Mask-based diagnostic system using breath condensate

Abstract

To provide an exhalation-based diagnostic device for detecting a biomarker contained in the exhalation of a subject.SOLUTION: A breath based diagnostic device for detecting biomarkers contained in the breath of a subject, the device comprising: an exhaled breath condensate (EBC) collector for converting exhaled breath vapour received from the lungs and airways of a subject into a fluid biosample, the EBC collector comprising a thermal mass, a condensate forming surface and a fluid conductor disposed on the condensate forming surface; a fluid transfer system for receiving a fluid biosample from the EBC collector; and a biomarker testing unit for receiving the fluid biosample from the fluid transfer system and testing the fluid biosample for a target biomarker contained in the fluid biosample.SELECTED DRAWING: Figure 155

Description

[Technical Field]

[0001] Related Applications: This international application claims the benefit of priority to U.S. Utility Patent Application No. 17,189,711, entitled "Mask-Based Diagnostic System Using Exhaled Breath Condensate," filed March 2, 2021 (which is a continuation-in-part of and claims priority to co-pending U.S. Patent Application No. 17,065,488, entitled "Mask-Based Test System for Detecting Biomarkers in Exhaled Breath Condensate, Aerosols, and Gases," filed October 7, 2020; and to co-pending U.S. Utility Patent Application No. 16,882,447, entitled "Use of Exhaled Breath Condensate, Aerosols, and Gases for Detecting Biomarkers," filed May 23 .... Co-pending U.S. Utility Patent Application No. 16,876,054, filed May 17, 2020, entitled "USE OF EXHALED BREATH CONDENSATE FOR TESTING BIOMARKERS FOR COVID-19," and U.S. Provisional Application No. 63,012,247, filed April 19, 2020, entitled "LOW-COST, SCALABLE, ACCURATE, AND EASY-TO-USE TESTING SYSTEM FOR COVID-19"; U.S. Utility Patent Application No. 63,019,378, filed May 3, 2020; and U.S. Utility Patent Application No. 63,026,052, filed May 17, 2020, entitled "USE OF EXHALED BREATH CONDENSATE FOR TESTING BIOMARKERS FOR COVID-19," the disclosures of which are incorporated herein by reference in their entireties.

[0002] Exemplary, non-limiting embodiments of the present invention relate generally to diagnostic systems, methods, apparatus, and computer programs, and more particularly to digital diagnostic devices for detecting biomarkers of biological agents such as coronaviruses.

[0003] The present invention also relates to device architectures, specific uses, and computer algorithms used to detect biometric parameters for the treatment and monitoring of physiological conditions in humans and animals. [Background technology]

[0004] This section is intended to provide a background or context for example embodiments of the invention that are described in the claims. The description herein may include concepts that may be appealing, but not necessarily have been previously conceived, implemented, or described.

[0005] Therefore, unless otherwise indicated herein, the statements in this section are not prior art to the description and claims of this application and are not admitted to be prior art by inclusion in this section. Summary of the Invention [Problem to be solved by the invention]

[0006] Governments around the world have implemented stay-at-home orders and civil lockdowns to slow the spread of the COVID-19 virus. Billions of people worldwide are currently suspending normal employment, recreation, and social activities. Testing for biomarkers indicating COVID-19 exposure, infection, and recovery can be used to minimize the spread of the virus while allowing for a safer and more efficient reopening of economies. For example, active viral protein and RNA tests could indicate who is currently infected. Antibody tests could be used to identify members of a population who have recovered from the virus and are immune to reinfection. This knowledge could enable precise social distancing and more effective contact tracing, along with the re-employment of a growing workforce of protected individuals and consumers. People at risk of infection and transmission could remain isolated until other solutions, such as a vaccine or highly successful drug treatments, are developed. [Means for solving the problem]

[0007] The following summary section is intended to be exemplary only and not limiting. The foregoing and other problems are overcome, and other advantages are realized, by use of exemplary embodiments of the present invention.

[0008] According to a non-limiting exemplary embodiment, a mask-based diagnostic device for detecting biomarkers contained in a subject's exhaled breath is provided. An exhaled breath condensate (EBC) collector converts exhaled vapor received from the subject's lungs and airways into a fluid biosample. The EBC collector includes a thermal mass, a condensate-forming surface, and a fluid conductor disposed on the condensate-forming surface. A liquid transfer system receives the fluid biosample from the EBC collector. A biomarker testing unit receives the fluid biosample from the fluid transfer system and tests the fluid biosample for target biomarkers. A testing system support is provided to support the EBC collector, the fluid transfer system, and the biomarker testing unit. The testing system support is configured and dimensioned to fit inside a face mask. A face mask is provided that forms an exhaled vapor containment volume to hold the exhaled vapor near the EBC collector to allow the condensate-forming surface, cooled by the thermal mass, to coalesce the exhaled vapor into a fluid biosample.

[0009] According to a non-limiting exemplary embodiment, a mask-based test system for detecting biomarkers received from a subject's lungs and airways includes an exhaled breath condensate (EBC) collector integrated inside a face mask worn by the subject. The EBC collector converts exhaled vapors received from the subject's lungs and airways into a liquid biosample. A biosensor is secured inside the face mask for receiving the fluid biosample from the EBC collector and testing the fluid biosample for target analytes. The biosensor generates a test signal dependent on at least the presence or absence of the target analyte in the fluid biosample. Electronic circuitry is secured outside the mask for receiving the test signal, determining a test result signal from the test signal in response to detection or non-detection of the target analyte, and transmitting the test result signal to a remote receiver.

[0010] According to one aspect of the present invention, a device for detecting biomarkers includes a particle capture structure for receiving and capturing exhaled breath aerosol (EBA) particles from the lining of a user's airways. The particle capture structure has an aerosol particle inspection system for receiving the captured particles and detecting a first biomarker. The aerosol particle inspection system includes a dissolvable EBA sample collector film for capturing the EBA particles. The dissolvable EBA sample collector film includes a first reagent for reacting with at least one component of the captured particles in a detection reaction to detect the first biomarker. The detection reaction generates at least one of an optical signal and an electrical signal change dependent on the first biomarker. The first reagent binds to first nanoparticles and is retained in an insoluble test region. The EBA particles include insoluble particles and droplet particles. The dissolvable EBA collector film includes a sticky surface for adhering to and capturing the insoluble particles and a water-soluble bulk for capturing the droplet particles.

[0011] According to another aspect of the present invention, an apparatus includes at least one processor, at least one memory containing computer program code, the at least one memory and the computer program code being configured to, using the at least one processor, cause the apparatus to at least perform the following: detect one or more biometric parameters using a particulate capture structure for receiving and capturing exhaled aerosol (EBA) particulates from the lining of a user's airways (the particulate capture structure has an aerosol particulate inspection system for receiving the captured particulates and detecting a first biomarker, the aerosol particulate inspection system including a dissolvable EBA sample collector film for capturing the EBA particulates, the biometric parameter being a biomarker dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection); receive the one or more biometric parameters and apply probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change. The one or more biometric parameters can be further detected using a droplet collection structure for converting the exhaled vapor into fluid droplets to form a fluid sample, and a testing system having a biomarker testing zone for receiving the fluid sample and detecting the biometric parameters. A probabilistic analysis is applied to the one or more biometric parameters to determine whether at least one physiological change threshold has been exceeded depending on a probabilistic analysis of the one or more biometric parameters detected from both the captured particulate and the fluid sample.

[0012] According to one aspect of the invention, an apparatus includes a droplet collection and channeling structure for converting vapor into fluid droplets, and a fluidic biosensor, the fluidic biosensor comprising a sample source, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal.

[0013] According to another aspect of the present invention, an apparatus for detecting a biomarker includes a droplet collection and channeling structure for converting vapor into fluid droplets, and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal depending on changes in the bioreceptor in response to receiving the biomarker analyte from the sample source.

[0014] According to another aspect of the present invention, an apparatus for detecting biomarkers includes a droplet collection structure for converting exhaled breath vapor into fluid droplets to form a fluid sample, and a testing system having a biomarker testing zone for receiving the fluid sample and detecting biomarkers. The droplet collection structure can include at least one of a hydrophobic field for receiving the exhaled breath vapor and forming fluid droplets from the received exhaled breath vapor, and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets toward the testing system. A fluid dam member can be provided disposed between the droplet collection structure and the biomarker testing zone.

[0015] According to another aspect of the present invention, an apparatus for detecting a biomarker includes a droplet collection and channeling structure for converting vapor into fluid droplets and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal as a function of changes in the bioreceptor in response to receiving the biomarker analyte from the sample source.

[0016] According to another aspect of the present invention, a method of forming a biomarker testing system includes forming a breath condensate sample collector. Forming the breath condensate sample collector includes providing a substrate, coating a hydrophobic field on the substrate, and coating at least one hydrophilic channel on the substrate. The hydrophobic field is for receiving bodily fluid vapor and forming fluid droplets from the received bodily fluid vapor, and the hydrophilic channel is for receiving the fluid droplets and directing the fluid droplets toward a testing system. At least one fluid sample ejection hole may be formed at an end of the hydrophilic channel for ejecting the fluid droplets through the at least one fluid sample ejection hole onto a sample receiving structure of the testing system.

[0017] According to another aspect of the present invention, a system for detecting biological agents from a subject's exhaled breath is provided, the system including: a breath condensate droplet collector for coalescing exhaled vapors into droplets to form a fluid biological sample; a testing system for receiving the fluid biological sample from the breath droplet collector and testing it for target analytes; and wireless communication electronics for detecting the target analyte test results and communicating the results to a wireless receiver. The exhaled aerosol capture system can include a sheet member having a surface for receiving exhaled aerosol containing at least one of microparticles and droplets. The surface can be an insoluble pressure-sensitive adhesive or an exposed portion of a dissolvable film formed, coated, adhered, or integrated onto the sheet member. The dissolvable film has a composition effective to receive and capture at least one of microparticles and droplets by at least one of embedding or dissolving the at least one of the microparticles and droplets on the surface or within the dissolvable film. At least one of the surface and the dissolvable film includes a reagent for reacting with at least one microparticle and droplet to detect the presence of the target analyte in the at least one microparticle and droplet.

[0018] According to one aspect of the present invention, there is provided a computer program product including a computer-readable medium having computer program code embodied thereon for use on a computer, the computer program code including: code for: detecting one or more biometric parameters, the biometric parameters dependent on at least one physiological change to a patient responsive to a condition of concern, such as a viral infection; receiving the one or more biometric parameters and applying probabilistic analysis to determine whether at least one physiological change threshold has been exceeded responsive to the probabilistic analysis of the one or more biometric parameters; and taking action responsive to the at least one physiological change being determined to have been exceeded.

[0019] According to another aspect of the present invention, an apparatus includes at least one processor; and at least one memory containing computer program code, the at least one memory and the computer program code being configured, by the at least one processor, to cause the apparatus to at least perform the following: detect one or more biometric parameters using an examination system having a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample and a biomarker examination zone for receiving the fluid sample and detecting the biometric parameters (the biometric parameters being biomarkers dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection); receive the one or more biometric parameters and apply probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change.

[0020] According to one aspect of the invention, an apparatus includes a droplet collection and channeling structure for converting vapor into fluid droplets, and a fluidic biosensor, the fluidic biosensor comprising a sample source, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal.

[0021] According to another aspect of the present invention, an apparatus for detecting a biomarker includes a droplet collection and channeling structure for converting vapor into fluid droplets and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal as a function of changes in the bioreceptor in response to receiving the biomarker analyte from the sample source.

[0022] According to another aspect of the invention, an apparatus for detecting biomarkers includes a test system having a droplet collection structure for converting exhaled breath vapor into fluid droplets to form a fluid sample, and a biomarker testing zone for receiving the fluid sample and detecting biomarkers. The droplet collection structure can include at least one of a hydrophobic field for receiving the exhaled breath vapor and forming fluid droplets from the received exhaled breath vapor, and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets toward the testing system. A fluid dam member can be provided disposed between the droplet collection structure and the biomarker testing zone.

[0023] According to another aspect of the present invention, an apparatus for detecting a biomarker includes a droplet collection and channeling structure for converting vapor into fluid droplets and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal as a function of changes in the bioreceptor in response to receiving the biomarker analyte from the sample source.

[0024] According to another aspect of the present invention, a method of forming a biomarker testing system includes forming a breath condensate sample collector. Forming the breath condensate sample collector includes providing a substrate, coating a hydrophobic field on the substrate, and coating at least one hydrophilic channel on the substrate. The hydrophobic field is for receiving bodily fluid vapor and forming fluid droplets from the received bodily fluid vapor, and the hydrophilic channel is for receiving the fluid droplets and directing the fluid droplets toward a testing system. At least one fluid sample ejection hole may be formed at an end of the hydrophilic channel for ejecting the fluid droplets through the at least one fluid sample ejection hole onto a sample receiving structure of the testing system.

[0025] According to one aspect of the invention, an apparatus includes a droplet collection and channeling structure for converting vapor into fluid droplets and a fluidic biosensor, the fluidic biosensor comprising a sample source, bioreceptor regions functionalized with analyte-specific bioreceptors, and a transducer for generating a readable signal. According to another aspect of the invention, an apparatus for detecting biomarkers includes a droplet collection and channeling structure for converting vapor into fluid droplets and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, bioreceptor regions functionalized with analyte-specific bioreceptors, and a transducer for generating a readable signal depending on changes in the bioreceptor in response to receiving the biomarker analyte from the sample source.

[0026] The foregoing and other aspects of exemplary embodiments of this invention will become more apparent in the following detailed description when read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows a Lateral Flow Assay (LFA) testing system showing a biomarker sample applied to a sample pad. [Figure 2] FIG. 2 shows an LFA containing biomarker-labeled antibody complexes formed on a conjugate release pad. [Figure 3] FIG. 3 shows the binding of the biomarker at the test line indicating the presence of the biomarker. [Figure 4] Figure 4 shows the mechanism of the bioreceptor detection system. [Figure 5] FIG. 5 is a side view of a wearable electronic breath chemical sensor. [Figure 6] FIG. 6 is a top view of a wearable electronic breath chemical sensor. [Figure 7] FIG. 7 is an exploded view of the Exhaled Breath Condensate (EBC) droplet sample collector. [Figure 8] FIG. 8 is a top view illustrating the steps for forming an EBC droplet collector. [Figure 9] FIG. 9 is a top view showing another step for forming an EBC droplet sample collector. [Figure 10] FIG. 10 is a top view illustrating yet another step for forming an EBC droplet sample collector. [Figure 11] FIG. 11 is a top view showing yet another step for forming an EBC droplet sample collector. [Figure 12] FIG. 12 shows an EBC sample collector showing EBC droplets. [Figure 13] FIG. 13 shows an EBC sample collector applied to an LFA inspection system. [Figure 14] FIG. 14 is an exploded view showing the screen-printed hydrophilic channels, screen-printed hydrophobic fields, and thermal mass substrate of the EBC sample collector. [Figure 15] FIG. 15 is an exploded view showing the components of the LFA. [Figure 16] FIG. 16 shows one embodiment of an LFA including emitter / detector electronics. [Figure 17]FIG. 17 shows an EBC sample collector applied to a nanoscale biosensor testing system, showing the pull tab for retaining the collected droplet on the sample pad. [Figure 18] FIG. 18 is a perspective view showing an EBC sample collector applied to an inspection system. [Figure 19] FIG. 19 is an isolated view showing the pull table positioned between the sample pad and the conjugate release pad. [Figure 20] FIG. 20 is an exploded view of a screen-printed EBC sample collector with liquid transfer openings. [Figure 21] FIG. 21 is a cross-sectional view showing a fluid sample collected from an EBC sample collector flowing between an emitter / detector pair. [Figure 22] FIG. 22 shows a side view of the steps for building an LFA inspection system. [Figure 23] FIG. 23 shows a top view of the steps for building an LFA inspection system. [Figure 24] FIG. 24 shows a 4×9 interlocking multi-up sheet of LFA inspection systems formed as a batch. [Figure 25] FIG. 25 shows a roll-to-roll manufacturing process for forming a roll of bottom adhesive / backing substrate / top adhesive. [Figure 26] FIG. 26 is a perspective view showing a bottom adhesive / support substrate / top adhesive stack. [Figure 27] FIG. 27 shows a roll-to-roll manufacturing process for forming the components of an LFA on a roll of bottom adhesive / backing substrate / top adhesive. [Figure 28] FIG. 28 shows an LFA test system formed by a roll-to-roll process cut from a continuous roll and shows a portion of the top adhesive for adhering the LFA test system to a separately formed ENC sample collector. [Figure 29]FIG. 29 shows an LFA inspection system formed by a roll-to-roll process cut from a continuous roll, showing a section of bottom adhesive for application to a wearable garment such as a face mask. [Figure 30] FIG. 30 shows a sheet of substrate having a hydrophobic field coating on a thermal mass substrate with droplet collection holes. [Figure 31] FIG. 31 shows a sheet of substrate with a hydrophobic field coating on a thermal mass substrate with droplet collection holes having a coating of hydrophilic channels. [Figure 32] Figure 32 shows the EBC sample collector and testing system with electronics for wireless data acquisition and transmission, as well as a separate trusted receiver and public blockchain data path and storage. [Figure 33] Figure 33 shows the manufacturing process for a thermally bonded face mask. [Figure 34] FIG. 34 shows the fabric, filter, and other layers joined by a roll-to-roll lamination process ore individually cut into blanks to form a preform mask stack. [Figure 35] FIG. 35 shows the biologically reactive silver fabric and other materials such as hot melt adhesive in the preform mask stack. [Figure 36] FIG. 36 is an exploded view of the mask stack. [Figure 37] FIG. 37 shows the mask stack fragments for the first and second hot pressing operations. [Figure 38] FIG. 38 shows the mask folded, pressed and heat sealed. [Figure 39] FIG. 39 shows the attachment of the EBC collector and inspection system to the folded mask. [Figure 40] FIG. 40 shows the step of turning the folded mask inside out to place the EBC collector and inspection system inside the mask. [Figure 41]FIG. 41 shows the hot pressing operation for bonding the elastic straps to the folded mask. [Figure 42] FIG. 42 shows a mask with an EBC collector and testing system located inside the mask in a concentrated atmosphere of exhaled air. [Figure 43] FIG. 43 shows a conventional bendable metal nose seal positioned within the folds of the mask in a position corresponding to the bridge of the subject's nose. [Figure 44] FIG. 44 shows a replaceable adhesive nose strip placed outside the folds of the mask in a position corresponding to the bridge of the subject's nose. [Figure 45] FIG. 45 shows the components of a removable magnetic nose seal. [Figure 46] Figure 46 is an exploded view of a test system including a dissolvable fluid dam that is released onto a conjugate release pad and holds EBCs collected on the sample pad until a sufficient amount accumulates to flush the fluid sample through the components of the test system. [Figure 47] FIG. 47 is an isolated view showing a dissolvable dam inserted between the sample pad and the conjugate release pad. [Figure 48] FIG. 48 shows an isolated view after the dissolvable body pad has been dissolved away and the accumulated fluid sample has been released from the sample pad onto the conjugate release pad. [Figure 49] FIG. 49 is an isolated view showing the dissolvable EBC droplets and EBA particulate collector. [Figure 50] FIG. 50 is a cross-sectional side view showing a cross section of a dissolvable droplet and particulate collector with particulates and droplets penetrating the surface. [Figure 51] FIG. 51 is a side cross-sectional view showing a cross section of a dissolvable droplet and particle collector having particles embedded in a dissolvable capture film and droplets that dissolve in the detection reagent of the dissolvable capture film to cause a detection reaction. [Figure 52]FIG. 52 is a top view showing an inspection system of the present invention including a dissolvable EBC droplet and EBA particulate collector with trapped aerosol droplets and aerosol particulates. [Figure 53] FIG. 53 is an isolated perspective view showing a dissolvable EBC droplet and EBA particulate collector with trapped aerosol droplets and aerosol particulates. [Figure 54] FIG. 54 is a top view of an inspection system of the present invention including a dissolvable EBC droplet and EBA particle collector prior to capturing aerosol droplets and aerosol particles. [Figure 55] FIG. 55 is a top view showing an inspection system of the present invention including a dissolvable EBC droplet and EBA particle collector after capturing aerosol droplets and aerosol particles. [Figure 56] FIG. 56 is a top view illustrating a testing system of the present invention including a dissolvable EBC droplet and EBA particulate collector mounted on a face mask substrate with multiple gas sensors for detecting volatile and gas components of exhaled breath and / or ambient air. [Figure 57] FIG. 57 is a side cross-sectional view showing a cross section of a dissolvable droplet and particulate collector having droplets and particles impacting a surface disposed within a beaker of dissolving liquid. [Figure 58] FIG. 58 is a cross-sectional side view showing a cross section of a dissolvable droplet and particle collector with particles released into a beaker of dissolving liquid and the droplets dissolved. [Figure 59] FIG. 59 is a block diagram of one possible non-limiting exemplary system in which exemplary embodiments can be implemented. [Figure 60] FIG. 60 is a logic flow diagram of applied probabilistic analysis for determining exposure to COVID-19, illustrating the operation of an exemplary method, the results of execution of computer program instructions embodied on computer-readable memory, functions performed by hardware-implemented logic, and / or interconnected means for performing functions according to an exemplary embodiment. [Figure 61]FIG. 61 is a logic flow diagram for data acquisition and transmission for trusted recipient and contact tracing applications, illustrating the operations of an exemplary method, the results of execution of computer program instructions embodied on computer-readable memory, functions performed by hardware-implemented logic, and / or interconnected means for performing functions according to an exemplary embodiment. [Figure 62] FIG. 62 is a perspective view of one embodiment of an EBC / EBA collection system. [Figure 63] FIG. 63 is a perspective view of the EBC / EBA collection system showing the pipette and pipette guide. [Figure 64] FIG. 64 is an exploded view showing the components of an embodiment of an EBC / EBA collection system. [Figure 65] FIG. 65 is another exploded view showing the components of the EBC / EBA collection system. [Figure 66] FIG. 66 is a cross-sectional view of the EBC / EBA collection system. [Figure 67] Figure 67 illustrates the use of the EBC / EBA collection system to obtain biomarker samples from the lungs of a subject. [Figure 68] FIG. 68 is an exploded view showing the mouthpiece, cap, base, dissolvable EBA sample collector, and inner barrel of an embodiment of an EBC / EBA collection system. [Figure 69] FIG. 69 is an isolated view showing the dissolvable EBA sample collector and inner barrel with trapped EBA particles and droplets. [Figure 70] FIG. 70 shows the inner tube submerged in a solvent to dissolve the dissolvable EBA sample collector and obtain captured EBA particles and droplets for biomarker testing. [Figure 71] FIG. 71 is an isolated view of a portion of one embodiment of a dissolvable EBA sample collector forming an aerosol particulate test system having trapped EBA particulates, an insoluble test area, and a dissolvable capture film area. [Figure 72]Figure 72 shows a series of side views of an embodiment of a dissolvable EBS sample collector capturing EBA droplets and / or particulates, illustrating an aerosol particulate testing system with target biomarkers captured and coupled to an insoluble testing area. [Figure 73] FIG. 73 shows nanoparticles held within trenches in a substrate, the nanoparticles containing a capture antibody or other reagent immobilized thereon. [Figure 74] FIG. 74 shows EBA particles and droplets being rinsed from a dissolvable EBA sample collector to form a fluid sample containing any biomarkers contained in the particles or droplets. [Figure 75] FIG. 75 shows an EBA / EBC testing system having wireless communication electronics that detects the results of a test for at least one of a first and a second biomarker and communicates the results to a wireless receiver. [Figure 76] Figure 76 shows an EBC / e-NSB testing system integrated into a ventilator circuit. [Figure 77] Figure 77 shows elements of a continuous flow embodiment in which a capillary space is formed in the test region of the sensor between the sensor substrate and the capillary cap. [Figure 78] Figure 78 shows the inside of a disposable mask with an EBC collector, microfluidics, and electronic biosensors. [Figure 79] Figure 79 shows the outside of the disposable mask, showing the electrical connection from the electronic biosensor inside the mask to the z-axis conductive tape on the outside of the mask. [Figure 80] Figure 80 shows the components of a self-cooled EBC collector. [Figure 81] Figure 81 shows the inside of the open mask containing the components for collecting and inspecting EBCs and EBAs. [Figure 82] FIG. 82 is a block diagram of the basic components for testing an EBC and transmitting test results to a smartphone and / or cloud server. [Figure 83]FIG. 83 is a side cross-sectional view showing the disposable components on the inside of the disposable mask and the sanitizable components on the outside of the disposable mask. [Figure 84] FIG. 84 is a side cross-sectional view showing a magnetic system for holding and electrically connecting electronics to a disposable mask. [Figure 85] FIG. 85 is a cross-sectional view showing the z-axis conductive tape that holds the electronics and electrically connects them to the disposable mask. [Figure 86] Figure 86 shows a stretchable hot melt adhesive attached to a foam. [Figure 87] Figure 87 shows a pocket formed with a stretchable hot melt adhesive attached to foam. [Figure 88] FIG. 88 shows a heat-absorbing compound placed within a pocket formed with a stretchable hot melt adhesive. [Figure 89] Figure 89 shows a water bag added to the pocket holding the endothermic compound. [Figure 90] Figure 90 shows pre-laminated aluminum foil on an adhesive sheet over a stretchable hot melt adhesive. [Figure 91] Figure 91 shows the bottom surface of the foam after press lamination of the layers that form the interlocking sheet of EBC. [Figure 92] FIG. 92 shows a superabsorbent polymer placed in a pocket of stretchable hot melt adhesive. [Figure 93] Figure 93 shows the superabsorbent polymer after it has been swollen with water. [Figure 94] FIG. 94 shows the top view of the interlocking sheets of EBC in a heat press operation. [Figure 95] Figure 95 shows the completed EBC with hydrophilic channels over hydrophobic fields. [Figure 96] FIG. 96 shows the water bag and endothermic compound used in the self-cooling EBC. [Figure 97] Figure 97 shows a roll-to-roll process for forming a laminate of Al foil and adhesive sheet. [Figure 98]FIG. 98 shows a roll-to-roll process for forming an EBC. [Figure 99] FIG. 99 is a cross-sectional view of the EBC. [Figure 100] FIG. 100 is a perspective view of a roll-to-roll process for forming an EBC. [Figure 101] FIG. 101 is an enlarged perspective view showing a conveyor belt of foam for forming pockets in the stretchable adhesive to form the EBC. [Figure 102] FIG. 102 shows a cross section of a stretchable hot melt adhesive with a pocket formed therein. [Figure 103] Figure 103 shows a portion of a foam conveyor belt. [Figure 104] FIG. 104 shows a cross section of the stretchable hot melt adhesive and a cross section of the foam conveyor belt. [Figure 105] FIG. 105 shows a roll-to-roll process for forming aligned nanoparticles between electrodes fixed to a substrate to form an electronic biosensor. [Figure 106] FIG. 106 shows the steps for forming the electronic sensor. [Figure 107] FIG. 107 illustrates steps for forming an unfunctionalized electronic sensor having aligned carbon nanotubes held in place between electrodes on a substrate. [Figure 108] FIG. 108 shows steps for functionalizing an electronic sensor having aligned carbon nanotubes held in place between electrodes on a substrate. [Figure 109] FIG. 109 shows steps for forming an unfunctionalized sensor having aligned carbon nanotubes immobilized on a binding layer. [Figure 110] FIG. 110 shows a continuous process for forming an unfunctionalized sensor using wet electrodeposition / alignment of carbon nanotubes locked between parallel conductors. [Figure 111]FIG. 111 shows a sequential process for forming a functionalized sensor comprising wet bonding and incubation of linker / capture molecules on carbon nanotubes locked between parallel conductors. [Figure 112] FIG. 112 shows printed electrodes linked together to apply an electrical alignment force. [Figure 113] FIG. 113 shows examples of aligned nanotubes at different AC voltages and frequencies. [Figure 114] FIG. 114 shows the printed electrode pattern. [Figure 115] FIG. 115 shows an optional insulator formed over the printed electrode pattern. [Figure 116] FIG. 116 shows the step of printing an electrode pattern onto a substrate. [Figure 117] Figure 117 shows misaligned nanotubes in a solvent fluid carrier. [Figure 118] FIG. 118 shows the alignment of nanotubes in a fluid carrier by an applied AC voltage. [Figure 119] FIG. 119 illustrates the step of placing misaligned nanotubes in a fluid carrier. [Figure 120] FIG. 120 shows the step of applying an AC voltage to align the nanotubes. [Figure 121] FIG. 121 shows aligned nanotubes fixed in alignment after evaporation of the solvent fluid carrier. [Figure 122] Figure 122 shows the addition of linker / aptamer molecules to bind to aligned nanotubes. [Figure 123] FIG. 123 shows a step of aligned nanotubes fixed in place on a substrate between electrodes. [Figure 124] Figure 124 shows linker / aptamer molecules in a non-solvent fluid carrier added onto aligned nanotubes. [Figure 125] Figure 125 shows the incubation to bind the linker / aptamer on the nanotube. [Figure 126]FIG. 126 shows the addition of a fluid biosample for testing. [Figure 127] Figure 127 shows a linker / aptamer attached to aligned nanotubes. [Figure 128] Figure 128 shows the addition of a fluid biosample having target biomarkers captured by aptamers. [Figure 129] Figure 129 illustrates different electronic and electrochemical biosensor strategies known in the art (at least some of which can be utilized to form sensors constructed for the applications and processes described herein). [Figure 130] FIG. 130 shows a cross section of parallel conductors with gaps between the conductor pairs that can be used in some of the applications and processes described herein. [Figure 131] Figure 131 shows a cross section of parallel conductors with nanoparticles aligned in the gap between the conductors. [Figure 132] FIG. 132 shows electronic sensors singulated from a roll or sheet of electronic sensors formed using the processes described herein. [Figure 133] FIG. 133 shows an alternative screen-printed electrode structure, including a reference electrode, for use in forming at least some versions of the electronic and electrochemical sensors described herein. [Figure 134] FIG. 134 illustrates an embodiment of a mask-based diagnostic device for detecting biomarkers contained in a subject's exhaled breath. [Figure 135] FIG. 135 shows an exhaled breath condensate (EBC) collector, thermal mass, fluid transfer system, and biomarker testing unit installed as a retrofit into the exhaled vapor containment volume created by an existing face mask. [Figure 136] Figure 136(a) shows a face mask with externally attached electronics worn by a subject at the start of an EBC test, and Figure 136(b) shows externally attached electronics displaying the results of the EBC test. [Figure 137]FIG. 137 shows the configuration of a breath-based diagnostic device with an electronic biosensor. [Figure 138] FIG. 138 shows a configuration of a respiratory-based diagnostic device having a fluid biosample accumulation reservoir for pooling biosamples on an electronic biosensor or for immersing the sample pad of an LFA in the accumulated fluid biosample. [Figure 139] FIG. 139 shows a test system support that supports the EBC collector, fluid transfer system, and biomarker test unit. [Figure 140] FIG. 140 shows a wick located on the backside of the test system support. [Figure 141] Figure 141 shows the structure of a wick comprising a SAP layer attached to a microfluidic paper layer. [Figure 142] FIG. 142 is a cross-sectional view of a wick with SAP and a microfluidic paper structure. [Figure 143] FIG. 143 shows the connection pins for connecting the electronic biosensor inside the mask with the electronics outside the mask. [Figure 144] FIG. 144 shows an LFA configuration of a respiratory-based diagnostic device having a pool area formed by a fluid biosample accumulation reservoir with an LFA strip positioned with a sample pad within the fluid biosample pool area. [Figure 145] Figure 145 shows the LFA configuration and pool region ready to receive an LFA constructed for a particular target biomarker. [Figure 146] FIG. 146 shows an LFA configuration in which an inspection system is retrofitted to an existing mask. [Figure 147] FIG. 147 shows the sealed LFA test configuration and face mask. [Figure 148] Figure 148 shows the LFA test configuration, which is retrofitted to an existing face mask and worn by the subject at the start of the test. [Figure 149]FIG. 149 shows the LFA test configuration after the subject's exhaled vapor has been converted into a fluid biosample that is transported through the LFA, and shows a visual display of the EBC test results. [Figure 150] FIG. 150 shows an electronic biosensor testing configuration retrofitted to an existing molded face mask. [Figure 151] FIG. 151 is an enlarged view showing the contact pins of an electronic biosensor testing configuration that penetrate the wall of an existing mask. [Figure 152] FIG. 152 shows an existing molded mask with an electronic biosensor testing configuration in which the electronic circuitry is located on the outside of the mask, mechanically secured via connecting pins, and electrically connected to the electronic biosensor. [Figure 153] FIG. 153 shows an electronic circuit located outside the mask that displays the EBC test results. [Fig. 154] FIG. 154 shows a multi-biomarker test unit supported on a test system support. [Figure 155] FIG. 155 shows a fluid transfer system for providing a fluid biosample from the EBC collector to each electronic biosensor of the multi-biomarker test unit. [Figure 156] FIG. 156 shows the underside of the test system support with a wick for continuous flow of fluid biosamples over the multi-biomarker test unit and adhesive for retrofitting onto existing masks. [Figure 157] FIG. 157 shows a flow conductor with a hydrophilic pattern for transporting EBCs towards the examination zone. [Figure 158] FIG. 158 shows a thermal mass having a front surface that forms a condensation forming surface. [Figure 159] FIG. 159 shows a fluid transfer system for transporting EBCs towards the testing zone of the biomarker testing unit. [Figure 160] Figure 160 shows an electronic biosensor version of the biomarker testing unit. [Figure 161]FIG. 161 shows a test system support for supporting an EBC collector, fluid transfer system, and biomarker testing unit, configured and dimensioned to fit inside an existing face mask. [Figure 162] FIG. 162 illustrates an assembly of a respiratory-based diagnostic system. [Figure 163] FIG. 163 shows the components of a mask-based diagnostic system. [Fig. 164] FIG. 164 shows the inch dimensions and shape of one embodiment of the fluid conductor. [Figure 165] FIG. 165 shows an exhaled vapor containment volume defined by a face mask, with the EBC collector and other parts of the breath-based diagnostic system located within the containment volume. [Figure 166] Figure 166 shows a composite thermal mass. [Figure 167] Figure 167 shows the thermal mass of the water / SAP gel. [Figure 168] FIG. 168 shows the back side of the breath-based diagnostic system with water / SAP thermal mass and LFA biomarker testing unit. [Figure 169] FIG. 169 shows an embossed metal foil thermal mass having a condensate-forming surface and fluid conductor channels. [Figure 170] FIG. 170 shows an endothermic thermal mass for insertion into a holding pocket of a mask-based diagnostic system. [Figure 171] Figure 171 shows a soapstone powder / binder composite thermal mass. [Fig. 172] Figure 172 shows the thermal mass of a metal slug. [Figure 173] FIG. 173 shows a face mask constructed with an EBC collector and accumulated fluid biosample reservoir located inside the mask, with the sample pad of the LFA within the reservoir and at least the visual readout portion of the LFA located outside the mask. [Fig. 174]FIG. 174 shows an EBC collector with thermal located inside the exhaled vapor containment volume inside the mask. [Figure 175] FIG. 175 shows the construction of a fluid transfer system having a fluid dam containing a dissolvable adhesive. [Figure 176] FIG. 176 shows the assembly of a bifurcated version of the respiratory-based diagnostic system. [Figure 177] FIG. 177 shows an exploded view of the components of the bifurcated version of the respiratory-based diagnostic system. [Figure 178] FIG. 178 shows a branched version formed with an embossed metal foil condensate forming surface with contours that form fluid transport channels. [Figure 179] FIG. 179 is a cross-sectional exploded view of a branched version of the respiration-based diagnostic system. [Figure 180] FIG. 180 is an assembled cross-sectional view of a bifurcated version of the respiratory-based diagnostic system. [Figure 181] Figure 181 shows the retrofitting of an existing KN95 mask with the LFA version of the breath-based diagnostic system. [Figure 182] FIG. 182 shows a retrofit testing system placed inside a KN95 mask with an LFA placed inside the mask. [Figure 183] FIG. 183 shows an electronic biosensor configured as a field effect transistor with a graph showing the output signal as target molecules begin to bind to capture molecules. [Figure 184] FIG. 184 shows an electronic biosensor configured as a field effect transistor with more target molecules captured and a graph showing the output signal after target molecules begin to bind to the captured molecules. [Figure 185] FIG. 185 shows an electronic biosensor configured as a field effect transistor with more target molecules captured and a graph showing the output signal after target molecules begin to bind to the captured molecules. DETAILED DESCRIPTION OF THE INVENTION

