture, and Aβ42 recognition and signal generation.

[0247] To develop iME beads with highly selective separation capabilities, the target is the typical exosome marker CD63 ( Figure 2 Figure a; Figure 5 (Figure a). CD63 is a tetraspan membrane protein that is highly concentrated in the exosome membrane and is widely used for the immunoisolation of exosomes. 37,48,49 For exosome identification and enrichment, biotinylated anti-CD63 antibody was coupled to the surface of streptavidin-positive magnetic beads via non-covalent interactions. The immunofunctionalization efficiency at the individual bead level was characterized by flow cytometry. Figure 2 Figures b and c). Phycocyanin (APC) fluorescent dye was used to label the CD63 antibody, facilitating the characterization of functionalization efficiency. After gating, the quartile distribution within a scatter plot based on the fluorescence intensity of individual beads showed that blank beads were predominantly distributed in quadrant Q4, while iME beads were dispersed in quadrant Q3. The APC fluorescence signal of iME beads shifted significantly compared to the blank beads, while the forward scattering (FSC) signal remained unchanged. The APC signal shift confirms effective antibody immobilization on individual beads, while the unchanged FSC signal indicates that the immunocoating did not cause any detectable size change in the beads. Histograms of the APC signals from individual beads show narrow peak widths in the iME bead group, indicating uniformity of immunofunctionalization. No cross-reactivity was detected between blank streptavidin beads and APC antibody staining. Figure 5 Figures a through g). The functionalization properties were further confirmed by scanning electron microscopy (SEM), where AuNP-conjugated secondary antibodies were used to label the surface functionalization sites of the iME beads. Results showed that AuNPs were uniformly localized on the surface of the iME beads, indicating successful binding of the immunocoating (data not shown). Energy-dispersive X-ray spectroscopy obtained via SEM confirmed that Au was uniformly distributed and co-localized with Fe and O elements on the bead surface (…). Figure 2(d plot), while in the control group, Au element was not detected (data not shown).

[0248] To evaluate the exosome immune capture performance of iME beads, samples were taken from the human neuroblastoma cell line SH-SY5Y (a common cell model used to study molecular events leading to Alzheimer's disease). 50 ) isolated exosomes for in vitro studies ( Figure 2 (Figure e). Under transmission electron microscopy (TEM), the isolated vesicles exhibit the characteristic cup-shaped morphology of exosomes (Figure e). Figure 6 Figure a shows a uniform distribution of particle size, as measured by nanoparticle tracking analysis (NTA), with an average diameter of approximately 150 nm. Figure 6 Figure b, data not shown). Western blot confirmed that exosome-specific protein markers, including the target CD63 protein and the reference TSG101 protein, were significantly expressed in the isolated vesicles. Figure 6 Figure c). The properties of exosome captures were studied by flow cytometry using FITC exosome staining. Figure 2 f-graph and g-graph and Figure 7 Figures a through e). Quartile distribution shows that 99.15 ± 0.10% of the iME beads were located in region Q4 before exosome capture, while 98.15 ± 0.19% of the beads migrated to region Q3 after incubation with exosomes, indicating the effectiveness of exosome capture. Histograms of FITC signals from individual beads show that the average fluorescence intensity increased from 6.97 ± 0.04 to 18.55 ± 0.06, further demonstrating the immunoaffinity of iME beads for exosome separation. Confocal microscopy images show a uniform FITC signal on the surface of the exosome-captured beads (…). Figure 2 The h-plot shows that there was no FITC signal in the control group (data not shown), indicating the uniformity of iME bead capture capability.

