Rapid test system for viral and bacterial infections

Abstract

A method of detecting a viral or bacterial pathogen is disclosed. The method includes collecting a potentially pathogenic sample with a collector, binding a first portion of the potentially pathogenic sample to magnetic particles with a first coating on the magnetic particles, binding a second portion of the potentially pathogenic sample to fluorescently labeled particles with a second coating on the fluorescently labeled particles to produce aggregates comprising the potentially pathogenic sample, the magnetic particles, and the fluorescently labeled particles, magnetically separating the aggregates, detecting fluorescence of the separated aggregates, and estimating an amount of the pathogen based on the detected fluorescence.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 085,404, filed September 30, 2020, entitled “Rapid Testing System for Viral and Bacterial Infections,” the entire contents of which are incorporated herein by reference.

[0003] field

[0004] This disclosure generally relates to testing for viral and bacterial infections, particularly rapid tests using breath analyzer systems. It also relates to systems and methods for pathogen identification and quantification, especially those using fluorescent and magnetic labeling, and microelectromechanical systems (MEMS).

[0005] background

[0006] Recent global pandemics, including COVID-19, have highlighted the need for rapid testing and screening of individuals for infection. Preventing or mitigating the spread of disease requires the reliable identification and isolation of infected individuals before they can infect others. This is particularly challenging when infected individuals may be asymptomatic, as is the case with COVID-19.

[0007] Currently, there is a lack of rapid, accurate, and low-cost diagnostic tests for SARS-CoV-2 and other pathogens. Laboratories have deployed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays for virus detection. However, turnaround times for qRT-PCR used for screening and diagnosing patients can range from several hours to several days. These tests are also expensive and require skilled technicians to perform. A shortage of relevant skills can also lead to bottlenecks in the processing.

[0008] Most tests currently being developed by major healthcare companies focus on rapid diagnostic screening in a laboratory setting and are therefore unsuitable for point-of-care (POC) applications. These methods include molecular assays using real-time PCR, droplet digital PCR, and immunological assays. Many tests claiming to be POC still must be performed in a healthcare facility because samples are collected by swabbing the patient's nasopharynx, nasal cavity, middle turbinate, or oropharyngeal region. This is challenging for healthcare workers and uncomfortable for patients. As of this writing, only one such test has received an Emergency Use Authorization (EUA) from the FDA for home sample collection. Test kits (which include a nasal swab for self-collection and saline) must be returned to a clinical laboratory for analysis and are therefore not rapid. Swab samples are difficult to obtain and can reduce the sensitivity of the test, leading to false negative results. There is an urgent need for POC tests, especially for SARS-CoV-2, which can be performed on-site at the testing site, can be managed and handled without specialized equipment, and are low enough in cost to be used in economically disadvantaged areas or countries.

[0009] Faster tools for identifying and quantifying pathogens do exist, but are primarily limited to laboratory settings. For example, fluorescent molecules or particles can be linked to antibodies that can bind to pathogens. This labels the pathogen with a fluorescent marker so that it can be detected and quantified. Similarly, magnetic particles can be linked to cells via antibodies. The particles can then be separated and analyzed using magnetic fields and / or fluorescence imaging.

[0010] As discussed in more detail in U.S. Patent No. 6,623,894 to Fleischman et al. (which is incorporated herein by reference in its entirety), labeled particles can be manipulated and quantized using MEMS techniques that create high-precision microstructures on silicon and other materials. MEMS techniques enable relatively low-cost, high-capacity electromechanical systems integrating sensors, actuators, electronics, and other components at a microscale. Related techniques have already enabled the detection of malaria-infected red blood cells using MEMS microfluidic magnetic separators. See PAZimmerman, J.M.Thomson, H. Fujioka, W.E. Collins, and M. Zborowski, “Diagnosis of malaria by magnetic deposition microscopy,” Am. J. Trop. Me.d. Hyg., vol. 74, pp. 568-72, Apr 2006. As described above, this technique has successfully separated cells and particles exhibiting much weaker magnetism than those labeled with magnetic markers. Methods and apparatus for magnetically separating cellular flows into fractional flow streams are described by reference to U.S. Patent No. 5,968,820 to Zborowski et al., the entire contents of which are incorporated herein by reference. Through a combination of a flow chamber and one or more magnetic fields, heterogeneous cellular flows are separated into fractional flow streams based on the cellular magnetic dipole moment and analyzed. However, to date, these techniques and related technologies have not been integrated into convenient, cost-effective, and rapid screening tests for pathogens themselves.

[0011] For at least the reasons mentioned above, there is currently a need for rapid viral or pathogen screening tests. Advantageously, such a test would involve sampling a patient's breath, which can be collected relatively quickly and is minimally invasive. Ideally, the test would be sensitive to relatively small amounts of pathogens and provide relatively rapid results without requiring extensive laboratory analysis. It would utilize MEMS technology to integrate the diagnostics into a single, disposable package.

[0012] Overview

[0013] This article discloses a method for detecting viral or bacterial pathogens. The method includes collecting a potentially pathogenic sample via a collector, binding a first portion of the potentially pathogenic sample to magnetic particles via a first coating on magnetic particles, binding a second portion of the potentially pathogenic sample to fluorescently labeled particles via a second coating on fluorescently labeled particles, to generate an aggregate containing the potentially pathogenic sample, magnetic particles, and fluorescently labeled particles, magnetically separating the aggregates, detecting the fluorescence of the separated aggregates, and estimating the amount of pathogen based on the detected fluorescence.

[0014] Separation can be based on at least one of the following: the distance the aggregate moves in the microfluidic magnetic separator, the time the aggregate moves in the microfluidic magnetic separator, and the flow of the aggregate in the microfluidic magnetic separator.

[0015] Estimating the amount of pathogens based on detected fluorescence can include estimating the amount based on the spatial distribution of detected fluorescence in a microfluidic magnetic separator.

[0016] The spatial distribution of the detected fluorescence can be generated by separation.

[0017] Detecting the fluorescence of isolated aggregates may include irradiating the aggregates and detecting the amount of fluorescence of the fluorescently labeled particles excited by the irradiation.

[0018] Fluorescence can be the fluorescence of fluorescently labeled particles within an aggregate. This method may include estimating viral load in a patient based on an estimated amount of pathogen.

[0019] This document also discloses an apparatus for detecting viral or bacterial respiratory pathogens. The apparatus includes a respiratory capture section for capturing potentially pathogenic samples. The respiratory capture section includes a mouthpiece, an inlet tube, and a collection container connected to the mouthpiece via the inlet tube. The collection container includes magnetic particles coated with a first coating that binds to a first portion of the pathogen; and fluorescently labeled particles coated with a second coating that binds to a second portion of the pathogen. The apparatus also includes an outlet tube connecting the collection container to a microfluidic channel, the microfluidic channel forming part of a microfluidic magnetic separator.

[0020] At least one breathing capture portion may include a window configured to transmit fluorescence from the microfluidic channel to the outside of the breathing capture portion, and the window may be configured to allow light from the outside of the breathing capture portion to illuminate the microfluidic channel.

[0021] The device may include a detection system configured to detect fluorescence within a displacement range of fluorescently labeled particles in a microfluidic channel through a window, and to provide light to the microfluidic channel through the window. The detection system may include an LED detection system that illuminates the fluorescently labeled particles with LED light. The coronavirus may be SARS-CoV-2, and a first portion of SARS-CoV-2 bound to the first coating and a second portion of SARS-CoV-2 bound to the second coating may be the SARS-CoV-2 spike protein. The second coating may contain angiotensin-converting enzyme 2 (ACE2).

[0022] At least one surface of the inlet and outlet pipes may be hydrophobic. The device may include a filter positioned to remove debris from potentially pathogenic samples. The device may also include an exhaust filter to remove pathogens from vapors to be discharged from the device. A portion of the breath-capturing section is disposable. At least one of the disposable portions may be a mouthpiece detachable from the breath-capturing section, and the entire breath-capturing section may be disposable.

[0023] The device may include a base separate from the breathing capture portion, which includes an interface for physically housing at least a portion of the breathing capture portion, electronic equipment configured to obtain fluorescence data from the breathing capture portion, and a communication port for communication based on fluorescence data transmission.

