Luminescence cascade sensor for point-of-care pathogen detection

Abstract

An ultrasensitive and long-lasting luminescence cascade can be used for point-of-care detection. A reaction chamber can receive and incubate a volume of a sample and a volume of magnetic tag solution before the combination is washed. Then, a working solution including d-luciferin-6-O-β-d-galactopyranoside (LUGAL), firefly luciferase (Fluc), and a luminescence buffer comprising ATP can be added to the reaction chamber. When the reaction chamber also includes tagged target components including beta galactosidase (GAL) then the GAL supplies luciferin intermediates that catalyze the cleavage of a protecting group from the LUGAL, generating luciferin, and the Fluc catalyzes the oxidation of D-luciferin in a presence of the ATP and O2, yielding oxyluciferin and luminescence. Repeated catalytic cycles increase a measurable signal of the luminescence. An optical sensor can capture an image of the luminescence and the presence of tagged target components can be determined based on an amount of luminescence in the image.

Description

BWH2024-099NONPROVISIONAL APPLICATIONLUMINESCENCE CASCADE SENSOR FOR POINT-OF-CARE PATHOGEN DETECTIONGOVERNMENT SUPPORT

[0001] This invention was made with government support under R01 EB033866, R01 Al 138800, R33A1140489, and R61 Al 140489 awarded by the National Institutes of Health. The government has certain rights in the invention.Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 718,709, filed November 10, 2024, entitled “ULTRASENSITIVE AND LONG- LASTING LUMINESCENCE CASCADE SENSOR FOR POINT OF CARE VIRAL PATHOGEN DETECTION”. The entirety of this provisional application is hereby incorporated by reference for all purposes.Technical Field

[0003] This disclosure relates generally to point-of-care sensors, and more specifically to systems and methods that can employ an ultrasensitive and long- lasting luminescence cascade sensor for point-of-care detection of pathogens.Background

[0004] Point-of-care sensors can be used at a patient’s location to perform rapid diagnostics by detecting the presence of one or more pathogens (e.g., viruses, bacteria, etc.) within a biological sample. Luminescence, more specifically bioluminescence, can be used in image based point-of-care sensors to detect the presence of one or more pathogens due to a high signal-to-noise ratio and absence of incident radiation characteristic of luminescence. Imaging the luminescence can generate minimal phototoxicity and low background noise without a need for external excitation light or undue handling, surpassing the drawbacks of fluorescence and colorimetric techniques. However, imaging luminescence still suffers from a number of challenges including rapid signal decay and reduced enzyme activity when enzymes are linked to targeting molecules. These challenges limit the use and reliability of imaging luminescence by point-of-care sensors.BWH2024-099Summary

[0005] Described herein are systems and methods that can employ an ultrasensitive and long-lasting luminescence cascade sensor for point-of-care detection of pathogens. The luminescence cascade sensor utilizes an enzyme cascade with ultrahigh sensitivity and prolonged luminescence that can be used to detect pathogens (e.g., viruses, bacteria, etc.) in a sample reliably and accurately. The systems and methods can provide a decentralized, rapid, sensitive, specific, and cost-effective sensor that is a viable diagnostic tool for low-resource environments.

[0006] In an aspect, the present disclosure can include a system that can be used to provide point-of-care detection of pathogens. The system includes an assay appliance for detecting the presence of target components in a sample. The assay appliance can include a housing configured to block external light from entering an interior of the assay appliance, a magnet configured to provide a magnetic force; an optical sensor configured to capture an image of a result of an assay, and a processor configured to run the assay and determine if the sample comprises the target components. The system can also include an assay chip configured to be removably placed within the housing of the assay appliance. The assay chip can include a washing solution storage chamber configured to hold a volume of a washing solution, a working solution storage chamber configured to hold a volume of a working solution, and a reaction chamber configured to receive a volume of a sample and a volume of magnetic tag solution, comprising a volume of magnetic molecules and a volume of reactant tags, and to hold contents of the assay during the steps of the assay that add and remove the washing solution and the working solution based on instructions from the processor, wherein at least a portion of the reaction chamber is covered by the magnet.

[0007] In another aspect, the present disclosure can include a method for point- of-care detection of pathogens. The method includes mixing a working solution comprising a d-luciferin-6-O- -d-galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer comprising ATP with contents of a reaction chamber for a time period to create mixed contents. When the contents of the reaction chamber comprise a sample including tagged target components that include at least beta galactosidase (GAL), then: the GAL on the tagged target components supplies a substantial amount of luciferin intermediates that catalyze the cleavage of a protecting group from the LUGAL, thereby generating luciferin, and the FlueBWH2024-099 catalyzes the oxidation of D-luciferin in a presence of the ATP and O2, yielding oxyluciferin and bioluminescence, wherein repeated catalytic cycles increase a measurable signal of the bioluminescence. When the contents of the reaction chamber do not comprise a sample including the tagged target components, then the working solution does not react. The method also includes capturing an image of the mixed contents of the reaction chamber via an optical sensor; and determining if the tagged target components are present in the mixed contents of the reaction chamber based on an amount of bioluminescence detected in the image.

[0008] In a further aspect, the present disclosure can include an assay chip for point-of-care detection of pathogens. The assay includes a washing solution storage chamber configured to hold a volume of a washing solution, a working solution storage chamber configured to hold a volume of a working solution; and a reaction chamber configured to receive a volume of a sample and a volume of magnetic tag solution, comprising a volume of magnetic molecules and a volume of reactant tags, and to hold contents of the assay during the steps of the assay adding and removing the washing solution and the working solution based on instructions from the processor, wherein at least a portion of the reaction chamber is covered by the magnet. The assay chip is configured to be placed inside an assay appliance configured to block external light from entering an interior of the assay appliance.Brief Description of the Drawings

[0009] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0010] FIG. 1 shows a block diagram of an example point of care system for determining if tagged target components are in a sample;

[0011] FIG. 2 shows an illustration of an example exterior portion of the system of FIG. 1 ;

[0012] FIG. 3 shows a block diagram of an example assay appliance of FIG. 1 ;

[0013] FIG. 4 shows a block diagram of an example assay chip of FIG. 1 ;

[0014] FIG. 5 shows illustrations of sample incubation on the assay chip of FIG.1 ;

[0015] FIG. 6 shows illustrations of sample washing on the assay chip of FIG. 1 ;BWH2024-099

[0016] FIG. 7 shows illustrations of the enzyme cascade reaction when the working solution is added to a sample with a tagged target component on the assay chip of FIG. 1 ;

[0017] FIG. 8 shows an illustration of image capture and the following determination by the system of FIG. 1 ;

[0018] FIG. 9 shows illustrations of example portions of the assay appliance of FIG. 1

[0019] FIG. 10 is a process flow diagram of a method for determining if a tagged target component is present in a sample via a luminescence assay reaction;

[0020] FIG. 11 is a process flow diagram of a method for preparing a sample for a luminescence assay reaction;

[0021] FIG. 12 is a process flow diagram of a method for diagnosing a patient based on an amount of luminescence in a reacted sample;

[0022] FIG. 13 is a process flow diagram of a method for analyzing an image captured by an optical image sensor;

[0023] FIGS. 14-19 are experimental images and / or graphical representations of experimental results of Experiment 1 ;

[0024] FIGS. 20-24 are experimental images and / or graphical representations of experimental results of Experiment 2; and

[0025] FIGS. 25-28 are experimental images and / or graphical representations of experimental results of Experiment 3.Detailed DescriptionI. Definitions

[0026] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0027] As used herein, the singular forms “a,” “an,” and “the” can also include the plural forms, unless the context clearly indicates otherwise.

[0028] As used herein, the terms “comprises” and / or “comprising,” can specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups.BWH2024-099

[0029] As used herein, the term “and / or” can include any and all combinations of one or more of the associated listed items.

[0030] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0031] It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0032] As used herein, the term “point-of-care” when referring to a sensor can refer to a medical diagnostic device designed to perform tests at or near a patient’s location rather than in a clinical laboratory. Results of the test can be available quickly compared to laboratory testing and without the need to send the sample away for analysis.

[0033] As used herein, the term “cascade reaction”, also referred to herein as a “cascade”, can refer to a chemical process that includes at least two consecutive and spontaneously occurring reactions such that each subsequent reaction occurs only in response to the chemical functionality formed in the previous step.

[0034] As used herein, the term “sensor” can refer to a device that detects or measures a physical property and records, indicates, or otherwise responds to the physical property. An example of a sensor is a luminescence cascade sensor that can measure luminescence (e.g., bioluminescence) formed as the product of a cascade reaction as part of an assay. One example of a sensor is an “optical sensor” that converts light rays into electronic signals to measure a physical quantity of light in a form readable by an instrument such as a computer or other controller. In some instances an optical sensor can be an optical image sensor that can captureBWH2024-099 an image including the light rays Examples of optical sensors include, but are not limited to, complementary metal oxide semiconductor (CMOS) image sensor, a charge coupled device (CCD) sensor, a contact image sensor, a photodiode array, a phototransistor array, or a mobile-grade camera molecule.

[0035] As used herein, the term “assay” can refer to a procedure used to measure the presence, amount, or functional activity of a specific substance in a sample.

[0036] As used herein, the term “luminescence” can refer to a form of spontaneous light emission without heat production by a substance upon energetic excitation. A subtype of luminescence, bioluminescence, can refer to the biochemical emission of light be living organisms during a chemiluminescence reaction.Bioluminescence occurs in multifarious organisms ranging from marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some bioluminescent bacteria, dinoflagellates and terrestrial arthropods such as fireflies.

[0037] As used herein, the term “magnet” can refer to a material or object that can produce a magnetic field, allowing the material to attract or repel other magnetic materials. A magnet can be a permanent magnet that is magnetized to create its own persistent magnetic field (e.g., ferro magnetic elements such as iron, cobalt, their alloys, lodestones, etc.), a temporary magnet that behaves as a magnetic in the presence of another magnetic field, and electromagnets that are created by coiling a wire around a core material to create a solenoid and then passing an electric current through the solenoid.

[0038] As used herein, the term “pathogen” can refer to a biological, physical, or chemical entity capable of causing a disease. Examples of pathogens include, but are not limited to viruses, bacteria, parasites, protozoans, prions, viroids, and fungi. The target component can be a pathogen when used herein.

[0039] As used herein, the term “sample” can refer to at least a portion of tissue, fluid, or other biological material derived from a patient that can be used in an assay to test if the patient has a pathogen. Non-limiting examples of a sample include skin tissue, saliva, blood, urine, stool, muscle tissue, and organ tissue.

[0040] As used herein, the term “patient” can refer to any warm-blooded organism including, but not limited to, a human being, a pig, a rat, a mouse, a dog, a cat, a goat, a sheep, a horse, a monkey, an ape, a rabbit, a cow, etc.BWH2024-099II. Overview

[0041] Point-of-care sensors can be used at a patient’s location to rapidly detect the presence of one or more pathogens (e.g., viruses, bacteria, etc.) within a sample. Luminescence imaging offers a non-invasive, minimally phototoxic, low background, and quantitatively precise method of imaging. Luminescence imaging can be used to measure molecules in complex media with minimal handling and without the need for external excitation of fluorescence or colorimetric techniques, including the gold standard enzyme-linked immunosorbent assay (ELISA). Luminescence is a highly efficient chemical reaction that produces light without significant heat and creates a high signal-to-noise ratio. However, luminescence imaging still suffers from a number of challenges including from rapid signal decay and reduced enzyme activity when bioluminescent molecules are linked to targeting molecules. These challenges limit the use and reliability of luminescence in point-of-care diagnostic applications.

[0042] Accordingly, there is a need for a luminescence based assay with minimal signal decay and / or reduced enzyme activity. More specifically, the assay employed by the systems and methods described herein is a bioluminescence assay. The systems and methods described herein detail a highly sensitive and enduring bioluminescence assay that leverages a novel enzyme cascade reaction to overcome the current challenges. The assay can include p-Galactosidase (GAL) that has the ability to maintain enzymatic activity even after binding to antibodies and the ability to supply a substantial amount of luciferin intermediates when utilized as a trigger in an initial reaction step. The luciferin intermediates can be converted into a bioluminescent signal by an excess of the natural form of firefly luciferase (Flue), amplifying the yield of the final product. This heightened sensitivity is several hundred times greater than that observed in conventional bioluminescence systems (non-LUCAS). Additionally, the stepwise reaction mechanism of LUCAS enables the continuous generation of luciferin intermediates, followed by Flue-mediated oxidation to produce sustained luminescent signals. The luminescent signal generated through the cascade enzyme reaction can be maintained at a high level for at least an hour. A specialized assay appliance and assay chip for point of care use of the assay have also been developed. The systems and methods can detect, for instance, COVID, HIV, Hepatitis B and C, and the like with comparatively small samples sizes and viral loads in a rapid fashion (e.g., under 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or the like).BWH2024-099III. Systems

[0043] FIG. 1 shows a block diagram of a system 10 for determining whether a target component (e.g., pathogen, such as one or more viruses, one or more bacteria, or the like) is present in a sample (e.g., taken from a patent). The system 10 can be a point of care system configured to be used near the patient.Accordingly, the system 10 can be portable and easily used in a clinical setting, an at home setting, a rural setting, a field setting, or the like. The system 10 can include an assay chip 12 and an assay appliance 20 and can also optionally include an external device 30 that can be in communication with the assay appliance 20.

[0044] The assay chip 12 can be shaped and sized to be placed inside the assay appliance 20 for use such that the assay appliance can block external light from entering an interior of the assay appliance. The assay chip 12 can include a reaction chamber 14, a washing solution storage chamber 16, and a working solution storage chamber 18. The washing solution storage chamber 16 can be sized and shaped to hold a volume of a washing solution. In some instances, the washing solution storage chamber 16 can be pre-loaded with the volume of the washing solution. In other instances, the washing solution can be added to the washing solution storage chamber 16 before use (e.g., injected by a syringe or the like). The working solution storage chamber 18 can be sized and shaped to hold a volume of a working solution. In some instances, the working solution storage chamber 18 can be pre-loaded with the volume of the working solution. In other instances, the working solution can be added to the working solution storage chamber 18 before use (e.g., injected by a syringe or the like). The working solution can include at least d-luciferin-6-O-p-d-galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer including at least ATP (B. Buffer). The assay steps can occur in the reaction chamber 14, which can be sized and shaped to hold various volumes of the patient’s sample, a magnetic tag solution, the washing solution, and / or the working solution as contents throughout the steps of the assay (described in more detail below).

[0045] The assay appliance 20 can detect the presence of target components (e.g., pathogens) within the sample in the reaction chamber 14 of the assay chip 12. The assay appliance 20 can include a housing 22 that can block external light from entering an interior of the assay appliance. Inside the housing 22, the assay appliance can also include a magnet 24, an optical sensor 26, and a processor 28BWH2024-099(e.g., a microprocessor) that can be in bidirectional communication with at least the optical sensor. The magnet 24 can be positioned above, near, adjacent to, and / or in contact with at least a portion of a rection chamber 14 of the assay chip 12 when the assay chip is positioned within the assay appliance. The magnet 24 can be stationary and / or can be movable within the assay appliance 20 relative to a position of the assay chip 12. The magnet 24 can provide a magnetic force to the reaction chamber 14 of the assay chip 12 during at least a washing step of the assay. Optionally, the magnet 24 may be part of the assay chip 12 instead of the assay appliance 20. The magnet 24 can be a permanent magnet, a temporary magnet, or an electromagnetic device (connected to a current source). The optical sensor 26 can be positioned (and instructed) to capture an image of a result of an assay in the reaction chamber 14 of the assay chip 12. The optical sensor 26 can be, for example, a complementary metal oxide semiconductor (CMOS) image sensor, a charge coupled device (CCD) sensor, a contact image sensor, a photodiode array, a phototransistor array, or a mobile-grade camera molecule. The processor 28 can run the assay and determine whether or not a given sample comprises the target components.

[0046] Optionally, the processor 28 can be in communication (e.g., wired and / or wireless) with an external device 30 that can include a controller 30 (including a non- transitory memory and / or a processor, not shown) and a display / interface 34 (e.g., visual, audible, haptic, etc.). The external device 30 can be, for example, a smartphone, a tablet, a computer, or the like. The external device 30 may communicate further instructions to the processor 28 and / or can receive data from the processor 28 (e.g., images, alerts that determinations or diagnoses have been made, determinations of target component presence, diagnoses, or the like). Alternatively and / or additionally the assay appliance 20 can include a display and user interface (as shown in FIG. 2).

[0047] FIG. 2 shows an illustration of an example external portion of the system 10 (e.g., the assay chip 12 is not shown because it is positioned within the assay appliance 20). The assay appliance 20 can include a removable lid 40 that can be totally removable and / or attached by a hinge or the like. The removable lid 40 can be used to position the assay chip 12 inside the assay appliance 20 and remove the assay chip after testing. The removeable lid 40 and the housing 22 can block light from entering an interior of the assay appliance. It should be understood that theBWH2024-099 removable lid 40 can be any one or more sides of the assay appliance 20 (illustrated on top for ease of illustration and explanation but can also be any side or portion of a side of the housing 22). The assay appliance 20 can include a power source 44 that can be external and / or internal. The power source 44 can be a battery (rechargeable and / or replaceable) and / or can be a plug (as shown) for connected to a hardline power supply. The assay appliance 20 can include a display / user interface 42 that can communicate between a user and processor 28 (e.g., intake user instructions (e.g., start, etc.) and output images, determinations, and / or diagnosis). The assay appliance 20 can, as discussed with respect to FIG. 1 , be in communication with an external device 30 that can include a controller 32 and / or a display user interface 34.

[0048] FIG. 3 shows a block diagram of an example assay appliance 20, which can hold an assay chip 12 to test for a target component in a sample (e.g., a pathogen). As previously noted, assay appliance 20 can include a housing 22 and a removable lid 40 that can block external light from entering an interior compartment (e.g., interior 46) of the assay appliance. In the interior 46 the assay appliance 20 can include the magnet 24, the optical sensor 26, and a place for the assay chip 12 to be positioned (any holder and / or attachment mechanisms omitted for ease of illustration) relative the magnet and the optical sensor as previously described. The interior 46 can also include a motor 48 connected to a rotating valve 50. The rotating valve 50 can be connected to at least syringe(s) 52 and magnet 24 and / or can be connectable to the assay chip as described with respect to FIG. 4. The syringe(s) 52 can be controlled by the processor (via motor 48 and rotating valve 50) to actuate injection of washing solution to the reaction chamber of the assay chip and to then actuate injection of the working solution to the reaction chamber. The motor 48 can be a servo motor and can provide rotational and / or longitudinal force to the various movable parts of the assay appliance 20 (e.g., rotating force to rotating valve 50, rotating and / or longitudinal force to the syringe(s) 52 or the magnet 24, or the like) that interact with the assay chip.

[0049] The processor 28 can run the assay and determine whether or not a given sample comprises the target components. The processor 28 can be a part of a controller 54 with a non-transitory memory (e.g., memory 56). The processor 28 and the memory 56 can be embodied together as a microcontroller and / or can be housed separately within a controller 54. The memory 56 can store instructions for implementing the assay and the processor 28 can retrieve the instructions andBWH2024-099 execute the instructions to implement the assay (described in detail below with respect to FIGS. 5-8). In brief the instructions can include: incubate a volume of the sample combined with a volume of magnetic tag solution in the reaction chamber for an incubation time period, apply and / or maintain a magnetic field to the reaction chamber to magnetically retain tagged target components within the reaction chamber during washing; wash the contents of the reaction chamber after the incubation with the volume of the washing solution; inject the volume of the working solution into the reaction chamber during a reaction step of the assay; capture an image of the contents of the reaction chamber after the reaction step; and determine whether or not the target components were present in the sample based on the captured image.