[0249] To perform the Aβ42 recognition and enzyme signaling steps, a horseradish peroxidase (HRP)-conjugated capture antibody was prepared to target the second marker site, the Aβ42 protein (… Figure 2 (Figure i). Its substrate, 3,3'-diaminobenzidine tetrahydrochloride (TMB), is used to amplify the optical signal. For ease of in vitro experiments, exosome-amyloid β-binding compounds (Exo-Aβ42) were prepared by self-assembling Aβ42 aggregates onto the membrane of exosomes. Figure 8 (Figure a). The morphology of these complexes was confirmed by transmission electron microscopy and scanning transmission electron microscopy. Figure 8Figures b and c). Notably, both exosome-amyloid β-binding compounds and free amyloid β-aggregates were present in the prepared sample, implying that Exo-Aβ42 represents a subset within the total amyloid β environment. Using the prepared Exo-Aβ42 biomarker, the conditions used for the exosome capture process were examined, including the duration of immunofunctionalization ( ). Figure 2 (Figure j) and concentration ( Figure 2 The k-plot), which is quantified using a commercially available exosome quantitative kit ( Figure 9 Simultaneously, the enzyme signal generation process was analyzed to maximize the signal-to-noise ratio by varying the blocking buffer (PBS, PBST, and Block ACE buffer), washing buffer (PBS, PBST, and Block ACE buffer), enzyme concentration (500x, 1000x, and 2000x), the addition of immunoaffinity enhancers (with and without), and other variables. Figure 10 (Figures a through e).

[0250] Following the successful development of the iME bead-based Exo-Aβ42 identification assay, the specificity and sensitivity of the entire assay were tested on-chip. Figure 1 (Figure d). Compared with the three non-target proteins (Aβ42, CD63, and BSA), a significant signal was observed upon introduction of the target molecule Exo-Aβ42. Figure 2 (See Figure i). Notably, neither amyloid-β protein Aβ42 nor the exosome marker protein CD63 produced any background noise, indicating that free Aβ42 protein and exosomes do not affect the detection specificity of our assay. These results further confirm the two-site targeting mechanism of the Exo-Aβ42 identification assay based on iME beads. After establishing specificity, the sensing behavior of the assay was investigated by incorporating the prepared exosome-amyloid-β conjugate into PBS and measuring the results on the chip. Figure 11 (Figures a through d). Based on the quantitative results, the regression parameter Ra was used. 2 = 0.987 to obtain the platform's sensitivity ( Figure 2 The m-plot includes the reported concentration range of human plasma amyloid β42 protein. 51-53 .

[0251] (2) MEMOS integration and automation

[0252] To achieve fully automated immunosensing without user intervention, a MEMOS based on vertically stacked components is designed. These components include: 1) a μ-ExoBiochip embedded for identifying and determining the target molecule Exo-Aβ42; 2) a μ-Bead EMchip equipped with a programmable and addressable motor; and 3) an optical signal sensing model for signal readout. Figure 1 (Figure b).

[0253] The μ-Exo Biochip consists of a reagent reservoir for encapsulating various assay reagents, an oil zone for reagent separation and bead / droplet transport, and a detection chamber for signal readout. Figure 1 (Figure d). The structure of μ-Exo Biochip was laser-etched onto a polytetrafluoroethylene (PTFE) substrate due to its excellent resistance to molecular adsorption. 54 Its non-wetting properties (data not shown), along with its resistance to molecular adsorption, help prevent reagent residue, and the non-wetting property facilitates manipulation of beads and droplets. A transparent polyethylene terephthalate (PET) sheet is used to create a sealed environment for the reagents and forms a transparent detection area for signal readout. Different layers are assembled in a convenient and cost-effective manner using patterned double-sided tape. Figure 3A (Figure a).

[0254] To implement the developed iME bead-based Exo-Aβ42 identification assay on the μ-Exo Biochip in a fully automated manner, it is important to thoroughly investigate the key driving conditions controlling the operational behavior and movement dynamics of the beads and droplets. 44,55,56 The system utilizes magnetic force from a magnetic motor as the external driving force, while friction generated by sliding contact surfaces serves as the relative external resistance. The strength of the magnetic force is proportional to the mass of the loaded beads, as the magnetic field generated by the motor directly influences the bead cluster. Friction is proportional to the droplet transport velocity, which, in the current system, can be adjusted by the motor's movement speed.

