Diagnostic platform and universal biosensor for detecting exhaled gas condensate

Abstract

A mask-based diagnostic device for detecting a biomarker contained in a gas exhaled by a test subject. An exhaled air condensate (EBC) collector can convert exhaled air from the lungs and the respiratory tract into a fluid biological sample. The EBC collector includes a condensate forming surface and a thermal block thermally connected to the condensate forming surface. A fluid transfer system transfers the EBC to at least one of the test device and the EBC container. Before testing, the semipermeable membrane concentrates the target biomarker portion in the fluid biological sample to form a concentrated fluid biological sample for testing. The target biomarker release material, such as a cracking agent and / or mechanical cracking, can be used for acquiring the target biomarker in the virus envelope for detection. The test device may be a universal g-FET biosensor configured as a packaged semiconductor device that can receive and test EBC as a fluid biological sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application is related to U.S. Provisional Patent Application No. 63 / 245,295, filed on September 17, 2021, by Daniels et al., and U.S. Provisional Patent Application No. 63 / 233,473, filed on August 16, 2021, by Daniels et al. (Diagnostic Platform for Detecting Exhaled Breath Condensate), the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] Exemplary and non-limiting embodiments of the present invention relate generally to diagnostic systems, methods, devices and computer programs, and more specifically to digital and analog diagnostic devices for detecting biomarkers of biological agents such as coronavirus, lung cancer, tuberculosis, asthma and other respiratory diseases and conditions, and / or blood biomarkers and other biomarkers present in the exhaled breath of a test subject.

[0004] The present invention also relates to a device architecture, specific use applications and computer algorithms for detecting biometric parameters for treating and monitoring physiological conditions in humans and animals. Background Art

[0005] This section is intended to provide a background or context to the exemplary embodiments of the invention described in the claims.The description herein may include concepts that could be employed, but are not necessarily those that have been previously conceived, implemented, or described.

[0006] Therefore, unless otherwise indicated herein, what is described in this section and the description and claims in this application are not prior art and are not admitted to be prior art by inclusion in this section.

[0007] Testing for biomarkers that indicate exposure, infection, progression, and recovery from illnesses such as COVID-19 can be used to screen infected people and help slow the spread of the virus. For example, protein and RNA tests for active virus can show who is currently contagious. Antibody tests can be used to identify people who have recovered from the virus.

[0008] The use of real-time reverse transcription polymerase chain reaction (RT-PCR) with nasopharyngeal swabs to diagnose SARS-CoV-2 infection is now widely available, and saliva-based testing has recently become more widely used because it is more suitable for self-testing. The process of obtaining nasopharyngeal swab samples is not only uncomfortable, but also requires professional operation and has the potential to contaminate the tester. The advantages of saliva testing are simple operation, low invasiveness, limited risks, and the practice of RT-PCR testing of saliva samples is becoming more and more widespread. The viscosity of saliva and the presence of salivary proteases (salivary proteases are responsible for the proteolytic activity of saliva) make the direct application of saliva samples challenging. It is well known that the main transmission route of COVID-19 is airborne and contact transmission, which is mainly caused by aerosol droplets discharged from the lungs and respiratory tracts of infected people. Therefore, there is an increasing need for samples to be collected by patients themselves, and a simple and easy-to-use testing system is needed to detect target biomarkers indicative of pathogen infection from biological samples collected from the lungs and respiratory tracts. Summary of the invention

[0009] The following summary is intended to be illustrative only and not limiting. The above-mentioned problems and other problems may be overcome, and other advantages may be achieved, by using the exemplary embodiments of the present invention.

[0010] According to one non-limiting exemplary embodiment, an exhaled breath condensate (EBC) collector can convert exhaled breath vapor received from the lungs and airways of a test subject into a fluid biological sample. The EBC collector includes a condensate forming surface and a thermal mass thermally connected to the condensate forming surface. A fluid transfer system transfers the EBC to at least one of a test device and an EBC container.

[0011] According to another non-limiting exemplary embodiment, the electronic biosensor includes a substrate having water absorption properties provided by at least one of a selectively permeable membrane, a super absorbent polymer, a microfluidic material, and a wick. At least two electrodes are formed on the top surface of the substrate, forming a gap between the two. The functionalized detector in the gap includes an electron transport material and a capture molecule. The target molecule captured by the capture molecule causes at least one of the polarity and conductivity of the electron transport material to change. The target molecule is detected by detecting the change in the polarity or conductivity of the electron transport material. The target molecule detected depends on the capture molecule, and therefore, by changing the capture molecule to match the known biomarker of the corresponding disease or use, this electronic biosensor structure can be used to detect a variety of different diseases and other uses.

[0012] According to another non-limiting exemplary embodiment, an apparatus for detecting a target biomarker in exhaled breath condensate (EBC) includes an EBC collector for converting exhaled breath received from the lungs and airways of a test subject into an EBC biological sample. The EBC biological sample contains a target analyte. The biomarker concentrator includes a super absorbent polymer layer located in the EBC biological sample flow path. During the collection process, the EBC biological sample contacts the super absorbent polymer layer, and a portion of the water in the EBC biological sample is absorbed. The super absorbent polymer does not absorb the target analyte, thereby increasing the concentration of the target analyte in the remaining water in the EBC fluid biological sample. The biomarker detection device receives the concentrated EBC biological sample and performs a target biomarker (target molecule or target analyte) detection on the concentrated EBC biological sample.

[0013] According to another aspect of the present invention, a method for concentrating a target analyte in an exhaled breath condensate (EBC) sample comprises the following steps: collecting an EBC sample from the lungs and respiratory tract of a test subject. The EBC contains the target analyte. During the collection process, the EBC sample is contacted with a super absorbent polymer. The super absorbent polymer absorbs a portion of the water in the EBC sample without absorbing the target analyte, thereby obtaining a target analyte concentration in the remaining water in the EBC sample.

[0014] According to another aspect of the present invention, the capture molecule conjugate can detect the target analyte. The external field reaction capture molecule conjugate has at least one external field reaction end and at least one capture molecule end. Each capture molecule end binds to the target analyte and captures the target analyte. The binding of each capture molecule end to the target analyte increases the charge difference between at least one external field reaction end and at least one capture molecule end.

[0015] According to another aspect of the present invention, a method for detecting a target analyte comprises the following steps: providing a capture molecule structure having a ligand end and a polarizable end. When the capture molecule structure is placed in a carrier liquid, the capture molecule structure is a free floating element. The target analyte is another free floating element in the carrier liquid. The ligand end of the capture molecule structure binds to the target analyte to form a free floating polar conjugate having a positive end and a negative end. To detect the target analyte, the polar conjugate is arranged in the carrier liquid in an electric field, and the electrical properties of the arranged polar conjugate are measured.

[0016] According to another aspect of the present invention, a method is provided for forming a condensate collector having a fluid conductor channel on a substrate to direct the fluid flow in a desired direction. The surface of the substrate has a relatively low energy surface property. A texture structure is formed on the surface to form a fluid conductor channel having a relatively high energy surface property for directing the fluid flow in a desired direction.

[0017] According to another aspect of the present invention, a method for assembling an array of external field reaction capture molecule conjugates comprises the following steps: providing a soluble adhesive film. Providing a non-solvent carrier liquid for dissolving the adhesive film. The carrier liquid has randomly dispersed external field reaction capture molecule conjugates. Applying an alignment field to the carrier liquid to assemble the external field reaction capture molecule conjugates onto the soluble adhesive film. Removing the carrier liquid by evaporation and / or other processes, fixing the assembled external field reaction capture molecule conjugates onto the soluble adhesive film.

[0018] According to another non-limiting exemplary embodiment, the test system includes an exhaled breath condensate (EBC) collector for converting exhaled breath received from the lungs and respiratory tract of the test subject into a fluid biological sample. The biomarker concentrator partially concentrates the target biomarker in the fluid biological sample to form a concentrated fluid biological sample. The biomarker detection device receives the concentrated liquid biological sample and performs target biomarker detection on the concentrated liquid biological sample.

[0019] According to another non-limiting exemplary embodiment, an exhaled breath condensate (EBC) collector can convert exhaled breath vapor received from the lungs and airways of a test subject into a fluid biological sample. The EBC collector includes a condensate forming surface and a thermal mass thermally connected to the condensate forming surface. A fluid transfer system transfers the EBC to at least one of a test device and an EBC container.

[0020] According to another non-limiting exemplary embodiment, a method of forming a condensate collector having a fluid conductor channel on a substrate to guide fluid flow to a desired direction includes the steps of providing a substrate having a surface with relatively low energy surface properties, and forming a texture structure on the surface with relatively high energy surface properties to form a fluid conductor channel to guide the fluid flow to a desired direction.

[0021] According to another non-limiting exemplary embodiment, a device includes at least one processor, at least one memory including computer program code, and the at least one memory and the computer program code are configured to cause the device to perform at least the following operations through the at least one processor: provide a capture molecule structure having a ligand end and a polarizable end, wherein when the capture molecule structure is arranged in a carrier liquid, the capture molecule structure is a free floating element; provide a target analyte as another free floating element in the carrier liquid, wherein the ligand end of the capture molecule structure binds to the target analyte and forms a free floating polar conjugate, which has a positive end and a negative end; and test the electrical properties of the aligned polar conjugate to detect the target analyte.

[0022] According to another non-limiting exemplary embodiment, a computer program product includes a computer-readable medium carrying computer program code for a computer, the computer program code including: code for testing the electrical properties of a quasi-polar conjugate to detect a target analyte by providing a capture molecule structure having a ligand end and a polarizable end, wherein when the capture molecule structure is arranged in a carrier liquid, the capture molecule structure is a free floating element, and the target analyte is provided in the carrier liquid as another free floating element, wherein the ligand end of the capture molecule structure binds to the target analyte and forms a free floating polar conjugate having a positive end and a negative end.

[0023] According to another non-limiting embodiment, a sensor for detecting a target molecule in a fluid sample includes a detection area for receiving a fluid sample containing the target molecule. The detection area includes a detection interface functionalized with a capture molecule. A gap is formed between the top drive electrode and the bottom drive electrode. A fluid conductor disposed in the gap directs the fluid sample out of the gap. The potential applied to the top and bottom drive electrodes drives the target molecule toward the capture molecule, thereby concentrating the target molecule in a portion of the fluid sample received by the detection interface.

[0024] According to another non-limiting embodiment, a method for detecting a target molecule from a fluid sample comprises the following steps: receiving a fluid sample containing a target molecule in a microfluidic channel; transferring the fluid sample from the microfluidic channel to a detection interface of a sensor, the sensor comprising a detection area for receiving the fluid sample, the detection interface having a functionalized capture molecule, a top drive electrode and a bottom drive electrode defining a gap therebetween, and a fluid conductor disposed in the gap for conducting the fluid sample through the gap. The potential applied to the top and bottom drive electrodes can drive the target molecule toward the capture molecule, thereby concentrating the target molecule in a portion of the fluid sample received by the detection interface. The sensor may include a field effect transistor having a gate in electrical communication with the detection interface, the gate being functionalized with the capture molecule, and having a source and a drain on either side of the gate. At least a portion of at least one of the top and bottom drive electrodes is disposed in the detection area, wherein the gate of the sensor comprises at least one of the top and bottom drive electrodes. The electromotive force drives the target molecule toward the capture molecule, and is applied intermittently and periodically, while test readings are taken of changes in the electrical characteristics of the source, drain, and gate caused by the capture of the target molecule.

[0025] According to another non-limiting exemplary embodiment, an indoor biosensor includes an air inlet for drawing in ambient air. A condenser cools the ambient air and condenses water vapor into a condensate containing water and at least one target molecule. A condensate detection system detects whether the condensate contains at least one target molecule. The condensate detection system includes a biosensor, the biosensor including at least one g-FET biosensor, the biosensor having a detection interface, the interface including a graphene layer functionalized with a capture molecule, wherein the capture molecule is smaller than a Debye screening length.

[0026] According to another non-limiting exemplary embodiment, a field effect transistor sensor circuit is provided for detecting target molecules in a fluid sample. A semiconductor substrate of a conductive type has a source region and a drain region, and a channel region of a conductive type is defined between the source region and the drain region. The conductivity type of the source region and the drain region is opposite to that of the substrate region and the channel region. An insulator is formed on the channel region, wherein the channel region constitutes the back gate of the field effect transistor. A detection region is formed on the insulator for receiving a fluid sample. The detection interface is functionalized with capture molecules. The top electrode forms a gap with the detection region and constitutes a liquid gate electrode of the transistor. A fluid conductor is placed in the gap for introducing a fluid sample into the gap. The driving circuit applies an electromotive force of one polarity to the top electrode and applies an electromotive force of the opposite polarity to the back gate. The potential drives the target molecules toward the capture molecules, thereby concentrating the target molecules in the portion of the fluid sample received by the detection interface.

[0027] According to another non-limiting exemplary embodiment, a packaged semiconductor device includes a universal biosensor provided by a single g-FET chip on a wafer and encapsulated in epoxy. A detection hole is formed on top of the molded epoxy and placed above the graphene detection interface so that the fluid sample can contact the fixed capture molecules on the detection interface. The graphene layer can be formed at the wafer level and functionalized with the capture molecules. During the manufacturing process, the functionalized graphene is protected by a soluble mask so that the chip can be picked and placed and mounted on a lead frame and then encapsulated with epoxy. The detection hole can be pre-formed in the epoxy cap or formed in situ during the packaging process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To facilitate identification of the discussion of any particular element or act, the most significant digit or digits in the reference number refer to the drawing number in which the element is first introduced.

[0029] Figure 1A mask is shown and schematically illustrates an exhaled breath condensate (EBC) collector, fluid transfer system, biosensor testing system, and near field communication (NFC) antenna and signal conditioning circuitry, all arranged within a confined space inside the mask.

[0030] Figure 2 The open inner portion of the mask is shown, which contains components for collecting and detecting EBC and exhaled breath aerosol (EBA).

[0031] Figure 3 is a block diagram of one possible non-limiting example system in which certain exemplary embodiments may be implemented.

[0032] Figure 4 is a logic flow diagram for determining the application of probabilistic analysis for detecting target biomarkers and illustrates the operation of an exemplary method, a result 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 in accordance with exemplary embodiments.

[0033] Figure 5 is a logic flow diagram of data acquisition and transmission for trusted receiver and population research use cases and illustrates the operation of an exemplary method, the result of execution of computer program instructions embodied on a computer readable memory, the functions performed by logic implemented in hardware, and / or interconnected means for performing functions in accordance with the exemplary embodiments.

[0034] Figure 6 A block diagram of the basic components for testing EBC and transmitting the test results to a smartphone and / or cloud server.

[0035] Figure 7 A KN95 mask with a retrofittable testing system is shown, which includes an EBC collector with an aluminum foil condensation-forming surface, embossed fluid conductor channels, a fluid transfer system, and electronic biosensor electrodes.

[0036] Figure 8 Shown is the test system incorporated into a KN95 mask.

[0037] Fig. 9 A KN95 mask is shown with removable testing and communications electronics mounted on the outside.

[0038] Fig.10 An exhaled breath condensate collector and a testing area with a panel of syndromic test biosensors are shown.

[0039] Fig.11 The biosensor is shown immersed in the pooling area of ​​the collected EBC.

[0040] Fig.12 The assembled EBC collector and pooling region are shown.

[0041] Fig.13 The sizes of the engineered capture molecules, S protein, virus particles, and water molecules are shown.

[0042] Fig.14 An EBC concentrator is shown having a semipermeable membrane for separating excess water from the EBC containing concentrated viral particles.

[0043] Fig.15 The addition of superabsorbent polymers in an EBC concentrator is shown.

[0044] Fig.16 The addition of surfactant or lysing agent in an EBC concentrator is shown.

[0045] Fig.17 EBC droplets concentrating viral particles and cleaved proteins are shown.

[0046] Fig.18 is a cross section showing an EBC concentrator with a semipermeable membrane and wick adjacent to the thermal mass.

[0047] Fig.19 is a cross-sectional view showing an EBC concentrator with a fluid conductor containing dissolved material, a semi-permeable membrane and core in the EBC collection area.

[0048] Fig. 20 Capture molecule / linker / magnetic nanoparticle conjugates with free-floating and captured target molecules are demonstrated.

[0049] Fig.21 demonstrated an EBC collector with oriented magnetic nanoparticle conjugates on a dissolvable binder for capturing and concentrating target molecules in exhaled breath.

[0050] Fig. 22 demonstrated an EBC collector conjugate with oriented and aligned magnetic nanoparticles, forming an EBC that enables the binding of target molecules to capture molecules.

[0051] Fig.23 demonstrated that water-soluble binders in EBCs allow magnetically active molecules conjugated to target molecules to flow toward magnetic traps in the test area.

[0052] Fig.24 The binding of magnetically active molecules to target molecule conjugates, which are immobilized on magnetic traps in the test area, is shown.

[0053] Fig.25 A dissolvable adhesive film is shown supporting a carrier liquid containing free-floating and randomly dispersed magnetic nanoparticle conjugates prior to application of a magnetic alignment field, wherein the carrier liquid is a non-solvent for the dissolvable adhesive.

[0054] Fig.26 Shown is the alignment of magnetic nanoparticle conjugates on a dissolvable gel film in a carrier fluid containing free-floating conjugates following application of a magnetic alignment field.

[0055] Fig. 27 Well-organized magnetic nanoparticle conjugates immobilized on the dissolvable adhesive film after evaporation of the carrier liquid are shown.

[0056] Fig.28 The endothermic reaction components are shown.

[0057] Fig.29 A retrofittable internally heated EBC collector and an existing mask are shown.

[0058] Fig.30 An endothermic EBC collector with hydrophilic channels over hydrophobic domains is shown.

[0059] Fig.31 An internally heated EBC collector is shown retrofitted into an existing mask.

[0060] Fig.32 The internally heated EBC collector and the collected EBC droplets after the reaction are shown.

[0061] Fig.33 An internally heated EBC collector with a collection zone and dry buffer / surfactant is shown.

[0062] Fig.34 An exhaled breath condensate collector and a testing area with a panel of syndromic test biosensors are shown.

[0063] Fig.35 The biosensor is shown immersed in the pooling area of ​​the collected EBC with dried buffer / surfactant in the pooling area.

[0064] Fig.36 Shown is the assembled EBC collector and pooling region with dried buffer / surfactant in the pooling region.

[0065] Fig.37 A Teflon condensation surface with a textured fluid conductor is shown.

[0066] Fig.38 is a micrograph showing a tunable water-adhesive structure fabricated by laser ablation.

[0067] Fig.39 demonstrated the formation of tunable hydroadhesive structures in polytetrafluoroethylene sheets with polytetrafluoroethylene "spikes" that mechanically disrupt the viral envelope, yielding free-floating corona virus N and S proteins.

[0068] Fig.40 A roll-to-roll process for large-scale production of condensing surfaces with textured fluid conductors is shown.

[0069] Fig.41 Shown is a condensing surface with a textured fluid conductor.

[0070] Fig.42 A capture molecule structure with a tag attached to a ligand is shown.

[0071] Fig.43 A capture molecule structure is shown, which has a polarizable end and a ligand end for capturing the target analyte.

[0072] Fig.44 The capture molecule structure is shown to form a polar conjugate with the captured target analyte.

[0073] Fig.45 Shown is the arrangement and testing of polar conjugates to detect target analytes in fluid samples.

[0074] Fig.46 A carrier fluid consisting of a fluid sample in which free-floating target analyte molecules are mixed with capture molecule structures to form free-floating polar conjugates in the fluid sample is shown.

[0075] Fig.47 Shown is the alignment of free-floating polar conjugates in an applied electric field.

[0076] Fig.48 A polarized gold nanoparticle / linker / capture molecule / target molecule conjugate is shown.

[0077] Fig.49 A polarizable gold nanoparticle / linker / capture molecule conjugate is shown, where the linker can effectively functionalize the gold nanoparticle to form a positive gold nanoparticle end.

[0078] Fig.50 A polarizable gold nanoparticle / linker / capture molecule conjugate is shown, where the linker can effectively functionalize the gold nanoparticle to form a negative gold nanoparticle end.

[0079] Fig.51A test system is schematically shown, in which an EBC containing a target molecule is mixed with a polarizable capture molecule conjugate to form a polarized conjugate, the polarized conjugate is arranged in the gap between electrodes, and the electrical properties of the arranged polarized conjugate are used to test whether the target molecule is present in the EBC.

[0080] Fig.52 Results from testing of a proof-of-concept laboratory engineered mask and EBC collection system are graphically presented.

[0081] Fig.53 A diagnostic strategy for EBC-based detection of SARS-CoV-2 infectivity is shown.

[0082] Fig.54 The two-dimensional aptamer capture molecular structure was demonstrated, and the experiment proved that the structure was very effective in the EBC-based diagnostic strategy for SARS-CoV-2 infectivity detection.

[0083] Fig.55 Results of biolayer interferometry (BLI) of aptamers attached to activated BLI sensors with different concentrations of SARS-CoV-2 S protein are shown.

[0084] Fig.56 The surface attachment strategy of SARS-Cov-2 complex on the gold working electrode of screen-printed electrode is demonstrated.

[0085] Fig.57 Shown are the cyclic voltammograms (CVs) of a gold electrode functionalized with SARS-CoV-2 synthase using ferrocenylmethanol as a redox mediator.

[0086] Fig.58 Shown are (a) differential pulse voltammogram of the electrochemical SARS-CoV-2 ensemble sensor, (b) current response to increasing binding domain concentration, (c) Langmuir adsorption isotherm, (d) response curves of different virus variants, and (e) selectivity of the ensemble sensor.

[0087] Fig.59 is a table showing exploratory EBC studies performed on patients who were positive or negative for SARS-CoV-2 by nasopharyngeal swab RT-PCR.

[0088] Fig.60 The invention discloses an external magnetic field responsive capture molecule conjugate, which has an external magnetic field responsive end and a capture molecule end, wherein the connecting molecule has electrochemical properties and can change at least one of polarity and conductivity.

[0089] Fig.61 Capture molecule conjugates with magnetically attractive ends and capture molecule conjugates with AuNP-coated ends are demonstrated.

[0090] Fig.62 The process of forming capture molecule conjugates on a dissolvable binder that are aligned and oriented in a magnetic field is shown.

[0091] Fig.63 The process of forming capture molecule conjugates on a dissolvable binder that are aligned and oriented in an electric field is shown.

[0092] Fig.64 A lateral flow assay (LFA) system is shown in which a biomarker (analyte) sample is added to a sample pad having a magnetic label attached to a capture molecule that is aligned and oriented on a dissolvable adhesive film formed on a conjugate release pad.

[0093] Fig.65 The LFA with analyte-labeled capture molecule complex formed on the conjugate release pad is shown.

[0094] Fig.66 Binding of the biomarker to the test line is shown, indicating the presence of the biomarker.

[0095] Fig.67 Another detection system that can be used with the EBC collection system of the present invention is shown, which system uses an electronic biosensor.

[0096] Fig.68 A biosensor constructed on a water-absorbent substrate composed of at least one of a selectively permeable membrane and super absorbent polymer fibers is shown.

[0097] Fig.69 A sample pad of a microfluidic detection system (eg, lateral flow detection) is shown, wherein the target molecule concentration structure comprises at least one of a selectively permeable membrane, a SAP / wick, and a dissolvable fluid dam.

[0098] Fig.70 A flow-through sensor electrode structure is shown, in which one or more capture molecules are functionalized on an electron transport component (such as carbon nanotubes), and the electrode is formed on a water-absorbent substrate composed of a selectively permeable membrane and a SAP / capillary fiber composite core.

[0099] Fig.71 is a flow chart showing steps for concentrating a target biomarker in an EBC fluid biological sample using an electronic biosensor, the electronic biosensor comprising a substrate having water-absorbent properties provided by at least one of a selectively permeable membrane (SPM), a super absorbent polymer, a water-conducting fiber, a microfluidic material, and a wick.