[0024] At least one of the electronic devices may include a detection system for detecting fluorescence data, the electronic device including at least one of a camera and a photoelectric sensor, the at least one of the camera and photoelectric sensor being positioned to detect fluorescence within a particle displacement range of a fluorescent label within a microfluidic channel, the electronic device being configured to run software to analyze images of the fluorescence, a communication port including at least one of an Ethernet port, Bluetooth connectivity, WiFi connectivity, mobile phone connectivity, a barcode reader, or other methods for associating a patient and an exhaled breath sample, and an optical display on a base, the base also including a piston positioned in mechanical communication with a portion of the breath capture section, the portion of the breath capture section in mechanical communication with the piston including a plunger configured to move a potentially pathogenic sample within the breath capture section, the piston being configured to actuate the plunger to move the potentially pathogenic sample within the breath capture section, the base also including a motor configured to drive the piston, and the microfluidic magnetic separator having a high gradient magnetic separation (HGMS) configuration.

[0025] Microfluidic magnetic separators can be configured with open gradient magnetic separation (OGMS).

[0026] The advantages of the disclosed device and method include that they provide the only breath-based SARS-CoV-2 test to date, and the only such test combining the advantages of immunoassay and microfluidics. Its breath-based analysis is more economical, faster, more comfortable for consumers, and easier to perform than currently developed tests. Collected samples exhibit more consistent quality. Analysis requires virtually no laboratory testing and can be performed almost automatically at point of contact (POC). Furthermore, parts of the device (e.g., the breath analyzer section) are disposable, improving hygiene, efficiency, and speed. Parts of the device can be easily and inexpensively manufactured using techniques such as lamination, injection molding, and / or 3D printing. Additionally, the device has low power requirements, allowing it to be battery-powered.

[0027] Brief description of the attached figures

[0028] Figure 1 A pathogen collection and analysis system 100 according to aspects of this disclosure is shown.

[0029] Figure 2A The sample collection section 150 of the system 100 is shown after it has been removed from the interface or container 112 of the analyzer or base 110.

[0030] Figure 2B An exemplary channel 168 and magnet 170 setup 600 located inside portion 150 are shown.

[0031] Figure 3 An exemplary pathogen labeling system 300 that can be used in conjunction with system 100 is presented.

[0032] Figure 4 Another pathogen labeling system 400 that can be used in conjunction with system 100 is shown.

[0033] Figure 5A The system 300 and 400 are shown in system 500, which are used to label the target pathogen 510 for testing.

[0034] Figure 5B A schematic diagram of aggregate 520 is shown, in which magnetic particles 310 and fluorescently labeled particles 410 bind to different subunits S1 and S2 of the spike protein on pathogen 510, respectively.

[0035] Figure 6A A schematic diagram of the microfluidic channel 168 and magnet 170 portion of system 100 in an exemplary variant is shown.

[0036] Figure 6B The microfluidic magnetic separator 650 is shown. Figure 6B Photographs of the actual implementation of the microfluidic channel 168 and magnet 170.

[0037] Figure 7A An operational schematic diagram of an exemplary microfluidic magnetic separator configuration 700 combined with system 100 is shown.

[0038] Figures 7B-7D The operation of another exemplary microfluidic magnetic separator configuration 750 combined with system 100 is shown.

[0039] Figure 8 A schematic diagram of the operation of another exemplary microfluidic magnetic separator configuration 800 combined with system 100 is shown.

[0040] Figure 9A and 9B This is a flowchart of a method for detecting pathogens in patients using System 100.

[0041] Detailed description

[0042] Several illustrative embodiments will be described in detail, with the understanding that this disclosure is merely to illustrate the general inventive concept. Embodiments covering the general inventive concept can take various forms, and the general inventive concept is not intended to be limited to the specific embodiments described herein.

[0043] Test System Overview

[0044] Figure 1 A pathogen collection and analysis system 100 according to aspects of this disclosure is shown. System 100 can be divided into two main parts: an analyzer base 110 and a sample collection section 150. The sample collection section 150 is configured to obtain samples from a patient (e.g., through the patient's breath or saliva). The analyzer base 110 can then analyze the pathogens in the collected samples and report the results. Figure 1 As shown, the collection section 150 and the analyzer base 110 can be separate components. In this case, one or more sections (e.g., the collection section 150) can be designed for single use and be disposable. Other sections (e.g., the analyzer base 110) can be designed for multiple uses with different collection sections 150. However, it should be understood that such a configuration is merely exemplary. The collection section 150 and the analyzer base 110 can also be contained within the same housing. Both can be disposable.

[0045] like Figure 1 As shown, the analyzer base 110 may include an interface or container 112 for accommodating a sample collection section 150. The interface or container 112 may include electrical and / or mechanical connections (not shown) for interaction with the sample collection section 150. The electrical connections may, for example, provide power to a portion of the sample collection section 150 and / or retrieve data from the collection section 150. It may also charge a battery (not shown) in the collection section 150. The interface or container 112 may also include a motor or actuator 114 that can drive a piston 116, which is also part of the interface or container 112. The piston 116 may interact with a plunger 152 on the sample collection section 150 for driving the sample through the sample collection section 150, as discussed in more detail below.

[0046] The analyzer base 110 may also include electronics symbolically designated 118 (e.g., power regulation electronics, communication electronics, application-specific integrated circuits (ASICs) for exemplary purposes of communication and / or data processing). Electronics 118 may drive functions such as activation of piston 116 and motor 114, magnetic field strength generated by magnet 170 or magnet 801, and the function of any measuring device. It may operate heating or cooling devices in section 150 or base 110 and / or charge the battery on collection section 150. Electronics 118 may include a central processing unit (CPU) and other hardware capable of performing the data analyses described herein. Additionally or alternatively, base 110 may be connected to allow it to share data directly with a computer or other device. Analyzer base 110 may have Bluetooth functionality and / or be able to transmit results via Ethernet, WiFi, and / or mobile communication systems. Electronics 118 may also include barcode reader capabilities for collecting and correlating patient data.

[0047] For example Figure 1 As shown, the sample collection section 150 may include a sample extractor 154 for extracting patient samples for analysis (e.g., breath analysis). For example, the patient may place his or her lips on the end of the sample extractor 154 and breathe into the inlet tube 156. The sample extractor 154 may be any suitable type of container, such as a tube, orifice, nozzle, or mouthpiece. The inhalation section 150 may also be configured, or alternatively, to obtain samples from the patient in other forms. For example, the inhalation section 150 may be configured to obtain saliva- or mucus-based samples from the patient. The inhalation section 150 may also be configured to obtain samples from other bodily fluids and / or materials. If a portion of the sample collection section 150 is not disposable, the sample extractor 154 may be removable and itself disposable.

[0048] Section 150 may further include a series of tubes, such as inlet tube 156 and outlet tube 158, disposed on either side of collection container 160. Tubes 156 and 158 may include filters for filtering debris from the sample. Tubes 156 and 158 may also include valves and / or baffles to delay or stop flow. Tubes 156 and 158 may both be hydrophobic (e.g., their inner walls may be coated with a hydrophobic coating and / or the tubes themselves may be made entirely of hydrophobic materials) to facilitate the flow of water-based breath or other samples throughout section 150. Tubes 156 and 158 may have increased surface area for additional efficiency in the collected exhaled samples. Section 150 may also include a propellant device (e.g., a pouch) 162 that can spray saline, other liquids, and / or gases to propel the exhaled sample from sample extractor 154 through inlet tube 156. Any suitable type of propellant may be used in conjunction with propellant device 162. Suitable types of propellants are typically those that do not significantly interfere with, confuse, or reduce the accuracy of various measurement techniques (e.g., magnetic and / or fluorescence measurement techniques, which are discussed in more detail below).

[0049] Collection container 160 typically contains reagents 164, which may include magnetically and / or fluorescently labeled antibodies and / or particles, as discussed in more detail below. Reagents 164 may have various uses, one of which is to facilitate the collection and analysis of pathogen samples from patient samples provided by sample extractor 154. Collection container 160 may also contain chemicals for stabilizing and / or preserving various reagents before use. In variants where collection portion 150 is reusable, the chemicals in collection container 160 may stabilize and / or preserve reagents between uses. Reagents may be stored in powder or other solid form (e.g., lyophilized powder) and injected or released into the sample or other carrier liquid upon use of collection portion 150. Reagents may also be stored in liquid form (e.g., aqueous solution). Some reagents may be stored and / or provided as gases.

