Point-of-care saliva testing device and method

Abstract

According to some embodiments, a point-of-care saliva test device includes a biological fluid collection device, a cartridge device, a reader device, and / or a software-based detection interface system. The analyte detection system may be used to detect the presence and / or amount of one or more target analytes.

Description

Technical Field

[0001] The present disclosure relates to point-of-care saliva testing devices and uses thereof. Background Art

[0002] Determining and / or obtaining information about an individual's fitness level or general health and wellness and / or obtaining diagnostic information may be expensive, invasive, and / or inconvenient, such as requiring a visit to a physician.

[0003] For fitness measurements, solutions include wearable devices, step counters, and more complex treadmill and VO2max tests. For general health and wellness measurements, blood tests and mail-in tests using blood, saliva, stool, urine are used. For diagnostic determination of a condition, blood tests and specific scans (CAT, X-ray, MRI, etc.) may be required.

[0004] For fitness determination, current solutions are either only proxies for true health (step counters, wearables) or are very specific to one form of health and very expensive (treadmills, VO2max tests). In addition to wearables, professionals are currently needed to measure fitness, health and wellness or diagnostic biomarkers. General health and wellness tests performed through blood tests in a doctor's office are expensive, invasive and inconvenient. Mailing test kits, although more convenient, are still slow and expensive, and can still be invasive. Finally, diagnostic determination of the condition (blood tests, MRIZPET / CAT / X-ray scans, etc.), although considered the gold standard for sensitivity and specificity or the condition, are very expensive, inconvenient and invasive. In addition, any physical examination performed by a healthcare provider is also expensive, invasive and / or inconvenient. Summary of the invention

[0005] According to some embodiments, a point of care test (POCT) system is provided. In addition to other exemplary benefits, the exemplary POCT system can allow on-demand, portable, non-invasive (saliva) testing of fitness, general health and / or health care, and / or can be used as a diagnostic tool for detecting serious diseases. The exemplary POCT system can, for example, provide results on the order of seconds to minutes, and can provide results in a less expensive manner. The POCT system can operate without the need for medical professionals or other personnel.

[0006] According to some embodiments, a point-of-care testing (POCT) system allows for on-demand, portable, non-invasive (saliva) testing of fitness, general health and / or wellness, and / or can be used as a diagnostic tool to detect disease, with results available in seconds to minutes, at a cost of only a few dollars per test, and without the need for medical professionals or other personnel.

[0007] According to some embodiments, a point-of-care saliva testing device comprises a biological fluid collection device, a cartridge device, a reader, and / or a software-based detection interface system. The analyte detection system can be used to detect the presence and / or amount of one or more target analytes.

[0008] According to some embodiments, a biological fluid collection device intended for collecting biological fluids comprises a handle and a pipette tip. According to some embodiments, the biological fluid collection device further comprises a coupling mechanism by which the device can be coupled to a cartridge base. According to some embodiments, the biological fluid collection device further comprises an extendable element. According to some embodiments, the extendable element is a telescopic arm.

[0009] According to some embodiments, an electrochemically active sensor chip comprises a substrate comprising an electrically insulating material; a plurality of electrode holes arranged in different positions on the substrate, wherein each hole comprises a plurality of electrodes, the electrodes comprising a surface comprising a chemical substance, the chemical substance comprising a catalyst or a reactant corresponding to an analyte in saliva; wherein the electrodes are capable of sustaining a redox reaction with the chemical substance and the analyte to generate an electrical signal. According to some embodiments, the sensor chip further comprises a spacer layer coupled to the substrate, which allows saliva to enter the hole; and a hydrophilic top cover, which directs saliva to the hole and seals the chip.

[0010] According to some embodiments, an electrochemically active sensor chip comprises a substrate comprising an electrically insulating material; and a plurality of electrode holes arranged in different positions on the substrate, wherein each hole comprises a plurality of electrodes, the electrodes comprising a surface comprising a chemical substance, the chemical substance comprising a catalyst or reactant corresponding to an analyte in saliva; wherein the electrodes are capable of sustaining a redox reaction with the chemical substance and the analyte to generate an electrical signal. According to some embodiments, the sensor chip further comprises a spacer coupled to the substrate, which allows saliva to enter the hole; and a hydrophilic top cover, which directs saliva to the hole and seals the chip.

[0011] According to some embodiments, a cartridge base includes a cartridge body defining a collection device cavity therein, wherein a first side of the cartridge body has an opening leading to the cavity, wherein a portion of a biological fluid collection device is configured to be inserted into the collection device cavity of the cartridge body; and the cartridge body defines a sensor cavity; and a biosensor chip in the sensor cavity.

[0012] According to some embodiments, a reader unit comprises electrical components for wireless data transmission, a potentiostat, and a battery; wherein the reader unit is configured to collect data, wherein the reader unit is configured to interface with electrodes by contacting a cartridge.

[0013] According to some embodiments, a reader unit includes: a cartridge receptacle configured to receive a cartridge having a biosensor therein, the biosensor being configured to generate one or more electrical signals in response to a biological fluid contacting the biosensor; and a processor communicatively coupled to the biosensor of the cartridge received in the cartridge receptacle and configured to determine the concentration of one or more biomarkers in the biological fluid.

[0014] According to some embodiments, a point-of-care saliva testing system comprises: a biological fluid collection device configured to collect a biological fluid; a cartridge base configured to receive the collection device, the cartridge base having a biosensor therein, wherein the cartridge base is configured to transfer the collected biological fluid from the collection device to the biosensor having a plurality of electrical pads thereon after the collection device having the collected biological fluid thereon is received within the cartridge base; and a reader device configured to receive the cartridge base therein, wherein the reader device has a plurality of electrical contacts configured to electrically couple with the electrical pads of the biosensor when the cartridge base is received in the reader device, wherein the system is configured to detect the presence and / or amount of one or more target analytes in the collected biological fluid that has been transferred to the biosensor that is electrically coupled to the electrical contacts of the reader.

[0015] According to some embodiments, a method for detecting an analyte in saliva includes: reducing a chemical substrate in the saliva to a second product in a chemical reduction process, in which the second product obtains electrons; supplying the extracted electrical energy to electrodes of an electrochemical sensor; and detecting an electrical signal generated as a result of an oxidation-reduction reaction involving the analyte in the saliva and a chemical reagent coupled to at least one electrode of the electrochemical sensor from the electrodes of the electrochemical sensor in contact with the saliva.

[0016] The above summary is not intended to represent every embodiment or every aspect of the present disclosure. Instead, the above summary only provides examples of some of the novel aspects and features set forth herein. When taken in conjunction with the accompanying drawings and the appended claims, the above-mentioned features and advantages, whether individually or in any combination, which are inventive, as well as other features and advantages of the present disclosure, will become apparent from the following detailed description of representative embodiments and modes for implementing various embodiments of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1E A point of care testing ("POCT") system according to some embodiments of the present disclosure is shown.

[0018] Figure 2 A biosensor chip according to some embodiments of the present disclosure is shown.

[0019] Figure 3 An exemplary step-by-step protocol for using a POCT system comprising a handheld device and a disposable biological fluid or saliva test cartridge is shown according to some embodiments of the present disclosure.

[0020] Figure 4A is a perspective view of a disposable biological fluid or saliva test cartridge according to some embodiments of the present disclosure, showing both the biological fluid or saliva collection device and the cartridge base in an extended mode or position.

[0021] Figure 4B is a perspective view of some embodiments of a disposable biological fluid or saliva test cartridge according to the present disclosure, wherein a biological fluid or saliva collection device is in a collapsed position or mode and inserted into the cartridge base.

[0022] Figure 4C An exemplary biological fluid or saliva collection device according to some embodiments of the present disclosure is shown, along with an enlarged view of an interior portion of a telescoping arm.

[0023] Figure 5A A person's head is shown with a biological fluid or saliva collection device inserted into the person's oral cavity and the tip of the collection device in contact with the vicinity of the parotid gland and duct according to some embodiments of the present disclosure.

[0024] Figure 5B An external view of a person's head is shown according to some embodiments of the present disclosure, wherein a saliva collection device such as Figure 5A is shown inserted into the person's mouth, and the handle of the collection device is located outside the mouth.

[0025] Figures 6A-6F is an illustration of a biological fluid or saliva collection handle according to some embodiments of the present disclosure.

[0026] Fig. 7A is an exploded perspective view of a cartridge base according to some embodiments of the present disclosure, and Figure 7B This is its assembly perspective view.

[0027] Figure 7C is a perspective view of a latch for a cartridge base according to some embodiments of the present disclosure.

[0028] Figure 7D-7I is a view of a cartridge base (cartridge body) according to some embodiments of the present disclosure.

[0029] Fig. 8A is an exploded perspective view of subassemblies of a disposable biological fluid or saliva test cartridge or kit showing the collection device (with telescoping arm omitted), cartridge base, biosensor chip, and cartridge latch.

[0030] Figure 8B is an assembled perspective view of a biological fluid or saliva test cartridge according to some embodiments of the present disclosure, illustrating an exemplary method for packaging the subassemblies prior to use and during analysis.

[0031] Figure 8C-8D is an assembled perspective cross-sectional view of a disposable biological fluid or saliva test cartridge according to some embodiments of the present disclosure, and Fig.8F is a side cross-sectional view thereof when not assembled.

[0032] Fig. 8E is a top view of a biosensor chip according to some embodiments of the present disclosure.

[0033] Figure 8G is a perspective view of a cartridge according to some embodiments of the present disclosure, the cartridge containing a collection device that does not use a telescoping arm.

[0034] Fig.9A is an exploded perspective view of a collection device, a cartridge base, and a reader, which may take the form of a handheld POCT testing device, according to some embodiments of the present disclosure.

[0035] Fig. 9B is a cross-sectional view of a reader / POCT testing device according to some embodiments of the present disclosure, wherein a cartridge is inserted into a cartridge receptacle.

[0036] Fig. 9C is an exploded view of a reader / test device and some of its component parts according to some embodiments of the present disclosure.

[0037] Fig.9D is a perspective view of a cartridge receptacle having an aperture therein, according to some embodiments of the present disclosure.

[0038] Figure 10-12 is a plan view of a PCB (printed circuit board) circuit diagram according to some embodiments of the present disclosure.

[0039] Fig.13A is a top view of a biosensor chip according to some embodiments of the present disclosure.

[0040] Fig. 13Bis an exploded perspective view of a sensor chip according to some embodiments of the present disclosure, showing various constituent layers.

[0041] Fig. 13C A top view or a plan view of a screen-printed electrode layer of a biosensor chip according to some embodiments of the present disclosure is shown.

[0042] Fig.13D 1 is an exploded perspective view of a screen-printed electrode layer and a dielectric layer of a biosensor chip according to some embodiments of the present disclosure. The screen-printed electrode layer and the dielectric layer are collectively referred to as a sensor chip substrate.

[0043] Fig.13E yes Fig.13D A top view or a plan view of a sensor chip substrate according to some embodiments of the present disclosure is shown in FIG.

[0044] Fig.13F is a schematic side view showing various screen-printed layers of a sensor chip substrate according to some embodiments of the present disclosure.

[0045] Fig.14A is a diagram of a layer stack of an enzyme sensing membrane for a lactate sensing membrane or an enzyme sensing membrane in general according to some embodiments.

[0046] Figures 14B-14C An embodiment of a biosensor chip for measuring lactate is shown, along with an overall schematic diagram of the reactions used according to some embodiments.

[0047] Fig.15A is a diagram of a layer stack of an ion-selective hydration sensing membrane for use in a hydration sensor or generally an ion-selective electrode (ISE) according to some embodiments.

[0048] Fig. 15B is a graph showing the stability response of a sensor to an ion-selective reaction obtained by measuring different concentrations of NaCl over a 1 minute period, according to some embodiments.

[0049] Figures 15C-15E is a graph showing the response of a sensor at different electrolyte concentrations according to some embodiments, wherein Fig. 15C shows potassium, Fig.15D shows sodium, Fig.15E Chlorine is shown.

[0050] Fig.15F is a functional block diagram of a reader / test device and a sensor according to some embodiments of the present disclosure.

[0051] Fig.16Ais a top or plan view of a strip for automated manufacturing comprising a plurality of screen-printed pads according to some embodiments of the present disclosure.

[0052] Fig. 16B is a top or plan view of a spacer strip for automated manufacturing according to some embodiments of the present disclosure.

[0053] Fig. 16C is a top or plan view of a cap strip for automated manufacturing according to some embodiments of the present disclosure.

[0054] Fig.17A is an exploded perspective view of components of an immunoassay system according to some embodiments of the present disclosure.

[0055] Fig. 17B is an exploded perspective view of components of a Cube reader of an immunoassay system according to some embodiments of the present disclosure.

[0056] Figure 17B' is an exploded perspective view of components of a Cube reader of an immunoassay system according to some other embodiments of the present disclosure.

[0057] Fig. 17C is a perspective view of a test bench according to some embodiments.

[0058] Fig.17D is a perspective view of a magnetic gear assembly having magnetic gears according to some embodiments.

[0059] Fig.17E is a perspective view of a manifold according to some embodiments.

[0060] Fig.17F is a schematic diagram depicting fluid flow in a manifold and a microfluidic chip according to some embodiments.

[0061] Fig.18A is an exploded perspective view of an immunoassay biosensor chip according to some embodiments.

[0062] Fig.18B is a plan view of a biological fluid (eg, saliva) collector adapter for receiving a biological fluid collection device according to some embodiments.

[0063] Figures 18C-18E is a top view of the multiple layers of an immunoassay biosensor chip.

[0064] Figures 19A-19B and 19E-19F are top views of multiple layers of an immunoassay biosensor chip according to some embodiments.

[0065] Figures 19C-19DAn exemplary heating PCB is shown, with an exemplary wax valve thereon shown.

[0066] Figure 19G1 is a three-dimensional perspective view showing microfluidic pathways and wells within a disposable microfluidic chip according to some embodiments.

[0067] Figure 19G2 It means that the fluid passes through Figure 19G1 Block diagram of the physical flow.

[0068] Fig.19H is a perspective bottom view of an assembled microfluidic chip according to some embodiments, Fig.19I This is its top view. Fig.19J This is its bottom view.

[0069] Figure 19K is a functional block diagram of the electrical components of the immunoassay system 1700 according to some embodiments of the present disclosure.

[0070] Fig. 20 is a flow chart of a general immunoassay process performed during an analysis within an immunoassay device according to some embodiments.

[0071] Fig.21 is a flow chart of the chemical processes within an immunoassay device according to some embodiments.

[0072] Fig. 22 is a graph of the electrochemical reaction of Cortisol according to some embodiments of the present disclosure.

[0073] Fig.23 is a graph of sensor responses to different concentrations of Cortisol according to some embodiments of the present disclosure.

[0074] Figures 24A-24C is a flow chart illustrating a testing method according to some embodiments of the present disclosure.

[0075] Figures 25A-25C is a software flow chart illustrating a testing method according to some embodiments of the present disclosure.

[0076] Figures 26A-26F 26H-26P illustrate exemplary displays on a POCT device and / or a remote device such as a smartphone according to some embodiments of the present disclosure.

[0077] Figure 26G The collection of a biological fluid, such as saliva, according to some embodiments of the present disclosure is shown.

[0078] Figures 27A-27E are exemplary screenshots of a smartphone running an application (App) according to some embodiments of the present disclosure.

[0079] Fig.28A Various devices associated with different tests or general health categories are shown, with associated scores shown on displays of the devices, and examples of qualitative health categories with underlying biomarkers associated with samples are provided, according to some embodiments of the present disclosure.

[0080] Fig.28B A POCT device communicating test results and scores to a smartphone is shown according to some embodiments of the present disclosure.

[0081] Fig.29A is a graph of an accuracy study of a POCT sensor configured for measuring lactate according to some embodiments of the present disclosure compared to an industry standard lactate concentration analyzer from YSI (YSI Benchtop Analyzer).

[0082] Fig.29B is a graph showing the response of sialyllactate to incremental physical exercise, according to some embodiments of the present disclosure.

[0083] Fig.29C is a graph of a stability study obtained by graphically comparing measurements obtained using a POCT sensor configured to measure lactate from day 1 of sensor manufacturing until day 260 after the sensor manufacturing, according to some embodiments.

[0084] Fig.29D is a graph showing batch-to-batch manufacturing variability and robustness obtained by plotting the biomarker concentrations measured in three different production batches applied to a biosensor chip, according to some embodiments.

[0085] Fig.30 Disposable biofluid or saliva testing cartridges of the present disclosure are shown for various potential fitness categories (nutrition, energy, burn, hydration, etc.).

[0086] Fig.31 Shown are disposable biofluid or saliva testing cartridges of the present disclosure for health and wellness categories (ie, aging, defense, mood, GI health, stress) or gender-based cartridges (eg, women's health).

[0087] Fig.32 Disposable biofluid or saliva testing cartridges for specific diseases or medical conditions (eg, acidosis) or personalized medicine for which an individual (eg, "John Doe") has a dedicated cartridge are shown according to some embodiments of the present disclosure.

[0088] Figures 33A-33CA table showing examples of health categories and exemplary associated basic biomarkers according to some embodiments of the present disclosure is shown.

[0089] Fig.34A Some screens on a smartphone according to some embodiments of the present disclosure are shown, showing a hydration health category score (left) and related basal biomarker concentrations (center) and historical scores (right) displayed on a mobile application.

[0090] Fig.34B A mobile software application is shown that displays basic biomarker concentrations for the hydration health category (left) and self-guided actionable insights (center, right) according to some embodiments of the present disclosure.

[0091] Fig.34C A mobile software application is shown that displays actionable insights for self-guidance (left, right) and external links to advisors, coaches, medical professionals, or others (center) according to some embodiments of the present disclosure.

[0092] The present disclosure is susceptible to various modifications and alternative forms, and some representative embodiments have been shown by way of example in the accompanying drawings and will be described in detail herein. However, it should be understood that aspects of the present invention are not limited to the forms shown in the accompanying drawings. On the contrary, the present disclosure encompasses all modifications, equivalents, combinations and alternatives that fall within the spirit and scope of the present invention as defined by the appended claims. DETAILED DESCRIPTION

[0093] The term "about" means a possible variation of up to ±10%. Orientation terms such as up, down, top, bottom, left, or right are included in the present disclosure to aid in understanding the present disclosure, for example with reference to one or more of the accompanying drawings, but this is not limiting, as it is understood that the orientation of the objects and surfaces described herein can vary, and therefore the relative orientation terms can also vary.

[0094] Directional terms such as upward, downward, top, bottom, left, and right are exemplary and are made with reference to the various figures of the present disclosure to aid in understanding the various embodiments disclosed therein. It should be understood, however, that the orientation of components and devices described herein may change, for example, the device may be rotated or inverted relative to what is shown in a particular figure, so that what may be referred to as the top of a component or device may become located below what may have been referred to as the bottom of a component or device.

[0095] The various devices, systems, kits and methods disclosed herein are directed to detecting a target analyte within a biological fluid sample obtained from a specimen. In certain embodiments, a chemical reaction is employed to achieve such detection.

[0096] 1. Overview of POCT system

[0097] According to some embodiments, a point of care testing ("POCT") system is provided to allow testing or analysis of a biological fluid of a person and to allow determination of one or more characteristics of the biological fluid from which one or more characteristics about the person can be determined.

[0098] Figure 1A and 1B A point of care testing ("POCT") system 10 is shown according to some embodiments of the present disclosure. According to some embodiments, the POCT system 10 comprises a handheld POCT device or "Stone" or reader 100 including a display 110. Figure 1A In the example, the display 110 shows the score, in this case, a score of 63% for "energy". The system 10 also includes one or more cartridges 200. For example, in Figure 1A A single-use cartridge 200 for measuring energy is shown in FIG. 1 . Each cartridge 200 includes a cartridge base 700 and a cartridge biological fluid collection device 400 . Figure 1A is a perspective view of the POCT device 100 and the cartridge 200 of the system 10 .

[0099] According to some embodiments, the POCT system 10 is composed of a portable measurement and / or display device 100 and a disposable test cartridge 200. According to some embodiments, the device 100 includes electronic components, a display 110 and / or a communication mechanism. According to some embodiments, the POCT system 10 is designed for analyzing saliva, and the disposable cartridges each contain a device 400 for collecting a saliva sample and an embedded biosensor in a cartridge base 700, the biosensor being capable of sensing characteristics and / or concentrations of one or more substances in a saliva sample, such as lactate concentration in saliva and / or other biological fluids.

[0100] According to some embodiments, the point of care test (POCT) device 100 is referred to as "Stone". According to some embodiments, the system 10 comprises Stone 100 and a disposable cartridge 200. The system 10 uses the disposable cartridge 200 to collect a saliva sample and transfer the sample to a cartridge base 700 and / or Stone 100. The system 10 measures, for example, salivary biomarkers (analytes, compounds and / or elements), which may indicate, for example, health and wellness in a broader sense, and / or may also be used as a diagnostic screen with FDA or other government approval.

[0101] According to some embodiments, system 10 may be used to generate and provide a general health, fitness, and / or wellness score and / or basic biomarker concentration measurements.

[0102] According to some embodiments, Figure 1B As shown in FIG, the POCT system 10 comprises three main components: a handheld StonePOCT device 100, a single-use cartridge 200, and optionally a mobile or smartphone 191 or some other remote device (e.g., Fig.26J According to some embodiments, the App is not required for testing, but may be required for updating the POCT device 100 and for more complex interactions with the Internet and / or sharing of results.

[0103] Figure 1B A perspective view of a POCT device 100 and a cartridge 200 of a system 10 according to some embodiments is provided. Figure 1B A perspective view of a first end of the POCT device or reader 100 is provided, such as a downward perspective view of the top end of the POCT device or reader 100 . Figure 1C A perspective view of a second opposing end of the POCT device or reader 100 is provided, such as an upward perspective view of the bottom end of the POCT device or reader 100 and the uncoupled cartridge 200 . Figure 1D A perspective view of a first side of a POCT device or reader 100 and a cartridge 200 coupled thereto (eg, inserted therein) is provided. Figure 1E A perspective view of a second opposing side of the POCT device or reader 100 and a cartridge 200 coupled thereto (eg, inserted therein) is provided.

[0104] According to some embodiments, Figure 1B An exemplary analyte detection system 10 is shown. The detection system 10 can include a biological fluid collection device 400, a cartridge base 700, a Stone device 100, and / or a software-based detection interface system 190. The analyte detection system 10 can be used to detect the presence and / or amount of one or more target analytes.

[0105] The collection device 400 may be exposed to a biological fluid sample such as, but not limited to, blood, plasma, urine, saliva, mucus, or other biological fluid to determine the presence and / or amount of one or more target analytes in the sample.

[0106] According to some embodiments, the cartridge base 700 is configured to analyze a sample collected using the collection device 400. As described in Figure 1C As seen in the figure, the cartridge base 700 may contain a biosensor chip 1300, so that the collected sample can be analyzed within the cartridge base 700. Based on the analysis, the cartridge base 700 is configured to generate an electrical signal indicative of the presence and / or amount of one or more target analytes within the sample.

[0107] The Stone or reader 100 is configured to be electrically coupled to the cartridge base 700 to allow an electrical signal generated by the cartridge base 700 indicating the presence and / or amount of one or more target analytes in the sample to be transmitted to the Stone 100, and / or to provide an electrical signal and / or power to the cartridge base 700 and / or the chip 1300 therein. Figure 1B The cartridge base 700 is inserted into the cartridge receptacle 140 in the Stone 100 as shown in the figure, so that the corresponding electrical connectors of the cartridge base 700 and the Stone 100 contact each other, and the cartridge base 700 can be electrically coupled to the reader 100. According to some embodiments, the reader 100 can include a computer-readable medium having instructions, which, when executed by a processor of the reader 100, cause the electrical components of the reader 100 to perform steps for analyzing a sample on the biological fluid collection device 400 and / or receiving a signal from the biosensor chip 1300 located in the cartridge base 700. According to some embodiments, until the cartridge base 700 and / or the chip 1300 are electrically coupled to the reader 100 and the biological fluid collection device 400 is suitably disposed in the cartridge base 700, such as Figure 1D and 1E As shown in , the instructions are executed.

[0108] According to some embodiments, the biological fluid collection device 400 and the cartridge base 700 are each disposable and designed for one-time use, while the reader 100 is designed for multiple use and is used to receive many different cartridge devices 200 throughout the life of the reader 100 so that many samples can be analyzed by the reader 100 to determine the presence and / or amount of one or more target analytes in the corresponding samples. This configuration is expected to promote sanitary use of the system 10, because the components exposed to the biological fluid sample are disposable, while reducing costs, because components such as the reader 100 with more expensive electronics and parts can be reused.

[0109] like Figure 1B As shown in , the software-based detection interface system 190 can be installed and run on a mobile computing device 191 to allow a user to view analyte detection test results and actionable insights therein, for example, on a display 192 of the computing device 191. The computing device 191 can be, for example, a smart phone, a smart watch, a tablet or a wearable device or a computer such as a laptop. According to some embodiments, the computing device 191 is a mobile device. As shown in Figure 1BAs shown in , the reader 100 can communicate wirelessly with the computing device 191 to transmit data indicating the presence and / or amount of one or more target analytes based on the electrical signals generated within the cartridge base 700, although the connection can alternatively or additionally be wired. The communication can be direct (e.g., direct wireless or wired communication) or indirect (e.g., through a server, host device, or cloud-based system). The software-based detection interface system 190 can include a computer-readable medium having instructions that, when executed by a processor of the computing device 191, cause the display 192 to display information indicating the presence and / or amount of one or more target analytes.

[0110] Additionally or alternatively, the reader 100 may include a display 110, such as Figure 1A In such an embodiment, the reader 100 may include a computer-readable medium having instructions that, when executed by a processor of the reader 100, cause the display 110 to display information indicating the presence and / or amount of one or more target analytes and actionable insights thereof, and in some embodiments, wirelessly transmit data indicating the presence and / or amount of one or more target analytes to a computing device 191.

[0111] like Figure 1C As shown in , according to some embodiments, biosensor 1300 is contained in cartridge 200. According to some embodiments, multiple different biomarker chemistries can be combined in one biosensor / cartridge 1300 / 200 so that multiple biomarker tests can be done in parallel. According to some embodiments, specific biomarkers related to a broader health category such as "hydration" are combined. For example, a hydration cartridge can contain chemistries that can measure sodium, potassium, and chloride simultaneously.

[0112] According to some embodiments, when the test is complete (which occurs within seconds to minutes according to some embodiments), the biofluid or saliva test cartridge 200 can be ejected and a score, such as a health, fitness or wellness score, such as a hydration score, can be displayed on the display 110 of the Stone 100 and / or through an associated application (#1). According to some embodiments, the cartridge 200 is designed as a disposable, single-use cartridge. According to some embodiments, basal / relevant biomarker concentrations (e.g., sodium, potassium, chloride measurements or scores) can be displayed optionally and / or additionally on the Stone 100 or through an application or remote device (e.g., device 191).

[0113] Additionally, according to some embodiments, other biomarkers may be grouped in other ways to provide more meaningful health and fitness data to the everyday user - energy, fat burn, nutrition, stress, etc. For example, an "energy" category might measure basic biomarkers such as lactate, glucose, and testosterone in addition to a hydration category.

[0114] According to some embodiments, health categories are defined based on the test results, such as providing self-guided actionable insights to improve health scores, and / or additional information may be provided to the user through Stone100 or the app. According to some embodiments, scores and biomarker concentrations may also be shared with others - friends, family, coaches, trainers, or even healthcare professionals to gain even more insights.