[0255] To differentiate between different motion conditions, the internal forces between the beads and the droplets were considered. The internal forces generated by the deformation of the droplet surface are related to the curvature length of the deformed droplet. The maximum internal force can be estimated when the entire circumference of the bead cluster contributes to surface deformation, such that it is proportional to the cube root of the bead-loaded mass. In bead manipulation, magnetic force acts as the driving force, while internal forces act as the drag. In droplet manipulation, the interaction force of the internal forces (which has the same magnitude but opposite direction to the internal forces) acts as the driving force, while friction acts as the drag. Therefore, to facilitate bead manipulation, increasing the bead-loaded mass improves the magnetic force. Alternatively, reducing the driving speed can reduce friction, thus facilitating more efficient droplet manipulation.

[0256] By systematically balancing the three forces present in the current system, a model was simulated to explore the potential mechanisms of manipulation and interaction between bead and droplet operations. Figure 3A(Figure b, line). Simulation results show three distinct zones based on different programmed motor speeds and bead loading masses. In the no-connection zone (yellow), the magnetic force is insufficient to overcome the internal resistance of the bead or droplet friction, rendering motor movement ineffective for driving the bead or droplet motion. In the droplet-operated zone (pink), for the bead, the magnetic force equals the internal force, but the internal force has not yet reached its maximum value, rendering it ineffective for extracting the bead from the droplet. For the droplet, the internal force equals the frictional force, resulting in stable droplet motion. In the bead-operated zone (lake blue), for the bead, the magnetic force exceeds the maximum value of the internal force, causing the bead to be extracted from the droplet. Simultaneously, for the droplet, the internal force does not exceed the frictional force, preventing the motor from operating the droplet. Experiments were also conducted to confirm the conclusions drawn from the simulation. The results show that the simulation operation diagram (Figure b) Figure 3A The three different operating conditions are consistent with the points in Figure b. Figure 3A Figure c shows a series of optical images corresponding to two typical motion regions. Under droplet operation conditions ( Figure 3A (Figure c, bottom) Both the bead and the droplet stably follow the movement of the motor. Under bead operating conditions ( Figure 3A (Figure c, top) The bead clusters are extracted and always follow the motor drive, while the droplets are left behind. Based on simulation and experimental studies, it was found that adjusting the bead loading mass or adjusting the motor speed can effectively switch between bead operation and droplet operation modes, providing flexibility in terms of automated drive functions and biochemical reactions within the system.

[0257] The μ-Bead EMchip includes a programmable navigation matrix comprising electromagnetic coil elements and a magnetic motor having a size comparable to that of the coil unit. 57 ( Figure 1 (See diagram f). The coil elements are programmed and driven sequentially to generate temporary magnetic fields when current is applied to adjacent coil elements. Following the guidance of successive activated magnetic fields, the motor serves as the driving force for a series of continuous functions ( Figure 1 (g-image). To integrate the μ-Bead EMchip with the μ-ExoBiochip, the coil element pattern was designed to correspond to the structure in the microfluidic layer. To achieve full automation of bioassays, the motors were programmed to follow predetermined movement commands, facilitating non-contact manipulation of the beads and droplets for the assay procedure. Figure 12 ).