[0050] Collection container 160 may include or be adjacent to a cooler, such as a Peltier cooler. Other coolers may also be used, including various electric coolers, dry ice, liquid nitrogen, "blue ice gel" (typically pre-cooled or pre-frozen), and / or disposable cooler cartridges having one or more of these elements. The cooler may be located in base 110 and in thermal communication with container 160, or it may be located on section 150. The cooler preserves reagents and allows for the condensation or solidification of the exhaled sample. One function of the cooler is to condense the exhaled sample into exhaled condensate. Among other things, this helps the collector section 150 obtain a sufficient amount of exhaled sample for accurate testing.

[0051] After the sample is blown in or otherwise provided to the sample extractor 154 by the patient, the sample enters the inlet tube 156. The sample may settle into the collection container 160 under gravity and / or pressure. The sample may also be propelled into the collection container 160 by the emitter 162, as described in more detail below. Once the sample is in the collection container 160, it can be combined with reagent 164 for later identification. It may also be condensed by the aforementioned cooler to form exhaled breath condensate (EBC) to improve measurement sensitivity and detection.

[0052] Sample portions not collected in collection canister 160 can be transferred via outlet tube 158. The exhaled sample portion flowing in this manner is the uncaptured sample portion. The uncaptured sample portion can be filtered by exhaust filter 166. Exhaust filter 166 can remove pathogens (e.g., viruses) from the uncaptured sample portions before releasing them into the surrounding environment outside collection section 150. Exhaust filter 166 can be constructed from any suitable filter material, including but not limited to HEPA filters that can block or prevent the spread of nebulized respiratory pathogens.

[0053] A piston 116 in base 110 can actuate a plunger 152 of collection section 150 to push a capture portion of the expiratory sample through collection canister 160 and into microfluidic channel 168. Pistons 116 and 152 may be mechanically connected, while only plunger 152 is in fluid communication with the contents within tube 156 (potentially including the expiratory sample collected from the patient). The action of pistons 116 and plunger 152 can push condensed (or other) expiratory sample from collection canister 160 upward through microfluidic channel 168 into the vicinity of the analytical system (e.g., various microfluidic magnetic separators discussed herein). Specifically, plunger 152 can actuate the sample upward along a path marked “flow” in microfluidic channel 168 against gravity. Thus, the sample can flow upward from the bottom of system 100.

[0054] The plunger 152 may also be used to "prime" the portion of the system 100 that communicates with the microfluidic channel 168 (e.g., the analytical section). For example, the plunger 152 may push fluid into the channel 168 before adding a sample and / or before performing an analysis to displace gases (e.g., air) and / or bubbles that may have formed in the channel 168 or elsewhere in the device 100. The gas may be vented, for example, from an exhaust device 166 or some other outlet mechanism. Eliminating bubbles in the channel 168 is generally advantageous. Bubbles may impede or obstruct fluid flow. They may also confound certain types of measurements. Therefore, priming the system to remove or expel bubbles is generally useful. It should be understood that mechanisms other than the plunger 152 may be used to prime the system and remove bubbles in this manner. Any suitable configuration for priming is within the scope of this disclosure (e.g., a pump system or other systems that can actuate fluid flow).

[0055] Although Figure 1 The diagram shows flow in one direction (“flow”) due to the action of plunger 152, which can, in fact, be reversed. For example, due to gravity, the sample may flow downwards in the direction opposite to the “flow”. Furthermore, plunger 152 can be retracted, causing liquid to withdraw and creating fluid flow in the opposite direction of the “flow”. This countercurrent may be intentionally initiated. In some cases, periodically extending and retracting plunger 152 to create a circulating flow of the sample in channel 168 may be advantageous. Doing so may facilitate the collection and accumulation of the sample in the analytical section of system 100, discussed further below. It may also allow the sample to mix well with any carrier liquid or other components of the system described herein. Circulation can further increase the amount of sample exposed to the analytical section of system 100 and thus increase or amplify the signal-to-noise ratio of the measurement. This circulation process may be assisted by a sample collection section or container (not shown) located at the top of system 100 (e.g., near filter 166) for collecting samples near the top of system 100. This collection section or container can provide samples for the aforementioned flow circulation. In addition, the collection section or reservoir can simply contain the sample for later removal from system 100 for further analysis (e.g., where it is advantageous for more than one analytical technique and / or for repeat analysis) and / or for storage.

[0056] like Figure 1 As shown, the microfluidic channel 168 can be aligned with magnets 170 or 801 for magnetic separation and detection, as discussed in more detail below. Flow arrow 171 indicates an exemplary flow direction of the sample within the microfluidic channel 168, as shown. Figure 1As shown. One detection method includes using a detection system 172, which may be located on the base 110. In an alternative configuration, the detection system 172 may be located on the portion 150. The system 172 can detect particles by the fluorescence of fluorescently labeled antibodies, molecules, and / or synthetic or other particles. Figure 1 An exemplary location of the detection system 172 is shown. If the detection system 172 is located on the base 110, an adjacent portion of the collection portion 150 may have a window (not shown) that allows light to be transmitted to and from the detection system 172 and the channel 168.

[0057] If the collection section 150 is reusable, then its various components can be shut down and replaced. As described above, the sample extractor 154 can be shut down and replaced for multiple uses. Furthermore, the collection container 160 and reagent 164, as well as the exhaust filter 166, can be replaced. Parts of these components (e.g., the collection container 160 and reagent 164) can be replaced in modular or cylindrical form. During replacement, exposed portions of the collection section 150 can be covered with disposable caps to prevent contamination of the interior of section 150.

[0058] Figure 2A The sample collection section 150 is shown after being removed from the interface or container 112 of the analyzer or base 110. Figure 2A The housing of the plunger 152 of the sample collection section 150 is shown. The housing 174 can form a liquid-tight seal on the plunger 152, shielding the plunger 152 from external components. The housing 174 thus prevents contamination of the samples and reagents 164 inside the section 150 (e.g., samples within the inlet tube 156 and / or collection vessel 160). As described above, the housing 174 can move with the plunger 152 to actuate fluid flow within the section 150. When the housing 174 moves with the plunger 152, it maintains the shielding of the interior of the section 150 from the components and shields the piston 116 from the contents of the section 150. The latter prevents the piston 116, which may not be disposable, from being contaminated by the exhaled breath sample within the collection section 150. Figure 2A The outer shell 176 of the magnet 170 and the microfluidic channel 168 of the portion 150 are further shown.

[0059] illustration Figure 2B An exemplary channel 168 and magnet 170 configuration 600 located inside section 150 are shown, referred to herein as a “microfluidic magnetic separator”. Figure 2B The separator configuration 600 shown is in Figure 6A The following sections illustrate and discuss the separator configurations in more detail. Note that other separator configurations (e.g., those shown below) may also be considered. Figure 8 The configuration 800 discussed in the context can be used in conjunction with system 100, rather than Figure 2BThe configuration 600 shown. The housing 176 may include the aforementioned window (not shown) that allows light transmission between the microfluidic channel 168 of the collection portion 150 and the detection system 172.

[0060] Back Figure 2A The base 110 includes output reporting features 120 and 122. Exemplary output reporter 120 reports an "indicator" that can indicate the status of system 100 (e.g., power-on, ready, internal temperature, presence of blockages or malfunctions in section 150 or 110, etc.). Output reporter 122 can also indicate simple positive / negative reports. A positive report indicating pathogen infection can be indicated, for example, by illuminating a red LED. A negative report indicating no pathogen infection was detected can be indicated by illuminating a green LED. It should be understood that the colors and indicators are merely exemplary. Other configurations are possible and within the scope of this disclosure. For example, a touchscreen or other display may be provided.