[0050] FIG. 4 shows a block diagram of an example assay chip 12 that can be placed within the system 10. The assay chip 12 can be shaped and sized to be placed inside an assay appliance (e.g., assay appliance 20) for use such that the assay appliance can block external light from entering an interior of the assay appliance. The assay chip 12 can include a reaction chamber 14, a washing solution storage chamber 16, and a working solution storage chamber 18. The washing solution storage chamber 16 can be sized and shaped to hold a volume of a washing solution 66. In some instances, the washing solution storage chamber 16 can be pre- loaded with the volume of the washing solution 66. The washing solution 66 can be I xTBST or another washing buffer known to persons of ordinary skill in the art. In other instances, the washing solution 66 can be added to the washing solution storage chamber 16 before use (e.g., injected by a syringe or the like). The working solution storage chamber 18 can be sized and shaped to hold a volume of a working solution 68. In some instances, the working solution storage chamber 18 can be pre- loaded with the volume of the working solution 68. As an example, the working solution 68 can include at least d-luciferin-6-O-p-d-galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer including at least ATP (B. Buffer). The bioluminescence buffer can include, for example, ATP, BSA, TCEP, EDTA, MgSC>4-7H2O, Coenzyme A, and 1 xTris-HCl. The volume of the working solution 68 can be 100 pL of a gently mixed combination of LUGAL (5.0 pg / pL, 2.5 pL) and Flue (1 .0 pg / pL, 50.0 pL) diluted to 500 pL with the bioluminescence buffer (ATP (1 .0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSO4-7H2O (2.0 mM) and Coenzyme A (8.0 pM) were mixed with 9.8 mL of 1 xTris-HCl). In otherBWH2024-099 instances, the working solution 68 can be added to the working solution storage chamber 18 before use (e.g., injected by a syringe or the like).

[0051] The reaction chamber 14 can be the site of the assay steps and can be sized and shaped to hold various volumes of the sample, the magnetic tag solution, the washing solution 66, and / or the working solution 68 as contents throughout the steps of the assay. At the start of the assay the reaction chamber 14 can receive a volume of the sample combined with a volume of the magnetic tag solution (including a volume of magnetic molecules and a volume of (reactant) tag molecules) (e.g., through an inlet, via a syringe, or the like). The reaction chamber 14 can also include inlet(s) 60 to enable transfer of fluid into and / or out of the reaction chamber. As an example, the inlet(s) 60 can include (1) a first inlet that can enable injection of the volume of the sample combined with the volume of magnetic tag solution into the reaction chamber; (2) a second inlet that can enable injection of at least the portion of the volume of the washing solution 66 into the reaction chamber; and (3) a third inlet that can enable injection of the at least a portion of the volume of the working solution 68 into the reaction chamber.

[0052] Throughout the assay processes, the reaction chamber 14 can have the washing solution 66 and the working solution 68 added and removed based on instructions from the processor (e.g., processor 28). At least a portion of the reaction chamber 14 can be covered by, near, adjacent to, in contact with, or the like, a magnet 24 for at least a period of time (e.g., at least during washing). The magnet 24 can be a part of the assay chip 12 or a part of the assay appliance 20 (discussed in more detail above). As described in more detail above, the magnet 24 can be a permanent magnet, a temporary magnet, or an electromagnet; and can be constantly positioned covering, near, adjacent to, and / or in contact with the reaction chamber. While the magnet may stay in position over the assay chip 12, in some instances the magnet can be movable (e.g., via portions of the assay appliance 20 controlled by the processor 28). The magnet can apply and / or maintain a magnetic force to the reaction chamber 14 during at least a washing step of the assay.

[0053] The assay chip 12 can also include microfluidic channel(s) 58 (shown as dashed lines) and a rotating channel piece (connecting channel(s) 62). The microfluidic channel(s) 58 can include at least a microfluidic channel connected to the washing solution storage chamber 66 and another microfluidic channel connected to the working solution storage chamber 68. The rotating channel pieceBWH2024-099(connecting channel(s) 62) can connect with a rotating valve 50 of the assay appliance 20, which can be controlled by the processor 28 to be rotated into position to connect the microfluidic channel(s) 58 with the appropriate inlet(s) 60 of the reaction chamber to enable the injection first the washing solution 66 then the working solution 68 (e.g., rotated one direction for the washing step and the other direction for the reaction step). The connecting channel 62 can be rotated to connect the microfluidic channel (of microfluidic channel(s) 58) connected to the washing solution storage chamber 16 to the second inlet (of inlet(s) 60) of the reaction chamber 14 and then to connect the other microfluidic channel (of microfluidic channel(s) 58) connected to the working solution storage chamber 18 to the third inlet (of inlet(s) 60) of the reaction chamber.

[0054] In some instances, the assay chip 12 can also include a substrate and / or 3D housing including a cover (e.g., cartridge) that can include and / or at least partially hold all the components described herein and any number of holders and / or attachment mechanisms (not shown) to position the assay chip in the assay appliance 20. The assay chip 20 can be configured to be removably positioned within the assay appliance 20 with at least the reaction chamber 14 in a field of view of an optical sensor (e.g., optical sensor 26) such that after the reaction step of the assay the optical sensor can capture an image of a completed assay reaction, or of unreacted contents of the reaction chamber. It should be noted that the reaction chamber can be at least partially transparent to facilitate the image capture.

[0055] FIG. 5 shows example steps of mixing and incubating the sample with the magnetic tag solution. The sample can be a biological sample taken from a patient suspected of having a pathogen / disease caused by a pathogen. The sample can be, for example, blood, saliva, urine, stool, tissue (e.g., from skin, muscle, organs, or the like), or the like. If the sample is solid, then the solid may be suspended in a fluid to form a solution. The suspected pathogen is the target component (TO). It should be noted that multiple assays for the same or different target components can be performed with at least one of the system 10 (sequentially and / or at least partially concurrently - e.g., if multiple systems are utilized and / or the system is set up for multiplexing).

[0056] As shown in FIG. 5, element A the magnetic tag solution and the sample can be mixed. The magnetic tag solution and the sample can be mixed (e.g., introduced and then shaken, stirred, etc.) outside of the of the system 10 (e.g., in aBWH2024-099 container) and then injected into a reaction chamber. The magnetic tag solution can include magnetic molecules and tag molecules. A magnetic molecule can include a magnetic bead conjugated with a capture antibody for a specific target component (cAb-MgBead). A tag molecule can include the tag for the reaction (beta galactosidase (GAL), discussed in more detail with respect to FIG. 7) conjugated with a detection antibody for the specific target component (dAb-GAL). The sample may or may not contain the target component (TO) and can contain any number of other components (Other). The combined magnetic tag solution and the sample can be incubated in the reaction chamber for an incubation time period (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or the like depending on the specific target component and the detection and capture antibodies).

[0057] FIG. 5, element B shows example illustrations of the incubated contents of the reaction chamber after the end of the incubation time period and before the system proceeds to washing. It should be noted that one of each respective component is shown for ease of illustration and description, but when the components are present then any number one or greater should be understood. The contents shown on the left are an example of when the target component (illustrated as TC) is present in the sample. Accordingly, the incubated contents of the reaction chamber include at least the tagged target component (formed during the incubation time period) and the other components from the sample originally. The tagged target component can include the target component bound with the magnetic molecule (cAb-MgBead) and with the tag molecules (dAb-GAL). While not shown, it should be understood that any target molecules, magnetic molecules, and / or tag molecules that do not bind (e.g., excess of target molecules or excess of magnetic molecules or tag molecules) can remain in their unbound states. The contents shown on the right are an example of when the target component is not present in the sample.Accordingly, the tag molecules, the magnetic molecules, and the other components each remain unbound.

[0058] FIG. 6 shows example steps of implementing washing of the contents of the reaction chamber. FIG. 6, element A shows a magnet positioned near (can be adjacent, touching, near, or the like) the reaction chamber and the contents of the reaction chamber during washing. To start the washing the magnet can apply and maintain a magnetic field to the reaction chamber, such that any magnetized contents can be held in the reaction chamber during the washing and will remain inBWH2024-099 the reaction chamber following the washing. The washing solution (e.g., a volume of the washing solution) (e.g., 1x TBST or another known washing solution) can be added to the reaction chamber contents by connecting the washing solution storage chamber with the reaction chamber via a microfluidic channel and an inlet of the reaction chamber being connected with the connecting channel on the rotating valve (e.g., by the processor as discussed in more detail with respect to FIGS. 3 and 4). The magnet can be positioned near the reaction chamber permanently and / or moved to be near the reaction chamber for the washing portion of the assay (e.g., by the processor as discussed in more detail with respect to FIGS. 3 and 4).

[0059] The washing solution can remove all non-magnetized contents of the reaction chamber (e.g., to a waste receptacle, drain, or the like) when flowed through the reaction chamber. FIG. 6, element B shows an illustration of the contents of the reaction chamber when at least one tagged target component (one shown for ease of illustration only) was present and when no tagged target components were present. When the tagged target components (target components (TC) tagged with the magnetic molecule (cAB-MgBeads) and the tag molecules (dAb-GAL)) are present then the force of the magnetic field can hold them in the reaction chamber through the washing. When no target components were in the sample (see FIG. 5) then following washing the magnetized contents can be only the magnetic molecule (cAB-MgBead).

[0060] FIG. 7 shows illustrations of the enzyme cascade reaction that can occur in the reaction chamber when the target components are present in the reaction chamber following washing. FIG. 7, element A shows an illustration of adding the working solution (a volume of the working solution) to the washed contents of the reaction chamber. The working solution can include at least d-luciferin-6-O-p-d- galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer including at least ATP (B. Buffer). The bioluminescence buffer can include, for example, ATP, BSA, TCEP, EDTA, MgSO4'7H2O, Coenzyme A, and 1 xTris-HCl. The volume of the working solution can be 100 pL of a gently mixed combination of LUGAL (5.0 pg / pL, 2.5 pL) and Flue (1 .0 pg / pL, 50.0 pL) diluted to 500 pL with the bioluminescence buffer (ATP (1 .0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSO4-7H2O (2.0 mM) and Coenzyme A (8.0 pM) were mixed with 9.8 mL of 1 xTris-HCl). The working solution can be added to the reaction chamber by connecting the working solution storage chamber with the reaction chamber via aBWH2024-099 microfluidic channel and an inlet of the reaction chamber being connected with the connecting channel on the rotating valve (e.g., by the processor as discussed in more detail with respect to FIGS. 3 and 4).

[0061] FIG. 7, elements B and C show different pictorial examples of the reaction that occurs when the tagged target components are mixed with the working solution. Only one tagged target component is shown for ease of illustration, but it should be understood that any number of tagged target components can be present. When the tagged target components are not present in the washed contents, then this reaction cannot occur. The tagged target component can include the target component bound to the capture antibody conjugated magnetic molecules (e.g., cAb-MgBead) and the detection antibody conjected tag molecules (e.g., dAb-GAL). The LUGAL in the working solution can react with the GAL to form Luciferin (e.g., D- Luciferen). Specifically, the GAL on the tagged target components can supply a substantial amount of luciferin intermediates that catalyze the cleavage of a protecting group from the LUGAL to form the Luciferin (e.g., D-Luciferen). And the Flue can then react with the Luciferin in the presence of ATP to form oxyluciferin, which spontaneously luminesces (e.g., bioluminesces). Specifically, the Flue can catalyze the oxidation of D-luciferin in the presence of the ATP and O2 (e.g., from at least the B. Buffer), yielding oxyluciferin and bioluminescence. Repeated catalytic cycles increase the measurable signal of the bioluminescence. The reaction is a cascade reaction and can proceed stepwise to generate a sustained bioluminescent signal when the Flue is present in excess in a native and unconjugated form, which can intensify the signal intensity. Without wishing to be bound by theory the intensified bioluminescent signal can make the assay more reliable and accurate than traditional point of care assay methods.

[0062] FIG. 8 shows an example implementation of image capture and determination of a diagnosis (e.g., using as assay chip 12 within an assay appliance 20 having processor 28, it should be noted that the processor discussed herein can be the processor 28 and / or a processor of the external device 30). After the working solution and the contents of the reaction chamber have been mixed and allowed time to react, then the optical sensor can capture an image of the contents of the reaction chamber. The reaction chamber can be at least partially transparent such that the optical sensor can capture an image of the contents of the reaction chamber. The captured image may include luminescence (bioluminescence) or may not. If theBWH2024-099 target components are present as tagged target components, then the enzyme reaction as discussed in FIG. 7 can create luminescence, specifically bioluminescence. If the target components are not present (e.g., the tag molecules could not bind to the target components and were washed out during the washing process), then there may be no luminescence.

[0063] The optical sensor can be in communication (wired and / or wireless) with a processor that can receive the image (and can also instruct the optical sensor to capture the image at the given time). After receiving the image, the processor can determine if the target components are present (which, in some instances, can be above at least a preset limit of detection (LOD)) based on an amount of luminescence being above or below at least one threshold. Optionally, multiple thresholds may be included that may indicated if a disease associated with the target component is active, dormant, strengthening, weakening, or the like.

[0064] The processor can analyze the image to make the determination. First, the processor can isolate a portion of the captured image that includes at least the mixed contents of the reaction chamber from the captured image. Then, the processor can convert the portion of the captured image from RGB (red, green, blue) color space to HSV (hue, saturation, and value) color space to increase sensitivity and apply a binary color mask to the portion of the captured image to increase color sensitivity to the bioluminescence signal. For example, the yellow sensitivity can be increased for the reaction described herein as the bioluminescence signal appears yellow in the captured image. Optionally, the processor can further analyze the image by refining the binary color mask using morphological closing and opening operations to reduce pixel-level noise in the portion of the captured image and then segmenting a region of interest of the portion of the captured image with a value channel into individual pixels. Next, the processor can compute the total brightness of the portion of the captured image. For instance, the total brightness can be computed by summing pixel intensity values of the individual pixels in the region of interest if the optional processing steps were performed.

[0065] The processor can then compare the total brightness of the portion of the captured image (e.g., including or specifically the region of interest) with the threshold(s) for the target components (e.g., the tagged target components) that indicate the LOD. Then, the processor can classify the total brightness of the portion of the captured image as a positive or negative diagnosis for the disease associatedBWH2024-099 with the target components. And finally, the processor can output (by a display or the like not shown in this example) a diagnosis. The diagnosis can be a positive diagnosis when the amount of bioluminescence indicates that the tagged target component is present. The diagnosis can be a negative diagnosis when amount of bioluminescence indicates that the tagged target component is not present. The outputs can be visual (e.g., words, images, numbers, etc. on a screen, a positive and / or negative light turning on), audible (e.g., verbal positive or negative confirmation), and / or haptic (e.g., vibrate to indicate diagnosis was made, specific haptic signals indicated positive or negative, or the like). The diagnosis can also be output to a memory, a central medical record, or the like.

[0066] FIG. 9, element A shows an example of the assay appliance and assay chip and FIG. 9, element B shows an example of the signaling flow through the components of the assay appliance to perform the incubating, washing, working, and image capture steps described herein.

[0067] FIG 9, element A shows an example exterior and a transparent view of an open interior of the assay appliance with an assay chip (also referred to as a disposable microfluidic cartridge and / or microfluidic chip) attached to an interior portion of the assay appliance. To ensure stable and precise bioluminescent signal detection, the assay appliance can form a fully enclosed, light-shielded environment, minimizing external light interference. The exterior of the assay appliance can be configured with a housing and at least a removable and / or openable lid (e.g., on hinges as shown) that can entirely and / or substantially (e.g., 95% or more, 90% or more, 80% or more, 70% or more, 50% or more, or the like) block light when closed. The assay appliance can integrate seamlessly with the microfluidic chip, enabling accurate processing of samples and reagents. The assay appliance can include a compact 3D-printed frame (e.g., housing) (120 x 122 x 163 mm) designed to house the core electronic components, including a Raspberry Pi 4, Arduino Nano, servo motor, battery pack, CMOS image sensor, and an LCD display (85 x 55 x 5 mm), ensuring the device's autonomous functionality and blocking light The appliance can be portable.

[0068] The integrated display, processor, and battery pack of the appliance can allow the system to be operate independently in resource-limited environments, such as rural clinics, disaster relief zones, or low-infrastructure settings, where dependence on external devices for result interpretation may be impractical due toBWH2024-099 connectivity or equipment constraints. The diagnostic assay can be conducted using an assay chip, positioned within the assay appliance. The assay chip can be a disposable microfluidic cartridge fabricated by laser-cutting three layers of 1 .5 mm thick poly(methyl methacrylate) (PMMA) substrates, which can be subsequently bonded using double-sided adhesive (DSA). The assay chip and / or the assay appliance itself can also incorporate a 3D-printed polylactic acid (PLA) chip cover, designed to house a rotation valve, an integrated magnet for the reaction chamber, and syringes for fluid delivery. If part of the assay appliance the chip cover can be removably attached to the assay chip during use and can connect to at least a servomotor attached to the lid of the assay appliance as shown. The chip can include three fluidic inlets for sample input, washing solution input, and working solution input, and the sample, washing solution, and working solution can be directed into the reaction chamber through microfluidic channels as regulated by the processor (e.g., the raspberry pi 4) controlling a connecting channel within the rotation valve, enabling sequential reagent delivery throughout the assay process.

[0069] FIG. 9, element B shows an example of the automated processes of the assay workflow. After, the user collects the sample, mixes it with assay reagents, and introduces the mixture into the assay chamber via pin actuation, then the automated sequence can be initiated by pressing the start button on the integrated display (e.g., 3.5 inch LCD touch display). After an incubation period, the Raspberry Pi can signal the servo motor (via an Arduino nano) to actuate a lever mechanism to introduce the washing solution into the reaction chamber. The buffer can effectively wash away unbound reagents and can then be subsequently drained from the chamber. The servo motor can then rotate in the opposite direction to introduce the working solution to initiate the bioluminescent reaction (if tagged target components are present). Finally, the Raspberry Pi can trigger the CMOS image sensor to capture an image of any emitted bioluminescence. Signal intensity can be analyzed on-device, and the final diagnostic result can be displayed as either positive or negative based on a pre-defined signal threshold.IV. Methods

[0070] Another aspect of the present disclosure can include methods 100, 200, 300, and 400 (FIGS. 10, 11 , 12, and 13) for determining if a target component (e.g., a pathogen, such as one or more viruses, one or more bacteria, etc.) is present in aBWH2024-099 sample taken from a patient (e.g., using a point-of-care diagnostic device). Specifically, method 100 (FIG. 10) relates to determining whether the target component (which has been tagged) is present in the sample via a luminescence assay reaction (e.g., which can be executed within the point-of-care diagnostic device); method 200 (FIG. 11) relates to preparing the sample for the luminescence assay reaction; method 300 (FIG. 12) relates to diagnosing a patient (who provided the sample) based on an amount of luminescence in a reacted sample (e.g., exhibited because of the luminescence assay reaction); and method 400 (FIG. 13) relates to analyzing an image captured by an optical image sensor. The methods 100, 200, 300, and / or 400 can be executed by one or more components of the system 10.

[0071] For purposes of simplicity, the methods are shown and described as being executed serially; however, it is to be understood and appreciated that the present disclosure is not limited by the illustrated order as some steps could occur in different orders and / or concurrently with other steps shown and described herein. Moreover, not all illustrated aspects may be required to implement the methods, nor are the methods necessarily limited to the illustrated aspects. The methodsl OO, 200, 300, and / or 400 can be performed automatically by the system 10 (e.g., by processor 28) and / or manually, employing one or more components of the system.

[0072] FIG. 10 shows a method 100 for a determining whether target components (e.g., pathogens) are present in a patient sample based on a luminescence from an enzyme cascade. The method 100 can be utilized for accurate and reliable point-of-care patient diagnostics. At 102, a working solution can be mixed (e.g., combined) with contents of a reaction chamber (e.g., reaction chamber 14) of an assay chip (e.g., assay chip 12) for a time period to create mixed contents. The mixing can be done manually (e.g., by hand) or via an automated process (e.g., via a processor 28 controlling an assay appliance (e.g., assay appliance 20) to inject working solution from a working solution storage chamber (e.g., working storage chamber 18) into the reaction chamber of the assay chip)

[0073] The mixed contents of the reaction chamber can include the working solution and the contents of the reaction chamber post incubation and washing (described in more detail below). The contents of the reaction chamber can include a remainder of a sample that has been (1 ) incubated with a magnetic tag solution to tag any target components in the sample with a magnetic molecule and a tagBWH2024-099 molecule (e.g., to form tagged target components) and then (2) washed with a washing solution to remove any portions of the incubated contents that are not tagged target components (e.g., the tagged target components include the magnetic molecules or unbound magnetic molecules). During the washing a magnetic field can be applied to hold the magnetic contents inside the reaction chamber. The tag molecules (bound to target components during incubation if target components are present) can include beta galactosidase (GAL) such that the tagged target components can include at least the target component and the GAL. If not bound to the target components with the magnetic molecules, then the tag components are washed out during the washing.