[0258] With the help of the integration of μ-Exo Biochip and μ-Bead EMchip, Figure 3AFigure d illustrates the automated bioassay on the chip. In the automated testing workflow, bead and droplet manipulations are performed to complete the on-chip iME bead-based Exo-Aβ42 identification assay. Specifically, after sample loading, the bead clusters are first magnetically rotated and mixed within introduced sample droplets to capture exosomes carrying CD63 (…). Figure 3A (Figure d, step 1) provides a pathway for programmable control and interaction with subsequent reagents of the biomarker. Then, the bead extract is passed through the water-oil interface and transferred to a washing droplet to remove excess material (…). Figure 3A (See Figure d, step 2). Subsequently, the enzyme-specifically labeled target molecule Exo-Aβ42 (conjugated with anti-Aβ42 antibody) was used. Figure 3A (See d-plot, step 3). The next step in the sequence involves three washes to minimize background interference, followed by delivery of the labeled molecule to the detection chamber, which contains the substrate reagent to initiate the reaction (…). Figure 3A (See d-plot, step 4). After the redox reaction, the termination buffer droplet is transferred to the detection chamber, where it is combined and mixed with the reactive droplet to terminate the reaction and amplify the color development, thereby enhancing the signal. Figure 3A (See d-graph, step 5). To prevent the light from the scattering magnetic beads from interfering with the sensing accuracy... 44 Extract beads from the testing chamber. As demonstrated by the assay and in Figure 3A As illustrated in Figures b and 3C, magnetic beads can be used to extract from or transport droplets from a single droplet, depending on the loading mass of the beads and the speed of the magnetic motor. By carefully controlling these parameters, the system facilitates flexible droplet manipulation, enabling either bead-only extraction or bead-assisted droplet transport. In the context of the assay, magnetic beads are first extracted from the sample loading chamber, where the target molecules have been captured, facilitating the separation of the analyte from the sample droplet. The beads are then sequentially extracted through various processing chambers, including washing chamber 1, antibody labeling chamber, washing chambers 2, 3, and 4, and finally enter the detection chamber. A quantitative colorimetric reading is then taken and displayed in a sample-result manner. Figure 3A (See d-graph, step 6).

[0259] An optical sensor model was integrated directly beneath the detection chamber to measure the light absorption of the droplet readout via a signal. The detection accuracy of the optical sensor was adjusted to achieve R... 2 Linear regression with a value of 0.995 Figure 13 To verify the on-chip assay and automation performance of real samples, blood samples were collected and on-chip tests were performed using C57BL / 6J and 5×FAD transgenic mice. Figure 3B(Figure e). The 5xFAD transgenic mouse model exhibits early onset and age-dependent progression of aggressive Aβ accumulation in the brain, and is one of the most widely used mouse models in Alzheimer's disease research. 58 Immunohistochemical staining of Aβ42 aggregates in the hippocampus of 5xFAD mice was performed to confirm the viability and reliability of the transgenic model. Figure 3B (f-plots and data not shown). Immunoreactivity results from the brains of 5xFAD mice tested in all groups showed robust Aβ42 deposition compared to age-matched C57BL / 6J controls. Plasma samples (n = 20) were collected and tested on a chip, showing a significant difference in Exo-Aβ42 levels between the 5xFAD group and the C57BL / 6J controls (P = 0.0014, Mann-Whitney test). Figure 3B (g-plot and h-plot). Additionally, the total Aβ42 protein level in plasma samples (n = 20) was measured using ELISA. Figure 14 Figures a through c). Results showed that the mean total Aβ42 protein level was higher in 5xFAD mice compared to the control. However, this difference was not statistically significant. Receiver operating characteristic (ROC) curve analysis of plasma Exo-Aβ42 levels measured by MEMOS and plasma Aβ42 levels detected by ELISA (… Figure 15 This demonstrates the diagnostic potential of MEMOS-targeted plasma Exo-Aβ42 biomarkers for AD detection and further proves the feasibility of automating MEMOS detection.

[0260] (3) MEMOS clinical sample testing and verification

[0261] To improve the accessibility of MEMOS in various environments, including hospitals, clinics, and even homes, a user-friendly workflow has been adopted, consisting of the following three simple steps ( Figure 4A Figure a): (i) Load the collected microliter volume sample into the μ-ExoBiochip; (ii) Insert the μ-Exo Biochip to initiate the fully automated sample handling and detection process; (iii) View the reading signal and interpret the results on the screen or smart device. Compared with conventional laboratory-based methods ( Figure 4A(See Figure b) MEMOS provides a user-friendly workflow that requires no specialized training in sample handling. Furthermore, MEMOS reduces manual operation time from a typical 60 minutes to zero minutes due to its fully automated sample handling capabilities, eliminating the need for manual pipetting during washing, labeling, reaction, and stop steps. In addition, MEMOS provides on-chip readings and signal transmission, overcoming the limitations associated with bulky laboratory signal readers. Therefore, the integration of the μ-Exo Biochip and μ-Bead EMchip enables MEMOS to significantly reduce manual intervention, eliminate complex manual procedures, and remain feasible under normal conditions, thereby improving diagnostic accessibility.