[0061] Reagents and labeling systems

[0062] Figure 3 An exemplary pathogen labeling system 300 is presented that can be used in conjunction with this disclosure and system 100. Figure 3 Magnetic particles 310 coated with antibody 320 are shown. The coated magnetic particles 310 may be included in reagent 164 adjacent to collection vessel 160, such as... Figure 1 As shown. When the sample is introduced into the collection container 160, the patient sample can bind to or adhere to the coated magnetic particles 310, as described below. As described in more detail below, the adhesion between the sample and the magnetic particles 310 is achieved through a coating 320.

[0063] Coating 320 may include any relevant antibody, such as an antibody preferably binding to a known site on a known pathogen that is the test target of system 100. For example, coating 320 may bind to SARS-CoV-2 or other coronaviruses. Coating 320 may include monoclonal antibodies against the spike protein or other parts of such coronaviruses or other pathogens. However, it should be understood that, depending on the application, other antibody-like reagents are also possible, such as aptamers. Furthermore, or alternatively, element 320 may include other molecules, such as single-chain variable fragments (ScFv). Antibody 320 may bind to pathogens other than coronaviruses (e.g., tuberculosis or malaria, or various bacteria). Antibodies 320 may be modified, substituted, or altered such that they bind to viruses or other pathogens unknown at the time of writing or other pathogens not specifically discussed herein. Although Figure 3The diagram shows a generally uniform distribution of coating 320 relative to magnetic particles 310, but it should be understood that this is merely exemplary. Such a uniform distribution is only one example of system 300 and is not required for system 100 to operate as disclosed.

[0064] System 300 may include any suitable immunomagnetic marker, including magnetic particles 310. Such particles 310 may include, for example, magnetic nanoparticles. While magnetic particles of various sizes can be used in principle, characteristics that may affect the attachment of such particles (e.g., steric hindrance) should be considered. They may also include Dynabead M450, a monodisperse polystyrene bead doped with magnetite. Other exemplary forms of magnetic particles 310 include colloidal magnetic markers, such as MACS particles, which are dextran particles doped with magnetite. Molecular magnetic markers may also be used. These include ferritin, an iron storage protein. Immunomagnetic markers typically include, for example, paramagnetic compounds or molecules that bind to a primary or secondary antibody. Marking is performed by attaching an antibody to a marker of interest on a target pathogen (i.e., the pathogen being tested by system 100). Any suitable magnetic material may be used for magnetic particles 310, including materials containing cobalt, iron, nickel, neodymium, magnetite, etc. Magnetic materials may include nonmagnetic portions (e.g., embedding the magnetic material within a nonmagnetic matrix (e.g., a polymer or glass)).

[0065] Figure 4 Another pathogen labeling system 400 is shown, which can be used in conjunction with system 300 in test system 100. (Example) Figure 4 As shown, system 400 may include fluorescently labeled particles 410 (particles, including “microparticles,” which may be equivalently referred to as “microbeads,” as used herein) coated with another coating 420, which may include antibodies specific to aspects of the target pathogen that differ from those of coating 320. As discussed above in the context of coating 320, coating 420 may include molecules that are not antibodies (e.g., angiotensin-converting enzyme 2 (ACE2), aptamers, and ScFv), or another biomolecule capable of binding to the target pathogen. Coating 420 may also include antibodies specific to epitopes of the spike protein of SARS-CoV-2 or other coronaviruses that differ from those of coating 320.

[0066] The fluorescently labeled particles 410 can have a variety of different sizes. In some variations, particles 410 can be nanoparticles or microparticles. In any case, the size of particle 410 should be chosen to balance several practical considerations in microfluidics and magnetic measurements as described below (e.g., whether the magnetophoretic mobility caused by the paramagnetic tag / dipole moment in the field gradient of the magnetic separator / ferrospectrator configuration is greater than the viscous drag force proportional to the particle radius). Large drag forces should be avoided when the magnetic force is small. However, particles 410 should also be large enough to be easily observed by the detection system 172.

[0067] Although Figure 4 Not shown, but the fluorescently labeled particles 410 preferably include fluorescent materials, such as any fluorophore commonly used in immunofluorescence. Fluorescent materials may include fluorescent proteins, non-protein organic fluorophores, and certain dyes. Common fluorescent dyes include fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), or Alexa. Dye. This fluorescent material enables relatively rapid and easy detection of fluorescently labeled particles 410 and pathogens attached to them. The fluorescently labeled particles 410 may comprise glass, polymers, or some other suitable structural material that can contain the fluorescent material. In alternative variations, the fluorescently labeled particles 410 may comprise other materials detectable by optical, radiological, magnetic, chemical, or electrical means. Furthermore, system 400 may include any suitable immunofluorescent label. Immunofluorescent labels typically include, for example, fluorescent molecules linked to antibodies. System 400 may also include stained particles 410. This can have the advantage of making the particles 410 significantly brighter than the fluorophore.

[0068] Figure 5A The system 300 and 400 are shown in system 500 for labeling the target pathogen 510 in the test. In system 500, the pathogen 510 binds to both coating 320 and coating 420 to produce aggregate 520. Note that the term "aggregate" is used herein to refer to an aggregation of particles. According to this disclosure, the aggregate itself may also be referred to as a "particle". As described above, antibodies 320 and 420 can be selected such that magnetic particles 310 and fluorescently labeled particles 410 bind different subunits of the same pathogenic protein. Figure 5B This explains the situation. More specifically, Figure 5BThis diagram illustrates agglomerate 520, where magnetic particles 310 and fluorescently labeled particles 410 bind to different subunits S1 and S2 of the spike protein on pathogen 510, which is a coronavirus. Such antibodies are currently commercially available for SARS-CoV-2. Since the SARS-CoV-2 S protein is known to share approximately 76% homology with the SARS-CoV S protein, but lower homology with the S proteins of other common coronaviruses, it is crucial to develop antibody assay systems capable of recognizing target proteins with high affinity and minimal unwanted cross-correlation.

[0069] Aggregates 520 of magnetically labeled pathogens 510 and fluorescently labeled particles 410 will form a suspension (e.g., inside collection vessel 160), creating a magnetic dipole for each aggregate 520. This binding provides pathogens 510 with potential magnetic (310) and fluorescent (410) labels, facilitating detection or differentiation through two complementary methods (i.e., magnetic and fluorescent). This labeling complementarity can be advantageous. For example, it can amplify the expected relatively low level of pathogen signal in a breath sample. This is because using a sandwich assay (e.g., system 500) to bind fluorescently labeled particles 410 together with magnetic particles 310 to pathogens 510 allows for multi-site binding of particles 310 or 410. In other words, each pathogen 510 can be labeled with multiple labels, effectively doubling or tripling (or more) the magnetic effect induced by the magnetic field on a single pathogen 510. This increases the magnetophoretic mobility for displacement separation and the adhesion for binding separation. Studies have shown that exhaled droplets contain relatively low levels of pathogens (e.g., SARS-CoV-2) compared to samples obtained through other more invasive methods (e.g., swabbing). The two-stage enrichment process described above (i.e., first labeling the pathogen with magnetic particles 310, then labeling the magnetically labeled pathogen with fluorescently labeled particles 410) results in aggregates 520 of suspended magnetically and fluorescently labeled pathogens.

[0070] Back Figure 5A Although aggregates 520 may appear identical, it should be understood that this is not always the case. Aggregates 520 may differ in the amount of attached pathogens 510, fluorescently labeled particles 410, and, importantly, the number of attached magnetic particles 310. In particular, differences in the attachment of magnetic particles 310 to pathogens 510 can produce aggregates 520 with different magnetic dipole moments. As discussed in more detail below, these differences in magnetic dipole moments can be used to separate different particles and estimate quantities such as pathogen load.