[0028] Below is provided a further description of various non-limiting exemplary embodiments. The exemplary embodiments of the invention as described below may be implemented, practiced, or utilized in any combination (e.g., any combination that is suitable, practical, and / or feasible), including but not limited to only those combinations described herein and / or included in the appended claims.

[0029] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. All embodiments described in this detailed description are exemplary embodiments provided to enable any person skilled in the art to make or use the invention and do not limit the scope of the invention, which is defined by the claims.

[0030] Numerous configurations, embodiments, methods of manufacture, algorithms, electronic circuits, microprocessors, memory and computer software product combinations, networking strategies, database structures and uses, and other aspects are disclosed herein for a variety of wearable electronic digital therapeutic devices and systems for medical and non-medical applications.

[0031] Although embodiments are described herein for detecting biomarkers for the SARS-CoV-2 virus, the described systems, methods, and devices are not limited to any particular virus or disease. In most cases, when the term virus or COVID-19 is used, other health- or fitness-related biomarkers can be used instead. Therefore, the description, drawings, and claims herein are not intended to be limited to the detection of viruses. The described and claimed inventions can be used for many diseases, including lung cancer, diabetes, asthma, tuberculosis, environmental exposures, glucose, lactic acid, blood-borne infections, and other indications of a subject's illness or health condition. Furthermore, the electronic biosensors, test systems, use, and manufacturing methods described herein are not limited to the use of exhaled breath condensate. Wastewater, drinking water, environmental quality samples, and any bodily fluid can be used as test samples. In particular, the use of aptamers allows for the sensors of the present invention to have broad applications, as the properties of the selected aptamers can be adapted for specific manipulation and selection to have tailored binding affinities for the corresponding target analytes. Therefore, the description of the innovation is not intended to be limited to a specific application, capture molecule, biomarker, or analyte.

[0032] In immunochromatography, capture molecules, which may be, for example, aptamers, naturally occurring antibodies, or engineered antibodies, are placed on the surface of a porous membrane, and the sample passes along the membrane. As described herein, the terms antibody, aptamer, engineered antibody, or capture molecule are used interchangeably. In some cases, specific types of capture molecules may be described. Biomarkers in the sample are bound by capture molecules that are linked to detection reagents. As the sample passes through the region where the capture molecules are placed, the biomarker-detection-reagent complex is captured, generating a color proportional to the concentration or amount of biomarker present in the sample.

[0033] In a lateral flow assay, a fluid sample containing target biomarkers flows through a multi-zone transport medium by capillary action. The zones are typically made of a polymer strip that allows molecules attached to the strip to interact with the target biomarkers. An overlying membrane is usually attached to a backing card for improved stability and ease of use. The sample containing the target biomarkers and other components is finally received by an absorbent sample pad, which facilitates wicking of the fluid sample through the multi-zone transport medium.

[0034] The fluid sample is first received on a sample pad (which may have buffer salts and may be disposed or impregnated with surfactants) to improve the flow of the fluid sample and the interaction of the target biomarkers with various parts of the detection system. This ensures that the target biomarkers bind to the capture reagents as the fluid sample passes through the membrane. The processed sample then migrates from the sample pad through a conjugate release pad. The conjugate release pad contains labeled antibodies or other capture molecules specific for binding to the target biomarkers and are conjugated to colored or fluorescent indicator particles. The indicator particles are typically colloidal gold or latex microspheres.

[0035] In the conjugate release pad, the labeled antibody, indicator particle, and target biomarker bind to form a target biomarker-labeled antibody complex. If a biomarker is present, the fluid sample now contains indicator particles conjugated to the labeled antibody and bound to the target biomarker (i.e., target biomarker-labeled antibody complex), as well as separate labeled antibodies conjugated to indicator particles that are not bound to the target biomarker. The fluid sample migrates along the strip to the detection zone.

[0036] The detection zone is typically a nitrocellulose porous membrane with a specific biological component (usually an antibody or antigen) deposited or impregnated thereon, forming a test line zone and a control line zone. The biological component reacts with a target biomarker-labeled antibody complex. For example, the target biomarker-labeled antibody complex binds to a specifically selected primary antibody deposited at the test line by competitive binding. This causes colored or fluorescent indicator particles to accumulate in the test line zone, creating a detectable test line indicating the presence of the target biomarker in the fluid sample.

[0037] The primary antibody does not bind to another labeled antibody and continues to flow with the fluid sample. In the control line zone, the secondary antibody binds to a separate labeled antibody conjugated to an indicator particle, thereby indicating proper fluid flow through the test strip.

[0038] The fluid sample flows through the multi-zone transport medium of the test device due to capillary forces in the materials that make up the zones. To maintain this movement, absorbent pads are attached as end zones of the multi-zone transport medium. The role of the absorbent pads is to absorb excess reagent and prevent backflow of the fluid sample.

[0039] Components are selected and positioned on the membrane such that if the target biomarker is absent from the fluid sample, no target biomarker-labeled antibody complex will flow through the test line zone. In this case, no colored or fluorescent particles will accumulate and no detectable test line will form. Even if the biomarker is absent and therefore no test line is present, a comparison line will still form because the secondary antibody will bind to another labeled antibody that flows with the fluid sample.

[0040] The test and comparison lines may appear at different intensities depending on the device's construction, and the indicator particles can be evaluated visually or using an optical or other electronic reader. Multiple biomarkers can be tested simultaneously under the same conditions using additional test line zones with antibodies specific for different biomarkers arranged in the detection zone in an array format. Additionally, multiple test line zones loaded with the same antibody can be used for quantitative detection of target biomarkers. This is often referred to as a "ladder bar" assay, based on the stepwise capture of colorimetric conjugate-antigen complexes by antibodies immobilized at each successive line. The number of lines displayed on the strip is directly proportional to the concentration of the target biomarker.

[0041] What is needed now is a low-cost, scalable, accurate and easy-to-use testing system that can be deployed to the masses via mail or courier for home use.

[0042] Researchers have been able to detect biomarkers in the exhaled breath of patients with interstitial lung disease (see Hayton, C., Terrington, D., Wilson, AM et al. Breath biomarkers in idiopathic pulmonary fibrosis: a systematic review. Respir Res 20, 7 (2019). https: / / doi.org / 10.1186 / s12931-019-0971-8). Embodiments of the test system of the present invention detect COVID-19 specific biomarkers present in the exhaled breath of infected, infectious, or recovered individuals.

[0043] The COVID-19 testing system of the present invention, with its ability to coalesce exhaled vapor into droplets and then pass the droplet sample through a fluidic biosensor, such as a lateral flow assay (LFA) or electronic nanoscale biosensor (e-NSB), enables a very low-cost, manufacturable, large-scale testing system that can be distributed to the masses for at-home triage testing. The testing system of the present invention can also be used for other biometric and environmental testing applications beyond virus detection.

[0044] LFAs can be used to detect a wide range of biomarkers present in exhaled breath, including cytokines, proteins, haptens (which elicit antibody production), nucleic acids, and amplicons (fragments of RNA and DNA) (see Corstjens PL, de Dood CJ, van der Ploeg-van Schip JJ, et al. Lateral flow assay for simultaneous detection of cellular and humoral immune responses. Clin Biochem. 2011;44(14-15):1241-1246. doi:10.1016 / j.clinbiochem.2011.06.983).

[0045] Directed assembly techniques for high-throughput fabrication of e-NSBs have been demonstrated, which selectively assemble specific antibody-coated nanoparticles onto a single microchip surface for simultaneous detection of multiple biomarkers. Initial results suggest sensitivity to concentrations well below 1 ng / mL, representing a significant improvement over commercially available ELISA detection kits. The biosensors are extremely small, approximately 0.25 mm in diameter, offering advantages over traditional in vitro techniques by enabling detection of disease markers at very low detection limits with few false positives. This capability is highly useful for detecting very small changes in biomarker concentrations in disease monitoring (see "Highly sensitive micro-scale in vivo sensor enabled by electrophoretic assembly of nanoparticles for multiple biomarker detection," Malima et al., Lab Chip, 2012, 12, 4748-4754).

[0046] Breath collection has long been recognized as requiring the least invasive method and is therefore preferred for environmental and public health research. In contrast to blood and urine, breath sampling does not require trained medical personnel or privacy, does not create potentially infectious waste, and can be performed virtually anytime, anywhere. The exhaled breath condensate (EBC) format has the advantage of separating out most nonpolar VOCs while collecting polar compounds and heavier biomarkers, including semivolatile and nonvolatile organic compounds, cytokines, proteins, cell fragments, DNA, and bacteria. Exhaled breath also contains small aerosols (including both liquid and solid particles) generated by surface membrane disruption at the alveolar level and turbulence in the upper airways. These aerosols provide mobility to substances that would otherwise be relegated to the liquid layer in the lungs and are therefore the portion of the EBC that contributes to nonvolatile biomarkers.

[0047] Common methods for collecting clinical specimens from the respiratory tract include nasopharyngeal or oropharyngeal swabs, nasopharyngeal aspirates and nasal washes, tracheal aspirates, bronchoalveolar lavage, or sputum collection. Each of these techniques has drawbacks: nasopharyngeal and oropharyngeal swabs, aspirates, and washes provide mucus from the upper respiratory tract, which may not contain the same viral load or types as those in the lower respiratory tract. Collection of aerosol particles produced by patients during coughing or periodic breathing may provide a noninvasive method for collecting diagnostic specimens of respiratory viruses. Respiratory viruses, particularly influenza viruses, have been detected in exhaled and coughed aerosols from infected patients. Microbial aerosols may also be more representative of lower respiratory tract disease in viral illnesses where sputum production is uncommon.

[0048] Because exhaled aerosol collection is noninvasive, repeated sample collection should be more acceptable to patients than traditional methods. If limitations can be overcome, exhaled aerosol analysis could be a useful tool for diagnosing respiratory infections and monitoring disease progression and response to treatment (see Fennelly KP, Acuna-Villaorduna C, Jones-Lopez E, Lindsley WG, Milton DK. Microbial Aerosols: New Diagnostic Specimens for Pulmonary Infections. Chest. 2020;157(3):540-546. doi:10.1016 / j.chest.2019.10.012).

[0049] There are over 2,000 compounds identified in EBCs (see Montuschi P, Mores N, Trove A, Mondino C, Barnes PJ, The electronic nose in respiratory medicine. Respiration. 2013;85(1):72-84), many of which are thought to represent sensitive biomarkers of lung disease (see Sapey E, editor. Bronchial Asthma: Emerging Therapeutic Strategies. Rijeka: InTech). Biomarkers present in EBCs provide a more detailed description of processes occurring in the lungs than processes throughout the body.

[0050] Therefore, specific profiles of exhaled breath biomarkers can reveal information that is specifically applicable to the diagnosis of lung diseases. EBC is a biological matrix that reflects the composition of bronchoalveolar extracellular lung fluid. The main advantage of EBC as a matrix is ​​its specificity to the airways (this fluid is not affected by processes occurring in other parts of the body) (see Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate, Tereza Kacerova, Petr Novotny, Jan Boron, and Petr Kacer. Submitted: October 9, 2016. Reviewed: January 25, 2018. Published: September 5, 2018. DOI: 10.5772 / intechopen.7440).

[0051] The surfaces of all parts of the lungs leading up to the alveoli are coated with an aqueous mucus layer that can aerosolize and carry various nonvolatile components. EBC and EBA are different types of exhaled breath matrices used to assess human health and disease states. EBA represents a portion of the total EBC and targets larger molecules, such as fatty acids and cytokines, as well as cellular fractions, proteins, viruses, and bacteria instead of the gas phase. Exhaled breath condensate (EBC) and exhaled breath aerosol (EBA) contain a variety of compounds, including volatile organic compounds (VOCs), NO, CO2, NH3, cytokines, and hydrogen peroxide (H2O2). VOCs present in adipose tissue are released into the blood and exchanged with exhaled air via the alveoli and airways of the lungs. Some VOCs are also retained in the airways after exposure. Thus, exhaled breath concentrations of VOCs are representative of blood concentrations, but samples can be obtained noninvasively with little discomfort to the individual (see Wallace MAG, Pleil JD. Evolution of clinical and environmental health applications of exhaled breath research: Review of methods and instrumentation for gas-phase, condensate, and aerosols. Anal Chim Acta. 2018;1024:18-38. doi:10.1016 / j.aca.2018.01.069).

[0052] EBC and EBA are valuable noninvasive biological media used for quantification of biomarkers. In addition to exhaled water vapor, soluble gas-phase (polar) organic compounds, and ionic species, EBC contains semivolatile and nonvolatile organic compounds, proteins, cell fragments, DNA, dissolved inorganic compounds, ions, and dissolved microbiota (bacteria and viruses) in the co-collected EBA (see Inters BR, Pleil JD, Angrish MM, Stiegel MA, Risby TH, Madden MC. Standardization of the collection of exhaled breath condensate and exhaled breath aerosol using a feedback regulated sampling device. J Breath Res. 2017;11(4):047107. Published 2017 Nov 1. doi:10.1088 / 1752-7163 / aa8bbc).

[0053] Previous references have reported detection of influenza virus RNA in the exhaled breath of patients infected with influenza A and influenza B viruses. While EBC samples may contain lower concentrations of viral RNA than nasal swabs, these tests have detected detectable influenza virus RNA in exhaled breath. Exhaled breath sample concentrations ranged from 48 to 300 influenza virus RNA copies per filter for positive samples, corresponding to exhaled breath production rates ranging from 3.2 to 20 influenza virus RNA copies per minute (see Fabian P, McDevitt JJ, DeHaan WH, et al.).