[0115] Alternatively or in addition, outside of health and fitness, measurement of salivary biomarkers may also be used as a diagnostic screen for serious, chronic or transient health conditions: for example cancer, sepsis, congestive heart failure, etc.

[0116] According to some embodiments, the point of care testing (POCT) system 10 uses saliva to provide fitness, health, and / or wellness related and / or diagnosis related information and / or one or more scores or values.

[0117] According to some embodiments, the POCT system 10 determines and provides information about a person's fitness level or general health and wellness, and / or is used to provide diagnostic information in a manner that is low-cost, invasive, and / or inconvenient (eg, compared to having to visit a doctor).

[0118] In general, system 10 provides electrochemical measurements, thereby directly correlating the electrical signature of a chemical reaction to the concentration of a biomarker in a biological fluid.

[0119] Figure 2 A biosensor chip 1300 according to some embodiments of the present disclosure is shown.

[0120] Figure 3An exemplary step-by-step scheme for using POCT Stone 100 according to some embodiments of the present disclosure is shown. According to some embodiments, in step 1, the user inserts the entire test cartridge 200 (base 700, saliva collection tip 400) into the POCT device 100, and then (step 2) removes the biological fluid or saliva collection device 400 from the cartridge base 700 to collect a saliva sample from the oral cavity (e.g., his or her oral cavity). Once completed, the user then reinserts the collection device 400 into the cartridge base 700 (step 3). According to some embodiments, the system 10 automatically senses the chemical reaction between the saliva sample and the biosensor chip 1300, thereby starting the test. According to some embodiments, the automatic sensing works by maintaining a constant potential between any two electrode holes. After adding saliva, there is a resistance change on these electrodes, thereby changing the measured current. This change can be quantified and calibrated according to the biological fluid. This change triggers the processor or microcontroller 102 to start the test process. Once the test is completed, information about the test, such as the test score, is displayed on a display (step 4), such as the display 110 of the POCT device 100 and / or a different display, such as a display on a smartphone, tablet, watch, etc. In step 5, the used cartridge 200 can be ejected or removed from the POCT device 100 so that the POCT device 100 can receive a new cartridge 200.

[0121] 2. Exemplary Cartridges and Sample Collection Devices

[0122] The sample collection device of various embodiments is configured to collect a sample from a specimen. The sample collection device can be configured to collect biological fluids from any desired area or location, such as from the mouth, throat, urine, blood, plasma or saliva. An exemplary sample collection device includes a unit that collects biological fluids and can be compressed to distribute the sample into an analytical device.

[0123] The size and shape of the sample collection device of various embodiments are designed to collect a sufficiently large sample from an appropriate location of the sample so that the presence and / or amount of one or more target analytes in the sample can be detected using other devices described below. For example, for certain target analytes such as biomarkers contained in saliva, the collection device can be a telescopic rod with a liquid suction tip so that saliva from the parotid gland can be absorbed to optimize the analyte concentration. The sample collection device for collecting fluids (e.g., urine, blood, plasma, or saliva) can include a part for compressing the absorption portion of the device to discharge the sample absorbed on the absorption portion to analyze the discharged sample.

[0124] Figure 4Ais a perspective view of a disposable biological fluid or saliva test cartridge 200 according to some embodiments of the present disclosure, showing both the biological fluid or saliva collection device 400 in an extended mode or position and a cartridge base 700 serving as a collection container. According to some embodiments, the biological fluid collection device 400 automatically extends after being removed from the cartridge base 700.

[0125] Figure 4B is a perspective view of a disposable biological fluid or saliva test cartridge 200 according to some embodiments of the present disclosure, wherein a biological fluid or saliva collection device 400 is in a collapsed position or mode and inserted into a cartridge base 700.

[0126] Figure 4C An exemplary biological fluid or saliva collection device 400 is shown, along with an enlarged view of an interior portion of a telescoping arm, according to some embodiments of the present disclosure.

[0127] According to some embodiments, the disposable cartridge 200 consists of two parts: a cartridge base 700 that accommodates the electrochemical biosensor 1300 and a biological fluid or saliva collection device 400. In order to test, the user inserts the cartridge 200 into the Stone 100, and then removes the saliva collection device 400 from the cartridge base 700 to collect a saliva sample from the oral cavity of a human or other animal or mammal (e.g., the oral cavity of the user). According to some embodiments, a saliva sample from the oral cavity of a human is collected near the parotid duct of the human. Once the sample collection is completed (which takes less than 5-20 seconds in some embodiments), the saliva collection device 400 is inserted back into the cartridge base 700. When reinserted into the cartridge base 700, the moist collection tip 480 is compressed, releasing the saliva sample into the cartridge base 700, such as into the bottom chamber or reservoir in the cartridge base.

[0128] Figure 4A A cartridge collection device 400 is shown according to some embodiments. Figure 4A As shown in FIG. 4 , the cartridge collection device 400 comprises a handle 450, a connection mechanism 454 that secures the device 400 to a cartridge base 700, an extendable arm 460 that may take the form of a telescoping arm, and a swab tip 480. The cartridge collection device 400 is used as a biological fluid (eg, saliva) sample collector.

[0129] Figure 4C Shows Figure 4A A partial cross-sectional view of a portion of a telescoping arm 460 of a cartridge collection device 400 of the device illustrates an internal spring or spring mechanism 467.

[0130] When the cartridge collection device 400 is removed from its storage or cartridge base 700, it may or may not automatically expand to its full length. According to some embodiments, when the cartridge collection device 400 is removed from its storage or cartridge base 700, the spring or spring mechanism 467 automatically transitions the arm 460 from the retracted or shortened position to the extended position. After the arm 460 is in the extended position, the user can use the handle 450 to insert the cartridge collection device 400 into the oral cavity (e.g., mouth) for collecting saliva.

[0131] According to some embodiments, biological fluid collection device 400 is configured to collect a small amount of sample to be analyzed and is configured to be fully or partially inserted into cartridge base 700 after sample collection. Figure 4A and 4C As shown in , biological fluid collection device 400 includes a handle 450 , an extendable arm 460 , which may take the form of a hollow telescoping arm, and a biological fluid collection tip 480 . Figure 4C The biological fluid collection device 400 is further illustrated with a partial cross-sectional view of a portion of a telescoping arm 460 illustrating a telescoping arm spring or spring mechanism 467. The telescoping arm 460 is shown in an extended position.

[0132] According to some embodiments, the biological fluid collection tip 480 can be secured to the end of the telescoping arm 460 using a dental grade adhesive (eg, dental glue).

[0133] According to some embodiments, the biological fluid collection device 400 may not include the telescoping arm 460 , but may only include the biological fluid collection tip 480 .

[0134] like Figure 4C As shown in FIG. 4 , the telescopic arm 460 may include a spring or spring mechanism 467, a proximal joint 463, an intermediate joint 464, and a distal joint 465, wherein the proximal joint 463 and the intermediate joint 464 may be hollow cylinders, and the distal joint 465 may be a solid cylinder. The spring mechanism 467 may be composed of a proximal coil 416 and a distal coil 417. The spring mechanism 467 is located within the telescopic arm and engages with the slot 456 (see FIG. 467 ) of the threaded shaft 454 at the proximal coil 416. Figure 6B) and attached to the handle 450 on the slot 456. The distal coil 417 is connected to the distal portion of the telescopic arm at the distal joint 465, so that the spring mechanism 467 applies pressure to the distal joint 465 when at rest, keeping the telescopic arm 460 rigid and extended. According to some embodiments, the three joints have concentric diameters, wherein the distal joint 465 is assembled within the intermediate joint 464, and the intermediate joint 464 is assembled within the proximal joint 463, so that when the collection device 400 is inserted into the cartridge base 700 and rotated on the threaded shaft 454, the telescopic arm 460 can be retracted, starting with the distal joint 465 retracting into the intermediate joint 464, then the intermediate joint 464 retracting into the proximal joint 463, and then the proximal joint 463 retracting into the slot 456, until the telescopic arm 460 is completely retracted into the handle 450. When the telescoping arm 460 is retracted into the handle 450 , the spring mechanism 467 applies pressure that resists the torque of the handle 450 rotating on the cartridge base 700 , thereby compressing the collection tip 480 and dispensing any biological fluid contained therein into the cartridge base 700 .

[0135] According to some embodiments, telescoping arm 460 is extended to a length that allows for collection of samples directly at parotid salivary gland 524 (see Figure 5A ), to sample specifically at the gland, rather than by pooling indiscriminately from all glands, because saliva from the parotid glands has a higher concentration of useful biomarkers and a faster response to changes in blood biomarker concentrations. During analysis, the telescoping arm 460 is further retracted into the cartridge base 700, which reduces possible contamination from the user's fingers and allows for a smaller form factor prior to use.

[0136] According to some embodiments, the biological fluid collection tip 480 is a piece of absorbent material used to facilitate the collection, storage, and dispensing of the biological fluid sample in the cartridge base 700 during analysis. The biological fluid collection tip 480 can be sized to fit within the channel 720 of the cartridge base 700 (see Fig.7I ), so that when the biological fluid collection device 400 is inserted into the cartridge base 700, an appropriate amount of biological fluid for analysis can be dispensed into the sensor chip 1300 during the application of pressure.

[0137] According to some embodiments, the volume and absorbency of the biological fluid collection tip 480 are optimized to collect the precise saliva sample volume required for analysis.

[0138] According to some embodiments, the biological fluid or saliva collection tip 480 is made of some form of absorbent material, which may include, for example, cotton, tencel, or linen.

[0139] According to some embodiments, some components of the biological fluid collection device 400 are made from some form of plastic, preferably non-toxic (eg, the handle 450 , the telescoping arm 460 ).

[0140] According to some embodiments in which the telescoping arm 460 is used, the biological fluid collection device 400 is removed from the cartridge base 700 and inserted into the oral cavity at a specific location, such as near the parotid gland for saliva collection or more generally moved around for collection, such that the handle 450 remains outside the oral cavity, but the biological fluid collection tip 480 is on or near the parotid gland 524 and the telescoping arm 460 is fully extended, as shown. Figure 5A-5B Once the user has soaked the saliva into the collection tip 480 for an appropriate collection time, such as 5-20 seconds or until the tip is completely wet, the collection device 400 can be reattached to the cartridge base 700 for analysis.

[0141] According to some embodiments in which other biological fluids (e.g., blood or urine) are used, the biological fluid collection device 400 is removed from the cartridge base 700 and inserted into a sample of the biological fluid of interest, such as blood or urine, so that the biological fluid collection tip 480 may become wetted, at which time the collection device 400 can be reattached to the cartridge base 700 for analysis.

[0142] According to some embodiments in which the telescoping arm 460 is not used, the biological fluid collection device 400 is removed from the cartridge base 700 and shallowly inserted into the oral cavity so that the handle 450 remains outside the oral cavity but the biological fluid collection tip 480 is close to the user's lips. Once the user saturates the collection tip 480 with saliva for 5-20 seconds or until the tip is completely wet, the collection device 400 can be reattached to the cartridge base 700 for analysis.

[0143] The use of telescoping arm 460 can allow for sanitary testing, such as when using blood or urine, because the user does not come into contact with the sample, and / or can provide more direct contact with specific areas of the mouth (e.g., the parotid glands), and / or can allow for reduced contamination from the user that could distort the results, such as contamination from fingers that come into contact with collection tip 480.

[0144] Figure 5AA person's head is shown with the cartridge collection device 400 inserted into the person's mouth and clamped between the person's teeth with the tip 480 of the device 400 in contact with the parotid gland and duct vicinity. In this position, saliva can be collected by the swab tip 480 as it contacts the parotid gland and duct 524. Once the swab tip 480 remains in the person's mouth for a sufficient time to allow it to collect the desired amount of saliva, the device 400 is stored in a storage or collection container or cartridge base 700, see e.g. Figure 4B .

[0145] Figure 5B An external view of a person's head is shown, wherein a cartridge collecting device or tip 400 is shown. Figure 5A 4 is shown inserted into the person's oral cavity, showing the device 400 fully inserted, with the handle 450 of the collection device 400 located outside the oral cavity to reduce the risk of oral fluid contamination.

[0146] The concentrations of various biomarkers and proteins in saliva vary depending on whether the saliva is collected from a specific duct / site or from a dilute pool within the oral cavity. For example, the concentrations of salivary alpha-amylase and secretory IgA vary depending on the location within the oral cavity where the saliva sample is collected. Other problems with saliva collection include messiness associated with collection, overcollection, or contamination from both the user's or individual's fingertips and from contaminated saliva samples within the oral cavity. Therefore, current saliva collection methods and techniques are either dilute (pooled), indiscriminate, messy, unsafe, or result in excessive collection of saliva.

[0147] Saliva collection for diagnostic and other testing is typically performed by sampling pooled saliva in the oral cavity in the sublingual area, or pooling saliva in this area, and then spitting the pooled saliva into a container, thereby diluting key biomarkers and proteins. Methods that require spitting saliva also tend to be messy and may be unsafe because the collection container is often too small or too narrow. Other methods are to collect in vivo with a long swab, scrape the lining of the mouth, or collect from the inside of the mouth in an inconspicuous manner. Some previous methods result in more saliva being collected than needed. Previous saliva collection methods (both in vivo and in vitro) have failed to focus on collecting at the catheter entry point in the oral cavity. The catheter entry point into the oral cavity will have a greater or maximum biomarker and protein concentration. Previous saliva collection methods (both in vivo and in vitro) also tend to collect sample saliva in a messy or excessive amount.

[0148] According to some embodiments, an extendable or retractable saliva collection mechanism 400 having a handle 450 is provided. According to some embodiments, the collection device 400 allows for sample collection directly at the entrance of the conduit into the oral cavity for specific sampling, rather than by pooling or indiscriminate collection, and the collection device includes a swab tip 480 to optimize the saliva sample volume and is retracted into a reservoir base or collection container 700, thereby minimizing mess, improving safety, and reducing the possibility of contamination.

[0149] According to some embodiments, a saliva sample collection mechanism 400 is provided that is extended to allow compact storage but also allows for direct or targeted saliva sampling, has a centralized swab collection tip 480 for optimal, consistent collection volume, and a retraction / retraction mechanism 460 / 467 for applying pressure to the swab tip 480 directly to the biosensor 1300 located in the collection container or cartridge base 700. According to some embodiments, a saliva sample collection mechanism 400 is provided that reduces or minimizes mess and improves safety.

[0150] According to some embodiments, the saliva collection mechanism 400 contains 1) a handle 450 , 2) a locking mechanism 454 for connection or insertion into a collection container or cartridge base 700 , 3) ​​a telescoping / retracting arm 460 (with an internal spring design), and 4) an absorbent swab collection tip 480 .

[0151] Other embodiments may include different handle designs or handles designed to be used without human contact. In addition, in one embodiment, an attachment device 454 to the collection container or cartridge base 700 may not be included, or an optional attachment design such as magnetic, suction, form fit, threaded screw, etc. may be used. Other embodiments may include different means for extending the telescopic arm, may not consist of more than one segment, and / or may not use an internal spring design. In addition, other embodiments may use optional designs, shapes, and materials that are different from the swab tip 480 described above and / or shown in the accompanying drawings.

[0152] Other embodiments may use a similar device 400 to collect other biological fluids (blood, urine, sweat, tears, etc.).

[0153] Other embodiments may not focus on human saliva or other biological fluid collection, but may be directed to animal (pet, livestock) saliva or other biological fluid collection, and the length or structure of the device 400 may be different from these embodiments.

[0154] According to some embodiments, a collection device 400 may be provided having one or more or all of the following features:

[0155] - a telescopic collection arm 460 which concentrates the collection where it is most suitable, reducing the possibility of contamination and / or improving safety;

[0156] - A collection device closure with an associated collection container or base 700 that facilitates safe handling of a biological fluid sample while expressing or releasing saliva from the swab tip 480 to an appropriate location within the base 700, such as a biosensor.

[0157] -Optimized swab tip size to help minimize excess collection that causes safety and contamination issues and confusion. According to some embodiments, the swab tip 480 is made of some form of absorbent material, which may include, for example, cotton, tencel, or linen. The swab tip 480 may be optimized to contain an optimal saliva sample volume, and in some embodiments, the size of the tip may have a diameter of 3-7 mm and may have a length of 5-7 mm, or have such a shape that accommodates an acceptable saliva sample, wherein in some embodiments the optimal saliva sample volume may be in the range of 1-10 uL, and the size of the tip 480 may be configured to collect such a volume of saliva. In addition, other embodiments may employ optional designs, shapes, and materials different from the swab tip 480 described above and / or shown in the accompanying drawings.

[0158] According to some embodiments, some parts of the device 400 and / or the cartridge base are made of some form of plastic, preferably non-toxic (e.g., the handle 450, the collection container mechanism 454, the telescopic arm 460, the collection container or the cartridge base 700). According to some embodiments, the swab tip 480 is made of some form of absorbent material, which may include, for example, cotton, tencel, or linen.

[0159] According to some embodiments, the saliva collector 400 as a whole is contemplated to be integrated with the collection container or cartridge base 700. The collector 400 and the cartridge base 700 together form a saliva collection mechanism for the system 10. According to some embodiments, the length of the collection container or base 700 is preferably shorter than the length of the telescopic arm 460, so that just when the collection mechanism 400 is inserted and / or fixed into the collection container or base 700, the swab tip 480 is pressed against the inner surface of the base 700, for example, against the biosensor.

[0160] According to some embodiments, the saliva collector 400 is removed from the collection container or cartridge base 700 using the handle 450 (eg, by snapping, magnetism, twisting, etc.), thereby extending the saliva collector 400 to its full length after removal.

[0161] According to some embodiments, the collection mechanism 400 is then inserted into a specific location in the oral cavity, such as near the parotid gland, to collect saliva, or more generally moved around to collect. Once the user soaks the saliva into the collection tip 480 for 5-20 seconds or until the tip is completely wet, the collection mechanism 400 is reattached to the collection container or cartridge base 700. The collection container 700 may or may not be part of a larger saliva testing device 100 or system 10.

[0162] According to some embodiments, use of the collection device 400 and / or the cartridge base 700 may achieve one or more or all of the following advantages:

[0163] Directed saliva collection - Studies have shown that direct collection of saliva from specific salivary ducts (parotid or submandibular / sublingual glands) results in higher concentrations of salivary biomarkers or proteins (e.g., RNA, DNA, etc.). Pooled saliva generally maintains lower concentrations in comparison. Higher concentrations of target biomarkers or proteins in a sample increase the ability to make accurate measurements and / or reduce the likelihood that follow-up or additional sampling and testing will be required to obtain accurate test results.

[0164] Less contamination, safer – Fewer or limited interactions of the fingertips with the saliva sample means there is less chance of said sample being contaminated. Additionally, there may be less chance of transmission from saliva to the fingertips.

[0165] Less mess – Some current collection mechanisms rely on pooling the sample and then spitting it into a collection vessel. In addition to possible contamination, this leads to increased splashing and mess.

[0166] Optimized collection volume and high concentration of target molecules - Using optimized swab tips, only the amount needed is actually collected. In comparison, other current methods have excessive collection, causing mess, contamination and safety issues.

[0167] Figure 3 Step 2 illustrates the removal of the biological fluid or saliva collection device 400 from the inserted cartridge base 700 according to some embodiments of the present disclosure. Figure 5A The collection of a biological fluid (eg, saliva) according to some embodiments of the present disclosure is shown. Figure 3 Step 3 illustrates reinserting the biological fluid or saliva collection device 400 into the inserted cartridge base 700 , according to some embodiments of the present disclosure.

[0168] According to some embodiments, after the disposable biological fluid or saliva test kit or cartridge 200 is inserted into the reader device 100, only the biological fluid collection device 400 ( Figure 3 Step 2), the user collects a saliva sample ( Figure 5A ), and then reinsert the sample collection device 400 with the now wetted tip into the cartridge base 700. Figure 3 Step 2 illustrates removing the biological fluid collection device 400 from the cartridge base 700 in the reader device 100, wherein the biological fluid collection device 400 is unscrewed from the cartridge base 700. Once the collection device 400 has been removed from the cartridge base 700, the user can use the collection tip 480 to collect a saliva sample. Figure 5A 4 shows the collection tip 480 of the collection device being inserted into a cavity of a user or subject from whose body biological fluid is to be collected. Figure 3 In step 3 , the collection device 400 is reinserted into the cartridge base 700 .

[0169] 3. Cartridge

[0170] According to some embodiments, the disposable sensing unit or cartridge 200 used with the TRAQ Stone 100 consists of a biosensor chip 1300 integrated into a modular cartridge 200, which consists of a collection container / reservoir base 700 and an integrated saliva collection device 400 (e.g., a handle 450, a retractable cotton swab for saliva collection). The biosensor chip 1300 is located in the base 700 of the disposable cartridge 200.

[0171] A. Cartridge handle part 450

[0172] Fig. 6A is a side view of a handle 450 of a biological fluid collection device 400, Figure 6B is its bottom view, Figure 6C is its perspective view, Fig.6D is an enlarged view thereof, and Fig. 6E and Fig. 6F 450 are cross-sectional views of two embodiments of the handle 450. Fig. 6A As shown in , the handle 450 can include a curved handle knob 452 and a threaded shaft 454. The threaded shaft 454 can constitute an extrusion extending from the base 452b of the handle knob 452, which can have a diameter commensurate with the diameter of the insertion chamber 710 of the cartridge base 700, so that the threaded shaft 454 can lock the biological fluid collection device 400 to the cartridge base 500 while the collection tip 480 is located in the insertion chamber of the cartridge base 700. As shown in Fig.6DAs seen in the figure, the threaded shaft 454 can further include one or more vertical grooves 459 formed in the threaded shaft 454, for example, on each side of the threaded shaft 454, and the grooves are completely or approximately opposite to each other to help fix the biological fluid collection device 400 to the cartridge base 700, so that when the collection device 400 is pushed into the cartridge base 700, one or more protrusions 712 on the inner wall 713 of the insertion chamber 710 of the cartridge base 700 can be engaged with the inner wall 713 of the insertion chamber 710 of the cartridge base 700. The vertical grooves 459 are aligned and inserted therein until the protrusions 712 meet or engage with the flanges 455, whereby when the handle knob 452 is rotated, the protrusions 712 on the inner wall of the insertion chamber 710 of the cartridge base 700 can travel within the internal threads 453 located on the shaft 454, allowing the handle knob 452 to subsequently be used to apply torque to twist the collection device 400 until the protrusions 712 on the cartridge base 700 lock into the top grooves 451 formed in the shaft 454. The biological fluid collection device 400 may also include Figure 6B 4, which is configured to seal the insertion chamber 710 of the cartridge device 700 when the collection tip 480 of the biological fluid collection device 400 is inserted into the insertion chamber 710 and the collection device 400 is twisted into engagement with the cartridge base 700. According to some embodiments, the proximal sealing area 457 can be formed by a protrusion extending around the threaded shaft 454, which is larger than the opening of the insertion chamber 710 to seal the insertion chamber and any biological fluid therein from the collection tip 480. Thus, the protrusion 712 can further lock the distal end of the biological fluid collection device 400 within the insertion chamber 710 of the cartridge base 700. According to some embodiments, the threaded shaft 454 can further include a distal sealing area 458 formed near the distal end 454D of the threaded shaft 454, which can be pressed against, abutted and / or engaged with the bottom surface 722 of the insertion chamber 710 of the cartridge base 700 and when coupled with the O-ring 702 in the cartridge base 700 (see Figure 8C ), the biological fluid can be prevented from flowing back into the chamber 710 during the process of dispensing the biological fluid from the collection tip 480. Figure 6B As seen in FIG. 4 , the threaded shaft 454 may further include a handle slot or cavity 456 that is open on one side (e.g., the distal side or bottom side) of the shaft 454. According to some embodiments, the proximal end of the telescoping arm 460 is secured to the handle 450 near or within the handle slot 456. As shown in FIG. Fig. 6E As seen in FIG. 4 , according to some embodiments, handle slot 456 may extend into handle 450 a sufficient distance to allow telescoping arm 460 to be fully depressed into handle 450. As shown in FIG. Fig. 6F In other embodiments where telescoping arm 460 may not be used, slot 456 may be shallow and not extend into handle 450, as seen in FIG.

[0173] According to some embodiments, telescoping arm 460 may be secured to handle 450 in slot 456 using a dental grade adhesive, such as dental glue.

[0174] According to some embodiments, the biological fluid collection device 400 has the advantage of not having any electronics and / or batteries and / or other power sources therein. According to some embodiments, the biological fluid collection device 400 has the advantage of not having any electronics and / or batteries and / or other power sources on a portion thereof configured to be received in a cavity of a subject (e.g., an oral cavity in which saliva is collected) for collecting a biological sample using the biological fluid collection device 400. For example, according to some embodiments, the biological fluid collection device 400 has the advantage of not having any electronics and / or batteries and / or other power sources on the extendable arm 460 and / or the collection tip 480.

[0175] B. Cartridge base

[0176] The collection tip 480 of the collection device 400 can be locked into or seated into the cartridge base 700 to eliminate or reduce any chance of oral fluid leakage. According to some embodiments, the collection device 400 can be locked and / or seated into the cartridge base 700 to reduce or eliminate the chance of oral fluid leaking from the chamber 710 of the cartridge 200. According to some embodiments, the device 400 and the base 700 engage and seal with each other to reduce the chance of oral fluid leaking out of the cartridge 200.

[0177] The torque applied by the twisting motion of the collection device 400 into the cartridge base 700 allows a greater force to be applied to the collection tip 480 of the collection device 400 with less effort than would be the case using a simple insertion method.

[0178] Fig. 7A is an exploded perspective view of a cartridge base 700 according to some embodiments, Figure 7B 700 is an assembled perspective view thereof. The cartridge base 700 may include an O-ring 702, a cartridge base body 700A, a latch 760, and a sensor chip 1300. The cartridge base 700 is configured to accommodate the biological fluid collection device 400 before use, receive the biological fluid collection device 400 with the impregnated collection tip 480 during analysis, and be electrically connected to the reader device 100 during analysis. Based on the above analysis, the cartridge device 700 is configured to generate an electrical signal indicating the presence and / or amount of one or more target analytes in the sample.

[0179] According to some embodiments, the cartridge base body 700A is manufactured by injection molding, but can also be 3D printed (e.g., ABS, TPU, PLA, etc.). According to some embodiments, the cartridge base body 700A includes three main areas: a collection device insertion chamber 710, a saliva reservoir 720, and a sensor chip cavity 730. According to some embodiments, the collection device insertion chamber 710 has a circular cross-sectional shape and has two extruded small protrusions 712 on opposite sides near the first end 700-1 (e.g., the top end) of the cartridge base body 700A. The cartridge base body 700A also has a second end 700-2, such as a bottom end.

[0180] Figure 7C is a perspective view of a latch 760 for a cartridge base according to some embodiments of the present disclosure.

