Patent Publication Number: US-2022214277-A1

Title: Cartridge-based automated rapid test analyzer

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 63/163,027, filed Mar. 18, 2021, and U.S. Provisional Application No. 63/280,783, filed Nov. 18, 2021, and is a continuation-in-part of U.S. patent application Ser. No. 16/855,709, filed Apr. 22, 2020, which claims the benefit of U.S. Provisional Application No. 62/988,320, filed Mar. 11, 2020, U.S. Provisional Application No. 62/991,906, filed Mar. 19, 2020, U.S. Provisional Application No. 62/993,222, filed Mar. 23, 2020, U.S. Provisional Application No. 62/994,165, filed Mar. 24, 2020, and U.S. Provisional Application No. 63/001,291, filed Mar. 28, 2020, the contents of which are incorporated herein in their entirety. 
    
    
     BACKGROUND 
     The present invention relates to a rapid test device that provide rapid detection of substances, including those involved in pathogen infection, for example, using Microscale Affinity Chromatography (MAC), indirect ELISA, and optical molecular sensing technology. 
     For example, COVID-19, a disease caused by the novel coronavirus (SARS-CoV-2) that was first reported from Wuhan, China, on Dec. 31, 2019, has been declared an international pandemic by the World Health Organization. The virus is spread between people who are in close contact with one another through respiratory droplets produced by coughing or sneezing. The incubation period of COVID-19 is approximately 14 days, making it difficult to contain and contributing to its rapid spread. Current detection techniques rely on RT-PCR, which is a lengthy process, necessitating well-equipped laboratories and skilled personnel to perform the technique. Rapid screening methods are needed to detect the disease at points of entry, transportation hubs, schools, hospitals, and other areas at high risk for communicating the disease in order to limit the spread of the virus. Furthermore, rapid methods of assessing a person&#39;s immunity to the virus are becoming increasingly important as people plan for returning to schools and workplaces after the peak of the epidemic. The need to assess the efficacy of vaccines in development has been recognized and is essential in assessing the “herd immunity” to prevent a resurgence of the pandemic. 
     Accordingly, a need arises for techniques for rapid detection of substances, including those involved in pathogen infection. 
     SUMMARY 
     Embodiments may include a rapid test device that provide rapid detection of substances, including those involved in pathogen infection, for example, using Microscale Affinity Chromatography (MAC), indirect ELISA, and optical molecular sensing technology. 
     For example, embodiments may provide pathogen detection using a marked target substance and fluorescence detection for high sensitivity and fast test time. Such test may be on the order of seconds or minutes instead of hours. An embodiments of a test device may be compact and cost effective and may not need to be cleaned or serviced between tests. Embodiments may include sample cartridges that are pre-filled with the necessary compounds and are ready to accept a liquid test sample for immediate testing. 
     For example, in an embodiment, a system may comprise a cartridge comprising a first chamber configured to receive a test sample including a target substance, the first chamber pre-filled with micromagnetic particles treated so as to bind to the target substance, and a first reservoir pre-filled with at least one reagent labeled with at least one fluorescent compound, the reagent adapted to bind to micromagnetic particles that are bound to the target substance, and an apparatus comprising a light source disposed so as to excite the least one fluorescent compound with a light and an optical sensor disposed so as to detect an emitted spectrum of light from the excited least one fluorescent compound. 
     In embodiments, the light source may comprise at least one excitation light emitting diode disposed so as to excite fluorescence of the least one fluorescent compound. The optical sensor may comprise at least one photodiode disposed so as to detect the excited fluorescence of the least one fluorescent compound and to output a signal representing the detected fluorescence. The first reservoir may be pre-filled with a plurality of reagents each labeled with a different one of a plurality of fluorescent compounds. The light source may comprise a plurality of excitation light emitting diodes disposed so as to excite fluorescence of the plurality of fluorescent compounds. The optical sensor may comprise at least one photodiode disposed so as to detect the excited fluorescence of the plurality of fluorescent compounds and to output signals representing the detected fluorescences. Each of the plurality of fluorescent compounds may have a wavelength range that does not overlap with at least some of the others of the plurality of fluorescent compounds. The apparatus may further comprise a loading bay disposed on the apparatus to receive the cartridge, a door disposed on the apparatus to cover the loading bay, a plurality of prongs disposed on an interior of the door to provide actuation force to dispense blister reservoirs disposed on the cartridge when the door is closed, and a device disposed relative to the cartridge to move at least a portion of contents of the cartridge among a plurality of chambers of the cartridge. The device disposed relative to the cartridge to move at least a portion of the contents of the cartridge among chambers of the cartridge may comprise a motor having disposed on a drive shaft thereof a pinion, a rack meshed with the pinion, and a magnet disposed on the pinion and disposed relative to the cartridge so as to move at least a portion of the contents of the cartridge among chambers of the cartridge when the motor is driven. Driving of the motor may be controlled by a computer system comprising a processor, memory to store program instructions and data and accessible by the processor, and program instructions stored in the memory and executable by the processor to control driving of the motor. 
     In an embodiment, a method may comprise illuminating at least one fluorescent compound with a light from a light source so as to excite the least one fluorescent compound and detecting an emitted spectrum of light from the excited least one fluorescent compound with an optical sensor, wherein the at least one fluorescent compound is contained in a cartridge comprising a first chamber configured to receive a test sample including a target substance, the first chamber pre-filled with micromagnetic particles treated so as to bind to the target substance and a first reservoir pre-filled with at least one reagent labeled with the at least one fluorescent compound, the reagent adapted to bind to micromagnetic particles that are bound to the target sub stance. 
     In embodiments, the light source may comprise at least one excitation light emitting diode disposed so as to excite fluorescence of the least one fluorescent compound. The optical sensor may comprise at least one photodiode disposed so as to detect the excited fluorescence of the least one fluorescent compound and to output a signal representing the detected fluorescence. The first reservoir may be pre-filled with a plurality of reagents each labeled with a different one of a plurality of fluorescent compounds. The light source may comprise a plurality of excitation light emitting diodes disposed so as to excite fluorescence of the plurality of fluorescent compounds. The optical sensor may comprise at least one photodiode disposed so as to detect the excited fluorescence of the plurality of fluorescent compounds and to output signals representing the detected fluorescences. Each of the plurality of fluorescent compounds may have a wavelength range that does not overlap with at least some of the others of the plurality of fluorescent compounds. The method may further comprise moving at least a portion of the contents of the cartridge among a plurality of chambers of the cartridge using apparatus comprising a motor having disposed on a drive shaft thereof a pinion, a rack meshed with the pinion, and a magnet disposed on the pinion and disposed relative to the cartridge so as to move at least a portion of the contents of the cartridge among chambers of the cartridge when the motor is driven. Driving of the motor is controlled by a computer system comprising a processor, memory to store program instructions and data and accessible by the processor, and program instructions stored in the memory and executable by the processor to control driving of the motor. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The details of the present invention, both as to its structure and operation, can best be understood by referring to the accompanying drawings, in which like reference numbers and designations refer to like elements. 
         FIG. 1  is an exemplary schematic representation of viral detection. according to embodiments of the present techniques. 
         FIG. 2  are exemplary schematic representations of target antibody immobilization on a surface according to embodiments of the present techniques. 
         FIG. 3  is an exemplary block diagram of Microscale Affinity Chromatography (MAC) according to embodiments of the present techniques. 
         FIG. 4  is an exemplary schematic representation of optical detection according to embodiments of the present techniques. 
         FIG. 5  is an exemplary schematic representation of optical detection according to embodiments of the present techniques. 
         FIG. 6  is an exemplary flow diagram of a testing process according to embodiments of the present techniques. 
         FIG. 7 a    is an exemplary top view of sample test cartridge according to embodiments of the present techniques. 
         FIG. 7 b    is an exemplary bottom view of sample test cartridge according to embodiments of the present techniques. 
         FIG. 8  is an exemplary illustration of a cartridge movement apparatus according to embodiments of the present techniques. 
         FIG. 9  is an exemplary illustration of an embodiment of fluorescence detection according to embodiments of the present techniques. 
         FIG. 10  is an exemplary block diagram of a test cartridge according to embodiments of the present techniques. 
         FIG. 11  is an exemplary block diagram of a testing device according to embodiments of the present techniques. 
         FIG. 12 a    is an exemplary illustration of a front panel of a testing device according to embodiments of the present techniques. 
         FIG. 12 b    is an exemplary illustration of a front panel of a testing device according to embodiments of the present techniques. 
         FIG. 13 a    is an exemplary internal view of a testing device according to embodiments of the present techniques. 
         FIG. 13 b    is an exemplary internal view of a testing device according to embodiments of the present techniques. 
         FIG. 14  is an exemplary block diagram of a testing system according to embodiments of the present techniques. 
         FIG. 15  is an exemplary block diagram of a computing device, in which processes involved in the embodiments described herein may be implemented. 
         FIG. 16  is an exemplary block illustration of antibody types present at different stages of disease, as may be utilized by embodiments of the present techniques. 
         FIG. 17  is an exemplary internal view of a testing device according to embodiments of the present techniques. 
         FIG. 18  is an exemplary block diagram of a process of flow-based sandwich immunoassay according to embodiments of the present techniques. 
         FIG. 19  is an exemplary illustration of an apparatus for test sample collection according to embodiments of the present techniques. 
         FIG. 20  is an exemplary illustration of a cartridge for test sample analysis according to embodiments of the present techniques. 
         FIG. 21  is an exemplary illustration of a cartridge for test sample analysis according to embodiments of the present techniques. 
         FIG. 22  is an exemplary flow diagram of reagent processing according to embodiments of the present techniques. 
         FIG. 23  is an exemplary illustration of a cartridge for test sample analysis according to embodiments of the present techniques. 
         FIG. 24  is an exemplary illustration of a cartridge for test sample analysis according to embodiments of the present techniques. 
         FIG. 25  is an exemplary illustration of an analyzer device according to embodiments of the present techniques. 