[0071] Magnetic separation and analysis

[0072] The basic principles of magnetic particle separation are discussed in more detail in U.S. Patent No. 5,968,820 to Zborowski et al., which is incorporated herein by reference in its entirety. In short, the flow of magnetically labeled particles (e.g., magnetic particles 310 or aggregates 520) in a conduit such as microfluidic channel 168 will change direction in response to a changing magnetic field within channel 168. Conversely, the flow of non-magnetic particles will not change due to a change in the magnetic field. Therefore, introducing a change in the magnetic field into microfluidic channel 168 can distinguish magnetically labeled particles or aggregates from unlabeled particles. Furthermore, the flow change in response to the change in the magnetic field can provide insight into the amount of pathogens attached to aggregates 520. The flow of magnetically labeled particles (aggregates 520) will depend on… It is the square of the magnetic flux density gradient, weighted by the density (α) of the attached magnetic particles, among other things. See Equations 1 and 2 of U.S. Patent No. 5,968,820. In the case of system 500, the density (α) of the magnetic particles attached to aggregate 520 depends on the number of magnetic particles 310 bound to its surface by coating 420 (and pathogen 510). Therefore, passing the magnetically labeled patient sample through a varying magnetic field can allow the aggregate 520 to separate based on their magnetic attachment density. Because in this case, the number of attached magnetic particles depends on the attachment of pathogen 510, this particle separation is also related to the number of attached pathogens.

[0073] In principle, any suitable configuration that utilizes the magnetic particle adhesion density effect (hereinafter referred to as dipole moment) to sort particles 520 can be used to detect pathogens by system 100. That is, any dipole separator system disclosed in U.S. Patent No. 5,968,820 (e.g., system 200 or...) Figure 4 A dipole classifier may be used in system 100. Several specific systems that may be used in conjunction with test system 100 are described below. However, it should be understood that the systems described herein do not represent an exclusive or exhaustive list of such systems that may be used with test system 100. Test system 100 and this disclosure may be used with any suitable magnetic separation system, whether expressly disclosed herein or disclosed herein by reference.

[0074] Figure 6A This shows an internal schematic of the microfluidic channel 168 and magnet 170 portion of system 100 in an exemplary variant 600. Figure 6A The configuration shown is commonly referred to as high gradient magnetic separation (HGMS) and may also be called a "ferrometer" or "microferrometer". In this configuration, magnet 170 generates several regions with high magnetic field gradients in channel 168, which tend to trap aggregates 520 based on their magnetic properties.

[0075] The microfluidic channel 168 and the magnet 170 together form a microfluidic magnetic separator 600. For example... Figure 6A As shown, channel 168 is fabricated (e.g., via microfabrication or MEMS) within a closed microchannel assembly 610. Channel 168 may be pressed against the interpole gap of magnet 170. Magnet 170 may include a magnetic manifold containing permanent magnets (e.g., cobalt, iron, rare earth, NdFeB) or other magnets. Figure 6A In this example, magnet 170 comprises four trapezoidal magnetic elements 170a-170d arranged in series or in an array. Magnetic elements 170a-170d can be adjacent to each other using a non-magnetic material 620 (e.g., steel, such as low-carbon steel). In this configuration, the smaller of the two parallel sides of the trapezoid forms the inter-pole magnetic gap N / S, as shown in the case of magnetic element 170b. Element 630 is an optional base on which magnet 170 rests. Element 630 can be, for example, part of a housing 176 as shown in Figure 2.

[0076] To maximize the magnetic field gradient provided by magnet 170, magnetic elements 170a-170b can be positioned with alternating polarities (i.e., as shown in the image). Figure 6A The dimensions of channel 168 and magnets are shown as 170a N / S, 170b S / N, 170c N / S, and 170c S / N. The dimensions of channel 168 and magnets can vary and should be set according to the specific application (e.g., based on the type, weight, and volume of the particles / aggregates 310 / 410 / 520 used, their magnetic dipole moment, and the expected pathogen attachment on aggregate 520, as well as the properties of the carrier fluid (e.g., viscosity)). An exemplary thickness of the wall of channel 168 is 400 μm and a diameter of 250 μm. The height and width of magnetic elements 170a-170d can be, for example, on the order of millimeters. In this configuration, an exemplary magnetic gap width (i.e., the distance between N and S in magnetic element 170a) can be approximately 1 mm.

[0077] Figure 6AA schematic diagram of a detection system 172 in an exemplary placement adjacent to channel 168 is also shown. Detection system 172 can provide light to cause particles 410 (and thus aggregates 520) to fluoresce. If detection system 172 is located on base 110, it can transmit light through the window described above in the housing 176 of collection portion 150. System 172 can also resolve the detected fluorescence relative to distance along channel 168. In this case, the detected fluorescence is correlated with the number of aggregates 520. As discussed in more detail below, the correlation between fluorescence and distance along the channel can reveal, among other things, the viral load in the patient providing the sample. Microchannel assembly 610 can be at least partially transparent, thereby allowing light from the fluorescence of particles 410 in the channel to reach detection system 172. The entire microchannel assembly 610 can be transparent. Alternatively, a portion of assembly 610 (e.g., the portion adjacent to detection system 172 or the window described above) can be transparent.

[0078] Figure 6B The microfluidic magnetic separator 650 is shown. Figure 6B A photograph of an actual implementation of the microfluidic channel 168 and magnet 170. A penny 601 is included to show scale. The dimensions of the components in the microfluidic magnetic separator 650 correspond to the exemplary dimensions discussed in the context of the microfluidic magnetic separator 600 above.

[0079] Figure 7A This diagram illustrates the operation of an exemplary HGMS microfluidic magnetic separator configuration 700 (e.g., implemented via microfluidic magnetic separator 600 or 650) of the combined system 100. Figure 7A As shown, the microfluidic magnetic separator 700 includes a magnet array 170 and a microfluidic channel 168 (also as...). Figure 1 (As shown in system 100). Figure 7A The direction of the aggregate flow 710 and the direction of the microfluidic channel 168 have been changed from Figure 1 The flow direction of 171 and microfluidic channel 168 is rotated. This is for illustrative purposes only and does not imply any difference in design or configuration.

[0080] As mentioned above Figure 1 As discussed in the context, the direction of flow 710 in channel 168 can be reversed (e.g., by withdrawing plunger 152). Flow 710 can circulate multiple times (e.g., forward flow, then reverse flow, forward flow, then reverse flow, etc.). As in Figure 1 As discussed in the context, such cycling can be used to help the sample accumulate on the microfluidic magnetic separator 700, mix with the carrier liquid, and increase the signal-to-noise ratio. Although not shown in... Figure 7AHowever, the collection container can be placed at one or both ends of channel 168 (or anywhere else on channel 168) to collect samples for recirculation or storage. These principles of recirculation, reverse flow direction, and fluid storage can be applied to any variation described herein.

[0081] For the purpose of explanation, Figure 7A Four different types of aggregates 520 were shown, differing in their magnetic dipole moments. Figure 5A This refers to aggregate types 720a-720d. Aggregates 720a-720d are aggregates 520 formed by forcing a patient sample (obtained via sample extractor 154) into contact with reagent 164 in the collection container 160 of the sample collection section 150. Multiple magnetic elements (e.g., such as...) are used. Figure 7A The use of four of these elements can improve the collection efficiency of aggregates 720a-720d because employing more elements allows the aggregates more time to deposit. In some cases, the flow rate of aggregates 720a-720d in channel 168 can be increased without sacrificing sensitivity. It should be understood that any suitable number of magnetic elements can be used in magnet 720 and remain consistent with this disclosure. In some cases, only a single magnetic element may be used.

[0082] As the aggregates 720a-720d flow through the microfluidic channel 168, they are affected by a changing magnetic field B emitted from the magnetic elements 170a-170d. They are attracted to the elements 170a-170d and captured onto the stationary surface 168a of the channel 168. Since the attractive force depends on the dipole moment of the aggregates, the capture effectively classifies the aggregates 720a-720d according to the magnetic dipole moment. After capturing the aggregates 520, data can be collected and / or analyzed (e.g., by measuring the fluorescence of fluorescently labeled particles 410 within the aggregates 520).

[0083] Aggregates 720a have the highest magnetic dipole moment and are black. Aggregates 720a may have the highest magnetic dipole moment because they have a relatively large number of attached magnetic particles 310, which is associated with a relatively large number of attached pathogens 510. Aggregates with the lowest magnetic dipole moment are labeled 720d and are orange. Aggregates 720d may have the lowest magnetic dipole moment because they have a relatively small number of attached magnetic particles 310 due to a relatively small number of attached pathogens. Aggregates with intermediate magnetic dipole moments are labeled 720b and 720c and are brown and green, respectively. The different magnetic dipole moments of aggregates 720a–720d are associated with different numbers of magnetic particles 310 attached to each aggregate due to different numbers of attached pathogens, as discussed above.