[0074] The working solution can include d-luciferin-6-O- -d-galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer including at least ATP (e.g., including ATP, BSA, TCEP, EDTA, MgSO- HaO, Coenzyme A, and 1 xT ris-HCI). For example, the working solution can be 100 pL of a gently mixed combination of LUGAL (5.0 pg / pL, 2.5 pL) and Flue (1 .0 pg / pL, 50.0 pL) diluted to 500 pL with bioluminescence buffer (ATP (1.0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSCk HaO (2.0 mM) and Coenzyme A (8.0 pM) were mixed with 9.8 mL of 1 xTris-HCI).

[0075] When the contents of the reaction chamber do not include the tagged target components, then the working solution does not react and little or no luminescence can occur. When the contents of the reaction chamber do include the tagged target components (in some instances, an amount above a limit of detection (LCD), predetermined for the given target component), then the GAL on the tagged target components can react with the working solution to create a bioluminescent cascade reaction that can last for an extended time (e.g., at least an hour, at least 30 minutes, at least 20 minutes, or the like). The GAL can supply a substantial amount of luciferin intermediates that can catalyze the cleavage of a protecting group from the LUGAL in the working solution, thereby generating luciferin (e.g., D-luciferin). The Flue in the working solution can catalyze the D-luciferin in a presence of the ATP and oxygen (O2), yielding oxyluciferin and bioluminescence. Repeated catalytic cycles increase a measurable signal of the bioluminescence. The reaction among the GAL, the LUGAL, and the Flue proceeds stepwise to generate a sustained measurable signal of the bioluminescence when the Flue is present in excess (e.g.,BWH2024-099 at least greater than the amount of D-luciferin) in a native, unconjugated form to increase signal intensity.

[0076] At 104, an image of the mixed contents of the reaction chamber can be captured via an optical imaging sensor (e.g., optical sensor 26). The optical imaging sensor can be, for example a complementary metal oxide semiconductor (CMOS) image sensor, a charge coupled device (CCD) sensor, a contact image sensor, a photodiode array, a phototransistor array, a mobile-grade camera molecule, or the like. The image can be captured inside a darkened space (e.g., with minimal or no ambient light) such as an interior of an assay appliance (e.g., assay appliance 20). The optical imaging sensor can be in communication (wired and / or wireless) with a processor (e.g., processor 28) and the processor can receive the captured image. At 106, the processor can determine whether the target components (e.g., the tagged target components) are present in the mixed contents of the reaction chamber based on an amount of luminescence (e.g., bioluminescence) detected in the image. When the target components are present in the mixed contents of the reaction chamber, then the image can include luminescence (e.g., bioluminescence from the enzyme cascade reaction). When the target components are not present, then the image can include no luminescence. In some instances, when the target components are present, but beneath a limit of detection, then the image may include some luminescence that is below an amount to indicate the target component is present / to give a positive diagnosis.

[0077] FIG. 11 shows a method 200 for preparing a sample for a luminescence assay reaction as discussed with regard to FIG. 10 (please note that steps 202 and 204 are shown as dashed because both steps occur prior to incubation). Prior to incubation, at 202, the sample (a volume of the sample, which may or may not include the target component (e.g., pathogen) can be mixed with the magnetic tag solution (a volume of the magnetic tag solution). The magnetic tag solution can include magnetic molecules (e.g., magnetic beads conjugated with a capture antibody for the target component) and tag molecules (e.g., GAL conjugated with a detection antibody for the target component). The sample and the magnetic tag solution can be mixed (e.g., shaken, stirred, or the like) for a time (e.g., a time between 15 seconds and 60 seconds (like 30 seconds), but may be shorter or longer). Then at 204, the combined volume of the sample and the magnetic tag solution can be injected into a reaction chamber (e.g., on the assay chip 12 - whichBWH2024-099 can be inside the assay appliance 20 or external to the assay appliance at the injection time). Alternatively, the sample can be premixed with the magnetic tag solution and / or can be mixed within an assay chip and / or assay appliance.

[0078] At 206, the combination of the sample with the magnetic tag solution can be incubated in the reaction chamber for an incubation time period (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or the like depending on the specific target component and specific detection and capture antibodies). As noted, the magnetic tag solution can include magnetic molecules (e.g., magnetic beads) conjugated with one or more capture antibody for the target component and tag molecules (e.g., detection antibodies for the target component conjugated with the GAL). When the sample includes target components, then the magnetic beads conjugated with a capture antibody for the target component and the detection antibodies for the target component conjugated with the GAL can attach to the target components to form the tagged target components. When no target components are present in the sample, then the cAb-magnetic beads and the dAb-GAL remain unattached in the mixed contents in the reaction chamber.

[0079] At 208, a magnetic field can be applied to the reaction chamber and maintained for another time period (following the incubation time period). The magnetic field can be applied by a magnet (e.g., magnet 24). The magnet may be brought into proximity with the reaction chamber (e.g., by hand or by a motorized component (e.g., lever attached to servo motor)), unshielded (e.g., shield removed by hand or by a motorized component), and / or turned on (e.g., an electromagnet, when an electric current is added). The magnetic field can apply a force on any magnetic portions of the incubated contents of the reaction chamber (e.g., magnetic molecules and / or tagged target components bound to the magnetic molecules (and the tag molecules)). The force can hold the magnetic portions of the incubated contents within the reaction chamber during washing. At 210, the incubated sample in the reaction chamber can be washed with a washing solution (e.g., a volume of 1 x TBST, or other common washing buffer solutions) for at least a portion of the other time period. The washing solution can be added by hand and / or automatically (e.g., via a processor instructing a portion of an assay appliance to affect the washing solution storage chamber of an assay chip). The non-magnetic portions of the incubated contents of the reaction chamber can be washed away (e.g., to a trash receptacle, drain, or the like) by the washing while the magnetic tag molecules andBWH2024-099 any components bound to the magnetic tag molecules can remain in the reaction chamber.

[0080] When the sample includes the target components, then the tagged target components (e.g., target components bound to the magnetic molecules and the tag molecules (GAL)) can be held in the reaction chamber by the magnetic field during the washing and any other portions of the sample can be washed out with the washing solution. When the sample does not include the target components, then the magnetic molecules can be unbound and can be held in the reaction chamber by the magnetic field during the washing and the sample and the unbound tag molecules (GAL) can be washed out of the reaction chamber. Then the working solution can be added to the reaction chamber as described with respect to FIG. 10.

[0081] FIG. 12 shows method 300 for diagnosing a patient based on an amount of luminescence in a reacted sample in a reaction chamber. The method 300 can be performed by at least a processor (e.g., processor 28) and can be stored as instructions in a non-transitory memory (e.g., memory 56). At 302, the capture image can be received from the optical image sensor (e.g., optical sensor 26) that can be in communication (wired and / or wireless) with the processor. While not shown, the processor can instruct the optical image sensor to capture the image at a time after the working solution has been added to the reaction chamber. At 304 an amount of luminescence, specifically bioluminescence, in the captured image can be calculated by the processor (described in further detail with respect to FIG. 13). At 306, the processor can determine whether the amount of the luminescence, specifically bioluminescence, is above or below a threshold set for the given target component (e.g., tagged target component). The threshold can be set based on a predetermined limit of detection (LOD) for the given target component that can show, at 308, a disease is present when the luminescence (e.g., bioluminescence) is above the threshold and not present, at 310, when the luminescence is below the threshold. Optionally, multiple thresholds may be included that may indicate if a disease associated with the target component is active, dormant, strengthening, weakening, or the like. At 312, a diagnosis can be output (e.g., by a display and / or other visual, audible, and / or haptic output device). The diagnosis can be a positive diagnosis when the amount of luminescence indicates that the tagged target component is present. The diagnosis can be a negative diagnosis when amount of luminescence indicates that the tagged target component is not present. The outputs can be visualBWH2024-099(e.g., words, images, numbers, etc. on a screen, a positive and / or negative light turning on), audible (e.g., verbal positive or negative confirmation), and / or haptic (e.g., vibrate to indicate diagnosis was made, specific haptic signals indicated positive or negative, or the like). The diagnosis can also be output to a memory, a central medical record, or the like.

[0082] FIG. 13 shows method 400 for preparing and analyzing an image captured by an optical image sensor as part of the determination and diagnosis methods described above. The method 400 can be performed by at least a processor (e.g., processor 28) and can be stored as instructions in a non-transitory memory (e.g., memory 56). It should be noted that the processor and / or non- transitory memory, in some instances, may be in a device located remote from the assay appliance. At 402, a portion of the captured image that includes at least the mixed contents of the reaction chamber can be isolated from the captured image. At 404, the portion of the captured image can be converted from RGB (red, green, blue) color space to HSV (hue, saturation, and value) color space to increase sensitivity. At 406, a binary color mask can be applied to the portion of the captured image to increase color sensitivity to the bioluminescence signal. For example, the yellow sensitivity can be increased for the reaction described herein as the luminescence and / or bioluminescence signal appears yellow in the captured image.

[0083] Optionally, at 408 the binary color mask can be further refined using morphological closing and opening operations to reduce pixel-level noise in the portion of the captured image. Then, again optionally, at 410, a region of interest of the portion of the captured image can be segmented with a value channel into individual pixels. At 410, the total brightness of the portion of the captured image can be computed. If steps 408 and 410 have been performed, then the total brightness can be computed by summing pixel intensity values of the individual pixels in the region of interest. At 412, the total brightness of the portion of the captured image (e.g., including or specifically the region of interest) can be compared with the threshold(s) for the target components (e.g., the tagged target components). At 414, the total brightness of the portion of the captured image can be classified as a positive or negative diagnosis for the disease associated with the target components and then the diagnosis can be output (per FIG. 12).BWH2024-099V. Experimental

[0084] The following experiments were conducted to test the luminescence cascade-based sensor (LUCAS) assay and associated systems and devices developed for the utilization of the LUCAS assay for point of care diagnostics.

[0085] Experiment 1

[0086] This experiment introduced the luminescence cascade-based sensor(LUCAS) assay, an enzyme cascade system capable of detecting analytes with ultrahigh sensitivity and prolonged bioluminescence (see FIG. 14). Utilizing a sequential enzymatic reaction, the assay achieved a greater than 500-fold increase in bioluminescence signal and maintained an 8-fold improvement in signal persistence compared to conventional bioluminescence assays. Implemented on a portable, fully automated device designed for point-of-care settings, the system facilitated rapid (<23 min) sample-to-answer analysis of viruses without an external power supply. The system’s accuracy surpassed 94% in the qualitative classification of 177 viral-infected patient samples and 130 viral-spiked serum samples, various pathogens including the respiratory virus SARS-CoV-2, and blood-borne pathogens such as HIV, HBV and HCV as clinical models. The decentralized, rapid, sensitive, specific and cost-effective nature of LUCAS positions LUCAS as a viable diagnostic tool for low-resource environments.

[0087] 1.1 Methods

[0088] 1.1.1 Antibody pairs for infectious diseases

[0089] Paired antibodies (40150-D003 for Capture, 40150-D001 for detection) for SARS-CoV / SARS-CoV-2 Spike antibody and SARS-CoV Spike / S1 Protein (40150-V08B1 ), and paired antibodies (40150-D005 for capture, 40150-D004 for detection) for SARS-CoV-2 Spike and recombinant SARS-CoV-2 B.1 .1 .529 (Omicron) S1 +S2 trimer Protein (ECD, His Tag, 40589-V08H26-100) were purchased from Sino Biological. Paired antibodies (11695-MM08T for capture, 11695-MM15 for detection) for HIV p24 were purchased from Sino Biological and recombinant HIV p24 protein (11695-V08E) was purchased from Sigma-Aldrich. Paired antibodies (J343 for capture, J345 for detection) for HBV surface antigen and recombinant HBV surface antigen (LA549) were purchased from EastCoastBio, part of Medix Biochemica. Paired antibodies (MBS569238 for capture, MBS569240 for detection) for HCV core antigen were purchased from MyBioSource and recombinant HCV core antigen (8903) was purchased from ViroStat.BWH2024-099

[0090] 1 .1 .2 Data acquisition by CMOS sensor and quantification

[0091] Images were captured with a Complementary Metal-Oxide- Semiconductor (CMOS) sensor and cropped to 1 .5x1 ,5-inch size. The cropped images were analyzed using Imaged.

[0092] 1 .1 .3 Preparation of the bioluminescence buffer

[0093] Bioluminescence buffer as a working solution was prepared by following a recipe such that, briefly: ATP (1 .0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSO4'7H2O (2.0 mM) and Coenzyme A (8.0 pM) were mixed with 9.8 ml_ of 1 xTris-HCl.

[0094] 1 .1 .4 Preparation of working solution

[0095] D-Luciferin-6-O-beta-D-galactopyranoside (LLIGAL) (5.0 pg / pL, 2.5 pL) and firefly luciferase (Flue) (1 .0 pg / pL, 50.0 pL) were mixed and diluted to 500 pL with bioluminescence buffer. After gentle mixing, 100 pL of working solution were used per test for the bioluminescence assay.

[0096] 1 .1 .5 Comparison of signal persistency between LUCAS and non-LUCAS systems

[0097] For the LUCAS system, 7.5 nM of p-galactosidase (GAL) conjugated with streptavidin (ST-GAL) was incubated with biotin on the surface of a 96-well plate. In the case of the non-LUCAS system, 32.3 nM of biotinylated Flue was incubated with a streptavidin-coated plate. After five washes with 1 xTBST (200 pL) to eliminate unbound enzyme, the working solution for each system was applied to generate bioluminescence. The bioluminescence signal intensity was captured by a CMOS sensor at predetermined timepoints (0, 10, 20, 30, 40, 50, and 60 min) and quantified using Imaged software (n = 5, technical replicates). The signal decay rates of each system was compared by fitting a linear trendline to the graphs and comparing the slope values. The linear trendline were calculated for the LUCAS and non-LUCAS system as y = -0.0546% + 99.721 ( / ?2= 0.957) and y = -0.445% + 97.219 ( / ?2= 0.948), respectively.

[0098] 1 .1 .6 Comparison of enzyme activity of ST-GAL and ST-Fluc after conjugation to dAb-biotin

[0099] ST-GAL (266.7 nM) and Flux conjugated with conjugated with streptavidin (ST-Fluc) (800.1 nM) were mixed with 66.7 nM and 200.1 nM of dAb- biotin, respectively. A volume of 0.28 pL of ST-GAL was taken from the original stock and mixed with working solution (final cone. = 746 pM). 5 pL of ST-Fluc (final cone. =BWH2024-09940 nM) was taken from the original stock and mixed with bioluminescence buffer containing 56.5 pM of luciferin. The bioluminescence signal was observed daily from day 1 to day 4, and the intensity was quantified using Imaged software (n = 4, technical replicates).

[0100] 1 .1 .7 Determination of optimal Flue concentration for the generation of bioluminescence

[0101] To find optimal concentration of Flue for the generation of bioluminescence, 100 pM of ST-GAL and 56.5 pM of LUGAL were mixed with different concentration of Flue (8.0 pM, 6.4 pM, 4.8 pM, 3.2 pM, 1 .6 pM, 0.8 pM, 0.16 pM, 80 nM, and 16 nM) in the working solution. The bioluminescence signal was taken by luminometer (n = 5, technical replicates).

[0102] 1 .1 .8 Determination of optimal LUGAL concentration for the generation of bioluminescence

[0103] To find an optimal concentration of LUGAL for the generation of bioluminescence, 100 pM of ST-GAL and 4.8 pM of Flue were mixed with different concentration of LUGAL (56.5, 113.0, 169.5, 226.0, 282.5, 339.0, 395.5 pM). The bioluminescence signal was taken by luminometer (n = 5, technical replicates).

[0104] 1 .1 .9 Minimum concentration of ST-GAL for the generation of bioluminescence

[0105] To find minimum concentration of ST-GAL for the generation of bioluminescence, 4.8 pM of Flue and 282.5 pM of LUGAL were mixed with different concentration of ST-GAL (1 nM, 100 pM, 10 nM, 1 pM, 100 fM, 10 fM, and 1 fM). The bioluminescence signal was taken by luminometer (n = 5, technical replicates). The minimum concentration of ST-GAL was determined by calculating the mean of the blank plus three times the standard deviation obtained for the blank.

[0106] 1 .1 .10 Minimum concentration of Flue for the generation of bioluminescence

[0107] Various concentration of Flue (186 nM, 138 nM, 103 nM, 76 nM, 57 nM, 42 nM, and 31 nM) were mixed with bioluminescence buffer containing 56.5 pM of luciferin. The bioluminescence signal was taken by luminometer (n = 5, technical replicates). The minimum concentration of Flue was determined by calculating the mean of the blank plus three times the standard deviation obtained for the blank.

[0108] 1.1.11 Preparation of dAb-biotinBWH2024-099

[0109] The dAb was conjugated to biotin using SiteClick™ Antibody Labeling Kit (Thermo Fisher Scientific, S20033) by following the manufacturer’s instructions. The SiteClick™ conjugation workflow involves three key steps: modification of the antibody’s carbohydrate domain, attachment of azide to the antibody, and subsequent conjugation with the sDIBO-modified biotin using copper-free click chemistry. This process covalently links the biotin to the antibody.

[0110] 1.1.12 Investigation of dAb-biotin using TMB-ELISA

[0111] The function of dAb-biotin was confirmed using TMB-ELISA. The surface of 96-well plate was coated with 100 ng of spike protein, p24, HBV surface antigen or HCV core antigen and incubated at room temperature for 1 hour. After washing five times with 200 pL of 1 xTBST, the plate surface was blocked with 200 pL of blocking solution (5% skim milk in 1 xTBST) for 2 hour at room temperature.Following removal of the blocking solution, the wells were incubated with or without dAb-biotin (50 ng, 2.5% BSA in PBS) for 1 hour at room temperature. After five washes, the wells were incubated with streptavidin-HRP (10 ng) or anti-IgG-HRP (10 ng) in 2.5% BSA in PBS for 1 hour at room temperature. Following seven washes, 100 pL of TMB substrate were treated and the color change was monitored for 20 min. The reaction was stopped by adding Stop Solution (0.16 M sulfuric acid) and the absorbance of each well was analyzed at 450 nm wavelengths.

[0112] 1.1.13 Preparation of dAb-GAL

[0113] The dAb-biotin and ST-GAL (Thermo Fisher Scientific, S931 ) were added in 1 :4 ratio (by mass) and diluted using Tris (pH 7.0, 0.05 M) to a final antibody concentration of 0.05 mg / mL. This reaction mixture was incubated at 4°C overnight, with gentle rotation. For future use, dAb-GAL was stored in refrigerator at 4°C. The product was analyzed by western blotting with anti-hlgG-HRP and anti-p- galactosidase-HRP.

[0114] 1.1.14 Investigation of dAb-GAL using TMB-ELISA

[0115] The function of dAb-GAL was confirmed via TMB-ELISA. The surface of 96-well plate was coated with 100 ng of SARS-CoV-2 spike protein, p24 HIV surface antigen, HBV surface antigen or HCV core antigen and incubated at room temperature for 1 hour. After washing five times with 200 pL of 1 xTBST, the plate surface was blocked with 200 pL of blocking solution (5% skim milk in 1 xTBST) for 2 hours at room temperature. Following removal of the blocking solution, the wells were treated with or without dAb-GAL (100 ng, 2.5% BSA in PBS) and incubated forBWH2024-0991 hour at room temperature. After five washes, the wells were treated with anti- p- galactosidase-HRP (2 pg) or anti-IgG-HRP (10 ng) in 2.5% BSA in PBS and incubated for 1 hour at room temperature. Following seven washes, 100 pL of TMB substrate were treated and the color change was monitored for 20 min. The reaction was stopped by adding stop solution (0.16 M sulfuric acid) and the absorbance of each well was measured at 450 nm wavelength.

[0116] 1.1.15 Preparation of VTM Passivated cAb-Mg Beads.

[0117] The cAb-MgBeads were prepared using Dynabeads antibody coupling kit (Invitrogen, 14211 D) which uses an epoxy-amine reaction mechanism for antibody conjugation. The manufacturer’s instructions were followed for a reaction scale involving 5 mg of MgBeads and 100 pg of cAb. The cAb-MgBeads solution having a final volume of 500 pL was divided into 5 aliquots of 100 pL and stored at 4°C. For cAb-MgBeads surface passivation, 1 .5 mL of VTM was incubated with 20 pL of cAb- MgBeads solution at 37°C for 30 min with gentle rotation. After the incubation, the reaction mixtures were removed using magnetic rack pulldown and the supernatant was discarded. The MgBeads were washed twice by re-suspending them in 500 pL of 1 xTBST, pulling them out using magnetic rack and discarding supernatant. After the two washes, the VTM passivated cAb-MgBeads were resuspended in 20 pL of PBS.