[0262] The performance of MEMOS for clinical testing feasibility was investigated using a total of 60 clinical samples, including 20 Alzheimer's disease samples, 10 mild cognitive impairment samples, and 30 healthy control samples. Optical readings of the Exo-Aβ42 signal detected by MEMOS were used to differentiate between Alzheimer's disease (AD), mild cognitive impairment (MCI), and healthy controls (CON) (data not shown). CD63 levels in the clinical samples were analyzed in each group (n = 30), and no significant differences were found among the AD, MCI, and CON samples. Figure 16 Figures a and b show that the Exo-Aβ42 biomarker was evaluated under the assumption that exosomal CD63 levels were unchanged in clinical samples. To evaluate the clinical performance of the MEMOS, each plasma sample was loaded into the MEMOS for Exo-Aβ42 detection, while total plasma Aβ42 was measured on microplates using a commercially available ELISA kit for comparison. Figure 4A Figures c and e show the sensing signals of Exo-Aβ42 protein detected on the chip for 60 samples, while the results of total plasma Aβ42 detection are shown in... Figure 4A d-graph and Figure 17 In Figure a, in the MEMOS Exo-Aβ42 group, AD samples showed significantly lower reading signals compared to the healthy control group (P < 0.0001, Mann-Whitney test), and the difference between MCI and CON was also significant (P < 0.0027, Mann-Whitney test). Figure 4B (See f-plot). However, no significant differences were found in total plasma Aβ levels among the three groups (see f-plot). Figure 17 (Figures b and c). These results indicate a gradually increasing trend in plasma Exo-Aβ42 levels throughout the AD, MCI, and CON stages of Alzheimer's disease, suggesting the potential of plasma Exo-Aβ42 as a predictor of different stages of Alzheimer's disease progression.

[0263] ROC analysis was also performed to evaluate the current platform's diagnostic potential for AD and to determine the cutoff values ​​for the Exo-Aβ42 protein biomarker in the AD, MCI, and CON groups. Figure 4B (See graph g). For the comparison between the AD and CON groups, the area under the curve (AUC) was found to be 0.94, with a p-value less than 0.0001, based on established AUC interpretation guidelines. 59 This demonstrates excellent diagnostic accuracy for AD (AUC ≥ 0.9). Furthermore, for the MCI group compared to the CON group, the AUC value was 0.82 (P < 0.01), which was interpreted as demonstrating good and considerable test performance, further indicating the platform's ability to monitor AD progression. ROC curve analysis showed that the current platform can diagnose AD patients with 95% sensitivity and 90% specificity (cutoff value ≤ 1104), and can distinguish between MCI patients and healthy CON patients with 80% sensitivity and 80% specificity (cutoff value ≤ 1115). These results demonstrate the potential of MEMOS for automated blood testing of Alzheimer's disease and show that it provides an accessible and convenient approach for the diagnosis and monitoring of disease progression.

[0264] 3. Discussion

[0265] In this study, a fully integrated, automated system, MEMOS, was introduced for detecting the Alzheimer's disease (AD) blood biomarker, Exo-Aβ42 protein. MEMOS automates biomarker processing, analysis, and data transmission via a programmable drive system, thus eliminating the need for manual intervention. Unlike previous technologies for monitoring AD biomarkers, which typically operate in routine laboratory settings, MEMOS offers a more accessible solution for a wider range of environments. This allows non-professionals to perform testing at home while maintaining high sensitivity and specificity for diagnosing Alzheimer's disease. The advantages of MEMOS stem from the integrated combination of the following components: 1) a μ-Exo Biochip, encapsulated within a magnetic field-responsive assay, specifically designed to target dual biomarker sites of the Exo-Aβ42 protein, achieving detection limits as low as a few picograms per milliliter; 2) a μ-Bead EMchip characterized by a programmable navigation matrix designed for robust addressable magnetic motor actuation according to predetermined commands; and 3) an integrated system that facilitates non-contact control of both the bead and droplet movement, enabling fully automated operation of various functions and immunosensing procedures.