[0181] Fig.7D It is a top view. Fig. 7E It is a side perspective view. Figure 7F is the bottom view, Figure 7G This is another side perspective view. Figure 7H 71 is an inverted side perspective view, and FIG. 72 is a downward perspective view showing an internal view of the chamber 710 of the cartridge body 700A of the cartridge base 700 according to some embodiments of the present disclosure. The cartridge body 700A may include a collection device insertion chamber 710, a rod holding groove 704, one or more cutout grooves 705 (e.g., two cutout grooves 705), an island connector 706, one or more latch grooves 721 (e.g., four latch grooves 721), and a sensor chip cavity 730. The collection device insertion chamber 710 may include a channel 720 in which a hole 720H ​​is formed, an O-ring groove 740, and one or more protrusions or threaded pins 712 (e.g., two protrusions 712) protruding from the inner wall 713 of the insertion chamber 710. The hole 720H ​​in the channel 720 serves as a hole for the biological fluid to flow from the insertion chamber 710 to the chip 1300 during biological fluid analysis. The channel 720 leads to the underside of the cartridge body 700A, wherein, according to some embodiments, the size and / or location of the hole 720H ​​matches a similar hole on one side (e.g., the bottom side) of the sensor chip 1300, so that when the biological fluid collection device 400 is inserted into the cartridge base 700 and pressure on the collection tip 480 causes the biological fluid to be dispensed from the collection tip 480 into the channel 720, the biological fluid can flow to the sensor chip 1300 for analysis. According to some embodiments, the channel 720 and the O-ring groove 740 are concentric grooves or structures inserted into one end 710B (e.g., the bottom end) of the chamber 710 (see Fig.8F , 7I), such that the O-ring groove 740 surrounds the channel 720. The size and configuration of the O-ring groove 740 can accommodate an O-ring 702, which can be used to seal the interface between the collection tip 480 and the channel 720 when dispensing the biological fluid to prevent backflow from the channel 720. For example, when the biological fluid collection device 400 is inserted and screwed into the cartridge base 700, the collection tip 480 is pressed against the collection channel 720 between the distal sealing area 458 of the handle 450 and the channel 720 and the O-ring 702. The O-ring 702 deforms under the pressure to seal the interface. According to some embodiments, when the handle 450 of the saliva collection device 400 is twisted and locked into the base 700, the swab 480 is compressed, promoting the flow of saliva onto the biosensor chip 1300 contained therein. According to some embodiments, for safety reasons, the locking orientation also seals the cartridge 200 to prevent leakage.

[0182] According to some embodiments, the protrusion 712 is configured and positioned to engage with the threads 453 of the collection device 400 to allow a tightening motion and promote a tight seal (see, e.g., Figure 8C According to some embodiments, the protrusion 712 can travel through the vertical groove 459 of the handle 450 on the threaded shaft 454 and slide into the threads 453 on the threaded shaft 454, so that when the collection device 400 is inserted into the insertion chamber 710, the protrusion 712 can be inserted into the vertical groove 459 until it reaches the flange 455 of the vertical groove 459, at which time the biological fluid collection device 400 can be tightened and the protrusion 712 slides into the threads 453 of the threaded shaft 454 until it is locked into the top groove 451 of the collection device 400.

[0183] According to some embodiments, the cartridge body 700A may also include a rod groove 704, which may allow a release rod on the reader device 100 to connect with the cartridge base 700, so that when the cartridge base 700 is inserted into the cartridge receptacle 140 of the reader unit 100, the release rod may be inserted into the rod groove 704 and fix and / or hold the cartridge base 700 in the cartridge receptacle 140 of the reader unit 100 from a longitudinal force for analysis.

[0184] According to some embodiments, the cartridge body 700A may further include a pair of cutout grooves 705 and an island connector 706 (see, e.g. Figure 7F and 9A), which are located on the outside of the cartridge body and are sized and positioned to mate with a connector mechanism having protruding cartridge electrical connectors 145 and matching grooves 146 associated with the cartridge receptacle 140 / 140A of the reader device 100, such that when the cartridge base 700 is inserted into the cartridge receptacle 140 of the reader unit 100, the cutout grooves 705 of the cartridge 700 align with a pair of protruding cartridge electrical connectors 145 in the cartridge receptacle 140 / 140A. Likewise, the island connector 706 aligns with the cutout grooves 146 in the cartridge receptacle 140. When the cartridge 700 is inserted into the cartridge receptacle 140 / 140A, the island connector 706 can slide into and be received in the cut-out groove 146 in the cartridge receptacle 140 / 140A, and the protruding cartridge electrical connector 145 can be received in the cut-out groove 705 of the cartridge 700, securing and / or holding and / or locking the cartridge base 700 in the cartridge receptacle 140 to resist rotational forces, so that during use and / or sample analysis, the handle 452 of the collection device 400 can be rotated or screwed in and out of the cartridge base 700 while the cartridge base 700 is secured in the receptacle 140A.

[0185] According to some embodiments, the cartridge body 700A may further include a sensor chip cavity 730, which is located on one side of the cartridge body 700A, such as the lower side, and whose shape and size may be designed to accommodate and position the biosensor chip 1300 therein. According to some embodiments, when the biosensor chip 1300 is placed in the sensor chip cavity 730, it is fixed and / or retained therein. According to some embodiments, when the sensor chip is located in the sensor chip cavity 730, the hole 720H ​​of the channel 720 may be aligned with the central hole 1339 (see Fig. 8E and 13E ) to align.

[0186] According to some embodiments, the cartridge body 700A may further include one or more latch grooves 721, such as four latch grooves 721. According to some embodiments, the latch grooves 721 are formed in one or more latch engagement protrusions or flanges 714, which are located on one side (e.g., the lower side) of the cartridge body 700A surrounding the sensor chip cavity 730, and may be arranged in pairs, cross each other to form a cross, and may dock with the legs 766 of the latch 760, so that when the biosensor chip 1300 is placed in the sensor chip cavity 530, each leg of the latch 760 may be inserted into the corresponding latch groove 721, so that the latch 760 may be placed near the chip 1300 to fix the biosensor chip 300 in the sensor chip cavity 730.

[0187] According to some embodiments, the sensor chip 1300 faces downward relative to the cartridge bottom 710A so that saliva flows down through the hole 720H ​​and the bottom sensor chip hole 1339 onto the hydrophilic cover 1320, causing the saliva to diffuse along the electrodes 1311, 1312, 1321, 1322, 1323.

[0188] As in Figure 7C As seen in FIG. 7 , according to some embodiments, the latch 760 may include a hub 762, one or more elbows 764 (e.g., four elbows 864), and one or more legs 766 (e.g., four legs 768). According to some embodiments, the legs 766 of the latch 760 extend farther than the diameter of the cartridge body 700A and / or extend a sufficient distance to allow for secure insertion into the latch groove 721 and engagement with the latch engagement protrusion or flange 714. When the biosensor chip 1300 is placed in the sensor chip cavity 730, the latch 760 may be used to further secure the biosensor chip 1300.

[0189] According to some embodiments, the latch 760 may further include one or more (e.g., four) elbow fittings 764 that radiate outward from the hub 762 and couple the legs 766 of the latch 760 to the hub 762. According to some embodiments, the elbow fittings 764 may be formed of a flat material that is compressed into an elbow or curved shape, which allows the elbow fittings 764 to be compressed toward or away from the hub 762 on a radial axis, so that when the latch 760 is inserted into the sensor chip cavity 730 near the biosensor chip 1300, when the corresponding elbow fitting 764 of the legs 766 is compressed radially inward, each leg 766 may be inserted into a corresponding latch groove 721, and once the pressure on the elbow fittings 764 is released and the legs 766 are inserted into the latch grooves 321, the elbow fittings 864 may extend to their resting position and lock the latch 760 on the cartridge body 700A.

[0190] C. Cartridge handle and base

[0191] After manufacture, the cartridge base 700 and the collection device 400 are stored together in a sterile package, wherein the collection device 400 is partially compressed into the cartridge base 700 , and this complete assembly may be referred to as a biological fluid testing kit or cartridge 200 .

[0192] Fig. 8A is an exploded perspective view of a subassembly of a complete portion of a disposable biological fluid test kit or cartridge 200, Figure 8B 2 is an assembled perspective view thereof, wherein the cartridge 200 has a handle 450 of the collection device 400, a cartridge body 700A, a sensor chip 1300 and a latch 760. Fig. 8AIn the figure, the extendable element 460 and the pipette tip 480 are omitted from the view of the collection device 400. Figure 8B It shows how the cartridge 200 subassemblies are assembled and packaged prior to use.

[0193] Figure 8C-8D is an assembled perspective cross-sectional view of a disposable biological fluid or saliva test cartridge 200 according to some embodiments, Fig.8F is a side cross-sectional view thereof when not assembled. Fig. 8E is a top view of a biosensor chip 1300 according to some embodiments. Figure 8C The cartridge 200 is shown with a sleeved or compressed biological fluid or saliva collection device 400 and how the handle 452 is locked to the cartridge base 700 by means of a protrusion 712 on the cartridge base 700 which seats in a top groove 451 formed in the threaded shaft 454 (see also Figure 4C , 6A , 6D). Fig.8D The biological fluid or saliva collection device 400 is shown compressed into the cartridge base 700 , whereby the biological fluid can be expelled from the absorbent tip 480 and onto the biosensor chip 1300 , wherein the arrows show the flow of fluid from the tip 480 onto the biosensor chip 1300 . Fig. 8E Further shown, according to some embodiments, when the tip 480 is pressed against the channel 720 and the collected biological sample flows through the hole 720H ​​in the channel 720 and the central hole 1339 of the chip 1300 (see Fig.13D and 13E ) when the biosensor chip 1300 and the exemplary flow of the sample fluid therein.

[0194] Fig.8F The biological fluid or saliva test cartridge 200 is shown with the collection device 400 separated from the cartridge base 700 .

[0195] Figure 8G 200 according to some embodiments of the present disclosure, the cartridge 200 contains a collection device 400 that does not use a telescopic arm. Instead, the pipette tip 480 is directly coupled to the shaft 454 of the handle 450.

[0196] D. Cartridge Overview

[0197] According to some embodiments, the cartridge base 700 not only houses the biosensor 1300, but also serves as a storage location for the saliva collection device 400, i.e., a reservoir for the saliva sample, and also contains electrical connections 1316 of the biosensor 1300 to connect the cartridge 200 to the POCT device 100, for example through electrical contacts 160 located in the cartridge receptacle 140.

[0198] According to some embodiments, the cartridge 200 and cartridge base 700 have the advantage of not having batteries and / or other power sources therein.

[0199] 4. Reader device

[0200] Fig.9A is an exploded perspective view of a collection device 400, a cartridge base 700, and a reader 100, which may take the form of a handheld POCT testing device, according to some embodiments of the present disclosure.

[0201] Fig. 9B is a cross-sectional view of a reader / POCT test device 100 according to some embodiments of the present disclosure, wherein a test cartridge 200 is inserted into a cartridge receptacle 140 (e.g., as in use). The cartridge receptacle 140 has a cartridge cavity 140A therein for receiving a cartridge seat 700 of a cartridge 200.

[0202] Fig. 9C is an exploded view of the reader / test device 100 and some of its components according to some embodiments of the present disclosure. The cartridge receptacle 140 serves as a receptacle for a biological fluid or saliva test cartridge 200 .

[0203] Fig.9D is a perspective view of a cartridge receptacle 140 having an aperture 1406 according to some embodiments of the present disclosure.

[0204] According to some embodiments, reader 100 may include a release lever 170 and a power switch 180 .

[0205] According to some embodiments, the reader 100 is a handheld point-of-care testing device that contains computing capabilities (e.g., a CPU or processor), electrical capabilities, and wireless (e.g., Bluetooth) communication capabilities. According to some embodiments, the reader 100 includes a cartridge receptacle 140, an analysis computer, a CPU or processor, and a power source such as a battery (not shown) located on one or more printed circuit boards ("PCBs") 150.

[0206] According to some embodiments, the cartridge receptacle 140 may also include a cutout or aperture 140B (see Fig.9D), the release rod 170 can pass through the cutout or aperture so as to contact and / or abut the cartridge 200 located in the cartridge receptacle 140. For example, when the cartridge 200 is inserted into the cartridge receptacle 140, a portion of the release rod 170, such as the cartridge engagement protrusion 170A, can pass through the cartridge receptacle aperture 140B and enter the retaining rod groove 704 of the cartridge base 700, thereby retaining the cartridge 200 in the cartridge receptacle 140. The reader 100 can also include an internal rod spring 171 for applying a biasing pressure to bias the rod 170 to engage with the cartridge 200 located in the cartridge receptacle 140, thereby retaining the cartridge base 700 in the cartridge receptacle 140.

[0207] According to some embodiments, the cartridge receptacle 140 itself may be made of plastic. According to some embodiments, the inner surface of the cartridge receptacle 140 defining the cartridge cavity 140A of the cartridge receptacle 140 has a geometry complementary to the exterior of the cartridge base 700 to help properly position and / or retain the cartridge base 700 in the cartridge receptacle 140.

[0208] According to some embodiments, the cartridge receptacle 140 may contain a reader electrical contact 160 to facilitate electrical and / or communication coupling between the cartridge base 700 and / or the biosensor chip 1300 located therein (e.g., via electrical contacts 1316) and the reader 100. According to some embodiments, the reader electrical contact 160 is a spring-loaded (or magnetic) contact. According to some embodiments, the reader electrical contact 160 is located within the cartridge cavity 140A, for example, on or near the bottom of the cartridge receptacle 140, and is positioned to contact or couple with a corresponding cartridge electrical contact located on the cartridge base 700 (e.g., a cartridge electrical contact or pad located on or near the bottom of the cartridge base 700). According to some embodiments, the cartridge electrical contact or pad is located on the sensor chip 1300 within the cartridge base 700.

[0209] According to some embodiments, the reader 100 may include communication hardware and may be configured to communicate with one or more external devices 191, such as a smart phone, a watch, a tablet computer, and / or a computer, so as to allow communication (e.g., sending and / or receiving data) with such external devices. According to some embodiments, the reader 100 includes Bluetooth hardware such as Bluetooth Low Energy (BLE) and is configured to communicate with the external device via a Bluetooth protocol. According to some embodiments, the reader 100 sends data to a phone (e.g., a smart phone) via Bluetooth.

[0210] According to some embodiments, instead of, and / or in addition to, transmitting information to remote device 191 and / or facilitating display of information on remote device 191 , reader 100 may include display 110 and be configured to display information on display 110 .

[0211] According to some embodiments, the cartridge base 700, the saliva collection device 400, and the swab 480 are stored together in a closed position. When the user uses the device 100, they place the cartridge 200 in the Stone 100 and press it until it engages with the latch 170 and is flush with the surface of the socket 140. Once the cartridge 200 is seated and locked in the reader socket 140, the user can then unscrew the collection device 400 from the cartridge base 700. The user can then saturate the swab 480 with a biological fluid (e.g., saliva) and reposition the collection device 400 into the cartridge base 700 located in the socket 140 of the reader 100. During the process of repositioning the collection device 400 into the base 700, the twisting motion of the collection device 400 will force the saliva down to contact the sensor 1300 to begin analysis.

[0212] According to some embodiments, hub 762 (see Figure 7C ) can be located in the center of the latch 760 and configured to mate with the button 120 located in the cartridge receptacle 140 (see Fig. 9B ) so that when the cartridge base 700 is inserted into the cartridge receptacle 140 of the reader unit 100, the button 120 in the cartridge receptacle 140 of the reader 100 can press against the hub 762, which applies pressure to the biosensor chip 1300, keeping it fixed and aligned with the hole 720H ​​in the channel 720 of the cartridge base 700, and the sample fluid can flow into the biosensor chip 1300. The pressure applied by the button 120 in the cartridge receptacle 140 of the reader 100 on the hub 762 may also force the cartridge base 700 into the release lever 170 on the reader device 100. For example, when the cartridge base 700 is inserted into the cartridge receptacle 140 of the reader unit 100 and the release rod 170 of the reader unit 100 is inserted into the rod groove 704, the pressure of the button 120 of the cartridge receptacle 140 on the reader unit 100 (e.g., upward pressure) can cooperate with the opposing pressure (e.g., downward pressure) applied by the release rod 170 of the reader 100 to hold and / or secure the cartridge base 700 within the cartridge receptacle 140 of the reader unit 100. According to some embodiments, the button 120 is biased into the cavity 140A of the cartridge receptacle 140 by a biasing element 122 (e.g., a spring).

[0213] refer to Fig. 9C According to some embodiments, the reader 100 can include a housing formed by one or more housing parts 130. According to some embodiments, the housing parts can be secured to each other by one or more fastening means, such as screws 132.

[0214] Figure 10-12is a plan view of a PCB circuit diagram according to some embodiments of the present disclosure. According to some embodiments, the various analytical methods described herein (e.g., enzymatic methods and ISE) can have their own corresponding PCBs that are integrated into the reader / stone 100 to work together. According to some embodiments, Figure 10-12 The multiple PCBs shown in can be combined into one PCB to maintain stability.

[0215] exist Fig.10 , circuit 10-01 shows a system on chip microcontroller circuit according to some embodiments. Circuit 10-02 is a temperature sensor that can be used to correct the hydration measurement if there is a temperature dependency. Circuit 10-03 is a battery management system that handles the charging of the lithium-ion battery and distributes the power to the device.

[0216] exist Fig.11 , circuit 12-01 is the potentiostat channel. As shown, three channels can be monitored and / or recorded at one time. These channels may correspond to different wells.

[0217] exist Fig.12 , circuit 11-02 can be used to generate clean 5V power from a variable battery voltage. Circuit 11-04 can be used to generate a pair of clamping voltages for ESD protection. Circuit 11-05 multiplexes the analog output of each channel signal conditioning into the ADC. Circuit 11-01 is the ADC and reference that convert the analog voltage from the potentiostat into a digital signal to be read in microcontroller 10-01.

[0218] According to some embodiments, the ISE PCB 150 (see Fig. 9C ) is a mixed signal board that handles the potentiometry / voltage measurement, analysis, uplink, and power processing of the reader 100. According to some embodiments, the digital components include a serial debug / programming port, a temperature sensor to detect overheating, a power switch, and a battery charger / controller, as well as a connector and charging port. According to some embodiments, the digital domain also includes a BLE module that is configured to communicate with an external device 191 (e.g., a user's smartphone). The BLE module is contained within a microcontroller, such as the CYBLE-214015-01 module (although other microcontrollers may also be used). According to some embodiments, the analog components include a 5V analog power supply, a digital power supply, and a protection clamp voltage. According to some embodiments, the analog components may also include a reference voltage chip and a multiplexer that allows signals from different channels being measured to be brought together. According to some embodiments, the ADC is also used to convert analog sensor information into digital signals that can be used by the microcontroller.

[0219] According to some embodiments, when the biosensor 1300 is configured to detect the presence and / or amount of an analyte enzymatically or by immunoassay, three data points are obtained at the beginning, middle, and near the end of the amperometric analysis. The calculated concentration of the analyte is determined by the processor 102, for example, by using a calibration curve.

[0220] According to some embodiments where the biosensor chip 1300 is configured to detect the presence and / or amount of an ionic analyte, periodic voltage measurements are taken and then converted to ion concentrations via the Nernst equation in the CPU of the reader / Stone 100 without requiring any calibration steps.

[0221] 5. Biosensor Chip

[0222] According to some embodiments, the cartridge base 700 further comprises a sensor chip (eg, biosensor) chip 1300, which can be configured to analyze a sample and interface with the reader 100 to determine the presence and / or amount of one or more target analytes within the sample.

[0223] Fig.13A is a top view of a biosensor chip 1300 according to some embodiments. The chip 1300 includes a plurality of electrical contacts 1316.

[0224] Fig. 13B An exploded perspective view of a biosensor chip 1300 that may include various layers is shown. According to some embodiments, the chip 1300 includes a sensor chip substrate 1330, a spacer layer 1340, and a cap layer 1350, and according to some embodiments of the present disclosure, the biosensor chip 1300 may also include a sensing film on top of a printed layer not shown here.

[0225] exist Fig. 13B , a sensor chip 1300 according to some embodiments of the present disclosure is shown having a single spacer layer 1340 and a single cap layer 1350 added to a sensor chip substrate 1330. According to some embodiments, during the manufacturing process, the layers are added in a stripe design to make multiple sensors (see, e.g. Figures 16A-16C ), rather than adding them separately as shown here.

[0226] Fig. 13C A top view of a screen-printed electrode layer 1310 of a biosensor chip 1300 is shown according to some embodiments of the present disclosure. Fig. 13C A screen printed underlayer 1310 (Ag, carbon or another material) is shown according to some embodiments of the present disclosure.

[0227] Fig.13D1 is a partially exploded perspective view of a screen-printed electrode layer 1310 and a dielectric layer 1320 of a biosensor chip 1300 according to some embodiments of the present disclosure. The screen-printed electrode layer 1310 and the dielectric layer 1320 are collectively referred to as a sensor chip substrate 1330. Fig.13D A hydrophobic dielectric layer 1320 is shown added on top of a screen-printed electrode layer 1310 according to some embodiments of the present disclosure.

[0228] Fig.13E According to some embodiments of the present disclosure Fig.13D A top view or plan view of the sensor chip substrate 1330 is shown in FIG.

[0229] refer to Fig. 13C , the printed electrode layer 1310 may include a plurality of electrical contacts or pads 1311, 1312. Fig.13E and 13C , pads 1311 can be combined in different holes 1332, 1333, 1334, 1335 and 1336. These holes are generally referred to as holes 1331. According to some embodiments, three-pad hole 1336 and three-pad hole 1331 are composed of three carbon pads 1311, and distal hole 1332 and proximal hole 1333 are composed of two carbon pads 1313. Another three-pad hole 1335 contains two carbon pads 1311 and one Ag / AgCl pad 1312.

[0230] According to some embodiments, the pads 1311, 1312 in each well 1331 and each well 1331 generally have different uses, depending on what configuration the biosensor chip 1300 is manufactured for, allowing modular design when designing a new biosensor chip 1300 within this existing framework to determine the presence and / or amount of an analyte, wherein the well 1331 used or the sensing film deposited in the well 1331 can be changed to accommodate a specific configuration. Some sample configurations of the biosensor chip 1300 will be discussed. In some embodiments, all three pads in well 1334 or well 1336 or two pads in well 1332 or well 1333 can be used for the same purpose, in which case the well 1331 will be referred to according to its use. For example, if all three pads in well 1334 or well 1336 are used together as working electrodes, the well will be referred to as a "working well", without using one pad as a "working electrode".

[0231] According to some embodiments, electrical contacts 1316 and holes 1331 are formed by methods such as screen printing, where multiple screen printed electrical and dielectric layers are printed on substrate 1313.

[0232] refer to Fig. 13C, the carbon pads not shown with hatching are combined with similar pads so that when the dielectric layer 1320 is placed over the printed layer 1310, the formed well 1331 can be used as a single measurement unit. In this case, the well 1331 can contain a complete electrochemical measurement system, such as but not limited to a three-electrode system, in which there can be a working electrode, a reference electrode, and a counter electrode.

[0233] Fig.13E A top view of a sensor chip substrate 1330 is shown, which may include holes 1339, electrical contacts 1316, and, for example, five holes 1331, including a distal double pad hole 1332, a proximal double pad hole 1333, and three pad holes 1336, three pad holes 1335, and three pad holes 1334 that continue in a clockwise manner from the proximal double pad hole 1333. According to some embodiments, the holes 1339 may match the holes 720H ​​of the cartridge base 700, so that when the biosensor chip 1300 is placed in the sensor chip cavity 730, the holes 720H ​​allow the fluid discharged from the absorbent tip 480 to enter the middle of the sensor chip cavity 730 and eventually pass through the holes 1339 of the biosensor chip. Thus, according to some embodiments, the biological fluid sample has an unobstructed flow path from the channel 720 to the hydrophilic top cover 1350 of the biosensor chip 1300. The hydrophilic cap 1350 facilitates diffusion of the biological fluid along the aperture 1331 for analysis therein.

[0234] Fig.13F 1 is a schematic side view showing various screen printed layers of a sensor chip substrate 1330 according to some embodiments of the present disclosure. According to some embodiments, the sensor chip substrate 1330 is printed with a first layer 1314 of Ag (silver), a second layer 1315 of carbon forming an electrode pad 1311 (where Ag / AgCl forms one electrode or pad 1312, and Fig. 13C 1312) and a dielectric third layer 1320. In some embodiments, the Ag layer acts as a conductor running through the chip 1300, electrically coupling the pads 1311, 1312 to the outside of the chip 1300. According to some embodiments, the carbon is printed on top of a PET substrate 1313 or any other suitable material used as a substrate as traces or pads for measurement. The dielectric layer 1320 placed over the screen-printed electrode layer 1310 has an orifice therein that defines a hole 1331 over the pads 1311, 1312 for performing biological fluid sample analysis therein. The dielectric layer 1320, along with the hydrophilic top cap 1350 and the spacer 1340, acts as a hydrophobic barrier to help push or guide the fluid toward the hole 1331. When the dielectric layer 1320 is printed on top of the other two layers 1315, 1314, the Ag trace 1314 is covered, leaving only the exposed edge area as an electrical contact 1316. Reference Fig.13FAccording to some embodiments, the biosensor chip 1300 is composed of multiple screen-printed electrical and dielectric layers on a PET substrate 1313, although according to some embodiments, any suitable flexible but stable polymer may work.

[0235] According to some embodiments, the Ag layer 1314 acts as a conductor for the entire chip 1300, and wires electrically connect contacts 1316 (which are in electrical contact with electrical contacts 160 within Stone 100) to enzyme / ion selective electrodes (ISE) 1311, 1312. According to some embodiments, the carbon or silver is printed on top as contact pads 1316 for the electrodes 1311, 1312 themselves.

[0236] Fig.14A FIG. 1 is a layer stack diagram of an enzyme sensing membrane 1300E for a lactate sensing membrane or a general enzyme sensing membrane. The membrane 1300E can be drop cast as a layer into the hole 1331 (see FIG. 1 ). Fig.13E ), wherein the film covers the carbon pads 1311, 1315 of the chip 1300 (see Figures 13C-13F ).

[0237] According to some embodiments, the biosensor chip 1300 can be configured to determine the presence and / or amount of an analyte using an enzyme reaction, wherein the working electrode counts the electrons added to the pore 1331 by the mediator during the redox reaction. Specifically, according to such embodiments, a chronoamperometric measurement is used between the reference electrode and the working electrode. According to some such embodiments, three-electrode pores 1334 and 1336 are used to determine the presence and / or amount of an analyte and contain a sensing membrane 1300E (e.g. Fig.14A ), the remaining holes are not used. According to some embodiments, in order to obtain better accuracy, the measured values ​​of the presence and / or amount of the analyte determined between the two holes are averaged. In such embodiments, a pad 1321 (e.g., the outermost pad) in each hole 1334, 1136 acts as a sample loading detector pad, which has a small constant current flowing through it, so that when the sample is dispensed into the cartridge base 700 and further enters the biosensor chip 1300, the resistance on the sample loading detector pad 1321 may change, thereby changing the current. According to some embodiments, when the circuit (e.g., processor 102) in the reader 100 detects this current change, the processor causes a set of instructions (e.g., stored in the memory 106) to run on the reader 100 (e.g., processor 102 therein), so that the signal associated with the other pads in the holes 1334, 1336 is measured. The other two pads 1322 and 1323 are used as the working electrode and reference electrode of the relevant holes 1334, 1336, respectively.

[0238] According to some embodiments, accuracy is determined using blood or saliva samples measured by commercial grade machines, which are the same samples used on the sensors, devices and systems described herein. Thus, comparisons can be made between results obtained using the devices, sensors and systems described herein and commercial grade machines, and the results can be calibrated.