         FIG. 26  is an exemplary illustration of an analyzer device according to embodiments of the present techniques. 
         FIG. 27  is an exemplary illustration of an analyzer device according to embodiments of the present techniques. 
         FIG. 28  shows exemplary flow diagrams of processes of operation of an analyzer device according to embodiments of the present techniques. 
         FIG. 29  is an exemplary flow diagram of a process of operation according to embodiments of the present techniques. 
         FIG. 30 a    is a portion of an exemplary flow diagram of a process of operation according to embodiments of the present techniques. 
         FIG. 30 b    is a portion of an exemplary flow diagram of a process of operation according to embodiments of the present techniques. 
         FIG. 30 c    is a portion of an exemplary flow diagram of a process of operation according to embodiments of the present techniques. 
         FIG. 31  is exemplary format of a memory of a cartridge authentication chip according to embodiments of the present techniques. 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments may include techniques that provide, rapid detection of COVID-19 infection and techniques for rapid assessment of a person&#39;s immunity to the virus. For example, embodiments of the present techniques may provide rapid, accurate antibody and viral load testing using Microscale Affinity Chromatography (MAC), indirect ELISA, and optical molecular sensing technology. 
     Due to the nature of the coronavirus and its ability to spread quickly, there is an immediate need for a portable, instant, non-invasive test that does not require skilled technicians or lab equipment. Currently, only patients who experience severe symptoms are tested in hospitals because tests are too expensive and short in supply. Meanwhile, unscreened patients who have mild or no symptoms continue their daily life, increasing the scale of contamination. The current gold standard for SARS-CoV-2 detection is real time RT-PCR (reverse transcription-polymerase chain reaction). The drawbacks of RT-PCR are multi-faceted in that the equipment is expensive, conducting tests requires expertise, and results take hours to acquire. A complete reaction can be performed in as little as 4 hours, however, the collection of samples, transportation to the lab, preparation of equipment and analysis of results mean significantly longer time is required. Increasing demand for samples to be tested, along with a limited supply of reagents, skilled staff and equipment, can extend the entire RT-PCR process from sample collection to final result to several days. Furthermore, not only is RT-PCR time consuming (average turnaround 3-6 days), but it is also highly labor and cost intensive charging patients and insurance companies up to $4,000 per test. Therefore, there is a recognized need for more rapid, inexpensive tests. 
     More rapid diagnostic tests have recently come to the market. However, these tests lack reproducibility and have a higher risk of providing false positives. These technologies also utilize forms of sampling that require proximity to the patient, leaving healthcare workers at a higher risk of contracting the disease, such as a nasotracheal swab or a blood sample. The risk to healthcare workers can be minimized by simply acquiring a pooled saliva sample, as SARS-CoV-2 has been detected in saliva of infected patients. 
     Additionally, evidence shows that convalescent plasma from patients who have recovered from viral infections can be used as a treatment for infection without severe adverse events. The rationale behind convalescent plasma therapy is that the antibodies from a recovered patient&#39;s serum might suppress viraemia in an ill patient. The ability to quickly screen recovered patients and administer their convalescent plasma to ill patients is an area lacking in proper diagnostic tests. 
     After the threat of the pandemic has been mitigated, a need to screen the population to assess the efficacy of vaccines towards the virus to build “herd immunity” has been recognized. Herd immunity (also called community immunity) is an important mechanism by which the larger community is protected. For some diseases, if enough people are immune, transmission of the disease is reduced or eliminated. In the case of protecting against a resurgence of another global pandemic, herd immunity will need to be assessed to determine vulnerable sites with the potential to become hotspots. 
     Embodiments of the present techniques may include a process to rapidly detect COVID-19 through an alternative method of testing crude saliva samples. Embodiments may utilize lab-on-a-chip technology, where a patient sample containing saliva may be analyzed for antibodies capturing the virus with a simultaneous release of a fluorescent marker ligand. The lab on a chip technology may have the capability to produce a read out in real time using a fluorescent marker passing through a fluorometer. The lab on a chip technology may provide reusable capability, allowing for multiple testing opportunities from one device. 
     Embodiments may provide a completely self-contained device, with single-use sampling chips, which will reduce the risk of carry-over and minimize error as the sample does not have to be handled after it is loaded. All reagents needed for buffering, dilutions, or washing may be fully contained in a multiple use cartridge, which can be replaced or refilled as needed. Embodiments may be utilized at transportation hubs, such as airports, schools, clinics, and hospitals to limit the spread of the virus, and the technology may be expanded to screen for immunity towards other infectious diseases. In embodiments, the sampling chips and devices may be designed to concurrently detect the presence of both antibodies and virus in the same sample by utilizing the methods described below on the same sampling chip and in the same device. 
     In embodiments, the testing device may be an automated sandwich immunoassay with fluorescence detection intended for detection of IgA, IgM, IgG antibodies toward SARS-CoV-2 in patients with an active immune response to the virus. Results may identify an immune response to SARS-CoV-2. The screened antibodies are generally detectable in saliva during all phases of infection. Positive results from the embodiments of the testing device are indicative of an active immune response to the virus, that is, that an individual has been infected with SARS-CoV-2 and facilitated a targeted immune response to the virus. 
     In embodiments, the testing device is intended for use by consumers, clinicians, and point-of-care facilities. In embodiments, the testing device may be used by a variety of consumers in both traditional healthcare settings and elsewhere, for example, in clinics and hospitals, point of entry locations, and private settings, such as at home and in commercial locations. A trained technician is not required to operate the device, and results can easily be read by both licensed healthcare professionals and private individuals. Such access significantly increases the likelihood to determine transmission, resulting on an immediate impact on local and global health. 
     Embodiments may include a self-contained device that may utilize single-use disposable test cartridges, containing microfluidic chips. Each disposable test cartridge may be self-contained, and the sample may be magnetically moved through the cartridge&#39;s microfluidic chip. The test cartridge comes with all necessary reagents, and cleaning supplies are not applicable due to the disposable nature of the cartridges. Additional testing cartridges can be ordered as needed. 
     Embodiments may include a testing device utilizing automated sandwich immunoassay with fluorescence detection in a microfluidic device for the detection of anti-SARS-CoV-2 IgA, IgM, and/or IgG antibodies in saliva samples. S1 protein from the SARS-CoV-2 virus or a mixture of proteins or their subunits from the virus may be immobilized on, for example, magnetic particles. The magnetic particles may be moved through the use of electromagnets to mix with the saliva sample. In embodiments, magnetic particles that may be used may include, for example, Epoxy Silica Magnetic Particles, 6 μm, Ni-NTA Silica Magnetic Particles, 6 μm, etc. 
     Embodiments may use the principle of a sandwich immunoassay on magnetic beads. Spike protein specific to the SARS-CoV-2 virus or a subunit of this protein may be immobilized on the surface of silica-coated magnetic beads. Anti-SARS-CoV-2 IgA, IgM, or IgG antibodies from the saliva sample will attach to the antigen immobilized on these beads. Examples of the antigen may include a recombinant SARS-CoV-2 spike protein or the s1 subunit of the spike protein, etc., these proteins may be his-tagged. The magnetic particles will be moved through the cartridge to mix with secondary antibodies labeled with a fluorescent molecule. Examples of fluorescent molecules may include QUANTABLU™ Fluorogenic Substrate, having an excitation maximum at about 325 nm and an emission maximum at about 420 nm, QUANTARED™ Enhanced Chemifluorescent Substrate having an excitation maximum at about 570 nm and an emission maximum at about 585 nm, fluorescein: having an excitation maximum in a range of about 475-495 nm and an emission maximum in a range of about 510-520 nm, etc. The anti-IgA, anti-IgM, and anti-IgG secondary antibodies will serve to detect the binding of antibodies from a positive sample to the immobilized spike protein. Examples of antibodies may include anti-Goat IgG, anti-Goat IgG-FITC labeled, anti-human IgG, anti-human IgM, anti-rabbit IgG, anti-rabbit IgG-FITC labeled, etc. After a wash step with a neutral buffer, to remove excess antibodies, the beads may be moved under a fluorescence detector in the device to detect the signal. Examples of buffers may include tris-buffer, pH ˜7 (for wash), phosphate buffer, pH ˜7 (for wash), etc. A solution of 10 N HCl may be used for regeneration of the cartridge if desired. 
     The antibodies raised against SARS-CoV-2 that are present in the saliva from patients with an active immune response to the virus will bind to the immobilized antigen on the particles. The magnetic particles will then be mixed with a fluorescent-labeled secondary antibody, which will bind to anti-virus antibodies, which were present in the sample. In embodiments, the magnetic particles will then be passed into a washing area, then passed under a fluorescent detector, which will detect the signal. 
     In embodiments, the results may be indicated by a colored light, such as green, yellow or red. Green indicates a positive result, meaning anti-SARS-CoV-2 antibodies have been detected at or above a level that represents current or past infection by SARS-CoV-2 and an active immune response. Yellow indicates an indeterminate result (i.e., user error), and red indicates a negative result, meaning that no anti-SARS-CoV-2 antibodies are present. 
     In embodiments, testing device capacity may be, for example, several hundred tests per day. In embodiments, the total time required to perform the test may be, for example, five (5) minutes. In embodiments, the number of tests that can be performed per testing device may be one per run. 
     In embodiments, the estimated shelf life of reagents may be approximately six (6) months with refrigeration. Without refrigeration, shelf life is expected to be shortened, but still likely to be suitable for intermediate to long-term use, for example, approximately three (3) to six (6) months. In embodiments, nM concentrations of antibodies may be used, which are typical concentrations for enzyme based assays. Specific materials to be used may include IgA, IgM, and/or IgG antibodies. Cross-reactivity with other pathogens or antibodies towards other pathogens is not expected. The S1 subunit of the SARS-CoV-2 virus is expected to react specifically with antibodies against the virus. 
     Detection of Antibody. Embodiments may provide a rapid COVID-19 screen that utilizes a sandwich assay or indirect enzyme-linked immunosorbent assay (ELISA) method. A sandwich immunoassay is a method using two antibodies, which bind to different sites on the antigen or ligand, as shown in  FIG. 1 . The capture antibody, which is highly specific for the antigen, is attached to a solid surface. The antigen is then added, followed by addition of a second antibody referred to as the detection antibody. The detection antibody binds the antigen at a different epitope than the capture antibody. As a result, the antigen is ‘sandwiched’ between the two antibodies. 