[0266] Using MEMOS, we assessed blood Exo-Aβ42 levels in both animal models (including healthy mice and AD transgenic mice) and clinical samples (plasma from healthy individuals, patients with mild cognitive impairment, and Alzheimer's disease patients). These findings demonstrated a significant correlation between blood Exo-Aβ42 levels and the progression stage of AD, highlighting the potential of MEMOS as an accessible point-of-care testing device for monitoring AD development. Currently, this system provides a convenient method for assessing blood Exo-Aβ42 levels. Despite the urgent need for blood testing for AD, such assessments are not widely implemented in routine clinical practice, primarily due to a lack of established protocols rather than a lack of demand. 23,25,26,28,60 Therefore, MEMOS can serve as a practical tool for providing clinical insights during early medical evaluation, reducing the burden on healthcare systems and facilitating the rational allocation of medical resources. Furthermore, results from MEMOS can be interpreted in conjunction with other diagnostic modalities, such as cerebrospinal fluid analysis and positron emission tomography (PET), to improve clinical decision-making.

[0267] At a broader level, the versatility of MEMOS can be enhanced to accommodate large-scale, high-throughput, and multiplex biomarker detection. From a system perspective, the current configuration is designed to perform iME bead-based assays for Exo-Aβ42 identification. The pattern arrangement in the μ-Exo Biochip and the navigation matrix in the μ-Bead EMchip can be easily modified to accommodate different assay protocols. Additionally, the integrated readout module currently relies on a colorimetric sensor. Future improvements could include the automation and integration of other optical and electrochemical signal readout models to meet diverse signal generation requirements. Based on configurable assay-driven modes and signal acquisition methods, MEMOS can theoretically support multiple parallel detection channels, enabling large-scale, high-throughput screening. From an application perspective, MEMOS can be customized for on-demand analysis of other disease-related exosomes and / or protein biomarkers. Furthermore, leveraging the technological capabilities for large-scale and high-throughput driving, MEMOS has the potential for simultaneous analysis of multiple targets, thereby improving healthcare efficiency. The anticipated integration of artificial intelligence algorithms could further simplify the operational steps required for multiplex assays, facilitating more complex bioanalytical procedures.

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[0329] It should be understood that the disclosed methods and compositions are not limited to the specific methods, schemes, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0330] Those skilled in the art will recognize or be able to determine numerous equivalent embodiments of the methods and compositions described herein using no more than conventional experiments. Such equivalent embodiments are intended to be included in the appended claims.

Claims

1. A microfluidic device for automated analyte detection, characterized in that, The microfluidic device includes: (a) Biochip; (b) An electromagnetic chip including a programmable navigation matrix; The biochip and the electromagnetic chip are oriented parallel to each other, with the electromagnetic chip directly below the biochip.

2. The microfluidic device according to claim 1, wherein, The biochip includes: (i) Sample loading chamber; (ii) One or more washing rooms; (iii) Combination chamber; and (iv) Testing room; The sample loading chamber, one or more washing chambers, binding chamber, and detection chamber are in fluid communication with each other.

3. The microfluidic device according to claim 2, wherein, The microfluidic device also includes multiple immunomagnetic exosome beads; Optionally, the plurality of immunomagnetic exosome beads are provided as separate components or pre-loaded in the sample loading chamber.

4. The microfluidic device according to claim 1, wherein, The biochip includes a first washing chamber and a second washing chamber. The first washing chamber is directly fluidly connected to both the sample loading chamber and the binding chamber, and The second washing chamber is in direct fluid communication with both the detection chamber and the bonding chamber.

5. The microfluidic device according to claim 2, wherein, The biochip also includes a termination buffer chamber, and the detection chamber is in direct fluid communication with the termination buffer chamber.

6. The microfluidic device according to claim 1, wherein, The electromagnetic chip includes a programmable navigation matrix, which includes: (1) Multiple electromagnetic coils, (2) One or more switching integrated circuits electrically connected to the plurality of electromagnetic coils, (3) One or more magnetic motors, and (4) A microcontroller configured to control the activation of the plurality of electromagnetic coils to electromagnetically drive the magnetic motor.