[0118] 1.1.16 Investigation of cAb-MgBeads conjugation

[0119] The function of cAb conjugated on the surface of MgBeads was evaluated by mixing the cAb-MgBeads (2 pL), MgBeads (2 pL) without cAb conjugation, cAb (0.4 pg) without MgBeads or PBS (1 pL) with anti-IgG-HRP (0.01 pg in 100 pL of PBS). After 20 min incubation at room temperature, the mixture was washed with 200 pL of 1 xTBST followed by removal of supernatant and the washing step was repeated 4 more times. The pellet was re-suspended with TMB (100 pL) and the absorbance of each signal at 450 nm was measured using a microplate reader after treatment of stop solution (100 pL) at 20 min time point.

[0120] 1 .1 .17 Evaluating the dose effect response of Flue with CMOS sensor

[0121] A concentration of ST-GAL (0.4 nM) was mixed with different concentrations of Flue (3.2 pM, 1 .6 pM, 0.8 pM, 0.16 pM, 80 nM, and 16 nM) in the working solution. The images of bioluminescence were taken via CMOS sensor and the intensity was measured using Imaged software.BWH2024-099

[0122] 1.1.18 Evaluation of the functionality of the individual components of theLUCAS

[0123] cAb-MgBeads (0.2 pg of cAb, 1 pL) and VTM with or without four different antigens (100 ng, 100 pL) including SARS-CoV-2 spike, HIV p24, HBV surface antigen and HCV core antigen, and dAb-GAL (0.05 pg of dAb, 1 pL) were mixed. After incubation for 20 min at room temperature, the washing step described in ‘Protocol of LUCAS’ was followed and the mixture was re-suspended with or without bioluminescence buffer, LUGAL and Flue. The bioluminescence results were evaluated via CMOS sensor.

[0124] 1.1.19 Design of the disposable microfluidic cartridge

[0125] The disposable microfluidic cartridge is prepared by cutting three identical 1 .5 mm thick PMMA substrates with a laser cutter. These substrates are then attached to each other using double-sided adhesive (DSA). Additionally, the cartridge includes a 3D printed chip cover made from PLA, which houses the rotation valve, a magnet for the reaction chamber, and syringes connected to the inlets. The assay chip itself features two prepacked chambers, one containing washing buffer (400 pL of 1 xTBST), and the other containing the working solution (150 pL). It also includes a separate reaction chamber where the assay takes place. The chip has a magnet attached, covering one side of the reaction chamber area. The cartridge is designed with three distinct inlets, enabling the injection of the sample, washing buffer, and working solution. These inlets are connected to the reaction chamber via a series of channels. The connection between the channels and the reaction chamber is regulated by a small connecting channel in the rotation valve, which facilitates the sequential entry of the desired solution into the reaction chamber at specific stages of the assay.

[0126] 1 .1 .20 Design of the detection module

[0127] The detection module, which has dimensions of 122 x 112 x 158 mm, is specifically tailored to cover the assay chip entirely. It effectively blocks external light and creates a dark environment for precise monitoring of bioluminescence. The detection module comprises a body and a lid that are attached to each other using two M3 bolts on each side. Within the module, various components are housed, including the cartridge, Raspberry Pi 4, Arduino Nano, servo motor, battery pack, and a CMOS image sensor. The CMOS sensor is positioned at the center of the bottom of the module, precisely 56.5 mm below the assay chip, to capture a focusedBWH2024-099 image of the bioluminescence signal within the assay chamber. To automate the assay process, a servo motor is affixed to the lid, and a rotation valve lever is connected to it. The Raspberry Pi sends a signal to the Arduino Nano, which prompts the servo motor to rotate. The lever, attached to the motor, pushes the syringe in the chamber containing the buffer solution. Additionally, the Raspberry Pi sends signals to the CMOS sensor to capture photos with the desired parameters for further analysis.

[0128] 1.1.21 Development of smartphone applications

[0129] In the detection module, the Raspberry Pi was the central processing unit and host controller and served as the core computational unit responsible for executing the image processing algorithms and coordinating the data acquisition process. To facilitate the interaction between the image processing system and the user, an Android application was developed using Android Studio (v2022.2.1 ). This Android app enabled seamless communication with the Raspberry Pi and allowed us to remotely trigger the sample insertion and image capture process while maintaining incubation time and waiting time. The communication between the Android app and the Raspberry Pi was established through a Flask server hosted on the Raspberry Pi. This server acted as the intermediary, facilitating the exchange of commands and data between the two devices. In addition to its role as a computational unit, the Raspberry Pi also controlled the physical components of the setup. To send samples into the imaging system at the designated times, servo motors were controlled by an Arduino board. The Raspberry Pi communicated with the Arduino to precisely control the movement of these servo motors, ensuring the accurate delivery of samples into the microfluidic channel setup. This level of automation and control enhanced the consistency and reliability of the image capture process. A Pi camera module was utilized to capture images from Raspberry Pi. For image processing the primary goal was to assess where the sample is positive or negative using bioluminescence property of the sample. To achieve this, OpenCV version 4.8.0, a widely used computer vision library that provides essential tools for image processing was utilized. The approach involved several steps. First, the images were cropped to isolate the region of interest. This cropping ensured that the analysis focused exclusively on the relevant areas. Next, the cropped images were converted to the HSV (Hue, Saturation, Value) colour space. To detect the target colour pattern - in this case, yellow -lower and upper bounds were defined within the HSV colourBWH2024-099 space that corresponded to the desired colour range. A mask was created to extract the pixels falling within these boundaries. To refine the results and remove unwanted noise, morphological operations were applied to the mask, namely closing and opening operations. Subsequently, the mask was used to segment the relevant region within the HSV image. By applying this mask, the yellow colour pattern within the region of interest was effectively isolated. This segmentation step was crucial for the analysis. Finally, the brightness of the segmented yellow region was calculated by summing the values in the Value channel of the HSV image. This value served as a quantitative measure of the bioluminescence property of the sample.

[0130] 1 .1 .22 Automated assay protocol of LUCAS

[0131] Step 1 . The test sample was introduced into the Eppendorf tube, which contains cAb-MgBeads (0.4 pg of cAb in PBS) and dAb-GAL (0.05 pg of dAb in 20 mM Tris-HCI) and was swirled for 30 seconds. The mixture was transferred to the sample injection syringe. Step 2. Push the solution with injection bar. Step 3. A disposable cartridge was attached to the detection module and the lid of the detection module was closed. Step 4. Press “Start” button in the smartphone app and wait until the assay result was displayed.

[0132] 1 .1 .23 Biosafety and human participant statement

[0133] The research work reported was approved and performed in adherence to guidelines and procedures approved by the Institutional Biosafety Committee of Mass General Brigham (parent organization of Massachusetts General Hospital and Brigham and Women’s Hospital) and under appropriate institutional review boards (IRB nos. 2015P000454, 2019P002209, 2019P001996, 2019P001489, 2023P000538, 2020P001065, and 2020P001405). In this study de-identified secondary use research patient samples or de-identified leftover patient samples were used. The authors did not have access to any identified patient information. All the experiments with SARS-CoV-2, HIV, HBV, and HCV were performed in BSL2+. SARS-CoV-2 inactivated sample (isolate USA-WA1 / 2020-Washington Strain) was provided through the NIH RADx Program. HIV stock-cultured samples were received from the division of infectious diseases at Brigham and Women’s Hospital. SARS-CoV-2-infected patient samples in VTM were purchased from Boca BioListics. The viral loads of SARS-CoV-2-infected patient serum samples were measured using a standard RT-PCR system and reported by the vendor (Boca BioListics).HBV-infected and HCV-infected patient serum / plasma samples were purchased fromBWH2024-099Discovery Life Sciences Inc. The viral loads of HBV-infected and HCV-infected patient serum samples were measured using a standard RT-PCR system and reported by the vendor (Discovery Life Sciences Inc.). The spiked samples that were used in system testing throughout the study were prepared by serial dilutions of stock virus samples with known viral loads measured by standard PCR approaches. In these cases, the control samples were virus-free samples that were processed for the detection assay with the same protocol as the viral samples, except for the addition of viral-stock dilutions.

[0134] 1 .1 .24 Statistical Analysis

[0135] Statistical analyses were performed using Origin software version 8.5 (Origin Lab Corporation. Northampton, MA, USA). All viral load values were converted into Iog10 copies / ml. The mean and SDs were calculated for each data point from at least a total of two to three independent experiments unless indicated in the text. The level of significance was set at P > 0.05. ROC and scattering plots analysis were used to define performance of LUCAS to the standard analytical technique. The strength of the linear and non-linear relationship was evaluated using Pearson’s correlation and Spearman correlation, respectively.

[0136] 1.2 Results

[0137] 1 .2.1 Comparing sensitivity and persistency: LUCAS vs. non-LUCAS

[0138] The stability of the LUCAS system was evaluated at the optimal Flue concentration as compared to non-LUCAS system, a conventional bioluminescence system where Flue is conjugated to the dAb for labelling the captured target molecules (see FIG. 15, elements A and B). The purpose of this experiment was to evaluate the activity of enzymes in each system after conjugation to the antibody. The decrease in enzyme activity was monitored by measuring the reduction of the bioluminescence signal. For the comparison, GAL conjugated with streptavidin (ST- GAL) or Flue conjugated with streptavidin (ST-Fluc) was mixed with or without detection antibody-biotin. Portions from the reaction stock were taken and mixed with a working solution to generate a bioluminescence signal immediately before measurement at each predetermined timepoint (see FIG. 15, element C). The bioluminescence signal on Day 1 was set as 100% and the signal at other timepoints was normalized accordingly. The bioluminescence signal of unconjugated ST-GAL remained comparable to that of ST-GAL conjugated to antibody, suggesting over 98% retention of activity over four days. In contrast, when Fluc-ST combined with theBWH2024-099 antibody, a substantial 77% decrease in bioluminescence was observed by day 2, with no signal detected by day 3, indicating complete loss of activity in the ST-Fluc system upon conjugation. This underscores the superior stability and functionality of the LUCAS system over non-LUCAS system. These results strongly support GAL’s retention of enzyme activity post-conjugation and highlight its higher stability compared to Flue under conjugation conditions.

[0139] To assess the bioluminescence decay of the LUCAS system’s signal, ST-GAL was utilized on the biotin-coated surface of a 96-well plate. The conjugated ST-GAL for LUCAS and ST-Fluc for non-LUCAS on the surface of the plate were mixed with a working solution at the 0-minute time point, and the intensity of the bioluminescence signal was tracked at predetermined intervals (see FIG. 15, element D). The signal intensity at each timepoint was normalized to the intensity at the 0-minute time point. The bioluminescent signal emitted by ST-GAL, once connected with biotin, remained at over 96% even after 1 hour. In contrast, the signal from Flue attached to the plate’s surface dropped to 74% in the same period see FIG. 15, element D). The signal decay rate was assessed by comparing the slopes of the trendlines for each system. For the LUCAS assay, the trendline was y = -0.055% + 99.71 (R2= 0.96), while for the non-LUCAS system, it was y = -0.445% + 97.219 (R2= 0.95). These slopes indicate that the LUCAS system’s signal decay rate is approximately 8 times slower than that of the non-LUCAS system, demonstrating improved signal persistence in LUCAS. The incorporation of GAL within the LUCAS system appears to play a role in sustaining enzyme activity, potentially enabling the continuous production of bioluminescence signals. This discovery underscores GAL’s beneficial characteristics in preserving enzyme functionality and stability, further solidifying its appropriateness for utilization within the cascade system.

[0140] The cascade system demonstrates a remarkable feature by optimizing assay output through the precise control of enzyme concentrations at individual stages. Rhe concentration of Flue was varied during the second step and gauging the subsequent bioluminescence intensity with a luminometer. Given the dependency of ST-GAL concentration on the binding of target molecules, the Flue concentration was adjusted within a range from 16 nM to 8.0 pM, maintaining ST- GAL at a fixed concentration of 100 pM. This led to a gradual increase in signal intensity. The signal intensity reached a plateau when the Flue concentration hit 4.8BWH2024-099 iM, as detected by the luminometer, indicating the optimal condition for generating strong bioluminescence. These results confirm that an excessive amount of native Flue in the cascade system can accelerate the overall reaction rate and boost assay sensitivity. Subsequently, the quantity of D-Luciferin-6-O-beta-D-galactopyranoside (LUGAL) was optimized in the working solution by adjusting its concentration from 56.5 pM to 395.5 pM when GAL and Flue were maintained at fixed concentrations of 100 pM and 4.8 pM, respectively. With increasing LUGAL concentration, the bioluminescence intensity also rose and stabilized at 282.5 pM, demonstrating the most effective substrate concentration for generating the brightest signal.

[0141] Having established the optimal concentrations for Flue and LUGAL, the concentration of ST-GAL was reduced to determine the minimum required for bioluminescence generation. As enzymes bind to target molecules with a finite capacity based on available conjugation sites, the enzymes’ necessary concentration to produce a particular bioluminescent signal intensity directly correlates with the assay’s sensitivity. For evaluating the sensitivity contrast between the LUCAS and non-LUCAS systems, the bioluminescence signal intensities were measured by employing a dynamic range of enzymes in each approach — GAL for LUCAS and Flue for non-LUCAS. The findings showed that to produce detectable bioluminescent signals, LUCAS required 6.2 atto-moles of GAL, while the non-LUCAS system necessitated 3.2 femto-moles of Flue. This reveals a substantial 515-fold difference in enzyme concentration between the two systems (see FIG. 15, element E). These results strongly indicate that GAL holds the capacity to generate a bioluminescence signal over 515 times stronger than that of the non-LUCAS system, marking it as a substantially more sensitive approach. Overall, these outcomes underscore that LUCAS displays enhanced assay sensitivity, primarily attributed to GAL’s stability and the incorporation of an excessive amount of natural Flue.

[0142] 1 .2.2 Preparation of detection antibody-conjugated GAL (dAb-GAL) and capture antibody-conjugated MgBeads (cAb-MgBeads), and assay optimization for point-of-care setting

[0143] To tailor the LUCAS system for point-of-care testing, the assay materials were synthesized. For site-specific biotinylation of the dAb (dAb-Biotin), the Invitrogen SiteClick biotin antibody labeling kit (no. S20033; Thermo Fisher) was utilized following the manufacturer’s guidelines. Confirmation of the conjugation was carried out using ELISA, wherein a noticeable color change of TMB (3, 3’, 5,5’-BWH2024-099Tetramethylbenzidine) was specifically observed for the dAb-Biotin treated with streptavidin conjugated to horseradish peroxide (ST-HRP). Conversely, no color change was noted for the unconjugated antibody. This clear differentiation indicates that the biotin conjugation on the antibody does not disrupt its functionality for target proteins, and the attached biotin molecules remain accessible for subsequent streptavidin conjugation. Post site-specific modification, the recovery yield of dAbs using the BCA (Bicinchoninic acid) assay. The results revealed conjugation yields of 59%, 68%, 53%, and 70% for the SARS-CoV-2, HIV, HBV, and HCV antibodies, respectively, were determined.

[0144] The dAb-GAL was synthesized through a straightforward process involving the mixing of dAb-Biotin and ST-GAL. The confirmation of successful conjugation of dAb-GAL was established using western blotting. The protein bands detected with anti-IgG-HRP were found to correspond precisely to those detected with anti-p-galactosidase-HRP, validating the successful synthesis of dAb-GAL. This confirms the successful preparation of the dAb-GAL conjugate. The functionality of dAb-GAL was further validated using 3,3’,5,5’-Tetramethylbenzidine-ELISA (TMB- ELISA). In this assay, dAb-GAL was treated with the SARS-CoV-2 spike antigen, resulting in a noticeable color change. However, when ST-GAL, dAb-GAL, or the spike antigen were absent from the reaction conditions, no color change was observed. These findings confirm that the dAb-GAL conjugate retains its functional activity.

[0145] To prepare the cAb-MgBeads, the Dynabeads® Antibody Coupling Kit were utilized following the supplier’s instructions. Using a BCA assay, the concentration of cAb covalently coupled to the MgBeads’ surface was determined to be 15.0 pg / mg for SARS-CoV-2, 14.3 pg / mg for HIV, 15.3 pg / mg for HBV, and 15.2 pg / mg for HCV. To block the surface of the conjugated MgBeads and prevent nonspecific binding, the cAb-MgBeads were further incubated with viral transport medium (VTM) (Methods). The successful conjugation of cAb on the surface of the MgBeads was confirmed by observing the color change of TMB upon the addition of anti-IgG-HRP. Specifically, in the presence of cAb-MgBeads, the color of TMB changed to yellow, whereas no change was detected when MgBeads without cAb conjugation were used. This confirms the successful conjugation of cAb on the surface of the MgBeads.BWH2024-099

[0146] A bioluminescence buffer formulation was specifically designed for optimal functionality with a portable CMOS sensor ideal for point-of-care diagnostics. By adjusting the concentration of Flue within a range from 16.0 nM to 3.2 pM while maintaining ST-GAL at a fixed concentration of 0.4 nM, a continuous improvement in signal intensity occurred. The signal intensity plateaued at a Flue concentration of 1 .6 pM as detected by the CMOS sensor, indicating the optimal condition for robust bioluminescence generation. Following the optimization of Flue concentration, the amount of LUGAL substrate was adjusted in the working solution. With an increase from 0 to 339.0 pM of LUGAL, the bioluminescence intensity showed a proportional rise, ultimately saturating at 56.5 pM of LUGAL. These results guided the selection of the optimized LUGAL concentration for subsequent bioluminescence assays. The buffer composition of this working solution was then employed for the development of the reported point-of-care LUCAS.

[0147] After preparing and optimizing the assay materials, LUCAS efficacy in capturing a target protein and generating bioluminescence using an immunoassay was assessed with the recombinant SARS-CoV-2 spike antigen as a clinical model. Strong bioluminescence was observed when all components were present, whereas no signal was detected in the absence of the target antigen. Similar results were obtained for various viral antigen targets such as HIV, HBV, and HCV. These findings validate the selected dAbs’ ability to efficiently capture the target molecule and demonstrate that ST-GAL, conjugated to the dAb, triggers a cascade reaction leading to bioluminescence production.

[0148] 1 .2.3 Performance of LUCAS using a manual process

[0149] Before developing a fully automated point-of-care LUCAS system, a series of tests were conducted using a manual-based LUCAS assay to evaluate its performance in detecting different target viral antigens including SARS-CoV-2, HIV, HBV, and HCV (see FIG. 16). The manual-based LUCAS assay included a dark container and a CMOS sensor for bioluminescence image data acquisition from samples placed in an Eppendorf tube. The manual process of the LUCAS system is outlined in FIG. 16, element A, comprising the following steps: (1) capturing target viral antigens in samples using assay materials, (2) conducting washing and signal generation procedures, (3) capturing an image with a CMOS sensor, and (4) analyzing image data utilizing Imaged software.BWH2024-099

[0150] The dynamic range and LOD of manual-based LUCAS assay were assessed using five distinct known standard concentrations of recombinant proteins representing the S1 subunit of SARS-CoV-2, p24 for HIV, the surface antigen for HBV, and the core antigen for HCV. These proteins were serially diluted in VTM. A dynamic range of target antigen concentrations spanning from 10 fg to 100 ng was tested. For each target protein, a dose-response curve was plotted, and a strong correlation was observed between the target concentration and the corresponding bioluminescence intensity (see FIG. 16, elements B - E). The obtained r-squared values exceeded 0.95, indicating high reliability and accuracy of the manual-based LUCAS system in quantifying the target proteins. The LOD of the manual-based LUCAS assay was determined by calculating the mean of the blank plus three times the standard deviation obtained for the blank. The theoretical LOD values were estimated as 26.7 fM for SARS-CoV-2, 3.9 fM for HIV, 0.4 fM for HBV, and 0.8 fM for HCV, respectively. This variation is due to differences in antibody affinity to target antigens and the number of available antigens per viral target. Following the successful assessment of recombinant antigens, the evaluation was extended to encompass samples containing inactivated viruses. These samples, which closely mimic actual clinical scenarios, were subjected to the same experimental workflow. The SARS-CoV-2 samples inactivated with Ultraviolet (UV), underwent serial dilution, and the resulting bioluminescence signal was observed (Fig. 3f). A detectable signal was noted at concentrations as low as 1 x103viral RNA copies / mL, and this signal continued to escalate until reaching a minimum of 1 x106copies / mL of viral load. The LOD of the manual-based LUCAS for SARS-CoV-2 as a clinical model was calculated as 518 copies / mL, indicating a higher level of sensitivity compared to the existing FDA-EUA approved antigen test kits (see FIG. 16, element F). In further investigating the detection sensitivity of LUCAS with a more high- performance detector, specifically a luminometer, the calculated LOD was approximately 1 19 copies / mL (see FIG. 16, element G).