[0054] Influenza virus in human exhaled breath: An observational study. PLoS One. 2008;3(7):e2691. Published 2008 Jul 16. doi:10.1371 / journal.pone.0002691). This reference indicates that nasal and throat swabs may typically have greater RNA concentrations than EBCs. However, viral RNA is clearly present in EBCs, and an EBC testing system with sufficient sensitivity should be effective in detecting viruses, bacteria, and other disease- and health-related biomarkers.

[0055] Scanning electron microscopy (SEM), polymerase chain reaction (PCR), and colorimetric analysis of bacteria and viruses (VITEK2) have shown that bacteria and viruses in EBCs can be rapidly collected with an observation efficiency of 100mL EBCs within 1 minute (see Xu Z, Shen F, Li X, Wu Y, Chen Q, et al. (2012) Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method. PLoS ONE 7(7): e41137. doi:10.1371 / journal.pone.0041137).

[0056] Exhaled breath contains volatile organic compounds (VOCs), a collection of hundreds of small molecules associated with several physiological and pathophysiological processes. Analysis of exhaled breath by gas chromatography and mass spectrometry (GC-MS) has led to the accurate diagnosis of ARDS in several studies. Most of the identified markers are related to lipid peroxidation. Octane is one of the few validated markers of ARDS and may be pathophysiologically increased in ARDS (see Bos LDJ. Diagnosis of acute respiratory distress syndrome by exhaled breath analysis. Ann Transl Med. 2018;6(2):33. doi:10.21037 / atm.2018.01.17).

[0057] The testing system of the present invention is designed to be self-administered; simply put on the mask and open the smartphone app to transmit and store data.

[0058] Alternatively, data transmission can be avoided by not storing the data, and instead results can be privately displayed via an on-board indicator such as an LED or a smartphone app. When a test result signal is transmitted, the data is encrypted at the source, the electronic device attached to the mask, before wireless transmission. Privacy concerns are addressed at least as well as government requirements for electronic medical records. The testing system of the present invention can include wireless communication capabilities that allow test data, along with GPS location information, to assist in backward and forward contact tracing, for use in the event of an epidemic or pandemic, further enhancing the ability to increase the portion of the population that can safely return to work and reopen the economy, and allowing those potentially exposed to the virus to be identified through real-time contact tracing as soon as a positive test result is obtained.

[0059] Although biometric data can be acquired and used for the public good, the collection of biometric information carries the burden of privacy issues. Patient biometric data has two potential uses: patient monitoring for prevention and treatment; and population research to improve global healthcare. The testing system of the present invention can be configured with software and hardware for a separately created and maintained database shared only with trusted recipients (e.g., healthcare providers who access patient data through a secure two-step authentication process), and demographic-only data that stores anonymized data used for big data analysis to identify patterns and trends related to outbreaks. To maximize compliance, subjects can be allowed to choose the level of data reporting (self-reporting; sharing only with the subject's registered HCP; or automated data reporting for contact tracing and electronic medical records). Captured data can be anonymized and encrypted at source (e.g., by electronic devices associated with the testing system). Using a smartphone app, subjects can always control how their test data is reported, opting out or in to the level of data sharing.

[0060] Figure 1 shows a lateral flow assay (LFA) test system showing a biomarker sample added to the sample pad. Figure 2 shows an LFA containing a biomarker-labeled antibody complex formed in the conjugate release pad. Figure 3 shows binding of the biomarker at the test line, indicating the presence of the biomarker.

[0061] Another testing system that can be used with the EBC collection system of the present invention uses an electronic nano-scale biosensor (e-NSB). Like LFAs, e-NSBs can be much more sensitive and can be used, for example, to provide a direct electrical signal, allowing for simple wireless connectivity. The EBC collection system of the present invention with e-NSB testing can be easily deployed as a complement to existing contact tracing apps. The nanoscale dimensions mean that many detectors can be fabricated at once on a single wafer, or as described herein, through a high-volume roll-to-roll manufacturing process for low-cost, high-throughput manufacturing.

[0062] Figure 4 illustrates the mechanism of a biosensor detection system. Briefly, the key components of a fluidic biosensor include a sample source (a); a biosensor region functionalized with biomarker-specific bioreceptors (b); and a transducer (c) that generates a readable signal. The bioreceptors match specific target biomarkers for lock-and-key selectivity screening. A fluid sample containing a concentration of the target biomarker (which can be as small as a single molecule) flows into the biosensor field. A portion of the biosensor "lock" receives the biomarker's "key." This causes a detectable change in the transducer's output, which is converted into a readable signal for amplification and data processing.

[0063] For example, a desirable biomarker would be an antibody that indicates recovery from a Covid-19 infection. A fluid sample can be received as droplets of sweat, exhaled breath, or other bodily fluid, and if the target antibody is present in the sample, it will interact with a biomarker-specific bioreceptor. The bioreceptor outputs a signal with a defined sensitivity, and the transducer generates a change in an electrical property, such as conductivity, that indicates the presence of the antibody biomarker in the fluid sample.

[0064] According to one embodiment, an apparatus for detecting a biomarker comprises a droplet collection and channeling structure for converting a vapor into a fluid sample source having a biomarker, a biosensor region functionalized with a biomarker-specific bioreceptor, and a transducer for generating a readable signal as a function of changes in the bioreceptor in response to receiving the biomarker from the sample source.

[0065] The use of nanoscale sensor technology allows for the detection of very low concentrations of target biomarkers, such as viral RNA, proteins, and / or antibodies, eliminating the need for blood draws. According to one embodiment of the inventive test system, the droplet collection and channeling mechanism uses a hydrophobic field for fluid collection and a hydrophilic channel for droplet movement onto the nanosensor. This mechanism makes the inventive system practical for creating a very inexpensive, scalable, manufacturable COVID-19 test that does not require blood or for the test to be administered by a skilled technician, nurse, or healthcare provider.

[0066] Embodiments of the mask-based inspection system use nanoscale fluidic biosensor technology with a unique aqueous droplet collection and channeling structure that potentially enables the use of nanoscale sensors to detect down to single molecules of target biomarkers, enabling the detection of very low concentrations of antibodies, proteins, and other chemical biomarkers present in bodily fluids without the need to draw blood.

[0067] Non-limiting embodiments are based on the sweat chemical sensor technology described in PCT / US19 / 45429 (Method and Apparatus for Wearable Electronic Digital Therapeutic Devices, inventor Daniels, published April 10, 2020), which is incorporated herein by reference in its entirety. Embodiments of the invention described herein include COVID-19 testing systems that can be mass-produced in readily available mass production facilities in the millions required for mass population testing. Embodiments of the testing system use nanoscale fluidic biosensors with unique moisture droplet collection and channeling structures.

[0068] This architecture enables the use of nanoscale sensors to detect COVID-19 biomarkers in bodily fluid samples, such as exhaled breath condensate. The system can detect antibodies, protein, RNA biomarkers, and other COVID-19 chemical biomarkers without the need for blood draws, expensive equipment, or technically trained personnel. The proposed system can be configured as at least a first-pass no-obligation test to determine who should be more accurately tested by traditional testing methods.

[0069] We need a low-cost, accurate, and easy-to-use COVID-19 testing system that would ideally be mailed and self-administered at home. For example, current testing protocols require individuals to use nasal swabs for RNA testing to indicate active infection or to take a blood sample to determine whether they have sufficient antibodies to the COVID-19 virus for immunity. These tests typically require individuals to leave isolation and travel to a testing site where a technician, nurse, or other healthcare provider administers the test. We propose a testing system that can be used as an initial need / no-need assessment to first determine whether more elaborate and costly testing methods are justified. For example, an inexpensive, easy-to-use testing system that can be administered at home and detects low concentrations of COVID-19 antibodies in breath or sweat could subsequently trigger an individual to visit a testing facility to more accurately determine their immunity to further COVID-19 infection.

[0070] Figure 5 shows a side view of a wearable electronic breath chemical sensor. Figure 6 shows a top view of a wearable electronic breath chemical sensor. The biometric sensor is tuned to detect at least one biometric indicator associated with the presence of COVID-19 antigens, RNA, and / or antibodies. The droplet collector draws EBC droplets into the transport opening. Once the sensor is wetted by the droplets, they are drawn into the wicking / evaporation material via wicking. A continuous stream of fresh droplets passes the sensor. The hydrophobic field promotes EBC beading and migration into the hydrophilic channel. The tapered hydrophilic channel uses surface tension to draw sweat into the sweat transport opening. Hydrophobic and hydrophilic screen-printable inks are available from companies such as Cytonix and Wacker.

[0071] Figure 7 is an isolated view of an exhaled breath condensate (EBC) droplet sample collector. Figure 8 is a top view showing steps for forming an EBC droplet collector. Figure 9 is a top view showing another step for forming an EBC droplet sample collector. Figure 10 is a top view showing yet another step for forming an EBC droplet sample collector. Figure 11 is a top view showing yet another step for forming an EBC droplet sample collector. Figure 12 shows an EBC sample collector showing EBC droplets. According to a non-limiting exemplary embodiment, an at-home triage COVID-19 testing system uses exhaled breath condensate (EBC) for biomarker fluid samples. Exhaled breath is an exceptional source of viral antigens, antibodies, and RNA. EBCs can be analyzed using established methods such as lateral flow assays, nanoscale bioreceptors, and photonic quantitative assays. EBCs produce a much cleaner sample for testing than nasal swabs, are non-invasive, and are easier than blood draws. However, collection of EBCs typically requires large, expensive chillers and is always performed in a clinical setting.

[0072] There is a significant push worldwide to develop appropriate tests for the COVID-19 virus. Traditional PCR tests detect dead virus fragments in nasal swabs or sputum. These tests determine whether a person is infected. These tests are expensive, require trained personnel and machinery, and result in delays in obtaining test results due to sample collection, transportation, and processing. PCR also requires many chemical reagents, resulting in many false negatives. Antibody tests detect the body's immune response to the virus. They typically require a blood sample. Antibody tests can be performed relatively quickly and necessarily require trained personnel. False positives occur frequently because other viruses may be causing the antibodies.

[0073] EBC has been used for the rapid detection of microbial DNA and RNA to demonstrate bacterial and viral lung infections (Xu Z, Shen F, Li X, Wu Y, Chen Q, Jie X, et al. Molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method. PLoS One 2012;7:e41137. See https: / / www.ncbi.nlm.nih.gov / pubmed / 22848436).

[0074] Nasal swab samples often contain a lot of background biological material, making it difficult to identify viral RNA due to the presence of other molecules in the sample. Exhaled breath condensate is naturally enriched with viruses and has much lower concentrations of interfering molecules (see https: / / www.zimmerpeacocktech.com / products / electrochemical-sensors / covid-19-and-pcr-on-the-breath / ).

[0075] Antibodies are present in exhaled air. IgA antibodies are found in areas of the body such as the nose and respiratory passages. IgG antibodies are found in all body fluids and are the most common antibody (75%-80%), and are very important in fighting bacterial and viral infections. IgE antibodies are found in the lungs, skin, and mucous membranes (see https: / / www.uofmhealth.org / health-library / hw41342).

[0076] Viral antigens are contained in airway lining fluid (ALF). EBC is a noninvasive method for sampling airway lining fluid (ALF). The ALF component is representative of the lining of the respiratory tree. ALF is a measure of the concentration of biomarkers directly affected by respiratory cells (see Exhaled breath condensate: a comprehensive update, Ahmadzai, et al., Clinical Chemistry and Laboratory Medicine (CCLM) 51, 7; 10.1515 / cclm-2012-0593). EBC may be an exceptional source of biomarkers indicative of COVID-19 virus infection and recovery stages, as well as other medical and fitness applications. However, conventional equipment for obtaining EBC fluid samples is large, expensive, and only used in clinical settings. Conventional devices require chillers and are designed for relatively large sample collection volumes. This makes conventional EBC sampling devices unsuitable for home testing.

[0077] An embodiment of the EBC sample collector of the present invention includes a hydrophobic field that breaks vapor into droplets. Hydrophilic channels coalesce and transport the droplets to form an accessible EBC fluid sample. The hydrophobic field and hydrophilic channels can be screen-printed or otherwise coated onto a thermal mass aluminum sheet substrate. The polished aluminum sheet itself can be made into a hydrophobic field without further processing. This substrate can be cooled before use to improve EBC collection. The EBC sample collector of the present invention enables low-cost lateral flow assays, electronic biosensors, and other testing systems for home triage testing. The CDC has stated that rapid development of affordable screening tests is essential. The EBC sample collector of the present invention makes such screening tests feasible for large-scale deployment to large segments of the population. No isolation break is required. No skilled technicians, clinics, or laboratory equipment are required. Existing manufacturing methods can be modified to commercialize a multi-up (many at a time) screen-printed EBC sample collector for very high production volumes. The low-cost aluminum substrate acts as a thermal mass and can be cooled to collect droplets more quickly. Using batch manufacturing, multiple LFA modules can be manufactured on a sheet in a format that is rapidly adaptable for ultra-high volume roll-to-roll manufacturing.

[0078] Figure 13 shows an EBC sample collector applied to an LFA test system. Figure 14 is an exploded view showing the screen-printed hydrophilic channel, screen-printed hydrophobic field, and thermal mass substrate of the EBC sample collector. A first emitter / detector pair is used to determine whether the novel coronavirus N protein at the test line (T) is bound by an IgM-IgM complex. A second emitter / detector pair is used to determine whether free anti-human IgM antibodies are bound to anti-mouse antibodies at the comparison line (C), confirming that the fluid sample has passed through the transport medium and that the test has been performed correctly.

[0079] According to one embodiment, a method of forming a biomarker testing system includes forming a breath condensate sample collector. Forming the breath condensate sample collector includes providing a substrate, coating a hydrophobic field on the substrate, and coating at least one hydrophilic channel on the substrate. The hydrophobic field is for receiving bodily fluid vapor and forming fluid droplets from the received bodily fluid vapor, and the hydrophilic channel is for receiving the fluid droplets and directing the fluid droplets toward the testing system. At least one fluid sample ejection hole can be formed at an end of the hydrophilic channel through which the fluid droplets can be ejected onto a sample-receiving structure of the testing system.

[0080] At least one light emitter and one photodetector can be provided, with the light emitter emitting radiation toward the biomarker testing zone and the photodetector receiving radiation from the biomarker testing zone. FIG. 15 is an exploded view showing the components of an LFA. FIG. 16 shows one embodiment of an LFA including the light emitter / detector electronics. In immunochromatography, a capture antibody is placed on the surface of a porous membrane and a sample is passed along the membrane. Biomarkers in the sample are bound by the antibody and then bound to a detection reagent. As the sample passes through the area where the capture reagent is placed, biomarker-detection reagent complexes are captured and a color is developed that is proportional to the biomarker present in the sample. The photonics light emitter / detector pair allows for proportional quantitative measurement of the biomarker, and the biomarker concentration in the fluid sample is determined from the intensity or count of photons received by the detector.

[0081] Solid-phase lateral flow testing platforms are an example of immunochromatography, widely used for home pregnancy testing. Lateral flow tests benefit from the use of sol particles as labels. Inorganic (metal) colloidal particles are typically used as labels in immunoassays, and several techniques are used to measure the amount of bound conjugate. These include visual detection, colorimetry, and atomic absorption spectrophotometry. Colorimetry applies the Beer-Lambert law, which states that the concentration of a solute is proportional to its absorbance. At higher antigen concentrations, immunochromatographic results can be read with the naked eye (e.g., a typical home pregnancy test). At low concentrations, colorimetry has been shown to be over 30 times more sensitive than visual detection.

[0082] According to one embodiment, immunochromatography is used to detect the presence of COVID-19 biomarkers. Generally, immunochromatography involves separating components in a mixture through a medium using capillary forces and the specific and rapid binding of antibodies to antigens. A dry transfer medium is separately coated with novel coronavirus N protein (the "T" test line) and anti-mouse antibody (the "C" comparison line). Free colloidal gold-labeled anti-human IgM is present in the release pad section (S). A vapor coalescence and droplet collection structure of the present invention is used to obtain a fluid sample of exhaled breath condensate. This fluid sample is applied to the release pad section. The anti-human IgM antibody binds to at least a portion of the IgM antibody (if present), forming an IgM-IgM complex. The fluid sample and antibody move through the transfer medium by capillary action. If coronavirus IgM antibody is present in the fluid sample, the novel coronavirus N protein at the test line (T) is bound by the IgM-IgM complex, resulting in color development. If the sample does not contain coronavirus IgM antibodies, the free anti-human IgM will not bind to the test line (T) and no color will develop. The free anti-human IgM will bind to the anti-mouse antibody at the comparison line (C), causing the comparison line to develop color, confirming that the fluid sample has passed through the transport medium and that the test was performed correctly.

[0083] Figure 17 shows an EBC sample collector applied to a nanoscale biosensor testing system, showing a pull tab for retaining droplets collected on the sample pad. Figure 18 shows a perspective view of an EBC sample collector applied to a testing system. Figure 19 shows an isolated view of a pull table positioned between the sample pad and the conjugate release pad. The EBC fluid sample is collected over a period of time from the hydrophobic field through the hydrophilic channel, flows through the fluid sample discharge hole, and accumulates in the sample pad. A typical LFA uses approximately three drops of buffer sample added to the sample pad. According to an exemplary embodiment, buffer materials and surfactants can be incorporated into the sample pad in dry form, making the EBC sample, which is mostly water, suitable as a directly applied fluid sample without the need for the addition of a fluid buffer. When a person is at rest, approximately 17.5 ml of EBC is produced per hour (see "How much water is lost during breathing?", Zielinski et al., Pneumonol Alergol Pol 2012;80(4):339-342). There are 20 drops in one milliliter. Therefore, approximately 350 drops of EBCs can be collected for each hour of resting time. A collection efficiency by the EBC sample collector of only about 3% should provide a sufficient number of EBC droplets for a fluid sample in about 15 minutes from an individual at rest.

[0084] According to one embodiment, an apparatus for detecting biomarkers includes a droplet collection structure for converting exhaled breath vapor into fluid droplets to form a fluid sample, and a testing system having a biomarker testing zone for receiving the fluid sample and detecting biomarkers. The droplet collection structure can include at least one of a hydrophobic field for receiving the exhaled breath vapor and forming fluid droplets from the received exhaled breath, and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets toward the testing system. A fluid dam member can be provided disposed between the droplet collection structure and the biomarker testing zone.

[0085] The testing system may include a fluidic lateral flow assay comprising a sample pad for receiving a fluid sample potentially containing biomarkers, a conjugate release pad, a flow membrane, and an absorbent pad for receiving and flowing the fluid sample and detecting potential biomarkers from the sample source. A fluid dam member is disposed between the sample pad and the conjugate release pad, the fluid dam including a pull-tab structure such that a subject can remove the fluid dam member to allow the fluid sample to flow from the sample pad to the conjugate release pad.

[0086] To flush the EBC fluid sample through the test system, a fluid dam is provided to prevent EBC accumulation on the sample pad. The fluid dam may be, for example, but not limited to, a silicone-coated release paper forming a pull tab positioned between the sample pad and the conjugate release pad. The pull tab is held in place by an adhesive on top, allowing the EBC fluid sample to accumulate. After sufficient time has passed to saturate the sample pad with a sufficient amount of EBC fluid sample, the subject pulls the pull tab, allowing the EBC fluid sample to flow from the sample pad to the conjugate release pad. This allows the EBC fluid sample to flush through the various components of the test system by capillary action. Because the EBC fluid sample can accumulate on the sample pad, removing the pull tab releases the sample flow all at once, ensuring proper sample flow and promoting test consistency.

[0087] Figure 20 is an isolated view of a screen-printed droplet sample collector with fluid transfer openings. Figure 21 is a cross-sectional view showing a collected fluid sample from an EBC sample collector flowing between a photonics emitter / detector pair. In this embodiment, instead of coalescing exhaled vapor into exhaled condensate, another bodily fluid, such as sweat, can be used with an EBC sample collector configured to collect sweat droplets from the skin. Note that any of the embodiments and innovations described herein may be useful in other medical and fitness applications, for other disease or virus testing or biometric detection, in addition to or instead of the described use for COVID-19 testing.

[0088] The manufacturing techniques, equipment, and materials for most components of embodiments of the COVID-19 testing system of the present invention are readily available and very well known. For example, to create the fluid collection and droplet channeling structures, screen printing is used to pattern hydrophilic and hydrophobic inks provided by companies such as Cytonix and Wacker. There is no shortage of manufacturing capacity required to rapidly screen-print the hundreds of millions of test units required. Fluidic biosensor components can be manufactured using high-throughput equipment available from companies such as Conductive Technologies, Inc., York, PA, USA, and chemicals for functionalizing the biosensor are available from companies such as RayBiotech, Peach Tree Corners, GA, USA. Other required manufacturing steps, such as wire bonding and printed circuit board fabrication, utilize the same ubiquitous machines used for similar purposes for semiconductor and circuit board electronics.

[0089] Figure 22 shows a side view of the steps for constructing an LFA test system, although they do not necessarily follow the order shown. Figure 23 shows a top view of the steps for constructing an LFA test system. Figure 24 shows a 4x9 interlocking multi-up sheet of LFA test systems formed as a batch. Figure 25 shows a roll-to-roll manufacturing process for forming a roll of bottom adhesive / backing substrate / top adhesive. Figure 26 is a perspective view of a stack of bottom adhesive / backing substrate / top adhesive. Figure 27 shows a roll-to-roll manufacturing process for forming the bottom adhesive / backing substrate / top adhesive LFA components on a roll. Figure 28 shows an LFA test system cut from a continuous roll in a roll-to-roll process, showing a section of the top adhesive for adhering the LFA test system to a separately formed ENC sample collector. Figure 29 shows an LFA test system cut from a continuous roll formed by a roll-to-roll process, showing a section of the bottom adhesive for attaching to a wearable garment such as a face mask. Figure 30 shows a sheet of substrate having a hydrophobic field coating on a thermal mass substrate with droplet collection holes. FIG. 31 shows a sheet of substrate having a hydrophobic field coating on a thermal mass substrate with droplet collection holes having a coating of hydrophilic channels.

[0090] Figure 32 shows EBC's sample collector and testing system with electronics for wireless data acquisition and transmission, as well as a separate trusted receiver and data path and storage on the public blockchain. Villanova University recently published an example using blockchain to enable medical facilities to track coronavirus cases globally. A private blockchain is shared among medical facilities around the world, publishing coronavirus test results between doctors on a trusted, immutable ledger. IoT and AI are used to survey public spaces for potential high-risk gatherings and trigger alerts via the blockchain. (See https: / / www1.villanova.edu / university / experts / spotlight-detail.html?spotlight=7180)

[0091] According to exemplary embodiments, the EBC collection system with biomarker detection can be enabled for use in new or existing apps for contact tracing and electronic medical records using self-reporting or automated data collection. The acquired data can be anonymized and encrypted at the source (e.g., at the electronic device associated with the testing system). A first data stream / database allows trusted recipients to access patient-identifying data, while a second data stream / database provides anonymized data that can be offered as an open-source or other data transfer, storage, and usage mechanism without identifying the source of the data.

[0092] The testing system of the present invention is very low cost and could potentially be shipped in conventional envelopes for mass distribution to every household in a target region, state, or country, allowing a much higher percentage of the population to receive at least a baseline test that indicates whether they need to follow up with a visit to a drive-thru, hospital, or clinic testing facility for more detailed testing.

[0093] The COVID-19 testing system of the present invention may have the ability to simultaneously test for two or more viral biomarkers. For example, an RNA or protein test can be combined with an antibody test. Testing for these two biomarkers statistically significantly reduces the chance of false negatives and may be a more preferable method.

[0094] The proposed COVID-19 testing system can be integrated into personal protective equipment such as masks, provided as a patch to be worn on the body, or provided as a standalone testing unit, similar to a home pregnancy test. The biosample can be obtained from saliva, blood, urine, EBC, tears, sputum, feces, or other materials that may contain the target analyte. Buffers and diluents can be used, and, if necessary, incubation and amplification techniques similar to those used in conventional PCR testing can be employed.

[0095] Testing systems can include wireless communication capabilities such as RFID, near field communication, WiFi, cellular, and Bluetooth, which could allow test data to be used, for example, along with GPS location information to assist with contact tracing, further increasing the ability to get a larger portion of the population back to work and reopen the economy, and identify those who may have been exposed to the virus through real-time contact tracing.

[0096] As an enhancement to the basic system, biometric data can be captured and used for the public good. Collecting biometric information raises privacy concerns. Patient biometric data has two potential uses: patient monitoring for prevention and treatment; and population research to improve global healthcare. The system of the present invention uses a separately created and maintained database.

[0097] Biometric parameters, such as those described herein with respect to embodiments, can also be detected, recorded, and / or transmitted, allowing for a detailed history of a patient's disease progression, treatment, course of treatment, treatment measurements, etc., and can be made available to improve the care provided to a particular patient, and collectively provide important data, along with other patient data, created by the detection, transmission, storage, and analysis of biometric data obtained directly from patients during the course of drug therapy or other treatment, to aid in the discovery of new drugs, therapy changes, and many other benefits of a beneficial cycle.