     An overview of a flow-based sandwich immunoassay process  1800  according to the present systems and methods is shown in  FIG. 18 . In process  1800 , a Sandwich Immunoassay may be conducted based on two antibodies and binding agents to measure a target compound located in the cartridge of the device. At  1802 , a sample may be sample with a first binding agent on a support in a flow-based system. For example, The first binding agent, a COVID-19 (SARS-CoV-2) specific antigen, may be attached to magnetic beads and may be used to capture the anti-COVID-19 IgA, IgM, and IgG antibodies from the saliva sample. At  1804 , one or more secondary labeled antibodies may be added (this may instead be done at  1082 ). Each secondary antibody may contain a fluorescent label that will be used to detect and measure the amount of the capture agent. At  1806 , labeled antibodies may be detected, either on the support or after their release with an elution buffer. This approach is highly advantageous as it allows for the detection of any stage of infection (early/late/resolved) by identifying at least three isotypes of antibodies. 
     As shown in  FIG. 1 , a crude saliva sample  102  may be injected into a capillary  104  etched onto a silica microfluidic chip  106 . For example, saliva may be collected into a 1 mL sterile tube, such as an Eppendorf tube, and transferred with a sterile plastic Pasteur pipette, which will be included with the cartridge. This device is already FDA cleared and widely available. In embodiments, once the cartridge with the saliva sample is put into the device, the remaining steps may be performed automatically by the device. The anti-SARS-CoV-2 antibodies in the sample will be selectively separated by the magnetic beads with spike protein antigen attached. Any particulate matter will be left behind in the initial port where the sample is introduced. In embodiments, there is no sample preparation that needs to be done by the user. 
     Capillary  104  contains an immobilized SARS-CoV-2 specific antigen  108  to detect SARS-CoV-2 specific antibodies in the sample. If a sample contains antibodies towards the virus, indicating an immune response has occurred, these antibodies  110  will attach to the immobilized antigen. After the sample is run through the capillary, a detection reagent containing a secondary antibody  112  tagged with a fluorescent fluorophore  114  will be introduced. Secondary antibody  112  will recognize blood or saliva-borne antibodies  110  which have attached to immobilized antigen  108 . A fluorometer (not shown) will then detect whether secondary antibody binding has occurred. A fluorometer may be used to measure parameters of visible spectrum fluorescence such as its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. This is described further below with reference to  FIG. 4 . 
     This approach is highly advantageous as it allows for the detection of any stage of infection (early, late or resolved) by identifying three isotypes of antibodies. 
     Immobilization of Antigen. Embodiments may include disposable silica chips  106  be pre-packaged with SARS-CoV-2 specific antigen  108 , preferably the S1 protein, directly immobilized onto etched capillaries or onto magnetic particles or beads. Antigen will be diluted in binding solution (0.2 M carbonate-bicarbonate), added to the chip, and incubated. Deactivated surfaces will be used to prevent the need for a blocking step to prevent the non-specific binding of antibodies to the chip. The user need only collect a crude saliva sample, which can then be introduced to the column for detection. 
     Use of Saliva Sample. Not only is the use of pooled saliva less invasive than blood or nasopharyngeal swabs, but it also minimizes exposure for healthcare workers. Some virus strains have been detected in saliva as long as 29 days after infection. SARS-CoV-2 can present in the saliva in at least three ways. First, SARS-CoV-2 in the lower and upper respiratory tract can enter the oral cavity with the liquid droplets frequently exchanged by these organs. Second, SARS-CoV-2 in the blood can access the mouth via crevicular fluid. Third, major- and minor-salivary gland infection, with subsequent release of SARS-CoV-2 particles in saliva via salivary ducts can cause SARS-CoV-2 to present in the saliva. 
     Recent tests for other viral diseases, such as HIV, are employing saliva in a similar fashion as they have advantages over blood-based tests in terms of quality, rapidity and convenience. The sensitivity, specificity, positive predictive value and negative predictive value of such HIV tests may be quite high. 
     Pooled saliva samples can be used to detect both IgG and IgM antibodies, which pass into the mouth through the mucosa, and IgA which are secreted in the mouth. The production of IgM, IgA and IgG antibodies against COVID-19 were found in patient serum as early as day 1 after symptom onset. IgA antibodies were detected in 92.7% of patient samples collected within 0-7 days of symptom onset. IgM and IgA antibodies were both detectable at day 5, and the detection time of IgM, IgA, and IgG against COVID-19 ranged from day 1 to 39 PSO. 
     One study found the sensitivity and specificity of a lateral flow kit utilizing blood to detect COVID-19 IgM and IgG was 88.66% and 90.63%, respectively (Li, Z., et al., 2020). As the present techniques will additionally test IgA, and since the profile of antibodies in the saliva is similar to that in blood, embodiments of the present techniques should yield similar, if not higher, sensitivity and specificity, as well as a higher accuracy in detecting patients at any stage of infection. 
     Detection of Antibody. Along with the crude saliva sample, a detection reagent including a secondary antibody labeled with a fluorophore will be introduced to the chip. In embodiments, non-captured sample components and any non-bound secondary antibody can be effectively washed from the device using an application buffer, with the possible use of additives to minimize non-specific binding, as antibodies against the viral antigen are captured by the support. Alternatively, in embodiments, the sample may be applied first to the support, followed by application of the labeled secondary antibodies, with the non-captured or non-bound components again being washed from the support during this process. The conjugated antibodies that are used for this process may be obtained from existing sources or prepared according to well-established procedures for adding fluorescent tags to antibodies or other secondary binding agents. The performance of this device over extended use can be monitored by analyzing positive and negative control samples along with samples. 
     In addition to detecting immunity, embodiments may be used to verify vaccine immunogenicity. Embodiments may assist researchers in assessing whether the correct antibody profile necessary to protect a patient from reinfection is present. The overall estimated effectiveness of seasonal influenza vaccine for preventing medically attended, laboratory-confirmed influenza virus infection in the 2019 to 2020 flu season was only 45% (Dawood, F. S., et al.). In clinical trials, this method can be used to confirm that antibodies have been raised to the pathogen, meaning the patient has had effective coverage from infection with the pathogen, and has been applied in confirming the effectiveness of the rabies vaccine in dogs and cat (Servat, A., et al., 2007). In addition, immunity to multiple diseases can be screened using a tunable laser and different fluorescent markers. 
     Rapid COVID-19 Viral Detection. Embodiments may combine viral detection and antibody detection onto one device, or embodiments may provide stand-alone viral or antibody detection product depending on the needs of the market. In embodiments, a rapid COVID-19 viral detection test may employ Microscale Affinity Chromatography (MAC) technology, a separation technique that combines the specificity of antibody recognition and binding with the power, efficiency and speed of modern liquid-phase separations. An example of Microscale Affinity Chromatography  300 , according to embodiments of the present techniques, is shown in  FIG. 3 . A saliva sample  302  is loaded  304  onto a silica microfluidic chamber  306  etched with columns  308  on which primary antibodies  309  towards SARS-CoV-2 are immobilized. Low-affinity fluorophores  310  are weakly bound to these antibodies and are displaced  312  when a sample containing SARS-CoV-2 is introduced. Displaced fluorophores  312  can be considered an indication of a positive result. 
     In embodiments, antibodies  309  towards SARS-CoV-2 specific proteins will be immobilized on the surface of micro-capillaries  308 , microcolumns or etched silica channels on a lab-on-a-chip technology and then tagged with a low affinity competitive ligand  310  containing a fluorophore. A crude patient sample  302 , such as saliva, oral or nasopharyngeal swab or blood, can be introduced onto the channel  308  and migrated through the device either by electric charge or a flow system. If SARS-CoV-2 is present in the sample, the virus antigens will be captured on the columns  312  by the antibodies  309  releasing the low affinity ligand  310  to the end of the column. The viral antigens in sample  302  have a higher affinity or liking for the antibodies  309  and therefore will bind to the antibodies  309  and elute the ligand  310  which has a lower affinity. If the low affinity ligands  310  are released from the antibodies  309 , a fluorescent marker ligand  310  will be released to indicate a positive sample. If no fluorescence is seen, the sample is negative, because the low affinity ligand  310  was never released from the antibodies. 
     Sampling. Due to ease of sampling, and lack of necessity for sample preparation, embodiments may use a crude saliva sample  302 . The subject will spit into a tube, and a plastic pipette or dropper may be used to transfer the saliva to the etched chip  306 . Embodiments may use alternative sampling techniques, such as a nasopharyngeal swab, oral swab or blood sample. In embodiments, a breathalyzer may be interfaced with the COVID-19 detection device. In embodiments, samples from surfaces or air may also be tested using swab methods, or air sampling methods, respectively. 
     Although the example of Microscale Affinity Chromatography shown in  FIG. 3  is described in terms of detection of SARS-CoV-2, the described techniques and apparatus are equally applicable to detection of other pathogens, such as viruses, bacteria, etc. 
     Antibody Immobilization to Silica Chip. Exemplary schematic representations of target antibody immobilization on surface, such as gold, using (a) direct target antibody  202 , (b) protein A/G-mediated  204 , and (c) secondary antibody-mediated immobilization strategies  206  are shown in  FIG. 2 . In embodiments, antibodies can be attached directly  202  to the walls of a capillary column or microchip channel, however, the orientation of the stationary antibody is key to the binding activity. Antibodies can be immobilized covalently, using a thiol group  208 , to a surface or connected to a solid support, although the oriented immobilization of antibodies is considered to be optimal for their effectiveness. An antibody is considered to be properly oriented and perfectly active  210  when the fragment crystallizable region (Fc), which has no antigen binding affinity, is immobilized on a surface, rather than the antigen-binding sites  214  being immobilized on the surface  212 . This situation can be produced by a covalent immobilization method, such as carbohydrate groups in an antibody&#39;s Fc region. Directly immobilized antibodies do not allow for specific orientation of the antibody, thus, embodiments may use other methods to immobilize antibodies to a silica microfluidic chip, as described below. 
     It is also possible to achieve proper immobilization through secondary molecule protein A/G-mediated immobilization  204 , or secondary Ab-mediated immobilization  206 . In protein A/G-mediated immobilization  204 , the biomolecules used for antibody immobilization are proteins A and G  216 . Protein A is the most successful surface protein able to bind with animal immunoglobulin G (IgGs), but is not effective in certain animal IgGs, such as goat, sheep, cow, and horse. Protein G reacts more with IgGs than protein A and reacts less with other antibody types. A recombinant protein A/G that combines four immunoglobulin-binding domains from protein A and two from protein G can be employed to modify silane-functionalized silicon nitride surfaces. 