[0100] Fig.72An electronic biosensor with a detection interface built on a carbon nanotube immobilized (CNIM) superabsorbent polymer structure for concentrating target biomarkers in EBC fluid biospecimens is shown.

[0101] Fig.73 A cross-sectional view of a target molecule concentration flow electrode structure is shown, wherein the electrode of the structure is formed on a waterproof substrate and a drainage hole is provided in the detection interface area.

[0102] Fig.74 The flow path of a fluid sample is shown, with the target biomarker blocked at the sensor electrode and excess water flowing past the electrode.

[0103] Fig.75 The nanoCLAMP capture molecules immobilized on magnetically active nanoparticles are shown, with two capture molecules binding to different binding sites of a protein molecule.

[0104] Fig.76 Shown are two gold nanoparticles immobilized with nanoCLAMP capture molecules binding to the same target protein molecule.

[0105] Fig.77 A comparison of the relative sizes of nanoCLAMP and classical antibody capture molecules with the Debye length is shown.

[0106] Fig.78 Shown is a single layer of graphene functionalized with nanoCLAMP capture molecules via a pyrene linker, wherein example capture molecules (eg, two or more nanoCLAMP capture molecules) bind to different binding sites of example target molecules (eg, proteins).

[0107] Fig.79 is a cross-sectional view showing a biosensor having an electric field application structure for concentrating target molecules to a detection interface, and a local heating element provided on the biosensor for accelerating a binding reaction between capture molecules and target molecules.

[0108] Fig.80 is a magnified image showing that the electric field drives the charged target molecules to move toward the capture molecules at the detection interface.

[0109] Fig.81 is a stand-alone view showing SAP beads on the top drive electrode grid used to absorb excess water in the fluid sample.

[0110] Fig.82 A schematic diagram of the structure of a field effect transistor sensor is shown.

[0111] Fig.83A schematic diagram of the structure of a field effect transistor sensor is shown, in which the bottom driving electrode gate of the sensor is also the liquid gate of the field effect transistor sensor.

[0112] Fig.84 Shown is a fluid conductor applied on top of the bottom drive electrode grid / gate.

[0113] Fig.85 A top drive electrode is shown disposed on a fluid conductor.

[0114] Fig.86 Shown are SAP microbeads used to remove excess water from a fluid sample on a top drive electrode grid.

[0115] Fig.87 A wick is shown in fluid communication with the SAP beads for removing excess water from the fluid sample.

[0116] Fig.88 is a side view of a sensor for detecting target molecules in a fluid sample, the sensor having a detection area for receiving the fluid sample and a top drive electrode and a bottom drive electrode, on which the electric potential drives the target molecules toward the capture molecules, thereby concentrating the target molecules in a portion of the fluid sample received by the detection interface.

[0117] Fig.89 is a flow chart of the steps for concentrating target molecules in a fluid sample and testing the changes in the electrical properties of a functionalized transistor sensor.

[0118] Fig.90 is a side view of a back-gate field effect transistor sensor having a detection area for receiving a fluid sample and a top drive electrode working in conjunction with the back-gate electrode to apply an electric potential to drive target molecules toward capture molecules, thereby concentrating the target molecules in a portion of the fluid sample received at the detection interface.

[0119] Fig.91 is a flow chart of the steps for concentrating target molecules using an applied electric field and testing changes in the electrical properties of a functionalized field effect transistor sensor.

[0120] Fig.92 is a side view of a sensor for detecting target molecules in a fluid sample, the sensor having a detection region for receiving a downstream portion of the fluid sample, a top drive electrode and a bottom drive electrode being provided in the region for applying an electric potential to separate the target molecules from excess water in the fluid sample before the sample enters the detection interface.

[0121] Fig.93A room-scale biosensor is schematically shown for detecting target molecules in a fluid sample obtained from the ambient humidity of a room.

[0122] Fig.94 is a block diagram showing the components of one embodiment of a chamber scale biosensor.

[0123] Fig.95 Schematic diagram of a field effect transistor biosensor with a fluid sample flow conductor and capture molecules at a distance less than the Debye screening length from the conductive surface of the detection interface.

[0124] Fig.96 Schematic diagram of a field-effect transistor biosensor showing the flow direction of a fluid sample with target molecules and ions randomly dispersed in the flow conductor above the detection interface.

[0125] Fig.97 is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a driving circuit for driving the target molecules toward capture molecules immobilized on a detection interface.

[0126] Fig.98 is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a liquid gate detection circuit for detecting the presence and quantity of target molecules captured at a detection interface.

[0127] Fig.99 is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a back-gate detection circuit for detecting the presence and quantity of target molecules captured at a detection interface.

[0128] Fig.100 A graphene detection interface with nanoCLAMP capture molecules fixed by linker molecules is shown, where some capture molecules are fixed at a greater distance from the detection interface than others.

[0129] Fig.101 An electric field potential applied at a detection region is shown to drive target and non-target molecules toward capture molecules, wherein the target molecules are captured by capture molecules extending on linker molecules relatively far from the detection interface.

[0130] Fig.102 The electric field potential removed from the detection region is shown.

[0131] Fig.103 An electric field potential of opposite polarity is demonstrated, which drives the target and non-target molecules away from the detection interface, where the target molecules are still tethered by the capture molecules fixed by relatively long linkers on the detection interface.

[0132] Fig.104 More target and non-target molecules flowing into the detection area are shown.

[0133] Fig.105 An electric field potential applied to the detection area is shown, which causes the tethered target molecule to be captured in place by a capture molecule fixed by a relatively short linker molecule, and another target molecule to be captured by another capture molecule extending from the linker molecule, which is at a relatively long distance from the detection interface, thereby concentrating the target molecules in a portion of the fluid sample received by the detection interface.

[0134] Fig.106 Figure 1 is a block diagram of a g-FET sensor integrated circuit with a functionalized sensing interface.

[0135] Fig.107 Shown are groups of g-FET sensors fabricated in a functionalizable detection interface.

[0136] Fig.108 A g-FET semiconductor device encapsulated in an epoxy substrate is shown, with the sensing interface of the g-FET exposed on the surface of the epoxy substrate.

[0137] Fig.109 An epoxy top cap is shown with a test well in the epoxy base through which the test interface is accessible.

[0138] Fig.110 A fluid conductor is shown in fluid communication with the detection interface through the detection well.

[0139] Fig.111 A mesh drive electrode / liquid grid is shown disposed on a fluid conductor.

[0140] Fig.112 The SAP on top of the mesh drive electrode / liquid gate electrode is shown.

[0141] Fig.113 The wicks are shown disposed on the SAP and mesh drive electrode / liquid gate electrode.

[0142] Fig.114 A g-FET semiconductor device encapsulated in epoxy is shown, the device having source, gate, and drain leads.

[0143] Fig.115 Shown are Z-axis conductors used to connect the source, gate, and drain leads of a packaged g-FET semiconductor device to external circuitry.

[0144] Fig.116A packaged g-FET semiconductor device is shown coupled to source, gate, and drain circuit lines of an external circuit.

[0145] Fig.117 A packaged g-FET semiconductor device is shown having a drive electrode / liquid gate lead for connecting the mesh drive electrode / liquid gate electrode to a lead of an external circuit.

[0146] Fig.118 is a wireframe of a packaged g-FET semiconductor device showing the drive electrode / liquid gate leads on the top of the epoxy and the bottom of the epoxy base, and the leads for connecting the mesh drive electrode / liquid gate electrode to the external circuit.

[0147] Fig.119 A packaged g-FET semiconductor device is shown with a drive electrode / fluid gate lead located on top of a fluid conductor and connecting the top of the fluid conductor to leads of an external circuit.

[0148] Fig.120 The bottom of a packaged g-FET semiconductor device is shown, showing the source and drain leads, as well as the back gate and drive electrode / liquid gate leads. DETAILED DESCRIPTION

[0149] Various non-limiting exemplary embodiments are further described below. Exemplary embodiments of the present invention, such as the embodiments described below, may be implemented, practiced or used in any combination (e.g., any suitable, feasible and / or feasible combination), and are not limited to the combinations described herein and / or included in the appended claims.

[0150] 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. In any case, all embodiments described in this detailed description are exemplary embodiments intended to enable persons skilled in the art to make or use the invention, rather than to limit the scope of the invention as defined by the claims.

[0151] The following defined words and phrases are listed to define some components or elements of the disclosed embodiments. These definitions are provided for the convenience of the reader and are not intended to limit the scope of the invention, which is defined by the claims.

[0152] "Adhesive" means a pressure-sensitive, heat-activated, two-component or other bonding material used to adhere one part to another.

[0153] "Aligned polarizable conjugate" refers to an electrically aligned polarizable capture molecule conjugate.

[0154] "Antenna" refers to the transmitter and receiver of wireless communication signals.

[0155] The "externally applied magnetic field responsive end" refers to the end of a molecule that responds to an external electric field or magnetic field.

[0156] "Back gate" refers to the back gate of a semiconductor biosensor.

[0157] "Biosensor" refers to a detection device used to sense the presence of a predetermined biomarker.

[0158] "Biosensor on a water permeable substrate" refers to a biosensor disposed on a water permeable substrate for selectively absorbing water from an EBC sample.

[0159] "Bottom drive electrode" refers to the lower drive electrode.

[0160] "BTS" refers to Biomarker Detection System.

[0161] "Bus" refers to the signal communication structure.

[0162] A "capture molecule" is a ligand that is selected for its ability to bind to a target molecule.

[0163] "Capture molecule 1" refers to the first capture molecule in a multi-capture molecule system.

[0164] "Capture molecule 2" refers to the first capture molecule in a multi-capture molecule system.

[0165] "Capture pool" refers to a pool used to collect and preserve electronic biodiversity.

[0166] "Carrier fluid" refers to the liquid used to hold and carry particles or solutes.

[0167] "Cellulose sponge" refers to a compressed cellulose sponge.

[0168] "Condensate-forming surface" refers to an actively cooled surface that condenses breath vapor into breath condensate.

[0169] The "control line" is the area in the lateral flow assay where excess labeled capture molecules accumulate, indicating that the assay is working.

[0170] "DAS module" refers to the data acquisition and storage module.

[0171] The "Debye screening length" refers to the optimal distance from the transport surface for the capture molecule to bind to the target molecule.

[0172] "Detection interface" refers to the area of ​​the biosensor used to detect target molecules.

[0173] "Detection well" refers to a well used to receive a liquid biological sample and contact it with a detection area.

[0174] "Dissolvable adhesive" means a pressure-sensitive adhesive or other adhesive that is available as a tape or coating.

[0175] "Dissolvable fluid dam" refers to a water-soluble film used to block the flow of EBC.

[0176] "Drain" refers to the semiconductor drain feature.

[0177] "Drain hole" refers to a hole in the substrate that is used to pass a portion of the liquid sample pad.

[0178] "Dry buffer / surfactant" refers to chemical additives that can be added to the EBC sample flow path.

[0179] "EBC Collector" means an actively cooled structure used to convert exhaled breath vapor into exhaled breath condensate.

[0180] "EBC Tank" means a tank used to store EBC.

[0181] "Electric field" refers to an externally applied electric field used to align and / or attract the electric-field responsive ends of molecules.

[0182] "Electrode" refers to a conductive component used to apply electrical energy.

[0183] "Electronics" refers to the printed circuit board that has components such as processors and communications circuits used to determine and communicate test results.

[0184] "Intrinsic heat materials" are chemical components that, when mixed together, absorb heat from their surroundings.

[0185] "Epoxy base" refers to the base on which semiconductor devices are packaged.

[0186] "Epoxy resin top cover" refers to the top cover of the packaged semiconductor device.

[0187] "Exhaled breath condensate" refers to a liquid sample obtained by cooling exhaled breath to form liquid droplets.

[0188] "Exhaled water vapor" refers to the relatively warm, moist breath that leaves the lungs through the mouth and nose and cools on relatively warm, actively condensing surfaces.

[0189] "Face mask" means a face covering that forms a volume for containing exhaled breath in contact with an actively cooled condensing surface.

[0190] "Fluid transport" refers to materials or structures used to transport liquid samples.

[0191] "Fragment" refers to a fragment of a virus or other organism that can be detected as a biomarker.

[0192] "Free floating molecules" refers to polar conjugated molecules that float freely in a carrier fluid.

[0193] A "set of biosensors" refers to two or more biosensors that share common electronics and are used to detect one or more biomarkers.

[0194] "Gate" refers to the gate feature of a semiconductor biosensor.

[0195] "Nanogold particles" refer to gold particles of nanometer size.

[0196] "Graphene" refers to the charge transport material placed at the detection interface.

[0197] "Heating element" refers to a small heat source used to increase the temperature to improve molecular bonding.

[0198] "Hydrophilic structure" refers to a surface or structure that is easily wetted by water.

[0199] "Hydrophobic surface" refers to a surface that is not easily wetted by water.

[0200] "Tag" refers to a molecule that is tagged to a capture molecule.

[0201] "Linker" refers to the molecule that binds to the charge transport layer and the capture molecule.

[0202] "Liquid gate electrode" refers to an electrode used to apply voltage to a liquid sample to form a liquid gate of a semiconductor biosensor.

[0203] "Lysing material" refers to the chemical or mechanical mechanism that opens the viral particle to release the biomarker.

[0204] "Magnetic field" refers to a magnetic field applied as an attraction field and / or an alignment field.

[0205] "Magnetic nanoparticles" refer to small particles of magnetic material.

[0206] "Magnetic trap" refers to a permanent or electronically controlled magnetic source used to trap magnetic nanoparticles.

[0207] "Membrane" refers to the sample flow membrane of the lateral flow assay.

[0208] "Memory" refers to computer information storage.

[0209] "MME / SGW" refers to Mobility Management Entity / Serving Gateway.

[0210] "N protein" refers to the biomarker protein of a virus, such as the SARS-CoV-2 virus.

[0211] "NanoCLAMP" refers to a small size capture molecule that is particularly suitable for use in g-FET biosensors.

[0212] "NFC antenna" refers to the near field communication antenna and signal conditioning circuitry.

[0213] "Node" refers to a communications network element.

[0214] The "non-solvent carrier liquid" refers to the liquid containing the capture molecules and the field-directed nanoparticles, which is a non-solvent that can dissolve the binder.

[0215] "Non-target proteins" refer to unwanted proteins in the EBC sample.

[0216] "Patterned Teflon sheet" refers to Teflon sheet that has been treated with laser, calendering or sandblasting.

[0217] "Polarizable terminus" refers to a portion of a molecule or conjugate that has an increased polarity upon binding to a target molecule.

[0218] "Collection zone" refers to the structure used to collect EBC droplets.

[0219] "Processor" means a microprocessor or a microcontroller.

[0220] "RRH" means Remote Radio Head.

[0221] "S protein" refers to the biomarker protein of viruses such as SARS-CoV-2.

[0222] "Sample pad" refers to the sample receiving portion of a lateral flow analyzer.

[0223] "SAP" refers to super absorbent polymer.

[0224] "SAP beads" refers to super absorbent polymer beads.

[0225] A "selectively permeable membrane" is a membrane that allows one substance to pass through while preventing another substance from passing through.

[0226] "Separator" refers to the component used to separate wicks with lower and higher concentrations of target molecules.

[0227] "Source" refers to a semiconductor source feature.

[0228] "Baseplate" means the thin plate component used to support the test system.

[0229] "Layer" refers to a laminate component upon which a biosensor or test area is formed or supported.

[0230] "Surfactant" refers to a chemical substance used to disrupt the structure of lipids.

[0231] "Target molecule" refers to a biomarker or other chemical species selected for detection in a sample.

[0232] "Target molecule-labeled capture molecule complex" refers to a labeled capture molecule bound to a target molecule.

[0233] "Target protein" refers to the target molecule protein contained in the EBC sample.

[0234] "TBCA Module" refers to the Target Biomarker Collection and Analysis Module.

[0235] "Teflon spikes" means a textured Teflon or other plastic or metal surface with spiked protrusions used to affect the flow of liquid biological specimens and / or to disrupt the shell or cell wall of microorganisms.

[0236] "Detection line" refers to the area in a lateral flow assay where labeled capture molecule and target molecule conjugates accumulate to indicate detection of the target molecule.

[0237] "Detection zone" refers to the location where target molecules are detected in a sample.

[0238] "Test system bracket" means the bracket structure used to fix the EBC collector and other components inside the mask.

[0239] "Thermal mass" means a structure which may be suitably used to absorb heat from vapor, thereby condensing the vapor into a liquid.

[0240] "Timed-release lysis beads" refer to dry lysis materials that can be provided in a fluid path for lysing microorganisms to release biomarkers.

[0241] "Top drive electrode" refers to the upper drive electrode.

[0242] "Transceiver" refers to the electronic circuitry of a transmitter and / or receiver.

[0243] "Urea crystals" refers to an endothermic chemical composition that, when mixed with water, can remove heat from the surrounding environment.

[0244] "Virus" is an example of an organism that spreads infectious diseases.

[0245] "Virus-enriched EBC" refers to an EBC sample from which a portion of the water has been removed to concentrate the viruses remaining in the EBC collector.

[0246] Water" refers to a drop or a pool of water.

[0247] "Water pocket" means a container, pouch, or space between adhesive sheets that contains water or other liquid heat-generating material or thermal mass.

[0248] "Wick" refers to the absorbent material used to receive a liquid sample.

[0249] "Wireless link" refers to a wireless communication link.

[0250] "Z-axis conductive tape" refers to a tape with a Z-axis conductor, which is used to conduct electrical signals only in the Z-axis direction.

[0251] Disclosed herein are numerous configurations, implementations, manufacturing methods, algorithms, electronic circuits, microprocessors, memory and computer software product combinations, network strategies, database structures and uses, and other aspects, for diagnostic or detection platforms, devices, methods and systems having a variety of medical and non-medical uses.

[0252] Although the embodiments described herein are used to detect biomarkers of the SARS-CoV-2 virus, the systems, methods, and devices are not limited to any specific virus or disease, or to biological use cases. In most cases, when the term virus or COVID-19 is used, any other biomarker related to health or fitness can be used. Therefore, the description herein, as well as the drawings and claims, are not limited in any way to virus detection, and the invention described and claimed can be used for a variety of diseases, including lung cancer, diabetes, asthma, tuberculosis, environmental exposure, glucose, lactate, blood-borne diseases and other diseases or health indicators of the test subject. In addition, the electronic biosensors, test systems, uses and manufacturing methods described herein are not limited to the use of exhaled gas condensate. Wastewater, drinking water, environmental quality samples, environmental samples and any body fluids can be used as test samples. In particular, the use of aptamers and engineered capture molecules makes the sensors of the present invention have a wide range of uses because the properties of the selected aptamers can be adjusted through specific engineering design and selection to have customized binding affinity with the corresponding target analyte. Therefore, the description of the innovation is not intended to be limited to a specific use case, capture molecule, biomarker or analyte.

[0253] In immunochromatography, capture molecules (which may be, for example, aptamers, natural antibodies, or engineered antibodies) are placed on the surface of a porous membrane and the sample is passed along the membrane. As described herein, antibodies, aptamers, engineered antibodies, or capture molecules are used interchangeably. In some cases, a specific type of capture molecule may be described. In a lateral flow assay (LFA) type detection system, biomarkers in the sample bind to capture molecules, which are coupled to detection reagents. When the sample passes through the area where the capture molecules are located, the biomarker-detection reagent complex is captured and produces a color proportional to the concentration or amount of the biomarker in the sample. In electronic or electrochemical detection systems, the captured biomarkers are typically electrically connected to two or more electrodes to cause a detectable change in the obtained signal.

[0254] Figure 1 A system including an EBC collector 102 , a fluid transport 104 , an NFC antenna 106 , and a mask 108 is shown.

[0255] The figure shows a mask and a mask-based diagnostic platform, wherein an exhaled breath condensate (EBC) collector, a fluid transfer system, a biosensor test system, and a near-field communication (NFC) antenna and a signal conditioning circuit are arranged in a closed space inside the mask. The biosensor shown in the figure is an electrical or electrochemical biosensor, such as a g-FET or other electronic biosensor structure. In addition, the exemplary embodiments disclosed herein can also be used in other detection systems, such as LFA, cellulose biosensors, color-changing reagent solutions, etc. In some cases, the EBC collector and mask are used to collect fluid biological samples, which are then tested on a desktop or laboratory detection system (such as a traditional PCR test) outside the mask; in other cases, the detection system is directly integrated into and / or on the mask as a stand-alone detection device. In most cases, the test subject only needs to wear a mask and breathe normally while collecting biological samples, and technicians or other trained personnel are usually not required to complete the test or sample collection.

[0256] According to a non-limiting embodiment, a mask-based diagnostic platform is provided for detecting biomarkers received from the lungs and airways of a test subject. An EBC collector is mounted on the inside of a mask worn by a test subject. The EBC collector converts the breath vapor received from the lungs and airways of the test subject into a fluid biological sample. The EBC collector has a thermal mass or other cooling system, such as a frozen water / super absorbent polymer gel, a frozen metal plate, or an internal thermal reaction (as described herein), to cool a condensation surface that receives exhaled gas at a temperature higher than the condensation surface temperature. The exhaled vapor condenses into droplets on the condensation surface. The EBC collector may include a droplet collection structure, including a field for receiving breath vapor and forming droplets, and a channel for receiving droplets from the field and introducing the droplets into a fluid transport system (such as a microfluidic, capillary, or other fluid conducting structure) in the form of a collected fluid biological sample.

[0257] The biosensor is secured to the mask and is configured to receive a fluid biosample from the EBC collector and detect a target biomarker in the fluid biosample. The test signal generated by the biosensor depends at least on the presence or absence of the target biomarker in the fluid biosample. If desired, a signal conditioning circuit, such as an amplifier, may be provided to receive the test signal transmitted to a wireless receiver (e.g., a cell phone) via a near field communication (NFC) antenna. Additionally, the signal conditioning circuit may further include energy harvesting electronics for receiving radio frequency energy, such as that transmitted from a cell phone and received by the NFC antenna. For example, the energy harvesting antenna may include a capacitor or other circuit element such that the biosensor may operate using the harvested energy without the need for an onboard battery or other energy source.

[0258] Figure 2 A system consisting of an NFC antenna 106, a mask 108, a hydrophobic surface 202, a hydrophilic structure 204, a wick 206, and a biosensor 208 is shown.

[0259] The mask-type diagnostic platform shown in the figure is open inside and has components for collecting and testing EBC. The EBC collection and detection system can be installed on existing masks or integrated into the structure of the mask. Figure 2 A simple, low-cost disposable mask structure is shown. The mask substrate can be a multi-layer N95-type mask material, filter material, cloth or paper, or a breathable non-woven polymer material with micropores to facilitate air exchange. An EBC collector with hydrophobic areas and hydrophilic channels is fixed to the mask material. The fluid sample collected by the EBC collector is transferred to the biosensor via a microfluidic transport material, as described in this article, which can allow the fluid sample to be collected on the biosensor or flow through the biosensor using a wick material located downstream of the biosensor test area. The biosensor test area is small in area, typically a few millimeters square or less in surface area, but larger areas and multiple biosensors or test areas can also be provided. The electrodes on the biosensor device have wires that allow electrical communication with the signal conditioning circuit and NFC antenna.

[0260] This configuration creates a low-cost biospecimen detection and communication system that can be used with an app running on a mobile phone to determine a wirelessly transmittable test result from the test signal based on the detection or non-detection of the target biomarker. The test result can be transmitted from the mobile phone to a remote receiver, such as a cloud server or a local server. Other wired or wireless communication systems can also be used. When using relatively expensive test readers and communication electronics, it is best to place the electronics and batteries on the outside of the mask and place the EBC collector, microfluidic transport material, biosensor, and wick material on the inside of the mask. After use, the electronic components can be removed from the outside of the mask and disinfected for the next use. Disposable masks and components located inside the mask (most likely to be contaminated) can be sealed in a suitable bag and thrown away.