[0098] Figure 33 shows the manufacturing process for thermally bonded face masks. Figure 34 shows fabric, filter, and other layers bonded by a roll-to-roll lamination process, individually cut into blanks to form a preform mask stack. Figure 35 shows other materials, such as biologically reactive silver fabric and hot melt adhesive, in the preform mask stack. The contagiousness and deadly effects of COVID-19 have increased the need for personal protective masks. Disposable masks are an excellent solution for healthcare providers, police, and other workers who are constantly in contact with individuals, both those with and without the virus. For example, the ability to replace disposable masks for each patient allows doctors and nurses to use fresh, clean, and uncontaminated masks to better protect themselves and their patients from the spread of the virus. However, disposable masks are not a good solution for the general public. The cost and waste associated with disposable masks make them an insufficient solution for most people. Rather, what is needed are masks that are low-cost, easy to manufacture, and ideally, can be sterilized in conventional home clothes washers and dryers.

[0099] Figure 36 is an exploded view of the mask stack. Figure 37 shows the folds of the mask stack for the first and second hot pressing operations. Figure 38 shows the folded, pressed, and heat-sealed mask. Figure 39 shows the attachment of the EBC collector and inspection system to the folded mask. Figure 40 shows the step of turning the folded mask inside out and placing the EBC collector and inspection system inside the mask. Figure 41 shows the hot pressing operation of bonding the elastic straps to the folded mask.

[0100] FIG. 42 illustrates a mask with an EBC collector and testing system positioned inside the mask in a concentrated atmosphere of exhaled breath. According to a non-limiting embodiment, a mask-based testing system for detecting biomarkers received from a subject's lungs and airways is provided. The exhaled breath condensate (EBC) collector is positioned inside a face mask worn by the subject. The EBC collector converts exhaled vapor received from the subject's lungs and airways into a liquid biosample. The EBC collector has a thermal mass and a front surface, where the front surface receives exhaled vapor at a temperature higher than the surface temperature of the front surface and converts the exhaled vapor into a liquid at a temperature lower than the temperature of the exhaled vapor. The EBC collector includes a droplet collection structure on the front surface, the droplet collection structure including a field for receiving exhaled vapor and forming fluid droplets from the received exhaled vapor, and a channel for receiving the fluid droplets from the field and flowing the fluid droplets together to form a collected fluid biosample.

[0101] The biosensor is secured to the face mask for receiving a fluid biosample from the EBC collector and testing the fluid biosample for target biomarkers. The biosensor generates a test signal that depends at least on the presence or absence of the target biomarker in the fluid biosample. Electronic circuitry secured to the face mask receives the test signal, determines a test result signal from the test signal in response to detection or non-detection of the target biomarker, and transmits the test result signal to a remote receiver.

[0102] Figure 43 shows a conventional bendable metal nose seal positioned within the folds of the mask in a position corresponding to the bridge of the subject's nose, Figure 44 shows a replaceable adhesive nose strip positioned outside the folds of the mask in a position corresponding to the bridge of the subject's nose, and Figure 45 shows the components of a magnetic detachable nose seal.

[0103] Figure 46 is an exploded view of a testing system including a dissolvable fluid dam, which holds collected EBCs on the sample pad until a sufficient amount accumulates to be released onto the conjugate release pad, and then flushes the fluid sample through the components of the testing system. A fluid dam member can be disposed between the droplet collection structure and the biomarker testing zone, and the fluid dam member includes at least one of a removable moisture-resistant sheet member and a dissolvable film for accumulating the fluid sample from the droplet collection structure and releasing the accumulated fluid sample to flow into the biomarker testing zone. The fluid sample testing system includes a fluid lateral flow assay, which includes a sample pad for receiving a fluid sample potentially containing a biomarker as a second biomarker, a conjugate release pad, a flow membrane, and an absorbent pad for receiving and flowing the fluid sample and detecting potential biomarkers from the sample source. A fluid dam member can be disposed between the sample pad and the conjugate release pad, and the fluid dam includes a pull-tab structure, allowing the fluid sample to flow from the sample pad to the conjugate release pad when the subject removes the fluid dam member. At least one light emitter and one light detector may be provided, the light emitter emitting radiation towards the biomarker testing zone and the light detector receiving radiation from the biomarker testing zone.

[0104] Figure 47 is an isolated view showing the dissolvable fluid dam inserted between the sample pad and the conjugate release pad. Figure 48 is an isolated view showing the dissolvable fluid dam after it has been dissolved and removed, releasing the accumulated fluid sample from the sample pad to the conjugate release pad. The home testing system of the present invention can be used for COVID-19, other viral, bacterial, environmental, cancer, asthma, diabetes, fitness, or other medical applications. The basic premise is to use a face mask to collect exhaled breath condensate (EBC) and exhaled breath aerosol (EBA).

[0105] EBCs are collected via a hydrophobic / hydrophilic droplet collection structure and delivered to a testing system (e.g., a lateral flow assay or electronic biosensor). To effectively collect and accumulate EBCs, dissolvable materials can be used to adjust the capillary filling time. This allows the flow of the fluid sample (EBC) from the droplet collection structure to be restricted until a sufficient sample volume has accumulated and capillary action allows the liquid to pass through the testing system. To capture EBAs, droplets and aerosol particles can be suspended on a surface or in a dissolvable film. The film's adhesive surface allows particles exhaled during breathing or coughing to adhere to the adhesive surface. If the film is also water-soluble, exhaled droplets can also be adsorbed onto the film. This COVID-19 testing system uses EBCs for screening (i.e., triage testing). If the EBC test indicates a positive detection of target biomarkers (e.g., COVID-19 antibodies or RNA), the mask is shipped to a testing laboratory where the captured EBAs are analyzed.

[0106] Figure 49 is an isolated view of the dissolvable EBC droplet and EBA particulate collector. Figure 50 is a side cross-sectional view showing a cross section of the dissolvable droplet and particulate collector with the particulates and droplets impacting the surface. In an expanded version of the proposed testing system, an aerosol particulate collection system is provided to capture viral biomarkers exhaled or coughed up by the test subject. The surfaces of all parts of the lung leading up to the alveoli are coated with an aqueous mucus layer that can aerosolize and carry various non-volatile components. EBC and EBA are different types of exhaled breath matrices used to assess human health and disease states. EBAs represent a fraction of total EBCs and target non-gas-phase cellular fractions, proteins, viruses, and bacteria, as well as larger molecules such as fatty acids and cytokines (see Wallace MAG, Pleil JD. Evolution of clinical and environmental health applications of exhaled breath research: Review of methods and instrumentation for gas-phase, condensate, and aerosols. Anal Chim Acta. 2018;1024:18-38. doi:10.1016 / j.aca.2018.01.069).

[0107] Figure 51 is a side cross-sectional view showing a cross section of a dissolvable droplet and particle collector with droplets embedded in a dissolvable capture film that dissolve into the detection reagents in the dissolvable capture film to trigger a detection reaction. Figure 52 is a top view showing a test system of the present invention including a dissolvable EBC droplet and EBA particle collector that captures aerosol droplets and aerosol particles. The particle capture mechanism can be a dissolvable film with a sticky surface and can include a visual detection reaction for one or more target biomarkers. Soluble biomarkers that react with the visual detection chemicals visually indicate the presence of the biomarkers in the EBA. Insoluble particles are captured on the sticky surface and embedded in the capture film, allowing for easy transport to a lab for analysis. Dissolvable adhesives can be obtained, for example, from Adhesives Research in Pennsylvania. As an example, if the EBC test system is used for at-home screening, a positive test result for the EBC target biomarkers can be used to prompt the subject to return the test system so that the captured particles from the EBA sample can be further analyzed using more sophisticated laboratory equipment.

[0108] A system of the present invention for detecting biological agents from the exhaled breath of a subject includes a breath condensate droplet collector for coalescing exhaled vapor into droplets to form a fluid biological sample, a testing system for receiving the fluid biological sample from the breath droplet collector and testing it for target biomarkers, and wireless communication electronics for detecting the test results for the target biomarkers and communicating the results to a wireless receiver.

[0109] An exhaled aerosol capture system can be provided, comprising a sheet member having a surface for receiving exhaled aerosol containing at least one of fine particles and liquid droplets. The surface can be an exposed portion of an insoluble pressure-sensitive adhesive or a dissolvable film formed, coated, adhered, or integrated onto the sheet member. The dissolvable film has a composition effective to receive and capture at least one of fine particles and liquid droplets by at least one of embedding or dissolving the at least one of the fine particles and liquid droplets onto the surface or within the dissolvable film.

[0110] At least one of the surface and the dissolvable film includes a reagent for reacting with the at least one microparticle and droplet to detect the presence of a target biomarker in the at least one microparticle and droplet.

[0111] Figure 53 is an exploded perspective view showing a dissolvable EBC droplet and EBA particulate collector capturing aerosol droplets and aerosol particulates, Figure 54 is a top view showing an inventive test system including a dissolvable EBC droplet and EBA particulate collector prior to capturing aerosol droplets and aerosol particulates, and Figure 55 is a top view showing an inventive test system including a dissolvable EBC droplet and EBA particulate collector after capturing aerosol droplets and aerosol particulates.

[0112] A further enhanced version of the proposed COVID-19 testing system could include a nanosensor array along with the EBC and / or EBA collection system to also test for virus-specific VOCs, nitric oxide, other gaseous biomarkers, and / or changes in the body in response to exposure to COVID-19. Figure 56 shows a top view of the inventive testing system, including a dissolvable EBC droplet and EBA particulate collector mounted on a face mask substrate with multiple gas sensors for detecting volatile and gaseous components of exhaled breath and / or ambient air. A common feature of the inflammatory response in patients infected with influenza is the production of numerous volatile products by the alveolar and airway epithelium. These products include many volatile organic compounds (VOCs) and nitric oxide (NO). These could potentially be used as biomarkers to detect disease. One research team has demonstrated that a portable three-sensor array microsystem-based tool can detect biomarkers of influenza infection (see, e.g., Gouma PI, Wang L, Simon SR, Stanacevic M. Novel Isoprene Sensor for a Flu Virus Breath Monitor. Sensors (Basel). 2017;17(1):199. Published 2017 Jan 20. doi:10.3390 / s17010199). The gas sensor can be connected to the same electronics and wireless communication system used by other biometric detection functions of the testing system of the present invention.

[0113] FIG. 57 is a side cross-sectional view showing a cross section of a dissolvable droplet and particle collector with particles and droplets impacting a surface placed in a beaker of dissolving liquid. FIG. 58 is a side cross-sectional view showing a cross section of a dissolvable droplet and particle collector with particles released into a beaker of dissolving liquid, resulting in dissolved droplets. In a proposed use case, the test system of the present invention can be distributed on a large scale through national, state, or local postal or courier systems. The test system of the present invention can be incorporated into a mask as shown, or can be provided as a stand-alone system that can be easily retrofitted to existing masks. As an alternative to EBC droplet collectors, alternative mechanisms can be used to collect EBCs. For example, in a hospital environment, EBCs can be collected from face masks used to administer oxygen or other gases to patients. At home, EBCs can be collected by exhaling into a chiller tube (not shown) or other exhaled breath condensation system.

[0114] The dissolvable droplet and particle collector can be mailed to a laboratory where it is analyzed for captured biomarkers. The particles and / or droplets can be expelled from the subject by forceful coughing, deep exhalation, sneezing, or other respiratory maneuvers. In triage or screening procedures, multiple test systems can be distributed to an entire population or to a statistically meaningful sample of the population. If the EBC test system indicates possible current or prior COVID-19 infection (or other biological condition), the entire test system kit or just the dissolvable droplet and particle collector can be sent to a laboratory for more rigorous analysis.

[0115] The lysis solution used by a laboratory (or other testing facility) to test for target biomarkers may contain reagents that cause discoloration, precipitation, amplification, or otherwise aid in the identification of target biomarkers captured by the lysable droplets and particulate collector.

[0116] According to a non-limiting exemplary embodiment, a system for detecting biological agents from a subject's exhaled breath is provided, the system including: a breath condensate droplet collector for coalescing exhaled vapor into droplets to form a fluid biological sample; a testing system for receiving the fluid biological sample from the breath droplet collector and testing it for target biomarkers; and wireless communication electronics for detecting the test results of the target biomarkers and communicating the results to a wireless receiver. The exhaled aerosol capture system can include a sheet member having a surface for receiving exhaled aerosol containing at least one of microparticles and droplets. The surface can be an insoluble pressure-sensitive adhesive or an exposed portion of a dissolvable film formed, coated, adhered, or integrated onto the sheet member. The dissolvable film has a composition effective to receive and capture at least one of microparticles and droplets by at least one of embedding or dissolving the at least one of the microparticles and droplets on the surface or within the dissolvable film. At least one of the surface and the dissolvable film includes a reagent for reacting with at least one microparticle and droplet to detect the presence of a target biomarker in the at least one microparticle and droplet.

[0117] Referring to FIG. 59, this figure shows a block diagram of one possible, non-limiting, exemplary system in which exemplary embodiments can be implemented. In FIG. 59, a COVID-19 testing system (C19TS) 110 is in wireless communication with a wireless network 100. The C19TS is a wireless COVID-19 testing system that can access the wireless network. The C19TS 110 includes one or more processors 120, one or more memories 125, and one or more transceivers 130 interconnected via one or more buses 127. Each of the one or more transceivers 130 includes a receiver Rx 132 and a transmitter Tx 133. The one or more buses 127 may be address, data, or control buses and may include any interconnection mechanism, such as a series of lines on a motherboard or integrated circuit, or other optical communication devices. The one or more transceivers 130 are connected to one or more antennas 128. The one or more memories 125 include computer program code 123. The C19TS 110 includes a target biomarker collection and analysis (TBCA) module 140, which comprises the COVID-19 testing system of the present invention described herein. TBCA embodiments also include wireless communication capabilities, including one or both of portions 140-1 and / or 140-2, which can be implemented in many ways. TBCA module 140 may be implemented in hardware as TBCA module 140-1, such as implemented as part of one or more processors 120. TBCA module 140-1 can also be implemented as an integrated circuit or via other hardware, such as a programmable gate array. In another example, TBCA module 140 can be implemented as TBCA module 140-2, which is implemented as computer program code 123 and executed by one or more processors 120. For example, one or more memories 125 and computer program code 123 can be configured to cause the COVID-19 testing system 110, using one or more processors 120, to perform one or more of the operations described herein.The C19TS 110 communicates with the node 170 via a wireless link 111 .

[0118] The node 170 is a base station (e.g., 5G, 4G, LTE, Long Term Evolution, or any other cellular, internet, and / or wireless network communication system) that provides access to the wireless network 100 by wireless devices such as the C19TS 110. The node 170 includes one or more processors 152, one or more memories 155, one or more network interfaces (N / W / I / F) 161, and one or more transceivers 160, interconnected via one or more buses 157. Each of the one or more transceivers 160 includes a receiver Rx 162 and a transmitter Tx 163. The one or more transceivers 160 are connected to one or more antennas 158. The one or more memories 155 include computer program code 153. Node 170 includes data acquisition and storage (DAS) module 150, which includes one or both of portions 150-1 and / or 150-2, which can be implemented in many ways. DAS module 150 may be implemented in hardware as DAS module 150-1, such as as part of one or more processors 152. DAS module 150-1 may also be implemented as an integrated circuit or via other hardware, such as a programmable gate array. In another example, DAS module 150 may be implemented as DAS module 150-2, which is implemented as computer program code 153 and executed by one or more processors 152. For example, one or more memories 155 and computer program code 153, together with one or more processors 152, are configured to cause node 170 to perform one or more of the operations described herein. One or more network interfaces 161 communicate over a network, such as via links 176 and 131. Two or more nodes 170 communicate using, for example, link 176. Link 176 may be wired or wireless or both, and may implement, for example, an X2 interface.

[0119] The one or more buses 157 may be address, data, or control buses and may include any interconnection mechanism, such as a series of lines on a motherboard or integrated circuit, optical fiber or other optical communication equipment, wireless channels, etc. For example, one or more transceivers 160 may be implemented as a remote radio head (RRH) 195, with the other elements of node 170 physically located differently from the RRH, and the one or more buses 157 may be implemented in part as fiber optic cables to connect the other elements of node 170 to the RRH 195.

[0120] The wireless network 100 may include a network control element (NCE) 190, which may include MME (Mobility Management Entity) / SGW (Serving Gateway) functionality, providing connectivity to additional networks, such as a telephone network and / or a data communication network (e.g., the Internet). The node 170 is coupled to the NCE 190 via a link 131. The link 131 may be implemented, for example, as an S1 interface. The NCE 190 includes one or more processors 175, one or more memories 171, and one or more network interfaces (N / W / I / F) 180, interconnected via one or more buses 185. The one or more memories 171 include computer program code 173. The one or more memories 171 and the computer program code 173, together with the one or more processors 175, are configured to cause the NCE 190 to perform one or more operations.

[0121] Wireless network 100 may implement network virtualization, which is the process of combining hardware and software network resources and network functions into a single software-based management entity, a virtual network. Network virtualization includes platform virtualization and is often combined with resource virtualization. Network virtualization can be categorized as either external, combining many networks or portions of networks into virtual units, or internal, providing network-like functionality to software containers on a single system. It should be noted that the resulting virtualized entity of network virtualization is still implemented at some level using hardware, such as processor 152 or 175 and memory 155 and 171, and such virtualized entity still produces a technical effect.

[0122] Computer-readable memory 125, 155, and 171 may be of any type suitable for the local technology environment and may be implemented using any suitable data storage technology (such as semiconductor-based memory devices, flash memory, magnetic memory devices and systems, optical memory devices and systems, fixed and removable memory, etc.). Computer-readable memory 125, 155, and 171 may be a means for performing storage functions. Processors 120, 152, and 175 may be of any type suitable for the local technology environment and may include, by way of non-limiting example, one or more of a general-purpose computer, a special-purpose computer, a microprocessor, a digital signal processor (DSP), and a processor based on a multi-core processor architecture. Processors 120, 152, and 175 may be a means for performing functions such as controlling C19TS 110, node 170, and other functions described herein.

[0123] In general, various embodiments of the COVID-19 testing system 110 may include, but are not limited to, wireless communication components used for Bluetooth, mobile phones such as smartphones, tablets, personal digital assistants (PDAs) with wireless communication capabilities, portable computers with wireless communication capabilities, image capture devices such as digital cameras with wireless communication capabilities, gaming consoles with wireless communication capabilities, music storage and playback devices with wireless communication capabilities, internet appliances that enable wireless internet access and browsing, tablets with wireless communication capabilities, and mobile devices or terminals incorporating a combination of such capabilities.

[0124] Figure 60 is a logic flow diagram of applied probabilistic analysis for determining exposure to COVID-19. This diagram further illustrates the operation of an exemplary method, the results of execution of computer program instructions embodied on a computer-readable memory, functions performed by logic implemented in hardware, and / or interconnected means for performing functions according to exemplary embodiments. For example, the TBCA module 140 may include multiple circuit elements for implementing the functions illustrated in the blocks of Figure 59, with each included block being an interconnected means for performing the function within the block. It is envisioned that at least some of the blocks of Figure 59 are executed by the C19TS 110, for example, at least in part under the control of the TBCA module 140.

[0125] In the probability analysis applied to determine COVID-19 exposure, biomarker 1 is tested (step 1), biomarker 2 is tested (step 4), and biomarker N is tested (step 3). N can be any number of biomarkers tested using the testing system of the present invention. If no target biomarkers are detected (step 3), a negative test report is generated (step 4). If any target biomarkers are detected (step 3), a probability analysis can be performed (step 5) depending on the detected presence (yes / no) or quantitative analysis (e.g., concentration) of one or more detected biomarkers. If the probability analysis does not exceed a threshold (step 6) (e.g., low concentration of a specific target biomarker or the presence of only one weak biomarker indicating possible infection), a false test report is generated (step 7). If the probability analysis exceeds a threshold (step 6) (e.g., high concentration of a specific target biomarker or the presence of two or more biomarkers indicating possible infection), a positive test report is generated (step 8). The inspection report is then transmitted (step 9) (eg, via methods described herein or other suitable transmission mechanisms, including oral, digital, written, or other communication transmission).

[0126] The logic flow of FIG. 60 is implemented by a non-limiting embodiment of a device that includes at least one processor; and at least one memory containing computer program code, the at least one memory and computer program code configured, using the at least one processor, to cause the device to perform at least the following: detect one or more biometric parameters using a testing system having a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample and a biomarker testing zone for receiving the fluid sample and detecting the biometric parameters (the biometric parameters are biomarkers dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection); receive the one or more biometric parameters and apply probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change.

[0127] According to one embodiment, a digital testing device is provided, including a biomarker testing device having one or more biometric detectors each for detecting biomarkers as one or more biometric parameters. The biometric parameters depend on at least one physiological change in a patient or subject (such as the production of immune response chemicals, the presence in the body of activated or inactivated viruses or viral components, antibodies, antigens, viral RNA or DNA, or other biomarkers that induce the change). A microprocessor receives the one or more biometric parameters and determines whether at least one physiological change threshold has been exceeded as a function of the one or more biometric parameters. An activation circuit takes action in response to the determined physiological change. The action includes at least one of sending an alert, modifying a therapeutic treatment, and transmitting data dependent on the at least one physiological change, the one or more biometric parameters, and the therapeutic treatment.

[0128] The at least one physiological change may also be responsive to an applied therapeutic treatment, causing a change in the patient's condition that allows monitoring of the body's response to the applied treatment. Actions may include sending an alert, modifying the therapeutic treatment, and transmitting data dependent on at least one of the at least one physiological change, the one or more biometric parameters, and the therapeutic treatment. The microprocessor may analyze the one or more biometric parameters using probabilistic analysis, including determining, from a dataset of the one or more biometric parameters, whether the dataset is acceptable for determining that at least one physiological change threshold has been exceeded. The probabilistic analysis may further include applying a statistical weighting to each of the one or more biometric parameters, the statistical weighting depending on a predetermined value of a ranking of importance in detecting each of the at least one physiological change for each of the one or more biometric parameters compared to others of the one or more biometric parameters.

[0129] FIG. 61 is a logic flow diagram for data acquisition and transmission for trusted recipient and contact tracing applications. This diagram further illustrates exemplary method operations, results of execution of computer program instructions embodied on computer-readable memory, functions performed by hardware-implemented logic, and / or interconnected means for performing functions according to exemplary embodiments. The execution of the data acquisition and transmission flow for trusted recipient and contact tracing applications can occur in a test system, a node, a smartphone, or a combination of components located on or associated with the test subject, through the ultimate subject or ultimate repository of acquired data. Acquired data can include information identifying the patient or subject, such as name, GPS location, list of known contacts, previous medical history, demographics, etc. Acquisition and transmission of data for trusted recipient and contact tracing applications can occur on any secure server on a network. For example, the DAS module 150 can include multiple circuit elements for implementing the functions illustrated in the blocks of FIG. 59, each of which is an interconnected means for performing the functions within the block. It is envisioned that at least some of the blocks of FIG. 59 are executed, for example, by a base station, such as node 170, at least partially under the control of the DAS module 150.

[0130] The architecture, manufacturing method, and application of the digital testing system can be used to capture biometric data from a subject's or patient's breath. The biometric data can be captured and transmitted continuously or at selected times, providing data access directly to healthcare providers to enable early diagnosis and continuous monitoring, and to researchers to gain valuable insights and assistance through AI analysis. This data detection can occur directly from the breath and be provided via wireless connectivity for collection, access, and analysis in blockchain and AI databases. The digital testing system of the present invention for biometric capture is adapted for mass production as a roll-to-roll manufactured testing device with sensors and transducers embedded.

[0131] A test report is received (e.g., from a smartphone transmission from a patient or subject) (Step 1). If the report is intended for transmission to a trusted recipient, such as the patient's healthcare provider or insurance company (Step 2), an encrypted report can be generated (Step 3) and sent to the trusted recipient, including the patient's identifying information. If the report is not intended for a trusted recipient (Step 2) but is instead used for contact tracing (Step 4), only the data necessary for contact tracing is sent to a contact tracing app (Step 5). A contact tracing app can be, for example, a system provided to identify and notify people who have come into contact with a subject or patient within a predetermined time period or since testing positive or suspect for one or more target biomarkers. If the report is used for population census research (Step 6) rather than for a trusted recipient (Step 2) or contact tracing (Step 4), only minimal patient identifying information, compliant with privacy regulations and / or agreements, is transmitted and / or stored with the received test report (Step 7). If the report is not for trusted recipient, contact tracing, or census purposes (step 6), it is determined whether there is a legitimate use for the test report data and action is taken accordingly or the data is automatically deleted from storage.

[0132] Figure 62 is a perspective view of one embodiment of an EBC / EBA collection system. Figure 63 is a perspective view of an EBC / EBA collection system showing a pipette and pipette guide. Figure 64 is an exploded view showing the components of an embodiment of an EBC / EBA collection system. Figure 65 is another exploded view showing the components of an EBC / EBA collection system. Figure 66 is a cross-sectional view of an EBC / EBA collection system. Figure 67 illustrates use of an EBC / EBA collection system to obtain a biomarker sample from a subject's lungs.