[0084] like Figure 7AAs shown, the aggregate 720a with the highest magnetic dipole moment travels along path 730a and eventually collects near magnetic element 170a. Aggregate 720a travels from the starting point S of microfluidic channel 168 across a minimum average distance 740a. As mentioned above, these aggregates may have a higher dipole moment because they have increased magnetic particle 310 attachment due to a higher number of attached pathogens 510. Conversely, aggregate 720d travels from the starting point S of microfluidic channel 168 along path 730d across a maximum distance 740d. This is because aggregate 720d has the lowest magnetic dipole moment, which is likely due to less attachment with magnetic particles 310. Intermediate aggregates 720b and 720c may travel intermediate distances 730b and 730c, respectively, due to intermediate levels of magnetic particle 310 attachment and pathogen attachment.

[0085] It should be understood that although magnetic elements 170a-170d are shown as identical in Figure 6, they need not be identical. Magnetic elements 170a-170d may differ in terms of the magnetic field source (e.g., permanent magnet or electromagnet), field strength, and / or other characteristics. Furthermore, although four magnetic elements are shown, other suitable numbers of magnetic elements may be used. In some cases, only one magnetic element may be required.

[0086] Once aggregates 720a-720d are collected near the magnetic element, they have been effectively classified. Their relative distribution can then be assessed using optical or other detection techniques. As described above, one such technique is to collect fluorescence data from fluorescently labeled particles 410 in aggregates 520 at different locations along microfluidic channel 168. Higher concentrations of aggregates at shorter distances from the channel origin S (e.g., 740a or 740b) may indicate higher pathogen loads in patients. Higher concentrations of aggregates at farther distances from the channel origin S (e.g., 740d or 740c) may indicate lower pathogen loads in patients. The fluorescence of the fluorescently labeled particles 410 in aggregates 720a-720d can be excited by any suitable means (e.g., by using...). Figure 1 and 6A The detection system 172 in the example uses LED light, for instance. However, LED lights are merely exemplary. Other sources of illumination can be used in the context of this disclosure to, for example, cause the fluorescently labeled particles 410 to fluoresce. Fluorescence data can be obtained, for example, by irradiating the fluorescently labeled particles 410 with UV radiation, lasers, and / or any other suitable type of radiation.

[0087] Fluorescence intensities at different locations in channel 168 can be collected by a camera (e.g., for digital image processing), a light sensor, or any other suitable means, including but not limited to sensing modalities such as giant magnetoresistance, Hall sensors, resonant sensors, and impedance. Obtaining digital images of the collected aggregates for further analysis of their distribution in regions adjacent to one or more of the magnetic elements 170a-170d may be advantageous. For example, the data may reveal whether the aggregates are monodisperse in terms of their attachment to the fluorescently labeled particles 410. This data may also be used for system diagnostics or fine-tuning of the devices and processes described above (e.g., to determine whether a change in the magnetic field in a region of channel 168 will produce a change in that region). Alternatively, each collection region may be assigned a single average brightness based on the collected fluorescence intensity. The latter approach may be used in versions of system 100 where one or more portions (e.g., collection portion 150) are simplified and / or disposable. Single-valued average fluorescence readings may also allow for rapid positive / negative testing.

[0088] Once fluorescence intensity data is obtained, it can be collected and analyzed by system 100 (e.g., via electronics 118 included in base 110). For example, as described above, digital image processing can be performed on the fluorescence intensity images. Digital image processing can be performed by any suitable software platform, such as Image Pro Plus. Examples of image processing that can be used include edge analysis to distinguish individual aggregates / particles / cells / other collected objects, filtering to remove noise and thresholding image intensity (e.g., to remove debris and large objects inconsistent with aggregate or particle size), and generating binary spectra of aggregates to separate them from the background. The latter technique can help to better quantify aggregates, for example, through more accurate aggregate counting algorithms. It can also further improve data accuracy by identifying objects in the image that are too large or too small to be counted as aggregates being examined (e.g., debris).

[0089] Figures 7B-7D Another magnetic separation configuration 750 is shown. In configuration 750, at least two magnetically distinct aggregates 720e and 720f are present. The first type, aggregates 720e, have a significant magnetic dipole moment because they have a large number of attached magnetic particles 310 (e.g., like...). Figure 5A Aggregate 520 in the sample). This may indicate that aggregate 720e has a large number of pathogens 510 attached (see Aggregate 520 in the sample). Figure 5A Aggregates 720e are orange. The second type, aggregates 720f, do not have a significant dipole moment because they lack a large number of attached magnetic particles 310. Aggregates 720f are blue. Aggregates 720f may have a lower pathogen 510 content than aggregates 720e.

[0090] like Figure 7B As shown, aggregates 720e and 720f are introduced together into channel 168 at 751. Typically, aggregates 720e and 720f flow downwards through channel 168 in the direction of 752. Flow 752 can pass through plunger 152 ( Figure 1 The movement of aggregates 720e and 720f is the driving force. However, aggregates 720e and 720f may also settle onto the channel surface 168a due to their interaction with forces acting on them within the channel. These forces may include gravity G, such as... Figure 7B As shown. They may also include the effective attraction of the aggregates 720e to the magnet 170e by the dipole moment passing through them. In this arrangement, both G and magnetic interactions tend to pull the aggregates toward the surface 168a. The magnet 170e can have any suitable form, including Figure 7A The trapezoidal form of the magnetic element 170a-170d.

[0091] like Figure 7C As shown, both aggregates 720e and 720f follow an average path 753 from inlet 751 to the vicinity of magnet 170e. The exact paths followed by aggregates 720e and 720f may differ slightly because they may be subjected to different forces. In any case, as Figure 7C As shown, aggregates 720e and 720f settle at position 755a on the channel surface 168a near magnet 170e.

[0092] like Figure 7D As shown, magnet 170e can then be moved along direction 760 to a second position 755b. This movement of magnet 170e can cause the magnetic aggregate 720e to flow along direction 760. Aggregate 720e will tend to move with magnet 170e because they effectively attract it by their dipole moment. Aggregate 720e will re-sink onto channel surface 168a at the new position of magnet 170e (i.e., 755b). On the other hand, although magnet 170e moves, aggregate 720f remains at position 755a. This is because aggregate 720f lacks a significant dipole moment and therefore lacks an effective attraction to magnet 170e.

[0093] As discussed above in the context of Figure 5, the aggregates 720e possess a significant dipole moment because they are attached to the magnetic particles 310 via pathogen 510. Therefore, the aggregates 720e move in response to their dipole interaction with the magnet 170e. Figure 7DThis effectively distinguishes aggregates 720f from those with a large amount of pathogen attachment based on the attachment of pathogen 510. In other words, in response to the movement of magnet 170e, aggregates with a large amount of pathogen attachment (i.e., magnetic aggregates 720e) are effectively separated or classified from aggregates without a large amount of pathogen attachment (i.e., non-magnetic aggregates 720f). This separation can be used in conjunction with fluorescence detection (e.g., using detection system 172) to detect whether a patient is positive for pathogen 510. As mentioned above, this separation of aggregates can also be used to estimate the pathogen 510 load in a patient. Any detection method described in the context of configuration 700 above can also be used with configuration 750.

[0094] Figure 8 This diagram illustrates the operation of another exemplary microfluidic magnetic separator configuration 800 (e.g., implemented via microfluidic magnetic separators 600 or 650) of the combined system 100. Configuration 800 is commonly referred to as Open Gradient Magnetic Separation (OGMS) and contrasts with HGMS configurations 600, 650, 700, and 750. Figure 8 As shown, the microfluidic magnetic separator 800 includes Figure 1 The magnet 801 and microfluidic channel 168 are shown. An exemplary advantage of configuration 800 is that it may be potentially easier to use to determine viral expression levels rather than binary (positive or negative).