[0151] The specificity of the assay was also evaluated using SARS-CoV-2 samples and non-target samples including Influenza A (IAV), HBV, HCV, and HIV with virus concentrations of around 1 x106copies / ml (see FIG. 16, element H). The bioluminescence of the non-target viral samples generated an intensity equal to or less than 2,500 Relative Light Unit (RLU), which is comparable to blank without the presence of any target virus. However, the SARS-CoV-2 samples with an averageBWH2024-099 viral load of 1 x106copies / ml generated an intensity greater than 8,500 RLU. In the same manner, the specificity of other virus samples such as HBV, HCV, and HIV was evaluated using non-target samples and confirmed that LUCAS showed specificity towards all the target viruses (see FIG. 16, element l-K).

[0152] 1 .2.4 System integration: a fully automated LUCAS

[0153] A fully automated LUCAS system was developed, incorporating a microfluidic cartridge with pre-loaded reagents and a portable, cost-effective reader designed for straightforward sample handling (see FIG. 17, element A). By a mere press of a button within a custom smartphone application, all essential procedures, encompassing washing sequences, signal generation, data capture, and result reporting, are seamlessly initiated and managed. The functionality of the automated LUCAS was initially assessed using three distinct food-colored dyes: red to denote the sample with cAb-MgBeads and dAb-GAL, blue for the washing buffer (400 pL of 1 x Tris-buffered saline with 0.1% Tween® 20 detergent, TBST), and green for the working solution. The process commences by blending the collected sample with assay materials (red), seamlessly transferring the mixture to the assay chamber within the microfluidic cartridge. Activating the procedure involves opening a smartphone application and initiating the sequence by pressing the start button, commencing a 20-minute incubation period within the assay chamber. After this incubation, a rotating valve automatically pivots counterclockwise, aligning with the chamber containing the blue dye (washing buffer). The valve then propels a syringe loaded with washing buffer, facilitating the washing of the captured mixture while expelling excess washing solution and air into a drain chamber. Following the washing step, the rotating valve shifts clockwise to access the chamber housing the green dye (working solution). It injects a syringe filled with the working solution into the chamber, triggering bioluminescence, captured by the CMOS sensor for result analysis. The captured image undergoes analysis within the smartphone application to determine the sample’s positive or negative status. The entire sample-to-answer assay process was completed in approximately 23 minutes, encompassing the virus capture incubation time.

[0154] The cartridge and the detection module were fabricated using a 3D printer and a laser cutter. To guarantee precise device performance and the accuracy of assays, multiple optimization measures were carried out. Initial optimization focused on adjusting the volume of air within the syringe linked to theBWH2024-099 sample, washing buffer, and working solution chambers. This adjustment aimed to achieve precise delivery of the solution into the assay chamber. The ideal air volumes for transferring the sample, efficiently eliminating all the washing buffer to the waste outlet, and ensuring the working solution reaches the assay chamber were determined to be 0.3 mL, 1.2 mL, and 0.2 mL, respectively (see FIG. 17, element B). Maintaining the optimum air volumes for the sample and working solution is crucial to ensure proper liquid distribution within the assay system. Deviations from these volumes caused issues, such as the liquid not reaching the assay chamber or passing through to the waste outlet. When employing a smaller air volume (<1 .2 mL) to inject the washing buffer, a decrease or absence of the bioluminescent signal was observed. This decline was linked to potential interference from residual washing buffer remaining in the assay chamber or channel. These findings confirm that having more than 10 pL of residual washing buffer (1 xTBST) can result in a weaker bioluminescence signal. Hence, it is imperative to thoroughly remove the washing buffer post-sample washing to ensure precise and dependable results.

[0155] To mitigate the risk of false positive signals, VTM was applied to the cartridge chamber and channels. cAb-MgBeads and dAb-GAL within the assay chamber were incubated under assay conditions, but without the presence of any viral biotargets. After a washing step, a working solution was introduced to assess the occurrence of bioluminescence. Cartridges treated with VTM showed no bioluminescent signals, whereas untreated cartridges exhibited detectable bioluminescent signals. These findings affirm that VTM effectively prevents potential false positive signals by adequately coating the poly(methylmethacrylate) (PMMA) surface.

[0156] After fine-tuning the parameters, the performance of the fully automated LUCAS assay was first evaluated using samples containing a representative target protein, the SARS-CoV-2 omicron variant spike antigen (see FIG. 17, element C). Evident and vibrant bioluminescent signals were observed in the presence of the target protein, accompanied by a positive indication on the smartphone application as depicted in FIG. 17, element C. Conversely, in the absence of the target virus, no bioluminescent signal was detected, and a negative result was displayed on the smartphone application (see FIG. 17, element D).

[0157] The microfluidic cartridge was designed for single use, while the detection module can be employed repeatedly for multiple tests. The material costsBWH2024-099 for manufacturing the microfluidic cartridge amount to approximately $2.3, while the cost for the detection module is approximately $86.8. These findings validated the successful integration of all facets of the reported LUCAS technology, encompassing sample handling, signal amplification, and readout, within a portable and costefficient cartridge.

[0158] To assess the frequency of user errors during sample processing using the fully automated LUCAS system, experiments were conducted involving a diverse group of individuals. These tests utilized samples containing the recombinant SARS- CoV-2 omicron variant spike antigen as the test material. The participant pool comprised a total of 13 individuals: five with specialized training and expertise related to the developed LUCAS system, and eight untrained individuals with diverse backgrounds, including non-PhD graduates and undergraduate students (see FIG. 17, element E). The untrained participants were provided with a user instruction sheet as their only reference to conduct the testing procedure. Notably, both groups achieved consistent and reproducible results, underscoring the user-friendly nature of the automated LUCAS system. This outcome emphasizes the ease of operation and the robustness of the automated LUCAS assay, making it accessible and effective for a broad spectrum of users.

[0159] 1 .2.5 Assay validation with patient samples

[0160] Initially, the LCD of the automated LUCAS system was assessed by conducting experiments involving serially diluted SARS-CoV-2 nasopharyngeal swabs samples and serum samples spiked with HIV, HBV, or HCV (see FIG. 18, elements A-D). To determine the LODs, Receiver Operating Characteristic (ROC) curve analyses were conducted on serially diluted samples spiked with SARS-CoV- 2, HIV, HBV, and HCV, with virus concentrations ranging from 0 to 1 ,000,000 copies / mL (see FIG. 18, elements E-H). The experimental LODs were 655, 753, 641 , and 396 copies / mL for SARS-CoV-2, HIV, HBV, and HCV. To further evaluate the determined LODs of LUCAS, the assay was tested per FDA guidelines with 20 viral- spiked samples with viral loads near the calculated LODs. The assay accurately classified these samples based on the established viral load thresholds in over 95% of the cases, confirming that the determined LODs of the LUCAS system are valid. Subsequently, the automated LUCAS system was modified to create a qualitative assay. This was achieved by leveraging the LODs established for each viral target or clinically relevant viral load threshold as the system’s qualitative assessmentBWH2024-099 threshold. Specifically, following viral load thresholds were used for the automated qualitative LUCAS: 655 copies / mL for SARS-CoV-2, 1000 copies / mL for HIV as per WHO guidelines, 641 copies / mL for HBV, and 396 copies / mL for HCV. This qualitative system was then evaluated using a total of 307 biosamples including 177 viral-infected patient samples and 130 viral-spiked serum samples. The sample set included 44 positive and 44 negative samples for SARS-CoV-2, 31 positive and 35 negative serum samples for HBV, and 50 positive and 33 negative serum samples for HCV. Additionally, the assay was evaluated with 35 positive and 35 negative serum samples for HIV.

[0161] To assess the performance of the automated qualitative LUCAS system in categorizing the viral-infected samples, vertical scatterplots (see FIG. 18, elements l-L) were employed. The vertical scatterplots display the correlation between the automated LUCAS results and PCR qualitative outcomes for patient samples, highlight specific discrepancies. These consist of five false positives and one false negative in SARS-CoV-2 patient samples, two false negatives in HIV- spiked samples, two false positives in HBV patient samples, and two false positives and one false negative in HCV patient samples. The specificities and sensitivities of the automated LUCAS in qualitatively classifying viral-infected samples with cl inically-relevant viral load thresholds were 85% and 97% for SARS-CoV-2 (n = 68 nasopharyngeal swab samples), 100% and 92% for HIV (n = 50 HIV-spiked serum samples), 92% and 100% for HBV (n = 46 HBV-infected patient serum samples), and 91% and 98% for HCV (n = 63 HCV-infected patient serum samples).Additionally, samples with viral loads near the detection limit were tested to assess the reliability of the assay. Specifically, HIV-spiked samples with concentrations below 20,000 copies / mL were evaluated. A total of 32 samples with viral loads ranging from 770 to 15,000 copies / mL were tested, resulting in 29 true positives and 3 false negatives. These results provide strong evidence for the system’s capability to accurately distinguish between infected and virus-free samples using clinically relevant viral load thresholds, thereby reinforcing its diagnostic utility. The substantial discriminatory capacity demonstrated by the automated LUCAS system underscores its suitability for clinical use, highlighting its valuable role in enhancing the accuracy of virus detection. Concordance was evident when assessing the correlation between the RLU values measured by the automated LUCAS system (y-axis) and the viral loads of viral-infected samples (x-axis) (Fig. 5m-p). This alignment wasBWH2024-099 supported by both Pearson’s and Spearman’s correlation coefficients, underscoring the robustness of the association between these variables.

[0162] To highlight the advantages of LUCAS over other assays, a performance evaluation was conducted comparing LUCAS, non-LUCAS, and the commercially available HIV p24 ARCHITECT assay. This evaluation focused on their ability to detect the HIV p24 antigen in serum, using it as a clinical model biotarget (see FIG.19, elements A-D). Serially diluted HIV p24-spiked serum samples were first used to determine the LODs of LUCAS, non-LUCAS, ELISA, and HIV p24 ARCHITECT. The calculated LODs for detecting recombinant p24 antigen were as follows: 3.9 fM for LUCAS, 4.7 pM for non-LUCAS, 1 .8 pM for ELISA, and 18.2 pM (visible to the naked eye) for ARCHITECT. The performance of LUCAS, non-LUCAS, ELISA, and ARCHITECT were then evaluated by calculating the clinical sensitivities and specificities of the assays in classifying 50 HIV-spiked serum samples at the clinically relevant viral load threshold of 1 ,000 copies / mL (see FIG. 19, elements E- H). All assays were evaluated for specific viral loads simultaneously using the same stock solutions prepared for those viral loads. The clinical sensitivities and specificities were confirmed as follows: 52% and 100% for the non-LUCAS system, 64% and 100% for ELISA, and 48% and 100% for the ARCHITECT assay, respectively. The sensitivity and specificity of LUCAS were 92% and 100%, respectively. Accuracy was also evaluated, resulting in 96% for LUCAS, 76% for non-LUCAS, 82% for ELISA, and 74% for ARCHITECT. These results demonstrated that LUCAS outperformed non-LUCAS, ELISA, and ARCHITECT in classifying HIV serum samples when using a clinically relevant low viral load threshold of 1 ,000 copies / mL. These results are consistent with previous reports on the capabilities of fourth and fifth-generation assays on various HIV subtypes using panels of diluted HIV isolates. Point-of-care tests like Alere Determine and SD Bioline were found to be less effective, especially for low viral load detection.

[0163] To further clarify why the LUCAS system outperforms the non-LUCAS conventional bioluminescence approach, HIV-spiked samples were tested using the non-LUCAS system with a Flue concentration of 1 .6 pM (dAb-Fluc), matching the concentration used in the LUCAS (natural Flue). Despite the higher concentration of dAb-Fluc, the dose-response curve remained consistent with the results obtained using a lower 13.3 nM dAb-Fluc concentration. Additionally, the bioluminescent signals generated by the LUCAS system and the non-LUCAS system for p24 antigenBWH2024-099(10 pM p24 recombinant antigen) detection using a luminometer. In this experiment, the Flue concentration used in the non-LUCAS system (800 pM) was 500 times higher than that in the LUCAS system (1 .6 pM). Nevertheless, the LUCAS system produced a bioluminescent signal over 500 times stronger. However, in the non- LUCAS system, the condition with 500 times excess Flue (800 pM) and the condition with the same concentration as LUCAS (1.6 pM) produced similar bioluminescence signal intensity. These results suggest that any excess dAb-Fluc not bound to the virus is removed during the washing step, leaving only the virus-bound dAb-Fluc to contribute to the bioluminescent signal.

[0164] Experiment 2

[0165] This study reported a highly sensitive and user-friendly automated point of care (POC) hepatitis C (HCV) Ag assay utilizing enzyme cascade reaction-based bioluminescence imaging (see FIG. 20). This rapid magnetic bead (MgBead)-based immunoassay enhanced the bioluminescence signal by incorporating excess natural luciferase in the second enzyme cascade step, generating high-yield luminescence without reducing enzyme activity. The cascade produced an intermediate substrate oxidized by luciferase, ensuring continuous bioluminescent signals and reproducibility. Integrated into a portable system, this assay enabled rapid sample- to-answer HCV analysis in under 30 minutes without external power, making the assay ideal for on-site diagnostics in underserved areas. The assay’s sensitivity, simplicity, and portability position it as a promising alternative for expanding HCV testing access among high-risk populations, particularly American Indians and Alaskan Natives (AI / AN).

[0166] 2.1 Materials and Methods

[0167] 2.1.1 Materials

[0168] All the reagents and solvents employed were commercially available and used as supplied without further purification. Dynabeads® Antibody Coupling Kit (14311 D), Streptavidin -galactosidase conjugate (S931 ), SiteClick™ Antibody Labelling Kits (S20033), MagJET Separation Rack for 12 x 1 .5 mL tube (MR02), Low Protein Binding Microcentrifuge Tubes (2 mL, 88380), 1 -Step™ Slow TMB-ELISA Substrate Solution (34024), Rabbit anti-Human IgG (H+L) Secondary Antibody (HRP, A18903) were purchased from Thermo Fisher Scientific. The concentration of antibody was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Adenosine 5'-triphosphate (ATP) disodium salt hydrate (A1852-BWH2024-0991 VL) Tris(2-carboxyethyl) phosphine hydrochloride, Powder, >98% (C4706-2G), Ethylenediaminetetraacetic acid (EDS-100G), Coenzyme A sodium salt hydrate (C4780-25MG), Magnesium sulfate heptahydrate (230391 -25G), Luciferase from Photinus pyralis (firefly, SRE0045), D-Luciferin (L9504) were purchased from Sigma-Aldrich. D-Luciferin-6-o-p-D-galactopyranoside (ab275053) was purchased from Abeam. Viral Transport Medium was purchased from Innovative Research (IGVTM500ML). Tween™ 20 (BP337100) was purchased from Fisher Chemical. TBST 20x (pH 7.5, 40120065-1 ) was purchased from bioWORLD. p-galactosidase antibody (HRP, GTX26646) was purchased from GeneTex. 96W Half Area UV-Star Plates Flat Bottom were purchased from Thomas Scientific. Clear Flat-Bottom Immuno Nonsterile 96-Well Plates were purchased from Thermo Fisher Scientific. Microplate reader (Infinite M nano+, Tecan) was used for TMB-ELISA.PMMA (12x12 1.5 mm Clear cast), O-rings, bolts (M3 x 0.5 mm), nuts (M3 x 0.5 mm) and ball bearing 6802 RS were purchased from MCMASTER-CARB. DSA (3M™ Optically Clear Adhesive 8146-2 50 pm) was purchased from 3M. Syringes (1 mm, 3 mm, 10 mm) were purchased from BD Syringe. Arduino Nano was purchased from AITEXM Robot. Raspberry Pi 4 was purchased from Raspberry pi foundation. CMOS sensor (Arducam IMX519 PDAF&CDAF Autofocus Camera Module for Raspberry Pi) was purchased from Arducam. Servo Motor (MG995 180 degree) was purchased from Towerpro. U Shape Type C Male to Female 40 Gbps Connector was purchased from AreMe. Type A Male to USB 3.1 Type C Male Up Opposite U Shape Back Angled 90 Degree Charge Adapter was purchased from Xiwai. Jumper wires (male to female) were purchased from EDGELEC. Laser cutter (VLS3.60DT, UNIVERSAL LASER SYSTEMS) was used for cutting PMMAs.

[0169] 2.1 .2 Preparation of the bioluminescence buffer

[0170] A bioluminescence buffer was prepared as a working solution. Briefly, ATP (1 .0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSO4-7H2O (2.0 mM), and Coenzyme A (8.0 pM) were mixed in 9.8 ml of 1 x Tris-HCl.

[0171] 2.1 .3 Preparation of working solution

[0172] LUGAL (5.0 pg / pL, 2.5 pL) and Flue (1 .0 pg / pL, 50.0 pL) were mixed in 447.5 pL of bioluminescence buffer. After gentle mixing, 100 pL of the working solution was used for each assay.

[0173] 2.1 .4 Information of antibody pairs and recombinant antigenBWH2024-099

[0174] Paired antibodies (MBS569238 for capture, MBS569240 for detection) for HCV core antigen were purchased from MyBioSource and recombinant HCV core antigen (8903) was purchased from ViroStat.

[0175] 2.1 .5 Preparation of dAb-biotin

[0176] The SiteClick™ Antibody Labeling Kit (Thermo Fisher Scientific, S20033) was used to conjugate the dAb with biotin according to the manufacturer's guidelines. The main points of the SiteClick™ conjugation process are altering the antibody's carbohydrate domain, conjugating azide to the antibody, and then attaching it with the sDIBO-modified biotin using copper-free click chemistry. This method results in the formation of a covalent bond between the biotin and the antibody, allowing the dAb conjugated with biotin to be obtained.

[0177] 2.1 .6 Investigation of dAb-biotin using TMB ELISA

[0178] To confirm the function of dAb-biotin, TMB-ELISA was utilized. Initially, the surface of a 96-well plate was coated with 100 ng of HCV core antigen and incubated at 4°C overnight. Subsequently, the plate was washed four times with 200 pL of 1 xTBST and then blocked with 200 pL of blocking solution (5% BSA in Milli Q) for 90 minutes at room temperature. After removing the blocking solution, the wells were incubated with or without 50 ng of dAb-biotin in 2.5% BSA in 1 xTBST for 1 hour at room temperature. Following four washes, the wells were incubated with 50 ng of streptavidin-HRP or 10 ng of anti-IgG-HRP in 2.5% BSA in 1 xTBST for 1 hour at room temperature. After five washes, 100 pL of TMB substrate was added, and the color change was observed for 20 minutes. Upon addition of 100 pL of Stop Solution (0.16 M sulfuric acid), the reaction was stopped, and the absorbance of each well was analyzed at 450 nm wavelengths.

[0179] 2.1 .7 Preparation of dAb-GAL

[0180] The dAb-biotin produced following the SiteClick™ Antibody Labeling Kit, and ST-GAL (Thermo Fisher Scientific, S931 ) were mixed at a 1 :4 mass ratio and diluted using Tris (pH 7.0, 0.05 M) to achieve a final antibody concentration of 25 ng / pL. This mixture was then incubated at 4°C for 2 days with gentle rotation. Following incubation, the conjugation of dAb-biotin with ST-GAL was confirmed by western blotting using anti-IgG-HRP and anti-p-galactosidase-HRP.