[0239] According to some embodiments, the chip 1300 may include one or more saliva wells 1331, one or more reference electrodes 1323, one or more electrical contacts 1316, and one or more working electrodes 1322. According to some embodiments, the biosensor chip 1300 consists of a screen-printed working electrode 1322 and a physical depression or well 1331 containing an enzyme chemistry for detecting specific biomarkers in saliva, and electrical connection points or contacts 1316 for interacting with the measurement tool or device 100.

[0240] According to some embodiments, when the saliva sample comes into contact with the biosensor 1300 in the cartridge base 700, the electrochemical test can be automatically started. According to some embodiments, each biosensor 1300 contains both electrical and chemical elements. According to some embodiments, the broader chemical aspects can be divided into three categories of reactions: enzymatic, ion selective, and immunoassay categories. According to some embodiments, immunoassay testing may require a different form factor of the reader 100 and / or the cartridge base 700 (this will be described in more detail below).

[0241] According to some embodiments, the sensor chip 1300 faces "downward" relative to the cartridge bottom 700A so that saliva flows downward through the reservoir hole 720H ​​and the bottom sensor chip hole 1339 onto the hydrophilic cover 1350, which helps the saliva spread along the electrodes 1311, 1312.

[0242] According to some embodiments, the different categories of chemical tests that are partially or completely present in the chip 1300 are distinguished by adding a small RFID tag to the top of the chip 1300, which sends information such as sensor type, sensor ID, test ID, test type on the sensor, sensor model and / or serial number, and / or calibration data to the processor 102 of the reader unit 100. In some embodiments, the distinction is not done through RFID, but rather through a mobile application, where such information about the cartridge to be inserted or inserted into the cartridge receptacle of the reader 100 is manually selected on a remote device such as a smartphone and then communicated to the processor 102 in the reader unit. In some embodiments, the distinction is made through a physical "lock and key" mechanism, whereby a cartridge 200 having certain physical or mechanical shapes or protrusions ("keys") is mated into corresponding physical receiving shapes ("locks") in the cartridge receptacle 140. Electrical contacts on the cartridge 200 and cartridge receptacle 140 can then electrically transmit messages to the processor 102 indicating the lock and key fit for the various cartridge test types. According to some embodiments, the chip 1300 may have memory therein, and when the corresponding cartridge base 700 is received in the cartridge receptacle 140, such information (e.g., information about the sensor type and / or the tests contained therein) is stored in the memory and communicated to the processor 102 of the reader unit 100.

[0243] A. Example Measurement Types

[0244] According to some embodiments, chemically, biosensor 1300 may contain chemicals related to measuring concentrations in any of a variety of chemical reactions, such as three or more of the following chemical reaction categories: enzymatic, ion selective, or immunoassay.

[0245] Enzymatic:

[0246] Detection method: Amperometric method

[0247] According to some embodiments, the enzyme sensor 1300 works by measuring the electrons added to the working electrode by a redox mediator (e.g., hexaamine ruthenium (II)) during the redox reaction. Specifically, a chronoamperometry is used between each electrode. According to some embodiments, a plurality of current data readings (e.g., three data points) at the beginning, middle, and near the end of the downslope of the analysis are averaged, and the related measurements and averages can be performed by one or more processors 102 in the reader 100 coupled to the sensor 1300 communication. According to some such embodiments, the average current value is converted to a potential, and then the concentration of the analyte is determined by the processor 102 using a standard calibration curve. For lactate, the potential (voltage) used is 100 to 300mv. Any potential (voltage) can be used as long as it reaches the reduction potential range of the specific biomarker.

[0248] Ion selectivity:

[0249] Detection method: open circuit potential measurement

[0250] According to some embodiments, the ion selective (ISE) sensor 1300 uses open circuit potentiometry (OCP) to detect ionic analytes in biological samples (e.g., saliva). According to some embodiments, there are two types of electrodes used for the measurement: a screen-printed Ag / AgCl reference electrode 1312 and a screen-printed carbon electrode 1311. According to some embodiments, the electrode 1311 collects electrons, and one or more potentiostats 104 compare the voltage (potential) between the electrode 1311 and the reference electrode 1312 and maintain a constant current between them. The processor 102 converts the voltage measurement value into a measurement value of the ion concentration through the Nernst equation without any calibration.

[0251] Immunoassay:

[0252] Detection method: Amperometric method

[0253] According to some embodiments, the amount of tetramethylbenzidine (TMB) of the chronoamperometric detection reaction on the unmodified screen-printed carbon electrode 1800-E. For TMB, the potential (voltage) used is 100 to 300mv. Any potential (voltage) can be used, as long as it reaches the reduction potential range of this particular substrate. The amount detected is inversely proportional to the amount of cortisol (or other analytes) present in saliva. According to some embodiments, the processor 102 uses the known cortisol concentration in the buffer to draw a standard curve, and then the processor 102 can use the standard curve to associate current with cortisol concentration. According to some embodiments, the processor uses 4-PL curve fitting (cortisol competitive immunoassay), but other parameterized logistic curves can also be used. See below for more information.

[0254] Regardless of the type of chemical reaction, according to some embodiments, the electronics in the reader / POCT device 100 measures the electrical signature of the chemical reaction on the biosensor 1300 in the cartridge 200. The relationship between the electrical signature of the chemical reaction and, for example, the lactate concentration is a known quantity. Therefore, according to some embodiments, when the reaction is complete (e.g., within 30 seconds to 12 minutes), the firmware and / or software in the reader / POCT device 100 converts the electrical signature into a biomarker concentration.

[0255] B. Enzyme and / or ion-selective sensors

[0256] In embodiments where the biosensor chip 1300 is configured to determine the presence and / or amount of an analyte using an enzymatic reaction, the sensing membrane 1300E according to some embodiments may be described as a second generation biosensor, in which a redox mediator is used to transfer electrons from the enzymatic reaction to the electrode surface to reduce reliance on oxygen mediation, thereby obtaining a better signal, such as Fig. 14B and 14C as shown in . Figures 14B-14C An embodiment of a biosensor chip 1300 for measuring lactate and a general schematic diagram of the reaction used according to some embodiments are shown. According to some such embodiments, the sensing film 1300E includes reagents, which preferably include enzymes, redox mediators, at least one adhesive and a surfactant. According to some embodiments, the enzyme can be lactate oxidase (LOx) for lactate detection, glucose oxidase or dehydrogenase for glucose, or lactose dehydrogenase for pyruvate. Some embodiments of the biosensor configured to determine the presence and / or amount of analytes using enzymatic reactions include lactate as a target analyte and lactate oxidase as a redox mediator. In general, as long as there is a suitable chemical reagent, any analyte that can be detected by enzymatic method, such as glucose, cholesterol, uric acid, alcohol, ketones, triglycerides, etc., can be measured. According to some embodiments, the enzyme used is from an oxidase, a dehydrogenase, a reductase or an esterase category. These enzymes may include glucose dehydrogenase (GDH), glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbate oxidase, alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase, and the like.

[0257] According to some embodiments, the redox mediator is a small redox active molecule that reacts with the enzyme active site and the electrode active surface 1315. The mediator is reduced by the electrons generated from the enzymatic primary reaction, and then oxidized at the working electrode surface, so that the electrons are transferred to the working electrode, and a current signal proportional to the detected analyte concentration is generated. The mediator can include a transition metal complex and a polymer backbone wherein one or more ligands are coordinated with a transition metal, such as selected from one or more of polyvinyl pyridine (PVP) or polyvinyl imidazole (PVI) and polyallyl glycidyl ether (PAGE), and a selectively connected polymer backbone and a transition metal complex joint structure. The transition metal used can include Os, Rh, Ru, Ir, Fe and Co, and the ligand can be a monodentate, bidentate, tridentate or tetradentate ligand. These ligands generally contain nitrogen and are heterocyclic (ammine compounds, pyridine, imidazole derivatives). For example, hexaammine ruthenium (III), ferrocyanide, ferrocene, dichloride pentaammine chlororuthenium (III) etc. According to some such embodiments, the redox mediator used is hexaammineruthenium(III) chloride (hereinafter referred to as "HR3") due to its structure and chemical reversibility between (II) and (III). HR3 converts from (III) to (II) and back after gaining or losing one electron, respectively, so it is perfect for single electron reactions such as the reaction of LOx.

[0258] According to some embodiments, the binder may include an inorganic polymer and / or an organic polymer. In some such embodiments, the inorganic polymer is PVP (polyvinyl pyridine), and the organic polymer is trehalose and Methocel (methylcellulose). Typically, the binder may include an inorganic polymer (PVC, PVP, etc.) and an organic polymer (trehalose, Methocel, cellulose, etc.). According to some embodiments, the use of trehalose, Methocel, and PVP as a binder for the sensing membrane 1300E allows for a shelf life greater than 12 months and greater stability.

[0259] According to some embodiments, the surfactant helps to evenly spread the sensing membrane comprising the enzyme, mediator and polymer backbone on the electrode. According to some embodiments, the surfactant used is Triton X-100. Other surfactants such as sodium dodecyl sulfate, perfluorooctane sulfonate and sodium stearate can be used.

[0260] According to some embodiments, the sensing membrane 1300E of the biosensor chip 1300 configured to detect the amount of lactate in a sample can be manufactured in the following manner. HEPES and BTP are mixed in an aqueous solvent to make a buffer solution. NaCl and Triton X are similarly dissolved in an aqueous solution to prepare a corresponding storage solution. PVP and hydroxyethyl cellulose are centrifuged and vortexed in an aqueous solution to form a polymer solution. Trehalose and sodium succinate are added to the polymer solution and stirred. The surfactant (Triton X, NaCl) solution is added, and the polymer solution is centrifuged and vortexed. METHOCEL is added to the buffer solution and stirred for at least 1 hour to be dispersed in the solution. Six amminoruthenium is added to an aliquot of the polymer solution, centrifuged / vortexed, and stirred for one hour. Six amminoruthenium is added to an aliquot of the polymer solution, and the combination is stirred for about one hour, then vortexed and centrifuged, and then final mixing can be performed to produce an intermediate mixture. LOx is then added to the mixture and stirred for at least 20 minutes.The resulting viscous solution is then cast into the corresponding wells 1331 in 2 uL or smaller droplets and cured at a maximum of 55°C for at least 10 minutes.

[0261] According to some embodiments, the selection of various reagents in the sensing membrane 1300E may allow for the following advantages:

[0262] Detection time: 30 seconds

[0263] Shelf life>6 months

[0264] Sample volume ≤5uL

[0265] Repeatability: <5% between sensors and <5% between batches

[0266] Sensitivity: 0.1mM

[0267] LOD: 0.2mM (millimolar)

[0268] Linear 3.5mM

[0269] Fig.15A 1300HS for hydration sensors or general ion selective electrodes (ISE) according to some embodiments. The membrane 1300HS can be drop cast in layers into the hole 1331, where the membrane 1300HS is coated with carbon mats 1311, 1315 (see Figures 13D-13F). According to some embodiments, the first layer is a hydrogel layer 1508, which adheres the ion-selective hydrated sensing membrane 1300HS to the carbon pads 1311, 1315 and contributes to overall stability. Next is the sensing layer 1518, which contains ion carriers and corresponding salts of target ions, which helps to generate a membrane potential for analyzing ion concentrations. The top layer is a PDMS cover layer 1528, which protects the sensing layer 1518 from external interference and damage and increases overall stability while allowing ions to diffuse from the solution into the sensing layer for use in the analysis process.

[0270] According to another embodiment, the biosensor chip 1300 can be configured to determine the presence and / or amount of an analyte using an ion selective reaction, wherein a working electrode detects the activity of an ionic species passing through a selective membrane. According to some such embodiments, an open circuit potentiometry (OCP) method can be used to detect ionic analytes. Fig.13E According to some such embodiments, holes 1334 and 1336 may contain a sensing layer 1300HS to measure the concentration of sodium (Na) (1334) and potassium (K) (1336) ions, respectively. One of holes 1334 and 1336 serves as a working hole for sodium (Na) ions and the other serves as a working hole for potassium (K) ions. According to some embodiments, hole 1335 contains two carbon pads used as common counter electrodes for some such embodiments, and an Ag / AgCl electrode 1312 as a common reference electrode. In some such embodiments, no other holes are used. The working electrode is where a chemical reaction (including oxidation or reduction) occurs. The counter electrode or reference electrode undergoes an opposite chemical reaction at its surface, for example, if the working electrode is undergoing reduction, oxidation occurs at the counter electrode or reference electrode. This completes the circuit between the two cells and the potentiostat 104. According to some embodiments, the reference electrode measures the potential of the working electrode in the absence of a current passing through the reference electrode and maintains a constant potential throughout the experiment.

[0271] According to some embodiments, the sensing membrane 1300HS is an ion selective electrode (ISE) that can be configured to measure key hydration biomarkers such as Na+, K+, and Cl-, although the techniques herein can be used to create any selective ISE for any ionic analyte. Fig.15A As shown in FIG, the ion selective hydrated sensing membrane 1300HS may include a cover layer 1528 containing only a polyvinyl alcohol (PVA) film, a sensing layer 1518, and a polymer film 1508 located on top of a screen printed carbon electrode 1315. The cover layer 1528 is used to protect the sensing layer 1518 and improve stability, as well as to facilitate diffusion of the analyte into the sensing layer 1518.

[0272] According to some embodiments, the covering layer 1528 material is Dowsil 3140, a form of PDMS. According to some embodiments, other types of PDMS can be used if they can be dissolved and drop-cast onto the sensor surface 1518. Other polymers such as Nafion, polyurethane, etc. can be used. According to some embodiments, the covering polymer is dissolved in a solvent such as THF, although other solvents such as 2-methyltetrahydrofuran, cyclopentyl methyl ether, etc. can also be used. According to some embodiments, a plasticizer such as DOS is also used in the mixture to drop-cast the polymer. Any other plasticizers such as DEHP, adipates, citrates, phthalates, etc. can also be used.

[0273] According to some embodiments, the sensing layer 1518 is composed of a polymer, an ion carrier, a lipophilic salt and a plasticizer. The ion carrier is used to capture the analyte ions and allow them to pass through / be captured in the sensing layer 1518, allowing a potential proportional to the analyte concentration to be generated. According to some embodiments, the ion carrier for the Na+ analyte is a sodium ion carrier X (4-tert-butyl calix [4] arene tetraacetic acid tetraethyl ester), but other ion carriers for sodium can be any one of the Selectophore series of Sigma Aldrich (i.e., sodium ion carriers I, II, etc.). In general, other ion carriers for other analytes will be similar derivative molecules that have a high affinity for the ions and cannot diffuse outside the layer 1518. Their examples include ETH 1001 for calcium, inactive bacteria for ammonium, valinomycin for potassium, etc. According to some embodiments, the polymer used for the sensing layer 1518 is PVC. Other polymers such as PVA can be used, as well as conductive polymers such as PEDOT:PSS, PPy, etc. The solvent for layer 1518 is THF, and the aforementioned solvents may also be used. According to some embodiments, the plasticizer is also DOS, and the aforementioned substitutes are also applicable. According to some embodiments, the lipophilic salt in layer 1518 is Na TTFPB (sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate hydrate). The salt has an increased solubility in layer 1518, which facilitates solvent extraction of cations and improves the sensitivity of the sensor. Other lipophilic salts of sodium may also include sodium tetrakis(4-fluorophenyl)borate dihydrate, etc. In general, lipophilic salts containing analyte ions will be used for general ISE. For example, for potassium, potassium tetrakis(4-chlorophenyl)borate salt may be used.

[0274] According to this embodiment, the sensing membrane 1300HS and the electrode 1315 form a solid-state electrode, without the need for a liquid inner membrane, which allows for easy manufacturing and greater stability during storage. It can produce instant results (<1 second) instead of taking up to 10 minutes of stabilization time like conventional ISEs. Due to its structure, it has a wide sensitivity range and gives a logarithmic Nernst response.

[0275] According to some embodiments, the following advantages can be obtained using the sensing membrane 1300HS: Stability: 3 months or more at room temperature, expected stability is more than one year after manufacturing, sensitivity: 0.1-100mM in saliva, no calibration required, no pre-conditioning required.

[0276] According to some embodiments, the sensing membrane 1300HS is manufactured in the following manner. 2.5% PVA is prepared in DI water and methanol is carefully added. The solution is drop-cast in the corresponding holes 1331 and then baked at no more than 100°C for at least 10 minutes. The separate solutions of sodium ion carrier X, Na-TFPB and THF are then stirred for 30 minutes or more. PVC is added to the solution and stirred for at least 1 hour. DOS is added to the final solution and stirred for at least 2 hours. The solution is then drop-cast onto the base layer 1508 and baked at no more than 40°C for at least 5 hours. The final solution consists of Dowsil 3140 in THF and DOS, which is stirred for at least 2 hours. It is drop-cast on the electrode 1315 and left at room temperature for one hour.

[0277] According to another embodiment, the biosensor chip 1300 contains an enzyme hole and an ion hole. In this embodiment, holes 1332 and 1333 are used to determine the presence and / or amount of an analyte using an enzymatic reaction, wherein hole 1332 is used as a background correction hole without a sensing layer, and hole 1333 is used as a measurement hole with a sensing layer 1300E drop cast. According to some embodiments, background correction can be achieved by subtracting the signal at the background electrode from the signal (current) obtained at the working electrode. In addition, both holes 1334 and 1336 and hole 1335 contain a sensing layer 1300HS, wherein holes 1334 and 1336 are used as working holes for measuring Na and K ions, respectively, and hole 1335 contains a common counter electrode and a reference electrode used in the measurement.

[0278] Fig. 15B is a graph showing the stability response of the sensor to ion selective reactions obtained by measuring different concentrations of NaCl over a 1 minute period according to some embodiments. According to some embodiments, the POCT device 100 measures ion selective chemical class biomarkers electrochemically by displaying changes in electromotive force (EMF, volts), where different concentrations of target ions are monitored under nearly zero current conditions. Fig. 15B The response of sensor 1300 to different concentrations of potassium ions (0.1 to 1000 mM) in the sample is shown. The stability of the response of sensor 1300 was evaluated by measuring the response potential as a function of time (over 60 seconds). The results show that sensor 1300 reaches a stable potential immediately and is stable during the detection period. In addition, the ion-selective electrode of the sensor shows high repeatability in the response of the sensor to detecting different concentrations of target ions.

[0279] Figures 15C-15E is a graph showing the response of sensor 1300 at different electrolyte concentrations according to some embodiments, wherein Fig. 15C shows potassium, Fig.15D shows sodium, Fig.15E Chloride is shown. Fig. 15C The response of the sensor to different concentrations (0.1 to 1000 mM) of potassium ions (K+) is shown in Fig.15D Sodium ions (Na+) are shown in Fig.15E Chloride ion (Cl-) is shown. For this specific example, the slope of the linear portion of the response curve for K+, Na+ and Cl- ions is 48.5mV, 59mV and 61mV per decade, respectively. Other examples may show alternative response curves. The slope of the calibration curve is close to the Nernst response and is within the experimental error range.

[0280] Fig.15F1 is a functional block diagram of a reader / test device and sensor according to some embodiments of the present disclosure. The reader or Stone 100 presses a processor 102 electrically and / or communicatively coupled to a potentiostat circuit 104, which in turn is electrically and / or communicatively coupled to an electrical contact 160 in a cartridge receptacle 140. The processor 102 may include one or more processors and may have one or more forms, such as a microcontroller. According to some embodiments, the potentiostat circuit 104 includes one or more potentiostats. The potentiostat measures and controls the potential difference between any two electrodes. In some embodiments employing enzymatic or immunoassay chemical tests, the potentiostat drives a single voltage to two electrodes and measures the resulting current over time. In some embodiments employing ion selective tests, the potentiostat changes the potential to maintain a constant current between a working electrode and a reference electrode. The potentiostat then measures the resulting potential change. The electrical contacts 160 in the cartridge receptacle 140 are electrically coupled to the sensor pads or contacts 1316 on the sensor 1300. In turn, these pads or contacts 1316 are electrically coupled to electrical contacts or pads 1311, 1312, 1321, 1322, 1323 associated with holes 1332, 1333, 1334, 1335, and 1336 of sensor 1300. Processor 102 may also be communicatively coupled to memory 106, which may have operating instructions stored therein and may store information or data received from processor 102 and / or chip 1300 and / or an external device (e.g., smartphone 191). According to some embodiments, electrodes 1311, 1312, 1321, 1322, 1323 on chip 1300 form part of a circuit monitored by processor 102 and / or potentiostat 104. When a chemical reaction occurs in one or more wells 1331 on the chip 1300, the number of electrons on the electrodes 1311, 1312, 1321, 1322, 1323 changes and can be sensed, and the characteristics of the biological sample (e.g., saliva) are determined by the processor 102. The change in the number of electrons can be sensed as a change in voltage and / or current in the electrical signal received by the reader 100 from the electrodes 1311, 1312, 1321, 1322, 1323, such a change is detected by the potentiostat 104 and / or the processor 102. According to some embodiments, the change in one or more signals received from the one or more electrodes 1311, 1312, 1321, 1322, 1323 is proportional to the amount of one or more substances present in the biological sample, and the proportional signal is used by the potentiostat 104 and / or the processor 102 to determine and / or calculate the amount of such substances as described above.According to some embodiments, when one or more specific substances are present in the biological sample, one or more signals received from one or more electrodes 1311, 1312, 1321, 1322, 1323 will change, and the signal is used by the potentiostat 104 and / or the processor 102 to determine the presence of such substances as described above. The reader 100 may also include a memory 106 coupled to the processor 102 in communication. The reader 100 may also include a display 110 coupled to the processor in communication, wherein the processor may cause data or test results to be displayed on the display. The reader 100 may also include an antenna or communication module 108 to allow the reader to be coupled to one or more external devices such as a smartphone, a smart watch, or a computer in communication. In some embodiments, the communication module includes Bluetooth hardware, such as Bluetooth Low Energy (BLE). The reader 100 may also include a power regulation controller 109. The power regulation controller allows the use of a battery 107; and the battery can be charged through a port 111 in the main chassis of the reader. The reader 100 may also include a signal conditioning circuit 112 to amplify the detected electrical signal from the biosensor chip.

[0281] C. Fabrication of enzyme and / or ion-selective sensors

[0282] Fig.16A is a top view or plan view of a printed layer strip 1610 according to some embodiments of the present disclosure, the strip comprising a plurality of screen-printed electrode layers 1310, which can be used to manufacture a plurality of sensor chips 1300. The manufactured strip 1610 comprises Fig. 13C . According to some embodiments, the plurality of screen-printed electrode layers 1310 are printed in strips 1610 with alignment holes 1612 for precise mounting to add spacers 1340 and capping layers 1350. Printing in strips allows for mass production and ease of automation in the manufacture of electrodes 1311, 1312 and chip 1300, as they can be cut to standard sizes from very large laser-cut strips of material 1610. During manufacturing, one or more alignment holes 1612 of the printed layer strips 1610 are placed around one or more alignment posts.

[0283] Fig. 16B is a top view or plan view of a strip 1640 of spacer layer 1340 for automated manufacturing according to some embodiments of the present disclosure. The manufactured spacer strip 1640 includes Fig. 13B. According to some embodiments, the spacer strip 1640 has one or more alignment holes 1642 for precise alignment with other strips, such as the print layer strip 1610 and the cap layer strip 1650. During the manufacturing process, the one or more alignment holes 1642 of the spacer strip 1640 are placed around one or more alignment posts, and the spacer strip 1640 is covered with a thin layer of adhesive and pressed onto the print layer strip 1612. Fig. 16B The exemplary dimensions shown in are in millimeters (mm). According to some embodiments, the spacer strip 1640 is made of PET and is laser cut to allow for the formation of grooves above each hole 1331 to increase saliva retention and strengthen the electrode material by making it more rigid. The spacer 1340 is also Fig. 13B Space is left between the screen printed electrode layer 1310 and the top cover 1350 shown in FIG. 1 to allow saliva to flow properly.

[0284] Fig. 16C is a top view or plan view of a cap strip 1650 for automated manufacturing according to some embodiments of the present disclosure, which includes multiple Fig. 13B The top cover 1350 shown in . According to some embodiments, the top cover strip 1650 is made of a hydrophilic plastic film. The hydrophilicity of the film redirects saliva along the film into each hole 1331 and protects the electrodes 1311, 1312 in each hole from external interference. According to some embodiments, the top cover strip 1650 has one or more alignment holes 1652 for precise alignment with other strips such as the printed layer strip 1610 and the spacer strip 1640. According to some embodiments, during the manufacturing process, the one or more alignment holes 1652 of the top cover strip 1650 are placed around one or more alignment posts, and the top cover 1350 is attached to the spacer strip 1640 using a pressure-activated adhesive.

[0285] According to some embodiments, the biosensor chip 1300 is manufactured in a manner that makes the chip 1300 cheap and reproducible. In such embodiments, the printed layer 1310 is first manufactured in a strip design 1610 using screen printing. The design is then installed so that the holes 1612 of the strip 1610 can be aligned on all three layers (including 1640 and 1650) to be pressed, which aligns the holes 1612, 1642, and 1652 of the corresponding layers. The spacer strip 1640 is pressed onto the printed layer strip 1310 so that the pressure-activated adhesive bonds the layers 1610, 1640 together. Similarly, the top cover strip 1650 is pressed onto the spacer layer 1640, and then the combined layer 1610 is cut or divided into individual biosensor chips 1300.

[0286] D. Immunoassay Sensors

[0287] Fig.17A 17 is an exploded perspective view of an immunoassay system or platform 1700 according to some embodiments of the present disclosure. According to some embodiments, the immunoassay system 1700 comprises a Cube reader 1710, a disposable microfluidic chip 1800, and a biological fluid (eg, saliva) collector 400.

[0288] Fig. 17B is an exploded perspective view of components of a Cube reader 1710 according to some embodiments of the present disclosure. Figure 17B' is an exploded perspective view of components of a Cube reader 1710 according to some embodiments of the present disclosure. According to some embodiments, one component of the immunoassay system 1700 is a Cube reader 1710 that houses electronics and actuators for performing analysis of a biological fluid such as saliva. According to some embodiments, the system 1700 may include disposable devices such as a microfluidic chip 1800 and a saliva collector 400.

[0289] According to some embodiments, the immunoassay system 1700 and Cube reader 1710 include a base 1712 , a manifold 1714 , and a test station 1716 .

[0290] According to some embodiments, base 1712 contains tubing and wiring.

[0291] Fig. 17C 1716, according to some embodiments. According to some embodiments, the test stand 1716 includes a test stand housing 1716-1, a magnetic gear assembly 1716-2, and a Pogo pin 1716-4. The test stand housing 1716-1 has a plurality of air ports 1716-5 therein, which correspond to the air inlet 1800-2 (see Fig.19J ) and valve 1714-2.

[0292] Fig.17D 1800-1 is a perspective view of a magnetic gear assembly 1716-2 with a magnetic gear 1716-2g according to some embodiments. The magnetic gear 1716-2g is inserted into the bottom chip port 1800-1 (see Fig.19H , 19J ) and rotate to mix the substrate in the reaction chamber 1931 by the action of the ferromagnetic beads (see Fig. 18C , 18E, 19F, 19G1). Neodymium magnet 1716-j is located in magnetic gear 1716-2g, which affects the changing magnetic field on reaction chamber 1931 by rotation to mix the reactants by moving ferromagnetic beads within reaction chamber 1931. In some embodiments, magnets can also be placed in bed 1716-1 to tightly attach chip 1800 to test bench 1730 so that there is a seal along air port 1716-5. Pogo pins 1716-4 correspond to contacts 1928 on chip 1800 (see Fig.19C ). When the chip 1800 is clamped to the test bench surface 1716-1s due to the force from the magnet 1716-3, the pogo pins 1716-4 are compressed. These pins 1716-4 transmit the voltage from the processor or microcontroller 102 to melt the wax valve 1925.