[0261] In order to capture aerosol droplets and particles, a dissolvable adhesive patch can also be optionally provided on the inner surface of the mask. The mask-based diagnostic platform can be equipped with such a particle capture structure for receiving and capturing exhaled gas aerosol (EBA) particles from the user's airway lining. The particle capture structure includes a dissolvable EBA sample collection film for capturing EBA particles. In this case, for example, when a forceful cough is applied while wearing a mask, aerosol droplets and particles in the lungs and airways are ejected and captured by the dissolvable film. The fluid biological sample detection assembly described herein can be used to provide a rapid screening test, and if a positive infection is detected, the mask can be placed in a sealed envelope and then taken to a laboratory for more rigorous testing of the fluid biological sample, aerosol droplets and particles contained in the mask and sealed in the envelope. In addition, a container for holding a fluid biological sample can be provided, or a pressure-sensitive adhesive strip can be included in the collection area to seal the collected EBC for transportation to the testing laboratory.

[0262] Figure 3 A system consisting of antenna 302, TBCA module 304, processor 306, memory 308, MME / SGW 310, and DAS module 312 is shown.

[0263] The figure shows a possible, non-limiting example system block diagram in which an exemplary embodiment can be implemented to deploy ultra-large-scale detection of target biomarkers (such as viruses spreading among people) using installed wireless communication networks and the Internet. Coordinated testing and data collection of people can be carried out across cities, states, countries, and even the world. For example, interconnected cellular, local area networks, wide area networks, and the Internet can be used to quickly obtain important data in airports, small communities, large cities, or larger populations, showing the presence of pathogens (such as epidemic or pandemic viruses). Test result data from many diagnostic tests can be automatically and conveniently collected and aggregated into large data sets for big data analysis by machine learning and artificial intelligence agents, so that pattern recognition and predictive models can be quickly generated to indicate the location of virus outbreaks, the speed of spread of outbreaks, etc. to governments, hospital managers, non-governmental organizations, and other authorities. In this exemplary embodiment, the communication infrastructure of a traditional cellular communication system is utilized to quickly obtain test results for a large number of test subjects, and the test results are transmitted in the form of at least two data streams: 1) a data stream containing patient identity information is transmitted to a trusted recipient (e.g., the patient's doctor, so that the individual patient can be properly monitored and treated); 2) a data stream that does not contain patient identity information is transmitted to a trusted recipient (for the purpose of protecting patient privacy) for aggregation and big data group analysis.

[0264] The biomarker detection system (BTS) communicates wirelessly with a wireless network. The BTS is a wireless biomarker detection system that can access a wireless network, such as the communication-based mask diagnostic platform described herein. The BTS includes one or more processors, one or more memories, and one or more transceivers, which are interconnected via one or more buses 127. Each of the one or more transceivers includes a receiver Rx and a transmitter Tx. In some use cases, the mask-based diagnostic unit may not require an onboard receiver. The one or more buses can be an address bus, a data bus, or a control bus, and can include any interconnection mechanism, such as a series of lines on a motherboard or integrated circuit, or other communication devices. The one or more transceivers are connected to one or more antennas. The one or more memories include computer program code. The base station 110 includes a target biomarker collection and analysis (TBCA) module, which includes the inventive biosensor test system described herein. An embodiment of the TBCA also includes wireless communication functionality, including one or both parts thereof, which can be implemented in a variety of ways. The TBCA module can be implemented in hardware as a TBCA module, such as as part of a mask-based diagnostic system including one or more processors. The processor of the TBCA module can also be implemented as an integrated circuit or through other hardware such as a programmable gate array. In another example, the TBCA module can be implemented as a TBCA module, which is implemented as a computer program code and executed by one or more processors, where the test results are obtained in the form of analog or digital electrical signals generated by a biosensor for testing a sample (such as an EBC sample). For example, one or more memories and computer program codes can be configured to work with one or more processors to cause the biomarker detection system to perform one or more operations described herein. The base station communicates with the node via a wireless link.

[0265] In one exemplary cellular communication mode, a near field communication system is used to obtain test results from a biosensor of a mask-based diagnostic system. The near field communication system can conveniently and cost-effectively obtain test results using a conventional handheld cellular phone, computer, communication pad, or dedicated wireless communication reader / transmitter / receiver.

[0266] The test results are transmitted to a node, which is typically a base station of a wireless communication network (e.g., 5G, 4G, LTE, Long Term Evolution, or any other cellular, Internet, and / or wireless network communication system) that provides wireless device (e.g., cell phone base station) access to the wireless network. For example, at a sports stadium or airport, the venue's wireless communication infrastructure can be utilized to communicate directly with the mask-based diagnostic system, and / or the user's cell phone can be used as a repeater.

[0267] The node includes one or more processors, one or more memories, one or more network interfaces (N / WI / F), and one or more transceivers interconnected by one or more buses. Each transceiver includes a receiver Rx and a transmitter Tx. One or more transceivers are connected to one or more antennas. One or more memories include computer program codes. The node includes a data acquisition and storage (DAS) module, which is composed of one or more components and can be implemented in a variety of ways. The DAS module can be implemented in hardware as a DAS module, for example, as part of one or more processors. The DAS module can also be implemented as an integrated circuit or through other hardware such as a programmable gate array. In another example, the DAS module can be implemented as a DAS module, and the DAS module is implemented as a computer program code and executed by one or more processors. For example, one or more memories and computer program codes are configured to enable the node to perform one or more operations described herein together with one or more processors. One or more network interfaces 161 communicate through a network such as a link. Two or more nodes communicate through a link or the like. The link can be wired or wireless or both, and can implement an X2 interface, etc.

[0268] The one or more buses may be an address bus, a data bus, or a control bus, and may include any interconnection mechanism, such as a series of wires on a motherboard or integrated circuit, an optical fiber or other optical communication device, a wireless channel, etc. For example, one or more transceivers may be implemented as a remote radio head (RRH), with the other elements of the node physically located at a different location from the RRH, and one or more buses may be implemented in part as a fiber optic cable to connect the other elements of the node to the RRH.

[0269] The wireless network 100 may include a network control element (NCE), which may include MME (mobile management entity) / SGW (serving gateway) functions and provide connections to other networks such as telephone networks and / or data communication networks (such as the Internet). The node is connected to the NCE via a link. The link can be implemented by means such as an S1 interface. The NCE includes one or more processors, one or more memories, and one or more network interfaces (N / WI / F), which are interconnected through one or more buses. The one or more memories include computer program code. The one or more memories and the computer program code are configured to enable the NCE to perform one or more operations together with the one or more processors.

[0270] The wireless network 100 can implement network virtualization, which is the process of combining hardware and software network resources and network functions into a single, software-based management entity (virtual network). Network virtualization involves platform virtualization, usually combined with resource virtualization. Network virtualization can be divided into external virtualization and internal virtualization. External virtualization refers to merging many networks or parts of networks into a virtual unit, and internal virtualization refers to providing network-like functions to software containers on a single system. It should be noted that the virtualized entities generated by network virtualization still need to be implemented using hardware such as processors and memory to some extent, and these virtualized entities will also produce technical effects.

[0271] The computer readable memory may be of any type suitable for the local technical 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 memory, and removable memory. The computer readable memory may be a device that performs a storage function. The processor may be of any type suitable for the local technical environment and may include 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, as non-limiting examples. The processor may be used to perform functions such as controlling C19TS, nodes, and other functions described herein.

[0272] In general, various embodiments of the biomarker detection system 110 may include, but are not limited to, wireless communication components for Bluetooth, mobile phones such as smartphones, tablet computers, 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 devices with wireless communication capabilities, music storage and playback devices with wireless communication capabilities, Internet devices that allow wireless Internet access and browsing, tablet computers with wireless communication capabilities, and portable devices or terminals that combine these functions.

[0273] Figure 4 is a logic flow diagram of an applied probability analysis for determining target biomarker detection and illustrates the operation of an exemplary method, the result of execution of computer program instructions embodied on a computer readable memory, the 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 Figure 3 The circuit elements of the functions shown in the blocks are each included, where each block is an interconnected device for performing the function in the block. Figure 3 At least some of the blocks in are assumed to be executed by the BTS, for example at least partially under the control of the TBCA module.

[0274] The world is currently dealing with a pandemic caused by the novel coronavirus SARS-CoV-2. This viral pathogen has killed millions of people, shut down many national economies, and caused trillions of dollars in global economic losses. Billions of people around the world have stopped their usual employment, recreational, and social activities to combat the spread of the virus. Testing for biomarkers that indicate exposure, infection, and recovery from viral pathogens could allow economic activity to restart more safely and efficiently while minimizing viral transmission and keeping a close eye on the development of viral variants at a community, national, or global level.

[0275] For example, protein and RNA tests for active virus can show which people are currently contagious. Antibody tests can be used to find members of a population who have recovered from the virus and may be immune to reinfection. During the current and future pandemics, this knowledge could enable precise social isolation and more effective contact tracing, as well as the reemployment of increasing numbers of protected workers and consumers. Until a vaccine or other solutions (such as highly successful drug therapies) are developed and widely available, those who remain at risk of infection and transmission could be quarantined. In the future, rapid deployment of detection systems, such as the mask-based diagnostic platform described in this article, especially when used Figure 3-6 When combined with the wireless communications infrastructure described elsewhere in this article, it is possible to quickly determine where and how quickly a pathogen is spreading among a population and identify infected individuals before they travel and bring the virus to uninfected areas of the world.

[0276] The COVID-19 pandemic is an example of a viral pathogen that has required each country to deploy significant resources to combat the disease and shut down its economy to limit the spread of the virus while balancing the economic and social impacts on the population. Testing the population for the virus, tracking the spread of the pandemic, and estimating the size of the infected population are essential tools for responding to any current and future local disease or pandemic outbreaks.

[0277] Comprehensive testing can simultaneously detect multiple pathogens with overlapping symptoms. According to an exemplary embodiment, multiple biosensors can be used to simultaneously detect multiple biomarkers, thereby improving the accuracy of detection.

[0278] Stochastic analysis of data is used to handle variation involving randomness and uncertainty, aspects that are particularly difficult to manage during rapidly spreading viral outbreaks. More specifically, stochastic analysis of partially observables can account for an incomplete understanding of the spread of infectious pathogens in a population. For example, partial observations can provide insight but only with a certain degree of certainty. In the case of biological invasions or viral transmission, infection testing often results in false negative results, and therefore only detects infected individuals with a certain probability.

[0279] The application of probability analysis can be used to improve the prediction model of personal infection status, and in general, it helps to improve the threshold of the test results of objective quantitative or qualitative detection systems. According to an exemplary embodiment, when applying probability analysis to determine pathogen exposure, biomarker 1 is first detected (first step), and then biomarker 2 is detected (second step). In addition, biomarker N is also detected, where N can be any number of multiple biomarkers detected using the detection system of the present invention. If the target biomarker is not detected (third step), a negative test report is generated (fourth step). If any target biomarker is detected (step three), a probability analysis can be performed based on the presence (yes / no) or quantitative analysis (such as concentration) of one or more biomarkers detected (step five). Probability analysis can be performed using an updated probability model in which the probability multiplier of the detected biomarker is determined for a group. For example, if the virus outbreak occurred earlier and occurred in a different region or country from the current application detection location, the probability multiplier of the detected biomarker can be determined based on the confirmed cases that occurred during the earlier outbreak. Based on the probability multiplier obtained from the confirmed cases, the threshold of the probability analysis result can be determined, and it helps to improve the accuracy of the detection system. For example, in an electronic biosensor, the threshold voltage for considering a test result as positive can be adjusted based on the probabilistic analysis of previously tested and confirmed positive cases. Over time, the accuracy and confidence of both positive and negative determinations will improve based on the history of confirmed cases and the threshold voltages obtained. As the database of tested cases grows, the overall testing solution with interconnected, shared, and analyzed test results will be used to automatically improve the accuracy and confidence of future tests.

[0280] For examples of probabilistic analysis used to simulate the SARS-CoV-2 pandemic, see Modeling the dynamics of the COVID-19 population in Australia: A probabilistic analysis, Eshragh et al., October 2, 2020, https: / / doi.org / 10.1371 / journal.pone.0240153; Estimated Incidence of Coronavirus Disease 2019 (COVID-19) Illness and Hospitalization-United States, February-September 2020, Reese, et al., CID 2021:72, June 2021; and Güemes, A., Ray, S., Aboumerhi, K. et al. A syndromic surveillance tool to detect anomalous clusters of COVID-19 symptoms in the United States. Sci Rep 11, 4660 (2021). https: / / doi.org / 10.1038 / s41598-021-84145-5 , the disclosures of which are incorporated into this article in their entirety.

[0281] If the probability analysis result does not exceed the threshold (step six) (for example, the concentration of a specific target biomarker is low, or there is only one weak biomarker indicating possible infection), a "possible" test report is generated (step seven). If the probability analysis result does exceed the threshold (step six) (for example, the concentration of a certain target biomarker is high, or the presence of two or more biomarkers indicates possible infection), a "positive test report" is generated (step eight). The test report is then transmitted (step nine) (for example, the test result is added to the accumulated database using the methods described herein or other suitable transmission mechanisms, including oral, digital, written or other communication transmission methods).

[0282] Figure 4The logic flow is implemented by a non-limiting embodiment of a device, which includes at least one processor; and at least one memory including computer program code, wherein the at least one memory and the computer program code are configured to, together with the at least one processor, cause the device to perform at least the following operations: detect one or more biometric parameters using a droplet collection structure, the structure being used to convert respiratory vapor into fluid droplets to form a fluid sample; the detection system having a biomarker detection area for receiving the fluid sample and detecting the biometric parameters, wherein the biometric parameters are biomarkers of at least one physiological change depending on the patient's response to a related condition such as a viral infection; receive one or more biometric parameters, and apply a probability analysis, and determine whether at least one physiological change threshold is exceeded based on the probability analysis of the one or more biometric parameters; and initiate an operation based on the determination that the at least one physiological change threshold is exceeded.

[0283] According to one embodiment, the biosensor detection device has one or more biometric detectors, each detector is used to detect a biomarker as one or more biometric parameters. The biometric parameter depends on at least one physiological change of the patient or test subject, such as the production of immune response chemicals, the presence of active or inactivated viruses or viral components in the body, antibodies, antigens, viral RNA or DNA or other changes caused by other biomarkers (including immune response or viral load counts). The microprocessor receives one or more biometric parameters and determines whether at least one physiological change threshold is exceeded based on the one or more biometric parameters. The activation circuit activates an action based on the determined physiological change. The operation includes sending an alarm, modifying a treatment method, and sending data based on at least one physiological change, one or more biometric parameters, and a treatment method.

[0284] The mask-based diagnostic platform of the present invention and / or the components of the platform described herein can also be used to monitor the disease progression of patients, for example, hospitalized patients who are experiencing Covid-19 disease progression. At least one physiological change can also be a response to an applied treatment that causes a change in the patient's condition so that the body's response to the applied treatment can be monitored. The operation may include sending an alarm, modifying a treatment method, and sending data that depends on at least one of the at least one physiological change, one or more biometric parameters, and a treatment method. The microprocessor can use a probabilistic analysis to analyze the one or more biometric parameters, including determining from a data set of one or more biometric parameters whether the data set can be used to determine that at least one physiological change threshold has been exceeded. The probabilistic analysis may further include applying a statistical weight to each of the one or more biometric parameters, wherein the statistical weight depends on a predetermined value for ranking the importance of other parameters in the one or more biometric parameters when detecting at least one physiological change in each of the one or more biometric parameters.

[0285] Figure 5 is a logic flow diagram of data collection and transmission for trusted receiver and population studies and illustrates the operation of an exemplary method, the result of execution of computer program instructions embodied on a computer readable memory, the functions performed by logic implemented in hardware, and / or interconnected means for performing functions according to the exemplary embodiments

[0286] The data collection and transmission process for trusted receiver and population research purposes can be performed on a test system, node, smartphone, or combination of components with or remote from the test subject, such as at a storage location for the acquired data. The acquired data may include patient or subject identification information such as name, GPS location, a list of known prior or future contacts (pre-infection and / or post-infection), prior medical history, demographic data, etc. The data collection and transmission for trusted receiver and population research purposes can be performed on a secure server anywhere on the network. For example, the DAS module 150 may include multiple Figure 3 The circuit elements of the functions shown in the blocks are interconnected devices for performing the functions in the blocks. Figure 3 At least some of the blocks in are assumed to be executed by a base station such as node 170, for example, at least in part under the control of DAS module 150. Blockchain or other data security, storage, and distribution technologies can be used to enhance privacy protection and controlled access to de-identified patient data while making such data available to researchers and authorities anywhere in the world.

[0287] The architecture, manufacturing methods and applications of digital detection systems can be used to capture biometric data from the exhaled breath of a test subject or patient. Biometric data can be collected and transmitted continuously or at selected times, and data access can be provided directly to caregivers for early diagnosis and continuous monitoring, and to researchers for valuable insights and assistance through artificial intelligence analysis. This data detection is directly from the exhaled breath and can be provided through wireless connectivity for collection, access and analysis of blockchain and artificial intelligence databases.

[0288] like Figure 5As shown, a test report is received (step one) (e.g., transmitted from a patient or test subject's smartphone). If the report is to be sent to a trusted recipient (step two), such as the patient's healthcare provider or insurance company, an encrypted report (step three) can be generated and transmitted to the trusted recipient, including the patient's identification information. Two-step verification or better authentication protocols (such as those used to ensure online banking) can be used to ensure that the recipient of the patient data is indeed a trusted recipient. If the report is not for a trusted recipient (step two) but is used for contact tracing (step four), only the data required for contact tracing is transmitted. In addition, for contact tracing, a contact tracing app can be used (step five). For example, a contact tracing app can be a system for identifying and notifying people who have been in contact with a test subject or patient within a given time before or after the detection of one or more target biomarkers. If the report is not for a trusted recipient (step two) or contact tracing (step four), but is used for population research (step six), only the minimum patient identification information that complies with privacy regulations and / or protocols will be transmitted and / or stored with the received test report (step seven). If the report is not intended for trusted receivers, contact tracing, or population research (steps two, four, and six), determine whether there are any legitimate uses for the test report data and take appropriate action, or automatically purge the data from storage (step eight).

[0289] Figure 6 The following is a block diagram of the basic components for testing EBC and transmitting the test results to a smartphone and / or cloud server. The EBC collector provides a fluid sample, which is received by a biometric detector or biosensor. An electrical signal conditioner, such as a signal amplifier, filter, etc., may be provided to condition the raw test signal from the biosensor before the microprocessor or analysis circuit determines the test result signal. After processing the conditioned signal, the test result signal is transmitted via a communication circuit, which may be the NFC system described above, or Bluetooth or other wired or wireless communication methods and structures. A smartphone or access point repeater may be used to receive the wireless test result signal and transmit it to the cloud.

[0290] According to one embodiment, the electronic circuit includes an amplifier circuit for receiving a test signal from a biosensor and amplifying the test signal into an amplified electrical signal. A comparator circuit compares the amplified electrical signal to a predetermined value based on at least one of a computer model-derived and empirically-derived electrical signal calibration of the biosensor. The calibration may be determined using at least one of a known presence and a known concentration of a target analyte in a calibration sample. The comparator circuit generates a test result signal based on the comparison of the amplified electrical signal to the predetermined value.

[0291] The electronic circuit may also include an analyte concentration circuit for determining a concentration value of the target analyte based on the amplified electrical signal. In this case, the amplified electrical signal changes value based on the number of target analyte molecules in the liquid biological sample, and the test result signal depends on the determined concentration value.

[0292] According to one embodiment, the electronic circuit further comprises a wireless communication circuit for wirelessly transmitting the test result signal to at least one of a smartphone, a tablet, a computer, a repeater, an access point, and a computer network.

[0293] Figure 7 A system consisting of a mask 702, an EBC collector 704, a condensate forming surface 706, a fluid transport 708, and a biosensor 710 is shown. A KN95 mask is shown with a prototype of a retrofittable test system, including an EBC collector with an aluminum foil condensate forming surface, an embossed fluid conductor channel, a microfluidic transport system, and electronic biosensor electrodes.

[0294] Figure 8 A system consisting of a mask 802 and an EBC collector 804 is shown, showing a prototype test system modified onto a KN95 mask.

[0295] Fig. 9 A system consisting of an electronic component 902 and a mask 904 is shown, showing a KN95 mask with removable test and communication electronics mounted on the outside of the mask

[0296] Fig.10A system consisting of a test system support 1002, a biosensor 1004, an EBC collector 1006, a hydrophilic structure 1008, and a hydrophobic surface 1010 is shown. An exhaled breath condensate collector and a test area with a group syndrome test biosensor are shown. According to an exemplary prototype embodiment, the mask-based EBC collector is made of a 30 cm wide roll of 120 micron thick natural virgin polytetrafluoroethylene (PTFE) sheet (eplastics.com., USA), a 10 cm wide 3M 465 double-sided tape (uline.com, USA), and super absorbent polymer (SAP) powder MediSAP 715 (M2 Polymer Technology, Illinois, USA). For example, a stamping fixture can be made from a 0.315 cm polytetrafluoroethylene sheet (eplastics.com, USA) and cut on a 100 watt CO2 laser cutter (Orion Motor Tech, China). A digital combination heat press (Geo Knight, Massachusetts, USA) can be used to stamp a 127 micron thick polytetrafluoroethylene sheet using a fixture to form a pocket on the polytetrafluoroethylene sheet for receiving the thermal mass mixture of water and SAP. A second layer of 127 micron polytetrafluoroethylene sheet can be bonded to the heat-stamped 127 micron polytetrafluoroethylene sheet using 3M 465 adhesive to sandwich the water / SAP thermal mass between the polytetrafluoroethylene sheet layers. The completed polytetrafluoroethylene / thermal mass / 3M 465 / polytetrafluoroethylene laminate sandwich can be cut into the final shape of the EBC collector using a laser or die cutter, and the configuration and size of the collector can be inserted into an existing mask or built into a newly built mask. The EBC collector constructed as described above can be retrofitted into disposable masks of various styles and structures, including N95 and KN95 masks produced by 3M and other manufacturers.

[0297] Fig.11 A system consisting of a collection area 1102 and an adhesive 1104 is shown, where the collection area forms a volume for accommodating a biosensor immersed in the collected EBC.

[0298] Fig.12A system consisting of an EBC collector 1202, a test system support 1204, and a collection area 1206 is shown. The exhaled gas condensate (EBC) collector converts exhaled gas received from the lungs and respiratory tract of the test subject into a fluid biological sample. The EBC collector includes a condensate forming surface and a heat block thermally connected to the condensate forming surface. The fluid transfer system transfers the EBC to at least one of the test device and the EBC container. The detection unit can be one or more electronic biosensors, one or more lateral flow detectors or other microfluidic type detection systems, or a combination thereof. The configuration and size of the test system support for the EBC collector are suitable for installation in a mask. The mask forms an exhaled gas volume to contain the exhaled gas near the EBC collector so that the exhaled gas is effectively condensed into a fluid biological sample.

[0299] As described in more detail herein, a detection device can include a g-FET biosensor whose detection interface includes a graphene layer functionalized with capture molecules, wherein the capture molecules are smaller than the Debye screening length. As an example of a syndromic detection device, a biosensor can be designed to bind to FluA, FluB, SARS N-protein (the more conserved, slower mutating protein in the SARS virus), and S-protein (the faster mutating, causative of the SARS-CoV-2 variant).

[0300] Fig.13 The relative sizes of an engineered capture molecule called nanoCLAMP, the S protein, the virus particle, and water molecules are shown.

[0301] Fig.14 An EBC concentrator is shown with a semi-permeable membrane for separating excess water from the EBC, which is then tested with concentrated virus particles. The system includes virus 1402, a selectively permeable membrane 1404, and water 1406.