[0181] 2.1 .8 Investigation of dAB-GAL using TMB-ELISA

[0182] The surface of a 96-well plate was coated with 100 ng of HCV core antigen and incubated at 4°C for overnight. Subsequently, the plate was washed fourBWH2024-099 times with 200 pL of 1 xTBST and then mixed with 200 pL of blocking solution (5% BSA in Milli Q) for 90 minutes at room temperature. After removing the blocking solution, the wells were incubated with 50 ng of dAb-GAL in 2.5% BSA in 1 xTBST or without it for 1 hour at room temperature. After four washes, the wells were incubated with or without 50 ng of anti-beta-galactosidase antibody in 2.5% BSA in 1 xTBST for 1 hour at room temperature. Following four washes, the wells were incubated with 100 ng of anti-human IgG-HRP or 10 ng of anti-mouse IgG-HRP in 2.5% BSA in 1 xTBST for 1 hour at room temperature. After five washes, 100 pL of TMB substrate was added, and the color change was observed for 20 minutes. After adding 100 pL of Stop Solution (0.16 M sulfuric acid), the absorbance of each well was analyzed using a microplate reader at 450 nm wavelengths.

[0183] 2.1 .9 Investigation of cAB-MgBeads conjugation

[0184] To evaluate the function of cAb conjugated on the surface of MgBeads, cAb-MgBeads (2 pL), MgBeads (2 pL) without cAb conjugation, cAb (0.4 pg) without MgBeads, or PBS (1 pL) with anti-IgG-HRP (0.01 pg in 100 pL of PBS) were mixed. After 20 min incubation at room temperature, the mixtures were washed with 200 pL of 1 x TBST, and the supernatant was removed. This washing step was repeated three more times. The pellet was re-suspended with 100 pL of TMB, and absorbance was measured at 450 nm using a microplate reader after adding 100 pL of stop solution at the 20 min time point.

[0185] 2.1 .10 Preparation of VTM Passivated cAb-MgBeads

[0186] The cAb-MgBeads were prepared using the Dynabeads antibody coupling kit (Invitrogen, 14211 D), which utilizes an epoxy-amine reaction mechanism for antibody conjugation. According to the manufacturer’s instructions, the reaction involved 5 mg of MgBeads and 100 pg of cAb. The final volume of the cAb-MgBeads solution was 500 pL, which was divided into 5 aliquots of 100 pL each and stored at 4°C. For surface passivation of the cAb-MgBeads, 20 pL of the solution was incubated with 1 .5 ml of VTM at 37°C for 30 minutes with gentle rotation. After incubation, the mixtures were fixed using a magnetic rack pulldown, and the supernatant was removed. The MgBeads were washed twice with 500 pL of1 xTBST. Following washing, the VTM-blocked cAb-MgBeads were stored in 20 pL of PBS.

[0187] 2.1 .11 Evaluation of the functionality of the individual components of the enzyme cascade-based bioluminescence systemBWH2024-099

[0188] To evaluate the functionality of the individual components of the enzyme cascade-based bioluminescence system 1 pL of cAb-MgBeads (0.2 pg of cAb in PBS), 1 pL of dAb-GAL (0.025 pg of dAb in pH 7.0, 0.05 M Tris), and VTM were mixed with or without 100 ng of HCV core antigen. After a 20 minutes incubation at room temperature, the supernatant was removed by using a magnet to pull down the mixture. The resulting pellet was resuspended in 200 pL of 1 xTBST twice. The washing step was repeated once more. The pellet was resuspended in 100 pL of working solution and the bioluminescence signal was detected using a CMOS sensor.

[0189] 2.1 .12 Evaluation of clinical LOD

[0190] Clinical samples of HCV were serially diluted in plasma to concentrations of 1.5x107, 106, 105, 104, 5x103, 103, 5x102copies / ml. Prior to testing, a lysis step was performed for the HCV clinical samples by incubating at room temperature with 0.1% Tween™20 for 1 minute. The samples were then mixed with assay materials and processed following the protocol outlined in the "automated assay protocol of the enzyme cascade-based bioluminescence system". The resulting bioluminescence intensities under each condition were used as references to determine the limit of detection of the system. n=5 technical replicates were conducted, and the LOD of the automated enzyme cascade-based bioluminescence system was calculated as the mean of the blank plus three times the standard deviation obtained for the blank.

[0191] 2.1 .13 Development of smartphone applications

[0192] In the detection module, the Raspberry Pi was the central processing unit and host controller and served as the core computational unit responsible for executing the image processing algorithms and coordinating the data acquisition process. To facilitate the interaction between the image processing system and the user, an Android application was developed using Android Studio (v2022.2.1 ). This Android app enabled seamless communication with the Raspberry Pi and allowed remote triggering of the sample insertion and image capture process while maintaining incubation time and waiting time. The communication between the Android app and the Raspberry Pi was established through a Flask server hosted on the Raspberry Pi. This server acted as the intermediary, facilitating the exchange of commands and data between the two devices. In addition to its role as a computational unit, the Raspberry Pi also controlled the physical components of the setup. To send samples into the imaging system at the designated times, servoBWH2024-099 motors controlled by an Arduino board were integrated. The Raspberry Pi communicated with the Arduino to precisely control the movement of these servo motors, ensuring the accurate delivery of samples into the microfluidic channel setup. This level of automation and control enhanced the consistency and reliability of the image capture process. A Pi camera module was utilized to capture images from Raspberry Pi. For image processing the primary goal was to assess where the sample is positive or negative using bioluminescence property of the sample. To achieve this, OpenCV version 4.8.0 (a widely used computer vision library that provides essential tools for image processing) was used. The approach involved several steps. First, the images were cropped to isolate the region of interest that was utilized. This cropping ensured that the analysis focused exclusively on the relevant areas. Next, the cropped images were converted to the HSV (Hue, Saturation, Value) color space. To detect the target color pattern - in this case, yellow -lower and upper bounds were defined within the HSV color space that corresponded to the desired color range. Then a mask was created to extract the pixels falling within these boundaries. To refine the results and remove unwanted noise, morphological operations were applied to the mask, namely closing and opening operations. Subsequently, the mask was used to segment the relevant region within the HSV image. By applying this mask, the yellow color pattern within the region of interest was effectively isolated. This segmentation step was crucial for the analysis. Finally, the brightness of the segmented yellow region was calculated by summing the values in the Value channel of the HSV image. This value served as the quantitative measure of the bioluminescence property of the sample.

[0193] 2.1 .14 Specificity test

[0194] The specificity of the enzyme cascade-based bioluminescence assay was evaluated using AI / AN HCV-spiked plasma samples and non-target samples, including HIV and HBV, each at virus concentrations of approximately 1 x106copies / ml. The assay was performed following automated assay protocol described in FIG. 22 and the intensity of bioluminescence was quantified using smartphone application.

[0195] 2.1 .15 Biosafety and human participant statement

[0196] The research work reported was approved and performed in adherence to guidelines and procedures approved by the Institutional Biosafety Committee of Mass General Brigham (parent organization of Massachusetts General Hospital andBWH2024-099Brigham and Women’s Hospital) and under appropriate institutional review boards (IRB nos. 2015P000454, 2019P002209, 2019P001996, 2019P001489, 2023P000538, 2020P001065, and 2020P001405). This study was approved by the Cherokee Nation IRB. Patient enrollment was voluntary, and informed consent was required for participation. Except for the member of Cherokee Nation Health Services, researchers did / do not have access to identifiable information. All the experiments with HCV were performed in BSL2+. AI / AN HCV-infected patient plasma samples were provided by the Cherokee Nation. HBV-infected patient serum / plasma samples were purchased from Discovery Life Sciences Inc. The viral loads of HBV-infected and HCV-infected patient serum samples were measured using a standard RT-PCR system and reported by Mass General Hospital and the vendor (Discovery Life Sciences Inc.). HIV stock-cultured samples were received from the colleagues at the division of infectious diseases at Brigham and Women’s Hospital. The spiked samples that were used in system testing throughout the study were prepared by serial dilutions of stock virus samples with known viral loads measured by standard PCR approaches. In these cases, the control samples were virus-free samples that were processed for the detection assay with the same protocol as the viral samples, except for the addition of viral-stock dilutions.

[0197] 2.1.16 Statistical Analysis

[0198] Statistical analyses were performed using Origin software version 8.5 (Origin Lab Corporation. Northampton, MA, USA). All viral load values were converted into Iog10 copies / ml. The mean and SDs were calculated for each data point from at least a total of two to three independent experiments unless indicated in the text. The level of significance was set at P > 0.05. ROC and scattering plots analysis were used to define performance of the enzyme cascade-based bioluminescence system to the standard analytical technique. The agreement between categorical variables was evaluated using Cohen's kappa coefficient.

[0199] 2.2 Results

[0200] 2.2.1 Material characterization

[0201] To adapt the automated enzyme cascade-based bioluminescence system for point-of-care testing, the assay materials were synthesized, including the site-specific biotinylation of the detection antibody (dAb-Biotin) using the Invitrogen SiteClick biotin antibody labeling kit (S20033; Thermo Fisher), following the manufacturer's instructions. The conjugation was confirmed through ELISA, whichBWH2024-099 demonstrated a distinct color change of TMB (3,3',5,5'-Tetramethylbenzidine) specifically in the presence of dAb-Biotin treated with streptavidin-HRP (Horseradish Peroxidase). In contrast, no color change was observed for the unconjugated antibody. This clear distinction indicates that the biotin conjugation on the antibody does not impair its functionality towards target proteins, and the attached biotin molecules remain accessible for subsequent streptavidin binding. Following sitespecific modification, the recovery yield of the dAbs was assessed using the BCA (Bicinchoninic Acid) assay, which revealed conjugation yields of 92% for the HCV antibody.

[0202] The synthesis of the detection antibody conjugated beta-galactosidase (dAb-GAL) conjugate was achieved through a straightforward procedure by mixing dAb-Biotin with streptavidin conjugated beta-galactosidase (ST-GAL) (see FIG. 21 , element A). The successful conjugation of dAb-GAL was confirmed with western blotting, where protein bands detected with anti-IgG-HRP precisely matched those detected with anti-beta-galactosidase-HRP, validating the synthesis of dAb-GAL (see FIG. 21 , element B). The functionality of the dAb-GAL conjugate was further assessed using a 3,3',5,5'-Tetramethylbenzidine-ELISA (TMB-ELISA). In this assay, a distinct color change was observed when dAb-GAL was treated with the HCV core antigen. In contrast, no color change was noted in the absence of ST-GAL, dAb- GAL, or the core antigen. These results confirm that the dAb-GAL conjugate retains its functional activity (see FIG. 21 , elements C and D).

[0203] To prepare the capture antibody conjugated magnetic beads (cAb- MgBeads), the Dynabeads® Antibody Coupling Kit was applied according to the supplier's instructions (see FIG. 21 , element E). The concentration of cAb covalently coupled to the MgBeads' surface was quantified using a bicinchoninic acid (BCA) assay, yielding 14.9 pg / mg. To block the surface of the conjugated MgBeads and prevent non-specific binding, the cAb-MgBeads were further incubated with viral transport medium (VTM) (Methods). The successful conjugation of cAb on the MgBeads' surface was confirmed by the color change of TMB upon the addition of anti-IgG-HRP. Specifically, a yellow color change in TMB was observed in the presence of cAb-MgBeads, whereas no change was detected when MgBeads without cAb conjugation were used (see FIG. 21 , element F). This confirms the successful conjugation of cAb on the MgBeads' surface.BWH2024-099Following the preparation and optimization of assay materials, the efficacy of automated enzyme cascade-based bioluminescence system in capturing a target protein and initiating bioluminescence was evaluated using an immunoassay with the recombinant HCV core antigen as a clinical model. Strong bioluminescence was observed in the presence of all components, while no signal was detected in the absence of the target antigen (see FIG. 21 , element G). These results affirm the efficient capability of the selected dAbs to capture the target molecule and indicate that ST-GAL, conjugated to the dAb, initiates a cascade reaction leading to bioluminescence production.

[0204] 2.2.2 System integration: a fully automated enzyme cascade-based bioluminescence system

[0205] A fully automated enzyme cascade-based bioluminescence system was developed, incorporating a microfluidic cartridge with pre-loaded reagents and a portable, cost-effective reader designed for simple sample handling. By pressing a single button on a custom smartphone application, all essential procedures, including washing sequences, signal generation, data capture, and result reporting, were seamlessly initiated and managed (see FIG. 22, element A).

[0206] The hardware part of the assay comprises a disposable microfluidic cartridge and a reusable standalone detection module, both fabricated using a 3D printer and a laser cutter, as illustrated in FIG. 22, elements A and C. The disposable microfluidic cartridge was fabricated by laser cutting three identical 1 .5 mm thick poly(methyl methacrylate) (PMMA) substrates (see FIG. 22, elements B and D). These substrates were then assembled using double-sided adhesive (DSA).Additionally, the cartridge included a 3D-printed chip cover made from polylactic acid (PLA), incorporating a rotation valve, a magnet for the reaction chamber, and syringes connected to the inlets. The cartridges also included two pre-loaded chambers: one containing 400 pL of washing buffer (1 xTBST) and the other containing 150 pL of the working solution. Additionally, the cartridge has a separate reaction chamber where the assay is conducted. The cartridge features a magnet attached to one side of the reaction chamber. The cartridge was designed with three distinct inlets, allowing for the injection of the sample, washing buffer, and working solution. These inlets are connected to the reaction chamber through a network of channels. The flow between these channels and the reaction chamber is controlledBWH2024-099 by a small connecting channel within the rotation valve, allowing the sequential entry of each solution into the reaction chamber at specific stages of the assay.

[0207] The standalone optical reader, measuring 122 x 112 x 158 mm, was specifically designed to fully encompass the microfluidic cartridge (see FIG. 22, elements C and E). It effectively blocks external light, creating a dark environment for precise monitoring of bioluminescence. The standalone optical module comprises a body and a lid that are attached to each other using two M3 bolts on each side. Within the module, various components are housed, including the cartridge, Raspberry Pi 4, Arduino Nano, servo motor, battery pack, and a complementary metal oxide semiconductor (CMOS) image sensor. The CMOS sensor is centrally positioned at the bottom of the module, precisely 56.5 mm below the cartridge, to capture a focused image of the bioluminescence signal within the assay chamber. To automate the assay process, a servo motor is mounted on the lid, with a rotation valve lever connected to it. The Raspberry Pi sends signals to the Arduino Nano, which activates the servo motor. The lever attached to the motor then pushes the syringe in the chamber containing the buffer solution. Additionally, the Raspberry Pi controls the CMOS sensor to capture images with the desired parameters for further analysis.

[0208] A multistep approach was implemented to optimize the performance of the device and assay. Initially, the optimization focused on the allowed air volume in multiple components of the device in order to deliver the correct volume of various reagents reliably. Proper device function is dependent on the correct distribution of reagents with air pressure control. Based on this analysis, the optimal air volumes were determined to be 0.3 ml for sample transfer, 1 .2 ml for wash buffer removal, and 0.2 ml for working solution injection. Too small of a volume for the washing buffer ejection (<1 .2 ml) diminished the final assay signal due to interference from the remaining wash buffer. Volumes as low as 10 pL of residual washing buffer (1 xTBST) can result in a weaker bioluminescence signal. Therefore, it is crucial to thoroughly remove the washing buffer after sample washing to ensure accurate and reliable results.

[0209] To further reduce the chance of signal interference, a blocking agent, VTM, was applied to the cartridge chamber and channels. cAb-MgBeads and dAb- GAL were incubated in the assay chamber under assay conditions without viral biotargets. After washing, working solution was added to assess the occurrence ofBWH2024-099 bioluminescence. Cartridges treated with VTM showed no bioluminescent signals, whereas untreated cartridges exhibited detectable bioluminescent signals demonstrating the value of VTM blocking to prevent false positives.

[0210] The functionality of the automated system was initially assessed using three distinct food-colored dyes: red to represent the sample with cAb-MgBeads and dAb-GAL, blue for the washing buffer (400 pL of 1 x Tris-buffered saline with 0.1% Tween™ 20 detergent, TBST), and green for the working solution. The process begins by mixing the collected sample with assay materials (red) and transferring the mixture to the assay chamber within the microfluidic cartridge. By opening the smartphone application and pressing the start button, the sequence is initiated, starting a 20-minute incubation period within the assay chamber. After incubation, a rotating valve automatically pivots clockwise to align with the chamber containing the blue dye (washing buffer). The valve then propels a syringe loaded with washing buffer to wash the captured mixture, expelling excess washing solution and air into a drain chamber. Following the washing step, the rotating valve shifts counterclockwise to access the chamber housing the green dye (working solution). It injects a syringe filled with the working solution into the chamber, triggering bioluminescence, which is captured by the CMOS sensor for result analysis. The captured image is analysed within the smartphone application to determine the sample's positive or negative status. The entire sample-to-answer assay process is completed in approximately 23 minutes, including the virus capture incubation time.

[0211] The automated assay protocol for the enzyme cascade-based bioluminescence system is as follows. Step 1 . The test sample is introduced into an Eppendorf tube containing cAb-MgBeads (0.4 pg of cAb in PBS) and dAb-GAL (0.05 pg of dAb in 20 mM Tris-HCI) and is swirled for 30 seconds. The mixture is transferred to the sample injection syringe. Step 2. A disposable pipette is used to transfer the mixture of the sample and assay materials into a disposable microfluidic cartridge. An injection bar is used to push the solution into the microfluidic cartridge. Step 3. The disposable cartridge is inserted into the reusable standalone detection module. Step 4. The “Start” button was pressed in the smartphone application to initiate the automated sample handling.

[0212] The performance of the fully automated assay was first evaluated using samples containing recombinant HCV core antigen. Evident and vibrant bioluminescent signals were observed in the presence of the target protein,BWH2024-099 accompanied by a positive indication on the smartphone application as depicted in FIG. 22, element F. Conversely, in the absence of the target virus, no bioluminescent signal was detected, and a negative result was displayed on the smartphone application (see FIG. 22, element G)).

[0213] The microfluidic cartridge was designed for single use, while the detection module can be employed repeatedly for multiple tests. The material costs for manufacturing the microfluidic cartridge amount to approximately $2.3, while the cost for the detection module is approximately $86.8. These findings validated the successful integration of all facets of the reported automated enzyme cascade-based bioluminescence technology, encompassing sample handling, signal amplification, and readout, within a portable and cost-efficient cartridge.

[0214] To assess the frequency of user errors during sample processing using the fully automated system, experiments were conducted involving a diverse group of individuals. These tests utilized samples containing the recombinant HCV core antigen as the test material. The participant pool comprised a total of 10 individuals: five with specialized training and expertise related to the developed the enzyme cascade-based system, and five untrained individuals with diverse backgrounds, including non-PhD graduates and undergraduate students (see FIG. 22, element H). The untrained participants were provided with a user instruction sheet as their only reference to conduct the testing procedure. The t-test analysis revealed no significant difference between the results obtained by the trained and untrained participants (p = 0.56 for positive, p = 0.35 for negative), underscoring the user- friendly nature of the automated enzyme cascade-based system. This outcome emphasizes the ease of operation and the robustness of the automated assay, making it accessible and effective for a broad spectrum of users.

[0215] 2.2.3 Assay validation with HCV-spiked plasma samples

[0216] Initially, the limit-of-detection (LOD) of the automated enzyme cascadebased bioluminescence system was assessed by conducting experiments with serially diluted AI / AN HCV samples at virus concentrations of 1 .5x1 o7, 1 xl o6, 1 xl o5, 1 x104, 5x103, 1 x103, and 5x102copies / ml (see FIG. 23, element A). The r-squared value was estimated at 0.9329, indicating a robust linear correlation between the bioluminescence intensity from the enzyme cascade-based system and the viral load measured by PCR.BWH2024-099

[0217] To determine the experimental LOD, Receiver Operating Characteristic (ROC) curve analysis was conducted using 92 serially diluted samples spiked with AI / AN HCV, covering virus concentrations ranging from 0 to 1 .5x107copies / ml (see FIG. 23, element B). The ROC analysis revealed that a threshold HCV viral concertation of 768 copies / ml provided an optimal sensitivity of 97% (Confidence Interval (Cl), 88.43% to 100.00%) and specificity of 95% (Cl, 83.30% to 98.21 %) for the enzyme cascade-based system in identifying viral infection (n = 92). The overall accuracy was 96% (Cl, 89.24% to 98.80%), with an area under the curve (AUG) of 0.995 and a binomial exact Cl ranging from 0.9871 to 1 .0000. The threshold value represents the experimental LOD of the enzyme cascade-based bioluminescence system for the detection of HCV viral pathogen.