     In Ab-mediated immobilization  206 , secondary antibodies  218  are attached to the support and used to recognize the Fc region of the primary antibodies against the target. In this situation, the binding ability of the secondary antibodies should match with the class or subclass of the primary antibody that is to be used immobilized. For example, if the primary antibody is one of mouse IgG subclasses or rabbit IgG, an anti-mouse IgG or anti-rabbit IgG could be used as secondary antibodies. After immobilizing the thiolated-secondary antibody on a silica surface, the target antibody can be captured by the secondary antibody in the correct orientation by binding between the Fab region of the secondary antibody and the Fc region of target antibody. 
     The example of target antibody immobilization on surface shown in  FIG. 2  is not described in terms of detection of any particular pathogen. Rather, the described techniques and apparatus are applicable to detection of many pathogens, such as viruses, such as SARS-CoV-2, bacteria, etc. 
     Primary Antibody. There are 4 conserved structural proteins across CoVs: the spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein. The S protein is responsible for binding to host cell receptors and viral entry to host cells. The M, E, and N proteins are part of the nucleocapsid of viral particles. S and N genes are under episodic selection as the virus is transmitted between humans. Mutations and adaptation in the S and N genes may affect virus stability and pathogenicity. 
     Embodiments may use a single primary antibody towards a conserved portion of any of the aforementioned proteins can be used, or embodiments may use a mixture of primary antibodies, which are specific to different mutations of the virus. Monoclonal antibodies towards a conserved portion of the S1 spike surface proteins of SARS-CoV-2 may be a primary target of embodiments. However S2, M, E, or N antibodies may be tested in embodiments. 
     An example of Microscale Affinity Chromatography with Competitive Affinity Ligand and optical sensing  400 , according to embodiments of the present techniques, is shown in  FIG. 4 . A saliva sample  402  is loaded  404  onto a silica microfluidic chamber  406  etched with columns  408  on which primary antibodies  410  towards SARS-CoV-2 are immobilized. In embodiments, a low-to-moderate affinity, competitive ligand fluorophore  412  will be attached to the primary antibody  410  before the sample  402  is run through the column  408 . This fluorescent compound will attach to the primary antibody immobilized on the chip, as visualized in  FIG. 2 . As the sample  402  is run  414  through the column  408 , SARS-CoV-2 within the sample will compete for binding to the primary antibody  410  and, because it has a higher affinity towards the antibody  410 , will displace the fluorophore  412 . The displaced fluorophore  412  will then be used as an indication of a positive result, and lack thereof can be regarded as a negative result. The concentrated sample may then be eluted  416  for analysis. 
     In embodiments, fluorescein, a xanthene dye that is highly fluorescent, and detectable even when present in minute quantities may be used. Embodiments may use one of numerous fluorescent markers that are available to serve this function. For example, fluorescein has an excitation maximum in a range of about 475-495 nm and emission in a range of about 510-520 nm, QUANTABLU™ Fluorogenic Substrate, has an excitation maximum at about 325 nm and an emission maximum at about 420 nm, QUANTARED™ Enhanced Chemifluorescent Substrate having an excitation maximum at about 570 nm and an emission maximum at about 585 nm, etc. 
     Rapid Optical Detection and Improved Sensing. In embodiments, after the concentrated sample containing the biomolecules of interest is eluted  416 , additional analysis may be performed using a novel lab-on-chip utilizing wavelength backscattering with at least two wavelengths of light. For example, a light source  418  capable of emitting at least two wavelengths of light, such as a plurality of light emitting diodes, laser diodes, or other lasers, or a tunable laser, may be used to illuminate the eluted sample, exciting the fluorophore  412  and causing light emission  420 . The emission spectrum of the light emitted  420  from the fluorophore  412  may be optically sensed  420  and analyzed by, for example, an optical sensing “box” or circuit  422 . Optical sensing circuit  422  may perform wavelength and amplitude analysis on the emitted light  420  and may determine the absence, presence, and/or quantity of SARS-CoV-2, or other pathogen, or antibody in the sample  402 . In embodiments, such determination may be made in optical sensing circuit  422  and communicated to a computing device  426 , such as a smartphone, tablet computer, laptop computer, personal computer, workstation computer, cloud computing service, etc. In embodiments, optical sensing circuit  422  generate data representing the performed wavelength and amplitude analysis and may transmit that data to a computing device  426 , such as a smartphone, tablet computer, laptop computer, personal computer, workstation computer, cloud computing service, etc., for determination of the absence, presence, and/or quantity of SARS-CoV-2, or other pathogen, or antibody in the sample  402 . 
     An example of an embodiment of detection of antibodies raised against SARS-CoV-2 with optical sensing  500 , according to embodiments of the present techniques, is shown in  FIG. 5 . It is best viewed in conjunction with  FIG. 6 , which is a flow diagram of an embodiment of the testing process  600 . Process  600  begins with  602 , in which a saliva sample  502  may be collected. The specimen volume may, for example, be less than 1 mL. For example, saliva may be collected into a 1 mL sterile tube, such as an Eppendorf tube, and transferred with a sterile plastic Pasteur pipette. In embodiments, once the cartridge with the saliva sample is put into the device, the remaining steps may be performed automatically by the device. 
     At  604 , particulate matter may be removed from the sample  502  during sample preparation. In embodiments, the anti-SARS-CoV-2 antibodies in the sample will be selectively separated by the magnetic beads with spike protein antigen attached. Any particulate matter will be left behind in the initial port where the sample is introduced. In embodiments, there is no sample preparation that needs to be done by the user. At  606 , a portion of saliva sample  502  may be introduced  504  into the cartridge loaded  506  including at least one silica microfluidic chamber  508 . For example, about 20-100 μL of the sample  502  may be introduced  504  into the cartridge  506 . In embodiments, only a crude approximation of the sample and insertion of the sample by a sterile disposable pipette is necessary. The device may only accept a controlled amount of sample (˜30 μL), so the amount of sample measured by the user and inserted into the device need to be exact. The pipette may collect at least 100 μL, which is more than needed. The excess saliva may be discarded with the pipette. 
     At  608 , cartridge  506  may be inserted into the device, and magnetic particles  510  in the cartridge may be moved to the sample in chamber  508  and mixed  512  with the sample. Magnetic particles  510  may have one or more antigens  514  to antibodies raised against SARS-CoV-2 immobilized on the particles. Antibodies present in the sample will bind to the antigens  514  immobilized onto magnetic particles  510 . Magnetic particles  510  may be moved by application of electric current by testing device circuitry to magnetic coils. In embodiments, the magnetic coils may be formed on cartridge  506 . In embodiments, the magnetic coils may be present in the test device and may be adjacent to or in the vicinity of cartridge  506 . 
     At  610 , secondary antibodies (such as IgA, IgM, and IgG)  516  labeled with a fluorescent compound  518 , such as fluorescein, QUANTABLU™, QUANTARED™, etc., will also be combined  520  with the magnetic particles and mixed to detect captured antibodies from the sample. At  612 , the magnetic particles  510  with attached IgA, IgM, and IgG  516  and fluorescent compound  518 , together indicates as  522 , may be moved to a washing station  524 , and washed  526  using a neutral buffer. At  614 , the magnetic particles, etc.  522  may be moved to a detection region  528  to obtain the signal  530  from the fluorescent compound  518  labelling the secondary antibodies  516  on the magnetic particles  510 . Antibody isotypes may be distinguished using the color or light emission spectrum of fluorescent compound attached to the secondary antibody. 
     In embodiments, fluorescein, a xanthene dye that is highly fluorescent, and detectable even when present in minute quantities may be used. Embodiments may use one of numerous fluorescent markers that are available to serve this function. For example, fluorescein has an excitation maximum in a range of about 475-495 nm and emission in a range of about 510-520 nm, QUANTABLU™ Fluorogenic Substrate, has an excitation maximum at about 325 nm and an emission maximum at about 420 nm, QUANTARED™ Enhanced Chemifluorescent Substrate having an excitation maximum at about 570 nm and an emission maximum at about 585 nm, etc. 
     In embodiments, analysis may be performed using a novel lab-on-chip utilizing wavelength backscattering with at least two wavelengths of light. For example, a light source  532  capable of emitting at least two wavelengths of light, such as a plurality of light emitting diodes, laser diodes, or other lasers, or a tunable laser, may be used to illuminate the washed sample  522 , exciting the fluorophore  518  and causing light emission  530 . The emission spectrum of the light emitted  530  from the fluorophore  518  may be optically sensed  534  and analyzed by, for example, an optical sensing “box” or circuit  536 . Optical sensing circuit  536  may perform wavelength and amplitude analysis on the emitted light  530  and may determine the absence, presence, and/or quantity and isotype of antibody in the sample  522 . In embodiments, such determination may be made in optical sensing circuit  536  and communicated to a computing device  538 , such as a smartphone, tablet computer, laptop computer, personal computer, workstation computer, cloud computing service, etc. In embodiments, optical sensing circuit  536  may generate data representing the performed wavelength and amplitude analysis and may transmit that data to a computing device  538 , such as a smartphone, tablet computer, laptop computer, personal computer, workstation computer, cloud computing service, etc., for determination of the absence, presence, and/or quantity of antibody in the sample  522 . 
     In embodiments, no interpretation of results is needed by the user. The response may be given by a green, yellow, or red indicator per antibody isotype. Green may indicate a positive result, meaning the antibody has been detected against SARS-CoV-2, representing current or past infection by SARS-CoV-2 and an active immune response. An indeterminate or inconclusive result may be shown with a yellow indicator, which is likely due to user error or device malfunction. Red may indicate a negative result, meaning no immune response to SARS-CoV-2 was detected. 
     In embodiments, the testing device may be a self-contained device that does not require a laboratory for interpretation of results. In embodiments, the positive and negative controls may either be included with the device for consumer use or may be directly built into the self-contained device. For example, a separate channel with magnetic particles labeled with a fluorescent molecule can act as an internal control, which will be directly built into the disposable testing cartridge. 
     Because each testing cartridge is a single use device, representative cartridges from a given batch may be tested with positive or negative controls and if these are found to be valid and acceptable, other cartridges in the batch may be used. This process can be performed periodically to confirm the validity of the devices in the same batch. The testing device itself may be tested using special-purpose cartridges. Different types of cartridges may have different authentication chips embedded in them, allowing the device to work in “live” or “testing” mode as required. Such authentication chips may include authentication circuitry, identification circuitry, data storage circuitry, etc., and may identify the type of chip, the type of testing being performed, the patient being tested, etc. 