[0302] Fig.15 The addition of super absorbent polymer in an EBC concentrator is shown. The system includes SAP 1502, virus 1504, water 1506 and a selectively permeable membrane 1508.

[0303] nanoCLAMPs (nano-CLostridial Antibody Mimetic Proteins) are capture molecules developed by Nectagen (Kansas City, KS). Like aptamers and engineered antibodies, these capture molecules can be designed to bind to specific antigens. nanoCLAMPS are based on an immunoglobulin-like, thermostatic carbohydrate binding module from Clostridium hyaluronidase. nanoCLAMPS can be designed and enhanced to bind to different locations on the same antigen, to different antigens, and to large and small target molecules within the Debye screening length range.

[0304] Especially for larger macromolecules, experiments with capture molecules of different sizes showed that the effectiveness of FET-based sensors (where the antigen is captured via specific binding to a capture molecule-functionalized surface) is strongly influenced by the Debye screen length.

[0305] For example, a nanoCLAMP can be designed to bind to biomarkers of the virus such as FluA HA, FluB NA, SARS N-protein (a more conserved and slower mutating protein in the SARS virus), and SARS S-protein (a faster mutating protein and responsible for the mutating SARS-CoV-2). Thus, a multi-biomarker syndrome detection system can be obtained through a combined biosensor array that receives collected EBC biospecimens. Such grouped biosensors can be made into independent biosensor arrays with printed electrodes and / or semiconductor (e.g., g-FET) designs and functionalized to meet specific use cases by selecting the respective capture molecules for the corresponding target molecules. In the case of a mask-based diagnostic platform, products made from this platform can share most, if not all, components (mask, EBC collector, concentrator, fluid transport, etc.). Since the blood-gas exchange that occurs in the lungs generates many of the biomarkers contained in the EBC, specific use cases for mask-based diagnostic systems (e.g., tuberculosis, lung cancer screening, environmental exposures, health and wellness, fitness, fatigue, vitamin deficiency, etc.) can be identified by adding biosensors functionalized with appropriate commercially available or custom-designed capture molecules.

[0306] According to an exemplary non-limiting embodiment, the biomarker concentrator may include a super absorbent polymer for preferentially absorbing water in the EBC into the polymer chains of the super absorbent polymer. The target biomarker is not absorbed by the polymer chains, but flows through the SAP and microfluidic structure of the diagnostic platform together with the EBC. When the EBC flows through the SAP, the water in the EBC is removed, while the content of the target molecule remains unchanged, thereby increasing the concentration of the target molecule in the test sample.

[0307] The test system includes an exhaled gas condensate (EBC) collector for converting exhaled gas received from the lungs and respiratory tract of the test subject into a fluid biological sample. Prior to testing, the elements on the path can be customized to condition the biological sample. For example, since most of the EBC is water, the concentration of target molecules in the test sample can be significantly increased by removing excess water. The biomarker concentrator can partially concentrate the target biomarker in the fluid biological sample to form a concentrated fluid biological sample. Other test interfering components in the EBC can also be removed from the test sample. For example, dissolved salts in the EBC can be removed by the SAP and semi / selective permeable membrane action described in this article, or other component removal techniques such as precipitation reactions and filtration can be used. The biomarker detection device receives the concentrated fluid biological sample and performs target biomarker detection on the concentrated fluid biological sample.

[0308] Fig.16 The addition of a surfactant or lysing agent in an EBC concentrator is shown. The system includes surfactant 1602, SAP 1604, virus 1606, selectively permeable membrane 1608 and water 1610.

[0309] Fig.17 An EBC test sample with concentrated viral particles and cleaved proteins is shown. The system includes fragments 1702, exhaled breath 1704, S protein 1706, N protein 1708, and virus 1710.

[0310] There is a selectively permeable membrane downstream of the superabsorbent polymer in the EBC sample flow path. The membrane has a certain pore size, and its configuration and size allow the portion of water in the EBC sample that is not absorbed by the superabsorbent polymer mixture and the target analyte to flow through the selectively permeable membrane. The pore size prevents the superabsorbent polymer from flowing through the selectively permeable membrane, thereby achieving a certain concentration level of the target analyte in the water remaining in the EBC sample on the permeate side of the membrane. The membrane allows viral particles and viral fragments to pass through, and the water that is not absorbed in the SAP to continue to flow to the biosensor.

[0311] Fig.18 is a cross-sectional view showing an EBC concentrator with a semipermeable membrane and wick adjacent to a thermal mass. The system includes a selectively permeable membrane 1802, a wick 1804, a thermal mass 1806, an exhaled breath condensate 1808, a collection area 1810, and a virus 1812.

[0312] Viruses are concentrated in the EBC sample and accumulate in the pooling area formed by the barrier. Excess water in the EBC passes through the semipermeable membrane and is expelled, so as the exhaled water vapor is cooled into the EBC formed on the surface of the EBC collector, excess water is continuously expelled.

[0313] Fig.19is a cross-sectional view showing an EBC concentrator with lysing material in the fluid conductor and a semipermeable membrane and core in the EBC collection area. The system includes thermal mass 1902, SAP 1904, exhaled gas condensate 1906, N protein 1908, S protein 1910, virus 1912, virus concentrated EBC 1914, core 1916 and selectively permeable membrane 1918.

[0314] The biomarker concentrator may include a selectively permeable barrier that allows excess water in the fluid biological sample to pass through. The selectively permeable barrier may prevent target biomarkers in the fluid biological sample from passing through the selectively permeable barrier, thereby allowing a higher concentration of the target biomarker in the measured fluid. The excess water absorbent core may be used to absorb excess water that passes through the selectively permeable material.

[0315] Alternatively, a selective permeable membrane may be provided upstream of the EBC sample flow path from the super absorbent polymer. The pore size and dimensions of the membrane allow a portion of the water in the EBC sample to flow through the selective permeable membrane toward the super absorbent polymer. The pores prevent the target analyte from flowing through the membrane, thereby allowing the target analyte to reach a certain concentration level in the remaining water in the EBC sample on the membrane inlet side.

[0316] According to an exemplary embodiment, an apparatus for detecting a target biomarker in exhaled breath condensate (EBC) includes an EBC collector for converting exhaled breath received from the lungs and respiratory tract of a test subject into an EBC biological sample. The EBC biological sample contains a target analyte. The biomarker concentrator includes a super absorbent polymer layer located in the EBC biological sample flow path. During the collection process, the EBC biological sample contacts the super absorbent polymer layer, which absorbs part of the water in the EBC biological sample. The SAP layer does not absorb the target analyte, thereby increasing the concentration of the target analyte in the remaining water in the EBC biological sample. The biomarker detection device receives the concentrated EBC biological sample and performs target biomarker detection on the concentrated EBC biological sample.

[0317] Fig. 20 Capture molecule / linker / magnetic nanoparticle conjugates (magnetically active molecules) with free floating and captured target molecules are shown. The system includes magnetic nanoparticles 2002, linkers 2004, capture molecules 2006 and target molecules 2008.

[0318] An external irradiation-field reaction capture molecule conjugate for capturing a target biomarker is provided. The external irradiation-field reaction capture molecule conjugate has one or more external irradiation-field reaction ends and one or more capture molecule ends. The capture molecule end binds to the target biomarker and captures the target biomarker. For syndrome detection, there can be multiple capture molecule types for capturing different target molecules (e.g., S protein, N protein of SARS-CoV-2; FluA protein, FluB protein, SARS protein, inflammatory immune response markers, cytokines, etc.).

[0319] The applied field responsive capture molecule conjugate may include a capture molecule immobilized on a magnetic nanoparticle to form a magnetic nanoparticle conjugate having a magnetically attractive nanoparticle end (mag.NP). Fig.21 As shown, the soluble adhesive can fix the magnetic nanoparticle conjugate in the path of the fluid biological sample (i.e., collected EBC). When the water in the fluid biological sample dissolves the soluble adhesive, the magnetic nanoparticle conjugate will float freely in the fluid biological sample. The wick and fluid transport system (such as microfluidic material, as needed) for absorbing excess EBC components can allow the EBC to flow continuously through the magnetic capture device, so that the magnetic nanoparticle conjugate and the captured target biomarker are concentrated in the magnetic capture device, while the excess EBC components not captured by the magnetic capture device flow through the biomarker detection device to the wick.

[0320] Fig.21 An EBC collector having oriented and aligned magnetic nanoparticle conjugates on a dissolvable adhesive for capturing and concentrating target molecules in exhaled breath is shown. The system includes exhaled breath 2102, target molecules 2104, wick 2106, thermal mass 2108, testing area 2110, dissolvable adhesive 2112, magnetic capturer 2114, magnetic nanoparticles 2116, and capture molecules 2118.

[0321] Fig. 22 An EBC collector is shown with oriented and aligned magnetic nanoparticle conjugates, with EBC formed on its surface called by thermal mass, binding target molecules in the EBC to capture molecules. The system includes exhaled breath condensate 2202, target molecules 2204, dissolvable binder 2206, thermal mass 2208, wick 2210, magnetic capturer 2212, collection area 2214 and testing area 2216.

[0322] Fig.23The system includes a dissolvable binder 2302, exhaled breath condensate 2304, capture molecules 2306, target molecules 2308, a testing area 2310, a magnetic capturer 2312, a thermal mass 2314, a core 2316, a collection area 2318, and magnetic nanoparticles 2320.

[0323] The soluble binder fixes the external field effect capture molecule conjugate in the path of the fluid biological sample. Water in the fluid biological sample dissolves the soluble binder, allowing the external field response capture molecule conjugate to float freely in the fluid biological sample. The magnetic capturer attracts and holds the particle end with magnetic attraction, fixing the magnetic nanoparticle conjugate and the captured target biomarker on the magnetic capturer.

[0324] Fig.24 The magnetic active molecule and the target molecule conjugate captured by the magnetic capturer in the test area are shown. The system includes a thermal mass 2402, a magnetic capturer 2404, magnetic nanoparticles 2406, target molecules 2408, exhaled breath condensate 2410, a collection area 2412 and a wick 2414.

[0325] The magnetic capturer is located near the biomarker detection zone and holds the magnetic nanoparticle conjugate. As complexes containing the captured target molecule accumulate, the detectable signal generated by the captured target molecule increases. Knowing or approximating the flow rate or volume of the EBC reaching the detection zone can be used to quantitatively measure the concentration of the target biomarker. Viral load, environmental exposure, or disease progression can be determined as a function of flow rate and detectable signal.

[0326] Fig.25 The formation process of a well-organized conjugate array of magnetic nanoparticle capture molecules immobilized on a dissolvable adhesive film is shown. Fig.25 A thin film of a dissolvable adhesive is shown supporting a carrier liquid, which is a non-solvent for the dissolvable adhesive, prior to application of a magnetic alignment field.

[0327] Fig.26 Shown is the alignment of magnetic nanoparticle conjugates on a dissolvable adhesive film in a carrier fluid upon application of a magnetic alignment field.

[0328] Fig. 27 Well-organized magnetic nanoparticle conjugates fixed in place on the dissolvable adhesive film are shown after evaporation of the carrier liquid and removal of the magnetic alignment field.

[0329] Fig.28Components of the endothermic reaction are shown, including water and urea crystals. Other chemicals that react endothermally with water include ammonium nitrate and calcium ammonium nitrate. The system includes a water bag 2802 and urea crystals 2804, which are examples of endothermic materials 2806.

[0330] Fig.29 A retrofittable internally heated EBC collector and a pre-existing mask are shown. The system includes a mask 2902, an EBC collector 2904, and a test system stand 2906.

[0331] Fig.30 An endothermic EBC collector is shown with hydrophilic channels on hydrophobic domains, into which dyes are added to simulate surfactants, precipitation reactions and / or buffer additives. The system includes a hydrophobic surface 3002 and a hydrophilic structure 3004.

[0332] Fig.31 An internally heated EBC collector is shown retrofitted into an existing mask. The system includes a mask 3102 and a test system stand 3104.

[0333] Fig.32 The reacted internally heated EBC collector and the dye-colored collected EBC droplets formed from the exhaled breath condensate 3202 are shown.

[0334] According to an exemplary embodiment, the EBC collector includes a condensate forming surface and a thermal mass thermally coupled to the condensate forming surface. The thermal mass may include at least a first chemical reagent and a second chemical reagent that may be combined to form an internal thermochemical reaction for absorbing thermal energy from the condensate forming surface to convert the exhaled gas vapor into the EBC. Fig.33 An internally heated EBC collector with a collection zone and dry buffer / surfactant is shown. The system includes a collection zone 3302 and an EBC collector 3304.

[0335] Fig.34 An exhaled breath condensate collector and a test area with a panel of syndromic test biosensors are shown. The system includes a segment 3402 and a test system support 3404.

[0336] Fig.35 The biosensor is shown immersed in a collection area of ​​collected EBC with dried buffer / surfactant in the collection area. The system includes a collection area 3502.

[0337] Fig.36 Shown is an assembled EBC collector and collection zone with dry buffer / surfactant in the collection zone. The system includes a collection zone 3602 and a dry buffer / surfactant 3604.

[0338] The fluid transfer system may include at least one of a fluid conductor, a collection area, and a microfluidic transfer path for controlling the flow of a fluid biological sample received from the EBC collector. The target biomarker release material is placed in or on at least one of the fluid conductor, the collection area, and the microfluidic transfer path. The target biomarker release material may include at least one of a surfactant and a chemical lysing agent. The EBC collector may also include a target biomarker release structure for mechanically lysing at least one of a cell wall, an encapsulation structure, and a viral envelope containing the target biomarker.

[0339] Fig.37 A Teflon condensation surface with a textured fluid conductor is shown. The system includes a patterned Teflon sheet 3702.

[0340] Fig.38 A micrograph showing tunable water adhesion structures fabricated by laser ablation is taken from a research paper, Superhydrophobic polytetrafluoroethylene surfaces with accurately and continuously tunable water adhesion fabricated by picosecond laser directablation, Qin et al., Materials and Design 173 (2019) 107782. The system includes polytetrafluoroethylene pins 3802 and a patterned polytetrafluoroethylene sheet 3804.

[0341] Fig.39 Tunable hydroadhesive structures formed in a polytetrafluoroethylene sheet with polytetrafluoroethylene "spikes" that mechanically disrupt the viral envelope to produce free-floating viral N and S proteins are shown. The system includes virus 3902, Teflon spikes 3904, and fragment 3906.

[0342] The target biomarker release structure may include a lysis structure protruding from the condensate forming surface and / or the surface in the EBC flow path. The lysis structure can mechanically disrupt the cell wall, encapsulation structure and / or viral envelope containing the target biomarker. For example, in order to release the N-protein from the SARS-CoV-2 virus, the lipid viral envelope can be destroyed by the action of micro- or nano-structures. When the EBC is formed and flows along the textured surface, the spike-like elements that form the texture interact with the virus and destroy the viral envelope. The EBC carrier fluid includes at least one of water, a buffer, a surfactant, a lysis material, a preservative, and a body fluid (including at least one of saliva, urine, exhaled breath condensate, blood and sweat). In any case, the biological sample is a fluid, and the lysis effect can be enhanced by providing other micro / nano abrasive materials, and a certain amount of pressure and friction may need to be applied (for example, the user's thumb presses the structure on the outside of the mask to rub the virus particles against the texture structure and / or micro / nano abrasive material).

[0343] Fig.40 A roll-to-roll process for mass producing a condensing surface with a textured fluid conductor is shown. The system includes a patterned polytetrafluoroethylene sheet 4002.

[0344] Fig.41 A condensation surface sheet with a textured fluid conductor is shown. The system includes a patterned polytetrafluoroethylene sheet 4102.

[0345] The sheets can be mass produced and become part of a material stack for forming an EBC collector in a mass production process, and then the EBC collectors are individually separated from a roll of finished EBC collectors. The condensate forming surface can have relatively low energy surface properties to limit the adhesion of target biomarkers to the condensate forming surface. The fluid conductor on the condensate forming surface can be a textured structure formed on the condensate forming surface. The textured structure has relatively high energy surface properties that can guide the EBC to flow in a desired direction.

[0346] A method of forming a condensate collector having a fluid conductor channel on a substrate for directing fluid flow in a desired direction includes providing a substrate having a surface with relatively low energy surface characteristics. A textured structure forms a fluid conductor channel on the surface with relatively high energy surface characteristics for directing fluid flow in a desired direction.

[0347] The relatively low energy surface features restrict the attachment of target analytes to the surface, making the surface relatively hydrophobic, while the high energy surface features of the texture structure make the channels relatively hydrophilic. The texture structure can be formed by at least one of laser ablation, sandblasting, etching and calendaring.

[0348] Fig.42A capture molecule structure is shown with a tag attached to a ligand. The system includes a tag 4202, a linker 4204, and a capture molecule 4206.

[0349] Fig.43 A capture molecule structure is shown having a polarizable end and a ligand end for capturing a target analyte. The system includes a polarizable end 4302, a capture molecule 4304, a target molecule 4306, and a polarizable end 4308.

[0350] Fig.44 The capture molecule structure is shown, and the captured target analyte forms a polar conjugate. The system includes a target molecule 4402.

[0351] Fig.45 Shown is the arrangement and testing of polar conjugates to detect target analytes in fluid samples.

[0352] Fig.46 A carrier fluid is shown, including a fluid sample with free-floating target analyte molecules, mixed with capture molecule structures to form free-floating polar conjugates in the fluid sample. The system includes a carrier fluid 4602, target molecules 4604, capture molecules 4606, and free-floating molecules 4608.

[0353] Fig.47 Shown are free floating polar conjugates before alignment in an applied electric field and free floating polar conjugates aligned in an applied electric field.

[0354] A method for detecting a target analyte includes providing a capture molecule structure having a ligand end and a polarizable end. When the capture molecule structure is placed in a carrier fluid, the capture molecule structure is a free floating element. The target analyte is placed in the carrier fluid as another free floating element. The ligand end of the capture molecule structure binds to the target analyte to form a free floating polar conjugate having a positive end and a negative end. The polar conjugate is aligned in the carrier fluid in an electric field, and the target analyte is detected by measuring the electrical properties of the aligned polar conjugate.

[0355] The carrier fluid may be a bodily fluid including at least one of saliva, urine, exhaled breath condensate, blood and sweat. The measuring step may include applying the pulsed electric field for a period of time and measuring the electrical characteristic for a period of time after the pulsed electric field has ended, wherein the period of time is short enough to detect the target analyte.

[0356] Prior to mixing the capture molecule structure with the carrier fluid, the carrier fluid may be a biological fluid sample and the capture molecule structure is provided in a dry powder form. The target analyte may be a component of the biological fluid sample. Alternatively, the carrier fluid may be an environmental fluid sample and the target analyte is a component of the environmental fluid sample.

[0357] Fig.48 Polarized gold nanoparticle / linker / capture molecule / target molecule conjugate is shown. The system includes gold nanoparticles 4802, capture molecules 4804, and target molecules 4806. There is affinity between the capture molecules and the target molecules, wherein the polarized gold nanoparticles have a stronger electrochemical affinity, which can promote the binding of the capture molecules to the corresponding target molecules.

[0358] Fig.49 A polarized gold nanoparticle / linker / capture molecule conjugate is shown, wherein the linker can effectively functionalize the gold nanoparticle to form a positive gold nanoparticle end. The system includes gold nanoparticle 4902, capture molecule 4904, target molecule 4906, target molecule 4908, capture molecule 4910 and gold nanoparticle 4912.

[0359] Fig.50 A polarizable gold nanoparticle / linker / capture molecule conjugate is shown, where the linker can effectively functionalize the gold nanoparticle to form a negative gold nanoparticle end.

[0360] Fig.51 A test system is schematically shown, wherein an EBC containing a target molecule is mixed with a polarizable capture molecule conjugate to form a polarizable conjugate, the polarizable conjugate is arranged in the gap between electrodes, and the electrical properties of the arranged polarizable conjugate are used to test the presence of the target molecule in the EBC. The system includes an electrode 5102, an EBC collector 5104, an exhaled breath condensate 5106, a wick 5108, and a fluid transfer device 5110.

[0361] The captured target molecule complexes can be arranged and oriented in the EBC carrier fluid in the test area. The organized array of captured target molecule complexes can improve the detection signal and test results. For example, the external magnetic field response capture molecule conjugate may include a connecting molecule arranged between the external magnetic field response end and the capture molecule end. The connecting molecule gives the external magnetic field response capture molecule conjugate electrochemical properties, and when the capture molecule end is bound to the target biomarker, at least one of the polarity and conductivity of the external magnetic field response capture molecule conjugate will change.

[0362] According to an exemplary embodiment, a capture molecule conjugate for detecting a target analyte includes an applied field responsive capture molecule conjugate, wherein the capture molecule is part of a larger molecular conjugate that responds to an applied field. For example, a polar molecular conjugate having a positively charged end and a negatively charged end will be oriented along the electric field lines between two parallel conductors, which form an electronic circuit, wherein one conductor is an anode and the other conductor is a cathode. An applied magnetic field responsive capture molecule conjugate has at least one applied magnetic field responsive end and at least one capture molecule end. The capture molecule end binds to and captures the target analyte. The binding of each capture molecule end to the target analyte will increase the charge difference between the applied electric field responsive end and the capture molecule end.

[0363] The connecting molecule disposed between the external electric field reaction end and the capture molecule end gives the external electric field reaction capture molecule conjugate electrochemical properties. When the capture molecule end binds to the target analyte, at least one of the polarity and conductivity of the external electric field reaction capture molecule conjugate changes. For example, if the gold nanoparticle end of the external electric field reaction capture molecule conjugate is positively charged (e.g., AuNP conjugated with nanoCLAMP capture molecule), when the negatively charged target molecule (SARS-CoV-2 S protein) is captured, a polar analyte-labeled capture molecule complex will be generated, and the degree of polarity of the positive end (AuNP) and the negative end (captured S protein) of the complex will change.

[0364] The external field responsive end may include at least one of nanoparticles, gold nanoparticles, magnetically active nanoparticles, carbon tubes, and graphene. At least one capture molecule end includes at least one of engineered antibodies, antibodies, aptamers, nanobodies, nanoCLAMPs, small molecules, and the like.

[0365] Proof-of-concept experiment results:

[0366] Fig.52 Shown: (a) The interior of a proof-of-concept laboratory engineered mask showing the exhaled breath condensate (EBC) collector (cold trap) used to convert exhaled breath into a fluid sample. The EBC collector is made of a polytetrafluoroethylene condensate-forming surface, (b) An image of the EBC formed on the polytetrafluoroethylene collector after breathing into the mask for 5 minutes, (c) A graph showing the amount of EBC collected in 5 minutes using an EBC mask (n=14), (d) A graph showing the amount of EBC collected in 5 minutes using a commercial RTube condenser (n=14).

[0367] The present exhaled breath condensate (EBC)-based diagnostic platform can be used to detect SARS-CoV-2 infectivity as well as other pathogens and environmental exposures. In proof-of-concept testing, laboratory-designed masks can collect EBCs by first cooling the mask in a refrigerator for 30 minutes and then wearing the cooled mask and breathing into it for 5 minutes. EBCs formed in the polytetrafluoroethylene lining on the inside of the mask are collected and directly deposited onto an electrochemical sensor modified with a SARS-CoV-2-specific aptamer that uses the receptor binding domain (RBD) region of the S1 spike protein as a surface receptor. Using ferrocene methanol as a redox mediator before and after viral interaction, positive and negative EBC samples can be distinguished.

[0368] The mask-based EBC collection system is based on a commercial mask, on which is mounted an engineered EBC collection system based on a polytetrafluoroethylene film cooled trap (a). To improve the efficiency of EBC collection, the mask is placed in a freezer at -20°C for 30 minutes before being placed on the subject's mouth. This polytetrafluoroethylene (PTFE) trap cools down and allows the sample to liquefy on its surface, and the droplets formed can be collected with a pipette and used directly for analysis. The presence of a collection pool allows further collection of EBC (b) without the need for specialized technical knowledge. During the collection of EBC, the inside of the mask is not exposed to the air, so the risk of contamination of the EBC sample is minimal. Using this collection system, 400±150μL of EBC can be collected in 5 minutes (c).