[0293] Fig.17E 1 is a perspective view of a manifold 1714 according to some embodiments. According to some embodiments, the manifold 1714 includes a micropump 1714-1 that moves fluid throughout the device 1700. According to some embodiments, the micropump 1714-1 uses low voltage and is piezoelectric. For example, the micropump 1714-1 can be a Bartels mp6 micropump. According to some embodiments, the manifold 1714 includes a valve manifold 1714-2, which includes individual microvalves 1714-2v. These microvalves 1714-2 can be shape memory alloy valves (such as valve SMV-2R-AN1F manufactured by Takasago Electric, Inc.) that open in response to a voltage signal from the microcontroller 102. They allow the input and output of fluids and air throughout the device 1700. These microvalves 1714 - 2v may be stacked in the manifold 1714 and correspond to the number of each port in the test station 1716 - 1 , for example valve number 0 may correspond to supply line 1716 - 5 which is also labeled number 0.

[0294] Fig.17F 1 is a schematic diagram depicting fluid flow in the manifold 1714 and the microfluidic chip 1800 according to some embodiments. Fig.17FAs shown in , pump 1714-1 is connected to valve manifold 1714-2 via a single line, by which the air pumped from the pump is delivered to valve 1714-2v and corresponding air port 1716-5 in chip 1800 through a separate line. In some embodiments, if valve 1714-2v in valve manifold 1714 is labeled, for example, "0", then the number corresponds to the entire line in the device that terminates at the corresponding air port 1716-5 labeled "O", and this is true for all lines from 0 to 5. Pump 1714-1 and / or valve 1714-2v can be communicatively coupled to a processor that can operate them. This control from pump 1714-1 to chip 1800 allows different routes in the microfluidic chip to be selectively used for steps in an experiment.

[0295] An exemplary system may employ one or more or all of the following features: small sample volumes (e.g., ≤ approximately 20uL), 30 second measurement times, in situ flow metering with processing times of 12 minutes rather than hours, active pumping in microfluidic devices rather than capillary flow, self-contained disposable cartridges, and heating to melt wax seals on reagent containers.

[0296] According to some embodiments, a system is provided having one or more or all of the following features:

[0297] Small-scale multilayer microfluidic designs for small-scale immunoassays;

[0298] POC immunoassays using saliva rather than in a laboratory;

[0299] Unique processing steps (reagents, mixing, etc.);

[0300] Small sample volume (20uL vs. 200uL (nearest competitor));

[0301] 30 seconds measurement time;

[0302] Processing time is less than 12 minutes;

[0303] A locking mechanism for integrating the handle into the cartridge;

[0304] A self-contained disposable cartridge that performs all analysis; and / or

[0305] · Melt the wax using the heating electrodes on the substrate.

[0306] Microfluidic Biosensor Chip 1800:

[0307] Fig.18A1 is an exploded perspective view of an immunoassay biosensor chip 1800 according to some embodiments. As shown, the biosensor chip 1800 includes a biological fluid (e.g., saliva) collector adapter 1810 for receiving a biological fluid collection device 400, a first or top microfluidic layer 1820 (see also Fig.18D , 19A -19B), a first or top adhesive layer 1830 (see also Fig.18E , 19A -119B), wax valve PCB 1840 (see also Fig.19C ), second or bottom adhesive layer 1850 (see also Figures 19E-19F ) and a second or bottom microfluidic layer 1860 (see also Figures 19E-19F ).

[0308] Fig.18B 1 is a plan view of a biological fluid (e.g., saliva) collector adapter 1810 for receiving biological fluid collection device 400. Biological fluid collector adapter 1810 includes one or more threaded pins 1812. The purpose of threaded pins 1812 is similar to that of protrusions or threaded pins 712 described above (see Fig.7D ) similarly, allowing the handle 450 to be tightened and apply pressure to the cotton swab tip 480 to release biological fluid such as saliva into the chip 1800.

[0309] Figures 18C-18E is a top view of the multiple layers of the immunoassay biosensor chip 1800 according to some other embodiments of the chip 1800. According to some embodiments, the chip has three layers: a microfluidic channel layer 1860, a wax sealing layer 1840, and a sample storage layer 1819.

[0310] Figures 18C-18E is a top view of multiple layers of an exemplary chip 1800 according to a first implementation of the chip 1800 .

[0311] Figures 19A-19B 19E-19F are top views of various layers of an exemplary chip 1800 according to the second embodiment. Figures 19C-19D An exemplary heating PCB 1840, 1840b is shown with an exemplary wax valve 1925 (individually labeled AI) thereon. In some versions of the second embodiment, only the Fig.19C The heating PCB 1840 is shown in FIG.

[0312] The first embodiment of chip 1800 will refer to Figures 18C-18E Give a description.

[0313] Sample storage layer 1819 ( Fig. 18C): Storage layer 1819 contains reagents and stores waste after sample and antibody analysis and mixing. According to some embodiments, sample storage layer 1819 includes a wash buffer reservoir 1811, a waste collection chamber 1815, a TMB reservoir 1812, a sample mixing chamber 1811, a plurality of sample storage chambers 1814-1, 1814-2, 1814-3, a saliva collector interface 1816, and a saliva metering port 1814-4.

[0314] Wax seal 1840( Fig.18D ): Wax sealing layer 1840 houses valve 1825 (labeled AI) located between reservoir layer 1819 and microfluidic layer 1860. Wax valve 1845 melts during the analysis process to release the reagents stored thereon.

[0315] Microfluidic layer 1830 ( Fig.18E ): The microfluidic layer 1860 contains the "laboratory" for analysis. According to some embodiments, this layer 1860 includes a reaction chamber 1861, a serpentine mixing channel 1863 for mixing a biofluid sample with TMB, a detection chamber 1862, a sample inlet port 1864, an outlet 1867 to a waste reservoir, an air / wash buffer inlet 1865, and a TMB inlet 1866.

[0316] refer to Figures 18A-18E and Figures 19A-19J According to some embodiments, each chip 1800 contains a microfluidic "laboratory" complete with microfluidic mixing, reaction and storage channels and its own reagents. The reagents are stored in the chip 1800 and released into the cartridge microfluidic system during analysis using a paraffin valve 1845 / 1925, which is melted by using a heated PCB 1840, 1840b in the chip 1800.

[0317] The second embodiment of chip 1800 will refer to Figures 19A-19J Give a description.

[0318] Fig.19A shows the adhesive layer, which is cut to match Fig.19B The perimeter of the microfluidic paths shown in FIG. 1 is sealed together to seal the chip. Fig.19A The adhesive layer is located at Fig.19B and 19D Between the layers shown in .

[0319] Fig.19BThe first half or upper half of the immunoassay chip 1800 is shown. This layer of the chip contains a main waste chamber 1913, a serpentine channel 1918 for mixing and TMB reaction and controlling signal reading delay by slowing fluid flow. The layer also contains a wash buffer storage chamber 1911, a TMB storage chamber 1916, and a pre-mixing chamber 1915 for antibodies, ferromagnetic beads, and HRP. The layer also contains a long serpentine mixing channel 1917 for thoroughly mixing the solutions before the reactants enter the reaction chamber, an antibody storage chamber 1914-1, a magnetic bead storage chamber 1914-2, and an HRP storage chamber 1914-3. The layer also contains an inlet 1800-S for receiving saliva or other biological fluids from a biological fluid collector adapter 1810.

[0320] Fig.19C A first side of the wax valve PCB layer is shown, which responds to electrical input to melt the wax, releasing the sealed fluid for reaction. Electrical contacts 1928 are connected to wax valves 1925. When an electrical signal is transmitted along the corresponding connection, the wax seal in the corresponding valve 1925 melts, thereby releasing the corresponding stored fluid.

[0321] Fig.19D Shows Fig.19C The second opposite side of the wax valve PCB layer.

[0322] Fig.19E shows the adhesive layer, which is cut to match Fig.19F The perimeter of the microfluidic paths shown in FIG. 1 is sealed together to seal the chip. Fig.19F The adhesive layer is applied to Fig.19E On one half of the immunoassay chip 1800 shown in FIG. Fig.19E The adhesive layer and Fig.19F The combination of half of the immunoassay chip 1800A shown in FIG. Fig.19C The wax valve is shown on the first half of the PCB layer.

[0323] Fig.19F The second or lower half of the immunoassay chip 1800 is shown. This half of the immunoassay chip 1800 contains chamber 1932, a waste chamber 1913 for collecting reacted TMB, a mixing channel 1933 for mixing TMB and slowing the flow of solution to chamber 1932, a sample metering chamber 1939, and a main reaction chamber 1931. Air ports 1800-2 allow air to flow from the pump into the chip fluid network. These ports correspond to their respective air lines (0-5).

[0324] refer to Figure 19G1 and 19G2, saliva is input at 1800-S. Capillary force moves the saliva into the mixing channel 1917 together with the antibodies, magnetic beads and HRP from chambers 1914-1, 1914-2, and 1914-3, respectively. They are first mixed in chamber 1915 and then pumped to the mixing channel 1917. The mixed sample fluid is pumped into the sample metering chamber 1939 and then pumped to the main reaction chamber 1931. The magnetic motor 1716-2 rotates below or near the chamber 1931, thoroughly mixing the mixed fluid and attaching the antibody-bead-HRP-cortisol complex to the bottom of the reaction chamber 1931. Buffer is then pumped from the buffer storage chamber or the wash chamber 1911 to remove uncoupled materials and pump them to the waste chamber 1913. TMB is then pumped from the TMB storage chamber 1916 into the main reaction chamber 1931 to react with the coupled materials. The solution mixed with TMB is then passed into the serpentine mixing channel 1918 for further mixing and reduced flow rate, where it is pumped toward the electrode 1800-E for measurement. Any excess fluid is pumped into the waste chamber 1913.

[0325] According to some embodiments, the chip 1800 works as follows: in response to a signal from the microprocessor 102, a 5V signal is sent through a trace to a 25Ω heating resistor rated at 1.5W. The electricity generated and dispersed across each resistor is distributed as heat to the copper through a hole containing a thin paraffin layer. The heat generated thereby melts the corresponding wax valves 1845, 1925, allowing the reagents or fluids contained in the storage layer of the microfluidic chip 1800 to be distributed into the microfluidic reaction layer 1860. Each valve 1845, 1925 is controlled by a separate trace of the microcontroller 102 leading to the immunoassay Cube reader 1710, allowing each chemical to be accurately distributed when needed in the reaction sequence.

[0326] Go to Figure 19G1 , which is a perspective view showing microfluidic pathways and wells within a disposable microfluidic chip 1800 according to some embodiments. These pathways correspond to Figures 19A-19B The paths shown in FIG. 1 are not shown in FIG. 1 , but when the chip is sealed, they will form these paths and chambers.

[0327] Figure 19G2 It means through Figure 19G1 A block diagram of the physical flow of fluid.

[0328] Fig.19H is a perspective bottom view of an assembled microfluidic chip 1800 according to some embodiments, Fig.19I This is its top view. Fig.19Jis a bottom view thereof. The bottom chip port 1800-1 allows the magnetic gear 1716-2 to be inserted and very close to the reaction chambers 1831, 1931 located on the opposite side of the surface. The chip air inlet 1800-2 allows the micro pump 1714-1 to move fluid around the microfluidic chamber. The electrode port 1800-3 can be inserted with a standard carbon electrode 1800-E for analyzing the reaction in the chip 1800.

[0329] According to some embodiments, the top microfluidic layer 1820 of the microfluidic chip 1800 may include a main waste chamber 1913. Reactant waste is transported to and contained in the main waste chamber 1913 for processing. The top microfluidic layer 1820 may also include a wash chamber 1911 - this is where a wash step is performed, where reagents are passed over the magnetically attached antibody complexes to remove unattached reactants. The top microfluidic layer 1820 may also include a sample metering and overflow chamber 1939 (see Fig.19B ). Excess saliva (exceeding the saliva required for analysis) is sent to the sample waste chamber 1939. The top microfluidic layer 1820 can also include an antibody storage chamber 1914-1, a magnetic bead storage chamber 1914-2, and an HRP storage chamber 1914-3. The antibody storage chamber 1914-1 stores antibodies. When its valve melts, the antibodies enter the mixing chamber 1915. The magnetic bead storage chamber 1914-2 stores magnetic beads for mixing. The HRP storage chamber 1914-3 stores HRP for mixing. These three chambers are released into the mixing chamber 1915 through separate wax valves and then sent to the mixing channel 1917 to mix with the sample. The top microfluidic layer 1820 can also include a premixing chamber 1915 and a premixing chamber 1916 in which premixed reactants / samples are stored. The top microfluidic layer 1820 may also include a sample metering chamber 1939 to help monitor the amount of sample loaded into the reaction chamber 1931. The top microfluidic layer 1820 may also include a TMB mixing channel 1918, which may be a serpentine profile channel for mixing TMB and HRP-antibody-bead-cortisol conjugate within the reaction chamber 1931.

[0330] According to some embodiments, the top adhesive layer 1830 of the microfluidic chip 1800 can be made of plastic (PET or other thin flexible plastic) covered with pressure sensitive adhesive on both sides. The plastic can be laser cut into a pattern as shown (see Fig.18A , 19A 19E). The adhesive bonds the top layer 1820 to the PCB layer 1940. The adhesive layer 1830 and the top layer 1820 are hydrophobic, which keeps the liquid in the microfluidic channels instead of leaking out.

[0331] According to some embodiments, the wax valve PCB 1840 includes a wax valve 1925 and a contact 1928. The PCB 1840 can be a thin (0.6 mm) board containing a wax through hole 1925. The PCB 1840 is located between the top 1820 and bottom 1860 microfluidic layers and forms the surface of the cavities and channels on each layer. The wax through hole 1925 can be a copper through hole of various diameters, which is coated in liquid paraffin and allowed to dry. When a signal is passed from the processor or controller 102 through the contact 1928, the voltage runs through the resistor connected to the corresponding one of the through holes 1925, and when the resistor has a small resistance value (<10 ohms), the resistor generates heat. The heat is transferred to the corresponding through hole by direct contact, and the wax in the through hole melts within one second, allowing the fluid to flow through the through hole, such as the corresponding specific chemical or sample flowing through the top microfluidic layer 1820 to the bottom layer 1860. The opening of the respective wax valves 1925 can be controlled, for example triggered one at a time, thereby releasing and using a series of chemicals / fluids throughout the process (documented in the flow chart - see Fig. 20 ).

[0332] According to some embodiments, the bottom adhesive layer 1850 of the microfluidic chip 1800 can be made of plastic (PET or other thin flexible plastic) covered with pressure-sensitive adhesive on both sides. The plastic can be laser cut into a pattern as shown (see Fig.19A and 19E ). The adhesive bonds the bottom layer 1860 to the PCB layer 1940. The adhesive layer 1850 and the bottom layer 1860 are hydrophobic, which keeps the liquid in the microfluidic channel instead of leaking out.

[0333] According to some embodiments, the bottom microfluidic layer 1860 of the microfluidic chip 1800 may include a reaction chamber 1931. According to some embodiments, the TMB reaction occurs in the reaction chamber 1931, and the chamber 1931 allows the fluid of the final reaction to be pumped to the electrode 1800-E. Chamber 1931 allows the insertion of a standard carbon screen-printed electrode, and the fluid is pumped into the chamber so that the electrode is covered in liquid so that the chamber can act as an electrochemical cell for measurement. The bottom microfluidic layer 1860 may also include a TMB mixing channel 1933, which may have a serpentine profile. The bottom microfluidic layer 1860 may also include an overflow chamber 1932, which may provide additional storage space for the overflow of any liquid exceeding the amount required for measurement on 1800-E. 1800-E may be located on the mixing channel 1933 before the chamber 1932. The bottom microfluidic layer 1860 may also include a main waste chamber 1938 and a sample waste chamber 1939.

[0334] According to some embodiments, the chip 1800 works as follows: a 5V signal is sent through a trace to a heating resistor of <25Ω with a rated power of at least 1.5W. The electricity generated and dispersed across each resistor is distributed as heat to a copper through-hole containing a thin paraffin layer. The heat thus generated melts the wax valve 1925, allowing the reagents or fluids contained in the storage layer 1820 of the microfluidic chip 1800 to be distributed into the microfluidic reaction layer 1860 (composing the entirety of Figure 9G1), and the reactants / substrates can be released into the system. Each valve 1825 is controlled by a separate trace leading to the microcontroller 102 of the immunoassay Cube (1700), allowing each chemical to be accurately distributed when needed in the reaction sequence.

[0335] Figure 19K 1712-d is a functional block diagram of the electrical components of the immunoassay system 1700 according to some embodiments of the present disclosure. 1712-d is a functional block of a driver for operating the valves / pumps in 1714 and 1716. The processor 102 sends signals / instructions to 1712-d to drive the pump, valve, or melt wax through hole based on the parameters set during execution. It also sends data to the communication module 108. The potentiostat 104 measures the output of the chemical reaction at the electrode 1800-E and sends the data / signal back to the processor 102.

[0336] Chemical Process:

[0337] The immunoassay platform operates by microfluid mixing and sampling. First, a sample (saliva, although other biological fluids such as blood or urine may be used in other embodiments) is added to the system using a biofluid collection device 400 through a collector adapter 1810. The stored reagents (Ab, enzyme-labeled competitive reagents, immobilized substrate particles) are mixed with the sample. These are moved to a reaction chamber 1831 and reacted for 7 minutes, during which the antibody is coupled to a streptavidin-labeled magnetic particle. Then, a binding competition occurs between the analyte present in the saliva and the competitive reagent (analyte labeled with an enzyme). The reaction chamber 1831 is then purged with air and cleaned with a phosphate buffer, leaving only the coupled magnetic particles. TMB is added and incubated with the particles for at least 5 minutes, and then pumped into a mixing channel 1833 for uniform mixing. The mixture is then sent to electrode 1800-E for analysis.

[0338] Exemplary reagents include: biotinylated polyclonal rabbit anti-cortisol antibody in stabilization buffer, cortisol-3-CMO-HRP in HRP stabilizer, magnetic particles with streptavidin conjugate in phosphate buffer, phosphate buffered saline with 0.05% Tween 20, [3,3',5,5']-tetramethylbenzidine.

[0339] The universal immunoassay platform 1700 comprises a combination of reagents to facilitate electrochemical detection. Typically, these reagents are biotinylated antibodies specific for the target analyte, competitive reagents (in the case of competitive assays), labels on competitive reagents (in competitive assays), secondary antibodies (sandwich assays only), chromogenic substrates, immobilized substrates, and labels on the immobilized substrates. Some embodiments of the platform 1700 are used to detect cortisol in saliva, but the platform can be used to detect any analyte for which antibodies can be found, and other reagents will be customized according to the specific target analyte. Some embodiments of the platform 1700 use competitive immunoassays to sense analytes, but other forms of immunoassays, such as sandwich, direct or indirect assays, may also be used.

[0340] According to some embodiments, biotinylated rabbit polyclonal anti-cortisol Ab is used, but any animal background such as mouse monoclonal antibody, rat antibody, etc., and some other conjugates mentioned above can also be used, which are also bispecific. In some exemplary embodiments, the buffer for stabilizing the antibody is Surmodics Research assay diluent, but any stabilizing buffer for the selected antibody can be used. For the exemplary competitive assay, the competitive reagent is cortisol-HRP, which is a cortisol labeled with horseradish peroxidase (HRP). In general, any HRP-labeled analyte that matches the target analyte can be used, such as testosterone-HRP for testosterone sensing, etc.; enzymes can also be different, such as alkaline phosphatase (AP), biotin or fluorescein. The enzyme used will have a corresponding enzyme stabilizer, which is an HRP stabilizer (StabilZyme TMHRP conjugate stabilizer), but any stabilizer for the enzyme can be used. In the preferred embodiment, there is no second antibody, but in the sandwich assay format, any antibody from the host (e.g., rat, mouse, etc.) can be used and labeled with HRP, AP or other tags. The chromogenic substrate in the preferred embodiment is 3,3',5,5'-tetramethylbenzidine (TMB), but other substrates such as ABTS, AEC, DAB, ECL and Amplex Red (if HRP is used as the enzyme tag) can also be used; otherwise, if AP is used as the labeling enzyme, PNPP and the like can be used. The immobilized substrate in the preferred embodiment is ferromagnetic particles labeled with streptavidin, but other particles such as agarose beads, polystyrene beads, etc. can also be used; and the conjugate can be changed to neutravidin, protein A, protein G, etc. The buffer used in the washing steps is phosphate buffered saline (PBS) and Tween 20 (polyoxyethylene sorbitan monolaurate), although other buffer types such as BSS, DPBS, etc. may also be used, and another detergent such as Tween 40, Triton X100, etc. may be used.

[0341] Fig. 20 is a flow chart of the chemical processes within an immunoassay device according to some embodiments. Fig.19H The process is also shown in the block diagram above. In step 2010, saliva is collected using, for example, the collection device 400. When the collection device 400 is reinserted into the cartridge base 700, the saliva on the tip 480 is squeezed out of the tip 480 and enters the microfluidic system ( Figure 7F , 7I ).

[0342] In step 2020, the saliva sample is magnetically mixed with reagents in the reaction chamber 1831 for 7 minutes.

[0343] In step 2030, the reaction chamber 1831 is flushed with air to remove the solution.

[0344] In step 2040, according to some embodiments, the reaction chamber 1831 is cleaned with phosphate buffer and / or detergent and flushed again with air, and this process is repeated three times, leaving only the coupled magnetic particles in the reaction chamber.

[0345] In step 2050, TMB is added to the reaction chamber 1831 containing the coupled sample and incubated with the particles for 5 minutes.

[0346] In step 2060, the sample is pumped to the mixing channel 1833 for uniform mixing.

[0347] In step 270, after mixing, the substrate is pumped to electrode 1800-E for analysis. According to some embodiments, the electrode used to quantify the subsequent reaction with TMB is composed of screen-printed carbon ink, and any type of carbon ink can be used.

[0348] Fig.21 is a flow chart of the chemical processes within an immunoassay device according to some embodiments.

[0349] Fig.21 is a flow chart of an immunoassay process according to some embodiments. In step 2110, the substrate of the reaction is pumped to the electrode for analysis by an internal micropump. In step 2120, the current measurement is run at a potential of -50 mV. In step 2130, the standard curve (see, e.g., Figure 22-23 ) Extrapolate the concentration of the analyte (in the preferred embodiment, Cortisol). According to some embodiments, in step 2140, the concentration data is sent to an application for display to a user.

[0350] Fig. 22 is a graph of the electrochemical reaction of Cortisol according to some embodiments of the present disclosure. Fig. 22 A salivary cortisol 4-parameter curve fit for detecting cortisol in saliva is shown.

[0351] Fig.23 It is a diagram of the response of the sensor to different concentrations of cortisol according to some embodiments of the present disclosure. The figure also shows the binding percentage (B / Bo) of each saliva sample. B / Bo is obtained by dividing the sensor response (B) of each sample by the average background value (Bo) of zero point. The logical range of cortisol measurement in saliva is 0.01 to 30ng / mL, and the linear range of measurement is 0.1 to 10ng / mL. Cortisol enters saliva through intracellular mechanisms and is independent of saliva flow rate. In saliva, most of the cortisol remains unbound to proteins. It reflects the circadian rhythm and the early morning peak, and responds quickly and reliably to changes in plasma cortisol concentrations. The average salivary cortisol concentration in the morning is between 3.6nmol / L and 8.3nmol / L, and the late night salivary cortisol value is less than 3nmol / L.

[0352] Detection Method: Chronoamperometry is performed on an unmodified screen-printed carbon electrode, which detects the amount of TMB reacted. This amount is inversely proportional to the amount of cortisol (or other analyte) present in saliva. A standard curve is prepared using known cortisol concentrations in phosphate buffered saline (PBS) ( Fig.23), which can then be used to correlate the current with the Cortisol concentration. According to some embodiments, a 4-PL curve fit is used (Cortisol competition immunoassay), but other parameterized logistic curves may also be used if they fit the data well.

[0353] 6. Overview

[0354] FIG. 24 is a flow chart illustrating a testing method according to some embodiments of the present disclosure. According to some embodiments, the steps of FIG. 24 may be implemented in firmware and / or software in the POCT device 100. In an exemplary operation:

[0355] 2401-Launch application (manually). Open the application.

[0356] 2402-The application software requires the user to open the POCT Stone

[0357] 2403 - Stone on? (Answer yes or no). If no, return to step 2402. If yes, go to steps 2404 and 2405.

[0358] 2404-Application software requests saliva sample.

[0359] 2405–Stone Fig.15F 250 mv is applied to the potentiostat 104, and then to 160 (the electrical contacts of the sensor in the cartridge), and then to 1316 (the contacts on the sensor 1300).

[0360] 2406-Enough saliva? (Answer yes or no). If no, return to step 2404. If yes, go to steps 2407 and 2408.

[0361] 2407 – Chemical reaction starts at the electrode.

[0362] 2408 – Display chemical reaction countdown on Stone.

[0363] 2409 - Perform immunoassay reaction? (Answer yes or no). If no, jump to step 2411. If yes, go to step 2410.

[0364] 2410-One / more of several steps to ensure:

[0365] ·-In Fig.15F Wait 10-20 seconds before applying an AC potential to the sensor 1300 in the cartridge by applying the AC potential to 160 (the electrical contacts of the sensor in the cartridge) and then to 1316 (the contacts on the sensor 1300).

[0366] - Clean / flush test reservoir

[0367] - Add the second reagent

[0368] -Mixed reaction

[0369] - Start the second reaction

[0370] 2411-Reaction Complete? (Answer Yes or No). If No, return to step 2408. If Yes, go to steps 2412 and 2413.

[0371] 2412-The firmware reads the electrical characteristics of the circuit. Specifically, Fig.15F Firmware embedded in the controller 102 senses changes in current, for example, from the potentiostat 104.

[0372] 2413-Countdown stopped

[0373] 2414-Firmware converts to biomarker concentration and general health category score (e.g., energy). Specifically, according to some embodiments, the firmware instructs the processor 102 to measure the change in current on one or more electrodes 1311, 1312 via the potentiostat 104. Within the firmware, the current is then converted to a biomarker concentration using a known relationship between current and concentration specific to that biomarker, such as the exemplary Fig.29D In addition, as shown in FIG33, the biomarker concentration is also converted into a health score in its related health category. For example, a specific lactate concentration reading on electrodes 1311, 1312 and potentiostat 104 may be 800nA. Using Fig.29D The known relationship between the mid current and lactate concentration would yield a lactate concentration of approximately 1.8 nM. This lactate concentration would then be associated with an energy score of approximately 50% based on the relationship shown in row 7 of Figure 33, as it is in the middle of the normal human lactate concentration range, where lactate concentrations above 3.4 nM would likely equate to an energy score of 0% and lactate concentrations below 0.2 nM would likely equate to an energy score of 100%.

[0374] 2415 – Firmware stores data and sends it to the application

[0375] 2416-Stone Display Data

[0376] 2417-Application Display Data

[0377] 2418-The application synchronizes data with the cloud database

[0378] 2419-Cartridge ejection

[0379] 2420 - Perform new test? (Answer yes or no). If no, skip to step 2421. If yes, go to step 2404.