     An example of a test cartridge  700  is shown in  FIGS. 7 a  and 7 b   . An exemplary top view of sample test cartridge  700  is shown in  FIG. 7 a   . In this example, test cartridge  700  may include inlet port  702 , mixer chamber  704 , wash chamber  706 , optional amplification chamber  708 , mixer reservoir  712 , wash reservoir  714 , and optional amplification reservoir  716 . Reservoirs  712 ,  714 , and  716  may contain solvents, reagents, buffers, etc., in hermitically sealed blisters for storage. Mixer reservoir  712  may be pre-filled with the necessary reagent solvent and micromagnetic beads or particles having antigens to antibodies raised against SARS-CoV-2 immobilized on the particles. Wash reservoir  1004   1005  may be pre-filled with buffer agent. Optional amplification reservoir  716  may be prefilled with enzymatic amplification agents or buffer. Test cartridge  700  may also include a plurality of passages, such as passage  718  between inlet port  702  and chamber  704 , passage  720  between chamber  704  and chamber  706 , and passage  722  between chamber  706  and chamber  708 . 
     In operation, the testing device may process test cartridge  702 , for example, as described in conjunction with  FIG. 6 . Process  600  begins with  602 , in which a saliva sample may be collected. At  606 , a portion of saliva sample may be introduced into via inlet port  702  into chamber  704 . At  608 , cartridge  700  may be inserted into the device, and magnetic particles or beads in chamber  704  may be mixed with the sample. The magnetic particles may have one or more antigens to antibodies raised against SARS-CoV-2 immobilized on the particles. Antibodies present in the sample will bind to the antigens immobilized onto magnetic particles. Mixing may be facilitated by movement of the magnetic particles. The magnetic particles may be moved by application of electric current by testing device circuitry to magnetic coils or by movement of a magnetic field produced by magnetic coils or by a permanent magnet relative to cartridge  700 , as described below. 
     At  610 , a reagent solvent in chamber  712 , including secondary antibodies (such as IgA, IgM, and IgG) labeled with a fluorescent compound, such as fluorescein, QUANTABLU™, QUANTARED™, etc., may be combined with the magnetic particles and mixed in chamber  704  to detect captured antibodies from the sample. At  612 , the magnetic particles with attached IgA, IgM, and IgG and fluorescent compound may be moved to a washing station in chamber  706 , and washed using a neutral buffer from chamber  714 . At  613 , the magnetic particles may optionally be moved into optional enzymatic amplification chamber  708  and optionally mixed with enzymatic amplification agents or buffer from optional amplification reservoir  716 . At  614 , the magnetic particles may be moved to a detection region in chamber  710  to obtain the signal from the fluorescent compound labelling the secondary antibodies  516  on the magnetic particles. Antibody isotypes may be distinguished using the color or light emission spectrum of fluorescent compound attached to the secondary antibody. 
     An exemplary bottom view of test cartridge  700  is shown in  FIG. 7 b   . In this example, test cartridge  700  may include Authentication Chip  718 . 
     An exemplary cartridge movement apparatus  800 , for performing reagent washing and mixing is shown in  FIG. 8 . In this example, apparatus  800  may include a servo motor  802 , a rack  804  and pinion  806  gear mechanism, and a permanent magnet  808 . Servo motor  802  may be controlled by control circuitry, as described below, and may turn pinion gear  806 , causing movement of rack  804  and thus, movement of permanent magnet  808 , which is attached to rack  804 . Movement of permanent magnet  808  may be used to move the magnetic particles in cartridge  700 . Permanent magnet  808  may, for example, be a neodymium alloy magnet. Using rack and pinion mechanism to move micromagnetic beads inside the cartridge to provide manipulation and movement of micromagnetic beads to achieve mixing and washing actions inside the cartridge reservoirs and for transporting the “washed” micromagnetic beads to the detection reservoir. 
     An exemplary embodiment of fluorescence detection apparatus  900  is shown in  FIG. 9 . As shown in this example, Multispectral photodiode chips  902  and excitation LEDs  904  may be arranged adjacent to the detection region in chamber  710 , which contains the washed magnetic particles with attached IgA, IgM, and IgG and fluorescent compound. Excitation LEDs  904 , as controlled by control circuitry, as described below, may illuminate the contents of chamber  710 , causing excitation of fluorescent compounds and emission of light from the fluorescent compounds. Multispectral photodiode chips  902  may receive and detect the emission spectrum of light emitted from the fluorescent compounds, which may be analyzed, for example, by optical sensing circuitry and/or computing devices, as described herein, to determine the presence or absence of COVID-19 infection, antibodies, etc. 
     An exemplary block diagram of a test cartridge  1000  is shown in  FIG. 10 . In this example, test cartridge  1000  is a disposable sample cartridge made, for example, from clear glass or silica, transparent acrylic, other plastic, or other transparent material. Cartridge  1000  may include mixer chamber  1002 , mixer reservoir  1003 , wash chamber  1004 , wash reservoir  1005 , optional amplification chamber  1006 , optional amplification reservoir  1007 , detection reservoir  1010 , and authentication chip  1008 . Reservoirs  1003 ,  1005 , and  1007  may contain solvents, reagents, buffers, etc., in hermetically sealed blisters for storage. Mixer reservoir  1003  may be pre-filled with the necessary reagent solvent and micromagnetic beads or particles having antigens to antibodies raised against SARS-CoV-2 immobilized on the particles. A patient saliva sample may be introduced into mixer chamber  1002  during testing and the reagent solvent and micromagnetic beads may be moved to mixer chamber  1002 . Antibodies present in the sample will bind to the antigens immobilized onto magnetic particles. Mixing may be facilitated by movement of the magnetic particles. The magnetic particles may be moved by application of electric current by testing device circuitry to magnetic coils or by movement of cartridge  1000  relative to a magnetic field produced by magnetic coils or by a permanent magnet. 
     Wash reservoir  1005  may be pre-filled with buffer agent. Micromagnetic beads may be magnetically transported from mixer chamber  1002  to wash chamber  1004 , and buffer agent may be moved to wash chamber  1004 . In wash chamber  1004 , the micromagnetic beads may be “cleaned” to ensure only bound analytes are detected. Optional amplification reservoir  1007  may be prefilled with enzymatic amplification agents or buffer. Micromagnetic beads may be magnetically transported from wash chamber  1004  to amplification chamber  1006 , and enzymatic amplification agents or buffer may be moved to amplification chamber  1006 . In amplification chamber  1006 , optional enzymatic amplification may be performed. Detection reservoir  1010  may be pre-filled with buffer agent. Micromagnetic beads may be magnetically transported from wash reservoir  1004  or optional amplification chamber  1006  to detection reservoir  1010 , where, for infected patients, fluorescence from micromagnetic beads may be detected by way of illuminating LEDs and multi-spectrum photodiodes, as described herein. 
     An exemplary block diagram of a testing device  1100  is shown in  FIG. 11 . In this example, testing device  1100  may include power supply  1102 , user interface  1104 , such as an LED display and membrane switch buttons, communication interface  1106 , such as a USB port or wireless communications adapter, microcontroller  1108 , servo motor drivers  1110 , detection and authentication circuitry  1112 , LED driver  1114 , sample excitation LEDs  1116 , servo motor, rack and pinion, and magnet assembly  1118 , light detector  1120 , and insertable sample cartridge(s)  1122 . Power supply  1102  may provide electrical power to the other components of testing device  1100  and may include batteries or other electrical and electronic components, such as voltage regulators to provide different voltage supply levels as needed. User interface  1104 , may include, for example, an LED display and membrane switch buttons, or other display and/or input/output devices. Embodiments may include other configurations of front panel, as well as other display devices, such as LCD displays, numeric displays, etc., which may display additional information, such as concentration, amount, percentage, etc., of antibodies, fluorescent indicator, threshold levels, etc. 
     Communication interface  1106 , may include, for example a USB port or wireless communications adapter, such as Wi-Fi, Bluetooth, cellular data, or other wireless communications technique. Microcontroller  1108  may include one or more processors, memory, input/output circuitry, and other circuitry to control the operation of testing device  1100  and to provide interfacing to external computers for data transfer of information including test results, patient information, etc., via USB port or wireless communications adapter and to provide interfacing to the authentication chip on the sample cartridge. Servo motor drivers  1110  may include electronic circuitry to provide electrical current to drive the operation of servo motor  1118  as controlled by microcontroller  1108 . Detection and authentication circuitry  1112  may include circuitry to interface microcontroller  1108  with authentication chip  718 ,  1008 , shown in  FIGS. 7 and 10 . Such authentication chips may include authentication circuitry, identification circuitry, data storage circuitry, etc., and may identify the type of chip, the type of testing being performed, the patient being tested, etc. Detection and authentication circuitry  1112  may allow microcontroller  1108  to access this information, allowing configuration of testing device  1100  based thereupon, for example, allowing the device to work in “live” or “testing” mode as required. 
     LED driver  1114  may include electronic circuitry to provide electrical current to drive the operation of sample excitation LEDs  1116 , as controlled by microcontroller  1108 . Sample excitation LEDs  1116  may provide light to excite the fluorescent compounds in the sample under test, as described herein. For example, sample excitation LEDs may emit light for excitation of fluorescent compounds at 500 nm wavelength, or other suitable wavelength for excitation of the fluorescent compounds. Multiple LEDs may be provided to increase the intensity, as well as to achieve a more uniform illumination of the sample. Servo motor, rack and pinion, and magnet assembly  1118  may include, for example, the apparatus shown in  FIG. 8 , and described in reference thereto. Light detector  1120  may include electronic circuitry to detect light emitted by the excited fluorescent compounds in the sample under test, as described herein. Light detector  1120  may, for example, include multispectral photodiodes chips, such as the AMS® multi-channel AS7265x chipset, or other suitable light detectors. Sample cartridge  1120  may include, for example, a test cartridge  700  similar to that shown in  FIGS. 7 a , 7 b   ,  9 ,  10 , etc. 
     An exemplary front view of a front panel  1200  of testing device  1100  is shown in  FIG. 12 a   . In this example, front panel  1200  may include test result indicators  1202 , test in progress indicator  1204 , and operation switch  1206 . In this example, test result indicators  1202  may include three by three LEDs (red, yellow, green) to provide intuitive test result based on predetermined threshold levels for IgA, IgM, and IgG. In embodiments, only one color (green, yellow, or red) will result for each antibody. For example, a green light may indicate the user sample is positive for the respective antibody, a red light may indicate the user does not have the respective antibody in high enough quantities to be detected by the device, a yellow light may indicate an indeterminate result. Research may show (for example, see  FIG. 16  and Table 1) that IgA and IgM are produced and persist in early stages of the disease (Days 0-14), and IgG is produced starting on Day 0 of the disease and levels plateau after Day 20 (Guo, et al., 2020). 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
             