[0369] The collection efficiency is comparable to EBC collected using a commercial RTube condenser (Respiratory Research, Inc., USA) (d). As can be seen in (c), the collection efficiency varies from person to person. However, in most cases, the 300 μL required for further analysis was obtained in this way. While it is indeed more efficient to collect an equal volume of saliva over a 5-minute time span, saliva is a complex sample matrix containing proteases and other variable components that can affect most tests. This includes potential degradation of the SARS-CoV-2S1 protein targeted by the aptamer used in this test. Therefore, purer EBC samples are considered more suitable for rapid testing and more reliable.

[0370] Fig.53 An EBC-based diagnostic strategy for detecting the infectivity of SARS-CoV-2 is shown.

[0371] To demonstrate the potential of the diagnostic platform of the present invention for diagnosing SARS-CoV-2 infection, experimental tests were conducted. In the experimental tests, the mask-type diagnostic platform of the present invention was used to detect exhaled gas condensate (EBC) collected by a mask-type sampling device and detected using a proprietary electrochemical biosensor with a modular structure that can quickly and specifically detect and quantify COVID-19. The mask forms an exhaled gas volume that contains the exhaled gas near the EBC collector, allowing the condensate-forming surface to condense the exhaled gas into a 200-500 μL liquid sample in about 2 minutes under thermal cooling. Samples from 7 SARS-CoV-2 positive and 7 SARS-CoV-2 negative patients were tested for SARS-CoV-2 genes (E, ORF1ab) by EBC RT-PCR. The presence of SARS-CoV-2 was detected in 5 of the 7 SARS-CoV-2 positive patients. Additionally, EBC samples were screened on an electrochemical aptamer biosensor that can detect SARS-CoV-2 viral particles as low as 10 pfu mL-1 in cultured SARS-CoV-2 suspensions. Results on the patient's infection status can be obtained within 10 minutes using a ferrocene methanol redox mediator for "off" detection.

[0372] While other human coronaviruses (such as HCoV-229E and HCoV-OC43) cause only mild common colds, the SARS-CoV-2 pandemic has caused more than 4.47 million deaths worldwide (as of August 26, 2021) and 214 million cases. Diagnosis of SARS-CoV-2 infection is done worldwide by nasopharyngeal swab samples and, more recently, saliva samples using real-time reverse transcription polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 RNA. The process of obtaining nasal swab samples is not only uncomfortable, but also requires specialized personnel and has the potential to contaminate the tester. The advantages of saliva testing are simple operation, low invasiveness, limited risks, and the practice of RT-PCR testing of saliva samples is becoming more and more widespread. The viscosity of saliva and the presence of salivary proteases (saliva has proteolytic activity) make direct application of saliva samples challenging. It is well known that the main transmission routes of COVID-19 are airborne and contact transmission, mainly due to the strong resistance of the virus in aerosol droplets expelled by infected people. Given the increasing need for patients to collect their own samples, exhaled breath condensate (EBC) may be another important sample type for diagnosing SARS-CoV-2.

[0373] The experimental results presented in this article demonstrate the utility of aptamer-based electrochemical biosensors. Aptamers have the advantages of small size, strong chemical stability, and low production cost compared to traditional antibodies. In the experimental tests, electrochemical sensing was targeted to the spike protein (S) embedded in the lipid membrane of the outer wall of the SARS-CoV-2 virus. The spike protein protrudes from the viral membrane, and the virus enters the host cell by recognizing the host receptor ACE2 through the receptor binding domain (RBD) of the spike protein. Because the spike protein is repeated approximately 50-200 times on the surface of the virus, the RBD region of the S protein is an excellent diagnostic target. The aptamer selected for this experimental test is a 32-nucleotide aptamer produced by Base Pair Biotechnologies (Pearland, Texas, USA).

[0374] Fig.54 A two-dimensional aptamer capture molecule structure (sequence adapted) was presented, which was experimentally proven to be highly effective in the EBC-based diagnostic strategy for SARS-CoV-2 infectivity detection.

[0375] Fig.55 Shown are the results of biolayer interferometry (BLI) measurements with biotinylated aptamers attached to streptavidin-activated BLI sensors with varying concentrations of SARS-CoV-2 S protein (3.13 nM, 6.25 nM, 12.5 nM, and 25 nM): running buffer: 1× PBS, 1 mM MgCl2).

[0376] Fig.56 A surface attachment strategy for attaching SARS-Cov-2 aptamer to the gold working electrode of screen-printed electrodes using maleimide-thiol-aptamer linkage is demonstrated.

[0377] Fig.57 Shown are cyclic voltammograms (CV) of a gold electrode before and after functionalization with SARS-CoV-2 synthase (10 μg mL-1 for 2 h) using ferrocene as a redox mediator (1 mM, 0.1 M PBS, pH 7.4, scan rate = 100 mV s-1).

[0378] SARS-CoV-2 aptamers against the S1 protein were screened from a combinatorial library of nucleic acid sequences by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process. The investigated aptamer is a 20-base aptamer "CFA0688T" (Base Pair Bio) in which one loop was modified with thiol-TTT-TTT at the 5' end, which allows the aptamer some flexibility when anchored to the gold interface. The binding affinity to the recombinant SARS-CoV-2 S1 spike protein was determined by biolayer interferometry (BLI) with KD = 3.52 ± 0.17 nM (R2 = 0.9985). This affinity value is comparable to other reported SARS-CoV-2 chimeras, such as the 51-base pair chimera with three hairpin structures that are selective for RBD, or the 58-base pair chimera with KD values ​​ranging from 5.8 ± 0.8 nM to 0.49 ± 0.05 nM.

[0379] The SARS-CoV-2 aptamer was attached to the screen-printed electrode (SPE) using a maleimide-functionalized poly(ethylene glycol) (PEG) spacer, a commonly used hydrophilic polymer to avoid biofouling and also used by other companies to integrate cysteine-modified aptamers. The spacer helps overcome potential steric barriers in virus detection. The success of the attachment strategy was verified using XPS.

[0380] A key concept in electrochemical systems is that the heterogeneous electron transfer kinetics of modified electrodes is strongly dependent on the surface coverage and thickness of the modified layer. Using ferrocenylmethanol as a redox mediator, cyclic voltammograms were recorded for a gold working electrode before and after modification with a chelator. This small mediator can penetrate into the monolayer modified gold electrode to a small extent or diffuse through the pinholes, and the electron transfer occurs at free sites on the electrode. As expected, the electron transfer is reduced with the presence of chelators on the electrode surface.

[0381] Analysis of 50 nM receptor domain binding from solution to the aptamer-modified electrode showed a significant decrease in current. At [RBD] = 10 nM, the current decreased linearly and reached complete saturation at 50 nM. The corresponding curve can be fitted with the Langmuir isotherm using the following equation:

[0382] Θ=j(c0) / j(c∞)=KA×c0 / (1+KA×c0)

[0383] Θ is the surface coverage, j(c0) is the current density at a given RBD concentration, and j(c∞) is the current density at infinite analyte concentration; assuming a 1:1 complex between the antigen (RBD) and the fused receptor in solution, the affinity constant KA can be estimated. Based on the expected S-shaped curve, the dissociation constant (i.e., half-saturation constant) KD = 1.6 ± 0.9 nM can be determined, indicating that the aptamer has a high affinity for RBD, which is consistent with the reported nanomolar dissociation constant of aptamer-protein interactions and the independent affinity measurement results of Base Pair using biolayer interferometry.

[0384] Fig.58 Shown are (a) differential pulse voltammogram of the electrochemical SARS-CoV-2 ensemble sensor, (b) current response to increasing binding domain concentration, (c) Langmuir adsorption isotherm, (d) response curves of different virus variants, and (e) selectivity of the ensemble sensor.

[0385] The potential of these interfaces to sense cultured SARS-CoV-2 viral particles was investigated. Immersion of the sensors in PBS (0.1 M, pH 7 = 4) containing different concentrations of SARS-CoV-2 isolates showed a limit of detection (LOD) (defined as the lowest level that can reliably distinguish the analyte from the background) of approximately 10 pfu mL-1 (associated with a current difference of 2 μA) and a saturation of 1.5 × 105 pfu mL-1. The limit of detection determined from five blank noise signals (95% confidence) was approximately 3 pfu mL-1. The analytical performance was compared to that of SPE with thiol-terminated aptamers directly attached to a gold surface. Analysis of the binding of the RBD to the aptamer determined a higher dissociation constant, KD = 6.2 ± 1.2 nM. More importantly, detection of cultured SARS-CoV-2 viral particles showed an LOD of approximately 200 pfu mL-1.

[0386] This sensing sensitivity of the maleimide thiol aptamer sensor is comparable to other electrochemical and electrical sensors reported in the literature to date. In addition, the potential for detecting variants 20I / 501Y.V1 (referred to as the "UK variant") and 20H / 501Y.V2 (referred to as the "South African variant") was also investigated using SARS-CoV-2 variant isolates. (c) shows that the aptamer-based sensor has the same sensing effect on the 20I / 501Y.V1e variant and the 20H / 501Y.V2 variant. This is consistent with the results obtained using commercial recombinant SARS-CoV-2 S protein (taking into account different variants). In fact, the affinity of the SARS-CoV-2 S protein UK variant to the aptamer was measured by BLI as KD = 6.0 ± 3 nM, kon = (1.52 ± 0.003) × 105 M-1 s-1, while the affinity of the South African variant was KD = 1.6.0 ± 0.1 nM, kon = (1.62 ± 0.05) × 105 M-1 s-1. This is in the same order as the affinity constant of the wild-type (Wuhan) variant of 3.52 ± 0.17 nM.

[0387] The reproducibility of the SARS-CoV-2 combined electrode is expressed as a relative standard deviation, which is 2.3% (n=5) when the virus concentration is 103pfu mL-1. In addition, the long-term stability of the sensor stored in PBS was evaluated, and the results showed that after the electrode was stored at 4°C for 1 month, the anode peak current loss was 2.5% when testing a 103pfu mL-1 virus solution. To illustrate the selectivity of the sensor, the sensitivity sensor was also incubated with other virus samples obtained from nasal swabs.

[0388] Tests performed on other coronaviruses, HCoVOC43 and HCoV NL63, showed reduced interactions with the aptamer-interface compared to the current difference values ​​recorded on positive nasopharyngeal swab samples. This was also the case for influenza A (H1N1) and influenza B samples.

[0389] Fig.59 is a table showing exploratory EBC studies performed on patients whose nasopharyngeal swab RT-PCR was positive (black numbers) or negative (red numbers) for SARS-CoV-2.

[0390] Given that the disease is spread through exhaled droplets and EBC is an established mode of sampling exhaled aerosols, detecting SARS-CoV-2 in EBC is a feasible method for safe and effective diagnosis of the disease. The experimental results validate the possibility of sensing SARS-CoV-2 using EBC collected using a commercial Rtube condenser and EBC mask system. EBC samples were collected using the cold trap described in this article. Nasopharyngeal swab samples were also collected.

[0391] In a proof-of-principle study, EBC samples were collected from 14 volunteers and analyzed by PCR, e.g. Fig.60 As shown. Of the 14 nasopharyngeal swab samples, 7 samples were identified as SARS-CoV-2 positive and 7 samples were identified as SARS-CoV-2 negative (cycle threshold (Ct)>40) by RT-PCR targeting N structural protein and RNA-dependent RNA polymerase (RdRp) nonstructural protein. Based on the experimental results of different dilutions of SARS-CoV-2 isolates, it is estimated that a Ct of 34 is approximately equivalent to about 104 viral RNA copies per ml, and this dilution is not infectious to Vero cells.

[0392] The lower Ct value of 22 correlates with approximately 107 viral RNA copies per ml.

[0393] Results of EBC RT-PCR on samples collected by Rtube condensers and EBC masks from patients who were negative for SARS-CoV-2 were completely consistent with the results of nasopharyngeal exchange samples (7 / 7, 100%). These samples were tested on an electrochemical sensor, and a current difference greater than 2 μA was considered to be associated with the presence of viral particles, further confirming that these samples were negative for SARS-CoV-2.

[0394] In EBC samples collected from patients identified as SARS-CoV-2 positive by nasopharyngeal RT-PCR, 3 out of 7 samples were identified as SARS-CoV-2 positive using a commercial RTube condenser; using a mask, 5 out of 7 samples were identified as SARS-CoV-2 positive with a Ct value of 40 as the cutoff. Compared with nasopharyngeal samples, the Ct values ​​of RdRp genes detected in EBS samples were always significantly lower than those of N genes. The different Ct values ​​between nasal swab samples and EBC are related to the different viral loads in these two fluids. In fact, it is speculated that the SARS-CoV-2 viral load in aerosol samples is several orders of magnitude lower than that in nasopharyngeal swab samples, which ranged from 6.41×102-1.34×1011 copies / mL. This suggests that N genes may be a more reliable target gene in EBC samples regardless of the collection strategy used. The results of testing the mask-collected EBC samples on the aptamer electrochemical sensor showed consistency with the EBC RT-PCR results using the mask. This shows that this sensor is well suited for detecting EBC viral samples and further confirms the presence of active viral particles in the exhaled breath of SARS-CoV-2 positive patients.

[0395] Fig.60An external field responsive capture molecule conjugate is shown, the conjugate has an external field responsive end and a capture molecule end, and the connecting molecule has an electrochemical property that can change at least one of polarity and conductivity. The system includes an external field responsive end 6002, a connector 6004, a capture molecule 6006, and a target molecule 6008.

[0396] Fig.61 A capture molecule conjugate with a magnetically attractive end and a capture molecule conjugate with an AuNP coated end are shown. The system includes magnetic nanoparticles 6102 and gold nanoparticles 6104.

[0397] Fig.62 The process of forming a capture molecule conjugate aligned and oriented in a magnetic field on a dissolvable binder is shown. The system includes a carrier fluid 6202, a dissolvable binder 6204, a magnetic field 6206, and an aligned magnetic conjugate 6208.

[0398] Fig.63 The process of forming a capture molecule conjugate aligned and oriented in an electric field on a dissolvable adhesive is shown. The system includes an electric field 6302, aligned polarized conjugates 6304, and a dissolvable adhesive 6306.

[0399] According to an exemplary embodiment, an array of external field reaction capture molecular conjugates is prepared by providing a soluble adhesive film on a substrate, a liner or an independent substrate. A carrier liquid as a non-solvent for the soluble adhesive film is randomly dispersed with external field reaction capture molecular conjugates. An alignment field is applied to the carrier liquid so as to assemble the external field reaction capture molecular conjugates onto the soluble adhesive film. The carrier liquid is removed (evaporated, dipped or spin-coated), and the assembled external field reaction capture molecular conjugates are fixed on the soluble adhesive film.

[0400] Figure 64-66 An example of a non-restrictive lateral flow assay is shown, where an EBC or other liquid sample containing a target biomarker flows through a multi-zone transfer medium by capillary action. The zones are typically made of polymer strips that allow molecules attached to the strips to interact with the target biomarker. Overlapping membranes are typically mounted on a base card to improve stability and handling. The EBC biological sample containing the target biomarker and other components is ultimately adsorbed to an absorbent sample pad, which facilitates the passage of the liquid sample through the multi-zone transfer medium.

[0401] like Fig.64As shown, the system includes a sample pad 6402, a dissolvable adhesive 6404, an aligned magnetic conjugate 6406, a conjugate release pad 6408, a membrane 6410, a test line 6412, a control line 6414, and a core 6416. The EBC fluid sample includes an analyte or target biomarker in a fluid carrier (such as water), which is first received on the sample pad. The sample pad can be placed or impregnated with a buffer salt and a surfactant to improve the fluidity of the fluid sample and the interaction of the target biomarker with various parts of the detection system. This ensures that the analyte binds to the capture reagent when the fluid sample flows through the membrane. The sample pad can be placed in the collection area of ​​the EBC collector, such as Fig.12 As shown, collected EBC samples can also be obtained from the pooling area using a pipette and then transferred to the LFA.

[0402] The processed sample migrates from the sample pad through the conjugate release pad. The conjugate release pad contains labeled antibodies or other capture molecules that have specificity for binding to the target biomarker or analyte, typically conjugated to colored or fluorescent indicator particles. The indicator particles are typically colloidal gold or latex microspheres. According to one embodiment, the indicator particles are included with a magnetic nanoparticle conjugate, and the magnetic nanoparticle conjugate array has a magnetically attractive particle end that is fixed to a soluble adhesive or otherwise removably fixed to the conjugate release pad. The magnetically attractive particles can be used as indicator particles, and the colloidal gold or latex microspheres can also be used as additional components of the magnetic nanoparticle conjugate.

[0403] like Fig.65 As shown, at the conjugate release pad, the labeled capture molecule, the indicator particles and the target analyte combine to form an analyte-labeled capture molecule complex. The system includes a target molecule-labeled capture molecule conjugate 6502.

[0404] The EBC fluid sample dissolves the soluble adhesive or otherwise displaces the biomarkers or analytes captured on the magnetic nanoparticle conjugate and the conjugate release pad. Therefore, if a biomarker or analyte is present, the fluid sample will contain an analyte conjugated to the labeled capture molecule conjugate. In other words, the capture molecule binds to the target biomarker to form an analyte-labeled capture molecule complex. These complexes, together with the indicator particles that are not bound to the target biomarker or analyte and the capture molecules individually labeled on the magnetically responsive particles, migrate along the membrane to the detection area with the EBC fluid sample.

[0405] like Fig.66 As shown, the detection area is usually a nitrocellulose porous membrane, on which a specific biological component (usually an antibody or an antigen) is placed or impregnated to form a detection line area and a control line area. The system includes a detection line 6602.

[0406] The biological component reacts with the analyte-labeled capture molecule complex. For example, the analyte-labeled capture molecule complex will bind to the selected primary antibody placed on the test line through competitive binding. This will cause the magnetic, colored or fluorescent indicator particles to gather in the test line area, forming a detectable test line, indicating the presence of the target biomarker in the fluid sample.

[0407] The first antibody will not bind to components of the EBC sample that are not bound to the analyte or target biomarker, which will continue to flow with the liquid sample. In the control line area, the second antibody will typically bind to individually labeled capture molecules bound to indicator particles, thereby indicating the appropriate liquid flow through the strip. However, according to one embodiment, a magnetic control line can be provided to replace or require the use of additional secondary antibody reagents, so that the independently labeled capture molecules bound to the indicator particles (without bound target molecules) will be attracted to the magnetically attractive particles of the magnetic nanoparticle conjugate and aggregate together.

[0408] The fluid sample flows through the multi-zone transfer medium of the test device by capillary forces of the materials that make up each zone. To maintain this flow, an absorbent pad is attached to the end area of ​​the multi-zone transfer medium. The function of the absorbent pad is to absorb excess reagents and prevent the liquid sample from flowing back.

[0409] The components are selected and placed on the membrane so that if there is no target biomarker or analyte in the fluid sample, no analyte-labeled capture molecule complex will flow through the test line area. In this case, the magnetic, colored or fluorescent particles will not aggregate and no detectable test line will form. Even if there is no biomarker, there will be no test line, but a control line will still form because the secondary antibody will still bind to the independently labeled antibody flowing with the fluid sample and / or the magnetically attracted particles of the magnetic nanoparticle conjugate will be adsorbed on the magnetic control line.

[0410] Depending on the device structure, the intensity of the test and control lines may also be different, and the indicator particles can be evaluated by eye or using an optical or other electronic reader. Under the same conditions, multiple biomarkers can be detected simultaneously, and additional test line areas with specific antibodies for different biomarkers are arranged in an array in the test area. In addition, multiple test line areas loaded with the same antibody can also be used for quantitative detection of target biomarkers. This is often referred to as the "ladder strip" detection method, and its principle is that the fixed antibody gradually captures the colorimetric conjugate-antigen complex on each consecutive test line. The number of lines appearing on the test strip is proportional to the concentration of the target biomarker.

[0411] Fig.67 Another detection system that can be used with the EBC collection system of the present invention is shown, which system uses an electronic biosensor.

[0412] Electronic biosensors have the potential for greater sensitivity and can be used to provide a direct electrical signal to enable features such as easy wireless connectivity. The inventive EBC collection system with electronic biosensors can be easily deployed as a complement to existing contact tracing apps. The nanoscale size means that many detectors can be produced at once on a single wafer or through high-volume roll-to-roll production processes, enabling low-cost, high-volume production.

[0413] Fig.67 The mechanism of the biosensor detection system is shown. Briefly, the main components of the biosensor include a sample source (a); a biosensor region that is functionalized with biomarker-specific bioreceptors (b); and a sensor for generating a readable signal (c). The bioreceptors are matched to specific target biomarkers for lock-and-key selective screening.

[0414] According to one embodiment, an applied-field reaction capture molecule conjugate is provided for capturing a target biomarker. The applied-field reaction capture molecule conjugate has at least one applied-field reaction end and a capture molecule end, which can bind to and capture the target biomarker. The applied magnetic field reaction capture molecule conjugate may include a connecting molecule disposed between the applied magnetic field reaction end and the capture molecule end. The connecting molecule gives the applied-field reaction capture molecule conjugate electrochemical properties, so when the capture molecule end binds to the target biomarker, at least one of the polarity and conductivity of the applied-field reaction capture molecule conjugate changes. For example, the capture molecule end binds to and captures the target analyte. Binding to the target analyte increases the charge difference between the applied electric field reaction end and the capture molecule end.

[0415] According to this embodiment, the capture molecular structure has a ligand end and a polarizable end. When the capture molecular structure is placed in a carrier liquid (such as EBC), the capture molecular structure is a free floating element. The target analyte in the EBC is another free floating element. The ligand end of the capture molecular structure combines with the target analyte to form a free floating polar conjugate with a positive end and a negative end. This free floating polar conjugate can be aligned with the EBC carrier liquid in an electric field, and the electrical properties of the aligned polar conjugate are measured to detect the target analyte.

[0416] With this capture molecule, biosensor, and test system configuration, a fluid sample containing a concentration of a target biomarker (perhaps as small as one molecule) flows into the biosensor field. Some of the biosensor "locks" receive the biomarker "keys." This causes a detectable change in the sensor's output, which converts the biosensor's output into a readable signal for amplification and data processing.

[0417] For example, a desired biomarker may be an antibody that can detect

[0418] Covid-19 infection. The body fluid sample can be a sweat drop, nasal swab, blood drop, breath condensate or other body fluid, and if the target antibody is present in the sample, it will interact with the biomarker-specific bioreceptor. The bioreceptor outputs a signal with a specified sensitivity, and the transducer produces a change in an electrical property such as conductivity, indicating the presence of the antibody biomarker in the fluid sample.

[0419] Fig.68 A biosensor constructed on a water-absorbent substrate composed of at least one of a selectively permeable membrane and super absorbent polymer fibers is shown, wherein excess water in a fluid sample is absorbed by the substrate, thereby leaving a higher concentration of a target biomarker in the measured fluid sample. According to an exemplary embodiment, an electronic biosensor includes a substrate having water-absorbent properties, the substrate being provided by at least one of a selectively permeable membrane, a super absorbent polymer, a microfluidic material, and a wick.

[0420] According to an exemplary embodiment, the concentration of a target analyte is increased in a fluid sample, such as an exhaled breath condensate (EBC) sample. An EBC sample containing the target analyte is collected from the lungs and respiratory tract of a test subject. During the collection process, a super absorbent polymer is placed in the flow path of the EBC. The EBC sample contacts the super absorbent polymer, which absorbs a portion of the water in the EBC sample. The super absorbent polymer does not absorb the target analyte, thereby deriving the concentration of the target analyte in the remaining water in the EBC sample.