7. The microfluidic device according to claim 6, wherein, The plurality of electromagnetic coils are arranged to align with the sample loading chamber, one or more washing chambers, bonding chambers, and detection chamber.

8. The microfluidic device according to claim 6, wherein, The plurality of electromagnetic coils are arranged to align with the termination buffer chamber.

9. The microfluidic device according to claim 1, wherein, The microfluidic device also includes a material exchange region; The material exchange area is in fluid communication with the sample loading chamber, one or more washing chambers, binding chamber, and detection chamber; and Optionally, the material exchange region is centrally located on the biochip.

10. The microfluidic device according to claim 2, wherein, Each of the sample loading chamber, one or more washing chambers, binding chamber, and detection chamber includes a mixing area and a flow area.

11. The microfluidic device according to claim 1, wherein, The microfluidic device also includes: (c) Optical sensing module; and (d) Optional operation modules.

12. The microfluidic device according to claim 11, wherein, The optical sensing module includes: (i) A light source, which is aligned with the detection chamber and is used to emit light that passes through the signal readout droplet; (ii) a photodetector configured to receive transmitted or absorbed light from a droplet in a detection chamber and to generate an output signal corresponding to the level of said transmitted or absorbed light; and (iii) An optional support structure for securing the optical sensing module to alignment with the detection chamber.

13. The microfluidic device according to claim 11, wherein, The light source of the optical sensing module is aligned below the detection chamber.

14. The microfluidic device according to claim 1, wherein the biochip comprises: (a) A barrier layer, the barrier layer comprising a top layer and a bottom layer; (b) One or more chamber floors; as well as (c) Optional one or more adhesive layers, When the biochip is assembled, the one or more chamber layers form one or more chambers and one or more flow paths. Furthermore, the one or more chamber layers and the one or more adhesive layers are located between the top layer and the bottom layer.

15. The microfluidic device according to claim 14, wherein, The barrier layer is formed of one or more polymers selected from the following: polyethylene terephthalate, polycarbonate, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polyethylene naphthalate, transparent polypropylene, fluoropolymers, cellulose acetate films, and thermoplastic polyurethane.

16. The microfluidic device according to claim 14, wherein, The one or more chamber layers are formed from one or more polymers selected from the following: polytetrafluoroethylene, DMS, cyclic olefin copolymers, cyclic olefin polymers, poly(methyl methacrylate), polycarbonate, transparent polypropylene, fluorinated ethylene propylene, polyethylene naphthalate, and TPU.

17. A microfluidic system for automated analyte detection, characterized in that, The microfluidic system includes the microfluidic device according to any one of claims 1 to 16.

18. The microfluidic system according to claim 17, wherein, The microfluidic system also includes: (1) A housing for enclosing internal components; and / or (2) A user display, wherein the user display is used to display real-time system status, measurement progress, and user prompts; and / or (3) One or more reagents used to detect analytes in biological samples.

19. A method for detecting an analyte in a biological sample, characterized in that, The method uses the microfluidic device of any one of claims 1 to 16 or the microfluidic system of claims 17 or 18 to detect analytes in biological samples.

20. The method according to claim 19, wherein, The method includes: (a) Loading the biological sample into the sample loading chamber of the microfluidic device of any one of claims 1 to 16 or the microfluidic system of claim 17 or 18; and (b) Activate the electromagnetic chip to generate an electromagnetic field.

21. The method according to claim 20, wherein, The biochip's sample loading chamber includes multiple immunomagnetic exosome beads pre-loaded therein; or multiple immunomagnetic exosome beads are provided by a separate component, and The method further includes loading the plurality of immunomagnetic exosome beads into the biochip before or during step (a).

22. The method according to claim 21, wherein, The method includes: functionalizing the plurality of immunomagnetic exosome beads with a binding agent, wherein the binding agent specifically binds to one or more exosome markers.

23. The method according to claim 22, wherein, The exosome markers are selected from CD63, CD9, CD81, CD82, CD151, anti-HSP70, anti-HSP90, or annexin V and Tim4 proteins or combinations thereof.

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