[0095] Figure 8 The magnet 801 in this configuration differs from the magnet 170 in the HGMS configurations (600, 650, 700, and 750) in that it does not include discrete magnetic elements, each with a unique magnetic pole (e.g., 170a-170d; see, for example, Figure 6) to impart a high field gradient region. Instead, the magnet 801 is positioned such that its two magnetic poles (N / S), labeled P1 and P2, are located on either side of the channel 168. In this way, the magnet 801 provides a moderate magnetic field gradient, or isomagnetic field, throughout the channel 168. As discussed in more detail below, the moderate field gradient applied by the magnet 801 causes the flow aggregates 520 in the flow to deflect rather than trap them on the surface 168b of the channel 168 (e.g., as shown below). Figure 7A (As shown). The deflection is a function of the magnetic dipole moment of the aggregate 520 and its geometry. One advantage of configuration 800 is that more aggregates 520 can be collected in this way to obtain an enhanced signal. Aggregates can be classified by magnetophoretic mobility related to the amount of pathogen 510. This can be used to determine the total dose per breath of a patient (assuming a single exhaled sample) and the patient-associated infectivity. This data can be collected, measured, and released for subsequent analyses, such as genomic analysis.

[0096] The placement of magnet 801 in configuration 800 creates region 800a, in which the aggregates are subjected to varying magnetic flux. There is another region in configuration 800, collection region 800b, in which aggregates 820a-820g are shielded from varying magnetic flux. The latter will be discussed in more detail below.

[0097] For example, in configuration 700, Figure 8 The direction of the flow 810 shown is relative to Figure 1 The direction of flow 171 rotates. This is for illustrative purposes only and does not imply any difference in design or configuration. Configurations 700 and 800 can both be used with system 100. Their channels can be oriented relative to section 150, such as... Figure 2B As shown, there is a flow of aggregation along the direction of arrow 171.

[0098] For the purpose of explanation, Figure 8 Seven different types of aggregates were shown (520). Figure 5A This refers to aggregate types 820a-820g. Aggregates 820a-820g differ in their magnetic dipole moments. They are aggregates 520 formed by forcing a patient sample (e.g., an expiratory sample obtained via sample extractor 154) into contact with reagent 164 in the collection container 160 of the sample collection section 150. Aggregates 820a-820g enter channel 168 through channel inlet 168a. However, the manner in which aggregates 820a-820g exit channel 168 depends on how magnet 170 influences their trajectory within the channel.

[0099] Orange aggregates 820a with the lowest magnetic dipole moment are virtually unaffected by magnet 170 throughout region 800a. They exit channel 168 through port 168c without significant deflection (i.e., following a basically straight path through channel 168). In this configuration, aggregates 820a collected or measured through port 168 can be considered negative relative to the target pathogen being tested. This is because the magnetic particles 310 they collect are too few to be affected by the microfluidic magnetic separator test performed by configuration 800, meaning they have almost no pathogen 510 adhesion. The trajectories of all other aggregates 820b-820g in channel 168 are affected to some extent, thus representing positive results. They are positive because their deflection indicates that aggregates 820b-820g have significant magnetic dipole moments, associated with significant pathogen adhesion.

[0100] In collection zone 800b, aggregates 820b-820g are separated and collected according to their various deflections. More specifically, positive aggregates 820b-820g are guided to one of channels 830b-830g in analyzer 830 (note that channel label 830a is omitted for convenience, so that the letter part of the channel label matches the aggregate label). Figure 8 As shown, for example, aggregate 800b is collected via output channel 830b, which separates it from other aggregates in analyzer 830. Similarly, aggregates 820c-820g are separated and directed to their respective output channels 830c-830g. Although not indicated as an output channel in analyzer 830, the output 168g from the collection of pathogenic negative aggregate 820a can also be fed to analyzer 830 for analysis.

[0101] Once the aggregates 820a-820g have been magnetically sorted according to configuration 800 or 750, their relative distribution can be evaluated using fluorescence techniques, as described in detail in the context of configuration 700. Any of the techniques discussed above in the context of configuration 700 or 750 can be used to collect and analyze data from configuration 800.

[0102] Operating method

[0103] Figure 9A and 9B A flowchart illustrating an exemplary method for detecting potential pathogens using System 100 is presented.

[0104] from Figure 9A Initially, in step 902, the sample collection section 150 is used to collect a sample from the patient. As described above, the sample can be collected in several ways, including by using a sample extractor 154 as a mouthpiece. In this case, the patient can blow his or her breath into the sample extractor 154, causing the breath to enter the inlet tube 156. The patient's breath may contain the target pathogen, as well as debris that can later be removed by filtration.

[0105] In step 904, the sample is pushed downwards through inlet tube 156 into collection container 160. This can be accomplished by gravity. In an exemplary case, the interior of inlet tube 156 will be hydrophobic, so that the sample (typically including water) will not adhere to the interior. Instead, the sample will settle toward the bottom of tube 156, where collection container 160 may be located. Figure 1 In some cases, the sample can be pushed downward through the inlet tube 156 by the firing device 162. For example, the firing device 162 can fire a salt solution (e.g., as a spray) to flush the inlet tube 156 downward toward the collection tank 160. In this step, the sample can also be filtered to remove debris.

[0106] In step 906, the sample is in collection container 160. While in container 160, the sample may be exposed to magnetic particles 310 and fluorescently labeled particles 410. Magnetic particles 310 bind to pathogens 510 in the sample via coating 320. Fluorescently labeled particles 410 bind to pathogens 510 in the sample via coating 420. As described above, antibodies 320 and 420 can be selected to bind to different sites on pathogens 510. For example, in the case of SARS-CoV-2, antibodies 320 and 420 can bind to different epitopes of the SARS-CoV-2 spike protein. For this purpose, coating 420 may include ACE2. Another advantage of using ACE2 is that it overcomes the problem of cross-reactivity with other SARS-type viruses mentioned in the literature for many antibody tests involving SARS-CoV-2. In other variants, two monoclonal antibodies that bind to different parts of the coronavirus S protein can be used. The latter variant may be advantageous in some cases because the affinity of monoclonal antibodies may be higher than that of ACE2 for the coronavirus S protein. In any case, binding to different sites of the pathogen in collection container 160 can generate aggregates 520, which include magnetic particles 310 and fluorescently labeled particles 410. Thus, the pathogen 510 is bound in a magnetically and fluorescently labeled manner. At higher pathogen concentrations, aggregates 520 may have increased binding of the pathogen 510 to the magnetic particles 310, and therefore a larger magnetic dipole moment. This property allows for magnetic separation, as discussed in more detail in the context of steps 912 and 914 below.

[0107] In step 908, the sample may be flushed out of collection tank 160. Although not shown in the figure, a valve may be present to prevent reagent 164 from entering outlet channel 158 or microfluidic channel 168. A flushing device uses plunger 152 to physically push the sample. Plunger 152 may be actuated by piston 116 located in interface portion 112 of base 110. A motor or actuator 114 on base 110 may actuate piston 116 to actuate plunger 152. This action may also push uncaptured portions of the sample into outlet tube 158 and through filter 166. It may further cause uncaptured portions to be discharged from portion 150 through filter 166. Filter 166 may remove pathogens from uncaptured portions, preventing pathogens from being released into the surrounding environment.

[0108] In step 910, the sample may flow into microfluidic channel 168, which may be part of a microfluidic magnetic separator (e.g., as shown in the image). Figure 2B , 6A(As shown in 6B). The movement of the sample from the outlet tube 158 into the microfluidic channel 168 can be actuated by the movement of the plunger 152. As part of this movement, any uncaptured portion of the sample can be forced through the exhaust filter 166 before leaving the collection section 150 into the surrounding environment.

[0109] Turning Figure 9B In step 912, the aggregate 520 of the sample is exposed to a varying magnetic field within the microfluidic channel 168. The exposure settings may conform to any suitable configuration of the microfluidic magnetic separator discussed herein (e.g., 600, 650, 700, 750, or 800), as well as other configurations of microfluidic separators incorporated herein by reference. Furthermore, other known forms of microfluidic separators not explicitly incorporated herein may be used with system 100 within the scope of this disclosure.