[0421] The super absorbent polymer is provided in the form of fibers. The fibers can be a melt-spun blend of conventional synthetic materials and super absorbent polymers. The fibers can be pressed into a substrate onto which the biosensor electrodes are deposited by vacuum deposition, screen printing, or other processes. The electrodes can be functionalized with capture molecule conjugates by the methods described herein or other sensor manufacturing techniques.

[0422] EP application: 90311692.9 filed on October 24, 1990 discloses a melt-spun fiber mixture of a conventional synthetic material and a super absorbent polymer and a process for producing the same. The mixture may be selected from a mixture of polyethylene, polypropylene, polyethylene copolymers, vinyl acetate and cellulose acetate with a super absorbent polymer. These super absorbent fibers can be used in wound dressings, sportswear and other applications requiring highly absorbent fibers.

[0423] According to one aspect of the present invention, there is provided a super absorbent fiber composed of a mixture of synthetic materials such as polyethylene, polypropylene, polyethylene copolymer, vinyl acetate and cellulose acetate copolymer, and spun together with a super absorbent polymer such as sodium polyacrylate, for concentrating analytes or target biomarkers in a fluid biological sample. The super absorbent fiber forms a wick that can absorb excess water from a fluid biological sample (such as exhaled condensed water).

[0424] The research paper Carbon nanotube-immobilized super-absorbent membrane for harvesting water from the atmosphere, Roy et al., Environ. Sci: Water Res. Technol., 2015, 1, 753, describes a carbon nanotube (CNT) immobilized membrane for harvesting pure water from the air. The carbon nanotubes were incorporated into a layer of superabsorbent poly(acrylamide-acrylic acid) that was cast on a porous hydrophilic polypropylene support. The superabsorbent polymer binds to water molecules to form water clusters. The paper reports that the addition of CNTs leads to the disruption of specific water-polymer and water-water interactions, resulting in more free water, making it easier to permeate through the membrane. The carbon nanotubes were functionalized with carboxyl groups to improve their dispersibility in the polymer matrix. The water vapor extraction efficiency reached more than 50%, and the presence of CNTs increased the water vapor removal rate by 45% and the mass transfer coefficient by 44%.

[0425] Fig.69 A sample pad of a detection system is shown, such as a lateral flow detection, whose target molecule concentration structure includes a super absorbent and selectively permeable membrane fixed with carbon nanotubes, a SAP / wick and a dissolvable fluid dam. According to a non-limiting embodiment, a sample pad is provided in the first stage of a lateral flow detection. The system includes a dissolvable fluid dam 6902, a conjugate release pad 6904, a wick 6906 and a sample pad 6908.

[0426] Adjacent to the sample pad is a selectively permeable membrane for allowing a portion of the fluid sample to pass through the sample pad and blocking at least some of the target molecules, causing the target molecules to accumulate in another portion of the fluid sample that has not passed through the selectively permeable membrane. The fluid absorber is in fluid communication with the selectively permeable membrane and is used to absorb the portion of the fluid sample that has passed through the selectively permeable membrane. The soluble fluid dam blocks the portion of the fluid sample that has accumulated the target molecules from flowing to the next stage of the lateral flow assay. Once the soluble fluid dam dissolves, the fluid sample with the accumulated target biomarkers flows through the various components of the lateral flow assay, and the target biomarker is detected for the fluid sample.

[0427] Fig.70A flow-through sensor electrode structure is shown, with one or more functionalized capture molecules on the electron transfer component (such as carbon nanotubes) of the detection interface, and the electrode is formed on a water-absorbent substrate composed of a selective permeable membrane and a SAP / capillary fiber composite core. The system includes a selective permeable membrane 7002, an electrode 7004, capture molecules 1 7006, capture molecules 2 7008, and SAP 7010.

[0428] Fig.71 The present invention is a flow chart showing the steps of concentrating a target biomarker in an EBC fluid biological sample using an electronic biosensor, the electronic biosensor including a substrate having water-absorbing properties provided by at least one of a selectively permeable membrane, a superabsorbent polymer, a microfluidic material, and a wick. The EBC sample flow containing the target biomarker is received (the first step), excess water is removed under the action of the selective water-absorbing structure (the second step), thereby concentrating the target molecule in the tested EBC sample received at the biosensor detection interface (the third step), and the target biomarker is bound to a capture molecule fixed to the detection interface (the fourth step).

[0429] Fig.72 An electronic biosensor with a superadsorption structure immobilized with carbon nanotubes for concentrating target biomarkers in EBC fluid biospecimens is shown.

[0430] According to a non-limiting exemplary embodiment, an electronic biosensor includes a substrate having water absorption properties provided by at least one of a selectively permeable membrane, a super absorbent polymer, a microfluidic material, and a wick. At least two electrodes are formed on the top surface of the substrate, forming a gap between the two. The functionalized detector in the gap includes an electron transport material and a capture molecule. The target molecule captured by the capture molecule causes at least one of the polarity and conductivity of the electron transport material to change. The target molecule is detected by detecting the change in the polarity or conductivity of the electron transport material. The target molecule detected depends on the capture molecule, and therefore, by changing the capture molecule to match the known biomarker of the corresponding disease or use, this electronic biosensor structure can be used to detect a variety of different diseases and other uses.

[0431] The target molecule can be an element of a capture molecule conjugate consisting of at least one connector and a capture molecule. The connector combines the capture molecule with the electron transport material. The electron transport material can include carboxyl carbon nanotubes incorporated into a super absorbent polymer layer, wherein the electron transport layer is cast on a porous hydrophilic polypropylene support and disposed on a substrate. The detected functionalized target biomarker can be patterned on a hydrophilic polypropylene support, and an electrode can be formed on the top surface of the hydrophilic polypropylene support. The biomarker concentrator can include a lysis material combined with a super absorbent polymer for releasing the target biomarker from a biological element including at least one of a virus, a cell, and a bacterium. The target molecule can be an element of a capture molecule conjugate consisting of at least one connector and a capture molecule. The connector combines the capture molecule with the electron transport material. The electron transport material can include carboxyl carbon nanotubes incorporated into a super absorbent polymer layer, wherein the electron transport layer is cast on a porous hydrophilic polypropylene support and disposed on a substrate. The detected functionalized target biomarker can be patterned on a hydrophilic polypropylene support, and an electrode can be formed on the top surface of the hydrophilic polypropylene support.

[0432] Fig.73 A cross-sectional view of a target molecule concentration flow electrode structure is shown. The system includes an electrode 7302, a stack 7304, a drainage hole 7306, a selectively permeable membrane 7308, a substrate 7310, a cellulose sponge 7312, and a SAP 7314.

[0433] Fig.74 The flow path of a fluid sample is shown, with the target biomarker blocked at the sensor electrode and excess water flowing past the electrode.

[0434] According to a non-limiting embodiment, the sensor includes a waterproof laminate having at least a first and a second electrode on the laminate, and a gap is defined between the two electrodes. The sensor detection interface includes a capture molecule disposed in the gap. A drain hole is formed on the substrate near the gap, and a selective permeable membrane or super absorbent polymer spun fiber is disposed at the drain hole to allow a portion of the fluid sample to pass through and block at least some of the target molecules. The fluid absorber is in fluid communication with the selective permeable membrane and is used to absorb a portion of the fluid sample.

[0435] Fig.75 The nanoCLAMP capture molecules immobilized on magnetically active nanoparticles are shown, with two capture molecules (such as nanoCLAMP) binding to different binding sites of a protein molecule.

[0436] Fig.76 Shown are two gold nanoparticles immobilized with nanoCLAMP capture molecules binding to the same target protein molecule.

[0437] Fig.77 A comparison of the relative sizes of nanoCLAMP and classical antibody capture molecules with the Debye length is shown.

[0438] Fig.78 A single layer of graphene for an electrolyte-gated graphene field-effect transistor is shown. The graphene layer is functionalized with nanoCLAMP capture molecules via a pyrene linker, where two or more nanoCLAMP capture molecules bind to different binding sites of a target protein molecule.

[0439] For example, a detection device provided in a mask-based diagnostic system may include a g-FET biosensor whose detection interface includes a graphene layer functionalized with capture molecules, wherein the capture molecules are smaller than the Debye screening length. Mask-based syndromic detection devices including biosensors may be designed to bind to biomarkers of FluA, FluB, SARS N-protein (a more conserved, slower-mutating protein in the SARS virus), and S-protein (a faster-mutating protein that is causative of the SARS-CoV-2 variant).

[0440] In one non-limiting example, an electrolyte-gated graphene field effect transistor is used as a biosensor, and nanoCLAMP capture molecules are fixed on a monolayer graphene structure provided on the detection interface of the sensor. In the electrolyte-gated graphene field effect transistor, an applied gate voltage causes interfacial charge separation between the electrolyte and the graphene channel, and there is a diffuse double layer on top of the graphene as a dielectric layer. The distance between the two charged layers is usually called the Debye length. This length is greatly affected by the ionic strength of the dielectric. If it is too high, counterions in the solution may shield the molecular charges away from the gate insulator. Therefore, this length is preferably wide enough to detect the interaction between the sensing probe and the charged analyte. The relationship between the Debye length at the solution / gate interface based on the electrical double layer (EDL) and the ionic strength of the electrolyte solution is expressed by the following formula:

[0441]

[0442] Where I is the ionic strength of the electrolyte, ε0 is the dielectric constant of free space, εr is the dielectric constant of the electrolyte, kB is the Boltzmann constant, T is the absolute temperature, NA is the Avogadro number, and e is the elementary charge. The ionic strength I of the electrolyte is expressed by formula (2):

[0443]

[0444] Among them, ∑ is the sum of all ions in the electrolyte; i is the total number of ion species; zi is the charge carried by the ion; and ci is the ion concentration, which determines the ionic strength of the electrolyte.

[0445] According to e.g. Fig.79 As a non-limiting example, the temperature T can be increased by a heating element near the sensor detection area. The heating element can be a resistive device, and the local temperature increase in the detection area can accelerate and improve the binding of the capture molecules to the target molecules.

[0446] According to formulas (1) and (2), the Debye length (λD) of 0.001×PBS solution (I=0.177mM) is estimated to be about 23nm, which is larger than that of 0.01×PBS solution (I=1.77mM, λD=7.2nm), 0.1×PBS solution (I=17.7mM, λD=2.3nm) and 1×PBS solution (I=177mM, λD=0.72nm). The lower the electrolyte concentration, the smaller the ionic strength, and the larger the λD.

[0447] EBC contains fewer ions than serum, saliva, and sweat, and its ionic strength is equivalent to 0.01×PBS (I=1.77mM, λD=7.2nm), which makes EBC an ideal biological sample matrix for GFET sensing, especially when the receptor / analyte interaction is smaller than about 7.2nm. Compared with traditional nanoantibody capture molecules, the 4nm-sized nanoCLAMP capture molecules can easily achieve this excellent receptor / analyte interaction. Compared with the use of smaller-sized nanoCLAMP as capture molecules, the classic nanoantibody with a length of about 13nm leads to poor GFET sensing effects.

[0448] Experimental Results

[0449] As an example, we constructed a graphene field effect transistor (g-FET) functionalized with a nanoCLAMP capture molecule designed to capture the S protein of the SARS-CoV-2 virus. The graphene was first synthesized in-house as described below. The insert microelectrode (ED-IDE1-Au w / o SU8) was provided by Micrux Technologies.

[0450] Synthesis of graphene: Single-layer graphene was grown by chemical vapor deposition (CVD) on commercial copper foil (purity up to 99.9999%) produced by Alpha Aesar. The growth of graphene was carried out in a Jipelec JetFirstRapid Thermal CVD (RTCVD) system. The system can heat and cool at high speed (10°C s-1). The growth itself includes heating, annealing, growth and cooling steps. In all steps, we used a mixture of 100 sccm of argon and 5 sccm of dihydrogen, and 20 sccm of methane as precursors during the growth stage. We first cut the copper foil into small pieces (2.5×2.5 cm) and then cleaned it with acetic acid, acetone and isopropanol under ultrasound for 5 minutes each to remove all possible copper oxide and make the surface as clean as possible. Then, we placed these copper pieces on the silicon wafer in the chamber. Before starting, we placed the sample in a high vacuum (<5×10-5 bar) environment, then heated from room temperature to 300°C for 5 minutes, then heated from 300°C to 1070°C for 2 minutes, annealed for 5 minutes, grew for 5 minutes, and finally used a water flow (from 1000°C to 700°C, decreasing at a rate of 60°C / s-1) to quickly cool the sample chamber to room temperature for 10 minutes.

[0451] Fabrication of field effect transistor sensors: Prior to graphene transfer, the plug-in microelectrodes (ED-IDE1-Au w / o SU8, Micrux Technologies) were cleaned in a UV-Ozone chamber (Jelight, USA) for 10 min and then soaked in 10 mL of acetone, isopropanol, and water for 15 min, respectively. Finally, each chip was rinsed with plenty of water and dried under a stream of nitrogen. The cleaned interfaces were placed in a plastic Petri dish and stored in a desiccator under vacuum. The electrodes were then soaked in ethanol for 15 min to remove excess silane compounds on the surface. Subsequently, the modified interfaces were nitrogen blown dry and stored under vacuum. The chips were placed on a hot plate at 120 °C and heated at ambient pressure for 1 h to anneal the formed monolayer and completely remove the solvent from the surface. Graphene was transferred directly to these interfaces. To transfer graphene, a 200 nm thick film of polymethyl methacrylate (PMMA) was spin-coated on the graphene / copper foil and then annealed at 110 °C with very slow heating and cooling rates (1 °C min-1) to prevent cracks in the graphene due to the difference in thermal expansion coefficients between copper and graphene. The graphene on the back of the copper foil was removed again by reactive ion etching (RIE) in oxygen plasma (50 W / 100 mT / 25 sccm / 1 min). The copper foil was etched in 0.2 M ammonium persulfate ((NH4)2S2O8) for 8 h, and then the floating PMMA / graphene sample was placed in deionized water. This operation was repeated about 10 times to rinse the graphene from the etching solution. The graphene was transferred to the IDE by immersing the IDE under the floating graphene / PMMA film. To remove the residual water traces between graphene and IDE and to increase the adhesion of graphene to IDE, the substrate was placed on a hot plate and annealed at 90 °C for 30 min with a slow heating and cooling rate (1 °C min-1). UV / ozone cleaning (28-35 mW cm-2) for 5 min followed by rinsing with hot acetone (30 °C, 30 min) effectively removed the PMMA layer.

[0452] Electrical sensing: Electrical measurements were performed using a probe station source meter device U2322A (Keysight Technologies, USA). All measurements were performed using a PMMA commercial flow cell (Micrux Technologies, Spain) with a fixed flow channel geometry (16 μL), ensuring a flow rate of 50 μL min-1 to minimize mass migration limitations of the analyte to the sensor surface in all experiments. The GFET devices were operated in a liquid gate configuration using a chlorided silver wire (1 mm diameter, Sigma-Aldrich) with a constant gate bias (VGS) of −0.1 V, a constant source-drain bias (VDS) of 0.05 V, and a gate voltage (VG) fluctuating between −0.8 V and +0.8 V. The general procedure for the sensing experiments was to first continuously flush with pure buffer (PBS, 0.01×) until a stable drain current baseline was established, and then inject the analyte at a constant flow rate of 50 μL min-1.

[0453] Researchers demonstrated an electric field-enhanced electrochemical CRISPR biosensor for DNA detection (Li et al., Electric field-enhanced electrochemical CRISPR biosensor for DNA detection, Biosensors and Bioelectronics, Volume 192, 2021, 113498, ISSN 0956-5663). The study tested a CRISPR electrochemical biosensor that can directly detect unamplified human papillomavirus-16 (HPV-16) DNA with a sensitivity of 1pM.

[0454] Fig.79 79 is a cross-sectional view showing a biosensor having an electric field application structure for concentrating target molecules to a detection interface and a local heating element provided on the biosensor for accelerating a binding reaction between a capture molecule and a target molecule. The system includes a time-release lysis bead 7902, an EBC reservoir 7904, a SAP bead 7906, a wick 7908, a heating element 7910, a biosensor 7912, a capture cell 7914, a top drive electrode 7916, and a bottom drive electrode 7918.

[0455] Fig.80 8002 , target molecules 8004 , SAP beads 8006 , top drive electrode 8008 , and bottom drive electrode 8010 .

[0456] The wick includes a superabsorbent polymer bead having a shell surface, and an electric potential applies a shell charge potential to the shell. The shell charge potential is opposite to the target molecule charge potential, which can drive the target molecule away from the superabsorbent polymer bead while allowing a portion of the fluid sample to enter through the shell and be absorbed by the bead.

[0457] Fig.81 8102, a top driving electrode grid and a bottom driving electrode 8106. In addition to the SAP beads, SAP powder or SAP / fluid conductive fiber composite materials can also be provided. The top driving electrode grid prevents the SAP material from entering the detection area while applying an electric field. Since the wires that make up the grid electrode are thinner, water tension can effectively absorb water in the fluid sample through the grid into the SAP material. If it is necessary to increase the concentration of the target molecule in the measured fluid sample, the SAP beads and / or the grid may include a selective permeable shell, coating or film to prevent molecules and particles larger than the desired size from being absorbed into the SAP material. According to a non-limiting embodiment, a sensor for detecting a target molecule in a fluid sample includes a detection area for receiving a fluid sample containing the target molecule and having a detection interface functionalized with a capture molecule. At least one of the top and bottom driving electrodes is composed of a grid electrode, and the grid electrode leaves space between the conductive elements to allow the fluid sample to flow. A gap is formed between the top driving electrode and the bottom driving electrode. A fluid conductor disposed in the gap conducts the fluid sample through the gap. A potential applied to the top and bottom drive electrodes drives the target molecules toward the capture molecules, thereby concentrating the target molecules in the portion of the fluid sample received by the detection interface.

[0458] Fig.82 The structure of a field effect transistor sensor is schematically shown. The system includes a drain 8202 , a source 8204 , a detection interface 8206 and a substrate 8208 .

[0459] Fig.83 The structure of a field effect transistor sensor is schematically shown, wherein the bottom drive electrode gate is also the gate of the field effect transistor sensor. The system includes a bottom drive electrode 8302.

[0460] Fig.84 A fluid conductor is shown applied to the bottom drive electrode grid / gate electrode top. The system includes a fluid transfer device 8402.

[0461] The fluid transport 8402 or fluid conductor includes at least one of a capillary channel, a microfluidic material, a super absorbent polymer, and a fluid conducting fiber sheet.

[0462] Fig.85A top drive electrode is shown disposed on a fluid conductor. The system includes a top drive electrode 8502.

[0463] Fig.86 Shown is a SAP bead used to remove excess water from a fluid sample on a top drive electrode grid. The system includes one SAP bead 8602.

[0464] Fig.87 A wick is shown in fluid communication with the SAP beads for removing excess water from the fluid sample. The system includes a wick 8702.

[0465] Fig.88 8804, a detection interface 8806, a SAP bead 8808, and a wick 8810.

[0466] The top and bottom drive electrodes can be placed at the detection area. The wick absorbs excess liquid in the sample. The fluid conductor conducts a portion of the fluid sample containing relatively fewer target molecules to the wick through the gap and another portion of the fluid sample containing relatively more target molecules to the capture molecules. The gate of the sensor includes at least one of the top and bottom drive electrodes. In the case where the sensor is configured as an electrolyte-gated graphene field effect transistor, the bottom drive electrode is also the top gate in contact with the fluid sample as an electrolyte. The applied gate voltage causes interfacial charge separation between the electrolyte and the graphene channel to form a diffuse double layer as a dielectric layer on top of the graphene.

[0467] Fig.89 is a flow chart of the steps for concentrating target molecules in a fluid sample and testing the changes in the electrical properties of a functionalized transistor sensor.

[0468] According to another non-limiting embodiment, a method for detecting a target molecule from a fluid sample comprises the following steps: receiving a fluid sample containing a target molecule in a microfluidic channel; transferring the fluid sample from the microfluidic channel to a detection interface of a sensor, the sensor comprising a detection area for receiving the fluid sample, the detection interface being functionalized with a capture molecule, a top drive electrode and a bottom drive electrode forming a gap therebetween, a fluid conductor being disposed in the gap for conducting the fluid sample through the gap, wherein an electromotive force applied to the top and bottom drive electrodes drives the target molecules toward the capture molecules, thereby concentrating the target molecules in a portion of the fluid sample received by the detection interface, the sensor comprising a field effect transistor having a gate electrically communicating with the detection interface, a source and a drain on both sides of the gate, wherein at least a portion of at least one of the top and bottom drive electrodes is disposed at or electrically communicating with the detection area, and the gate of the sensor comprises at least one of the top and bottom drive electrodes; intermittently and selectively applying an electromotive force to the drive electrode and the gate, the source and the drain to drive the target molecules, and reading a test reading of changes in electrical characteristics of the source, the drain and the gate.

[0469] like Fig.89 As shown, at the beginning of the test (step one), the microfluidic receives the collected EBC sample (step two). A voltage is applied to the drive electrode grid and the drive electrode grid / gate electrode (step three). The applied voltage drives the charged target molecules in the EBC sample toward the capture molecules on the biosensor detection interface, thereby concentrating the target molecules in the portion of the fluid sample received by the detection interface, and excess water is absorbed by the SAP and / or the wick, and the target molecules in the detected EBC sample are captured by the capture molecules fixed on the detection interface. Stop applying the electromotive force that drives the target molecules (step five). The test reading is the result of the captured target molecules affecting the electron charge mobility of the detection interface (for example, through the charge interaction between the target molecules and the capture molecules, whose distance is less than that fixed on Fig.78The method of the present invention is to detect the change of electrical characteristics caused by the Debye screening length of the capture molecules on the graphene layer shown in the figure. In other words, the electromotive force used to drive the target molecule is intermittently applied, and the change of electrical characteristics of the source / drain / gate of the reading transistor biosensor is tested (step 6). Other electrode and biosensor configurations can also be used, including printed electrodes with nanoparticles, nanotubes, metals, semiconductors and / or organic detection interface materials. If the change in electrical characteristics is greater than the threshold value (step 7), the test result is reported as positive (step 8) and the test ends (step 9). If the change is less than the threshold value (step 7), it is determined whether the test should be ended (step 10). For example, the test can be ended after a given time period or after a given number of fluid samples have been tested. If it is not time to end the test (step 10), the process returns to receive more collected EBC samples on the microfluidic (step 2). If it is time to end the test (step 10) and the change in electrical characteristics does not exceed the threshold value (step 7), the test result is reported as negative (step 11) and the test ends (step 9).

[0470] Fig.90 9002, a back gate 9004, a detection interface 9006, and a wick 9008.

[0471] The sensor includes a field effect transistor having a gate that is in electrical communication with a detection interface functionalized with a capture molecule and having a source and a drain on either side of the gate. At least a portion of at least one of the top and bottom drive electrodes is in electrical communication with the detection region, wherein the gate of the sensor includes at least one of the top and bottom drive electrodes. In the case where the sensor is configured as a back-gate field effect transistor, the bottom drive electrode may also be the back-gate electrode of the transistor. When a potential is applied to the top drive electrode gate and the back-gate electrode, the charged target molecule is driven toward the detection interface. For example, if the target molecule is the S protein of the SARS-CoV-2 virus, the target molecule is negatively charged. In this case, a negative potential is applied to the top drive electrode gate and a positive potential is provided to the back gate, thereby driving the negatively charged S protein target molecule toward the capture molecule fixed on the detection interface.