[0133] According to one aspect of the present invention, a device for detecting a biomarker includes a particle capture structure for receiving and capturing exhaled breath aerosol (EBA) particles from the lining of a subject's airways. The particle capture structure has an aerosol particle inspection system for receiving the captured particles and detecting a first biomarker. The aerosol particle inspection system includes a dissolvable EBA sample collector film for capturing the EBA particles. A first reagent is bound to first nanoparticles and retained in an insoluble inspection area. The EBA particles include insoluble particles and droplet particles. The dissolvable EBA collection film includes a sticky surface for adhering to and capturing the insoluble particles and a water-soluble bulk for capturing the droplet particles.

[0134] A droplet collection structure can be provided to convert exhaled vapor from the subject into exhaled breath condensate (EBC) fluid droplets to form a fluid sample. The subject exhales through a mouthpiece, and the exhaled vapor strikes the wall of the inner barrel. The inner barrel can include a thermal mass (e.g., made of aluminum or other suitable material, or including an interior space that can be filled with a low-temperature thermal mass). The wall of the inner barrel receives the exhaled vapor and forms droplets from the received exhaled vapor. The inner barrel ends with a sharp point, which helps direct the fluid droplets toward the sloped base. The sloped base is the end of the outer barrel that collects the fluid sample from the inner and outer walls of the outer barrel. A pipette is used to pass through the pipette hole in the cap and withdraw the accumulated fluid sample from the sloped base. Using the pipette, the subject expels the fluid sample droplets into the fluid sample testing system, which receives the fluid sample and has a biomarker testing zone for detecting a second biomarker. The cap may also include flow diverting structures to help maintain contact between the walls of the inner barrel and the exhaled vapor. All or part of the system can be integrally formed from an injection mold, or individual components can be assembled into a complete system. Placing the entire system, or just the inner barrel, in a freezer before use facilitates collection of droplets from the cooled walls in contact with the exhaled vapor.

[0135] Figure 68 shows an isolated view of the mouthpiece, cap, base, dissolvable EBA sample collector, and inner barrel of an embodiment of an EBC / EBA collection system. Figure 69 shows an isolated view of the dissolvable EBA sample collector and inner barrel with captured EBA particles and droplets. Figure 70 shows the inner barrel submerged in a solvent to dissolve the dissolvable EBA sample collector and obtain captured EBA particles and droplets for biomarker testing.

[0136] Figure 71 is an isolated view of a portion of one embodiment of a dissolvable EBA sample collector forming an aerosol particulate test system having captured EBA particulates, an insoluble test area, and a dissolvable capture film area. The dissolvable EBA sample collector film contains a first reagent for reacting with at least one component of the captured particulate in a detection reaction to detect a first biomarker. The detection reaction produces at least one of an optical signal and an electrical signal change that is dependent on the first biomarker. The detection reaction can be performed in situ, and because the soluble and insoluble test areas are spaced so closely together, the EBA droplets dissolve into the dissolvable film, where the biomarker in the droplet is picked up by, for example, a labeled antibody, forming a biomarker-labeled antibody complex that binds to the capturing antibody and is retained in the insoluble test area for visual or photonic detection (similar to the operation of a lateral flow assay described herein). In this case, Figure 72 shows a series of side views of an embodiment of a dissolvable EBS sample collector capturing EBA droplets and / or particulates, showing an aerosol particulate testing system with target biomarkers captured and coupled to an insoluble testing area.

[0137] Alternatively, the captured EBA particulates and droplets can be sent to a laboratory for analysis, where a technician or automated system rinses the dissolvable film and provides a fluid sample containing the captured EBA biomarker. For example, the inner tube can be washed with a flow or immersed in a solvent to dissolve the dissolvable EBA sample collector to obtain the captured EBA particles and droplets for biomarker testing.

[0138] Figure 73 shows nanoparticles held within trenches in a substrate, the nanoparticles including a capture antibody or other reagent immobilized thereon. In this case, a fluid sample testing system can include a fluid biosensor for receiving a fluid sample potentially containing a biomarker as a first or second biomarker, the fluid biosensor comprising a sample source having the biomarker, a bioreceptor region functionalized with a biomarker-specific bioreceptor, and a transducer for generating a readable signal in response to receiving the biomarker from the sample source based on a change in the bioreceptor. The biomarker-specific bioreceptor region includes a reagent for causing a detection reaction with the biomarker, and the fluid biosensor generates at least one of an optical signal and an electrical signal change dependent on the biomarker. The reagent binds to the nanoparticles and is held in the insoluble test region.

[0139] Figure 74 shows EBA particles and droplets being rinsed from a dissolvable EBA sample collector to form a fluid sample containing any biomarkers contained in the particles or droplets. This can be done by a subject using a solution containing buffer and surfactant (and other materials, or these materials may be contained in the dissolvable film). This can also be done in the laboratory by a technician or automated equipment.

[0140] 75 shows an EBA / EBC testing system having wireless communication electronics that detects results of a test for at least one of a first and a second biomarker and communicates the results to a wireless receiver. The wireless communication electronics is in communication with at least one of an aerosol particulate testing system and a fluid sample testing system to detect one or more biometric parameters, where the biometric parameter depends on at least one physiological change in the patient in response to a condition of concern, such as a viral infection, the one or more biometric parameters are received and a probabilistic analysis is applied by a microprocessor to determine whether a threshold value for at least one physiological change has been exceeded in response to the probabilistic analysis of the one or more biometric parameters, and the electronics transmits a signal in response to the determined exceeded at least one physiological change.

[0141] According to another aspect of the present invention, an apparatus includes at least one processor, at least one memory containing computer program code, the at least one memory and the computer program code being configured to, using the at least one processor, cause the apparatus to at least perform the following: detect one or more biometric parameters using a particulate capture structure for receiving and capturing exhaled aerosol (EBA) particulates from the airway lining of a subject (the particulate capture structure has an aerosol particulate inspection system for receiving the captured particulates and detecting a first biomarker, the aerosol particulate inspection system including a dissolvable EBA sample collector film for capturing the EBA particulates, the biometric parameter being a biomarker dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection); receive the one or more biometric parameters and apply a probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change. The one or more biometric parameters can be further detected using a droplet collection structure for converting the exhaled vapor into fluid droplets to form a fluid sample, and a testing system having a biomarker testing zone for receiving the fluid sample and detecting the biometric parameters. A probabilistic analysis is applied to the one or more biometric parameters to determine whether at least one physiological change threshold has been exceeded depending on a probabilistic analysis of the one or more biometric parameters detected from both the captured particulate and the fluid sample.

[0142] Figure 76 shows an EBC / e-NSB testing system integrated into a ventilator circuit. In this use case, EBCs are collected from the exhaled breath of a ventilator or ventilated patient.

[0143] Microfluidic and liquid channeling structures are used to induce continuous flow of EBCs over electronic or electrochemical biosensors. Continuous flow of EBCs is ensured by drainage or wicking structures, which allow for tracking of a patient's target biomarkers over time. As more target biomarkers are present in the EBC sample, the biosensor's capture molecules continue to capture and retain biomarker molecules, and the electrical readout from the biosensor continues to change proportionally to the number of captured biomarker molecules. When the biosensor's capture molecules become saturated with captured biomarkers, a new biosensor can be introduced, and the saturated biosensor can be regenerated or discarded.

[0144] Figure 77 shows elements of a continuous flow embodiment in which a capillary space is formed in the test region of the sensor between the sensor substrate and the capillary cap. An EBC collector delivers EBCs through the microfluidic material to the capillary space where the capture molecules of the biosensor bind to the target biomarker molecules. A wick or drainage structure downstream of the test region draws the EBCs from the capillary space, creating a continuous flow of EBCs over the test region.

[0145] Figure 78 shows the interior of a disposable face mask equipped with an EBC collector, microfluidics, and an electronic biosensor. The disposable face mask incorporates an EBC collector for cooling exhaled breath vapor into a fluid biosample. The EBC collector has a thermal mass that cools the exhaled breath vapor into droplets. The vapor cools onto the hydrophobic field of the EBC collector, forming droplets that are then transported along hydrophilic channels to a microfluidic system. The microfluidic system transports the collected droplets as a fluid biosample to an electronic biosensor testing system. The testing system determines the presence of target biomarkers in the fluid biosample and generates a test result signal. The test result signal is wirelessly transmitted by electronic circuitry to a remote receiver.

[0146] According to a non-limiting embodiment, a mask-based test system for detecting biomarkers received from a subject's lungs and airways is provided. An exhaled breath condensate (EBC) collector is positioned inside a face mask worn by the subject. The EBC collector converts exhaled vapor received from the subject's lungs and airways into a liquid biosample. The EBC collector has a thermal mass and a front face, where the front face receives exhaled vapor at a temperature higher than a surface temperature of the front face and converts the exhaled vapor into a liquid at a temperature lower than the temperature of the exhaled vapor. The EBC collector includes a droplet collection structure on the front face, the droplet collection structure including a field for receiving the exhaled vapor and forming fluid droplets from the received exhaled vapor, and a channel for receiving the fluid droplets from the field and flowing the fluid droplets together to form a collected fluid biosample.

[0147] An electronic biosensor is secured to the face mask for receiving the fluid biosample from the EBC collector and testing the fluid biosample for target biomarkers, the electronic biosensor generating an electrical test signal that depends at least on the presence or absence of the target biomarkers in the fluid biosample.

[0148] The electronic circuit is secured to the face mask for receiving the electronic test signal, determining a test result signal from the electronic test signal in response to detection or non-detection of the target biomarkers, and transmitting the test result signal to a remote receiver. The electronic circuit includes wireless communication circuitry for wirelessly transmitting the test result signal to at least one of a smartphone, a tablet, a computer, a relay, an access point, and a computer network.

[0149] According to this non-limiting exemplary embodiment, a mask-based test system for detecting biomarkers received from a subject's lungs and airways includes an exhaled breath condensate (EBC) collector integrated inside a face mask worn by the subject. The EBC collector converts exhaled breath received from the subject's lungs and airways into a liquid biosample. A biosensor is secured to the face mask for receiving the fluid biosample from the EBC collector and testing the fluid biosample for target biomarkers. The biosensor generates a test signal that depends at least on the presence or absence of the target biomarkers in the fluid biosample. Electronic circuitry secured to the face mask receives the test signal, determines a test result signal from the test signal in response to detection or non-detection of the target biomarkers, and transmits the test result signal to a remote receiver.

[0150] The EBC collector can include a droplet collection structure including a hydrophobic field for receiving exhaled vapor and forming fluid droplets from the received exhaled vapor. The hydrophilic channel receives the fluid droplets from the hydrophobic field and flows the fluid droplets together to form a collected fluid biosample. The EBC collector can further include a thermal mass and a front surface in thermal communication with the thermal mass. The front surface is positioned facing the subject's mouth and nose when the subject is wearing the face mask, and the hydrophobic field and hydrophilic channel are positioned as part of the front surface.

[0151] The front surface may comprise an aluminum or other metal foil, coated or uncoated to function as a hydrophobic surface. The hydrophilic channels may be screen printed or otherwise adhered to the metal foil. The thermal mass comprises at least one of a superabsorbent polymer, water, and an endothermic compound. In one embodiment of the EBC collector, the water is contained in the sealing structure and kept separate from the endothermic compound until an activation step, at which point the water is released from the sealing structure and mixes with the endothermic compound to cool the front surface, creating a relatively cool surface that facilitates the formation of droplets from the relatively hot exhaled vapor.

[0152] As an alternative to hydrophobic / hydrophilic structures, exhaled breath condensate (EBC) collectors are incorporated inside a face mask worn by a subject to convert exhaled vapor received from the subject's lungs and airways into a fluid biosample. The EBC collector includes a droplet collection structure comprising a field (e.g., a metal foil, plastic, or metal sheet) for receiving exhaled vapor and forming fluid droplets from the received exhaled vapor. A channel receives the fluid droplets from the field and channels the fluid droplets together to form a collected fluid biosample. The channel can be a fold, trough, raised surface, or other similar structure that collects and channels the collected fluid biosample toward a microfluidic or other structure, such as one that brings the biosample to a testing area of ​​a biosensor.

[0153] Figure 79 shows the outside of a disposable mask, illustrating the electrical connection from the electronic biosensor inside the mask to the z-axis conductive tape on the outside of the mask. This mask-based testing system is for detecting biomarkers received from a subject's lungs and airways. An exhaled breath condensate (EBC) collector is incorporated inside a face mask worn by the subject. The EBC collector converts exhaled vapor received from the subject's lungs and airways into a liquid biosample. The EBC collector has a thermal mass and a droplet collection structure including hydrophobic regions for receiving exhaled vapor and forming fluid droplets from the received exhaled vapor. The EBC collector includes hydrophilic channels for receiving the fluid droplets from the hydrophobic regions and channeling the fluid droplets together to form a collected fluid biosample. A biosensor affixed to the inside of the face mask receives the fluid biosample, tests the fluid biosample for target biomarkers, and generates a test signal. An electronic circuit affixed to the outside of the mask receives the test signal, determines a test result signal in response to detection or non-detection of the target biomarkers from the test signal, and transmits the test result signal to a remote receiver. This configuration allows the inexpensive disposable mask to be discarded along with any exhaled air and contamination from the mask's internal components. The more expensive electronics and battery are located on the outside of the mask during use and can be removed when the mask is discarded. The removable electronics can be sterilized for reuse.

[0154] In one embodiment, an electronic nanoscale biosensor is provided for providing direct electrical test results using electrical transduction of carbon nanotube chains functionalized with aptamer capture molecules, where the biosample is exhaled breath condensate (EBC) collected from a mask-based EBC collector. The EBC collector is integrated into a disposable mask and includes a thermal mass that facilitates cooling of the exhaled breath into liquid condensate. The thermal mass can be a gel made from water mixed with a superabsorbent polymer (SAP). To speed up the conversion of relatively hot exhaled breath vapor to liquid droplets on the relatively cool EBC collector surface, the mask can be first cooled in a refrigerator or freezer, or a cooler with dry ice or a chiller can be used. Alternatively, under most ambient conditions, exhaled breath is warm and moist, so the EBC collector functions to provide a sufficient EBC volume for testing without the need for cooling.

[0155] Figure 80 shows the components of a self-cooling EBC collector. A first stretchable hot melt layer, such as TPU Bemis 3914, is provided. As described in more detail below, a thermal mass (e.g., water held in a superabsorbent polymer) or a component of an endothermic chemical reaction is placed in a pocket formed in the stretchable hot melt layer. In Figure 80, a water bag and a heat-absorbing agent, such as ammonium nitrate, calcium ammonium nitrate, or urea, are placed in the pocket. A second stretchable hot melt layer seals the thermal mass or endothermic component between the layers of stretchable hot melt. A hydrophobic field material, such as aluminum foil, is bonded to the second stretchable hot melt layer. Hydrophilic channel structures are formed or placed on the hydrophobic regions of the aluminum foil. Other materials can be used to form the EBC collector; for example, the hydrophobic field can be formed by screen printing a hydrophobic ink onto a foil or plastic substrate, and the hydrophilic channel can be formed by screen printing a hydrophilic ink onto a foil or plastic substrate. The EBC collector can be assembled onto a disposable mask substrate (such as N95 mask filter sheet material) or can be pre-formed and then adhered to the mask via heat pressing, adhesive, stitching, or a fastening step. Existing off-the-shelf, commercially available masks can be used and the EBC and other components can be retrofitted to the inside and outside of the mask.

[0156] Figure 81 shows the inside of an open mask with components for collecting and testing EBCs. The EBC / e-NSB testing system can be retrofitted to existing masks or integrated into the mask design. Figure 81 shows the construction of a simple, low-cost disposable mask. The mask's base material can be N95 mask material, filter material, cloth or paper, or a breathable polymer material with micropores to allow air exchange. An EBC collector with hydrophobic fields and hydrophilic channels is fixed to the mask material. The fluid sample collected by the EBC collector is transported to the biosensor by a microfluidic transfer material and can pool in the biosensor area or flow over the biosensor area using a wicking material downstream of the biosensor area. The biosensor testing area is typically small, measuring a few square millimeters or less in surface area, but larger areas and multiple testing areas or zones can be provided. The biosensor device has electrodes with leads that enable electrical communication with the EBC / e-NSB testing system's electronics. Preferably, the electronics and battery are located on the outside of the mask during use, while the EBC collector, microfluidic transfer material, biosensor, and wicking material are located inside the mask. After use, the electronics can be removed from the outside of the mask and disinfected for subsequent use. Disposable masks and the components inside the mask (and most exposed to potential contamination) can be sealed in an appropriate bag and disposed of according to protocols for handling such materials. For home use, a bag containing a certain amount of alcohol or other virus-killing substance can be provided for disposing of the mask after use. Dissolvable adhesive patches can also be placed on the inner surface of the mask to capture aerosol droplets and particulates.

[0157] FIG. 82 is a block diagram of the basic components for testing an EBC and transmitting test results to a smartphone and / or cloud server. The EBC collector provides a fluid sample that is received by a biometric or biosensor. Electrical signal conditioners, such as signal amplifiers, filters, etc., can be provided to condition the raw test signal from the biosensor before a microprocessor or analytical circuit determines the test result signal. After processing the conditioned signal, the test result signal is transmitted over a communication line. The communication can be wireless, such as Bluetooth, cellular, or wifi. A smartphone or access point relay can be used to receive and transmit the wireless test result signal to the cloud.

[0158] According to one embodiment, the electronic circuit includes an amplifier circuit for receiving a test signal from the biosensor and amplifying the test signal into an amplified electrical signal. The comparator circuit compares the amplified electrical signal to a predetermined value based on at least one of a computer model of the biosensor and an empirically derived electrical signal calibration. The calibration can be determined using at least one of a known presence and a known concentration of the target analyte in a calibration sample. The comparator circuit generates a test result signal based on the amplified electrical signal compared to the predetermined value.

[0159] The electronic circuitry can also include an analyte concentration circuit for determining a target analyte concentration value in response to the amplified electrical signal, where the amplified electrical signal varies in value in response to the number of target analyte molecules in the fluid biosample, and the test result signal is dependent on the determined concentration value.

[0160] According to one embodiment, the electronic circuitry further includes wireless communication circuitry for wirelessly transmitting the test result signal to at least one of a smartphone, a tablet, a computer, a relay, an access point, and a computer network.

[0161] Figure 83 is a side cross-sectional view showing the disposable components inside a disposable mask and the disinfectable components outside the disposable mask. The components located inside the mask include an EBC collector designed for low-cost mass production. The COVID-19 or other biomarker testing system of the present invention features a unique mask-based exhaled breath condensate collector that coalesces exhaled vapor into droplets and passes the droplet sample to an electronic biosensor equipped with engineered capture molecules, enabling a very low-cost, manufacturable, large-scale testing system for mass distribution. This at-home testing system uses an electronic nanoscale biosensor (e-NSB) with a unique moisture droplet collection and channeling structure. This structure allows the e-NSB to detect COVID-19 or other biomarkers, for example, in exhaled breath condensate (EBC), without the need for blood draws, discomfort, expensive equipment, or technically trained personnel. Multiple simultaneously tested biomarkers can be tested using specifically functionalized biosensor testing areas, enabling many direct infectious disease control utilities, such as contact tracing, diagnostics, disease progression monitoring, and predictive machine learning population data analysis.

[0162] According to a non-limiting embodiment, a mask-based test system for detecting biomarkers received from a subject's lungs and airways is provided. An exhaled breath condensate (EBC) collector is placed inside a face mask worn by the subject. The face mask is composed of a mask material, which may be cloth, woven or nonwoven material, paper, fiber, plastic, or other suitable disposable or reusable material. The EBC collector, secured inside the mask, converts exhaled vapor received from the subject's lungs and airways into a liquid biosample.

[0163] A biosensor is also fixed to the inside of the face mask to receive the fluid biosample from the EBC collector. Alternatively, the biosensor can be located outside the mask with an appropriate fluid transfer mechanism (e.g., capillary action, pump, tubing, etc.) used to transport the collected fluid biosample to the biosensor. However, if the biosensor is an electronic biosensor capable of transmitting an electrical signal, the potentially contaminated biosensor and fluid transfer mechanism can be contained within the mask, making handling and disposal safer. The biosensor tests the fluid biosample for a target biomarker and generates a test signal that depends at least on the presence or absence of the target biomarker in the fluid biosample.

[0164] The electronic circuitry is secured to the exterior of the mask and is in electrical communication with the biosensor through the mask material to receive the test signal. For example, the electrical connection can be made via wires, printed conductors, magnets, conductive tape, conductive adhesive, or other mechanisms that allow electrical signals generated inside the mask to be transferred to the electronic circuitry outside the mask. The electronic circuitry determines a test result signal from the test signal in response to detection or non-detection of the target biomarkers and transmits the test result signal to a remote receiver.

[0165] 84 is a side cross-sectional view showing the electronics being held and electrically connected to the disposable mask. The disinfectable test electronics are held on the disposable mask by the electronics housing on the disposable mask and a magnet or magnet that can be disposed of with magnetic material. The magnetic connection can also provide electrical communication between the conductive leads of the biosensor and the input to the test electronics.

[0166] Figure 85 is a cross-sectional view of the z-axis conductive tape that holds the electronics and electrically connects them to the disposable mask. The z-axis conductive tape allows for a low-cost electrical connection between the leads of the electronic biosensor and the leads of the electronic circuitry.

[0167] Figure 86 shows a stretchable hot melt adhesive attached to a foam. The foam is a laser-cut Teflon sheet that allows the hot melt adhesive to be attached using a heat press. Figure 87 shows a pocket formed with the stretchable hot melt adhesive attached to the foam. The pocket is formed to receive a thermal mass or endothermic reactive component. Figure 88 shows an endothermic compound disposed within the pocket formed with the stretchable hot melt adhesive. The endothermic compound can be, for example, urea.

[0168] Figure 89 shows a water bag added to the pocket holding the endothermic compound. The water bag is added to the same pocket as the endothermic compound. When the EBC collector is compressed and the water bag ruptures, the urea and water react and remove heat from the EBC collector surface, effectively self-cooling the EBC collector and increasing its efficiency in converting exhaled vapor to liquid.

[0169] Figure 90 shows pre-laminated aluminum foil on an adhesive sheet over a stretchable hot melt adhesive. The aluminum foil and adhesive can be pre-laminated together in a simple roll-to-roll heated roller press lamination step. Figure 91 shows the bottom of the form after press lamination of the layers that form the interlocking sheet of EBC. The thermal mass of the water in the SAP can be seen in the pocket on the left side of the photo, and the water bag and urea self-cooling material are visible in part of the pocket on the left side of the photo.

[0170] Figure 92 shows superabsorbent polymer placed in pockets of stretchable hot-melt adhesive. Figure 93 shows the superabsorbent polymer after swelling with water. When water is added to the SAP, a gel-like thermal mass is formed that maintains its soft, flexible nature even after being chilled in a freezer. Figure 94 shows the top surface of the interlocking sheet of an EBC during the heat press operation. Figure 95 shows a finished EBC with hydrophilic channels over hydrophobic fields. Figure 96 shows the water bag and endothermic compound used in a self-cooling EBC. Figure 97 shows the roll-to-roll process for forming an aluminum foil and adhesive sheet laminate; in this first step for mass production of EBC collectors, the front surface of the EBC collector is constructed with an aluminum foil surface pre-laminated with stretchable hot-melt adhesive. This conversion process results in a roll of aluminum foil / adhesive / release sheet laminate. An in-line punching operation can be used to punch registration holes in the laminate. These registration holes are used to maintain registration in subsequent processing steps throughout the formation of the EBC collector.

[0171] FIG. 98 shows a roll-to-roll process for forming an EBC. FIG. 99 is a cross-sectional view of an EBC. The aluminum foil surface and adhesive are pre-laminated, for example, as described with reference to FIG. 97. The thermal mass is inserted into a pocket created in a separate adhesive layer at the start of the process described with reference to FIG. 98. This simple structure is adaptable to the ultra-high volume, highly automated manufacturing methods described herein. FIG. 100 is a perspective view of the roll-to-roll process for forming an EBC. FIG. 100 is an enlarged perspective view showing a foam conveyor belt for forming pockets in the stretchable adhesive to form the EBC.