[0110] In step 914, exposure to a changing magnetic field (step 912) should separate aggregates 520 based on their magnetic dipole moments. Since the magnetic dipole moment of aggregates 520 depends on the magnetic particles 310 attached to pathogens 510, the separation should also reflect the amount of pathogens 510 attached to aggregates 520. In this step, the separated aggregates 520 can be collected in different portions of channel 168 based on their dipole moments, such as... Figure 7A and 7D As shown. They can also be guided into different channels based on their magnetic deflection (e.g., Figure 8 (Channels 830b-830g in the document). It should be understood that other suitable methods for separating aggregate 520 are possible and within the scope of this disclosure.

[0111] In step 916, the spatial distribution of the aggregates 520 in the microfluidic channel 168 is then determined. This spatial distribution can be evaluated in various ways. For example, the spatial distribution can be obtained by using the fluorescence of a portion of the fluorescently labeled particle 410, as described above. In short, a detection system (LED, UV, or other) can irradiate the aggregates 520, causing them to fluoresce. For example, fluorescence can be detected by a detection system 172 located on the collection section 150. Fluorescence can also be detected by a detection system (not shown) located on the base 110. In either case, the detection system can include a camera and / or photodetectors positioned such that they can record fluorescence throughout the spatial distribution of the aggregates 520 in the channel 168. Another method for determining the spatial distribution of the aggregates 520 is simply by calculating the fluorescence of the collection channels at different intervals (e.g., Figure 8 Aggregates in channels 830b-830g. Aggregate counting can be performed using any suitable method, including aggregate counting by fluorescence measurement, as described above. Other examples include the use of a Coulter counter.

[0112] In step 918, the pathogen load (e.g., viral load, in the case of a virus) in the patient can be assessed based on the spatial distribution of aggregates 520 in channel 168 as evaluated in step 916. Since spatial distribution is associated with pathogen 510 attachment, it can be understood, as described above, as an indicator of pathogen load. For example, a higher concentration of aggregates 520 near magnetic element 170a ( Figure 7A and 7D This can indicate a higher pathogen load. This is because the result will indicate a higher degree of magnetic dipole moment in aggregate 520, corresponding to a higher degree of pathogen 510 attachment. Similarly, channel 820b ( Figure 8 A higher concentration or count of aggregate 520 in the cytokine cluster will indicate a greater deflection and dipole moment, and therefore a greater degree of pathogen 510 attachment. This may also be associated with a higher pathogen load in the patient.

[0113] In some cases, a threshold pathogen load may indicate a positive result. For example, in some situations, if the threshold pathogen load is within a certain distance (indicating the highest pathogen load), then a positive result may be indicated. Figure 7A The distance in the middle is 740a. Figure 7D The distance in the middle is 755b or Figure 8 If 50% or more of aggregates 520 are detected at a distance of 840d, this may indicate that the patient is testing positive for the pathogen. Otherwise, the patient is testing negative for the pathogen. The threshold can vary depending on the pathogen and other aspects of the experimental setup (e.g., characteristics of particles 320 and 420, strength of magnets 170 and 801, etc.). The threshold can also be a more complex function of the particle distribution on surface 168a or in channels 820b-820g. It should be understood that any such suitable threshold is within the scope of this disclosure. Specific quantifiable thresholds may vary depending on experimental details. Relevant parameters include the ferrographic configuration (e.g., configuration 700, 750, or 800), the size of channel 168, and the location where aggregates 520 are injected into channel 168. The balance between the effective magnetic attraction and other forces (e.g., shear stress relative to the particle size of channel 168) may also be important.

[0114] In step 920, the test results are reported. Any suitable method for reporting the results can be used. For example, the base 110 can be reported via a simple display (such as...). Figure 2AThe results (either via the LED in element 122 or other means) are reported to indicate whether a patient tests positive or negative for pathogen 510 based on a determined viral load. Raw or processed data (e.g., fluorescence data, image data, processed image data) can also be reported by any suitable means. Suitable means include reporting via Ethernet, Bluetooth, WiFi, mobile phone networks, disks, flash drives, or other storage media.

[0115] While various inventive aspects, concepts, and features of the invention may be described and illustrated herein as being embodied in combination in exemplary embodiments, these various aspects, concepts, and features may be used individually or in various combinations and sub-combinations in many alternative embodiments. All such combinations and sub-combinations are intended to be included within the scope of the invention unless expressly excluded herein. Furthermore, although various alternative embodiments of various aspects, concepts, and features of the invention (e.g., alternative materials, structures, configurations, methods, circuits, devices and components, software, hardware, control logic, alternatives in form, installation, and function, etc.) may be described herein, such description is not intended to be a complete or exhaustive list of available alternative embodiments, whether currently known or developed later. Those skilled in the art can readily adopt one or more inventive aspects, concepts, or features into additional embodiments and use them within the scope of the invention, even if such embodiments are not expressly disclosed herein. Moreover, even if some features, concepts, or aspects of the invention may be described herein as preferred arrangements or methods, such description is not intended to imply that such features are desired or necessary unless expressly stated otherwise. Furthermore, exemplary or representative values ​​and ranges may be included to aid in understanding this disclosure; however, such values ​​and ranges should not be construed as limiting and are intended to be critical values ​​or ranges unless so explicitly stated. Parameters identified as “approximately” or “about” are intended to include the specified value and values ​​within 10% of the specified value, unless otherwise explicitly stated. Furthermore, it should be understood that the accompanying drawings may, but need not, be drawn to scale and are therefore to be understood as teaching various ratios and proportions evident in the drawings. Moreover, while various aspects, features, and concepts may be explicitly identified herein as having inventive step or forming part of the invention, such identification is not intended to be exclusive, and there may be inventive aspects, concepts, and features of the invention fully described herein that are not explicitly identified as part of such or particular inventions but are shown in the appended claims. The description of the exemplary method or process is not limited to including all steps that are required in all cases, and the order in which the steps are presented should not be construed as necessary or required unless explicitly stated otherwise.

Claims

1. A method for detecting viral or bacterial pathogens, comprising: Potentially pathogenic samples are collected using a sample collection device configured to collect samples from at least one of the patient's breath, saliva, mucus, and other bodily fluids. A first portion of a potentially pathogenic sample is bound to a magnetic particle by a first coating on the magnetic particle, the first coating comprising at least one of an antibody, an aptamer, and a single-chain variable fragment (ScFv) that binds to a known site on a known pathogen. The second part of the potentially pathogenic sample is bound to the fluorescently labeled particles by a second coating on the fluorescently labeled particles that binds to the second part of the pathogen, to produce an aggregate containing the potentially pathogenic sample, magnetic particles and fluorescently labeled particles. Aggregates are magnetically separated using a gradient-based microfluidic magnetic separator; Detect the fluorescence of the separated aggregates; and The amount of pathogen is estimated based on the detected fluorescence.

2. The method of claim 1, wherein at least one of the following: The sample collection device is a breathalyzer; Collection includes obtaining potentially pathogenic samples from the patient's breath using a breath analyzer; The pathogen is a coronavirus; The coronavirus is SARS-CoV-2; The first part of SARS-CoV-2 bound by the first coating and the second part of SARS-CoV-2 bound by the second coating are different epitopes of the SARS-CoV-2 spike protein; The second coating contains angiotensin-converting enzyme 2 (ACE2); and Separation involves separating aggregates based on their magnetic dipole moment using a microfluidic magnetic separator.

3. The method of claim 2, wherein the separation is performed according to at least one of the following: The distance the aggregate moves within the microfluidic magnetic separator; The time it takes for the aggregate to move within the microfluidic magnetic separator; and Flow of aggregates in a microfluidic magnetic separator.

4. The method of claim 3, wherein estimating the amount of pathogen based on detected fluorescence comprises estimating the amount of pathogen based on the spatial distribution of detected fluorescence in the microfluidic magnetic separator.

5. The method of claim 4, wherein the spatial distribution of the detected fluorescence is generated by separation.

6. The method of claim 1, wherein detecting the fluorescence of the separated aggregates comprises: Irradiated aggregates; and The amount of fluorescence of fluorescently labeled particles excited by irradiation is detected.

7. The method of claim 6, wherein the fluorescence is the fluorescence of fluorescently labeled particles in the aggregate.

8. The method of claim 1, further comprising estimating the viral load in the patient based on the estimated amount of pathogen.

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