[0472] Fig.91 is a flow chart of the steps for concentrating target molecules using an applied electric field and testing the changes in the electrical properties of a functionalized field effect transistor sensor. Fig.89The flowchart shown is similar, and at the beginning of the test (the first step), the microfluid receives the collected EBC sample (the second step). In the case of a back-gate transistor biosensor, the back gate of the transistor can be used as both a bottom driving electrode to drive the target molecules to the detection interface and a gate electrode to detect whether the capture molecules fixed on the detection interface capture the target molecules. A voltage is applied to the driving electrode gate and the back gate layer of the biosensor (step three). The applied voltage drives the charged target molecules in the EBC sample to the capture molecules on the detection interface of the biosensor, thereby concentrating the target molecules in the portion of the fluid sample received by the detection interface, and the excess water is absorbed by the SAP and / or the wick, and the target molecules in the detected EBC sample are captured by the capture molecules fixed on the detection interface. Stop applying the electromotive force to drive the target molecules (the fifth step). Test and read the changes in the electrical characteristics of the source / drain / back gate layer / electrode of the transistor biosensor (the sixth step). If the change is greater than the threshold (the seventh step), the test result is reported as positive (the eighth step), and the test ends (the ninth step). If the change is less than the threshold (step 7) and it is not time to end the test (step 10), then the flow returns to the microfluidics to receive more collected EBC samples (step 2). If it is time to end the test (step 10) and the change in the electrical characteristic does not exceed the threshold (step 7), the test result is reported as negative (step 11) and the test ends (step 9).

[0473] Fig.92 9204, a bottom drive electrode 9206, a top drive electrode 9208, a detection interface 9210, and a separator 9212.

[0474] According to this embodiment, the top and bottom driving electrodes can be arranged upstream of the fluid sample flow in the detection area. Fluid conductors are arranged upstream and downstream of the detection area so that the fluid sample with the target molecule flows through the detection area. There is a filter element upstream of the detection area for removing excess water in the fluid sample, and the flow of excess water in the filter element is separated from the flow of the sample to be tested with the target molecule in the fluid conductor. The fluid conductor can have different components at different positions of the fluid flow path. For example, a hydrophilic surface can be provided on the condensation surface of the EBC collector, and a fibrous microfluidic material can be provided to attract the collected EBC to the detection area. In order to maximize the mobility of particles and ions so that they can align and move in the detection area, a capillary channel can be provided, in which the inherent surface tension of water in the fluid sample will attract the fluid sample to the detection interface and pass through the detection interface. A fiber core, SAP composite material or other absorbent material can be arranged downstream of the detection area.

[0475] Fig.93 An indoor biosensor for detecting target molecules in an ambient humidity fluid sample drawn from a room is schematically shown.

[0476] Fig.94 is a block diagram showing the chamber scale biosensor components.

[0477] According to one non-limiting exemplary embodiment, the indoor biosensor includes an air inlet for drawing in ambient air. A condenser cools the ambient air and condenses water vapor into a condensate containing water and at least one target molecule. A condensate detection system detects the target molecule in the condensate. The condensate detection system includes a biosensor, the biosensor comprising one or more g-FET biosensors, each biosensor having a detection interface, the interface comprising a graphene layer functionalized with a respective capture molecule. In a preferred embodiment, the capture molecule is smaller than the Debye screening length of the fluid sample.

[0478] The target molecule concentrator can be used to concentrate at least one target molecule in a fluid biological sample to form a target molecule concentrated condensate. The target molecule concentrator may include a selective permeability barrier for allowing excess water in the condensate to pass through and preventing the target molecules in the condensate from passing through the selective permeability barrier; and an excess water absorption core for absorbing excess water passing through the selective permeability material.

[0479] The target molecule concentrator may also include or optionally include a super absorbent polymer (SAP) for preferentially absorbing water in the condensate into the polymer chains of the super absorbent polymer, wherein the target molecule is not absorbed by the polymer chains but flows through the SAP together with the condensate. When the condensate flows through the SAP, the water content in the condensate is removed, thereby increasing the concentration of the target molecule in the test sample.

[0480] The biosensor may include an array of individual biosensors, each functionalized with a different corresponding capture molecule designed to bind to at least one FluA, FluB, SARS (SARS N-protein and SARS S-protein) biomarker. The biosensor may also include capture molecules for detecting environmental exposures, volatile organic compounds, bomb making materials and other markers that may be present in the sampled ambient air.

[0481] A fluid transfer system may be provided for transferring the condensate from the condenser to the condensate testing system, wherein the fluid transfer system includes at least one of a fluid conductor, a collection area, and a microfluidic transfer path for controlling the flow of the fluid biological sample received from the EBC collector. A target molecule releasing material may be placed in the fluid conductor, the collection area, and the microfluidic transfer path, or anywhere along the path from the condenser to the biosensor, wherein the target molecule releasing material includes at least one of a surfactant and a chemical lysing agent.

[0482] In addition to the surfactant and / or chemical lysing agent, a target molecule releasing structure may be provided for mechanically lysing at least one of a cell wall, an encapsulating structure, and a viral envelope containing the target molecule. The target molecule releasing structure may include a lysing structure extending from at least one of a condensate forming surface and an EBC flow path surface. The lysing structure may mechanically disrupt a cell wall, an encapsulating structure, and / or a viral envelope containing the target molecule.

[0483] The condenser may also include a condensation-forming surface having relatively low energy surface characteristics for limiting the adhesion of target molecules to the condensation-forming surface. The fluid conductor may be disposed on the condensation-forming surface, wherein the fluid conductor includes a texture structure formed on the condensation-forming surface, the texture structure having relatively high energy surface characteristics for guiding the EBC to flow in a desired direction.

[0484] During operation, an example embodiment of a room-scale biosensor is placed in a room or within a HVAC system of a building. An intake fan draws ambient air into a condenser channel, which is cooled by a cooling system, such as a coil with a working fluid (such as a traditional dehumidifier or air conditioner), or by other means (such as a Peltier cooler). The humidity in the cooled air is condensed into a fluid sample condensate containing particles, dissolved volatile organic compounds, non-target molecules and target molecules, etc. The condensate is transported to a target molecule concentrator via a fluid transport system (capillary action, surface flow, wick, etc.). The target molecule concentrated condensate is dripped onto or otherwise received by a condensate detection system containing at least one biosensor. The condensate can flow through the biosensor for testing and then be transferred to a condensate storage tank, where the sensor can also be located. The sensing electronics receive the detection signal emitted by the condensate detection system and determine whether the target molecule is detected. The sensing electronics can determine the presence and / or concentration of the target molecule in the ambient air. The microprocessor controls the operation of each component of the biosensor in the chamber and receives the detection result signal from the sensor electronic device. If the target molecule is detected, the microprocessor can control the condensate sterilizer to automatically inject the sterilization solution into the condensate holding tank and / or the entire system. The capture molecule refreshing system can be controlled to clean the capture molecule release agent, such as propylene glycol or glycerol, on the detection interface to refresh the biosensor array. The communication system may include at least some of the components shown and described herein, such as Figure 3-6 , the communication system is controlled by a microprocessor and automatically issues an alarm or report, which can include detailed information on the type of target molecule detected, the time of detection, and the concentration.

[0485] Fig.95 Schematic diagram of a field effect transistor biosensor with a fluid sample flowing through a conductor, and capture molecules falling off from the conductive surface of the detection interface, the falling height being less than the Debye screening length of the fluid sample being measured. The system includes a detection interface 9502, a fluid transport 9504, a back gate 9506, and a liquid gate electrode 9508.

[0486] According to a non-limiting exemplary embodiment, a field effect transistor sensor circuit for detecting target molecules in a fluid sample includes a semiconductor substrate of a conductive type. A source region and a drain region are located between a channel region of a conductive type. The conductivity type of the source region and the drain region is opposite to that of the semiconductor substrate (e.g., a PNP or NPN transistor structure). An insulator is formed on the channel region, and the channel region constitutes a back gate of the field effect transistor. A detection region is formed on the insulator for receiving the fluid sample and having a detection interface functionalized with a capture molecule.

[0487] Fig.96Schematic diagram of a field effect transistor biosensor showing a fluid sample stream with target molecules and ions randomly dispersed in a fluid conductor above a detection interface. The system includes a top electrode with a Debye screening length of 9602, which forms a gap with the detection region and constitutes the liquid gate electrode of the transistor.

[0488] The fluid conductor is disposed in the gap for guiding the fluid sample through the gap. For example, the fluid conductor may be composed of one or more components that flow smoothly with each other. For example, the fluid conductor may be made of microfluidic materials, capillary channels (thin empty spaces for the fluid sample to flow through capillary action), fiber cores, microchannels, etc.

[0489] Fig.97 is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a driving circuit for driving the target molecules toward capture molecules immobilized on a detection interface.

[0490] For example, the electronic components used to drive the circuit and read the detection signal may include a battery power source and a synchronous step-up DC / DC converter, such as the XC series discrete electronic components produced by Torex Semiconductor. The DC / DC converter can be used in series as needed to increase the battery voltage, thereby facilitating the alignment and driving of the target molecule to the detection interface. The boosted voltage can also be used in a biosensor structure in which the back gate is located below the insulating layer on which the detection interface is formed. Most or all of the discrete semiconductor devices that make up the drive circuit, the detection circuit, and the biosensor can be manufactured as an integrated circuit on a semiconductor wafer, thereby greatly reducing the cost, power consumption requirements and complexity, while improving the consistency and accuracy of the system. The drive circuit applies a potential of one polarity to the top electrode and a potential of the opposite polarity to the back gate. The potential drives the target molecule toward the capture molecule, thereby concentrating the target molecule in the portion of the fluid sample received by the detection interface. The detection interface includes at least one of graphene, nanoparticles, aligned carbon nanotubes, vacuum deposited conductive materials, transferred conductive materials, semiconductors, metals, and screen-printed conductive inks.

[0491] Fig.98 is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a liquid gate detection circuit for detecting the presence and quantity of target molecules captured at a detection interface.

[0492] Fig.99is a schematic diagram of a field effect transistor sensor sensing circuit for detecting target molecules in a fluid sample, showing a back gate detection circuit for detecting the presence and amount of target molecules captured on a detection interface. The detection circuit applies a voltage to the source region and at least one of the back gate and top electrode, and detects a change in current through the drain region that depends on the binding of the target molecule to the capture molecule. The drive circuit and the detection circuit can be controlled to cycle in sequence and can include test readings using the back gate and the liquid gate.

[0493] Fig.100 A graphene detection interface is shown, on which nanoCLAMP capture molecules are fixed via linker molecules, wherein a portion of the capture molecules are fixed to the detection interface at a greater distance than another portion of the capture molecules are fixed to the detection interface. The system includes a non-target protein 10002, a target protein 10004, a nanoCLAMP 10006, a Debye screening length 10008, and graphene 10010.

[0494] Fig.101 The system includes an electric field 10102, a target protein 10104, a linker 10106, and a capture molecule 10108.

[0495] Fig.102 The electric field potential removed from the detection area is shown. According to a non-limiting embodiment, a portion of the capture molecules are fixed on the detection interface, and they are farther away from the top surface of the detection interface than another portion of the capture molecules fixed on the detection interface. When the target molecule is driven toward the detection interface, it may encounter the capture molecule tethered at a relatively long distance and be captured, thereby being fixed and tethered to the detection interface.

[0496] Fig.103 A reverse polarity electric field potential is demonstrated, which can keep target and non-target molecules away from the detection interface.

[0497] The target molecule is still tethered to the capture molecule fixed by the relatively long connector on the detection interface. Non-target molecules and ions contained in the fluid sample and uncaptured target molecules are driven away from the detection interface by the electric field of opposite polarity. The drive circuit reverses the polarity of the applied potential to drive the non-target molecules out of the detection area (making room for another target molecule to migrate to the detection interface), while the target molecules captured by the capture molecules fixed on the detection interface are retained in the detection area.

[0498] Fig.104 More target and non-target molecules flowing into the detection area are shown.

[0499] Fig.105 The electric field potential applied to the detection area is shown to force the tethered target molecule into position, captured by a capture molecule fixed by a relatively short linker molecule. In addition, another target molecule is also captured by another capture molecule, which extends on the linker molecule and is at a relatively long distance from the detection interface.

[0500] This electric field polarity reversal cycle can be used to concentrate target molecules in a portion of the fluid sample received by the detection interface. Some target molecules may be directly captured by capture molecules at a shorter distance, or through the pumping action of cyclically applied electric field polarity, target molecules become more prevalent over time and captured by capture molecules at the detection interface, while non-target molecules and ions at the detection interface are reduced. Through the application of an electric field and the tethering and selective binding of capture molecules to target molecules within the Debye screening length range, the capture molecules are at different distances from the detection surface, and the sensor is at different distances from the antigen binding site. Capture molecules that bind to target molecules within the Debye screening length range have a stronger signal response to the detection circuit.

[0501] Fig.106 is a block diagram of a g-FET sensor integrated circuit with a functionalizable detection interface. An integrated circuit may be provided that includes a packaged biosensor semiconductor device to which a fluid sample may be connected to the detection interface of the biosensor. For example, a universal biosensor integrated circuit chip may include most, if not all, electronic circuit elements formed by conventional integrated circuit manufacturing techniques with the additional function of providing a detection interface. In one example of a universal biosensor that can detect target RNA and DNA molecules, a Cas9 complex is immobilized on a graphene detection interface. Adsorption and interaction of charged molecules on the surface of the graphene detection interface can cause changes in electrical properties between the source, drain, and gate. When a detection circuit applies a voltage to the surface of the detection interface, the capture of the target DNA molecule by the immobilized ribonucleoprotein (CRISPR Cas protein capture molecule complex) can be detected as a change in current and transmitted directly from the packaged integrated circuit semiconductor device as a direct electrical signal. Only a few wires are required to connect an external power supply and an antenna. In the case of near field communication and energy harvesting, the only wires required may be for connecting the RF energy harvesting and RF transmitting antennas.

[0502] Fig.107A group of g-FET sensors fabricated in a functionalizable detection zone including individually addressable channel regions is shown. A multi-biomarker syndrome detection system can be achieved through a group of biosensors arrays fabricated at the wafer level that can receive EBC biospecimens collected by a mask-based diagnostic system. This group of biosensors with different channel regions can be used for specific applications by selecting the respective capture molecules for the corresponding target molecules.

[0503] Fig.108 The g-FET semiconductor device is encapsulated in an epoxy substrate, and the detection interface of the g-FET is exposed on the surface of the epoxy substrate. The system includes a detection interface 10802, a drain 10804, a source 10806 and a detection interface 10808.

[0504] Fig.109 An epoxy resin top cover is shown, and a detection well opening is provided on the epoxy resin base, and the detection interface can be entered through the detection well opening. The system includes an epoxy resin top cover 10902, a detection well 10904, a detection interface 10906 and an epoxy resin base 10908.

[0505] Fig.110 A fluid conductor is shown in fluid communication with the detection interface through the detection well. The system includes a fluid transfer device 11002.

[0506] Fig.111 A mesh drive electrode / liquid gate electrode is shown disposed on a fluid conductor. The system includes a liquid gate electrode 11102.

[0507] Fig.112 Shown is an optional SAP on top of a mesh drive electrode / liquid gate electrode. The system includes SAP beads 11202.

[0508] Fig.113 A wick is shown disposed on a SAP and a mesh drive electrode / liquid gate electrode. The system includes a wick 11302.

[0509] According to one non-limiting embodiment, the capture molecules include at least one of antibodies, engineered antibodies, aptamers, nanoCLAMPS, and CRISPR Cas protein conjugates.

[0510] CRISPR-associated (Cas) proteins (such as Cas9, Cas12a, Cas13a) have recently been used for sequence-specific nucleic acid detection. In a specific structure of a non-limiting exemplary embodiment, the field effect transistor sensor is a g-FET sensor whose detection interface is a single-layer graphene sheet. The capture molecule includes a catalytically inactive Cas9 protein connected to the guide RNA and fixed on the graphene detection interface. The Cas9 protein complex serves as a capture molecule to form a CRISPR universal biosensor. The Cas9 capture molecule binds to the target DNA molecule, resulting in detectable changes in the electrical properties of the biosensor electrode.

[0511] When the target molecule is captured, the cleavage (gene editing) ability of CRISPR can be used to detect the suppressed current (Wei Xu, Tian Jin, Yifan Dai, Chung Chiun Liu, Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPRCas systems, Biosensors and Bioelectronics, Volume 155, 2020, 112100, ISSN 0956-5663).

[0512] CRISPR technology can be used to improve the sensitivity, selectivity and specificity of the biosensor disclosed herein, while simplifying the detection process. Due to the long distance between the linear ssDNA and the electrode including the detection interface, the efficiency of electron transfer will be affected, and a certain stereo hindrance will be produced, thereby affecting the cutting of Cas12a to linear DNA. To overcome this problem, the two ends of the hairpin-shaped ssDNA are respectively connected to the detection interface and receptors such as methylene blue (MB), which can directly improve the analytical performance of biosensors (g-FET, electrochemistry, etc.). According to the length and number of DNA fragments on the Cas hairpin, the size of ss DNA is the same as nanoCLAMP (but this depends on the length and number of DNA fragments thereon).

[0513] Separately, the researchers described a g-FET CRISPR universal biosensor in which the Cas9 protein is linked to a guide RNA and immobilized on a graphene layer at the detection interface (https: / / www.synthego.com / blog / crispr-electronic-biosensor). The catalytically inactive Cas9 protein conjugate recognizes and binds to target DNA molecules but does not cut the DNA. The target DNA is tethered to the detection interface. The catalytically inactive Cas9 protein can be immobilized on graphene using a PBA linker at a length less than the Debye screening length for detecting target DNA or RNA molecules in exhaled breath condensate biospecimens.

[0514] Fig.114 A g-FET semiconductor device encapsulated in an epoxy barrier material is shown with source, gate and drain leads. The system includes a sense well 11402, a drain 11404, a gate 11406 and a source 11408.

[0515] Fig.115 Z-axis conductors for connecting the source, gate, and drain leads of a packaged g-FET semiconductor device to external circuitry are shown. The system includes z-axis conductive tape 11502, source 11504, gate 11506, and drain 11508.

[0516] Fig.116 A packaged g-FET semiconductor device is shown connected to source, gate, and drain circuit lines of an external circuit. The system includes a drain 11602, a gate 11604, a source 11606, a sense well 11608, and a Z-axis conductive tape 11610.

[0517] Double-sided adhesive Z-axis tape (e.g., 3M 9703, 3M, Minnesota) can be used to establish simple and effective electrical connections between the leads of a packaged integrated circuit semiconductor device and the corresponding leads and connection pads of a detection circuit, power supply, or other external circuit. Z-axis tape also provides the possibility of manual placement of the packaged integrated circuit semiconductor device, allowing the end user to easily select the detection target biomarker based on the diagnostic use case.

[0518] Fig.117 A packaged g-FET semiconductor device is shown, which has a drive electrode / liquid gate lead for connecting the mesh drive electrode / liquid gate electrode to the lead of an external circuit. The system includes a detection well 11702, a liquid gate electrode 11704, a source 11706, a back gate 11708, and a drain 11710.

[0519] The packaged biosensor semiconductor device includes a semiconductor die mounted on a lead frame and encapsulated in a barrier material (such as epoxy resin). The semiconductor die includes a source region and a drain region, and a channel region is formed between the source region and the drain region. An insulator is formed on the channel region. The channel region forms the back gate of the field effect transistor. The detection region is located above the insulator and receives the fluid sample. The detection region has a detection interface, which is functionalized with a capture molecule.

[0520] The capture molecules can be deployed at the wafer manufacturing level by conventional masking / etching semiconductor manufacturing processes. Near the end of the manufacturing process, the detection interface is applied to at least one channel area, such as by vacuum deposition, transfer, spin plating or other deposition methods. Different capture molecules can be selectively applied to the channel by masking and etching. The final mask applied to the detection area, at least when the detection interface is formed, is a thin water-soluble layer so that the graphene detection interface decorated with fixed capture molecules can be retained. Spacer molecules such as PEG can also be fixed to form a desired capture molecule density at the detection interface. The water-soluble mask layer can protect the detection interface during testing, sorting, classification, packaging, etc. on the wafer. Appropriate solvents, treatment methods and materials need to be used during cleaning and other processes to prevent premature dissolution of the water-soluble mask layer.

[0521] The single chip is mounted on a die pad with wires for conducting electrical signals from the source, channel, and drain regions to external electronic circuits. The bottom barrier portion (epoxy bottom layer) encapsulates the semiconductor substrate and exposes the detection interface. The top barrier portion (epoxy top layer) has a detection well with an opening formed on the exposed detection interface for receiving a fluid sample (the bottom of the detection well is a detection interface decorated with capture molecules).

[0522] One end of the top gate lead is located on the top surface of the top gate portion. The top gate lead connects to the liquid gate electrode. The other end of the top gate lead connects the liquid gate electrode to an external electronic circuit. The liquid gate electrode forms a gap with the detection area and is in electrical communication with the fluid sample when the fluid sample is placed in the detection well. The geometry and volume of the detection well can accommodate and optimize the collected biological sample. For example, in an experimental test, EBC successfully tested unaltered biological samples flowing through a fibrous microfluidic material (filter paper) and contacting the detection interface of an electrochemical biosensor with aptamer capture molecules. In addition, the detection well can also form a capillary channel. Combinations of microfluidic materials and structures can be provided to allow sample flow from collection to the final wicking stage.

[0523] When the capture molecule binds to the target molecule, one or more of the source, drain, back gate and liquid gate electrodes undergoes a change in electrical properties, thereby detecting the target molecule in the fluid sample. Typically, the change in the detection signal is proportional to the number of target molecules bound and retained by the capture molecule. The capture molecules may include antibodies, engineered antibodies, aptamers, nanoCLAMPS and / or CRISPR Cas protein conjugates.

[0524] Fig.118 is a wireframe of a packaged g-FET semiconductor device showing drive electrode / liquid gate leads on the epoxy top and epoxy bottom, leads for connecting the mesh drive electrode / liquid gate electrode to external circuitry. The system includes a liquid gate electrode 11802.

[0525] The drive electrode / liquid gate leads on top of the epoxy can be connected to the detection circuitry in the integrated circuit package. Packaged semiconductor devices, including universal biosensors, are made from g-FET chips separated from a wafer and encapsulated in epoxy (a typical semiconductor discrete electronic device structure). A detection hole is formed on top of the molded epoxy and placed over the graphene detection interface so that the fluid sample can contact the immobilized capture molecules on the detection interface. The graphene layer can be formed at the wafer level and functionalized with the capture molecules. During the manufacturing process, the functionalized graphene is protected by a soluble mask so that the chip can be picked and placed and mounted on a lead frame, which is then encapsulated with epoxy (the detection hole can be pre-formed in the epoxy cover or formed in situ during the packaging process).

[0526] Fig.119 A packaged g-FET semiconductor device is shown with a drive electrode / liquid gate lead located on top of a fluid conductor for connecting leads to an external circuit. The system includes a top drive electrode 11902 and a fluid transport 11904.

[0527] Fig.120 The bottom of the packaged g-FET semiconductor device is shown, showing the source and drain leads, as well as the back gate and drive electrode / liquid gate leads. The system includes a back gate 12002 and a liquid gate 12004.

[0528] The packaged biosensor semiconductor device may include a liquid gate and / or back gate with optional connections, which may be mounted on a printed circuit board and connected to the outside of the circuit. The liquid gate, back gate, source and drain may be connected to other component semiconductor circuits on the same packaged integrated circuit device. The liquid gate may be formed of a mesh electrode to remove excess water from the liquid sample before it reaches the detection well, thereby allowing the concentrated capture molecule sample to continue to flow into the detection well as the liquid sample accumulates.

[0529] According to the above description, read in conjunction with the accompanying drawings, various modifications and adjustments of the above exemplary embodiments of the present invention may be obvious to those skilled in the relevant art. However, any and all modifications will still fall within the scope of the non-limiting and exemplary embodiments of the present invention.

[0530] Furthermore, some features of the various non-limiting and exemplary embodiments of the present invention may be used to advantage without the corresponding use of other features. Therefore, the above description should be regarded as merely illustrative of the principles, teachings and exemplary embodiments of this invention, and not in limitation thereof.