[0172] The process begins with a roll of elastic adhesive stretched over a first guide roller and a first heated shoe. The heated adhesive becomes tacky and adheres to the conveyor belt form under the compressive action of two sets of pressure rollers. The adhesive, attached to the conveyor belt, exits a second pair of pressure rollers, where the release sheet is removed and wound onto a release sheet roll. A vacuum station draws the heated, flexible adhesive into the conveyor belt form, creating pockets in the adhesive. Figure 102 shows a cross-section of the elastic hot melt adhesive with the pockets formed. Figure 103 shows a portion of the foam conveyor belt. Figure 104 shows a cross-section of the elastic hot melt adhesive and a cross-section of the foam conveyor belt. The cooling station cools the adhesive while the vacuum pressure is maintained, ensuring that the pockets remain in the adhesive while the adhesive adheres to the conveyor belt form. At the thermal mass / endothermic component station, a thermal mass material, such as a SAP / water gel or a water bag and endothermic compound, is inserted into the pockets created in the adhesive. SAP can also be added along with the water bag and endothermic compound to prolong the cooling effect of the endothermic reaction. The insertion step can be performed using automated deposition equipment or by robotic or human intervention. These steps are approximated for a sheet of 5 x 4 interlocking EBC units, as shown in photographs 86-96. The adhesive containing the thermal mass component within the created pocket advances toward a third pair of pressure rollers. The aluminum foil / adhesive from the release sheet laminate roll is a stripe of release sheet, which is wound onto a second release sheet roll. The adhesive laminate is guided by a guide roller over a second heated shoe, causing the adhesive to become tacky. As the aluminum / adhesive contacts the adhesive containing the thermal mass component within the created pocket, a continuous interlocking sheet of multiple EBC collectors is formed with the thermal mass component sealed between layers of stretchable hot-melt adhesive and a front surface of aluminum foil. The back of the EBC collector is a stretchable hot-melt adhesive, allowing the EBC collector to be secured to the inside of a face mask using a heat press operation.Interlocking sheets of multiple EBC collectors can be collected on a take-up roll, sheeted into individual sheets of interlocking EBC collectors, or sent directly to a singulation station such as a steel rule die press, slitter, or laser cutter.

[0173] Figure 105 illustrates a roll-to-roll process for forming aligned nanoparticles between electrodes fixed to a substrate to form an electronic biosensor. A roll of pre-printed or etched parallel conductors is provided. An example of a sheet of copper-etched parallel conductors is shown in Figure 130. In the case of printed parallel conductors, printing can be performed in-line, for example, using a rotary printing method. The roll substrate can include alignment and / or tractor feed holes to facilitate movement and alignment of the roll material during processing. The voltage application stage applies an AC (or DC) voltage to the parallel conductive lines. This applied voltage is used to align the conductive nanoparticles in later stages of the process. Because the parallel conductive lines are continuous, the applied voltage can be maintained throughout the processing steps. In the nanotube / solvent carrier matrix deposition / printing stage, nanotubes (or other nanoparticles) are dispensed onto the roll of parallel conductors. The nanotubes are randomly dispersed in a solvent fluid carrier matrix. The solvent fluid carrier allows the nanotubes to align in response to an applied voltage, and the solvent side of the fluid carrier softens the substrate (or a bonding film printed or placed on the substrate between the conductive lines). When the voltage is held to maintain the alignment of the nanotubes between the conductive lines, the extended polymer chains of the softened substrate partially encapsulate the nanotubes. When the voltage is held to maintain the alignment of the nanotubes, the solvent evaporates, and the aligned nanotubes are fixed in place and orientated and bonded to the re-hardened substrate or bonding layer.

[0174] Figure 106 illustrates steps for forming an electronic sensor with aligned nanotubes between conductors. Note: The drawing is not to scale; only the relative orientation and position of elements are shown. Also, note: This configuration of an electronic biosensor can have other forms and materials. For example, graphene sheets, semiconducting carbon nanotubes, colloidal gold nanoparticles, and other conductive materials in a solvent carrier fluid can be used. The drawing illustrates the use of aligned single-walled carbon nanotubes between two conductors. The conductors can be parallel lines (e.g., as described above with reference to Figure 105) or circular electrodes, such as those shown in Figures 114 and 133. As shown in Figure 106, in step 1, a substrate having a conductive pattern is provided. The conductive pattern is formed on the substrate, defining a gap between a pair of electrodes. In step 2, a mixture of unaligned carbon nanotubes in a fluid carrier matrix is ​​deposited in the gap. The fluid carrier matrix, for example, a solvent for the substrate, softens the region of the substrate within the gap, allowing the unaligned nanotubes to move and orient in response to an applied electric field or voltage. The layer thickness and aligned structure of the nanotubes can be controlled by controlling the concentration of nanotubes in the fluid carrier.

[0175] In step 3, a voltage is applied to the electrodes. The applied voltage causes the non-aligned nanotubes dispersed in the fluid carrier matrix to move and change orientation. The aligned carbon nanotubes are directed to self-assemble into aligned carbon nanotubes perpendicular to the electrodes. The voltage is maintained while the fluid carrier matrix evaporates in step 4, keeping the aligned carbon nanotubes aligned within the gap between the pair of electrodes. Step 5 shows the aligned carbon nanotubes between the electrodes after the fluid carrier matrix has evaporated. When the fluid carrier matrix softens the region of the substrate within the gap, the aligned carbon nanotubes bond with the extended polymer chains of the softened substrate and are locked in place when the fluid carrier matrix evaporates and re-hardens away from the softened surface of the substrate. Alternatively, or additionally, pressure and heat (e.g., above the glass transition temperature of the substrate, or lower depending on the softening of the solvent) can be used to embed the aligned carbon nanotubes into the gaps on the surface of the substrate. In either case, step 5 shows the aligned carbon nanotubes locked in position and orientation even when the voltage is removed from the electrodes and after the alignment process after rinsing. The functionalization process is shown schematically in steps 6-8, where capture molecule aptamers are modified with PBSE linkers and noncovalently adsorbed to the sidewalls of the aligned carbon nanotubes. Step 9 shows the aptamer in action to capture the biomarker. Step 10 shows the completed functionalized biosensor, with the aptamer capture molecules noncovalently adsorbed to the aligned carbon nanotubes.

[0176] Figure 107 illustrates the steps for forming an unfunctionalized electronic sensor with aligned carbon nanotubes held in place between electrodes on a substrate. In step 1, the gap between the electrodes exposes the substrate surface. In step 2, a droplet of solvent carrier fluid containing a concentration of unaligned nanotubes is placed in the gap, and in step 3, the solvent acts on the substrate surface, softening it and pulling the substrate's polymer chains away from the surface and into the solvent carrier fluid. Again, these drawings are not necessarily to scale. In step 4, a voltage (AC or DC, depending on the properties of the nanomaterial dispersed in the fluid carrier) is applied, causing the nanotubes to move and orient in response to the applied voltage. This voltage is maintained to keep the nanotubes aligned in step 5, when the solvent carrier begins to evaporate. Once the solvent carrier has completely evaporated, the voltage can be removed, leaving the carbon nanotubes embedded in the substrate surface in step 6.

[0177] Figure 108 illustrates steps for functionalizing an electronic sensor with aligned carbon nanotubes held in place between electrodes on a substrate. In step 1, a drop of a non-solvent carrier fluid containing a linker molecule is dispensed into the gap containing aligned nanotubes anchored to the surface of the substrate within the gap between the electrodes via microdeposition, screen printing, or other deposition process. The non-solvent carrier is formulated so as not to disturb the anchored nanotubes by softening the substrate surface or disrupting the nanotube alignment. The linker molecule is selected to bind to the outer wall of the carbon nanotube. For example, PBSE (1-pyrenebutanoic acid succinimidyl ester) is well known to form π-π non-covalent bonds with the sidewalls of single-walled carbon nanotubes and has a proven track record of use as a linker molecule for forming electronic biosensors.

[0178] According to the functionalization process described herein, a linker molecule is pre-linked at one end to a capture molecule, such as an aptamer, that has high affinity and selectively binds to a specific target biomarker. Aptamers are small molecules that can be designed to target almost any biomarker. Aptamers are short, single-stranded nucleic acid sequences that can bind to target molecules in a manner similar to antibodies. The process described herein decorates the sidewalls of carbon nanotubes with these capture molecules, which are designed to bind with high specificity to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells and tissues. Therefore, the test system of the present invention can be used to target nucleic acids, proteins, and other identifying biomarkers of viruses, such as the SARS-CoV-2 virus. However, because aptamers can be designed with the affinity and specificity to bind to many different target substances, the sensors described herein can be functionalized to test for many diseases, such as lung cancer, or other molecules present in biological or environmental samples.

[0179] As described in more detail herein, a two-sensor mask-based COVID-19 testing system allows for the selection of aptamers to detect the N and S proteins of the SARS-CoV-2 virus. Unfunctionalized biosensors can be functionalized in several different ways. For example, one end of a linker molecule, PBSE, can be first attached to the sidewall of a carbon nanotube in a first incubation step, and then a capture molecule can be attached to the other end of the linker in a second incubation step. Additionally, a spacer molecule, such as PEG, can be added to the linker molecule before terminating it with the capture molecule, increasing its distance from the carbon nanotube and preventing steric hindrance that would prevent the small molecule aptamer from contacting relatively large target molecules (e.g., viral proteins, virus particles, or cells).

[0180] In step 2, the unfunctionalized carbon nanotube-aligned biosensor is functionalized with a pre-attached linker / aptamer molecular structure. Because the aptamer is attached to one end of the PBSE linker in a previous chemical reaction, a single incubation step is required to form a π-π non-covalent bond between the other end of the PBSE linker and the carbon nanotube sidewall. This single incubation step is particularly useful for scalable wet-incubation fabrication processes, such as those described in Figure 111.

[0181] In step 3, linker / capture molecules are attached to the sidewalls of the carbon nanotubes. Depending on the target biomarker, other linker molecules, aptamers, spacer molecules, or other capture molecules, such as antibodies, can be combined as needed. The sensors described herein are not limited to biosensors; they can be used to detect environmental target molecules such as VOCs, gases, metals in drinking water, bacteria, antibodies, hormones, d-dimers, proteins, glucose, and lactate using a wide array of functionalized sensors made available for scalable production according to the structures and processes described herein. Post-functionalization steps, such as rinsing and drying, can also be performed, and additional layers, such as blockers, can be added to further enhance the functionality and stability of the sensor.

[0182] Step 4 shows the testing step, in which a droplet of a fluid sample containing a target biomarker is added onto the functionalized biosensor. As shown in step 5, capture molecules attached to the aligned carbon nanotubes by linkers capture and retain the target biomarker. When the electrodes are probed, changes in electrical properties caused by the capture of the target biomarker are determined, indicating the presence of the target biomarker in the fluid sample.

[0183] Figure 109 shows the steps for forming an unfunctionalized sensor with aligned carbon nanotubes immobilized on a binder layer. In this case, the steps are essentially similar to those described in Figures 106 and 107, except for the addition of the printed binder layer deposited in step 2. The chemistry of this binder layer is selected to be compatible with the solvent carrier, and because it is a layer added to the gap formed by the substrate and electrodes, it allows for greater flexibility in the selection of materials and chemistries for the substrate, electrodes, solvent carrier, etc. For example, the binder layer may be more easily softened by the solvent and / or may better hold the aligned nanotubes in place as the solvent evaporates. Other chemical mechanisms, such as catalysts, two-part systems, and thermal or low-temperature activation / compression, can be used to move and orient the nanoparticles and subsequently immobilize them in the gap between the two electrodes. Furthermore, in some systems, alignment can be achieved via a counter electrode formed on the opposite side of the substrate, and a separate conductive layer can be provided, allowing alignment to be in a direction other than perpendicular to the electrodes used for probing to test biomarkers when the biosensor is completed. Additionally, the insulating sidewalls can be patterned to provide a non-contact electric field where the aligned nanoparticles do not come into direct contact with the electrode (i.e., the patterned sidewalls prevent direct physical contact between the aligned nanotubes and the electrode that serves as the probe conductor). While the choice of materials, alignment direction, etc. depends on the desired structure of a particular biosensor, the general steps described here are adaptable to a wide range of materials and device architectures.

[0184] Figure 110 illustrates a continuous process for forming unfunctionalized sensors using wet electrodeposition / alignment of locked carbon nanotubes between parallel conductors. To enable a high-volume wet deposition process, a roll of pre-printed or etched conductor is provided at the input side of a roll-to-roll processing line. The conductors can be parallel conductors as described herein (e.g., as shown in Figure 133) or circular interconnected conductors, as shown in Figure 112. As described above with reference to Figures 106, 107, and 109, a voltage is applied during the voltage application stage, so that the nanoparticles in the fluid carrier / nanotube bath are induced by electromotive forces and become oriented and aligned in a directed assembly process. The energized conductor and substrate enter a wet deposition tank. As shown in the enlarged image toward the right of the figure, the deposition tank contains a concentration of unaligned nanotubes in the fluid carrier. As the substrate and conductor move through the nanotube-laden carrier fluid, the nanotubes are attracted and aligned in the gap between the electrodes. Note that, as described above, a counter electrode parallel to the roll of conductor can be provided to generate nanotube migration and alignment forces, for example, using a DC voltage. In either case, as the substrate and conductor approach the edge of the fluid carrier / nanotube tank, the nanotubes or nanoparticles are attracted and aligned in the gap between the electrodes, as shown in the zoomed-in image toward the left side of the drawing. Note: In some configurations, nanoparticle alignment and attraction may be perpendicular to or on the electrodes. The arrangement shown here is for illustrative purposes only. After exiting the fluid carrier / nanotube tank, some nanotubes that are not aligned with the fluid carrier remain attached to the substrate and conductor due to surface tension and other attractive forces. A guide roller can be used to provide a drip edge, where an air knife or other mechanism is used to remove the attached material and recapture the fluid carrier and excess nanotubes in the fluid carrier / nanotube tank. A post-positioning fixation step, using heat and pressure, rinsing, drying, and other processing steps, can further condition the unfunctionalized, aligned nanotubes locked onto the substrate between the electrodes before winding them into a roll.

[0185] Variables such as tank length and volume can be adjusted to optimize the alignment and voltage, substrate or tie layer softening (if a solvent fixation process is used as described herein), temperature, and alignment process for a given speed. In other words, the processes described herein are amenable to optimization through measurement, modeling, and adjustment of component chemistries, voltages, speeds, lengths, and other process steps and application characteristics of materials, biosensor construction equipment and process steps, process lines and processing steps, etc.

[0186] Figure 111 shows a sequential process for forming a functionalized sensor involving wet bonding and incubation of linker / capture molecules on carbon nanotubes locked between parallel conductors. The process begins with a preformed roll of unfunctionalized, aligned nanotubes locked onto a substrate between electrodes. Using a similar wet process, the linker / aptamer is incubated and attached to the sidewalls of the aligned carbon nanotubes. As described elsewhere herein, this process can be a multi-step incubation process, other nanoparticle materials can be used to form unfunctionalized biosensor rolls, and various process and structural attributes can be modified to optimize the process and performance of the resulting functionalized electronic biosensor.

[0187] The processes described herein can be used, for example, to create sensors that provide direct electrical test results. That is, the presence of a target biomarker captured by an aptamer or other capture molecule alters the electrical properties measured at the probe electrode. In the embodiments described herein for testing EBC, the electrical change is detected only if the biomarker is present in the EBC sample, creating an immediate opportunity to collect biometric information obtained from the EBC, helping to protect individuals and rapidly establish cloud-based data acquisition to facilitate rapid contact tracing. Because the biosensor is direct electrical and embodiments of the testing system include a wireless Bluetooth transmitter in the detection electronics, wireless test results can be transmitted to any database with appropriate encryption, privacy handling, etc. When used, for example, in workplaces, religious services, clinical settings, sporting events, etc., this digital data obtained directly from the testing system is an efficient means of providing employers, administrators, medical, and security professionals with data to track and maintain a safe environment.

[0188] Figure 112 shows printed electrodes linked together to apply an electrical alignment force. AC or DC voltage can be applied to all electrodes. For systems of more than two electrodes, insulation can be screen printed to cross the leads. The pattern can be repeated as needed to optimize sheet size or roll-to-roll manufacturing processes.

[0189] Figure 113 shows examples of nanotubes aligned at different AC voltages and frequencies (Source: Influence of AC Electric Field on Macroscopic Network of Carbon Nanotubes in Polystyrene, Yang, et al., Journal of Dispersion Science and Technology, 28:8, 1164-1168), which is incorporated by reference in its entirety. As an example of voltage and frequency for aligning single-walled carbon nanotubes in polystyrene, an AC electric field of 300 V and 450 Hz is suitable for aligning CNTs within a PS matrix.

[0190] Figure 114 shows a printed electrode pattern. This printed electrode pattern includes only a working electrode and a counter electrode. Other configurations are possible, including a third electrode, such as a reference electrode. Figure 115 shows an optional insulator formed on the printed electrode pattern. The insulator may be provided to aid in the electric field effect for more uniform alignment.

[0191] Figure 116 shows the step of printing an electrode pattern onto a substrate, which has a gap between two printed electrodes. Depending on the type of sensor being constructed, a conductive layer, a semiconductive layer, a patterned conductive layer, an insulating layer, and a semiconductive layer, or any combination thereof, can be printed into the gap and / or on the electrode surface.

[0192] Figure 117 shows unaligned nanotubes in a solvent fluid carrier. Figure 118 shows alignment of nanotubes in a fluid carrier with an applied AC voltage. Figure 119 shows the steps of placing unaligned nanotubes in a fluid carrier. Figure 120 shows the steps of applying an AC voltage to align the nanotubes.

[0193] Figure 121 shows aligned nanotubes fixed in alignment after evaporation of the solvent fluid carrier. Figure 122 shows the addition of linker / aptamer molecules to bind to the aligned nanotubes. Figure 123 shows a step of aligned nanotubes fixed in place on a substrate between electrodes. Figure 124 shows linker / aptamer molecules in a non-solvent fluid carrier added on top of the aligned nanotubes.

[0194] Figure 125 shows incubation to bind linkers / aptamers on nanotubes. Figure 126 shows addition of a fluid biosample for testing. Figure 127 shows linkers / aptamers attached to aligned nanotubes. Figure 128 shows addition of a fluid biosample with target biomarkers captured by aptamers. Figure 129 shows different electronic and electrochemical biosensor strategies known in the art (at least some of which can be utilized to form sensors constructed with the applications and processes described herein).

[0195] Depending on the desired cost, structure, manufacturing method, sensor characteristics, and target analyte, the biosensor can include at least one of a conductive and semiconductive base material disposed in a gap formed on a substrate between at least two probe electrodes.

[0196] The capture molecules are provided in electrical communication with the probe electrodes via the base material. The capture molecules can be immobilized to the base material via covalent or non-covalent bonds via at least one of π-π stacking, amine coupling, thiol-au bonds, click chemistry, electrostatic interactions, biotin-avidin affinity, and complementary DNA hybridization. The base material can include at least one of graphene, carbon nanotubes, gold, screen-printed conductive materials, and positively charged materials. The capture molecules can include at least one of aptamers and antibodies, or other suitable molecules with binding affinity for the target analyte.

[0197] The base material can include electric or magnetic field alignable microparticles locked in alignment by a binder layer formed on the top surface of the substrate, which can include at least one of a binder layer printed on the top surface and the top surface of the substrate.

[0198] Figure 130 shows a cross section of parallel conductors with a gap between the conductor pair that can be used in some of the applications and processes described herein. Figure 131 shows a second of the parallel conductors with nanoparticles aligned in the gap between the conductors. Figure 132 shows an electronic sensor singulated from a roll or sheet of electronic sensors formed using the processes described herein. Figure 133 shows an alternative screen-printed electrode structure, including a reference electrode, for use in forming at least some versions of the electronic and electrochemical sensors described herein.

[0199] According to one embodiment, the biosensor is a construct for testing multiple target analytes. For example, two biosensors may be provided, each functionalized to detect a different target analyte, or one biosensor may be used that has capture molecules with binding affinity for different analytes. In this case, the biosensor tests the fluid biosample for the target analyte and for at least one other target analyte, and the test signal depends on the presence or absence of at least the target analyte and the at least one other target analyte in the fluid biosample.

[0200] Figure 134 illustrates an embodiment of a mask-based diagnostic device for detecting biomarkers in a subject's exhaled breath. In this case, an existing commercially available N95 face mask is converted into a diagnostic tool for detecting biomarkers in exhaled breath. The breath-based diagnostic system is retrofitted inside the mask and connected to a test signal reader and wireless communication electronics on the outside of the mask.

[0201] According to an exemplary embodiment, a mask-based diagnostic device for detecting biomarkers contained in a subject's exhaled breath is provided, including an exhaled breath condensate (EBC) collector for converting exhaled vapor received from the subject's lungs and airways into a liquid biosample. The EBC collector includes a thermal mass, a condensate-forming surface, and a fluid conductor disposed on the condensate-forming surface. A liquid transfer system receives the fluid biosample from the EBC collector. A biomarker testing unit receives the fluid biosample from the fluid transfer system and tests the fluid biosample for target biomarkers. A testing system support is provided to support the EBC collector, the fluid transfer system, and the biomarker testing unit. The testing system support is configured and dimensioned to fit inside a face mask. A face mask is provided that forms an exhaled vapor containment volume for retaining exhaled vapor near the EBC collector, allowing the condensate-forming surface, cooled by the thermal mass, to coalesce the exhaled vapor into a fluid biosample.

[0202] Figure 135 shows an exhaled breath condensate (EBC) collector, thermal mass, fluid transfer system, and biomarker testing unit installed as a retrofit into the exhaled vapor containment volume created by an existing face mask. The shape and dimensions of the testing assembly, which is fixed inside the mask, and the low profile of the thermal mass and EBC collector allow for efficient retrofitting to existing masks or adding the diagnostic system during mask manufacturing.

[0203] Figure 136(a) shows a face mask with externally attached electronics worn by a subject at the start of an EBC test. The mask performs its intended filtering and barrier functions, and the subject only needs to breathe normally while wearing the mask to perform the diagnostic test.

[0204] Figure 136(b) shows externally mounted electronics that display the results of the EBC test. The exhaled vapors coalesce into a liquid biosample, which is collected and transported to a biomarker testing unit. In this embodiment, the biomarker testing unit outputs a signal that is read by electronic circuitry to determine the test result and illuminates an LED in a color that immediately visually indicates the determined test result.

[0205] Figure 137 shows the configuration of a breath-based diagnostic device with an electronic biosensor. The materials and geometry of the microfluidic fluid transfer system are designed to provide, for example, a microfluidic neck region that efficiently transports collected EBCs to the testing region of the electronic biosensor.

[0206] Figure 138 shows the configuration of a breath-based diagnostic device with a fluid biosample accumulation reservoir for pooling biosamples on an electronic biosensor or for immersing the sample pad of an LFA in the accumulated fluid biosample. Pooling EBCs allows the sample pad to be immersed in the liquid biosample, ensuring sufficient sample volume for the capillary action required for LFA operation. In the case of an electronic biosensor, the sample accumulation reservoir offers the possibility of pooling collected EBCs throughout the test area, allowing time for target molecules to bind to capture molecules.

[0207] Figure 139 shows the test system support that supports the EBC collector, fluid transfer system, and biomarker test unit. The support and other components are designed to fit a variety of existing masks, allowing for convenient and stable retrofitting of the diagnostic test system to a wide variety of disposable and reusable masks available worldwide.

[0208] Figure 140 shows a wick positioned on the backside of the test system support, Figure 141 shows a wick structure including a SAP layer attached to a microfluidic paper layer, and Figure 142 shows a cross-section of a wick with SAP and a microfluidic paper structure.

[0209] FIG. 143 shows connection pins for connecting the electronic biosensor inside the mask with electronics outside the mask. The fluid transfer system includes a wick for absorbing the fluid biosample flow after the biomarker testing unit tests the fluid biosample flow. The fluid biosample is allowed to flow over the electronic biosensor over time to allow target molecules to flow with the fluid biosample and the target molecules an opportunity to capture target molecules flowing with the fluid biosample on the electronic biosensor. The wick can include at least one of a superabsorbent polymer (SAP) and a flow transfer layer to receive and absorb the fluid biosample flow.

[0210] The layered structure of filter paper, 3M double-sided adhesive, SAP powder, and 3M double-sided adhesive forms a core with excellent EBC retention. The 3M double-sided adhesive is placed between two sheets or release papers. First, a hole is drilled in the 3M double-sided adhesive sheet and one release sheet is removed. A sheet of filter paper is then attached to the exposed adhesive. The other release sheet is then peeled off, SAP powder is sprinkled on the newly exposed adhesive, and excess SAP powder is shaken off. When used as a wick, EBCs are drawn from the test area into the filter paper by capillary action, and the hole provides access for the EBCs to flow from the filter paper to the SAP powder. The SAP powder absorbs the EBCs and swells. Relatively large amounts of EBCs can be retained in this layered wick, allowing for a continuous flow of EBCs throughout the test area. The layered wick also provides microfluidic drainage, allowing a continuous flow of EBCs throughout the test area, allowing target molecules to be captured over time and accumulated by the capture molecules. Knowing the flow rate set by the microfluidic material and geometry and the elapsed time of the flow of EBCs over the test area allows for the calculation of the viral load, which depends on the change in the electrical properties of the biosensor over time.