[0380] 2421-Application closed, stone closed.

[0381] FIG. 25 is a flow chart illustrating a testing method according to some embodiments of the present disclosure. According to some embodiments, the steps of FIG. 25 may be implemented in software executed by one or more processors in the POCT device 100. In an exemplary operation:

[0382] 2501-Launch application (manually). Open the application.

[0383] 2502-The application software requires the user to open the POCT Stone

[0384] 2503-Stone on? (Answer yes or no). If no, return to step 2502. If yes, go to step 2504.

[0385] 2504-User collects saliva sample. Is saliva sufficient? (Answer yes or no). If no, return to step 2503. If yes, go to step 2505.

[0386] 2505-Show response countdown timer

[0387] 2506-Received results? (Answer yes or no). If no, return to step 2505. If yes, go to step 2506.

[0388] 2507-Countdown stopped.

[0389] 2508-Application displays data / ratings.

[0390] 2509-The application synchronizes the data with a cloud database. Historical test data is also compared and used to improve the sensitivity and specificity of individual tests.

[0391] 2510-Did the user swipe the screen? (Answer yes or no). If no, jump to step 2517. If yes, go to step 2511. Swipe is a physical action taken by the user on the screen of their mobile device when operating the application. Swiping the screen left or right allows the user to navigate the application and view the test results and the actionable insights that the results may suggest. In this case, if the user swipes the screen, the next report (biomarker concentration) will be displayed.

[0392] 2511 - Stone / Application displays the basic biomarker concentration. Return to step 2510 or proceed to step 2512.

[0393] 2512-Stone / application displays historical test data. Return to step 2511 or proceed to step 2513.

[0394] 2513-Use the current test score, historical data and personal physiological data to display actionable insights on the Stone / app using artificial intelligence algorithms. Return to step 2512 or proceed to step 2515.

[0395] 2514-Internet. According to some embodiments, current test scores and / or other data produced by storage in the cloud on the Internet and / or in the Internet is a portal for retrieving information to provide actionable insights.

[0396] 2515-Stone / application displays the connected shared resources. Return to step 2513 or proceed to step 2516.

[0397] 2516 —Share data (friends / family, coach, medical professional). Return to step 2515 or step 2510 .

[0398] 2517-Cartridge ejected.

[0399] 2518-Run new test? (Answer yes or no). If no, jump to step 2519. If yes, go to step 2503.

[0400] 2519-Application closed, stone closed.

[0401] According to some embodiments, the steps of Figure 25 may be implemented in software, such as an application on a smartphone or remote device. According to some embodiments, some steps are optional, may not be performed and / or may be performed in a different order.

[0402] According to some embodiments, the POCT system 10 includes three main components, namely, the handheld Stone POCT device 100, the disposable biofluid or saliva test cartridge 200, and optionally a mobile or smart phone 191, 2600 or other remote device (e.g., smart watch 2620) with an application installed. According to some embodiments, the application is not required for testing, but may be required for updating the POCT device 100 and for more complex interaction with the Internet and / or sharing of results.

[0403] Figures 26A-26F26H-26P show exemplary displays on the POCT device 100 and / or remote device (e.g., smartphone 2600) according to some embodiments of the present disclosure. For illustrative purposes, both the Stone POCT device 100 and the application on the smartphone 2600 are shown in 26A-26F and 26H-26P. Figure 26G The collection of biological fluids (e.g., saliva) according to some embodiments of the present disclosure is shown. Exemplary embodiments are described below:

[0404] 1. Preparation instructions before user testing:

[0405] a. Avoid eating, drinking, exercise, or physical exertion for 90 minutes or more

[0406] b. Rinse your mouth with water between 2-5 minutes before the test.

[0407] 2. Testing.

[0408] a. Activate the device (application, POCT)

[0409] i. Open the TRAQ app on your mobile device or computer. You will see

[0410] TRAQ home page. [See Fig.26A ]

[0411] ii. The application will notify the user to turn on the POCT device 100. [See Fig.26B ]

[0412] iii. Open POCT 100. [See Fig.26C ]

[0413] b. The App can automatically connect to the POCT 100 (via Bluetooth) and instruct it to do so. [See Fig.26D ]

[0414] c. Insert the test cartridge and collect a saliva sample.

[0415] i. Insert the test cartridge into the POCT 100. [See Fig.26E ]. The app will indicate that the cartridge has been inserted and ask the user to apply a saliva sample. [See Fig.26F ]

[0416] ii. The POCT device 100 registers the test type and displays it (e.g., "Hydration"). [See Fig.26F ]

[0417] iii. The user removes the biological fluid collection device from the cartridge base (which has been inserted into the POCT). [See Fig.26F ]

[0418] iv. Collect saliva from the mouth. [See Figure 26G ]

[0419] v. When the entire tip is wet (typically 5-20 seconds), reinsert the biological fluid collection device 400 into the cartridge base 700. [See Fig.26H ]d. The test starts automatically.

[0420] i. According to some embodiments, when the device 100 detects saliva, the reaction has begun and the POCT / app displays a countdown until the test is complete (typically 30 seconds to 12 minutes). [See Fig.26I According to some embodiments, saliva detection can be achieved by maintaining a constant current across two electrodes and detecting when there is a change in resistance. Detection of a change in resistance can cause processor 102 to trigger the process to begin.

[0421] e. Results

[0422] i. When the test is complete, the results / category score will be displayed on the app on the POCT 100 and any of several devices to which the app is connected.

[0423] [See Fig.26J , 26K ]

[0424] ii. For health and wellness, the first screen displayed is the category score (eg hydration is 63%). For diagnostics, there is no category score, instead key biomarker concentrations are displayed as in iii) below.

[0425] iii. The user may then decide to drill down to view specific biomarker concentrations (e.g. sodium, potassium, etc.) used to determine a higher level health and wellness score (e.g. hydration) [see Figure 26L ];

[0426] iv. View historical scores and test data [See Figure 26M ];

[0427] v. View actionable insights (What does the score mean? What should I do to improve?) or share results. Link to the internet. [See Figures 26N-26O ]

[0428] iv. Review a list of shared resources: friends / family, counselors, coaches, or medical professionals, and share and connect. [See Fig.26O ]

[0429] 3. When finished, simply eject the cartridge to perform another test, or close the POCT device 100. [See Figure 26P ]

[0430] According to some embodiments, the disposable biomarker test cartridge 200 can be recycled / upcycled. If this has not already been done, another disposable biofluid test cartridge 200 can be inserted to perform another test, or the POCT device 100 and application can be shut down. [See Figure 26P ]

[0431] According to some embodiments, one or more or all of the following advantages may be achieved using the POCT system 10:

[0432] Non-invasive: The test uses saliva. No blood draw is required.

[0433] No medical professional is needed: users can test themselves.

[0434] On-demand, convenient and private: No appointment required. Test anytime, anywhere.

[0435] Fast: Results are available within seconds to minutes.

[0436] Safer: disposable cartridges, no blood, no masks, gloves, etc. required.

[0437] Accurate: Very similar to the approved FDA test, which is 10% accurate (within FDA guidelines)

[0438] Economical: The test costs only a few dollars.

[0439] Portable: handheld or desktop.

[0440] Easy: The solution is simple and has few steps.

[0441] Shareable: Ability to share results in near real-time with advisors, coaches, or medical professionals.

[0442] A single biomarker is rarely specific enough to fully reflect a person's fitness, health and wellness, or medical condition. Typically, several basic biomarkers are needed to provide a more complete picture, and although some embodiments of the device 100 are capable of making four unique biomarker measurements simultaneously, more biomarkers may be required to obtain an accurate picture, and other embodiments may include more than four simultaneous biomarker measurements. Contamination or dilution of saliva is possible, whether by food, drink (including water), or oral injuries that allow blood to enter the mouth. Such contamination can invalidate the test results.

[0443] Figures 27A-27E is an exemplary screenshot of a phone running an application (App) according to some embodiments of the present disclosure.

[0444] Among other components, an exemplary embodiment can provide a sample analysis reader configured to be electrically coupled to a sample analysis cartridge, the sample analysis reader comprising: a processor; and a non-transitory computer-readable medium having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to: detect the presence of a sample analysis cartridge electrically coupled to the sample analysis reader; detect identification information associated with the sample analysis cartridge; identify a suitable test protocol for the sample analysis cartridge based at least in part on the identification information; detect a sample collection device inserted into the sample analysis cartridge; and initiate a mixing protocol to mix a sample received from the sample collection device with a reagent in a fluid in a reservoir in the sample analysis cartridge.

[0445] In an exemplary embodiment, the processor 102 can be configured to initiate the mixing protocol by pumping reagents and sample from respective reservoirs into the mixing reservoir.

[0446] In an exemplary embodiment, the processor 102 may be configured to initiate the mixing protocol to promote the formation of a plurality of competitive complexes between the analytes present in the saliva and the competitive reagent format having the streptavidin-labeled magnetic particles.

[0447] In an exemplary embodiment, processor 102 may be configured to process signals from the internal electrode system that are indicative of the amount of one or more target analytes from the sample.

[0448] In an exemplary embodiment, the sample analysis reader 100 may be configured to be electrically coupled to a plurality of sample analysis cartridges 200, each of which is disposable.

[0449] The exemplary biosensors provided herein can provide one or more of the following benefits. However, providing these exemplary benefits does not mean that the exemplary biosensors are required to include any of these benefits. Exemplary biosensors:

[0450] No need to rely on batteries or internal power supply (intraoral);

[0451] There is no need for the device 100 or the sensor 1600 / 1800 to be wearable;

[0452] Possibility of using chemicals that are easy and cost-effective to manufacture;

[0453] Ability to measure multiple biomarkers / analytes simultaneously;

[0454] The accuracy may fully meet FDA requirements for tests based on non-saliva biofluids such as blood;

[0455] Potentially applicable to any biological fluid;

[0456] No medical professional is required to administer the test or read the results;

[0457] Possibly using AI (artificial intelligence) or machine learning to personalize tests for individuals;

[0458] Can be tested in a variety of environmental settings without the need for refrigeration or other special storage mechanisms;

[0459] Multiple types of reactions can be tested electrochemically simultaneously: enzyme, ion selective and / or immunoassay;

[0460] The potential for rapid, sensitive, robust and inexpensive testing using radically different chemical compounds;

[0461] The resulting overall categorical health score (energy, hydration, fat burning, etc.) can be provided;

[0462] A saliva collection device 400 may be used for safe and effective collection and insertion into the POCT test device 100;

[0463] Can make each test cost only a few dollars, compared to tens or hundreds of dollars per test;

[0464] Can remain stable for a long time (>12 months) without any special storage or refrigeration;

[0465] Ability to interface with software for display, storage, or interaction with a consultant, coach, or physician for actionable insights to improve overall health, wellness, or fitness:

[0466] May include associated firmware and software for independently performing computations; and / or

[0467] Potentially able to analyze repeated lactate tests to identify lactic acidosis with high specificity and sensitivity, thus serving as an early indicator of several serious diseases.

[0468] 7. Acidosis

[0469] According to some embodiments, a point-of-care testing (POCT) system 10 is provided that allows on-demand, non-invasive (salivary) lactic acidosis screening for serious illnesses, with results available in seconds and costing only a few dollars per test, allowing more frequent testing and therefore earlier detection.

[0470] Early detection of disease is desirable both for improving patient outcomes and for saving costs to address medical problems. Some of the most serious and costly diseases that would benefit from early detection include, for example:

[0471] Cancer (more than 20 million new cases and more than 10 million deaths worldwide each year);

[0472] Sepsis, which causes one in five deaths worldwide;

[0473] Kidney disease, which affects about 10% of people worldwide;

[0474] Liver disease, the 11th leading cause of death worldwide; and

[0475] Congestive heart disease (affects 33.5 million people each year).

[0476] Current diagnostics for the above medical issues rely on blood tests, scans (MRI, PET, mammography, etc.) or specific diagnostic tests (e.g., Pap smear for cervical cancer) and physical examinations by healthcare providers. All of these diagnostic procedures help diagnose the underlying disease with extremely high sensitivity and specificity. Serum test panels can scan for specific biomarkers that serve as early indicators. Other solutions include the use of alternative biofluids such as urine and pH test strips.

[0477] However, each of these procedures or tests is either expensive, time consuming, subjective in nature, or is prohibited after a problem has been detected, or has a prohibited testing frequency (e.g., annually or over a period of time). In addition, they are often invasive, cumbersome, or inconvenient, requiring trained medical professionals. In essence, they do not detect the disease as easily or earlier as other methods.

[0478] Most biomarkers in blood have a predictable and measurable relationship with their counterparts in saliva. Lactate is one such biomarker with a predictable relationship between its concentrations in serum and saliva.

[0479] Lactic acidosis is persistently elevated lactate levels that affects millions of people worldwide each year and is an early indicator of one of several serious underlying medical problems including cancer, sepsis, kidney disease, liver disease, and / or congestive heart failure.

[0480] Current diagnostic methods for lactic acidosis screening involve the use of serum tests in the laboratory to measure lactate levels, which require invasive blood extraction, time for laboratory processing, are cost-prohibitive, and require the presence of a medical professional.

[0481] The electronics in the POCT device 100 measure the electrical signature of the chemical reaction on the biosensor 1300 within the disposable biofluid or saliva test cartridge 200. The relationship between the electrical signature of the chemical reaction and the lactate concentration is a known quantity. Therefore, according to some embodiments, when the reaction is complete (e.g., within 30 seconds), the firmware and / or software in the POCT device 100 converts the electrical signature into the lactate concentration. In some embodiments, two lactate wells 1331 will be run simultaneously because the saliva covers all five (5) wells in each run, and the two simultaneous measurements are averaged and the POCT 100 arrives at a final measurement.

[0482] For example, according to some embodiments, two lactate wells 1331 may be read independently and / or simultaneously, and multiple measurements for each channel may be averaged and then each averaged measurement converted to a corresponding lactate concentration as follows:

[0483] a*X Avg 2 +b*X Avg +c

[0484] The resulting concentration conversion measurements a, b and c may then be:

[0485] a value = 0.0

[0486] b value = 0.0594424

[0487] c value = -72.71592

[0488] (Time limit = 30.0; Sample limit: 30; Unit = mg / dL)

[0489] The lactate concentration is:

[0490] a*X 2 +b*X+c

[0491] Where a = 0.0, b = 0.0594424, and c = -72.71592

[0492] and the electrical characteristic (voltage, current or resistance) from the circuit may be X Avg =1250. Therefore:

[0493] a*X Avg 2 +b*X Avg +c

[0494] 0.0*(1250) 2 +0.0594424*(1250)-72.71592

[0495] =1.59mg / dl

[0496] According to some embodiments, the lactate concentration obtained is:

[0497] - is displayed on the POCT device 100,

[0498] - are sent to a mobile application for display, off-device storage and / or possible sharing of results, and / or

[0499] - is stored on the POCT device 100 for later retrieval or display.

[0500] Once the test is completed, the disposable biological fluid or saliva test cartridge 200 may be ejected from the POCT device 100 for disposal or recycling.

[0501] According to some embodiments, the entire process typically takes less than 60 seconds, of which 5-20 seconds are for saliva collection, 5 seconds are for reinserting the collection tip into the POCT device 100, and 20-30 seconds are for completing the chemical reaction and displaying / communicating the results to an application and / or display on the POCT device 100. According to some embodiments, the disposable biofluid or saliva test cartridge 200 can be mass produced for only a few dollars.

[0502] While lactic acidosis testing is not new, some embodiments of the present disclosure provide and / or contain one or more or all of the following unique features:

[0503] - Non-invasive (saliva) lactate test;

[0504] - On-demand testing:

[0505] - Near real-time results;

[0506] - The test is based on electrochemistry;

[0507] -The test costs only a few dollars;

[0508] - Automatically start the test;

[0509] - The POCT device 100 communicates with the application;

[0510] - Ability to share results in near real time; and / or

[0511] - No trained medical professional is required to perform the test.

[0512] According to some embodiments, the intended single use of the system 10 is as described above; however, it is anticipated that 2-3 tests may need to be performed within an hour or two to confirm the results. For example, false high readings may come from: 1) food, biological matter contamination (to mitigate contamination, the system 10 may instruct the user to rinse the mouth with water within 5 minutes of the test, but no earlier than 2 minutes before the test, and this behavior may be part of the protocol to ensure that the saliva sample is not contaminated with food / biological matter or diluted with water), 2) recent physical activity or exercise that would increase the lactate reading (the testing protocol requires testing no earlier than 90 minutes of physical exertion or exercise).

[0513] After multiple tests, if a persistently elevated lactate level is detected, the subject can be counseled for follow-up services or testing. In this way, subjects can be easily, non-invasively, privately, quickly, and inexpensively tested for lactic acidosis.

[0514] Although some embodiments describe disposable biofluid or saliva test cartridges 200, other embodiments may contain chemicals that allow multiple uses (multiple tests) or non-disposable. According to some embodiments, within the POCT device 100, two measurements are taken on a single sample and then averaged. Other embodiments may use a single measurement or more than two measurements. In addition, it may be possible to use a pre-existing portable device (e.g., a mobile phone) to perform calculations currently performed on the stone, thereby eliminating the need for the separate stone device.

[0515] According to some embodiments, systems and / or devices related to saliva sensors, biosensors, saliva biomarkers, handheld medical devices, lactic acidosis testing, cancer screening, sepsis screening, kidney disease screening, liver disease screening, congestive heart failure screening, and / or diabetes screening are provided.

[0516] According to some embodiments, performing a lactose test using system 10 may require the following parameters.

[0517] 1. Preparation before the test:

[0518] a. Avoid physical activity for 90 minutes or more

[0519] b. Rinse your mouth with water 2-5 minutes before the test.

[0520] 2. Testing

[0521] a. Activate the device (application, POCT 100)

[0522] i. Open the TRAQ application on a mobile device or computer. The application will notify the user to turn on the POCT device 100. Fig.26B An exemplary screenshot of an application program is shown instructing a user to open the POCT device 100 according to some embodiments of the present disclosure.

[0523] ii. Turn on the POCT device 100. The app will automatically connect to the POCT device 100 (via Bluetooth) and indicate ready.

[0524] b. Insert the disposable biofluid or saliva test cartridge 200 and collect the saliva sample.

[0525] i. Insert the disposable biofluid or saliva test cartridge 200 into the POCT 100. The application will indicate that the cartridge has been inserted and ask the user to apply a saliva sample. See e.g. Fig.26F An exemplary application screen is shown in .

[0526] ii. Remove the collection tip 400 from the cartridge base 700 (already inserted into the POCT 100).

[0527] iii. Collect saliva from the mouth.

[0528] iv. When the entire tip is wet (usually 5-20 seconds), reinsert the collection tip into the cartridge base.

[0529] c. The test starts automatically.

[0530] i. When the device detects saliva, the reaction has begun and the POCT / app displays a countdown until the test is complete (usually 30 seconds or less). See e.g. Fig.26I An exemplary application screen is shown in .

[0531] d. Results

[0532] i. When the test is completed, the results will be displayed on the POCT and the app. See e.g. Figures 28A-28B An exemplary application screen is shown in .

[0533] ii. The user can then decide to view actionable insights (what does the score mean, what should I do?), share the results, or simply close the POCT device 100 and application.

[0534] 3. Eject the cartridge for disposal or recycling.

[0535] If this has not already been done, another test cartridge may be inserted and another test may be performed, or the POCT device 100 and App may be closed.

[0536] According to some embodiments, a system 10 is provided that has one or more or all of the following advantages:

[0537] Non-invasive: The test uses saliva. No blood draw is required.

[0538] No medical professional required: users can test themselves;

[0539] Convenient and private: No appointment required. Test anytime, anywhere;

[0540] Fast: Results can be obtained within seconds to minutes;

[0541] Safer: disposable biofluid or saliva test cartridge 200, no blood, no masks, gloves, etc. required;

[0542] Accurate: Very similar to the approved lactic acidosis test, which is 10% accurate (within FDA guidelines);

[0543] Economical: The test costs only a few dollars.

[0544] Portable: handheld or tabletop (i.e. not handheld but sits on the user's desk / counter like a toaster); and / or

[0545] Easy: The solution is simple and has few steps.

[0546] With these advantages, more frequent testing can be performed, resulting in earlier detection, saving more lives and more money.

[0547] In general, lactic acidosis tests (both blood and saliva) are non-specific as a marker for cancer or any of the other diseases listed above. However, the tests are indicative of general health issues that are best further diagnosed through existing tests and procedures listed above. This is true of our solution and others involving lactic acidosis testing.

[0548] Generally speaking, lactic acidosis tests (both existing and our solution) may give false readings if exercise or heavy physical activity has occurred within 30-90 minutes of the test.

[0549] According to some embodiments, contamination or dilution of saliva is possible, whether by food, drink (including water) or oral injury allowing blood to enter the mouth. Such contamination may invalidate the test results.

[0550] Fig.29Ais a graph of an accuracy study of a POCT sensor 1300 configured for measuring lactate according to some embodiments of the present disclosure compared to an industry standard lactate concentration analyzer from YSI (YSI benchtop analyzer). The statistical analysis of the accuracy of the sensor 1300 included a primary analysis and a secondary analysis. Saliva samples were collected and salivary lactate values ​​were measured using the reader / POCT device 100 and a YSI 2300Stat Plus (YSI Incorporated, Yellow Springs, OH). The YSI was selected as the reference instrument, and each YSI salivary lactate value was paired with the corresponding POCT sensor value. Correlation analysis of the POCT sensor readings and the YSI values ​​revealed a significant positive correlation (R 2 =0.98678, P <0.001). Clarke error grid plots were constructed using the salivary lactate values ​​shown in the figure. Scatter plots were generated based on YSI values ​​(horizontal coordinates) and POCT sensor readings (vertical coordinates), and the Clarke error grid was constructed as ±20% for ≥0.55 mM and ±0.055 mM for <0.55 mM. Clarke error grid analysis showed that all readings fell within the clinically acceptable range.

[0551] Fig.29B is a graph showing the response of sialy lactate to incremental physical exercise according to some embodiments of the present disclosure. The test protocol includes an incremental exercise test of continuous periods of 3 minutes, with a 1-minute rest period in the middle, during which saliva samples are collected and lactate values ​​are estimated. Typically, the test starts with a fixed pace of 3.8mph, and the speed gradually increases by 0.6mph every 3 minutes. The cycle of exercise / rest continues until the subject reaches his peak endurance (volitional failure). The sialy lactate curve constructed using sialy lactate values ​​during the incremental exercise test shows a response similar to the blood lactate curve. In all cases, a clear inflection point is observed in the lactate curve as the intensity increases. As the workload increases, the rate of change of sialy lactate is proportional to the rate of change observed in the blood.

[0552] Fig.29C1 is a graph of a stability study obtained by plotting and comparing measurements obtained using a POCT sensor configured to measure lactate from day 1 of the sensor manufacturing until day 260 after the sensor manufacturing, according to some embodiments. The real-time shelf life of the POCT sensor 1300 was evaluated by showing the sensor response to different concentrations of lactate on day 1 and day 260. The POCT lactate sensor was stored in a storage vial at room temperature on silica gel as a desiccant for various time periods, and the response of the sensor 1300 to different concentrations of saliva lactate (mg / dL) was measured at various time intervals. The vials were stored closed throughout the study and stored at room temperature. The results show that the response of the POCT sensor 1300 was comparable after 260 days of storage.

[0553] Fig.29D 1 is a graph showing the batch-to-batch manufacturing variability and robustness obtained by plotting the biomarker concentrations measured in three different production batches applied to the biosensor chip 1300 according to some embodiments. Batch-to-batch manufacturing variability can be defined as the change in the analytical performance of the POCT sensor 1300 from one production batch to the next production batch. POCT sensors 1300 from three different batches were selected and the response of the sensor to different concentrations of lactate was studied. The study was conducted within the analytical range of the lactate assay. On the same day, each sample was tested using the same lactate control solution and the same operator. No statistically significant variability in manufacturing between batches was observed.

[0554] According to some such embodiments, the biosensor 1300 consists of screen-printed electrodes 1311 within physical recesses or wells 1331 containing enzyme chemistry for detecting lactate in saliva (or other biological fluids) and electrical connection points 1316 for interacting with the handheld measurement tool 100.

[0555] According to some such embodiments, the system 10 collectively performs electrochemical measurements, whereby the electrical signature of the chemical reaction is measurable and directly correlated to the lactate concentration in the biological fluid. According to some embodiments, the user inserts the entire test cartridge 200 (saliva collection device 400, base 700 with embedded biosensor 1300) into the POCT device 100, and then removes the saliva collection device 400 from the cartridge base 700 to collect a sample from their mouth. Once the saliva collection device 400 collects saliva, the user reinserts the wet collection device 400 into the cartridge base 700. According to some embodiments, the system automatically senses the chemical reaction between the saliva sample and the biosensor 1300.

[0556] Specifically, according to some embodiments, the biosensor 1300 works by reading the electrons added to the working electrode by the mediator during the redox reaction. Specifically, according to some embodiments, chronoamperometry is used between the reference electrode 1323 and the working electrode 1322. According to some embodiments, three data points are measured at the beginning, middle and near the end of the chronoamperometry run and then averaged. The current average is converted to a potential and then converted to the corresponding concentration of the measured analyte.

[0557] According to some embodiments, biosensor 1300 is configured to measure lactate, whose chemistry consists of:

[0558] 50 mM HEPES, 50 mM BTP, DI water, polyvinylpyrrolidone, hydroxyethylcellulose, 0.9% NaCl, 5% Triton X, METHOCEL, trehalose, sodium succinate, hexaamineruthenium, and lactate oxidase.

[0559] The working electrode 1322 material has a redox mediator and lactate oxidase (LOx) thereon. The LOx can be from any source, such as a microorganism (e.g., toyobo, LCO_301), such as Aerococcus viridans. The electrode 1322 material and the analyte-related enzyme form an enzyme mixture capable of catalyzing a reaction. The redox mediator is capable of transferring electrons between the enzyme-catalyzed reaction and the working electrode 1322. The reference electrode 1323 is used as a voltage reference between the solution and the working electrode 1322, allowing the correct voltage difference to be read from the working electrode.

[0560] The following reaction is related to the above reaction: L-lactate + O 2 (LOx)-- Pyruvate + H 2 O 2

[0561] According to some embodiments, as part of the lactate biosensor 1300, the reagents preferably contain two enzymes, a redox mediator in reduced form, at least one binder, and a surfactant. According to some embodiments, the enzymes are the above-mentioned glucose oxidase (GOD) and peroxidase.

[0562] 8. Health Category

[0563] According to some embodiments, the system 10 and / or device 100 relates to health and wellness, biomarkers, health measurement and scoring, artificial intelligence, and / or diagnostics. Exemplary scoring methods that may be performed in part or in full by the exemplary system may include the following:

[0564] -Describes the various biomarkers known to be associated with the general categories of fitness, health, or wellness. They are referred to as foundational biomarkers.

[0565] - assigning (or receiving or retrieving (e.g., from a memory or storage) already assigned) a weighted importance factor (number) to each base biomarker as it relates to the optimal level of fitness, health, or wellness for that category. That is, one base biomarker may be more important or significant than another base biomarker. This relative importance score is assigned, e.g., as a percentage, with the combined importance factors of all base biomarkers totaling 100, expressed as a percentage.