            
               
                   
                 IgA 
                 early stage 
               
               
                   
                 IgM 
                 early stage 
               
               
                   
                 IgA + IgM 
                 early stage 
               
               
                   
                 IgA + IgG 
                 early stage 
               
               
                   
                 IgM + IgG 
                 early stage 
               
               
                   
                 IgA + IgM + IgG 
                 early stage 
               
               
                   
                 IgG 
                 late stage 
               
               
                   
                   
               
            
           
         
       
     
     This may roughly imply that a positive for IgA or IgM means the patient is in early stages of infection, and the presence of IgG alone means the patient is in later stages of infection. Thus, embodiments may detect which of the patients has a mounted immune response to the virus. As the test will be able to detect which antibodies bind to the spike protein, which is the protein that binds to the ACE receptors in the lungs to cause infection, this test may be able to detect which patients have raise neutralizing antibodies toward the virus. 
     Returning to  FIG. 12 a   , test in progress indicator  1204  may include a status LED indicating the test in progress. Operation switch  1206  may include a membrane switch for an operator to start the test process. Embodiments may include other configurations of front panel, as well as other display devices, such as LCD displays, numeric displays, etc., which may display additional information, such as concentration, amount, percentage, etc., of antibodies, fluorescent indicator, threshold levels, etc. 
     An exemplary rear view of a front panel  1200  of testing device  1100  is shown in  FIG. 12 b   . In this example, front panel  1200  may include a plurality of spring-backed actuators  1208  in a closable door portion  1210  of front panel  1200  to apply pressure, when the door is closed, to the reservoir blisters and move the reagents, solvents, and buffers, etc. into their associated chambers. 
     An exemplary internal view of testing device  1100  is shown in  FIGS. 13 a  and 13 b   . In this example, USB-B connector  1302 , authentication chip reader connector  1304 , and cartridge holder tray  1306  are shown. 
     An exemplary external view of testing device  1100  is shown in  FIG. 17 . In this example, after a sample cartridge is inserted into testing device  1100 , door  1210  may be closed and a latch  1702  may be activated, preventing removal of the cartridge during the test. When door  1210  is closed, spring-backed actuators  1208 , shown in  FIG. 12 b   , may apply pressure to the reservoir blisters and move the reagents, solvents, and buffers, etc. into their associated chambers. 
     An exemplary testing system  1400  is shown in  FIG. 14 . System  1400  may include testing device  1402 , computing device  1404 , network  1406 , and cloud computing system  1408 . Testing device  1402  may be a stand-alone or integrated device and may include testing hardware  1410 , control circuitry  1412 , and communications circuitry  1414 . Testing hardware  1410  may include the components shown in  FIGS. 3, 4 , and/or  5 , as described above, and that performs the testing functions described above. Testing hardware  1410  may include components to interface with an inserted test cartridge  1420 , which may include test components  1422 , such as a microfluidic silica chip with etched capillaries and chambers, and circuitry  1424 , which may include authentication circuitry, identification circuitry, data storage circuitry, etc. Testing hardware  1410  may further include components such as circuitry to apply, and to control the application of, electric current to magnetic coils that may be formed on cartridge  1420  or that may be present in the testing device  1402  and may be adjacent to or in the vicinity of cartridge  1420 . Control circuitry  1412  may include control logic, controller, or processor circuitry to control performance of the physical, optical, electrical, and computing processes involved in operating testing device  1402 , such as controlling the electric current in the magnetic coils, and in performing the functions described above. Communications circuitry  1414  may include circuitry to provide wired and/or wireless communications with one or more external or integrated devices, such as computing device  1404 . In embodiments, testing device  1402  may also include interface  1415 , which may include indicator or display components for direct display of test results from testing device  1402  and/or buttons, etc., for direct entry of information into testing device  1402 . 
     Computing device  1404  may be an integrated or stand-alone device, such as a smartphone, tablet computer, laptop computer, personal computer, workstation computer, cloud computing service, etc., to communicate with testing device  1402 . Computing device  1404  may provide processing and analysis of data received from testing device  1402 , as well as communications with testing device  1402  and with cloud computing system  1408  vis network  1406 . In embodiments, computing device  1404  may also include indicator or interface displays (not shown) for display of test results from testing device  1402  and/or entry of information into testing device  1402 . Network  1406  may be any public or proprietary LAN or WAN, including, but not limited to the Internet, carrier network, wireless network, etc. Cloud computing system  1408  may provide on-demand availability of computer system resources, such as database storage  1416  and computing power/data analysis  1418 , which is typically implemented in data centers available to many users over the Internet. 
     In embodiments, the optical analysis may result in imaging representative of sample  402 . The resulting imaging, using machine learning techniques, may be used to detect protein structure geometry (Daaboul, G. G., et al. 2017). Then, combined inputs from chemical lab-on-chip sensors and the optical sensors, both included in testing hardware  1410  may be analyzed  1418 , for example, using cloud computing system  1408 , using both commonly available and proprietary Artificial Intelligence/Machine Learning (AI/ML) architectures, such as a Deep Cognitive Neural Network (DCNN), such as that described in U.S. Patent Application Publication No. 2019/0156189, published May 23, 2019, which is hereby incorporated by reference herein. Machine learning algorithms may be trained to detect the coronavirus family of viruses, and over time, using additional sample data, will become more effective at identifying different strains of the virus. 
     In embodiments, the machine algorithms may be further trained to detect other classes of viruses and specific strains of viruses or other pathogens, or trained for detection of neurodegenerative disease markers. The computing device  1404  computational platform may interface with a cloud computing platform  1408 , opening up applications using anonymized patient and third party data. 
     An exemplary block diagram of a computing device  1500 , in which processes involved in the embodiments described herein, such as computing device  1406  or cloud computing system  1408 , may be implemented, is shown in  FIG. 15 . Computing device  1500  may be implemented using one or more programmed general-purpose computer systems, such as embedded processors, systems on a chip, personal computers, workstations, server systems, and minicomputers or mainframe computers, or in distributed, networked computing environments. Computing device  1500  may include one or more processors (CPUs)  1502 A- 1502 N, input/output circuitry  1504 , network adapter  1506 , and memory  1508 . CPUs  1502 A- 1502 N execute program instructions in order to carry out the functions of the present communications systems and methods. Typically, CPUs  1502 A- 1502 N are one or more microprocessors, such as an INTEL CORE® processor.  FIG. 15  illustrates an embodiment in which computing device  1500  is implemented as a single multi-processor computer system, in which multiple processors  1502 A- 1502 N share system resources, such as memory  1508 , input/output circuitry  1504 , and network adapter  1506 . However, the present communications systems and methods also include embodiments in which computing device  1500  is implemented as a plurality of networked computer systems, which may be single-processor computer systems, multi-processor computer systems, or a mix thereof. 
     Input/output circuitry  1504  provides the capability to input data to, or output data from, computing device  1500 . For example, input/output circuitry may include input devices, such as keyboards, mice, touchpads, trackballs, scanners, analog to digital converters, etc., output devices, such as video adapters, monitors, printers, etc., and input/output devices, such as, modems, etc. Network adapter  1506  interfaces device  1500  with a network  1510 . Network  1510  may be any public or proprietary LAN or WAN, including, but not limited to the Internet. 
     Memory  1508  stores program instructions that are executed by, and data that are used and processed by, CPU  1502  to perform the functions of computing device  1500 . Memory  1508  may include, for example, electronic memory devices, such as random-access memory (RAM), read-only memory (ROM), programmable read-only memory (PROM), electrically erasable programmable read-only memory (EEPROM), flash memory, etc., and electro-mechanical memory, such as magnetic disk drives, tape drives, optical disk drives, etc., which may use an integrated drive electronics (IDE) interface, or a variation or enhancement thereof, such as enhanced IDE (EIDE) or ultra-direct memory access (UDMA), or a small computer system interface (SCSI) based interface, or a variation or enhancement thereof, such as fast-SCSI, wide-SCSI, fast and wide-SCSI, etc., or Serial Advanced Technology Attachment (SATA), or a variation or enhancement thereof, or a fiber channel-arbitrated loop (FC-AL) interface. 
     The contents of memory  1508  may vary depending upon the function that computing device  1500  is programmed to perform. In the example shown in  FIG. 15 , exemplary memory contents are shown representing routines and data for embodiments of the processes described above. However, one of skill in the art would recognize that these routines, along with the memory contents related to those routines, may not be included on one system or device, but rather may be distributed among a plurality of systems or devices, based on well-known engineering considerations. The present systems and methods may include any and all such arrangements. 
     In the example shown in  FIG. 15 , memory  1508  may include, in the case of a testing device  1402 , testing control data and routines  1512 , testing analysis data and routines  1514 , in the case of a computing device  1404 , data analysis data and routines  1518 , and in the case of a cloud computing system  1408 , database data and routines  1520 , and data analysis data and routines  1522  and operating system  1524 . Testing control data and routines  1512  may include software routines to control performance of the physical, optical, electrical, and computing processes involved in operating testing device  1402 , as well as data obtained from such testing, as described above. Testing analysis data and routines  1514  may include software routines to perform initial analysis and derivation of data obtained from testing, as described above. Data analysis data and routines  1514  may include, which may include software routines to perform processing and analysis of data received from testing device  1402 , as described above. Authentication/matching routines  1514  may include modular proximity test routines  1518 , which may include software routines to perform modular proximity testing on received authentication data, as described above. Database data and routines  1520 , may include software routines to provide database storage of data on cloud computing system  1408 , as described above. Data analysis data and routines  1522  may include software routines to provide computing power/data analysis on cloud computing system  1408 , as described above. Operating system  1524  may provide overall system functionality. 
     As shown in  FIG. 15 , the present communications systems and methods may include implementation on a system or systems that provide multi-processor, multi-tasking, multi-process, and/or multi-thread computing, as well as implementation on systems that provide only single processor, single thread computing. Multi-processor computing involves performing computing using more than one processor. Multi-tasking computing involves performing computing using more than one operating system task. A task is an operating system concept that refers to the combination of a program being executed and bookkeeping information used by the operating system. Whenever a program is executed, the operating system creates a new task for it. The task is like an envelope for the program in that it identifies the program with a task number and attaches other bookkeeping information to it. Many operating systems, including Linux, UNIX®, OS/2®, and Windows®, are capable of running many tasks at the same time and are called multitasking operating systems. Multi-tasking is the ability of an operating system to execute more than one executable at the same time. Each executable is running in its own address space, meaning that the executables have no way to share any of their memory. This has advantages, because it is impossible for any program to damage the execution of any of the other programs running on the system. However, the programs have no way to exchange any information except through the operating system (or by reading files stored on the file system). Multi-process computing is similar to multi-tasking computing, as the terms task and process are often used interchangeably, although some operating systems make a distinction between the two. 
     The present invention may be a system, a method, and/or a computer program product at any possible technical detail level of integration. The computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present invention. The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. 
     The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire. 
     Computer readable program instructions described herein can be downloaded to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and/or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers, and/or edge servers. A network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device. 
     Computer readable program instructions for carrying out operations of the present invention may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, configuration data for integrated circuitry, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++, or the like, and procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program instructions may execute entirely on the user&#39;s computer, partly on the user&#39;s computer, as a stand-alone software package, partly on the user&#39;s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user&#39;s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present invention. 
     Aspects of the present invention are described herein with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and/or block diagrams, and combinations of blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer readable program instructions. 
     These computer readable program instructions may be provided to a processor of a general-purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function/act specified in the flowchart and/or block diagram block or blocks. 