[0211] Figure 144 shows an LFA configuration for a respiratory-based diagnostic device with a pooling area formed by a fluid biosample accumulation reservoir with an LFA strip positioned with a sample pad within the fluid biosample pooling area. The fluid transfer system can be configured and dimensioned to pool the accumulation of the fluid biosample on the electronic biosensor. The fluid biosample is pooled in contact with the capture molecules of the electronic biosensor for a period of time, providing time and opportunity for the capture molecules to bind to the target molecules while the fluid biosample accumulates. In this embodiment, the pooling of EBCs allows the capture molecules time to bind and access the accumulation of target molecules in the pool of EBCs, allowing for the detection of lower concentrations of biomarkers present in a given amount of EBCs as the amount of EBCs in the pool increases. Figure 145 shows an LFA configuration and pooling area ready to receive an LFA configured for a specific target biomarker. Figure 146 shows an LFA configuration in which a test system is retrofitted to an existing mask. Similarly, diagnostic systems can be configured to allow the electronic biosensor to be submerged in the accumulation pool of EBCs for extended periods of time.

[0212] According to this embodiment, the fluid transfer system includes a biosample pooling area for pooling the fluid biosample received from the EBC collector. The biomarker testing unit includes a lateral flow assay in which the fluid biosample flows through a multi-zone transport medium by capillary action. The lateral flow assay includes a sample pad disposed in the pooling area to receive the fluid biosample, a conjugate release pad where biomarker-labeled capture molecule complexes are formed, a detection zone, and a flow membrane for directing the fluid sample from the sample pad through the release pad to the detection zone for detecting potential biomarkers. During operation, a liquid biosample is generated by concentrating exhaled breath vapor into a fluid sample containing target biomarkers. The liquid biosample flows through the multi-zone transport medium by capillary action. The zones are typically made of a polymer strip that allows molecules attached to the strip to interact with the target biomarkers. An overlapping membrane is usually attached to a backing card for improved stability and operability. The sample containing the target biomarkers and other components is finally received by an absorbent sample pad, which facilitates wicking of the fluid sample through the multi-zone transport medium. This design is particularly adaptable to the breath-based diagnostic systems described herein, where the LFA strip can be simply inserted and configured with the remaining components of the diagnostic system, providing a visual indication (test and comparison lines) on the outside of the mask, and the sample pad is immersed in EBC accumulated on the inside of the mask. For point-of-care or at-home testing, these features are particularly useful, providing a simple way to test subjects for highly infectious diseases such as Covid-19.

[0213] According to another embodiment, the fluid transfer system includes a biosample pooling area for pooling the fluid biosample received from the EBC collector. The biomarker testing unit includes a lateral flow assay in which the fluid biosample flows through a multi-zone transport medium by capillary action. The lateral flow assay includes a conjugate release pad disposed in the pooling area to receive the fluid biosample. The conjugate release pad has capture molecules for capturing target molecules of the target biomarker and forming a biomarker-labeled capture molecule complex. The lateral flow assay further includes a detection zone for detecting potential biomarkers and a flow membrane for flowing the fluid sample from the conjugate release pad to the detection zone. The fluid transfer system further includes a fluid dam disposed in fluid communication between the conjugate release pad and the detection zone. That is, the fluid biosample can flow (i.e., fluid communication) from the conjugate release pad to the detection zone. In the conjugate release pad, a certain amount of fluid biosample is pooled in contact with the capture molecules for a certain period of time, allowing the capture molecules time and opportunity to bind to the target molecules. After this, a fluid dam releases the amount of fluid biosample, including the biomarker-labeled capture molecule complexes formed over time. The complexes flow from the conjugate release pad to the detection zone along with the accumulated biosample. The fluid dam comprises one of a dissolvable material that is dissolved and removed by the fluid biosample, and an impermeable material that is removed by a pull tab. Removal of the fluid dam releases the biomarker-labeled capture molecule complexes formed over time and allows at least a portion of the accumulated amount of fluid biosample to flow to the detection zone.

[0214] The conjugate release pad contains labeled capture molecules that have binding affinity for target biomarkers and are conjugated to colored or fluorescent indicator particles. By pooling the fluid biosample over the conjugate release pad while the fluid dam holds back the flow, the labeled antibody, indicator particle, and target biomarker have time and opportunity to bind to form target biomarker-labeled antibody complexes in the conjugate release pad. When the fluid dam is released, the fluid sample migrates along the strip to the detection zone.

[0215] Figure 147 shows a sealed LFA test setup and face mask. The sealed bag prevents moisture and contaminants from altering the effectiveness of the biomarker test unit and prevents the EBC collector and liquid transfer components from condensing ambient moisture. Ideally, the mask is stored in a refrigerator or freezer to cool the thermal mass. Figure 148 shows an LFA test setup retrofitted to an existing face mask and worn by the subject at the beginning of the test. Figure 149 shows the LFA test setup after the subject's exhaled vapor has been converted into a fluid biosample that is transported through the LFA, and shows a visual display of the EBC test results. The chilled thermal mass promotes the condensation of exhaled vapor into exhaled condensate. However, the described prototype diagnostic system has proven effective at collecting EBCs when all components are at room temperature before the mask is placed on the subject. With a thermal mass cooled in a home freezer, the LFA version of the diagnostic system has been shown to collect enough EBCs for a complete flow through the LFA system in approximately three and a half minutes. For room temperature diagnostic systems, EBC for LFA full flow typically takes less than 10 minutes to complete.

[0216] Figure 150 shows an electronic biosensor testing configuration retrofitted to an existing molded face mask. Figure 151 is a close-up view showing the connection pins of the electronic biosensor testing configuration penetrating the wall of the existing mask. Figure 152 shows an existing molded mask with an electronic biosensor testing configuration, in which the electronic circuitry is located on the outside of the mask, mechanically secured via the connection pins, and electrically connected to the electronic biosensor. The breath-based diagnostic system is adaptable to a wide range of existing masks. In the prototype configuration, a volume of approximately 5 mL of water / SAP gel is an effective freezing thermal mass for collecting more than a sufficient amount of EBCs in approximately 5 minutes for both the LFA and electronic biosensor versions of the diagnostic system. At room temperature, the same thermal mass takes approximately 20 minutes to collect adequate EBCs. Additional improvements in materials, geometry, microfluidics materials, etc. are expected to improve both room-temperature and refrigerated collection of useful amounts of EBCs for testing. For example, a Teflon surface can provide enhanced condensate formation and eliminate the need for a fluid conductor, especially if molded to include channels for directing collected EBCs to an examination area, etc.

[0217] Figure 153 shows electronic circuitry located on the outside of a mask that indicates EBC test results. In the prototype example shown, an LED indicates the presence of EBC. As described herein, functionalized devices use capture molecules with affinity for target molecules to alter electrical properties measured at two or more electrodes depending on the presence or absence of the target molecules in the test biosample.

[0218] Figure 154 shows a multi-biomarker test unit supported on a test system support. Figure 155 shows a fluid transfer system for providing a fluid biosample from the EBC collector to each electronic biosensor of the multi-biomarker test unit.

[0219] Figure 156 shows the backside of a test system support with a wick for continuous flow of a fluid biosample over a multi-biomarker test unit and an adhesive for retrofitting to an existing mask. In this embodiment, the fluid transfer system is configured and dimensioned to flow a predetermined volume of fluid biosample over the electronic biosensor for a predetermined time. For example, the selection of microfluidic materials and the geometry of the microfluidic pathway can be designed so that a predetermined volume of EBC drawn from the pooling region flows over the test region (i.e., functionalized electrodes) of the electronic biosensor. The concentration of the target molecule can be determined as a function of a predetermined volume of the fluid biosample flowing over the electronic biosensor within a predetermined time and the change in the electrical signal. The electronic biosensor outputs an electrical signal having a change in electrical properties depending on the captured molecule, which changes the electrical signal depending on the captured biomarker. The electronic circuitry can then calculate or look up in a lookup table stored in memory the calculated concentration of the target biomarker in a given volume of the collected biosample.

[0220] Figure 157 shows a flow conductor with a hydrophilic pattern for transporting EBCs toward the testing zone. Figure 158 shows a thermal mass with a front surface that forms a condensate-forming surface. Figure 159 shows a fluid transfer system for transporting EBCs toward the testing zone of the biomarker testing unit. Figure 160 shows an electronic biosensor version of the biomarker testing unit.

[0221] Figure 161 shows a test system support for supporting an EBC collector, fluid transfer system, and biomarker testing unit, configured and dimensioned to fit inside an existing face mask. Figure 162 shows the assembled diagnostic system.

[0222] FIG. 163 illustrates the components of a mask-based diagnostic system. In this embodiment, the EBC collector and components of the diagnostic system's fluid collection and transport elements are applied to a mask substrate using low-cost, mass-production techniques, including heat roll lamination, pressing, fusing, or pressure-sensitive adhesives. The disposable mask material can be selected to perform only the function of containing exhaled vapors during the testing procedure, or it can be selected to also provide barrier and absorption functions, for example, to allow the mask to be used outside of the testing process. The thermal mass can be provided integrally formed within the mask, or, as shown, the thermal mass can be removed from the freezer and placed in a holder or pouch integrated into the mask.

[0223] Figure 164 shows the inch dimensions and shape of one embodiment of the fluid conductor. Figure 165 shows the exhaled vapor containment volume defined by the face mask, with the EBC collector and other portions of the breath-based diagnostic system located within the containment volume.

[0224] The size and shape of the various components, as well as other physical properties such as specific heat, fluid flow rate, etc., can be selected depending on the particular face mask, location, time of year, indoor or outdoor use, etc.

[0225] The thermal mass can comprise at least one of a metal foil, a contoured shape with flow transfer channels, an endothermic chemical reaction, a metal slug, a composite material thermally enhanced to absorb heat energy from exhaled vapors, a water and SAP gel, a composite layer formed by a roll lamination process, etc. Figure 166 shows a composite thermal mass. Figure 167 shows a water / SAP gel thermal mass. Figure 168 shows the backside of a breath-based diagnostic system with a water / SAP thermal mass and an LFA biomarker testing unit.

[0226] Figure 169 shows an embossed metal foil thermal mass with a condensate-forming surface and fluid conductor channels. Figure 170 shows an endothermic thermal mass for insertion into a holding pocket of a mask-based diagnostic system. Figure 171 shows a soapstone powder / binder composite thermal mass. Figure 172 shows a metal slug thermal mass.

[0227] FIG. 173 shows a face mask constructed with an EBC collector and accumulated fluid biosample reservoir located inside the mask, with the sample pad of the LFA in the reservoir and at least the visual readout portion of the LFA located outside the mask. FIG. 174 shows an EBC collector with a thermal located inside the mask within the exhaled vapor containment volume. The fluid transfer system includes a biosample pool area for pooling fluid biosamples received from the EBC collector. According to this embodiment, the biomarker testing unit includes a lateral flow assay. The fluid transfer system includes a biosample pool area for pooling fluid biosamples received from the EBC collector. According to this embodiment, the biomarker testing unit includes a lateral flow assay.

[0228] The fluid transfer system can also include a fluid dam disposed in fluid communication between the EBC collector and the pooling area, which accumulates a volume of fluid biosample until the fluid dam releases the volume of fluid biosample, flooding the pooling area with the accumulated volume of fluid biosample. The accumulated volume of fluid biosample is provided to the sample pad as an overflow stream rather than being provided more slowly as EBCs are collected from the exhaled vapor to facilitate the LFA microfluidic flow process. The fluid dam can be a dissolvable material that is removed by being dissolved by the fluid biosample and / or a non-permeable material that is removed by a pull tab. Removal of the fluid dam releases the accumulated volume of fluid biosample, flooding the pooling area.

[0229] FIG. 175 illustrates the structure of a fluid transfer system having a fluid dam containing a dissolvable adhesive. The fluid dam is positioned in fluid communication between the EBC collector and the pooling region. After accumulating a certain amount of fluid biosample, the fluid dam releases this amount of fluid biosample, flooding the pooling region with the accumulated amount of fluid biosample and providing the accumulated amount of fluid biosample to the sample pad. The fluid dam can include a dissolvable material that is dissolved by the fluid biosample and releases the accumulated amount of fluid biosample. In an exemplary embodiment, the LFA strip has its sample end positioned within the sample accumulation reservoir that forms the pooling region. The fluid dam prevents EBCs from entering the pooling region until the appropriate amount of EBCs has been collected. When the fluid dam is released, the FLA sample pad is submerged, ensuring proper flow throughout the LFA microfluidics.

[0230] Figure 176 shows the assembly of a branched version of the respiratory-based diagnostic system. Figure 177 shows an exploded view of the components of the branched version of the respiratory-based diagnostic system. According to a non-limiting embodiment, the fluid conductor includes a transfer volume for absorbing a fluid biosample. The transfer volume has an absorption saturation point, and the fluid conductor conducts the fluid biosample at a slow rate before the absorption saturation point is reached and at a fast rate after the absorption saturation point is reached. In a prototype, the fluid conductor was constructed using filter paper attached to a pressure-sensitive adhesive. The pressure-sensitive adhesive does not significantly fill the microcapillary channels of the filter paper, so that when a fluid sample contacts the surface or exposed end of the filter paper, the fluid sample flows while being absorbed into the filter paper. The adhesive / filter paper layer structure is cut into the desired fluid conductor pattern. The adhesive provides a mechanism for adhering the fluid conductor of the patterned adhesive / filter paper layer to, for example, a condensation-forming surface, allowing fluid droplets that coalesce on the surface to migrate to the fluid conductor by gravity or shaking. A fluid conductor constructed with the adhesive / filter paper layered structure described above can hold large volumes of liquid, much like a water-filled sponge, and upon saturation, additional water simply flows out of the sponge instead of being absorbed and retained by the sponge. The volume is constrained by the surface to which the fluid conductor is attached (e.g., the surface on which condensation forms), providing a very fast transport channel for additional fluid that comes into contact with the saturated fluid conductor.

[0231] The condensation-forming surface is at least one of the following: a front surface of a thermal mass, a printed substrate having hydrophobic and hydrophilic channels, and a coating printed to form a boundary that defines a fluid conductor. The fluid conductor is at least one of a coating printed to form a boundary and define a condensation-forming surface, a contoured surface, a defined area on the front surface of the thermal mass having a hydrophilic texture, and a microfluidic assembly having a transfer volume for absorbing a fluid biosample. As an example of forming a hydrophilic texture, laser ablation or a patterned chemical etching process can be used to create areas of a surface (metal or plastic) that are more hydrophobic and other areas that are more hydrophilic.

[0232] In one embodiment, a flow initiation fluid is provided that is freezable within the fluid conductor to facilitate reaching the adsorption saturation point. The freezable solution includes at least one of a buffer and a calibration component for the fluid biosample. The calibration component allows the electronic circuit to determine a calibration value from the initiation fluid. Prior to use of the mask-based diagnostic device, the freezable solution is maintained in a frozen state. During use of the mask-based diagnostic device, the freezable solution thaws, wetting the surface of the EBC collector and facilitating fluid transfer of the EBC liquid biosample. By using a flow initiation fluid in this manner, the first liquid received at the test region (functionalized electrodes) of the electronic biosensor contains a known amount of the calibration component. The calibration component can be, for example, an electrolyte, salt, surfactant, or other chemical that produces a predicted change in the electrical properties between the electrodes of the electronic biosensor.

[0233] Figure 178 shows a branched version formed with an embossed metal foil condensate-forming surface with contours that form fluid transfer channels. Figure 179 shows a cross-sectional exploded view of the branched version of the respiratory-based diagnostic system. Figure 180 shows an assembled cross-sectional view of the branched version of the respiratory-based diagnostic system.

[0234] The bifurcated version is specifically designed for use with respirators that have symmetrical folds, such as a typical KN95 respirator. Figure 181 shows the retrofit of an existing KN95 respirator with the LFA version of the breath-based diagnostic system. Figure 182 shows the retrofit testing system placed inside a KN95 respirator, with the LFA placed inside the respirator.

[0235] The biomarker testing unit may include an electronic biosensor having capture molecules that capture target molecules contained in the fluid biosample and output an electrical signal that is dependent on the target molecules captured by the capture molecules.

[0236] Figure 183 shows an electronic biosensor configured as a field effect transistor with a graph showing the output signal as target molecules begin to bind to the capture molecules. Figure 184 shows an electronic biosensor configured as a field effect transistor with more target molecules captured and a graph showing the output signal after the target molecules begin to bind to the capture molecules. Figure 185 shows an electronic biosensor configured as a field effect transistor with more target molecules captured and a graph showing the output signal after the target molecules begin to bind to the capture molecules. A fluid biosample flows over the electronic biosensor over time, allowing the target molecules to flow with the fluid biosample and the capture molecules an opportunity to capture target molecules flowing with the fluid biosample over the electronic biosensor.

[0237] In the case of a field-effect transistor biosensor, an electrical and chemical insulating layer, such as silica, separates the fluid biosample from the semiconductor field-effect transistor device elements. A polymer layer, e.g., (3-aminopropyl)triethoxysilane (APTES), is used to chemically couple the binding surface to the capture molecule bioreceptor. For example, the capture molecule can be an aptamer or antibody engineered to have binding affinity for the target molecule. Binding of the capture molecule to the target molecule causes a change in electrostatic potential at the binding interface of the electrolyte-insulator layer, resulting in an electrostatic gating effect for the semiconductor device and a measurable change in the current between the source and drain electrodes.

[0238] The electronic biosensor can include a field-effect transistor structure including an electrode layer having at least a source electrode and a drain electrode. A binding surface is disposed between the source electrode and the drain electrode and functionalized with at least one capture molecule to capture a target biomarker. Capture of the target biomarker causes a change in at least one electrical property between the source electrode and the drain electrode, which is detected as a test result signal. In a multiple-biomarker configuration, each capture molecule has an infinity for each biomarker. For example, in a Covid-19 testing system, capture molecules can be provided to different electronic biosensors in a biomarker testing unit that receives an EBC sample and tests for different target molecules. An electronic circuit receives the output signal from the biomarker testing unit, determines affinity-dependent test signal values, and calculates result values ​​for each different capture molecule and its respective biomarker. This allows for the detection of multiple biomarkers, improving the statistical accuracy of the testing system and / or testing for multiple strains or types of the Covid-19 virus (or other diseases). The electronic biosensor further includes a substrate, where the binding surface is the top surface of a binding bulk, and the bottom surface of the binding bulk is diffusion-bonded to the substrate. The capture molecule may include at least one of an aptamer, an engineered antibody, an antibody, a protein, an antigen, a nucleic acid-based ligand, a small molecule modified to mimic a monoclonal antibody, and the like.

[0239] Various modifications and adaptations to the foregoing exemplary embodiments of the invention will become apparent to those skilled in the art in view of the foregoing description, when read in conjunction with the accompanying drawings, however, all changes will still fall within the scope of the non-limiting and exemplary embodiments of the invention.

[0240] The embodiments described herein are intended to be exemplary and non-limiting, and the selection of biometric, environmental, or other measurement conditions is not limited to the particular metric or metrics described herein but will depend on the particular application and processing, data collection, and / or other uses of the detected metric. Additionally, the treatment employed in any of the embodiments described herein is not limited to any particular treatment or action but will depend on the intended use and desired outcome of the combination of the detected metric and the treatment applied.

[0241] Moreover, some of the features of the various non-limiting exemplary embodiments of this invention may be used to advantage without the corresponding use of other features. Accordingly, the foregoing description should be considered as merely illustrative of the principles, teachings and exemplary embodiments of this invention, and not in limitation thereof.

[0242] Various modifications and adaptations to the foregoing exemplary embodiments of the present invention will become apparent to those skilled in the art in light of the foregoing description, when read in conjunction with the accompanying drawings. However, all variations will still fall within the scope of the non-limiting, exemplary embodiments of the present invention. The embodiments described herein are intended to be exemplary and non-limiting, and the selection of biometric, environmental, or other measurement conditions is not limited to the particular metric or metrics described herein, but rather depends on the particular application and processing, data collection, and / or other uses of the detected metric. Additionally, the treatment employed in any of the embodiments described herein is not limited to any particular treatment or action, but rather depends on the intended use and desired outcome of the combination of the detected metric and the treatment applied.

[0243] Moreover, some of the features of the various non-limiting exemplary embodiments of this invention may be used to advantage without the corresponding use of other features. Accordingly, the foregoing description should be considered as merely illustrative of the principles, teachings and exemplary embodiments of this invention, and not in limitation thereof.

Claims

1. An apparatus including: an exhaled breath condensate (EBC) collector for converting exhaled vapors received from the subject's lungs and airways into a fluid biosample; a biomarker testing unit for receiving the fluid biosample and testing the fluid biosample for target biomarkers contained in the fluid biosample; and A test system support for supporting the EBC collector, the test system support configured and dimensioned to fit inside a face mask, the face mask forming an exhaled vapor containment volume and holding the exhaled vapor close to the EBC collector so that the exhaled vapor coalesces into the fluid biosample.

2. Methods include: providing a substrate; providing a first conductor and a second conductor on the substrate, defining a gap between the first conductor and the second conductor; disposing a solvent carrier matrix within the gap, wherein the solvent carrier matrix is ​​a solvent for chemically softening the substrate and includes randomly dispersed nanoparticles; applying a voltage to the first conductor and the second conductor to align the nanoparticles; and evaporating the solvent carrier matrix to fix the aligned nanoparticles onto the substrate.

3. Methods include: providing a roll of unfunctionalized aligned nanotubes disposed on a substrate between electrodes; placing a first portion of the roll in a fluid bath containing linker / capture molecules randomly dispersed in a carrier fluid; functionalizing the aligned nanotubes with the linker / capture molecules; removing the first portion from the fluid bath having functionalized nanotubes disposed on the substrate between electrodes; and placing a second portion of said roll in said fluid bath;

4. 1. A mask-based diagnostic device for detecting biomarkers contained in the exhaled breath of a subject, comprising: an exhaled breath condensate (EBC) collector for converting exhaled vapors received from the subject's lungs and airways into a fluid biosample, the EBC collector including a thermal mass; a condensate-forming surface; and a fluid conductor disposed on the condensate-forming surface; a fluid transfer system for receiving the fluid biosample from the EBC collector; a biomarker testing unit for receiving the fluid biosample from the fluid transfer system and testing the fluid biosample for target biomarkers contained in the fluid biosample; a test system support for supporting the EBC collector, the fluid transfer system, and the biomarker testing unit, the test system support configured and dimensioned to fit inside a face mask; and a face mask that forms an exhaled vapor containment volume and holds the exhaled vapor close to the EBC collector, allowing the exhaled vapor to coalesce into the fluid biosample with the condensate-forming surface cooled by the thermal mass; 1. A mask-based diagnostic device comprising:

5. The mask-based diagnostic device of claim 4, wherein the fluid transfer system includes a biosample pooling area for pooling the fluid biosample received from the EBC collector; and the biomarker testing unit includes a lateral flow assay in which the fluid biosample flows through a multi-zone transport medium by capillary action, the lateral flow assay including a sample pad positioned in the pooling area to receive the fluid biosample, a conjugate release pad in which a biomarker-labeled capture molecule complex is formed, a detection zone, and a flow membrane for flowing the fluid sample from the sample pad, through the release pad, and into the detection zone to detect potential biomarkers.

6. 6. The mask-based diagnostic device of claim 5, wherein the fluid transfer system further comprises a fluid dam disposed in fluid communication between the EBC collector and the pool area, the fluid dam releasing a certain amount of the fluid biosample to flood the pool area with an accumulated amount of the fluid biosample and accumulating a certain amount of the fluid biosample until the accumulated amount of the fluid biosample is provided to the sample pad, the fluid dam comprising one of a dissolvable material that is removed by being dissolved by the fluid biosample and a non-permeable material that is removed by a pull tab, and removal of the fluid dam releases the accumulated amount of the fluid biosample to flood the pool area.

7. The fluid transfer system includes a biosample pooling area for pooling the fluid biosample received from the EBC collector; the biomarker testing unit includes a lateral flow assay in which the fluid biosample flows through a multi-zone transport medium by capillary action, the lateral flow assay including a conjugate release pad disposed in the pooling area to receive the fluid biosample, the conjugate release pad having capture molecules for capturing target molecules of target biomarkers to form biomarker-labeled capture molecule complexes, the lateral flow assay further including a detection zone and a flow membrane for flowing the fluid sample from the conjugate release pad to the detection zone to detect the target biomarker, the fluid transfer system being disposed in fluid communication between the conjugate release pad and the detection zone.

5. The mask-based diagnostic device of claim 4, further comprising a fluid dam, wherein the conjugate release pad allows a certain amount of the fluid biosample to pool in contact with capture molecules for a certain period of time, providing time and opportunity for the capture molecules to bind to target molecules, and then the fluid dam releases the certain amount of the fluid biosample, including the biomarker-labeled capture molecule complexes formed over time, to flow together with the accumulated biosample from the conjugate release pad to the detection zone, the fluid dam comprising one of a dissolvable material that is removed by being dissolved by the fluid biosample and a non-permeable material that is removed by a pull tab, and removal of the fluid dam releases at least a portion of the accumulated amount of the fluid biosample and allows the biomarker-labeled capture molecule complexes formed over time to flow to the detection zone.

8. The mask-based diagnostic device of claim 4, wherein the biomarker testing unit includes an electronic biosensor having capture molecules for capturing the target molecules contained in the fluid biosample and outputting an electrical signal dependent on the target molecules captured by the capture molecules.