[0218] The specificity of the assay was evaluated using AI / AN HCV-spiked plasma samples and non-target samples, including HIV and HBV, each at virus concentrations of approximately 1 x106copies / ml (see FIG. 23, element C). The bioluminescence of the non-target viral samples produced an intensity equal to or less than 7.5x1 o4Relative Light Units (RLU), comparable to the blank without any target virus present. In contrast, the HCV-spiked sample with the viral load of 1 x106copies / ml exhibited bioluminescence intensity exceeding 7 million RLU. These findings demonstrate the specificity of the enzyme cascade-based system towards the target HCV virus.

[0219] 2.2.4 Head-to-head comparison of the automated POC HCV Ag assay with conventional bioluminescence system and ELISA

[0220] To emphasize the advantages of the reported automated enzyme cascade-based bioluminescence system over other assays, a performance evaluation was conducted comparing it with a conventional bioluminescence system and ELISA. The performance of each approach was evaluated by calculating the clinical sensitivities and specificities in qualitatively classifying 50 AI / AN HCV-spiked plasma samples (23 positive and 27 negative samples) with a clinically relevant viral load threshold of 768 copies / ml (see FIG. 24). The clinical sensitivities and specificities were confirmed as follows: 61% and 100% for the conventional bioluminescence system and 74% and 100% for ELISA, respectively. The sensitivity and specificity of the POC HCV Ag assay were both 100%. Accuracy was also evaluated, yielding 100% for the POC HCV Ag assay, 78% for the conventional bioluminescence system and 84% for the ELISA. These results demonstrated thatBWH2024-099 reporting system outperformed conventional bioluminescence system and ELISA when using a low viral load threshold (see FIG. 24).

[0221] 2.2.5 Assay Validation with AI / AN HCV patient sample

[0222] The developed automated POC HCV Ag assay was evaluated using 71 AI / AN HCV patient plasma samples including 37 positive (viral load > 768 copies / ml) and 34 negative (viral load < 768 copies / ml) patient samples. The vertical scatterplots display the correlation between the automated enzyme cascade reaction results and PCR qualitative outcomes for patient samples, highlight specific discrepancies, which included 1 false positive and 2 false negatives (see FIG. 23, elements D and E. All positive AI / AN HCV patient samples were successfully detected. The specificity, sensitivity, and accuracy of the POC HCV Ag assay in qualitatively classifying viral-infected samples with clinically relevant viral load threshold of 768 copies / ml were 94%, 97%, and 96%, respectively.

[0223] The Cohen’s Kappa coefficient was employed to evaluate the agreement and reliability between the two diagnostic results, PCR and the enzyme cascadebased bioluminescence system. The coefficient is a probability value, and the closer it is to 1 , the higher the agreement between two different datasets. The coefficient turned out to be K = 0.875, which indicates concordance between two system is almost perfect

[0224] Experiment 3

[0225] Effective monitoring of HIV treatment remains constrained by limited access to affordable, sensitive, and user-friendly viral load diagnostics, particularly in resource-limited settings. To address this critical gap, a fully automated, point-of- care device was developed that integrates microfluidic sample processing with ultrasensitive bioluminescence-based viral load detection (see FIG. 25). Upon simple sample loading, the system automates all steps, capturing multiple HIV subtypes with antibody-coated magnetic beads without foldchange, amplifying bioluminescence via enzyme cascades, and quantifying viral load using a low-cost optical sensor. Analytical validation using 87 HIV-spiked plasma samples confirmed a 95 copies per mL detection limit and reliable quantification of viral loads. Clinical testing with 53 patient samples demonstrated 95% sensitivity and 100% specificity, exceeding the performance of currently available POC assays. The assay can be completed within 65 minutes at a cost of less than $3 per test, further supporting itsBWH2024-099 feasibility for widespread implementation. The platform's ease of use and robust performance across different user experience levels support its potential for expanding access to ART monitoring and improving HIV management worldwide.

[0226] 3.1. Methods

[0227] The workflow consists of the following three steps. 1 ) Sample collection. Plasma samples containing HIV are mixed with assay reagents and loaded into a disposable microfluidic chip, which is then inserted into the automated device equipped with an integrated display. 2) Automated assay execution. After the user initiates the process by pressing the start button, the system autonomously performs all diagnostic procedures, including fluid handling, biomolecular reactions, and bioluminescence signal acquisition without requiring further user intervention. 3) Result confirmation. The bioluminescence signal is automatically analyzed, and the diagnostic result is displayed on the screen as either positive or negative

[0228] 3.1 .1 Preparation of the bioluminescence buffer

[0229] Following the protocol described in the reported article, a bioluminescence buffer was prepared as a working solution. Briefly, ATP (1 .0 mM), BSA (7.6 mM), TCEP (1 mM), EDTA (0.1 mM), MgSO4-7H2O (2.0 mM), and Coenzyme A (8.0 pM) were mixed in 9.8 mL of 1 x Tris-HCl.

[0230] 3.1 .2 Preparation of working solution

[0231] LLIGAL (5.0 pg / pL, 2.5 pL) and Flue (1 .0 pg / pL, 50.0 pL) were mixed in 447.5 pL of bioluminescence buffer. After gentle mixing, 100 pL of the working solution was used for each assay.

[0232] 3.1 .3 Information of antibody pairs and recombinant antigen

[0233] Paired antibodies derived from mouse against HIV p24 antigen (11695- MM08T for capture and 11695-MM15 for detection), as well as recombinant HIV p24 (1 1695-V08E) and gp120 (11233-V08H) antigens, were purchased from Sino Biological. Recombinant HBV surface antigen (LA549) and HCV core antigen (V8903) were purchased from Medix Biochemica.

[0234] 3.1 .4 Preparation of dAb-biotin

[0235] SiteClick™ Antibody Azido Modification Kit (S20026) and Click-iT™ Biotin sDIBO Alkyne for Antibody Labeling(C20030) were used to conjugate the dAb with biotin according to the manufacturer’s guidelines. The main purpose of the SiteClick™ conjugation process is altering the antibody’s carbohydrate domain, conjugating azide to the antibody, and then attaching it with the sDIBO-modifiedBWH2024-099 biotin using copper-free click chemistry. This method results in the formation of a covalent bond between the biotin and the antibody, allowing to obtain the dAb conjugated with biotin.

[0236] 3.1 .5 Investigation of dAb-biotin using TMB-ELISA

[0237] TMB-ELISA was used to confirm the function of dAb-biotin. Initially, the surface of a 96-well plate was coated with 100 ng of HIV p24 antigen and incubated at 4°C overnight. Subsequently, the plate was washed four times with 200 pL of1 xTBST and then blocked with 200 pL of blocking solution (5% BSA in Milli Q) for 60 minutes at room temperature. After removing the blocking solution, the wells were incubated with or without 50 ng of dAb-biotin in 2.5% BSA in 1 xTBST for 1 hour at room temperature. Following four washes, the wells were incubated with 10 ng of streptavidin-HRP or 10 ng of rabbit polyclonal anti-mouse IgG-HRP in 2.5% BSA in 1 xTBST for 1 hour at room temperature. After five washes, 100 pL of TMB substrate was added, and the color change was observed for 20 minutes. Upon addition of 100 pL of Stop Solution (0.16 M sulfuric acid), the reaction was stopped, and the absorbance of each well was analyzed at 450 nm wavelength.

[0238] 3.1 .6 Preparation of dAB-GAL

[0239] The dAb-biotin produced following the SiteClick™ Antibody Azido Modification Kit and Click-iT™ Biotin sDIBO Alkyne for Antibody Labeling, and ST- GAL (Thermo Fisher Scientific, S931) were mixed at a 1 :4 mass ratio and diluted using Tris (pH 7.0, 0.05 M) to achieve a final antibody concentration of 25 ng / pL. This mixture was then incubated at 4°C for 2 days with gentle rotation. Following incubation, the conjugation of dAb-biotin with ST-GAL was confirmed by Western blotting using rabbit polyclonal anti-mouse IgG-HRP and human derived anti-p- galactosidase-HRP.

[0240] 3.1 .7 Investigation of dAb-GAL using TMB-ELISA

[0241] The surface of a 96-well plate was coated with 100 ng of HIV p24 antigen and incubated at 4°C for overnight. Subsequently, the plate was washed four times with 200 pL of 1 xTBST and then mixed with 200 pL of blocking solution (5% BSA in Milli Q) for 60 minutes at room temperature. After removing the blocking solution, the wells were incubated with 50 ng of dAb-GAL in 2.5% BSA in 1 xTBST or without it for 1 hour at room temperature. After four washes, the wells were incubated with or without 50 ng of anti-beta-galactosidase antibody derived from human in 2.5% BSA in 1 xTBST for 1 hour at room temperature. Following fourBWH2024-099 washes, the wells were incubated with 100 ng of rabbit polyclonal anti-mouse IgG- HRP or 10 ng of anti-mouse IgG-HRP in 2.5% BSA in 1 xTBST for 1 hour at room temperature. After five washes, 100 pL of TMB substrate was added, and the color change was observed for 20 minutes. After adding 100 pL of Stop Solution (0.16 M sulfuric acid), the absorbance of each well was analyzed using a microplate reader at 450 nm wavelength.

[0242] 3.1 .8 Investigation of cAB-Mg Beads conjugation

[0243] To evaluate the function of cAb conjugated to MgBeads, 2 pL of cAb- MgBeads (corresponding to 0.4 pg of cAb), 2 pL of unconjugated MgBeads, and 0.4 pg of free cAb without MgBeads, or PBS (1 pL) with rabbit polyclonal anti-mouse IgG-HRP (0.01 pg in 100 pL of PBS) were mixed. After 20 min incubation at room temperature, the mixtures were washed with 200 pL of 1 x TBST, and the supernatant was removed. This washing step was repeated three more times. The pellet was re-suspended with 100 pL of TMB, and absorbance was measured at 450 nm using a microplate reader after adding 100 pL of stop solution at the 20 min time point.

[0244] 3.1 .9 Preparation of cAB-MgBeads conjugation

[0245] The cAb-MgBeads were prepared using the Dynabeads antibody coupling kit (Invitrogen, 14211 D), which utilizes an epoxy-amine reaction mechanism for antibody conjugation. According to the manufacturer’s instructions, the reaction involved 5 mg of MgBeads and 100 pg of cAb. The final volume of the cAb-MgBeads solution was 500 pL, which was divided into 5 aliquots of 100 pL each and stored at 4°C. For surface passivation of the cAb-MgBeads, 20 pL of the solution was incubated with 1 .5 ml of VTM at 37°C for 30 minutes with gentle rotation. After incubation, the mixtures were fixed using a magnetic rack pulldown, and the supernatant was removed. The MgBeads were washed twice with 500 pL of1 xTBST. Following washing, the VTM-blocked cAb-MgBeads were stored in 20 pL of PBS. The final concentration of cAb conjugated to the beads was 0.2 pg / pL.

[0246] 3.1 .10 Evaluation of the functionality of the individual components of the enzyme cascade-based bioluminescence system

[0247] Mixed 2 pL of cAb-MgBeads (0.2 pg of cAb in PBS), 1 .5 pL of dAb-GAL (0.025 pg of dAb in pH 7.0, 0.05 M T ris), and plasma with or without 100 ng of HIV Ag. After a 20 minutes incubation at room temperature, the supernatant was removed by using a magnet to pull down the mixture. The resulting pellet in 200 pLBWH2024-099 of 1 xTBST was resuspended twice. The washing step was repeated once more. The pellet was resuspended in 100 pL of working solution and the bioluminescence signal was detected using a CMOS sensor.

[0248] 3.1.11 Development of sample-handling device

[0249] The bioluminescent detection system was developed as a fully automated POC platform comprising mechanical, optical, and computational subsystems that execute the assay workflow end-to-end with minimal user input. The core computational unit was a Raspberry Pi 4 Model B (8 GB RAM, Raspberry Pi Foundation, UK) responsible for regulating all device functions, including process timing, fluid actuation coordination, image acquisition, and bioluminescent signal analysis. Mechanical actuation was performed using an Arduino Nano V3.0 (Arduino LLC, Ivrea, Italy), which interfaced with a high-torque MG995 servo motor (TowerPro, China) to control the introduction of reagents into the microfluidic reaction chamber. The imaging subsystem consisted of a 16-megapixel camera module (Sony IMX519, Arducam, China) connected to the Raspberry Pi via the CSI interface for high-resolution image capture. All components were housed within a custom-fabricated, light-shielded enclosure. The control software was developed in Python (version 3.11 .2), with the graphical user interface implemented using Tkinter (Tk version 8.6). Real-time image handling was supported by the Python Imaging Library (PIL version 11.2.1 ), while numerical computations and array operations were performed using NumPy (version 2.2.4). Image acquisition and analysis were conducted using OpenCV (version 4.11 .0). The user initiated the assay through a touchscreen display (3.5-inch Resistive Touch Display (B) IPS, Waveshare, China), which guided the operator through sample introduction and assay initiation.

[0250] Once the sample and reagents were introduced into the microfluidic chip and the chip was mounted within the device, the user activated the assay by pressing a start button on the interface. This initiated an incubation period during which the Raspberry Pi maintained an internal timer and displayed a countdown for user feedback. At the conclusion of the incubation period, the Raspberry Pi communicated with the Arduino Nano to activate the MG995 servo motor, which delivered washing and reaction buffers to the assay chamber through syringe- actuated fluidics. The mechanical actuation was timed to rotate at 800 milliseconds per degree ensured stable flow without bubble formation or reagent overflow. Following reagent delivery, the Raspberry Pi triggered the camera module to captureBWH2024-099 an image of the bioluminescent reaction within the microfluidic detection zone. Image acquisition was conducted under light-isolated conditions to minimize background noise and captured images were analyzed immediately.

[0251] The image was first cropped to isolate the microfluidic reaction region, excluding all background and structural features. This cropped region was converted from the RGB to HSV color space to enhance sensitivity to the yellow bioluminescent signal emitted by the luciferase-luciferin reaction. To extract the relevant signal, a binary color mask was applied to the HSV image using optimized thresholds for yellow emission. This mask was refined using morphological closing followed by opening operations to reduce pixel-level noise. The refined mask was used to segment the region of interest within the Value channel of the HSV image, from which the total brightness was computed by summing the pixel intensity values. This scalar value reflected the magnitude of bioluminescent activity and was used as the analytical readout. A predetermined threshold intensity units was established during device calibration to differentiate between positive and negative samples. Samples with brightness values above this threshold were classified as positive, while those below were deemed negative. The final result was displayed to the user on the device’s screen alongside the acquired image, allowing for immediate interpretation without external hardware or software.

[0252] 3.1 .12 Biosafety and human participants statement

[0253] The research work reported was approved and performed in adherence to guidelines and procedures approved by the Institutional Biosafety Committee of Mass General Brigham (parent organization of Massachusetts General Hospital and Brigham and Women’s Hospital) and under appropriate institutional review boards (IRB nos. 2014P002784, 2015P000182, 2015P000454, 2019P002209, 2019P001996, and 2019P001489). All the experiments with HIV were performed in BSL2+. Fresh plasma samples from participants with HIV were obtained from the HIV Eradication and Latency (HEAL) Cohort, a longitudinal biorepository study of participants with HIV. Frozen HIV-infected patient serum / plasma samples were provided by Advancing Clinical Therapeutics Globally for HIV / AIDS (ACTG). HIV plasma samples were obtained from participants enrolled in the ACTG / IMPAACT A5257 clinical protocol. HIV stock-cultured samples were received from Dr. Li and Dr. Tsibris. The spiked samples used for system testing throughout the study were prepared by serial dilution of stock virus samples with known viral loads, asBWH2024-099 quantified by standard PCR-based methods. Control samples consisted of virus-free plasma processed using the identical assay protocol, with the only difference being the omission of viral stock during preparation

[0254] 3.1 .13 Specificity test

[0255] The specificity of the automated assay was evaluated using human plasma spiked with recombinant antigens, including HIV p24 as the target and HIV gp120, HBV surface antigen, and HCV core antigen as non-targets. The assay was performed as described in the main text, and bioluminescence intensity was analyzed via on-device decision-making.

[0256] 3.1 .14 Evaluation of clinical LOD

[0257] Clinical samples of HIV were serially diluted in plasma to concentrations ranging from 0 to 4.5x105copies / ml. Prior to testing, a lysis step was performed for the HIV clinical samples by incubating at room temperature with 0.1% Tween™20 for 1 minute. The samples were then mixed with assay materials and processed according to the general protocol for the automated enzyme cascade-based bioluminescence system described in the main text. The resulting bioluminescence intensities under each condition were used as references to determine the limit of detection of the system. n=5 technical replicates were conducted, and the LOD of the automated enzyme cascade-based bioluminescence system was calculated as the mean of the blank plus three times the standard deviation obtained for the blank.

[0258] 3.1 .15 Statistical analysis

[0259] Statistical analyses were performed using Origin software version 8.5 (Origin Lab Corporation. Northampton, MA, USA). The mean and SDs were calculated for each data point from at least a total of two to three independent experiments unless indicated in the text. The level of significance was set at P > 0.05. ROC and scatter plot analyses were used to define performance of the enzyme cascade-based bioluminescence system to the standard analytical technique.

[0260] 3.2 Results

[0261] 3.2.1 Synthesis and characterization of HIV p24 detection assay materials

[0262] To capture and detect HIV p24, a pair of monoclonal antibodies with high specificity for the p24 antigen were identified. For signal amplification, the enzyme p- galactosidase (GAL) was selected. GAL catalyzes the cleavage of the protecting group from a synthetic substrate, d-luciferin-O-p-galactoside (LUGAL), therebyBWH2024-099 generating luciferin. The liberated luciferin was subsequently oxidized by firefly luciferase (Flue) present in the working solution, producing a quantifiable bioluminescent signal.

[0263] To incorporate this bioluminescence system into the p24 antigen detection assay, p-galactosidase (GAL) was conjugated to the detection antibody (dAb, mouse monoclonal, IgG). To preserve the biological activity of both the antibody and the enzyme, site-specific conjugation was performed by targeting the Fc region of the antibody using a site-click chemistry approach, which introduced biotin moieties distal to both the antigen-binding sites and the enzyme’s active site. The successful biotinylation of dAb was confirmed by enzyme-linked immunosorbent assay (ELISA) using streptavidin-HRP and rabbit polyclonal anti-mouse IgG-HRP. Streptavidin-conjugated GAL was then incubated with the biotinylated dAb to generate the final dAb-GAL conjugate. The formation of the dAb-GAL complex was validated by observing colorimetric signals in ELISA when either rabbit polyclonal anti-mouse IgG-HRP (to detect the antibody component) or human-derived anti- GAL-HRP (to detect the enzyme component) was used, confirming the presence and functionality of both proteins in the conjugate. Furthermore, Western blot analysis revealed a band at a higher molecular weight in the dAb-GAL sample compared to the dAb alone, consistent with the expected size of the conjugated product. This result further confirms the successful synthesis of the dAb-GAL conjugate.

[0264] The capture antibody (cAb, mouse monoclonal, lgG1 ) was immobilized on magnetic beads using a commercially available coupling kit that facilitates oriented and stable antibody attachment. To prevent nonspecific interactions with the bead surface, the magnetic beads were blocked with viral transport medium (VTM) prior to use. The successful conjugation of cAb to the beads was confirmed using a magnetic pull-down assay. A colorimetric signal was observed only from beads conjugated with cAb following treatment with rabbit polyclonal anti-mouse IgG-HRP, indicating successful antibody immobilization.To assess the functionality of the assembled assay components in capturing and detecting HIV p24, the system was evaluated using recombinant p24 antigen. Robust bioluminescent signals were generated only under fully assembled conditions. In contrast, omission of any individual component, including dAb-GAL, cAb conjugated magnetic beads, GAL, LUGAL, or Flue, abolished detectable signal above background, apart from a faint background, attributable to low-level non-BWH2024-099 specific adsorption of dAb-GAL and minimized by washing. These results highlight the necessity of each constituent element and validate the assay as a fully integrated and highly specific platform for the sensitive detection of HIV p24.