Claims

1. A device comprising an exhaled breath condensate (EBC) collector for converting exhaled breath vapor received from the lungs and respiratory tract of a test subject into an EBC fluid biological sample; A biomarker concentrator, used to concentrate a target biomarker portion in a fluid biological sample to form a concentrated fluid biological sample; The biomarker detection device is used to receive the concentrated liquid biological sample and perform target biomarker detection on the concentrated liquid biological sample.

2. The apparatus of claim 1 further comprising a test system holder for the EBC collector, wherein the test system holder is configured and sized to fit inside a mask, wherein the mask forms an exhaled gas containing volume to contain the exhaled gas in proximity to the EBC collector, thereby condensing the exhaled gas into a fluid biological sample.

3. The device of claim 1, wherein the biomarker concentrator comprises a selectively permeable barrier for allowing excess water in the fluid biological sample to pass through the selectively permeable barrier and preventing target biomarkers in the fluid biological sample from passing through the selectively permeable barrier.

4. The device as claimed in claim 3 further comprises an excess water absorbing core for absorbing excess water passing through the selectively permeable material.

5. The device of claim 1, wherein the biomarker concentrator comprises a super absorbent polymer for preferentially absorbing water in the EBC into polymer chains of the super absorbent polymer, wherein the target biomarker is not absorbed by the polymer chains and flows together with the EBC through the SAP and microfluidic structure of the diagnostic platform, wherein when the EBC flows through the SAP, the water content in the EBC is removed while the content of the target molecule remains unchanged, thereby increasing the test sample concentration of the target molecule.

6. The device according to claim 1 further comprises an external field reaction capture molecule conjugate for capturing the target biomarker, the external field reaction capture molecule conjugate having at least one external field reaction end and one capture molecule end, wherein the capture molecule end binds to the target biomarker and captures the target biomarker.

7. The device as described in claim 6 further includes a soluble adhesive for fixing the external field reaction capture molecule conjugate on the path of the fluid biological sample, wherein the water in the fluid biological sample will dissolve the soluble adhesive, allowing the external field reaction capture molecule conjugate to float freely in the fluid biological sample.

8. The device as claimed in claim 6, wherein the external magnetic field responsive capture molecule conjugate comprises a magnetic nanoparticle conjugate, and the particle ends of the conjugate have magnetic attraction.

9. The device of claim 8, further comprising a magnetic capturer for attracting and holding the magnetic particle ends, and immobilizing the magnetic nanoparticle conjugate and the captured target biomarker on the magnetic capturer.

10. The device according to claim 9, wherein the magnetic capture device is close to the biomarker detection area; further comprises a wick for absorbing excess EBC components; and a fluid transport system for transporting EBC through the magnetic capture device, so that the magnetic nanoparticle conjugate and the captured target biomarker are concentrated in the magnetic capture device, while the excess EBC components not adsorbed by the magnetic capture device flow through the biomarker detection unit to reach the wick.

11. The device according to claim 6, wherein the external field responsive capture molecule conjugate includes a connecting molecule arranged between the external field responsive end and the capture molecule end, the connecting molecule gives the external field responsive capture molecule conjugate electrochemical properties, and when the capture molecule end binds to the target biomarker, at least one of the polarity and conductivity of the external field responsive capture molecule conjugate changes.

12. The device of claim 1, wherein the detection unit comprises a g-FET biosensor having a detection interface comprising a graphene layer functionalized with capture molecules, wherein the capture molecules are smaller than the Debye screening length.

13. A method for assembling an array of external field-responsive capture molecule conjugates, comprising providing a dissolvable adhesive film; providing a carrier liquid which is a non-solvent for dissolving the adhesive film, the carrier liquid having randomly dispersed epitaxial-field effect capture molecule conjugates; applying an alignment field to the carrier fluid to assemble the epitaxial-field effect capture molecule conjugate onto the dissolvable adhesive film; and evaporating the carrier liquid to fix the assembled epitaxial-field effect capture molecule conjugate on the soluble adhesive film.

14. An exhaled breath condensate (EBC) collector for converting exhaled breath vapor received from the lungs and airways of a test subject into a fluid biological sample, the EBC collector comprising: Condensate forms on the surface; and a thermal mass forming a surface thermal connection with the condensate; as well as A fluid transfer system is provided for transferring the EBC to at least one of a testing device and an EBC container.

15. The EBC collector of claim 14, wherein the thermal mass comprises at least a first chemical reagent and a second chemical reagent that can be combined to form an internal thermochemical reaction for absorbing thermal energy from the condensate forming surface to convert the exhaled breath vapor into EBC.

16. An EBC collector according to claim 14, wherein the fluid transfer system includes at least one of a fluid conductor, a collection area and a microfluidic transfer path for controlling the flow of a fluid biological sample received from the EBC collector; and also includes a target biomarker release material arranged in at least one of the fluid conductor, the collection area and the microfluidic transfer path.

17. The EBC collector of claim 16, wherein the target biomarker releasing material comprises at least one of a surfactant and a chemical lysing agent.

18. An EBC collector as described in claim 17, wherein the condensation forming surface includes relatively low energy surface properties for limiting the adhesion of target biomarkers to the condensation forming surface, and further includes a fluid conductor arranged on the condensation forming surface, wherein the fluid conductor includes a texture structure formed on the condensation forming surface, and the texture structure has relatively high energy surface properties for guiding the EBC to flow in a desired direction.

19. The EBC collector of claim 14, further comprising a target biomarker releasing structure for mechanically disrupting at least one of a cell wall, an encapsulating structure, and a viral envelope containing the target biomarker.

20. The EBC collector of claim 19, wherein the target biomarker release structure comprises a lysis structure protruding from at least one of a condensate forming surface and an EBC flow path surface, wherein the lysis structure mechanically disrupts at least one of a cell wall, an encapsulation structure, and a vial envelope containing the target biomarker.

21. A method of forming a condensate collector having a fluid conductor channel on a substrate for directing fluid flow in a desired direction, the method comprising: providing a substrate having a surface having relatively low energy surface characteristics; The texture is formed on the surface with relatively high energy surface characteristics to form fluid conductor channels for directing the fluid flow in a desired direction.

22. The method of claim 21, wherein the relatively low energy surface properties restrict the adhesion of target analytes to the surface and render the surface relatively hydrophobic, while the higher energy surface properties of the textured structure render the channels relatively hydrophilic.

23. The method of claim 22, wherein the texture is formed by at least one of laser ablation, sandblasting, etching, and calendaring.

24. A method for detecting a target analyte, comprising providing a capture molecule structure having a ligand end and a polarizable end, wherein when the capture molecule structure is placed in a carrier liquid, the capture molecule structure is a free floating element; providing the target analyte as another free floating element in the carrier liquid, wherein the ligand end of the capture molecule structure binds to the target analyte and forms a free floating polar conjugate having a positive end and a negative end; aligning the polar conjugate in the carrier liquid in an electric field; and measuring the electrical properties of the aligned polar conjugate to detect the target analyte.

25. The method of claim 24, wherein the carrier fluid comprises at least one of water, a buffer, a surfactant, a lysing material, a preservative, and a body fluid, the body fluid comprising at least one of saliva, urine, exhaled breath condensate, blood, and sweat.

26. The method of claim 24, wherein the measuring step comprises applying the pulsed electric field for a period of time and measuring the electrical characteristic for a period of time after the period of time, wherein the period of time is short enough to detect the target analyte.

27. The method of claim 24, wherein the carrier fluid is a biological fluid sample; the capture molecule structure is provided in a dry powder form prior to the step of mixing the capture molecule structure with the carrier fluid; and the target analyte is a component of the biological fluid sample.

28. The method of claim 24, wherein the carrier fluid is an environmental fluid sample and the target analyte is a component of the environmental fluid sample.

29. A capture molecule conjugate for detecting a target analyte, comprising: An externally applied electric field responsive capture molecule conjugate having at least one externally applied electric field responsive end and at least one capture molecule end, wherein each of the capture molecule ends binds to and captures a target analyte, and the binding of each of the capture molecule ends to the target analyte increases the charge difference between at least one externally applied electric field responsive end and at least one capture molecule end.

30. The capture molecule conjugate according to claim 27 comprises a connecting molecule arranged between at least one external magnetic field reaction end and at least one capture molecule end, wherein the connecting molecule enables the external magnetic field reaction capture molecule conjugate to have electrochemical properties, wherein when at least one capture molecule end binds to a target analyte, at least one of the polarity and conductivity of the external magnetic field reaction capture molecule conjugate changes.

31. The capture molecule conjugate of claim 29, wherein at least one applied field responsive end comprises at least one of nanoparticles, gold nanoparticles, magnetically active nanoparticles, carbon nanotubes, and graphene.

32. The capture molecule conjugate of claim 29, wherein at least one capture molecule end comprises at least one of an engineered antibody, an antibody, an aptamer, a nanobody, and a nanoCLAMP.

33. The capture molecule conjugate of claim 29, wherein the applied field responsive end comprises positively charged gold nanoparticles, the linking molecule comprises PEG, and the capture molecule comprises nanoCLAMP.

34. A method for concentrating a target analyte in an exhaled breath condensate (EBC) sample, comprising the steps of: An EBC sample is collected from the lungs and airways of a test subject, the EBC containing a target analyte; a super absorbent polymer is provided in a flow path of the EBC, and during the collection process, the EBC sample contacts the super absorbent polymer, which absorbs part of the water in the EBC sample but does not absorb the target analyte, thereby concentrating the target analyte in the remaining water in the EBC sample.

35. The method of claim 34, wherein the superabsorbent polymer is provided in the form of fibers.

36. A method as claimed in claim 34, wherein a selectively permeable membrane is provided downstream of the EBC sample flow path, the membrane being mixed with a super absorbent polymer, and having a pore size configuration and size that allows a portion of the water in the EBC sample that is not absorbed by the super absorbent polymer mixture and the target analyte to flow through the selectively permeable membrane, and prevents the super absorbent polymer from flowing through the selectively permeable membrane, resulting in a concentration of the target analyte in the remaining water in the EBC sample.

37. A method as claimed in claim 34, wherein a selectively permeable membrane is provided upstream of the EBC sample flow path from the super absorbent polymer, and its pore size configuration and size allow a portion of water in the EBC sample to flow through the selectively permeable membrane to the super absorbent polymer and prevent the target analyte from flowing through the selectively permeable membrane, thereby resulting in the concentration of the target analyte in the remaining water in the EBC sample.

38. An apparatus for detecting a target biomarker in exhaled breath condensate (EBC), comprising an EBC collector for converting exhaled air received from the lungs and respiratory tract of the test subject into an EBC biospecimen containing a target analyte; a biomarker concentrator, comprising a super absorbent polymer layer located in a flow path of an EBC biological sample, wherein during collection, the EBC biological sample contacts the super absorbent polymer layer, and the super absorbent polymer absorbs a portion of the water in the EBC biological sample without absorbing the target analyte, resulting in a concentration of the target analyte in the remaining water in the EBC biological sample; and The biomarker detection device is used to receive the concentrated EBC biological sample and perform target biomarker detection on the concentrated EBC biological sample.

39. The device of claim 38, wherein the biomarker concentrator further comprises a lysing material combined with the super absorbent polymer for releasing the target biomarker from biological elements, the biological elements comprising at least one of viruses, cells and bacteria.

40. The device of claim 38, wherein the biomarker concentrator further comprises a selectively permeable membrane having pores configured and sized to allow the target biomarker to flow through the selectively permeable membrane toward the detection unit.

41. An electronic biosensor, comprising a substrate with water absorption properties, provided by at least one of a selectively permeable membrane, a super absorbent polymer, a microfluidic material and a core; at least two electrodes formed on the upper surface of the substrate, forming a gap between the two; a functionalized detector arranged in the gap, comprising an electron transport material and a capture molecule, wherein the target molecule captured by the capture molecule will cause at least one of the polarity and conductivity of the electron transport material to change.

42. An electronic biosensor according to claim 41, wherein the target molecule is an element of a capture molecule conjugate, the conjugate comprising at least a linker and the capture molecule, wherein the linker binds the capture molecule to the electron transport material.

43. The electronic biosensor of claim 41, wherein the electron transport material comprises carboxylated carbon nanotubes incorporated into a super absorbent polymer layer, the electron transport layer being cast on a porous hydrophilic polypropylene support.

44. The electronic biosensor of claim 43, wherein the functionalized detectors are patterned on a hydrophilic polypropylene support and the electrodes are formed on a top surface of the hydrophilic polypropylene support.

45. A sensor for detecting a target molecule in a fluid sample, comprising: a detection zone for receiving a fluid sample containing target molecules and having a detection interface functionalized with capture molecules; A gap is formed between the top driving electrode and the bottom driving electrode; as well as A fluid conductor disposed in the gap is used to guide the fluid sample through the gap, wherein the potential applied to the top and bottom drive electrodes drives the target molecules toward the capture molecules, thereby concentrating the target molecules in the portion of the fluid sample received by the detection interface.

46. ​​A sensor according to claim 45, wherein the top and bottom driving electrodes are arranged upstream of the fluid sample flow in the detection area, wherein the fluid conductors are arranged upstream and downstream of the detection area to allow the fluid sample carrying the target molecules to flow through the detection area, and further includes a core for removing excess moisture from the fluid sample upstream of the detection area.

47. A sensor according to claim 45, wherein the top and bottom driving electrodes are arranged in the detection area; and also includes a conductive core for absorbing excess fluid in the sample, wherein the fluid conductor conducts a portion of the fluid sample containing relatively fewer target molecules to the conductive core through the gap, and conducts another portion of the fluid sample containing relatively more target molecules to the capture molecules.

48. A sensor as described in claim 47, wherein the wick includes superabsorbent polymer beads having a shell surface, and the potential applies a shell charge potential to the shell, wherein the shell charge potential is opposite to the target molecule charge potential and drives the target molecule away from the superabsorbent polymer beads, while allowing a portion of the fluid sample to enter through the shell and be absorbed by the beads.

49. The sensor of claim 45, wherein at least one of the top and bottom drive electrodes is comprised of a grid electrode that leaves spaces between conductive elements to allow fluid sample flow.

50. The sensor of claim 45, wherein the fluid conductor comprises at least one of a capillary channel, a microfluidic material, a superabsorbent polymer, and a fluid conducting fiber sheet.

51. A sensor according to claim 45, wherein the sensor includes a field effect transistor having a gate in electrical communication with a detection interface functionalized with a capture molecule, and a source and a drain located on both sides of the gate, and at least a portion of at least one of the top and bottom drive electrodes is arranged in the detection area, and the gate of the sensor includes at least one of the top and bottom drive electrodes.

52. A method for detecting a target molecule from a fluid sample, comprising the steps of: receiving a fluid sample containing a target molecule from a microfluidic channel; transferring a fluid sample from the microfluidic channel to a detection interface of a sensor, the sensor comprising a detection zone for receiving the fluid sample, the detection interface being functionalized with a capture molecule, a top drive electrode and a bottom drive electrode forming a gap therebetween, a fluid conductor disposed in the gap for directing the fluid sample through the gap, wherein an electrical potential applied to the top and bottom drive electrodes drives target molecules toward the capture molecules to concentrate the target molecules in a portion of the fluid sample received by the detection interface, the sensor comprising a field effect transistor having a gate in electrical communication with the detection interface, the gate being functionalized with the capture molecule, and a source and a drain disposed on either side of the gate, wherein at least a portion of at least one of the top and bottom drive electrodes is disposed in the detection zone, and the gate of the sensor comprises at least one of the top and bottom drive electrodes; as well as The potential is intermittently applied to drive the target molecule and the changes in the electrical characteristics of the source, drain and gate are read out.

53. A sensor comprising: substrate; at least one first and second electrode formed on the substrate and defining a gap therebetween, a detection interface including capture molecules disposed in the gap; a drainage hole formed in the substrate adjacent to the gap; a selectively permeable membrane disposed at the drainage hole for allowing a portion of the fluid sample to pass therethrough and blocking at least some of the target molecules; and a fluid absorber, disposed in fluid communication with the selectively permeable membrane, for absorbing a portion of the fluid sample.

54. A lateral flow analyzer, comprising a sample pad provided in a first stage of a lateral flow assay; a selectively permeable membrane adjacent to the sample pad, for allowing a portion of the fluid sample to pass through the sample pad and blocking at least some target molecules so that the target molecules accumulate in another portion of the fluid sample that has not passed through the selectively permeable membrane; a fluid absorber, in fluid communication with the selectively permeable membrane, for absorbing the portion of the fluid sample that has passed through the selectively permeable membrane; and a soluble fluid dam, for blocking another portion of the fluid sample that has accumulated the target molecules from flowing to the next stage of the lateral flow assay.

55. Indoor biosensors, including an air intake for drawing in ambient air; A condenser, used for cooling the ambient air and condensing the moisture in the ambient air into a condensate containing water and at least one target molecule; a condensate detection system for detecting whether the condensate contains at least one target molecule; The condensate detection system includes a biosensor including at least one g-FET biosensor, whose detection interface includes a graphene layer functionalized with capture molecules, wherein the capture molecules are smaller than the Debye screening length.

56. The chamber-scale biosensor of claim 55, further comprising a target molecule concentrator for concentrating at least one target molecule in a fluid biological sample to form a concentrated fluid biological sample.

57. A chamber-scale biosensor as described in claim 56, wherein the target molecule concentrator comprises a selective permeability barrier for allowing excess water in the fluid biological sample to pass through the selective permeability barrier and preventing target molecules in the fluid biological sample from passing through the selective permeability barrier; and an excess water absorption core for absorbing excess water passing through the selective permeability material.

58. A chamber-scale biosensor as described in claim 56, wherein the target molecule concentrator comprises a super absorbent polymer (SAP) for preferentially absorbing water in the EBC into polymer chains of the super absorbent polymer, wherein the target molecule is not absorbed by the polymer chains and flows through the SAP together with the EBC, and when the EBC flows through the SAP, the water content in the EBC is removed to increase the test sample concentration of the target molecule.

59. The chamber-scale biosensor of claim 58, wherein the biosensor comprises capture molecules designed to bind to at least one of FluA, FluB, SARS N-protein, and SARS S-protein.

60. The chamber-scale biosensor of claim 58, further comprising a fluid transfer system for transferring condensate from the condenser to a condensate testing system, the fluid transfer system comprising at least one of a fluid conductor, a collection area, and a microfluidic transfer path for controlling the flow of a fluid biological sample received from an EBC collector; and further comprising a target molecule releasing material placed in at least one of the fluid conductor, the collection area, and the microfluidic transfer path, wherein the target molecule releasing material comprises at least one of a surfactant and a chemical lysing agent.

61. A chamber-scale biosensor as described in claim 55, wherein the condenser includes a condensation-forming surface having relatively low energy surface properties for limiting the adhesion of target molecules to the condensation-forming surface, and further includes a fluid conductor disposed on the condensation-forming surface, wherein the fluid conductor includes a texture structure formed on the condensation-forming surface, the texture structure having relatively high energy surface properties for guiding the EBC to flow in a desired direction.

62. The chamber-scale biosensor of claim 55, further comprising a target molecule release structure for mechanically lysing at least one of a cell wall, a packaging structure and a viral envelope containing the target molecule, wherein the target molecule release structure comprises a lysis structure protruding from at least one of a condensate forming surface and a flow path surface of the EBC, wherein the lysis structure mechanically destroys at least one of a cell wall, a packaging structure and a vial envelope containing the target molecule.

63. A field effect transistor sensor circuit for detecting target molecules in a fluid sample, comprising: A conductive semiconductor substrate having a source region and a drain region, wherein a channel region of one conductive type is defined between the source region and the drain region, and the source region and the drain region have opposite conductive types; an insulator formed on the channel region, wherein the channel region forms a back gate of the field effect transistor; a detection region formed on the insulator, the detection region being used to receive a fluid sample and having a detection interface functionalized with a capture molecule; a top electrode forming a gap with the detection region and forming a liquid gate of the transistor; a fluid conductor disposed in the gap and configured to conduct the fluid sample through the gap; and a driving circuit for applying an electromotive force of one polarity to the top electrode and an electromotive force of the opposite polarity to the back gate, wherein the electromotive force drives the target molecules toward the capture molecules to concentrate the target molecules in a portion of the fluid sample received by the detection interface.

64. The field effect transistor sensor circuit of claim 63 further comprises a detection circuit for applying a voltage to the source region and at least one of the back gate and the top electrode, and detecting a change in current through the drain region that depends on the binding of the target molecule to the capture molecule.

65. The field effect transistor sensor circuit of claim 63, wherein the drive circuit applies an electromotive force periodically and the detection circuit detects changes in current.

66. The field effect transistor sensor circuit of claim 63, wherein a portion of the capture molecules are immobilized on the detection interface at a greater distance from the top surface of the detection interface than another portion of the capture molecules immobilized on the detection interface.

67. A field effect transistor sensor circuit according to claim 63, wherein the driving circuit reverses the polarity of the applied potential to drive non-target molecules out of the detection area, while retaining target molecules captured by capture molecules immobilized on the detection interface at a longer distance in the detection area.

68. The field effect transistor sensor circuit of claim 63, wherein the detection interface comprises at least one of graphene, nanoparticles, aligned carbon nanotubes, vacuum deposited conductive materials, transferred conductive materials, semiconductors, metals, and screen printed conductive inks.

69. The field effect transistor sensor circuit of claim 63, wherein the capture molecule comprises an antibody, an engineered antibody, At least one of aptamers, nanoCLAMPS and CRISPR Cas protein conjugates.

70. The field effect transistor sensor circuit of claim 63, wherein the capture molecule comprises a catalytically inactive CRISPR Cas protein linked to a guide RNA and immobilized on a graphene detection interface.

71. A packaged biosensor semiconductor device, comprising a semiconductor die, comprising at least a source region and a drain region, wherein a channel region is defined between the source region and the drain region; an insulator formed on the channel region, wherein the channel region forms a back gate of a field effect transistor; a detection area disposed on the insulator for receiving a fluid sample and having a detection interface functionalized with captured molecules; a lead frame including a die pad for receiving a semiconductor die, the die pad having leads for conducting electrical signals from a source region, a channel region, and a drain region to an external electronic circuit; and a bottom barrier portion of the packaged semiconductor substrate, exposing the detection interface to the outside; and a top layer barrier part, which has a detection well, wherein the detection well forms an opening and is arranged on the detection interface exposed to the outside for receiving a fluid sample.

72. The packaged biosensor semiconductor device according to claim 68; further comprises a top gate lead, one end of which is arranged on the upper surface of the top barrier portion for connecting to the liquid gate electrode, and the other end is used to connect the liquid gate electrode to an external electronic circuit, wherein the liquid gate electrode defines a gap with the detection area, and is used to electrically communicate with the liquid sample when the liquid sample is placed in the detection well.

73. The packaged biosensor semiconductor device of claim 71, wherein target molecules in a fluid sample are detected by changes in electrical properties occurring at one or more electrodes of the source, drain, back gate, and liquid gate.

74. The packaged biosensor semiconductor device of claim 71, wherein the capture molecules comprise antibodies, engineered antibodies, At least one of aptamers, nanoCLAMPS and CRISPR Cas protein conjugates.

75. The packaged biosensor semiconductor device of claim 71, wherein the capture molecule comprises a catalytically inactive CRISPR Cas protein conjugated to a guide RNA and immobilized on a graphene detection interface.

76. The packaged biosensor semiconductor device according to claim 71 further includes a Z-axis conductive adhesive for establishing electrical and mechanical connections between the leads of the packaged integrated circuit semiconductor device, the corresponding leads of the external circuit and the connecting pads, wherein the Z-axis conductive adhesive includes a manually removable protective film for protecting the Z-axis conductive adhesive before the packaged field effect transistor sensor establishes electrical and mechanical connections with the external circuit.

77. The packaged biosensor semiconductor device of claim 71 further comprising additional electronic semiconductor circuit elements packaged with the field effect transistor sensor to form a packaged integrated circuit semiconductor device.

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