9. 9. The mask-based diagnostic device of claim 8, wherein the fluid transfer system includes a wick for absorbing the flow of the fluid biosample after the biomarker testing unit tests the flow of the fluid biosample, whereby the fluid biosample flows over the electronic biosensor over time, allowing the target molecules to flow with the fluid biosample and the capture molecules an opportunity to capture the target molecules flowing with the fluid biosample over the electronic biosensor.

10. the wick comprises at least one of a super absorbent polymer (SAP) and a flow transport layer for receiving and absorbing the flow of the fluid biosample, and the thermal mass comprises at least one of a metal foil, a contoured shape with flow transport channels, an endothermic chemical reaction, a metal slug, and a thermally enhanced composite material for absorbing thermal energy from the exhaled vapor, water, a gel of water and SAP, and a composite layer structure; the condensation-forming surface is at least one of a front surface of the thermal mass, a printed substrate having hydrophobic and hydrophilic channels, and a coating printed to form a boundary defining the fluid conductor; the fluid conductor is at least one of a coating printed to form a boundary and define the condensation formation surface, the contoured surface, a defined area on the front surface of the thermal mass having a hydrophilic texture, a microfluidic assembly having a transfer volume for absorbing the fluid biosample; 10. The mask-based diagnostic device of claim 9, wherein the at least one capture molecule comprises at least one of an aptamer, an engineered antibody, an antibody, a protein, an antigen, a nucleic acid-based ligand, and a small molecule engineered to mimic a monoclonal antibody.

11. The mask-based diagnostic device of claim 8, wherein the electronic biosensor comprises: an electrode layer having at least a source electrode and a drain electrode; and a binding surface disposed between the source electrode and the drain electrode and functionalized with at least one capture molecule for capturing the target biomarker, wherein capturing the target biomarker changes at least one electrical property between the source electrode and the drain electrode, and each capture molecule has infinity for a respective biomarker; and further comprises an electronic circuit for receiving an output signal from the biomarker testing unit, determining an affinity-dependent test signal value, and calculating a result value for the at least one capture molecule and the respective biomarker.

12. The mask-based diagnostic device of claim 11 , wherein the electronic biosensor further comprises a substrate, the binding surface being a top surface of a binding bulk, and the bottom surface of the binding bulk being diffusion bonded to the substrate.

13. The mask-based diagnostic device of claim 8, wherein the fluid transfer system is configured and dimensioned to pool the accumulation of the fluid biosample over the electronic biosensor, and the fluid biosample is pooled in contact with the capture molecules of the electronic biosensor for a period of time, providing time and opportunity for the capture molecules to bind to target molecules while the fluid biosample accumulates.

14. The mask-based diagnostic device of claim 13, wherein the fluid transfer system is configured and dimensioned to flow a predetermined amount of the fluid biosample over the electronic biosensor for a predetermined time, and the concentration of the target molecule is determinable as a function of a predetermined volume of the fluid biosample flowing over the electronic biosensor within the predetermined time and a change in an electrical signal, and the electronic biosensor outputs an electrical signal having a change in electrical properties depending on the captured molecule, which changes the electrical signal depending on the captured biomarker.

15. 15. The mask-based diagnostic device of claim 14, wherein the fluid conductor includes a transfer volume for absorbing the fluid biosample, the transfer volume having an absorption saturation point, the fluid conductor conducting the fluid biosample at a slow rate before the absorption saturation point is reached and at a fast rate after the absorption saturation point is reached, and further including a flow starting fluid that is freezable within the fluid conductor to facilitate reaching the absorption saturation point during use, the freezable solution including at least one of a buffer solution and a calibration component for the test unit, the calibration component enabling the electronic circuit to determine a calibration value from the starting fluid, prior to use of the mask-based diagnostic device, the freezable solution is maintained in a frozen state, and during use of the mask-based diagnostic device, the freezable solution thaws to wet a surface of the EBC collector and facilitate fluid transfer of the EBC fluid biosample.

16. 1. A breath-based diagnostic device for detecting biomarkers contained in the exhaled breath of a subject, comprising: an exhaled breath condensate (EBC) collector for converting exhaled vapors received from the lungs and airways of a subject into a fluid biosample, the EBC collector including a thermal mass, a condensate-forming surface, and a fluid conductor disposed on the condensate-forming surface; a fluid transfer system for receiving a fluid biosample from the EBC collector; a biomarker testing unit for receiving the fluid biosample from the fluid transfer system and testing the fluid biosample for target biomarkers contained in the fluid biosample;

17. 17. The breath-based diagnostic device of claim 16, further comprising a test system support for supporting the EBC collector, the fluid transfer system, and the biomarker testing unit, wherein the test system support is configured and dimensioned to fit inside an existing face mask, the existing face mask forming an exhaled vapor containment volume and holding the exhaled vapor close to the EBC collector, allowing the condensation-forming surface cooled by the thermal mass to coalesce the exhaled vapor into the fluid biosample.

18. 17. The breath-based diagnostic device of claim 16, wherein the fluid transfer system includes a biosample pooling area for pooling a fluid biosample received from the EBC collector, and the biomarker testing unit includes a lateral flow assay in which the fluid biosample flows through a multi-zone transport medium by capillary action, the lateral flow assay including a sample pad positioned in the pooling area to receive the fluid biosample, a conjugate release pad in which a biomarker-labeled capture molecule complex is formed, a detection zone, and a flow membrane for flowing the fluid sample from the sample pad, through the release pad, and into the detection zone to detect potential biomarkers.

19. 20. The breath-based diagnostic device of claim 18, wherein the fluid transfer system further comprises a fluid dam disposed in fluid communication between the EBC collector and the pooling area, the fluid dam accumulating a certain amount of the fluid biosample until the fluid dam releases a certain amount of the fluid biosample to flood the pooling area with the accumulated amount of the fluid biosample and provides the accumulated amount of the fluid biosample to the sample pad, the fluid dam comprising a dissolvable material that is removed by being dissolved by the fluid biosample.

20. 17. The breath-based diagnostic device of claim 16, wherein the biomarker testing unit includes an electronic biosensor having capture molecules for capturing the target molecules contained in the fluid biosample and outputting an electrical signal proportional to the target molecules captured by the capture molecules; and the fluid transfer system includes a wick for absorbing the flow of the fluid biosample after the biomarker testing unit tests the flow of the fluid biosample, whereby the fluid biosample flows over the electronic biosensor over time and the target molecules flow with the fluid biosample, allowing the capture molecules an opportunity to capture the target molecules flowing with the fluid biosample over the electronic biosensor; and the wick includes a super absorbent polymer (SAP) for receiving and absorbing the flow of the fluid biosample.

21. The electronic biosensor includes an electrode layer having at least a source electrode and a drain electrode; and a binding surface disposed between the source electrode and the drain electrode and functionalized with at least one capture molecule for capturing the target biomarker, wherein capturing the target biomarker changes at least one electrical property between the source electrode and the drain electrode, and each capture molecule has an infinity for a respective biomarker. The electronic biosensor further includes an electronic circuit for receiving an output signal from the biomarker testing unit, determining an affinity-dependent test signal value, and calculating a result value for the at least one capture molecule and the respective biomarker. The electronic biosensor further comprises a substrate, the binding surface being a top surface of a binding bulk, and the bottom surface of the binding bulk being diffusion bonded to the substrate.

21. The breath-based diagnostic device of claim 20, wherein the at least one capture molecule comprises at least one of an aptamer, an engineered antibody, an antibody, a protein, an antigen, a nucleic acid-based ligand, and a small molecule engineered to mimic a monoclonal antibody.

22. 17. The breath-based diagnostic device of claim 16, wherein the fluid transfer system is configured and dimensioned to flow a predetermined amount of the fluid biosample over the electronic biosensor for a predetermined time, and the concentration of the target molecule is determinable as a function of a predetermined volume of the fluid biosample flowing over the electronic biosensor within the predetermined time and a change in an electrical signal, and the electronic biosensor outputs an electrical signal having a change in electrical property depending on the captured molecule, which changes the electrical signal depending on the captured biomarker.

23. 17. The breath-based diagnostic device of claim 16, wherein the fluid conductor includes a transfer volume for absorbing the fluid biosample, the transfer volume having an absorption saturation point, the fluid conductor conducting the fluid biosample at a slow rate before the absorption saturation point is reached and at a fast rate after the absorption saturation point is reached, and further including a flow starting fluid that is freezable within the fluid conductor to facilitate reaching the adsorption saturation point, the freezable solution including at least one of a buffer solution and a calibration component for the test unit, the calibration component enabling the electronic circuit to determine a calibration value from the starting fluid, prior to use of the mask-based diagnostic device, the freezable solution is maintained in a frozen state, and during use of the mask-based diagnostic device, the freezable solution thaws to wet a surface of the EBC collector and facilitate fluid transfer of the EBC fluid biosample.

24. 1. A mask-based examination system for detecting biomarkers received from the lungs and airways of a subject, comprising: an exhaled breath condensate (EBC) collector incorporated inside a face mask worn by the subject, the EBC collector for converting exhaled vapors received from the subject's lungs and airways into a fluid biosample; a biosensor secured inside the face mask for receiving a fluid biosample from the EBC collector, testing the fluid biosample for target biomarkers, and generating a test signal dependent upon at least the presence or absence of the target biomarkers in the fluid biosample; and an electronic circuit secured to the exterior of the mask for receiving the test signal, determining a test result signal in response to detection or non-detection of the target biomarker from the test signal, and transmitting the test result signal to a remote receiver; A mask-based inspection system, including:

25. 25. The mask-based inspection system of claim 24, wherein the EBC collector includes a droplet collection structure, the droplet collection structure including a hydrophobic field for receiving the exhaled breath vapor and forming fluid droplets from the received exhaled breath vapor, and a hydrophilic channel for receiving the fluid droplets from the hydrophobic field and channeling the fluid droplets together to form the collected fluid biosample.

26. 26. The mask-based inspection system of claim 25, wherein the EBC collector further includes a thermal mass and a front surface in thermal communication with the thermal mass, the front surface positioned facing the subject's mouth and nose when the subject is wearing the face mask, and the front surface includes the hydrophobic field and the hydrophilic channel.

27. 27. The mask-based inspection system of claim 26, wherein the front surface comprises a hydrophobic surface of a metal foil having hydrophilic channels printed thereon.

28. 27. The mask-based inspection system of claim 26, wherein the thermal mass comprises at least one of a superabsorbent polymer, water, and an endothermic compound.

29. 30. The mask-based inspection system of claim 28, wherein the water is contained in a sealed structure and kept separate from the endothermic compound until an activation step in which the water is released from the sealed structure to mix with the endothermic compound and cool the front surface.

30. 25. The mask-based inspection system of claim 24, wherein the biosensor includes at least one of a conductive and semiconductive base material disposed in a gap formed on a substrate between at least two probe electrodes, and capture molecules in electrical communication with the probe electrodes through the base material.

31. 31. The mask-based inspection system of claim 30, wherein the capture molecules are immobilized on the base material via at least one of π-π stacking, amine coupling, thiol-au binding, click chemistry, electrostatic interaction, biotin-avidin affinity, and complementary DNA hybridization.

32. 31. The mask-based inspection system of claim 30, wherein the base material comprises at least one of graphene, carbon nanotubes, gold, a screen-printed conductive material, and a positively charged material.

33. 31. The mask-based inspection system of claim 30, wherein the capture molecules include at least one of an aptamer and an antibody.

34. 31. The mask-based inspection system of claim 30, wherein the base material comprises electric or magnetic field alignable microparticles fixed in alignment by a binding layer formed on the top surface of the substrate.

35. 35. The mask-based inspection system of claim 34, wherein the bonding layer comprises at least one of a bonding layer printed on the top surface and the top surface of the base material.

36. 25. The mask-based inspection system of claim 24, wherein the electronic circuitry includes an amplifier circuit for receiving the test signal from the biosensor and amplifying the test signal into an amplified electrical signal, and a comparator circuit for comparing the amplified electrical signal to a predetermined value based on at least one of a computer model of the biosensor and an empirically derived electrical signal calibration using at least one of a known presence and a known concentration of the target biomarker in a calibration sample, and the comparator circuit generates the test result signal based on the amplified electrical signal compared to the predetermined value.

37. 37. The mask-based inspection system of claim 36, wherein the electronic circuit further includes a biomarker concentration circuit for determining a concentration value of the target biomarker in response to the amplified electrical signal, the amplified electrical signal varying in value depending on the number of target biomarker molecules in the fluid biosample, and the test result signal depending on the determined concentration value.

38. 37. The mask-based inspection system of claim 36, wherein the electronic circuitry further includes wireless communication circuitry for wirelessly transmitting the inspection result signal to at least one of a smartphone, a tablet, a computer, a relay, an access point, and a computer network.

39. 25. The mask-based inspection system of claim 24, further comprising a particulate capture structure for receiving and capturing exhaled aerosol droplets and particulates (EBA) from the inner airway lining of the subject, the aerosol particulate inspection system comprising a dissolvable EBA sample collector film for capturing EBA droplets and particulates.

40. 25. The mask-based inspection system of claim 24, wherein the EBA particulates include insoluble particulates and droplet particulates, and the dissolvable EBA collection film includes a sticky surface for adhering to and capturing the insoluble particulates and a water-soluble bulk for capturing the droplet particulates.

41. 25. The mask-based inspection system of claim 24, wherein the EBA particulates include insoluble particulates and droplet particulates, and the dissolvable EBA collection film includes a sticky surface for adhering to and capturing the insoluble particulates and a water-soluble bulk for capturing the droplet particulates.

42. 25. The mask-based inspection system of claim 24, wherein the biosensor tests the fluid biosample for the target biomarker and tests the fluid biosample for at least one other target biomarker, and the inspection signal depends at least on the presence or absence of the target biomarker and the at least one other target biomarker in the fluid biosample.

43. A device for detecting biomarkers, comprising a particle capture structure for receiving and capturing exhaled breath aerosol (EBA) particles from the lining of a user's airways, the particle capture structure having an aerosol particle inspection system for receiving the captured particles and detecting a first biomarker, the aerosol particle inspection system including a dissolvable EBA sample collector film for capturing EBA particles.

44. 44. The device for detecting a biomarker of claim 43, wherein the dissolvable EBA sample collector film comprises a first reagent for reacting with at least one component of the captured microparticles in a detection reaction to detect the first biomarker.

45. 45. The apparatus for detecting a biomarker of claim 44, wherein the detection reaction produces at least one of an optical signal and an electrical signal change that is dependent on the first biomarker.

46. 46. ​​The device for detecting a biomarker of claim 45, wherein the first reagent is bound to a first nanoparticle and retained in the insoluble test region.

47. 44. A device for detecting a biomarker as described in claim 43, wherein the EBA microparticles include insoluble microparticles and droplet microparticles, and the dissolvable EBA collection film includes a sticky surface for adhering to and capturing the insoluble microparticles and a water-soluble bulk for capturing the droplet microparticles.

48. 48. The device for detecting a biomarker of claim 47, further comprising a droplet collection structure for converting exhaled breath vapor from a user into exhaled breath condensate (EBC) fluid droplets to form a fluid sample.

49. 49. The device for detecting biomarkers described in claim 48, wherein the droplet collection structure includes at least one of a hydrophobic field for receiving the exhaled breath vapor and forming fluid droplets from the received exhaled breath vapor, and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets to a fluid sample testing system, the fluid sample testing system having a biomarker testing zone for receiving the fluid sample and detecting a second biomarker.

50. 50. The device for detecting biomarkers described in claim 49, further comprising a fluid dam member disposed between the droplet collection structure and the biomarker testing zone, the fluid dam member comprising at least one of a removable moisture-resistant sheet member and a dissolvable film for accumulating the fluid sample from the droplet collection structure and releasing the accumulated fluid sample to flow into the biomarker testing zone.

51. 50. The device for detecting biomarkers of claim 49, wherein the fluid sample testing system includes a fluidic lateral flow assay comprising a sample pad for receiving the fluid sample potentially containing a biomarker analyte as the second biomarker, a conjugate release pad, a flow membrane, and an absorbent pad for receiving and flowing the fluid sample and detecting the potential biomarker analyte from the sample source.

52. 52. The device of claim 51, further comprising a fluid dam member disposed between the sample pad and the conjugate release pad, the fluid dam comprising a pull-tab structure, wherein a user can remove the fluid dam member to allow the fluid sample to flow from the sample pad to the conjugate release pad.

53. 52. The device of claim 51, further comprising at least one light emitter and one photodetector, said light emitter emitting radiation toward said biomarker testing zone and said photodetector receiving radiation from said biomarker testing zone.

54. 51. The device for detecting biomarkers of claim 50, wherein the fluid sample testing system includes a fluid biosensor having a sample source having a biomarker analyte for receiving a fluid sample potentially containing the biomarker analyte as the second biomarker, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal according to a change in the bioreceptor in response to receiving the biomarker analyte from the sample source.

55. 55. The device for detecting a biomarker of claim 54, wherein the analyte-specific biomarker comprises a reagent for causing a detection reaction with the biomarker analyte, and the fluidic biosensor generates at least one of an optical signal and an electrical signal change that is dependent on the biomarker.

56. 56. The device for detecting a biomarker of claim 55, wherein the reagent is bound to nanoparticles and retained in the insoluble test region.

57. 57. The device of claim 56, further comprising wireless communication electronics for detecting a result of a test for at least one of said first and said second biomarkers and communicating said result to a wireless receiver.

58. 58. The system of claim 57, wherein the electronic circuit is in communication with at least one of the aerosol particle testing system and the fluid sample testing system to detect one or more biometric parameters, the biometric parameters being dependent on at least one physiological change in the patient in response to a condition of concern such as a viral infection, the one or more biometric parameters being received and a probabilistic analysis being applied by a microprocessor, and in response to the probabilistic analysis of the one or more biometric parameters, it is determined whether at least one physiological change threshold has been exceeded, and the electronic circuit transmits a signal in response to the determined excess of the at least one physiological change.

59. at least one processor; and at least one memory containing computer program code; 1. An apparatus comprising: The at least one memory and the computer program code are configured, by the at least one processor, to cause the device to perform at least the following: detect one or more biometric parameters using a particle capture structure for receiving and capturing exhaled aerosol (EBA) particles from the lining of a user's airways, the particle capture structure having an aerosol particle inspection system for receiving the captured particles and detecting a first biomarker, the aerosol particle inspection system including a dissolvable EBA sample collector film for capturing EBA particles, the biometric parameter being a biomarker dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection; receive the one or more biometric parameters and apply a probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change.

60. 60. The device of claim 59, wherein the one or more biometric parameters are further detected using a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample, and an inspection system having a biomarker inspection zone for receiving the fluid sample and detecting the biometric parameters, and wherein the probabilistic analysis is applied to the one or more biometric parameters to determine whether at least one physiological change threshold has been exceeded depending on the probabilistic analysis of the one or more biometric parameters detected from both the captured particulate and the fluid sample.

61. 1. An apparatus for detecting a biomarker, comprising: a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample; and a testing system having a biomarker testing zone for receiving the fluid sample and detecting a biomarker.

62. 62. The device for detecting biomarkers of claim 61 , wherein the droplet collection structure includes at least one of a hydrophobic field for forming the fluid droplets from the received exhaled vapor and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets to the testing system.

63. 62. An apparatus for detecting biomarkers as described in claim 61, including a fluid dam member disposed between the droplet collection structure and the biomarker testing zone.

64. 62. The device for detecting biomarkers of claim 61, wherein the testing system includes a fluidic lateral flow assay comprising a sample pad for receiving the fluid sample potentially containing a biomarker analyte, a conjugate release pad, a flow membrane, and an absorbent pad for receiving and flowing the fluid sample and detecting the potential biomarker analyte from the sample source.

65. 65. The device of claim 64, further comprising a fluid dam member disposed between the sample pad and the conjugate release pad, the fluid dam comprising a pull-tab structure, wherein a user can remove the fluid dam member to allow the fluid sample to flow from the sample pad to the conjugate release pad.

66. 62. The device of claim 61, further comprising at least one light emitter and one photodetector, said light emitter emitting radiation toward said biomarker testing zone and said photodetector receiving radiation from said biomarker testing zone.

67. 1. An apparatus for detecting biomarkers, comprising: a droplet collection and channeling structure for converting vapor into fluid droplets; and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal according to a change in the bioreceptor in response to receiving the biomarker analyte from the sample source.

68. 1. A system for detecting biological agents from the exhaled breath of a subject, the system comprising: a breath condensate droplet collector for coalescing exhaled vapors into droplets to form a fluid biological sample; a testing system for receiving the fluid biological sample from the breath droplet collector and testing it for target analytes; and wireless communication electronics for detecting test results for the target analytes and communicating the results to a wireless receiver.

69. 69. The system of claim 68, further comprising an exhaled aerosol capture system comprising a sheet member having a surface for receiving exhaled aerosol comprising at least one of fine particles and liquid droplets.

70. 70. The system of claim 69, wherein the surface is an exposed portion of a dissolvable film formed on or integral with the sheet member, the dissolvable film having a composition effective to receive and capture at least one of microparticles and liquid droplets by at least one of embedding or dissolving the at least one of microparticles and liquid droplets on the surface or within the dissolvable film.

71. 71. The system of claim 70, wherein at least one of the surface and the dissolvable film comprises a reagent for reacting with the at least one microparticle and droplet to detect the presence of a target analyte in the at least one microparticle and droplet.

72. 71. The system of claim 70, comprising: an electronic circuit in communication with the testing system for detecting one or more biometric parameters dependent on at least one physiological change in a patient in response to a condition of concern, such as a viral infection; receiving the one or more biometric parameters and applying a probabilistic analysis by a microprocessor to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and initiating an action in response to the determined at least one physiological change being exceeded.

73. at least one processor; and at least one memory containing computer program code; 1. An apparatus comprising: The at least one memory and the computer program code are configured, by the at least one processor, to cause the device to at least perform the following: detect one or more biometric parameters using an examination system having a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample, and a biomarker examination zone for receiving the fluid sample and detecting biometric parameters, the biometric parameters being biomarkers dependent on at least one physiological change in the patient in response to a condition of concern, such as a viral infection; receive the one or more biometric parameters and apply a probabilistic analysis to determine whether at least one physiological change threshold has been exceeded in response to the probabilistic analysis of the one or more biometric parameters; and take action in response to the determined exceedance of the at least one physiological change.

74. An apparatus for detecting a biomarker, comprising: a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample; and a testing system having a biomarker testing zone for receiving the fluid sample and detecting the biomarker.

75. 75. The device for detecting biomarkers of claim 74, wherein the droplet collection structure includes at least one of a hydrophobic field for receiving the exhaled breath vapor and forming the fluid droplets from the received exhaled breath vapor, and a hydrophilic channel for receiving the fluid droplets and directing the fluid droplets to the testing system.

76. 75. The apparatus for detecting biomarkers of claim 74, further comprising a fluid dam member disposed between the droplet collection structure and the biomarker testing zone.

77. 75. The device for detecting biomarkers of claim 74, wherein the testing system includes a fluidic lateral flow assay comprising a sample pad for receiving the fluid sample potentially containing a biomarker analyte, a conjugate release pad, a flow membrane, and an absorbent pad for receiving and flowing the fluid sample and detecting the potential biomarker analyte from the sample source.

78. 78. The device of claim 77, further comprising a fluid dam member disposed between the sample pad and the conjugate release pad, the fluid dam comprising a pull-tab structure, wherein a user can remove the fluid dam member to allow the fluid sample to flow from the sample pad to the conjugate release pad.

79. 75. The device of claim 74, further comprising at least one light emitter and one photodetector, said light emitter emitting radiation toward said biomarker testing zone and said photodetector receiving radiation from said biomarker testing zone.

80. 1. An apparatus for detecting biomarkers, comprising: a droplet collection and channeling structure for converting vapor into fluid droplets; and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal according to a change in the bioreceptor in response to receiving the biomarker analyte from the sample source.

81. 1. A method for forming a biomarker testing system, comprising the steps of: providing a substrate; coating a hydrophobic field on the substrate; and coating at least one hydrophilic channel on the substrate. Including, The method, wherein the hydrophobic field is for receiving body fluid vapor and forming fluid droplets from the received body fluid vapor, and the hydrophilic channel is for receiving the fluid droplets and directing the fluid droplets toward an inspection system.

82. 82. A method for forming a biomarker testing system as described in claim 81, further comprising forming at least one fluid sample ejection hole at an end of the hydrophilic channel for ejecting the fluid droplet through at least one fluid sample ejection hole onto a sample receiving structure of the testing system.

83. 1. An apparatus for detecting biomarkers, comprising: a droplet collection and channeling structure for converting vapor into fluid droplets; and a fluidic biosensor, the fluidic biosensor comprising a sample source having a biomarker analyte, a bioreceptor region functionalized with an analyte-specific bioreceptor, and a transducer for generating a readable signal according to a change in the bioreceptor in response to receiving the biomarker analyte from the sample source.

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