[0566] - The weight importance factor may be derived from any suitable source such as, but not limited to, historical ratings, the user's broader demographic or physiological parameters (age, gender, weight, height, etc.), or some combination thereof, e.g. by a suitable algorithm such as an artificial intelligence algorithm.

[0567] - Each biomarker is assigned a concentration score from 0 to 100 that correlates to its measured concentration within the biological fluids of the subject population, with healthier concentration numbers receiving concentration scores closer to 100 and unhealthy concentrations receiving concentration scores closer to 0.

[0568] - Combine the weighted importance score and concentration score for each underlying biomarker.

[0569] - These combined underlying biomarker scores are added together to calculate or arrive at a final score or value for that particular fitness, health or wellness category.

[0570] The exemplary salivary point-of-care biomarker measurement device may be configured to measure any of thousands of biomarkers, and the measurements may be used to generate an overall category health score, display the results on the device, and communicate the results to a software application.

[0571] An exemplary software application may communicate with a POCT device to display general health category scores, baseline biomarker concentrations, self-guided actionable insights to help users improve their general health category scores, and link to a counselor, coach, health professional, or others for external insights.

[0572] Current fitness, health and wellness scoring systems are unable to measure specific chemical biomarkers relevant to those areas of fitness, health and wellness. Instead, the various systems are limited to:

[0573] -Activity tracking – steps, miles, calories, weights lifted, etc. Quantifiable but not tied to specific biomarkers nor to the overall categories of fitness, health and wellness.

[0574] - Asked to rate non-chemical biomarkers (e.g., steps, heart rate, overall feeling). Quantifiable, not linked to actual physiological measures.

[0575] - Associates specific diseases with specific biomarker concentrations, such as diabetes and serum concentrations of glucose or insulin. Can be quantified, but not associated with overall categories of fitness, health, and wellness.

[0576] - Express fitness, health and wellness categories as relative qualities: good, bad, good, great, excellent, poor, fair, etc. Not numbers. Hard to compare.

[0577] No existing system attempts to digitally quantify (e.g., score) overall fitness, health, and wellness categories using relevant underlying chemical biomarkers. Using quantifiable scores, users can more easily compare fitness levels, health, and wellness categories within a larger group, or compare themselves against their own improvements or lack of improvement. Additionally, in non-human groups, scoring systems are worthless in understanding the fitness, health, and wellness of the subjects given the potential lack of communication. A scoring system for measuring "immunity" levels or "hydration" levels in pets or livestock would prove to be very valuable.

[0578] Furthermore, using quantitative metrics, more precise actions can be taken on whether or not a score is good or bad (actionable insights).

[0579] According to some embodiments, the value of system 10 relies on accurately assigning the underlying biomarkers to a category. Typically, no single biomarker can indicate a disease or broad category such as mental health. Typically, there are multiple underlying biomarkers that affect general fitness, health, or wellness categories.

[0580] According to some embodiments, the value of system 10 also relies on accurately measuring biomarker concentrations. Currently, blood is considered the "gold standard" because it correlates with biomarker concentrations, as a doctor's visit typically begins with collecting vital signs and possibly a blood sample for testing. Using other less consistent or reliable biofluids can also muddy the scoring results.

[0581] Finally, as long as there are numerical definitions of “normal,” “healthy,” “unhealthy,” and “abnormal” biomarker concentrations, there will be some subjectivity.

[0582] Some embodiments of the present disclosure provide a system that uses the concentrations of relevant basic biomarkers within saliva and / or other biofluids (blood, urine, tears, sweat) and general physical parameters of the user to quantitatively define health, fitness, and diagnostic health indicators that are typically non-quantifiable, including:

[0583] (a) concentrations of various biomarkers associated with qualitative health categories typically associated with the underlying biofluid;

[0584] (b) the weighting factor assigned to each biomarker as it relates to the overall importance of the qualitative assessment within the health category;

[0585] (c) an algorithm that combines the concentrations and weighting factors in one of several ways to calculate a final health category score;

[0586] (d) wherein the final health score may be a numerical scale (e.g. 1-10 or 1-100), binary (e.g. high-low) or any other hierarchical scale (e.g. high-medium-low, green-yellow-red) representing the quantitative difference between poor health and excellent health.

[0587] An exemplary intelligent algorithm may use the scores and relevant user health information to modify the weighting factors to obtain a personalized weighting table.

[0588] The exemplary system may additionally determine and provide to the user actionable insights related to the wellness category score that provide the user with suggestions on how to improve the overall wellness category score.

[0589] The POCT device or other device in the system may provide on-device display of basal biomarker concentrations, health category scores, and self-guided or external actionable insights related to improvements in health category scores.

[0590] A mobile software application displaying basic biomarker concentrations, health category scores, and actionable insights may be provided.

[0591] Some embodiments of the present disclosure provide a device with a microprocessor and a sensing element that is capable of measuring biomarker concentrations, storing and changing weighting factors manually or based on an artificial intelligence algorithm, and calculating a final health category score.

[0592] The device may further comprise means for communicating and transmitting biomarker concentrations, weighting factors, and general health category scores back and forth between the measurement device and a software application and / or database.

[0593] The device may further include means for sharing the quantitative health category scores with a coach, medical advisor, or others to gain further actionable insights or advice.

[0594] Health Category:

[0595] (Some) biomarkers serve as indicators of basic fitness categories, such as lactate as an indicator of energy levels. Other categories may include nutrition or hydration. Still other biomarkers indicate general health and wellness, such as heart health or cognitive health. In addition, some of these same biomarkers or other biomarkers serve as indicators of specific medical conditions. In general, these salivary biomarkers can help people determine their fitness, general health and wellness, and / or diagnose conditions in an on-demand, rapid, private, and cost-effective manner.

[0596] Once the biomarkers are measured:

[0597] -Fitness, health and wellness or diagnostic condition scores may be displayed on the POCT device 100 (and / or elsewhere);

[0598] - The concentration of the basic biomarker may be displayed on the POCT device 100 (and / or elsewhere);

[0599] - Results can be sent to a mobile app for display, off-device storage, providing actionable insights related to overall score or biomarker concentrations, and potentially sharing results;

[0600] - The results may be stored on the POCT device 100 (and / or elsewhere) for later retrieval or display.

[0601] Once the saliva test is complete, the associated cartridge can be ejected from the POCT device 100 for disposal or recycling / upcycling.

[0602] According to some embodiments, the entire process typically takes 30 seconds to 12 minutes, of which 5-20 seconds are used for saliva collection, 5 seconds are used to reinsert the collection tip into the POCT 100, and 20 seconds to 11 minutes are used to complete the chemical reaction and display / communicate the results to, for example, a related application. According to some embodiments, the large-scale production cost of the cartridge 200 is only a few dollars.

[0603] According to some embodiments, once the test is completed and the results are displayed, the subject (e.g., user) can further explore their fitness, health and wellness or medical conditions through the "actionable insights" available on the POCT device 100 and / or the supporting application. According to some embodiments, the actionable insights can allow users to learn more about their status and start taking action. In addition, according to some embodiments, the subject (e.g., user) can then be linked to each coach, consultant or health care professional to more easily enter the health care system. According to some such embodiments, stone100 is connected to another device (e.g., a coach's smartphone) via, for example, Bluetooth. In "coach mode", stone 100 can contain all the data of each team member and send it to the cloud or the main smartphone so that the coach can obtain a record of, for example, an increase in lactate threshold for each athlete in the coaching team.

[0604] According to some embodiments, the cartridge 200 may be biomarker specific (e.g., lactate, glucose, cholesterol) or tailored to any of several fitness categories (e.g., energy, stress, hydration, etc.).

[0605] Fig.30 A sample disposable biological fluid or saliva test cartridge 200 is shown for various potential fitness categories (nutrition, energy, burn, hydration, etc.) of the present disclosure.

[0606] Fig.31 A sample disposable biofluid or saliva test cartridge 200 of the present disclosure for health and wellness categories (eg, aging, defense, mood, GI health, stress) or a gender-based cartridge (eg, women's health) is shown.

[0607] Fig.32 A sample disposable biological fluid or saliva test cartridge 200 is shown for use in a specific disease or medical condition (eg, acidosis) or personalized medicine for which an individual (eg, "John Doe") has a dedicated cartridge, according to some embodiments of the present disclosure.

[0608] According to some embodiments, one or more or all of the following features are provided:

[0609] - Non-invasive (saliva) testing for specific biomarkers, fitness levels, health and wellness categories, or medical conditions;

[0610] - Test on-demand, anytime, anywhere, for full control and privacy:

[0611] - Portable testing using handheld or small benchtop devices:

[0612] - Testing one / multiple salivary biomarkers simultaneously;

[0613] - Simple, as no medical staff is required to perform, read or share the test;

[0614] - Near real-time results;

[0615] - Safe, using disposable cartridges;

[0616] - Accurate saliva testing, within current FDA requirements;

[0617] - The test is based on electrochemistry;

[0618] -The test costs only a few dollars;

[0619] - Automatically start the test;

[0620] - Communication between the POCT device 100 and the application; and / or

[0621] -Ability to share results in near real time.

[0622] The intended single use of the device is as described above. However, according to some embodiments, it is anticipated that several tests may need to be performed over a period of time to confirm that one or more results are falsely high readings that may be obtained from:

[0623] 1) Recent ingestion of food or fluids - Much like blood tests, according to some embodiments, subjects may need to refrain from eating for up to 2 hours prior to such a test;

[0624] 2) Food, biological matter contamination - According to some embodiments, the test protocol may require the subject to rinse the mouth with water within 5 minutes of the test, but no earlier than 2 minutes before the test, to ensure that the saliva sample is not contaminated with food / biological matter or diluted with water;

[0625] 3) Stress or recent physical activity or exercise can increase or decrease certain biomarker readings - According to some embodiments, the testing protocol may require testing to be performed no earlier than 90 minutes after physical activity or exercise or a stress-related event.

[0626] Some embodiments describe a single-use, disposable biological fluid or saliva test cartridge 200; however, other embodiments may contain chemistries that allow for multiple uses (multiple tests) or non-disposable use.

[0627] According to some embodiments, in the POCT system 10, a single saliva sample is measured twice and then averaged. Other embodiments may use a single measurement or more than two measurements. According to some embodiments, up to four saliva biomarkers can be measured simultaneously, but other embodiments may conceivably test fewer or more biomarkers simultaneously. In addition, it may be possible to use a pre-existing portable device (such as a smart phone) as the POCT device 100 itself, thereby eliminating a dedicated POCT device 100. According to some such embodiments, the functions (computation, analysis) of stone 100 can be divided between the smart phone and disposable biological fluid or saliva test cartridge 200, respectively. Other embodiments may not even conceive of human health and human saliva, but other animals or mammals such as pets or livestock. Finally, biological fluids other than saliva can be imagined.

[0628] According to some embodiments, health and wellness category definitions and quantitative scoring systems are provided.

[0629] General health claims (e.g., energy level, dehydration, recovery ability, etc.) provide high-level information about an individual's health, but are not directly linked to the specific underlying physiological biomarkers used to define these broad health categories. Furthermore, for comparison purposes, no quantifiable (numeric) metrics are assigned to these broad categories. For example, there is no numeric score to define "energy." This makes it very difficult to compare to historical energy "feelings" or energy feelings of other individuals.

[0630] Current solutions to this problem do link specific diseases to underlying biomarkers. For example, diabetes is often defined by the concentrations of glucose and insulin (as well as dozens of biomarkers that are less sensitive or specific to the disease).

[0631] Despite direct links between specific diseases and specific biomarkers, there remains a lack of links between higher level general health and wellness categories and specific underlying biomarkers, nor is there an assignment of the relative importance of these various underlying biomarkers to the health category in question. Additionally, there is currently no system for assigning numerical values ​​or scores to broad health and wellness categories in an attempt to quantify these categories and make actionable corrections to improve scores for individuals or comparison groups.

[0632] There are several general categories of health and wellness that are well known to both the medical community and laypeople. However, when people say they have “energy,” “a good mood,” or feel “dehydrated,” what does this mean on a physiological level?

[0633] According to some embodiments, various major chemical biomarkers indicative of these general health categories have been delineated through direct studies, clinical trials, and research reviews of published clinical papers.

[0634] First, the following health and wellness categories are defined (these categories are not exhaustive):

[0635] Burn, fat burning, calorie burning

[0636] ·Cognition and discrimination ability

[0637] ·defense

[0638] Detoxification

[0639] Energy and physical fitness

[0640] Gastrointestinal tract, digestive health, digestion

[0641] Heart and cardiovascular health

[0642] Hormone Health-Men

[0643] Hormone Health-Women

[0644] Hydration

[0645] Inflammation, immunity

[0646] Malnutrition

[0647] Mental health

[0648] ·Feeling

[0649] ·Nutrition

[0650] Oral health

[0651] ·pain

[0652] ·recover

[0653] Skin health

[0654] ·pressure

[0655] Next, various underlying chemical biomarkers were subsequently defined to broadly impact these health and wellness categories.

[0656] Figures 33A-33CAn example table of health categories and exemplary associated basic biomarkers according to some embodiments of the present disclosure is shown. The health categories can be defined in more detail by the concentrations of the listed associated and basic biomarkers. Each labeled biomarker has a varying degree of importance as an indicator of that health and wellness category. Some of the listed biomarkers are more important, while others are less important. The list is not exhaustive because in some cases, dozens or hundreds of individual biomarkers may have an impact on a general health category, although with varying degrees of importance.

[0657] refer to Figures 33A-33C ,Each annotated biomarker has a different degree of importance as an indicator of the corresponding health and wellness category.,Some biomarkers listed are more important, while others are less important.

[0658] Finally, according to some embodiments, a system is developed to assign numerical scores to general health and wellness categories based on: 1) the relative importance of the biomarker as an indicator of the health and wellness category in question, and 2) the relative concentration of the biomarker in relative biological fluids (blood, saliva, urine, sweat, tears, etc.), 3) as well as the user's physiological state and information such as gender, health, weight, age, etc.

[0659] To use this health scoring system, each relevant biomarker (when it relates to the higher-level health category) is assigned an importance score (IS) from 1 to 100. For more important biomarkers, their IS is closer to 100. For biomarkers that are less important as indicators within that category, their IS is closer to 0. However, in general, the IS scores of all biomarkers related to a particular health and wellness category add up to 100.

[0660] Fig.28A Various devices 100 are shown in association with different tests or general health categories according to some embodiments of the present disclosure, with associated scores shown on a display of the device 100 and providing examples of qualitative health categories with underlying biomarkers associated with the samples.

[0661] For example, for the broad measure of "energy", the following biomarkers were found to be indicative: lactate, glucose, testosterone, cholesterol, triglycerides, ketones, insulin, leptin. The energy IS score was assigned as follows:

[0662] Lactate: 60

[0663] Glucose: 30

[0664] Testosterone: 4

[0665] Cholesterol: 2

[0666] Triglycerides: 1

[0667] Ketone: 1

[0668] Insulin: 1

[0669] Leptin: 1

[0670] Total IS: 100

[0671] Individual IS assignments may fluctuate based on subjective and factual inputs. For example, it may be discovered that several additional biomarkers have an impact on the energy category, that the relative importance of one biomarker is greater or less than that noted here, or that the assigner of the IS may be biased toward one biomarker over another.

[0672] If a biomarker is not measured, it is removed from the IS list and the relative values ​​are recalculated proportionally between the remaining biomarkers. That is, if lactate and testosterone are the only biomarkers available for measurement, the unmeasured biomarkers are removed from the IS list and lactate is assigned an IS of 100*(60 / 64)=93.75 and testosterone is assigned an IS of 100*(4 / 64)=6.25, ensuring a total weight score of 100.

[0673] The IS score serves as an "importance weighting factor" in calculating the final health and wellness category score.

[0674] The second aspect of the scoring system involves a concentration score (CS), which is defined by the normal range of biomarker concentrations for a subject population (human, dog, cattle, etc.) and then plotted to a range of 0 to 100. CS values ​​closer to 100 represent biomarker concentrations within the normal or optimal range for the biological fluid in the subject population. CS scores further away from 100 represent concentrations below the optimal concentration for the biological fluid. Typically, the best CS score is in the middle of the biomarker concentration range, and lower scores are assigned to very low or very high biomarker concentrations because these generally indicate poor health conditions in terms of biomarkers, although this may not always be true. The CS score can be assigned manually or by extrapolation or fitting according to a variety of methods, such as linear regression, exponential, polynomial, cubic spline, a form of artificial intelligence (AI) assignment, or some other method or combination of methods or algorithms. The CS score may or may not be normalized.

[0675] The CS score was used as the primary health indicator for this biomarker.

[0676] However, it is important to point out that individual IS assignments may fluctuate due to subjective and factual inputs. For example, serum lactate may be found to be in a wider or narrower range, or "normal" may be broader than 0.5-2.3 mM. Furthermore, the assigner may be biased as it relates to the overall range or definition of healthy biomarker concentrations.

[0677] These ranges vary by gender and age. They also vary by weight. Using historical scoring data and this psychological information, weights can be calculated using a personalized artificial intelligence algorithm to define personalized weights.

[0678] The final aspect of the scoring system is the combination of the IS (Importance Score) and the CS (Concentration Score).

[0679] After both IS and CS scores are calculated, they are multiplied together, considering that CS is the primary health indicator and IS in percentage form is a weighted number as it relates to all other underlying biomarkers that are indicators of general health and wellness for this category.

[0680] For example, when calculating the energy score using only lactate, glucose, and testosterone:

[0681] The weighted IS scores for each might be lactate (60), glucose (30), and testosterone (10), ensuring that the total IS scores for all relevant biomarkers measured add up to 100.

[0682] If the concentration range of lactate in human serum is defined as 0-50 mM, and the normal range for healthy people is between 0.5-2.3 mM, then a serum lactate concentration of 1.5 mM may give a CS of 95.

[0683] If the concentration range of glucose in human serum is defined as 0-1000 mg / dl, and the normal range for healthy people is between 70-99 mg / dl, then a serum glucose concentration of 130 mg / dl may give a CS of 60.

[0684] If the concentration range of testosterone in human serum is defined as 0-2000 ng / dl, and the range for normal healthy men is between 300-1000 ng / dl, then a serum testosterone concentration of 600 might give a CS of 80.

[0685] Energy Exemplary IS and CS Scoring

[0686]

[0687] The energy score for this example is 83. Generally, the healthier the subject overall, the higher the value (closer to 100) the scoring system will assign.

[0688] Likewise, it is important to note that the various ranges considered healthy vary between groups, by gender, age, weight, overall fitness level, and for individuals, the ranges may change over time given their relatively changing fitness levels.

[0689] All actual biomarker concentrations, health category scores, and possible actionable insights are transmitted / communicated between the measurement device and the mobile software application to take further actions to improve the health category scores or to share with a coach, medical advisor, or others.

[0690] Fig.28B A device 100 is shown communicating test results and scores to a smartphone, such as via Bluetooth, according to some embodiments of the present disclosure. The device 100 is used as a biomarker measurement device (left), transmitting a health category score (physical strength) via Bluetooth to a software application on a mobile device (right).

[0691] Fig.34A Some screens on a smartphone according to some embodiments of the present disclosure are shown, showing a hydration health category score (left) and related basal biomarker concentrations (center) and historical scores (right) displayed on a mobile application.

[0692] Fig.34B A mobile software application is shown that displays basic biomarker concentrations for the hydration health category (left) and self-guided actionable insights (center, right) according to some embodiments of the present disclosure.

[0693] Fig.34C A mobile software application is shown that displays actionable insights for self-guidance (left, right) and external links to advisors, coaches, medical professionals, or others (center) according to some embodiments of the present disclosure.

[0694] According to some embodiments, a saliva sensor or biosensor is provided that can be used with a cartridge 200 and a POCT device 100. The system can be used to test saliva biomarkers, as a fitness sensor, a health and wellness sensor, a handheld medical device, and / or for remote health and / or disease screening.

[0695] Currently, urine or blood samples are used to obtain biomarkers / useful health information for different disease states. Blood sampling is invasive and usually requires complex laboratory machines to perform the test. Consumers cannot easily perform the test using point-of-care (POC) devices. Such tests are costly, time-consuming, and invasive.

[0696] Some previous solutions include the use of optional biofluids such as sweat, tears, and saliva. Current saliva testing solutions use a mail-in system where consumers can extract their own saliva and then return the saliva sample to the company so that they can run a laboratory diagnostic. These solutions are still time-consuming and expensive. Although easy to use, in addition to the cost and lack of real-time results (because this solution relies on mail-in and external laboratory testing), it also requires knowledge of precision mechanics and scientific training, so there is no real-time point-of-care testing (POCT) available. Finally, given that the samples are not self-contained, these professionals need safety training and equipment.

[0697] According to some embodiments, a point-of-care testing (POCT) device 100 is provided that allows for on-demand, portable, non-invasive (saliva) testing using a self-contained sample collection cartridge 200 containing biosensors capable of measuring a user's fitness, general health and wellness, and screening for serious diseases. According to some embodiments, results are available within seconds to minutes, cost only a few dollars per test, and no medical professional or other personnel are required.

[0698] 9. in conclusion

[0699] Although the concepts disclosed herein are susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are described in detail herein. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed, but rather, it is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Claims

1. An electrochemically active sensor chip, comprising: a plurality of electrode wells, wherein each well comprises a plurality of electrodes, the electrodes comprising surfaces comprising chemical substances including catalysts or reactants corresponding to analytes in the biological sample; The electrode is capable of sustaining a redox reaction with the chemical substance and the analyte to generate an electrical signal in response to the biological sample contacting the electrode.

2. The sensor chip according to claim 1, further comprising: a substrate comprising an electrically insulating material; and A plurality of electrode holes are arranged in different positions on the substrate.

3. The sensor chip according to claim 1 or 2, further comprising: a spacer coupled to the substrate that allows saliva to enter the wells; and A hydrophilic cap directs saliva to the wells and seals the chip.

4. The sensor chip according to any one of claims 1 to 3, wherein the biological sample is human saliva. 5 . The sensor chip according to claim 1 , wherein two of the electrode wells are used for two-electrode enzymatic analyte analysis. The sensor chip according to any one of claims 1 to 5 , wherein the first hole is used for background correction. The sensor chip according to claim 6 , wherein the second hole is used for electrochemical analysis.

8. The sensor chip according to any one of claims 1 to 7, wherein the well comprises three electrodes.

9. The sensor chip according to claim 8, wherein The first electrode in each well serves as a sample loading detector; The second electrode in each well serves as a working electrode; and The third electrode in each well served as a reference electrode.

10. The sensor chip according to any one of claims 1 to 9, wherein the electrodes comprise a conductive material such as carbon ink.

11. The sensor chip according to any one of claims 1-10, wherein the electrodes are configured to be electrically coupled to a reader device via contacts on an edge of the chip.

12. The sensor chip according to any one of claims 1 to 11, wherein the electrode surface in the well is coated with a polymer film containing an enzyme selective for a specific target analyte, a redox mediator and a surfactant.

13. The sensor chip of claim 12, wherein the enzyme reduces an oxygenated analyte substrate in saliva to a second product in a chemical reduction process in which the second product gains electrons; and Wherein the enzyme comprises lactate oxidase (LOx) and the analyte comprises lactate.

14. The sensor chip of claim 12, wherein the enzyme comprises glucose oxidase, and the analyte for detection by the device comprises glucose.

15. The sensor chip of claim 12, wherein the enzyme comprises lactose dehydrogenase, and the analyte comprises pyruvate.

16. The sensor chip of claim 12, wherein the enzyme comprises cholesterol oxidase, and the analyte comprises cholesterol.

17. The sensor chip according to claim 12, wherein the enzyme comprises ascorbate oxidase, and the analyte for detection comprises ascorbic acid.

18. The sensor chip according to claim 12, wherein the enzyme comprises alcohol dehydrogenase, and the analyte for detection comprises ethanol.

19. The sensor chip of claim 12, wherein the redox mediator comprises a transition metal complex wherein one or more ligands are coordinated to the transition metal; wherein the redox mediator transfers electrons from the enzymatic reduction process to at least one of the conductive electrodes. 20 . The sensor chip according to claim 19 , wherein the transition metal comprises Os, Rh, Ru, Ir, Fe and / or Co.

21. The sensor chip of claim 19, wherein the redox mediator comprises hexaammineruthenium (III) trichloride.

22. The sensor chip of claim 19, wherein the redox mediator comprises pentaamminechlororuthenium(III)dichloride.

23. The sensor chip of claim 19, wherein the redox mediator comprises ferric chloride cyanide.

24. The sensor chip according to any one of claims 12-23, wherein the surfactant promotes uniform distribution of membrane components.

25. The sensor chip according to claim 24, wherein the surfactant comprises Triton X-100.

26. The sensor chip of claim 24, wherein the surfactant comprises sodium dodecyl sulfate.

27. The sensor chip of claim 24, wherein the surfactant comprises sodium stearate.

28. The sensor chip according to claim 12, wherein the polymer film further comprises a polymer binder.

29. The sensor chip of claim 28, wherein the polymer binder comprises polyvinylpyridine (PVP).

30. The sensor chip according to claim 28 or 29, wherein the polymer binder comprises trehalose.

31. The sensor chip according to any one of claims 28 to 30, wherein the polymer binder comprises METHOCEL.

32. The sensor chip according to claim 12, wherein the polymer film is drop-cast onto the surface of the electrode hole.

33. The sensor chip according to claim C2, wherein the spacer layer comprises an opaque structural layer; wherein the spacer layer further comprises a pressure-sensitive adhesive layer on either side.

34. The sensor chip according to claim 33, wherein the spacer layer further forms a wall of the electrode hole and forms a space into which saliva flows.

35. The sensor chip of claim C2, wherein the top cover is coupled to the spacer layer, wherein the top cover comprises a hydrophilic polymer; and the top cover further comprises a pressure-sensitive adhesive layer on one side; and the top cover serves as a microfluidic channel for moving saliva into the electrode hole by capillary force.

36. An ion-selective hydration sensing membrane for a sensor, wherein the membrane can be drop-cast in layers in one or more wells of the sensor, wherein the membrane covers a carbon mat in the one or more wells, the membrane comprising: a first layer comprising a hydrogel layer that helps adhere the sensing membrane to the carbon mat; a sensing layer, which includes an ionophore and a corresponding salt of a target ion, and helps to generate a membrane potential for analyzing ion concentration, the sensing layer being applied to the first layer; and A top layer is applied to the sensing layer, the top layer comprising a polymer that protects the sensing layer from external interference and damage and increases overall stability while allowing ions to diffuse from solution into the sensing layer for use during analysis.

37. The sensing membrane of claim 36, wherein the top layer comprises PDMS.

38. The sensing film according to any one of claims 36-37, wherein the top layer contains only polyvinyl alcohol (PVA) film.

39. The sensing film of any one of claims 36-38, wherein the first layer comprises a polymer film.

40. The sensing film of any one of claims 36-39, wherein the top layer comprises Dowsil 3140, a form of PDMS.

41. The sensing film of any one of claims 36-40, wherein the polymer of the top layer is dissolved in a solvent.

42. The sensing membrane of claim 41, wherein the solvent is selected from THF, 2-methyltetrahydrofuran and cyclopentyl methyl ether.

43. The sensing film of any one of claims 41-42, wherein a plasticizer is also used in the solvent to drop cast the polymer of the top layer.

44. The sensing film of claim 43, wherein the plasticizer is selected from the group consisting of DOS, DEHP, adipates, citrates, and phthalates.