     The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions/acts specified in the flowchart and/or block diagram block or blocks. 
     The flowchart and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the blocks may occur out of the order noted in the Figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions. 
     Embodiments may employ an automated sandwich immunoassay method. The assay may be accomplished using affinity capture of antibodies through an immunoassay approach on magnetic beads, and the steps of the sandwich assay may be automated by moving the beads through the separate reservoirs using a magnet. 
     For example, a test subject may provide a saliva sample of ≥0.2 mL by a passive drool method into a collection vial  1902 , shown in  FIG. 19 , with an attachable funnel  1904 . After collection, a cap containing reagents  1906  may be attached to the vial, bursting a foil cover in the process, and releasing magnetic particles and secondary antibodies into the sample. In a positive test, SARS-CoV-2 viral antigen, the S1 subunit of the spike membrane protein immobilized on the magnetic beads, will selectively bind to the SARS-CoV-2 antibodies present in the saliva sample and fluorescent-labeled secondary antibodies will attach to the captured antibodies, forming a sandwich immunocomplex. 
     After incubation with the reagents, as shown in  FIGS. 20, 21, 23, and 24  the vial  1902  may be attached to port  2001  of a cartridge  2000  to be inserted into the analyzer device for analysis. The cartridge may include two low-volume reservoirs  2002 ,  2004  separated by a valve  2006 , which are filled with buffer solution via blisters  2008 ,  2010 ,  2012 . The magnetic beads containing the immunocomplex are transported in steps through each wash buffer reservoir  2002 ,  2004  in order to remove any components of the saliva sample that do not bind to the immobilized protein  2002 , as well as excess secondary antibodies  2004 . The washed magnetic beads may then be transported to detection region  2014 , where the fluorescence of the fluorescent-labeled secondary antibodies may be excited and detected. For example, as shown in  FIG. 24 , spectrometers  2402 ,  2404 , and  2406  may be located to detect the fluorescence of the fluorescent-labeled secondary antibodies, and thus, the presence of pathogens or antibodies to the pathogens in the sample. 
     Within the reagent cap  1906 , four different types of fluorescent dye may be used, which have been selected to avoid overlap in their wavelength ranges, optimizing the precision of fluorescence readings for each labeled protein. One of the dyes may be conjugated to bovine serum albumin and immobilized on a portion of the beads contained in the reagent cap for use as an internal standard. The internal standard allows for precise calibration of the detector, and ensures that the analyzer is functioning properly during each test. 
     An exemplary flow diagram of reagent processing  2200  is shown in  FIG. 22 . In this example, raw materials  2202  may include unconjugated lgG, lgA, and BSA, lgM preconjugated with dye, Alexa Fluor labelling kits, SARS-COV-2 S1 protein, epoxy silica magnetic beads, etc. Protein labeling  2204  may include labeling lgG Antibody with Alexa Fluor Dye A, labeling lgA Antibody with Alexa Fluor Dye B, labeling BSA with Alexa Fluor Dye C, etc. Bead preparation  2204  may include affixing BSA labelled with Alexa Fluor C to epoxy silica magnetic beads, affixing S1 protein to epoxy silica magnetic beads, etc. Mixing  2206  may include mixing lgG labelled with Alexa Fluor A, Labelled lgM, lgA labelled with Alexa Fluor B, etc. to form Labeled Antibody Solution  2208 , and mixing BSA beads, S1 beads, etc., to form bead solution  2210 . 
     An exemplary embodiment of an analyzer device  2500  is shown in  FIGS. 25, 26, and 27 . Analyzer device  2500  may, for example, be assembled using a combination of 3D-printed and/or injection molded plastic materials, glass, mirror, microprocessors, and various electronic components. This low cost, portable device accepts the disposable cartridge  2000 , performs the automated assay, and displays the results on an integrated display  2502 , such as an LCD screen. Test results can also be output using the built-in USB-C port  2504  and on-board firmware which allows for interfacing with a laptop computer or a mobile device. The device uses a latchable door  2506  which holds the cartridge in place during the test, blocks ambient light from entering the detector area, and provides the actuation force to dispense the blisters. When the door is closed, the wash reservoirs fill, and the device performs automated test functions. Actuation of a permanent magnet within the device moves the magnetic particles; implementing mixing and washing actions, as well as transferring the particles between chambers and positioning them in the detection area. The device then performs the automated fluorescence detection process and outputs the results. 
       FIG. 26  further shows a loading bay or area  2602  for receiving a cartridge  2000 . Loading bay or area  2602  may include a bracket to hold cartridge  2000  during testing. Also shown is latchable door  2506  with the plurality of prongs that provide the actuation force to dispense the blisters. 
     In embodiments, the analyzer device  2500  may be a low cost, reusable unit that accepts the disposable cartridge  2000 , performs the automated assay, and displays the results on an integrated display  2502 , such as a black and white LCD. The device may operate on an internal 5V power supply which may be plugged into a wall outlet. A latching door on top of the unit may provide three functions: it may hold the cartridge in place during the test, it may block ambient light from entering the detector area, and it may provide the actuation force to dispense the blisters at the beginning of the assay. A permanent magnet  808 , shown in  FIG. 8 , may be moved under the cartridge with a linear actuator  804  to move the magnetic particles, implementing mixing and washing actions as well as moving the particles between chambers and positioning them in the detection area. An array of three LEDs may provide excitation light at frequencies specific to the label dyes in the cartridge. Optical short-pass filters may further tune the wavelength cutoff characteristics of the LEDs to improve detector sensitivity. A pair of six-channel optical sensors may be used to detect twelve different emission wavelengths. The LED array and optical sensors may be positioned 90 degrees from each other to minimize detection of the excitation sources, improving sensitivity. LED intensity and sequencing, detector gain and integration time, actuator parameters such as chamber location, speed, and mixing time, and detection thresholds may be adjusted automatically by the device based on the requirements of the assay. These parameters may be selected during the test by identifying the cartridge type via an authentication integrated circuit  718 , shown in  FIG. 7 , embedded in the cartridge. 
     In embodiments, the analyzer device  2500  may include a built-in USB-C or other port, and or wireless communications connectivity, such as Bluetooth, WiFi, etc., and on-board firmware that allows for interfacing with, for example, a laptop computer or a mobile device. Through this interface, it may be possible to perform additional functions via connected applications. In its simplest form, an application may store the results of the tests and provide the patient with access to their test results after leaving the test site. The fully integrated system may assist infectious disease surveillance via connections to public health action initiatives and large-scale disease data collection. Analyzer device  2500  may also interface with other software for contact tracing and real-time data collection. Disease surveillance facilitation could help the NIH and CDC track and predict outbreaks as well as support their decision-making process during pandemic emergencies. A connected app could be individualized to give tailored, user-specific predictions of disease progression, contagiousness, quarantine, and suggestions or recommendations on resources for treatment. Embodiments may include hybrid tools that combine surveillance with large-scale data analysis for development of more accurate disease modeling and forecasting. 
     Processes of operation of analyzer device  2500  are shown in  FIGS. 28, 29, and 30   a - c . For example, a process of Startup or Cartridge Switch Open  2800  is shown in  FIG. 28 . Upon Startup or Cartridge Switch Open  2800 , device  2500  may display a message, such as “Preparing”  2801 , perform motor position calibration  2802 , and display message, such as. “Ready”  2803 . A process of Lid Switch Open  2810  is also shown in  FIG. 28 . When the Lid Switch Open  2810  occurs, at  2811 , if a test was in progress, the test is aborted. Device  2500  may display a message, such as “Test aborted”. The test cannot be resumed because the cartridge is ruined. If there was no test in progress, then at  2812 , device  2500  may display a message, such as “Insert New Cartridge”. 
     A process of On Cartridge Switch Close  2900  is shown in  FIG. 29 . When Cartridge Switch Close  2900  occurs upon insertion of a cartridge, at  2901 , the authentication chip may be read and the cartridge parameters obtained. At  2902 , if the cartridge parameters indicate that the cartridge is already used and that cartridge parameters contain test results, then at  2902   a , device  2500  may display those test results, and may display an indication that the results are for a previously performed test. If the cartridge parameters do not contain test results, then device  2500  may display a message such as “Cartridge Prev. Used”. If the cartridge is not previously used, then at  2903 , the magnet  808 , shown in  FIG. 8 , may be moved to pneumatic port sealing location to ready it to operate during the test. At  2904 , device  2500  may configure the test using parameters included in the cartridge parameters. At  2905 , device  2500  may display a message, such as “Close Lid”. 
     A process of On Lid Switch Close is shown in  FIGS. 30 a - c   . When Lid Switch Close  3001  occurs upon closing of the lid of device  2500 , at  3001 , device  2500  may display a message such as “Analyzing” and may further display a countdown timer indicating a time until completion of the test. At  3002 , the cartridge may be marked as used in the cartridge authentication chip memory. At  3003 , device  2500  may wait a short time for depression of the blisters  2008 ,  2010 ,  2012 , shown in  FIG. 20 . Waiting too long may pull down too many beads, but we do need to wait for dispensing to be complete. In embodiments, this wait time may be configured using parameters included in the cartridge parameters. 
     At  3004 , the magnet may be moved quickly away, possibly all the way to the detection area, but at least past the first mixing chamber. The magnet may be moved fast enough to not drag beads. At  3005  the magnet may be moved slowly back to the sample area  1902 , shown in  FIG. 19 , possibly oscillating to catch straggler beads. At  3006 , the magnet may be moved quickly away again, just to the first mixing area  2002 , shown in  FIG. 20 , for example. 
     At  3007 , an LED calibration run may be performed. a. Using the lime LED, which emits into a spectrometer channel that we can read, perform a series of measurements to characterize the LED intensity drift over the sampling period. At  3007   a , using the lime LED, which emits into a spectrometer channel that can be read, a series of measurements to characterize the LED intensity drift over the sampling period may be performed, for example, as follows. At  3007   a.i , an LED of a selected color, such as lime, may be turned on to a level near full brightness, for example, 80%. At  3007   a.ii , device  2500  may wait 1 integration time (for example, 180 ms) for any erroneous initial reading to clear. In embodiments, this integration time may be configured based on parameters of device  2500  and/or based on parameters included in the cartridge parameters. At  3007   a.iii , device  2500  may read, for example, 6 samples of channel R (610 nm) at that integration time (for example, total elapsed sampling time=6*2*180 ms=2.16 s). At  3007   a.iv , device  2500  may calculate a linear fit for the LED PWM that would generate a flat spectrometer reading of a certain amplitude over those points. At  3007   a.v , this same calculation may be applied to the other two LEDs, which, in embodiments, may not be read directly with the spectrometer. 
     At  3008 , when the incubation time has nearly passed, the magnet may be moved back under the sample area to pull down remaining beads that haven&#39;t already precipitated. In embodiments, the incubation time, magnet movement time, remaining precipitation time, etc., may be configured based on parameters of device  2500  and/or based on parameters included in the cartridge parameters. 
     At  3009 , a baseline fluorescence measurement may be performed as follows. At  3009   a , a baseline fluorescence measurement may be performed using the illumination intensity curve from the LED calibration run  3007  for each of the three LEDs. At  3009   b , for, for example, IgM: Channel G/560 nm, Blue LED, at  3009   b.i , the LED may be turned on using the calibration curve from  3007 . At  3009   b.ii , device  2500  may wait 1 integration time (for example, 180 ms) for any erroneous initial reading to clear. At  3009   b.iii , device  2500  may read 6 samples of spectrometer channel at that integration time. At,  3009   c , for, for example, IgG: ChannelR/610 nm, Lime LED, at  3009   c.i  the LED may be turned on using the calibration curve from  3007 . At  3009   c.ii , device  2500  may wait 1 integration time (for example, 180 ms) for any erroneous initial reading to clear. At  3009   c.iii , device  2500  may read 6 samples of spectrometer channel at that integration time. At  3009   d , for, for example, IgA: Channel J/705 nm, Deep Red LED &amp; Internal Standard: Channel U/760 nm, Deep Red LED, at  3009   d.i  the LED may be turned on using the calibration curve from  3007 . At  3009   d.ii , device  2500  may wait 1 integration time (for example, 180 ms) for any erroneous initial reading to clear. At  3009   d.iii , device  2500  may read 6 samples of spectrometer channel at that integration time. 
     At  3010 , device  2500  may perform bubble detection by looking for abnormally high spectrometer readings. At  3011 , device  2500  may move the bead mass to the first (mixing) chamber  2002 . Oscillating motion may be required to keep mass from breaking up. At  3012 , device  2500  may agitate by moving magnet  808  for the second incubation time, such as 120 seconds. In embodiments, the incubation time, etc., may be configured based on parameters of device  2500  and/or based on parameters included in the cartridge parameters. At  3013 , device  2500  may move the bead mass to the second (washing) chamber  2004 , shown in  FIG. 20 , by moving magnet  808 . Oscillating motion may be required to keep mass from breaking up. At  3014 , device  2500  may agitate for the washing time, such as 30 seconds, by moving magnet  808 . In embodiments, the incubation time, etc., may be configured based on parameters of device  2500  and/or based on parameters included in the cartridge parameters. At  3015 , device  2500  may move the bead mass to the detection area by moving magnet  808 . Oscillating motion may be required to keep the mass from breaking up. At  3016 , device  2500  may perform a fluorescence measurement using a procedure similar to that at  3009 . At  3017 , device  2500  may calculate the relative signal between the fluorescence and baseline measurements, for example, by subtracting the baseline measurements from the fluorescence measurements. At  3018 , device  2500  may calculate the three ratiometric signals by scaling to the internal standard measurement. At  3019 , device  2500  may map the three ratiometric signals to antibody or other readings. At  3020 , device  2500  may display the results. 