[0265] 3.2.2 Development of an automated testing device

[0266] The diagnostic assay was conducted using a microfluidic chip, following previously established protocols. Briefly, the disposable microfluidic cartridge was fabricated by laser-cutting three layers of 1 .5 mm thick poly(methyl methacrylate) (PMMA) substrates, which were subsequently bonded using double-sided adhesive (DSA). The assembly also incorporated a 3D-printed polylactic acid (PLA) chip cover, designed to house a rotation valve, an integrated magnet for the reaction chamber, and syringes for fluid delivery (see FIG. 26, element A). The chip includes three fluidic inlets for sample input, washing buffer (400 pL of 1 xTBST), and working solution (150 pL), which are directed into the reaction chamber through microfluidic channels. Solution entry into the reaction chamber at designated assay steps is regulated by a connecting channel within the rotation valve, enabling sequential reagent delivery throughout the assay process. To ensure stable and precise bioluminescent signal detection, a detection module was developed to operate within a fully enclosed, light-shielded environment, minimizing external light interference. The detection module integrates seamlessly with the microfluidic chip, enabling accurate processing of samples and reagents. A compact 3D-printed frame (120 x 122 x 163 mm) was designed to house the core electronic components, including a Raspberry Pi 4, Arduino Nano, servo motor, battery pack, CMOS image sensor, and an LCD display (85 x 55 x 5 mm), ensuring the device's autonomous functionality (see FIG. 26, element B). The integrated display allows the system to operate independently in resource-limited environments, such as rural clinics, disaster relief zones, or low-infrastructure settings, where dependence on external devices for result interpretation may be impractical due to connectivity or equipment constraints. The material costs for manufacturing the microfluidic cartridge amount to approximately $2.44, while the cost for the detection module is approximately $84.43, highlighting the potential for affordable deployment in resource-limited settings.

[0267] The assay workflow is divided into user-performed steps (see FIG. 26, element C) and automated processes (see FIG. 26, elements D-G). Initially, the user collects the sample, mixes it with assay reagents, and introduces the mixture into theBWH2024-099 assay chamber via pin actuation, ensuring accurate transfer. The automated sequence is then initiated by pressing the start button on the integrated display (see FIG. 26, element C). After an incubation period, the Raspberry Pi signals the servo motor to actuate a lever mechanism, introducing the washing buffer into the assay chamber (see FIG. 26, elements D and E). The buffer effectively washes away unbound reagents and is subsequently drained from the chamber. The servo motor then rotates in the opposite direction to introduce the working solution, initiating the bioluminescent reaction. Finally, the Raspberry Pi triggers the CMOS image sensor to capture an image of the emitted bioluminescence (see FIG. 26, element F). Signal intensity is analyzed on-device, and the final diagnostic result is displayed as either positive or negative based on a pre-defined signal threshold (see FIG. 26, element G).

[0268] To ensure stable and consistent reagent delivery, the fluid injection speed was precisely controlled. The injection speed was regulated by the rotation speed of the servo motor, which was defined as the time required to rotate the lever by one degree. An angular step duration in the range of 800-1000 ms per degree was found to provide the most stable performance, ensuring consistent reagent injection without bubble formation or flow irregularities within the assay chamber (see FIG. 26, element H). Improper actuation speeds, whether faster than 800 ms per degree or slower than 1000 ms per degree, led to incomplete washing due to abrupt or inefficient fluid injection into the assay chamber. At actuation speeds faster than 800 ms per degree, rapid lever movement caused a sudden influx of reagents, leading to air entrapment, bubble formation, and disrupted fluid flow. At actuation speeds slower than 1000 ms per degree, insufficient initial pressure delayed syringe actuation until pressure buildup triggered a sudden release, producing a similar burst effect. In both cases, imaging of the microfluidic chips revealed uneven dye distribution and the presence of residual solution localized within specific regions of the assay chamber (see FIG. 26, element I). Such incomplete washing may compromise assay performance by allowing cross-contamination or inducing nonspecific signals, underscoring the need for precise control of actuation speed to ensure reliable operation. These findings highlight the importance of maintaining an appropriate actuation speed to ensure consistent reagent delivery and reliable assay performance.BWH2024-099

[0269] To monitor device operation and verify fluidic control, food-grade dyes were incorporated into each solution: the sample mixed with assay reagents was labeled with red dye, the washing buffer with blue dye, and the working solution with green dye. These visual indicators enabled real-time observation of fluid movement and confirmed the correct execution of each microfluidic step. Following user- initiated injection via pin actuation, the red sample mixture successfully reached the assay chamber. After a 1 -hour incubation period, the servo motor automatically actuated the lever mechanism to dispense the blue washing solution. Complete flushing of the chamber was verified by the absence of residual red or blue dye, indicating effective waste removal. Subsequently, the servo motor reversed direction to deliver the green-labeled working solution into the assay chamber (see FIG. 26, element J). Bioluminescence signals were then captured in the light-shielded chamber, where strong signal intensities corresponding to positive results were observed (see FIG. 26, element K). The colocalization of the green dye and the bioluminescent signal within the reaction chamber further confirmed that the automated assay sequence was executed properly. Additionally, this observation demonstrated that the assay components retained their functionality throughout the automated process, successfully capturing the virus and generating a bioluminescent response. Collectively, these results validate that the device effectively automates the critical stages of the assay— including sample loading, incubation, washing, and signal development — with preserved assay integrity. The robust bioluminescent signals and clear positive outcomes confirm the successful integration of the enzyme cascade-based bioluminescence system with the fully automated platform.

[0270] 3.2.3 Analytical validation and detection reliability using HIV-spiked plasma samples

[0271] To evaluate the analytical performance of the automated device, the correlation between bioluminescent signal intensity and HIV RNA concentrations was assessed, showing high linearity ( ?2= 0.96) across the tested range of 10 to 10,000 copies / mL (n = 33) (see FIG. 27, element A).

[0272] To further assess the assay’s diagnostic performance across clinically relevant viral load levels, HIV-spiked plasma samples spanning a wide dynamic range (0 to 450,000 copies / mL; n = 87) were tested. A receiver operating characteristic (ROC) curve was then generated using these data to determine anBWH2024-099 optimal diagnostic threshold (see FIG. 27, element B). The optimal threshold for HIV positivity was identified by maximizing Youden’s Index, resulting in a cutoff value of 95 copies / mL. This threshold was selected to minimize false negatives while avoiding false positives. Based on this threshold, the assay demonstrated a sensitivity of 88% and a specificity of 100% (95% Cl: 0.951 -1.011 ), demonstrating the high accuracy of the system for HIV detection.

[0273] To evaluate the reliability of the assay near the LOD (95 copies / mL), 20 plasma samples spiked with low concentrations of HIV RNA were tested. This assessment aimed to determine the assay’s consistency and accuracy when detecting viral loads close to the LOD. The LOD is defined by the U.S. Food and Drug Administration (FDA) as the lowest analyte concentration distinguishable from background noise, and by the Clinical and Laboratory Standards Institute (CLSI) as the concentration detectable in at least 95% of replicates under defined conditions. By conducting this evaluation, the robustness of the system in detecting HIV RNA was confirmed at clinically relevant low concentrations, ensuring reliable performance even near the detection threshold. The system correctly identified all 20 HIV-spiked plasma samples ranging from 100 to 500 copies / mL as positive, achieving a detection rate of 100% (see FIG. 27, element C). These findings demonstrate that the assay reliably detects HIV RNA even at low concentrations near the detection limit, ensuring high sensitivity and consistency.

[0274] The specificity of the assay was evaluated using human plasma spiked with recombinant antigens, including the target HIV p24 and non-targets such as HIV gp120, HBV surface antigen (HBsAg), and HCV core antigen (HCcAg) (see FIG. 27, element D). The HIV p24-spiked sample generated a strong bioluminescence signal exceeding 6.0x1 o7relative light units (RLU), whereas non-target samples showed negligible signals comparable to the blank without target antigen. These results demonstrate the high specificity of the assay for HIV p24.

[0275] 3.2.4 Clinical validation using plasma samples from patients with HIV and HIV-negative controls

[0276] The assay was subsequently optimized for qualitative assessment using the predefined viral load threshold of 95 copies / mL. Plasma from 38 patients with HIV and 15 HIV-negative controls was analyzed and results were classified using the prespecified viral-load threshold. Samples with viral loads below 95 copies / mL were classified as negative, while those with viral loads equal to or greater than 95BWH2024-099 copies / mL were classified as positive. Based on this classification, the assay correctly identified 36 of 38 HIV-positive samples and all 15 HIV-negative controls, yielding an overall accuracy of 96%. The calculated sensitivity and specificity were 95% and 100%, respectively (see FIG. 28, elements A and B).

[0277] The classification performance of the assay was further evaluated using a confusion matrix. The positive predictive value (PPV) and negative predictive value (NPV) were 100% and 88%, respectively. As no false positives were observed, the positive likelihood ratio (LR+) approached infinity, while the negative likelihood ratio (LR-) was 0.053, supporting the reliability of the assay (see FIG. 28, element C). To complement these findings, a ROC curve was constructed using clinical HIV patient samples. The area under the curve (AUG) was 0.996 (95% Cl: 0.984-1 .007), confirming the high diagnostic accuracy of the assay across a range of thresholds (see FIG. 28, element D). These findings strongly suggest that the developed assay can detect HIV at clinically relevant levels with high precision and reliability. Moreover, the fully automated nature of the device eliminates the need for specialized training, enabling its use in POC settings. Given the high sensitivity, specificity, and reproducibility observed in both spiked and clinical samples, the assay offers a promising solution for rapid and accessible HIV screening, particularly in resource-limited settings.

[0278] 3.2.5 Assessment of user integration and device usability

[0279] To assess the ease of use and operational reliability of the automated device, a usability study was conducted using recombinant p24 antigen as the target analyte. The assay was performed by two user groups: five trained users with prior experience operating diagnostic devices, and five untrained users with no prior exposure to the system. The untrained users independently conducted the assay by following only the instructions outlined in the User Instruction Sheet, without any external assistance or prior demonstration. A box plot was used to compare the assay result distributions between trained and untrained users. The distributions were highly similar in both median values and spread. Consistent with this observation, there was no statistically significant difference between groups (p = 0.54), with both achieving highly consistent and reproducible outcomes. Additionally, the time required to complete the user-initiated steps at the beginning of the assay was comparable between groups. The trained users completed the steps in an average of 50 seconds (standard deviation: 9 seconds), while the untrained usersBWH2024-099 required 54 seconds (standard deviation: 8 seconds), with no statistically significant difference (p = 0.62).

[0280] The duration of the automated portion of the assay remained consistent across all users, further supporting the system’s robustness and efficiency.Collectively, these results demonstrate that the device enables users, regardless of prior experience, to achieve reliable and accurate results with minimal operational complexity. Based on these results, the fully automated diagnostic system, employing a self-contained disposable cartridge pre-loaded with reagents, serves as a reliable and accessible alternative to conventional laboratory-based viral load monitoring, especially for ART-treated patients. Existing FDA- and WHO-approved antigen-based diagnostics are limited by their reliance on laboratory infrastructure and suboptimal sensitivity. In contrast, the system delivers highly sensitive and fully automated detection in a decentralized setting. The system's high sensitivity, flexibility, and ease of use make it particularly valuable for point-of-care testing, home-based monitoring, and use in resource-limited settings, where access to centralized laboratory infrastructure is restricted.

[0281] From the above description, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims.

Claims

BWH2024-099The following is claimed:1 . A method comprising: mixing a working solution comprising a d-luciferin-6-O-p-d-galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer comprising ATP with contents of a reaction chamber for a time period to create mixed contents, when the contents of the reaction chamber comprise a sample including tagged target components that include at least beta galactosidase (GAL), then: the GAL on the tagged target components supplies a substantial amount of luciferin intermediates that catalyze the cleavage of a protecting group from the LUGAL, thereby generating D-luciferin, and the Flue catalyzes the oxidation of the D-luciferin in a presence of the ATP and O2, yielding oxyluciferin and bioluminescence, wherein repeated catalytic cycles increase a measurable signal of the bioluminescence; and when the contents of the reaction chamber do not comprise a sample including the tagged target components, then the working solution does not react; capturing an image of the mixed contents of the reaction chamber via an optical sensor; and determining if the tagged target components are present in the mixed contents of the reaction chamber based on an amount of bioluminescence detected in the image.

2. The method of claim 1 , further comprising: incubating a sample with a magnetic tag solution in the reaction chamber for an incubation time period, wherein the magnetic tag solution comprises magnetic molecules comprising magnetic beads conjugated with a capture antibody for the target component and tag molecules comprising detection antibodies for the target component conjugated with the GAL, and wherein when the sample comprises target components the magnetic beads conjugated with a capture antibody for the target component and the detection antibodies for the target component conjugated with the GAL attach to the target components to form the tagged target components.BWH2024-0993. The method of claim 2, further comprising: mixing the sample with the magnetic tag solution prior to the incubation time period; and injecting the sample with the magnetic tag solution into the reaction chamber.

4. The method of claim 2, further comprising: applying, via a magnet, a magnetic field to the reaction chamber for another time period; and washing the incubated sample in the reaction chamber with a washing solution during the other time period, wherein non-tagged components of the sample are washed away and the magnetic tag molecules remain in the reaction chamber.

5. The method of claim 1 , wherein the reaction among the GAL, the LLIGAL, and the Flue proceeds stepwise to generate a sustained measurable signal of the bioluminescence when the Flue is present in excess in a native, unconjugated form to increase signal intensity.

6. The method of claim 1 , wherein the determining further comprises: receiving the captured image from the optical image sensor; calculating an amount of bioluminescence in the captured image; determining if the amount of bioluminescence in the captured image is above or below a threshold for the tagged target component, wherein the tagged target component is present if the amount of bioluminescence is above the threshold.

7. The method of claim 6, further comprising: isolating a portion of the captured image comprising the reaction chamber; converting the portion of the captured image from RGB to HSV color space to increase sensitivity; applying a binary color mask to the portion of the captured image to increase yellow sensitivity, wherein the bioluminescence appears yellow in the captured image; computing a total brightness of the portion of the captured image; comparing the total brightness of the portion of the captured image with the threshold for the tagged target components; andBWH2024-099 classifying the total brightness of the portion of the captured image as positive or negative for the tagged target components.

8. The method of claim 7, further comprising: refining the binary color mask using morphological closing and opening operations to reduce pixel-level noise; segmenting a region of interest of the portion of the captured image with a value channel into pixels; and computing the total brightness of the portion of the captured image by summing pixel intensity values of the pixels.

9. The method of claim 1 , further comprising: outputting a positive diagnosis when the amount of bioluminescence indicates that the tagged target component is present; and outputting a negative diagnosis when amount of bioluminescence indicates that the tagged target component is not present.

10. A system comprising: an assay appliance for detecting the presence of target components in a sample, the assay appliance comprising: a housing configured to block external light from entering an interior of the assay appliance, a magnet configured to provide a magnetic force; an optical sensor configured to capture an image of a result of an assay, and a processor configured to run the assay and determine if the sample comprises the target components; and an assay chip configured to be removably placed within the housing of the assay appliance, the assay chip comprising: a washing solution storage chamber configured to hold a volume of a washing solution, a working solution storage chamber configured to hold a volume of a working solution, andBWH2024-099 a reaction chamber configured to receive a volume of a sample and a volume of magnetic tag solution, comprising a volume of magnetic molecules and a volume of reactant tags, and to hold contents of the assay during the steps of the assay that add and remove the washing solution and the working solution based on instructions from the processor, wherein at least a portion of the reaction chamber is covered by the magnet.11 . The system of claim 10, wherein the assay chip further comprises a microfluidic channel connected to the washing solution storage chamber; and another microfluidic channel connected to the working solution storage chamber; and the reaction chamber further comprises: a first inlet enabling injection of the volume of the sample combined with the volume of magnetic tag solution; a second inlet enabling injection of the at least the portion of the volume of the washing solution into the reaction chamber; and a third inlet enabling injection of the at least the portion of the volume of the working solution into the reaction chamber.

12. The system of claim 1 1 , wherein the assay appliance further comprises: a rotating valve controlled by the processor and comprising at least one connecting channel configured to rotate to connect the microfluidic channel connected to the washing solution storage chamber to the second inlet of the reaction chamber and then to connect the other microfluidic channel connected to the working solution storage chamber to the third inlet of the reaction chamber.

13. The system of claim 12, wherein the assay appliance further comprises: a plurality of syringes controlled by the processor and configured to actuate injection of the at least the portion of the washing solution to the reaction chamber and to actuate injection of the at least the portion of the working solution to the reaction chamber.BWH2024-09914. The system of claim 10, wherein the assay appliance further comprises: a controller comprising a non-transitory memory storing instructions and the processor or another processor configured to execute the instructions to: incubate the volume of the sample combined with the volume of magnetic tag solution in the reaction chamber for an incubation time period, wherein when the sample comprises the target component the magnetic molecules and the tag molecules combine with the target component to form a tagged target component, wherein incubation is a first step of the assay; maintain a magnetic field applied to the reaction chamber to magnetically retain the tagged target components within the reaction chamber during the assay; wash the contents of the reaction chamber after the incubation with the volume of the washing solution, wherein the tagged target components are held in the reaction chamber by the magnetic field and a remainder of the contents are washed out of the reaction chamber with the washing solution; inject the volume of the working solution into the reaction chamber during a third step of the assay, wherein when the tagged target components are present the tagged target components react with the working solution to bioluminesce; and capture an image of the contents of the reaction chamber after the third step.

15. The system of claim 14, further comprising another controller located external to the assay appliance and configured to communicate further instructions to the controller.

16. The system of claim 10, wherein the assay appliance further comprises a power source.

17. The system of claim 10, wherein the assay appliance further comprises a user interface and a display configured to facilitate communication between a user and the controller to start the determination.

18. The system of claim 10, further comprising the sample combined with the magnetic tag solution, wherein the magnetic tag solution comprises magnetic beads conjugated with a capture antibody for the target component and detection antibodies for the target component conjugated with beta-galactosidase (GAL).BWH2024-09919. The system of claim 10, further comprising the washing solution and the working solution, wherein the working solution comprises d-luciferin-6-O-p-d- galactopyranoside (LLIGAL), firefly luciferase (Flue), and a bioluminescence buffer.

20. The system of claim 10, wherein the optical image sensor is a CMOS, a CCD sensor, a contact image sensor, a photodiode array, a phototransistor array, or a mobile-grade camera molecule.21 . An assay chip comprising: a washing solution storage chamber configured to hold a volume of a washing solution, a working solution storage chamber configured to hold a volume of a working solution; and a reaction chamber configured to receive a volume of a sample and a volume of magnetic tag solution, comprising a volume of magnetic molecules and a volume of reactant tags, and to hold contents of the assay during the steps of the assay adding and removing the washing solution and the working solution based on instructions from the processor, wherein at least a portion of the reaction chamber is covered by the magnet, wherein the assay chip is configured to be placed inside an assay appliance configured to block external light from entering an interior of the assay appliance.

22. The assay chip of claim 21 , further comprising: a microfluidic channel connected to the washing solution storage chamber; and another microfluidic channel connected to the working solution storage chamber, and wherein the reaction chamber further comprises: a first inlet enabling injection of the volume of the sample combined with the volume of magnetic tag solution; a second inlet enabling injection of the at least the portion of the volume of the washing solution into the reaction chamber; and inBWH2024-099 a third inlet enabling injection of the at least the portion of the volume of the working solution into the reaction chamber.

23. The assay chip of claim 22, wherein the assay chip further comprises a rotating channel piece configured to connect with a rotating valve controlled by a processor of the assay appliance and comprising at least one connecting channel configured to rotate to connect the microfluidic channel connected to the washing solution storage chamber to the second inlet of the reaction chamber and then to connect the other microfluidic channel connected to the working solution storage chamber to the third inlet of the reaction chamber.

24. The assay chip of claim 21 , further comprising a magnet configured to maintain a magnetic force to the reaction chamber of the assay chip during a washing step of the assay.

25. The assay chip of claim 21 , wherein the assay chip is configured to be positioned within the assay appliance with at least the reaction chamber in a field of view of an optical sensor, wherein the optical sensor is configured to capture an image of a completed assay reaction.

27. The assay chip of claim 21 , further comprising the sample combined with the magnetic tag solution, wherein the magnetic tag solution comprises magnetic beads conjugated with a capture antibody for the target component and detection antibodies for the target component conjugated with beta-galactosidase (GAL).

28. The assay chip of claim 21 , further comprising the washing solution in the washing solution storage chamber and the working solution in the working solution storage chamber, wherein the working solution comprises d-luciferin-6-O-p-d- galactopyranoside (LUGAL), firefly luciferase (Flue), and a bioluminescence buffer.

Images