     An exemplary format of memory of a cartridge authentication chip  3100  is shown in  FIG. 31 . In this example, cartridge authentication chip memory  3100  may include authentication data  3102 , configuration data  3104 , test subject data  3106 , and test results data  3108 . Authentication data  3102  may include data that may be used to authenticate the cartridge, data stored on the cartridge, etc. Configuration data  3104  may include data to identify the type of cartridge, the testing to be performed, configuration data for initializing and performing the test, etc. Test subject data  3106  may include data relating to the person or animal being tested, for example, including identity, physical and medical information, etc. Test results data  3108  may include results data from a completed test or data indicated an aborted test. 
     The device and firmware architecture may allow for future expansion and upgrades, as well as adaptation to new versions of the single-use cartridge. The device may identify the type or version of the cartridge from the authentication chip. In embodiments, a connected application may also be used for updating the device firmware with support for additional types of cartridges, for example, adding new settings for magnet arm movement, LED brightness, optical sensor thresholds, and other parameters, etc. The versatility of the cartridges may provide expansion of this technology to screen for immunity towards other infectious diseases, such as seasonal influenza, as well as other conditions, whether infectious or not. 
     Embodiments may capture antibodies at a low limit of detection, while in embodiments, assay for viral antigen capture may be performed. This would involve substituting the immobilized capture protein with an immobilized antibody for a viral antigen on the magnetic beads. In addition, the labeled antibody that contains the fluorescent or enzymatic would be changed to a second antibody that binds to the virus and its viral antigen. The remaining components of the sandwich assay and the general assay format would be the same. 
     Embodiments may be extremely useful in emergency response and urgent care, informing doctors how to proceed with treatment without having to wait hours for a confirmatory test and informing decisions about whether or not to quarantine patients. Rapid determination of infectivity will also inform users of whether or not it is safe to return to school or work, gather in large groups, or travel. Moreover, individuals can feel confident that they will not bring the virus home to their families. These decisions, along with contact tracing methods, will allow us to track and slow the spread of pathogens. 
     Embodiments may provide sensitivity in the range of 95%. In embodiments, the assay may be optimized to have a linear response to the concentrations within the range of sensitivity. The assay may be reproducible within 5-10% and the instrument may self-calibrate down to the validated response range. Embodiments may provide specificity in the range of 97%. For example preliminary data suggest that SARS-CoV-2 antibodies bind to the S1 component of the virus, and that antibodies toward other viruses (such as RSV and MERS) show no statistically observable response vs a blank standard with no antibodies present for SARS-CoV-2. The dynamic range of the device may be set using a signal-to-noise ratio of at least 10 for quantitation or using a value of 3 for a qualitative screening assay. Embodiments may provide an analytical limit of detection when using direct fluorescence detection and 100-200 μL of saliva of &lt;1 nM (˜0.5-0.7 nM). Embodiments may provide a precision of ±5-8% over most of the calibration range. In terms of robustness, embodiments may provide less than 5% of variability between test cartridges. In terms of accuracy, embodiments may be no less than 95% accurate with a false positive and false negative output of no more than 5%. Embodiments may perform each test in less than 5 minutes, with a throughput of 12 tests per hour. 
     Embodiments may use beads containing human lgM, human lgG or human lgA (Int. Std., Alexa-568 BSA beads; 5 min incubation) may be placed into pH 7.4, 0.067 M phosphate buffer containing 0.1% sodium azide, for use transport and long-term storage at 4° C. The immunoglobulin-containing beads were found to still show a good response in a sandwich assay when stored under these conditions. These data are consistent with previous results indicating that sodium azide did not have any significant effect on the response for this type of sandwich assay. 
     Embodiments may use beads containing the new spike protein S1 beads (ACRO Biosystems) (Int. Std., Alexa 568 BSA beads; 2.5 min+2.5 min split incubation) were also placed into pH 7.4, 0.067 M phosphate buffer containing 0.1% sodium azide, for use transport and long-term storage at 4° C. The spike protein S1 beads were also found to show a good response in a COVID antibody sandwich assay when stored under these conditions. These data were again consistent with previous results indicating that around 0.1% sodium azide did not have any significant effect on the response for this assay. 
     Embodiments may use Alexa Fluor 750 BSA beads (using human lgM beads, FITC-labeled anti-human lgM antibodies and new Alexa-750 BSA beads as the internal standard (5 min incubation)) were prepared for use as an internal standard in a COVID-19 antibody assay with multi-channel detection. These beads gave a response and protein content that was consistent with previous batches of the same material. These beads were also examined for their response in a sandwich assay (demo format 1) after they were transferred to a pH 7.4 buffer containing 0.1% sodium azide for long term storage. A good response and low background signal was again seen when these beads were used in these assays. 
     Embodiments may use beads that contain immobilized lgG, lgA or lgM, combined with a mixture of secondary labeled antibodies, models binding of labeled antibodies with adsorbed targets on beads. Example may include FITC rabbit anti-human lgM (mu chain specific), Alexa 680 anti-human lgA (alpha chain specific, prescreened vs lgG &amp; lgM), Alexa 568 anti-human lgG (H+L chains) (commercial), Alexa 568 anti-human lgG (Fe specific chains), Alexa Flour 488 goat anti-human lgM (mu chain specific), etc. 
     Embodiments may use sandwich immunoassay that can be used to detect SARS-CoV-2 antibodies in biological samples and can detect multiple antibodies simultaneously in a tube-based assay. Embodiments may be easily repurposed for use with a viral detection assay. 
     Embodiments may include the capture component of the assay with a model assay using Human Serum Albumin (HSA). Incubation of diluted anti-HSA rabbit antiserum with fluorescein-labeled anti-rabbit IgG antibodies, and injection of 20 μL mixture at 1.0 mL/min onto 2.1 mm I.D.×5 mm HSA column may yield a column residence time of 0.5 seconds. This may indicate that the capture component can take less than a few minutes. This result has been verified in work with the magnetic beads. The estimated detection limit in work with the model system and the beads in a solution phase assay is currently 0.3 nM at a signal to noise ratio of 3 for the target. 
     Embodiments may be used without enzymatic amplification to detect SARS-CoV-2 antibodies at nanomolar concentrations. Beads containing approximately 10 mg fluorescent-labeled proteins per gram of beads may be easily be detected at final bead levels down to less than 0.01 mg/mL. The use of enzymatic amplification with horseradish peroxidase (HRP) may allow detection limits down into the picomolar range. HRP-labeled antibodies are detected down to 65-70 pM in 2.5 to 5 min. This detection limit is well below that needed to detect typical antibody levels in saliva (0.3-0.4 nM). The relative amount and type of secondary antibodies, fluorescent label, capture agents, and volume of sample may be modified to obtain lower limits of detection. 
     In embodiments, the final number of beads needed for detection are between 0.1-0.05 mg/mL in 20 μL. The content of capture protein on the beads are between 5 to 10 mg/g beads. Labeled secondary antibodies may be used in excess, &gt;2× the concentration of target antibodies. This assay gives a linear response with a mass-mol detection limit that is well below that needed with small samples of whole saliva (100-200 μL). 
     Typically, the beads do not have a significant background fluorescence which might interfere with the assay also do not have significant non-specific binding to components of the sample and reagents for the assay. Multiple dye labels, such as FITC, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 680, and Alexa Fluor 750 may be used with this assay format in the simultaneous detection of multiple analytes (e.g., multiple antibodies or viral strains plus a positive control) in a microchip-based device. 
     Good stability was seen for capture protein beads over at least 3 months of use, as well as good reproducibility in protein content and assay behavior over multiple batches of beads. Over the course of five days, the presence of BSA does not appear to contribute to bead aggregation during storage at 4° C. 
     Various pH 7.4 buffers may be used. Several types of buffers, such as potassium phosphate, or phosphate buffered saline, may be acceptable for use with the assay. In embodiments, rabbit IgG anti-spike protein antibodies may be used with either buffer (both with Tween 20 as an additive) in the sandwich assay for COVID spike protein antibodies. Embodiments may use up to 2.5 mM sodium azide as a preservative in the assay an may provide an acceptable assay response. 
     In embodiments, lowering the amount of assay beads on the response may cause some decrease in assay range, but a usable response may be obtained for a 2- or 4-fold decrease in assay bead levels. In terms of selectivity, the assay components may give no significant response to non-specific/generic antibodies (e.g., samples of human IgG) or the matrix components of normal pooled human saliva. Embodiments may have highly specific selection for SARS-CoV-2 antibodies compared to antibodies toward other viruses, such as SARS, MERS, RSV, HCV, and autoimmune antinuclear antibodies. No statistically observable response is seen with antibodies toward other viruses or toward ANA vs a blank standard with no antibodies present for SARS-CoV-2. 
     The ability to carry out multianalyte detection was confirmed and tested in a four-channel method by using 1) mixtures of labeled beads, 2) mixtures of labeled antibodies with immunoglobulin beads, and 3) various types of spike protein antibodies in a sandwich assay. We have successfully demonstrated the simultaneous detection of four analytes, IgA, IgM, IgG, and an internal standard, with cross-reactivity between the 3 antibodies or the internal standard, demonstrating high interclass specificity. Using a split incubation format (2.5 min in tube incubation and 2.5 min run time) gave the best improvement in assay response. Detection in the low nM range was seen, as noted previously, along with little or no hook effect at higher levels over the range of tested concentrations. Further analysis was also carried out on detection of these labels by a microchip-based detector in either the sample holder used originally vs a prototype cartridge, giving comparable trends in detection. The assay can be used with either a specific response at a given channel or with multiplexing for signals with a mixed response. 
     In embodiments, a sample collection kit may include on-board foil-sealed liquid reagents, including the functionalized magnetic beads and secondary antibodies needed for the detection assay. The kit also includes an attachable funnel to facilitate easy collection of the saliva sample, and a foil piercing system to interface with the cartridge. After providing the saliva sample, the user shakes the collection kit for 30 s to mix the pre-stored liquid reagents and saliva sample prior to insertion into the cartridge. 
     The compatible cartridge, with dimensions of, for example, 75 mm (L)×50 mm (W)×10 mm (H), may include a smart laminate, an injection molded part with blister piercing features, three blisters, and an injection molded case. The cartridge may be equipped with an inlet port for attachment of the sample collection kit, two 250 μl buffer solution blisters with adjacent 22 μL wash chambers, one air-filled blister for pneumatic control, a detection window, overflow passages, fluidic paths, and a valve to ensure proper fluid routing. The blisters of the cartridge are actuated by the closing of the top lid of the device. 
     Examples of pathogens or antibodies to pathogens that may be detected based on target substances may include West Nile virus, Malaria, Zika, Ebola, Tuberculosis, Hepatitis A, Hepatitis B, Hepatitis C, HIV/AIDS, Influenza A/B, etc. Example of other conditions that may be detected based on target substances may include Diabetes, Enzyme Markers, such as for CPK-1--+Brain injury after concussion, CPK-2, CPK-3, and Troponin, conditions that may be detected by a Coagulation Panel, such as hemophilia, acute myeloid leukemia, thrombosis, vitamin K deficiency, Prothrombin time (PT), Fibrinogen activity test, C-reactive Protein Test-inflammation, conditions that may be detected by a Cholesterol lipid panel including LDL level, HDL level, triglyceride level, conditions that may be detected by a Thyroid Panel including T3, T4, RU, TSH, Alzheimer&#39;s disease or other diseases based on, for example, Amyloid Beta Peptide, Tau protein, Lactoferrin, Alpha synuclein, DJ-1 Protein, Chromogranin A, and Huntingtin protein, Cognitive Decline based on, for example, Glucocorticoids, Cortisol, conditions such as Coeliac disease, Stomach Cancer, Breast Cancer based on, for example, CA 15-3 antigen for breast cancer, SPM, CAD, Ac-SPM, N1-Ac-SPD, N8-Ac-SPD polyamines in saliva, Prostate Cancer based on prostate-specific antigen (PSA), Colorectal cancer based on Carcino-EmbryonicAntigen (CEA), Gastrointestinal Cancer based on CA 19-9 antigen, Ovarian Cancer based on CA 125 antigen, etc. For example, types of cancer that may be detected based on known markers may include breast, prostate, gastrointestinal, ovarian, and colorectal cancers. Hepatitis A and B may be detected based on known markers. Thyroid disorders may be detected based on known markers such as T3, T4, RU, TSH, etc. Cholesterol issues may be detected based on known markers such as LDL, HDL, triglycerides, etc. Alzheimer&#39;s may be detected based on known markers such as Tau protein, Amyloid Beta, Peptide, Alpha synuclein, DJ-1 Protein, Chromogranin A, etc. 
     Embodiments may include analyzer devices which automate the assay using low cost, readily available components. Embodiments may control magnetic particles in a microfluidic cartridge using a permanent magnet on a linear actuator, for moving the particles between chambers on the cartridge as well as performing mixing and washing actions. Embodiments may perform fluorescent dye excitation and detection using cost-effective LEDs and multi-channel light sensors, and fluorescence detection from dye-labeled magnetic particles in concentrations representative of those present in the assay. Embodiments may include positioning and sequencing three light sources, combined with optical short-pass filters to excite fluorescence in four separate dyes, detecting a positive control and up to three separate analytes in a single sample. The adjustability of components in this system, as well as the ability to detect multiple analytes, allows for significant flexibility and expansion of assay development and testing. 
     Although specific embodiments of the present invention have been described, it will be understood by those of skill in the art that there are other embodiments that are equivalent to the described embodiments. Accordingly, it is to be understood that the invention is not to be limited by the specific illustrated embodiments, but only by the scope of the appended claims.