45. The sensing membrane according to any one of claims 36-44, wherein the sensing layer comprises a polymer, an ionophore, a lipophilic salt and a plasticizer.

46. ​​The sensing film according to any one of claims 36 to 45, which is manufactured in the following manner: Prepare 2.5% PVA in DI water and add methanol to form a first solution; drop casting the first solution into the hole of the sensor, and then baking at about 100° C. for about 10 minutes to form the first layer; The second solution of sodium ionophore X, Na-TFPB, and THF was stirred for about 30 minutes; Then add PVC to the second solution and stir for about 1 hour; Then, DOS was added to the second solution and stirred for about 2 hours; The second solution is then drop cast on top of the first layer in the wells of the sensor and baked at about 40° C. for at least 10 hours to form the sensing layer; A third solution was made of Dowsil 3140 in THF and DOS, which was stirred for 2 hours; The third solution was drop cast on the sensing layer and left at room temperature for about one hour.

47. A method for manufacturing a sensing membrane for a biosensor chip, the biosensor chip being configured to detect the amount of lactate in a sample, the method include: Mix HEPES and BTP in an aqueous solvent to prepare a buffer solution; Dissolving NaCl and Triton X in an aqueous solution to prepare the corresponding stock surfactant solutions; PVP and hydroxyethyl cellulose were centrifuged and vortexed in an aqueous solution to form a polymer solution; adding trehalose and sodium succinate to the polymer solution, and stirring the mixture of trehalose, sodium succinate and polymer solution; adding the surfactant (Triton X, NaCl) solution and the polymer solution, and centrifuging and vortexing the mixed solution; adding METHOCEL to the buffer solution and stirring the mixed METHOCEL / buffer solution for about 1 hour to disperse in the solution; adding hexaamineruthenium to an aliquot of the polymer solution and stirring the mixture for about one hour, and then vortexing and centrifuging the mixture to produce an intermediate mixture; and LOx was added to the intermediate mixture and the mixture was stirred for about 20 minutes to produce a viscous solution.

48. The method for manufacturing a sensing film for a biosensor chip according to claim 47, further comprising: include: The viscous solution was drop-casted in one or more wells of the biosensor chip in an amount of about 2 uL, and the viscous solution in the one or more wells was solidified at about 55° C. for about 10 minutes.

49. The method for manufacturing a sensing film for a biosensor chip according to claim 48, wherein the sensing film has a shelf life of more than 6 months.

50. The method for manufacturing a sensing membrane for a biosensor chip according to any one of claims 48-49, wherein the sensor produced by the sensing membrane has a repeatability of <5% from sensor to sensor and <5% from batch to batch.

51. The method for manufacturing a sensing film for a biosensor chip according to any one of claims 48-50, wherein the sensor produced by the sensing film can use a sample volume of ≤ about 5 uL.

52. The method of manufacturing a sensing film for a biosensor chip according to any one of claims 48-51, wherein the sensing film produces a sensor having a detection time of less than or equal to about 30 seconds.

53. The method of manufacturing a sensing film for a biosensor chip according to any one of claims 48-52, wherein the sensing film produces a sensor having a sensitivity greater than or equal to about 0.1 mM.

54. The method of making a sensing membrane for a biosensor chip according to any one of claims 48-53, wherein the sensing membrane produces a sensor having a LOD of at least about 0.2 mM.

55. The method for manufacturing a sensing film for a biosensor chip according to any one of claims 48 to 50, wherein the sensor produced by the sensing film has a linearity of at least 3.5 mM.

56. An oral fluid collection device for collecting oral fluid, comprising: handle; Extendible elements; a pipette tip; and A coupling mechanism, through which the device can be coupled to a cartridge base.

57. A biological fluid collection device for collecting a biological fluid, comprising: handle; Extendible elements; a pipette tip; and A coupling mechanism, through which the device can be coupled to a cartridge base.

58. A device according to claim 56 or 57, wherein the mechanism is configured to allow locking of the device to a cartridge base.

59. A biological fluid collection device for collecting a biological fluid, comprising: handle; an extensible element; and Aspiration tip.

60. The biological fluid collection device of claim 59, further comprising a coupling mechanism, through which the device can be coupled to a cartridge base.

61. The biological fluid collection device of any one of claims 57-60, wherein the biological fluid is saliva to be collected from a person's oral cavity by the device, and wherein the device is configured such that during collection of saliva, the handle remains outside the oral cavity while the extendable element and the pipette tip are located inside the oral cavity.

62. The fluid collection device of any one of claims 56-61, wherein the handle allows for collection of an oral fluid sample without any oral fluid coming into contact with the handle itself or a user of the device.

63. A fluid collection device according to any one of claims 56-62, wherein the extendable element is a telescopic arm.

64. A collection device according to any one of claims 56-63, wherein the extendable element can be extended or retracted in a manner to extend the pipette tip further into the oral cavity for sample collection, and can be retracted in a manner to keep the device more compact when coupled to the cartridge base.

65. A collection device according to any one of claims 56-64, wherein the extendable element has an extended position and a retracted position, and wherein the extendable element extends to its extended position when unconstrained, and retracts to the retracted position when the pipette tip and the extendable element are inserted into a cartridge base and the collection device is coupled to the cartridge base.

66. A collection device according to any one of claims 56-65, wherein the coupling mechanism provides a way to store the extendable element and the pipette tip in the cartridge base.

67. A collection device according to any one of claims 56-66, wherein the coupling mechanism allows the device to be stored in the cartridge base in a compact state, and wherein the coupling mechanism includes threads allowing the device to be screwed onto the cartridge base or a magnet that magnetically fixes the device to the base.

68. A collection device according to any one of claims 56-67, wherein when inserted into a cartridge base, the combination of the device and the base is considered a single cartridge, and wherein the device is configured to be seated or locked on the base in a manner to limit leakage of any biological fluid or oral fluid from the cartridge.

69. A collection device according to any one of claims 56-68, wherein when the device is inserted into a cartridge base before insertion into the mouth, the pipette tip is compressed at the bottom of the chamber in the base in a manner that allows closure of both the combined device and the cartridge base, and when the device is removed from the cartridge base, the pipette tip expands to allow collection of oral fluid or biological fluid.

70. A collection device according to any one of claims 56-69, wherein when the device is inserted into a cartridge base after being inserted into the oral cavity to collect saliva, the pipette tip is compressed at the bottom of the chamber in the cartridge base in a manner that allows closure of both the combined device and base and release of the collected saliva into the chamber in the base.

71. A method of using a collection device according to any one of claims 56-70, wherein when the device is inserted into a cartridge base after being inserted into the oral cavity to collect saliva, the pipette tip is compressed at the bottom of a chamber in the base in a manner that allows both the combined device and base (cartridge 200) to be closed and the collected saliva to be released into the chamber of the base.

72. A biological fluid collection device for collecting a biological fluid, comprising: handle; and Aspiration tip.

73. A reader unit comprising: Electrical components for wireless data transmission; Potentiostat; and Battery; wherein the reader unit is configured to collect data, Wherein the reader unit is configured to interface with the electrodes by contacting the cartridge.

74. A reader unit comprising: a cartridge receptacle configured to receive a cartridge having a biosensor therein configured to generate one or more electrical signals in response to a biological fluid contacting the biosensor; A processor is communicatively coupled to the biosensor of the cartridge received in the cartridge receptacle and is configured to determine a concentration of one or more biomarkers in the biological fluid.

75. A reader unit according to claim 74, wherein the cartridge includes one or more electrical contacts and the cartridge receptacle includes one or more corresponding electrical contacts, which are configured to contact the electrical contacts of the cartridge when the cartridge is received in the cartridge receptacle, thereby allowing the processor to communicate with the biosensor and receive one or more electrical signals from the biosensor.

76. The reader unit of claim 75, wherein the processor uses one or more electrical signals received from the biosensor to determine the concentration of one or more biomarkers in the biological fluid.

77. The reader unit of any one of claims 74-76, further comprising a memory communicatively coupled to the processor.

78. A reader unit according to any one of claims 73 to 77, wherein the unit is a handheld device which performs the data analysis and is configured to send the data to a mobile phone or display the data on an integrated screen.

79. A reader unit according to any one of claims 73-78, wherein the unit includes a chassis; and the chassis includes a body and a release lever.

80. A reader unit according to claim 79, wherein the release rod comprises a spring-loaded rod configured to engage with a recess in a cartridge body of the cartridge; wherein the rod is configured to retain the cartridge within the reader unit and allow the cartridge to be removed from the reader unit.

81. The reader unit of any one of claims 73-80, further comprising a printed circuit board (PCB) for performing biological sample analysis within the reader unit; and wherein the printed circuit board comprises a processor and a potentiostat; and the potentiostat is configured for performing chronoamperometry or potentiometry on a biological fluid sample.

82. The reader unit of claim 81, wherein the processor further comprises a logic gating function to determine when sample loading is detected in an electrode well of a biosensor.

83. A reader unit according to any one of claims 73-82, further comprising a wireless antenna for wireless data transmission to a remote device.

84. The reader unit of any one of claims 73-83, further comprising a display, wherein the processor is communicatively coupled to the display, wherein the processor can cause data or test results to be displayed on the display.

85. A reader unit according to claim 81, wherein the PCB further includes a power regulation controller; and the power regulation controller allows use of the battery; and the battery can be charged through a port in the reader chassis.

86. A reader unit according to any one of claims 73-85, wherein the potentiostat is coupled to the biosensor chip through internal pins or magnets in the cartridge receptacle of the reader unit; and these pins or magnets are configured to electrically couple with contacts on the biosensor chip to complete the circuit for electrochemical detection.

87. The reader unit of any one of claims 73-86, wherein the reader comprises a signal conditioning circuit to amplify the electrical signal detected by the biosensor chip.

88. The reader unit of any one of claims 73-87, further comprising a data processing unit comprising a processor for processing data based on the detected electrical signals and a memory for storing or buffering the data.

89. The reader unit of any one of claims 73-82, wherein the reader unit is configured as a handheld unit having a first dimension less than or equal to approximately 100 mm and a second orthogonal dimension less than or equal to approximately 50 mm.

90. A sample analysis reader unit configured to be electrically coupled to a sample analysis cartridge, the sample analysis reader comprising: processor ； and A non-transitory computer-readable medium having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to: detecting the presence of a sample analysis cartridge electrically coupled to the sample analysis reader; detecting identification information associated with the sample analysis cartridge; identifying a suitable testing protocol for the sample analysis cartridge based at least in part on the identification information; detecting a sample collection device inserted into the sample analysis cartridge; as well as A mixing protocol is initiated to mix a sample received from the sample collection device with a reagent in a fluid in a reservoir in the sample analysis cartridge.

91. The reader unit of claim 90, wherein the processor is configured to initiate the mixing protocol by pumping reagents and sample from respective reservoirs into a mixing reservoir.

92. The reader unit of claim 90, wherein the processor is configured to initiate the mixing scheme to promote the formation of a plurality of competitive complexes between analytes present in the saliva and the competitive reagent format having streptavidin-labeled magnetic particles.

93. The reader unit of claim 90, wherein the processor is configured to process one or more signals from one or more electrodes exposed to a biological sample, the one or more signals being indicative of an amount of one or more target analytes from the sample.

94. The reader unit of claim 90, wherein the sample analysis reader is configured to be electrically coupled to a plurality of sample analysis cartridges, each of the sample analysis cartridges being disposable.

95. A saliva based reading unit for measuring analytes and biomarkers associated with scanning of saliva to detect elevated and sustained lactate levels as it acts as an indicator of severe temporary and chronic diseases.

96. The reader unit of claim 95, wherein the reader unit is a point of care testing device (POCT) that provides rapid, real-time results.

97. The reader unit of claim 95 or claim 96, wherein the reader unit senses the concentration of a biological fluid biomarker with an accuracy of greater than 10%.

98. A reading unit according to claim 95 or claim 96, wherein the reading unit is capable of interfacing with software applications and databases for display, storage and communication means.

99. A reading unit according to claim 95 or claim 96, wherein the reading unit is capable of comparing previous scores of lactic acidosis tests with improved sensitivity and specificity.

100. A reading unit according to claim 95 or claim 96, wherein the reading unit is capable of performing artificial intelligence calculations based on the user's historical and current physiological state and generating actionable insights focused on recommendations or self-guidance to improve the accuracy of acidosis scoring.

101. A reading unit according to claim 95 or claim 96, wherein the reading unit is configured to share results and history with other individuals with the goal of improving health or initiating more comprehensive medical care.

102. A reading unit according to claim 95 or claim 96, wherein the reading unit operates in a variety of harsh physical environments at low cost without the use of medical professionals and without the use of batteries or power sources in an in vivo setting.

103. A reader unit according to claim 95 or claim 96, wherein the reader unit employs an electrochemical sensor that is inexpensive, scalable and stable.

104. A reading unit according to claim 95 or claim 96, wherein the reading unit has embedded firmware capable of independently performing tasks and calculations.

105. A reading unit according to claim 95 or claim 96, wherein the reading unit allows use by untrained personnel.

106. A reader unit according to claim 95 or claim 96, wherein the reader unit uses a chemical compound that does not require refrigeration or special storage mechanisms.

107. A reading unit according to claim 95 or claim 96, capable of providing robust and stable results for 12 months without the need for special storage or refrigeration.

108. The reader unit of claim 95 or claim 96, wherein the reader unit employs a disposable and retrievable, upgradeable test cartridge that allows for collection of sampled biological fluids without intervention by a medical professional or scientist using safety equipment or protocols.

109. A cartridge base comprising a biosensor chip according to any one of claims 1-35, configured to interface with a reader unit according to any one of claims 73-94, and configured to receive a saliva collection device according to any one of claims 56-72 or 185-186 and transfer the received saliva to the biosensor chip.

110. A cartridge base comprising a biosensor chip according to any one of claims 1-35, configured to receive a saliva collection device according to any one of claims 56-72 or 185-186, and transfer the received saliva to the biosensor chip.

111. The cartridge base of claim 109 or 110, wherein the base is configured to transfer received saliva to the biosensor chip using a microfluidic channel.

112. The cartridge base of any one of claims 109-111, configured to be inserted into a reader unit, and wherein the reader unit is configured to detect at least one analyte in the received saliva.

113. The cartridge base of any one of claims 109-112, comprising a cartridge body made of a rigid material; and wherein the cartridge body has a cylindrical shape having a cavity therein, wherein a first side of the cartridge body has an opening to the cavity; and wherein the cartridge body comprises a plurality of protrusions within the cavity, wherein the protrusions are configured to interface with the saliva collection device; wherein a portion of the collecting device is configured to be inserted into the cavity of the cartridge body, wherein the protrusion is configured to interface with the collection device in a manner so as to facilitate transfer of saliva initially on the collection device to the cavity of the cartridge base; wherein the cartridge base comprises an O-ring within a cavity of the cartridge housing; and Wherein a passage is located within the cavity of the cartridge housing, the passage has an orifice therein for allowing saliva to flow out of the cavity.

114. The cartridge base according to any one of claims 109-113, further comprising a sensor cavity configured to accommodate the biosensor chip; wherein the sensor cavity has a recess corresponding to the shape of the biosensor chip; and wherein the biosensor chip is fixed in the sensor cavity using a latch.

115. The cartridge base of any one of claims 109-114, further comprising one or more external recesses that interface with one or more opposing protrusions in the reader unit; and the recesses allow the cartridge base to be secured within the reader unit during sample analysis and released after use by a release latch mechanism on the reader unit.

116. A cartridge base comprising: a cartridge body defining a collection device cavity therein, wherein a first side of the cartridge body has an opening to the cavity, wherein a portion of a biological fluid collection device is configured to be inserted into the collection device cavity of the cartridge body; and The cartridge body defines a sensor cavity; A biosensor chip is in the sensor cavity.

117. The cartridge base of claim 116, further comprising an orifice extending from the wall of the collection device cavity to the wall of the sensor cavity, allowing biological fluid in the collection device cavity to flow onto the biosensor chip in the sensor cavity.

118. The cartridge base of any one of claims 116-117, wherein the collection device cavity is configured to collect biological fluid transferred from the biological fluid collection device along the wall of the collection device cavity when the biological fluid collection device is inserted into the collection device cavity.

119. A cartridge base according to claim 118, wherein the biological fluid collection device includes a pipette tip having a biological fluid therein, and wherein when the pipette tip is pressed against the wall of the collection device cavity when the biological fluid collection device is inserted into the collection device cavity, the biological fluid is transferred from the collection device to the wall of the collection device cavity.

120. A cartridge base according to claim 119, wherein the cartridge body and the collecting device include a device for fixing the collecting device to the cartridge base, which is within the pipetting tip of the collecting device within the collecting device cavity.

121. A cartridge base according to claim 120, wherein the device for fixing the pipette tip of the collection device within the collection device cavity causes the pipette tip to be pressed against the wall of the collection device cavity when the collection device is fixed to the cartridge base to help transfer the biological fluid from the collection device to the wall of the collection device cavity.

122. A cartridge comprising: A cartridge base according to any one of claims 109-121 or 187; and A collecting device according to any one of claims 56-72 or 185-186.

123. A system comprising: The cartridge of claim 122; and A reading unit according to any one of claims 73-89 or 90-94.

124. The system of claim 123, comprising a sensor chip according to any one of claims 1-35.

125. A point-of-care saliva testing system comprising: a biological fluid collection device configured to collect a biological fluid; a cartridge base configured to receive the collection device, the cartridge base having a biosensor therein, wherein the cartridge base is configured to transfer the collected biological fluid from the collection device to the biosensor having a plurality of electrical pads thereon after the collection device having the collected biological fluid thereon is received within the cartridge base; and a reader device configured to receive the cartridge base therein, wherein the reader device has a plurality of electrical contacts configured to become electrically coupled with the electrical pads of the biosensor when the cartridge base is received in the reader device, wherein the system is configured to detect the presence and / or amount of one or more target analytes in a collected biological fluid that has been transferred to the biosensor that is electrically coupled with the electrical contacts of the reader.

126. A method for detecting an analyte in saliva, the method include: reducing the chemical substrate in the saliva to a second product in a chemical reduction process, wherein the second product gains electrons during the chemical reduction process; supplying the extracted electrical energy to electrodes of an electrochemical sensor; and An electrical signal generated as a result of a redox reaction involving an analyte in the saliva and a chemical reagent coupled to at least one electrode of the electrochemical sensor is detected from the electrodes of the electrochemical sensor in contact with the saliva.

127. A method for detecting an analyte in saliva, the method include: receiving the saliva on electrodes of an electrochemical sensor; reducing the chemical substrate in the saliva to a second product in a chemical reduction process, wherein the second product gains electrons during the chemical reduction process; supplying the extracted electrical energy to electrodes of the electrochemical sensor; and An electrical signal generated as a result of a redox reaction involving an analyte in the saliva and a chemical reagent coupled to at least one electrode of the electrochemical sensor is detected from the electrodes of the activated electrochemical sensor in contact with the saliva.

128. The method of claim 126 or 127, wherein the saliva is transferred to the electrode via a saliva collection device.

129. The method of any one of claims 126-128, further comprising collecting the saliva from the oral cavity of a person using a saliva collection device.

130. The method of any one of claims 126-129, wherein the electrical signal is detected using amperometry, voltametry, or potentiometry.

131. The method of any one of claims 126-130, further comprising processing the electrical signal to determine a parameter of the analyte.

132. The method of claim 131, wherein the parameter comprises a concentration level of the analyte.

133. The method of any one of claims 126-132, further comprising using the electrical signals to quantitatively define health, fitness, and diagnostic health indicators using various parameters.

134. The method of claim 133, wherein the parameters include detected concentrations of one or more biomarkers associated with a qualitative health category generally associated with the base biological fluid.

135. The method of claim 134, wherein the parameters further comprise a weight assigned to each biomarker as it relates to its overall importance to the health category.

136. The method of claim 135, comprising changing the weight assigned to each biomarker based on previous measurements.

137. The method of claim 134, further comprising combining the detected concentrations and weights to calculate a final health category score.

138. The method of claim 137, wherein the health score may include a scaled numeric, binary or other scale to represent the quantitative difference between poor to excellent health.

139. The method of claim 137, further comprising providing actionable insights based on the health score.

140. The method of claim 139, wherein the actionable insights include recommendations for dietary or fitness changes to help improve the health score.

141. The method of any one of claims 126-140, further comprising using a mobile application running on a remote device.

142. The method of claim 141, further comprising sending data from a reader device that receives the electrical signal to the remote device using Bluetooth.

143. The method of claim 142, wherein the remote device uses the data to perform analysis.

144. The method of claim 141, wherein the mobile application further determines a health score, provides actionable insights, and / or displays the concentration of the measured analyte.

145. A biofluid-based point-of-care testing (POCT) system that provides rapid, real-time results for measuring analytes and biomarkers without the need for expensive blood tests or medical training, and at a fraction of the cost of current testing in a handheld device.

146. The system of claim 145, wherein the system allows for measurement of biomarker or analyte concentrations in several biological fluids: blood, saliva, tears, sweat, or urine. Some claims include using saliva as the primary biological fluid and using some or all of the chemistries and techniques described herein. The only differences with other biological fluids are the collection protocol, possible separation techniques, and the concentration of the biomarkers. For blood, the collection protocol may include a pinprick on a finger to draw the blood, deposit it on a cotton collection mechanism, and then dispense into the electrodes as usual. The concentration of lactate / other biomarkers in blood is higher than in saliva, so the calibration curve will be adjusted to reflect this. Blood also contains coagulants and other materials that may disrupt the analysis, so a filter for larger particles / blood cells in the test cartridge 200 before the electrode sensor 1300 will allow for accurate measurements. For tears, urine, and sweat, which share common characteristics with saliva, less care is needed. The most significant change will be the calibration curve to reflect the concentration of biomarkers in those fluids.

147. The system of claim 145, wherein the system senses the concentration of a biofluid biomarker with greater than 90% accuracy. According to some claims, this is achieved due to the following chemistry, wherein the redox mediator allows for more efficient transfer of electrons to the working electrode for measurement, and the polymer backbone composition of the sensing membrane allows for much higher stability over a useful temperature range. The inclusion of background correction holes 1332 within the sensing array eliminates any off-target sensitivity.

148. The system of claim 145, wherein the system is configured to aggregate biomarker concentration scores to obtain an average biomarker concentration.

149. The system of claim 145, wherein the system is configured to interface with software applications and databases for display, and / or with storage, analysis and communication devices.

150. The system of claim 145, wherein the system is configured to perform artificial intelligence calculations based on the user's historical and current physiological state and provide actionable insights focused on recommendations or self-guidance for improving overall or specific health.

151. The system of claim 145, wherein the system is configured to share results and / or history with other devices, such as devices associated with one or more individuals other than the user (e.g., a smartphone or computer). According to some claims, such sharing may be done to seek input from the user to improve their health or initiate a more comprehensive healthcare consultation.

152. A saliva-based point-of-care testing (POCT) system that provides rapid, real-time results for measuring analytes and biomarkers in a variety of harsh physical environments at low cost without the use of medical professionals and without the use of batteries or power sources in an in vivo setting.

153. The system of claim 152, wherein the system uses an inexpensive disposable biofluid or saliva test cartridge 200, and wherein the modular electrode well design of such sensor 1300 allows for inexpensive automated drop casting of the sensor polymer membrane.

154. The system of claim 152, wherein the system has embedded firmware capable of independently performing tasks and calculations.

155. The system of claim 152, wherein the system allows use by untrained or trained personnel.

156. The system of claim 152, wherein the system utilizes chemical compounds that do not require refrigeration or special storage mechanisms to render the device operable.

157. The system of claim 152, wherein the system is capable of providing robust and stable results over a 12 month period without the need for special storage or refrigeration.

158. The system of claim 152, wherein the system uses disposable and retrievable or upgradeable test cartridges that allow for collection of sampled biological fluids without intervention by a medical professional or scientist using safety equipment or protocols.

159. The system of claim 152, wherein the system utilizes a test cartridge configured for measuring multiple biomarkers or analytes simultaneously.

160. The system of claim 152, wherein the system is configured to aggregate multiple biomarker concentrations and calculate an overall quantitative health, wellness, or fitness score.

161. The system of claim 152, wherein the system is configured to scan for analytes and biomarkers that serve as indicators of specific temporary or chronic diseases.

162. A saliva-based point-of-care testing (POCT) system that provides rapid, real-time results for measuring analytes and biomarkers associated with scanning of saliva to detect elevated and sustained lactate levels, as lactate levels are used as an indicator of severe temporary and chronic diseases.

163. The system of claim 162, wherein the system senses the concentration of a biological fluid biomarker with an accuracy of greater than 10%.

164. The system of claim 162, wherein the system is capable of interfacing with software applications and databases for display, storage and communication devices.

165. The system of claim 162, wherein for lactic acidosis testing, the system is capable of comparing previous scores with improved sensitivity and specificity.

166. The system of claim 162, wherein the system is capable of performing artificial intelligence calculations based on the user's historical and current physiological state, and the resulting recommendations or self-guided actionable insights are focused on improving the accuracy of the acidosis score.

167. A system according to claim 162, configured to enable sharing of results and history with other individuals with the goal of improving health or initiating more comprehensive medical care.

168. The system of claim 162, wherein the system operates in a variety of harsh physical environments at low cost without the use of medical professionals and without the use of batteries or power sources in an in vivo setting.

169. The system of claim 162, wherein the system uses an electrochemical sensor that is inexpensive, scalable, and stable.

170. The system of claim 162, wherein the system has embedded firmware capable of independently performing tasks and calculations.

171. The system of claim 162, wherein the system allows use by untrained personnel.

172. The system of claim 162, wherein the system uses chemical compounds that do not require refrigeration or special storage mechanisms.

173. The system of claim 162, wherein the system is capable of providing robust and stable results over a 12 month period without the need for special storage or refrigeration.

174. The system of claim 162, wherein the system utilizes a disposable biological fluid or saliva test cartridge 200 that allows for collection of sampled biological fluid without intervention by a medical professional or scientist using safety equipment or protocols.

175. The system of claim 162, wherein the system utilizes a recyclable or upgradeable biofluid or saliva test cartridge 200 that allows for collection of sampled biofluid without intervention by a medical professional or scientist using safety equipment or protocols.

176. A system that uses the concentration of relevant basic biomarkers in saliva and / or other biofluids (blood, urine, tears, sweat) and the user's general physical parameters to quantitatively define normally non-quantifiable health, fitness and diagnostic health indicators, include: a. measuring the concentrations of various biomarkers associated with qualitative health categories typically associated with biological fluid samples; b. assigning a weighting factor to each biomarker as it relates to its overall importance to the qualitative assessment within the health category; c. an algorithm that combines the concentrations and weighting factors in one of several ways to calculate a final health category score; d. Calculate a final health score, which includes a numerical scale (e.g., 1-10 or 1-100), binary (e.g., high-low), or any other hierarchical scale (e.g., high-medium-low, green-yellow-red) representing the quantitative difference between poor health and excellent health.

177. The system of claim 176, further comprising: include: An intelligent algorithm that uses the score and relevant user health information to modify the weight factors of the personalized weight table.

178. The system of claim 176, wherein the system is further configured to provide actionable insights related to the health category score, the insights providing suggestions to the user on how to improve the overall health category score.

179. The system of claim 176, further comprising displaying on the device basal biomarker concentrations, health category scores, and self-guided or externally actionable insights associated with improvements in health category scores.

180. The system of claim 176, further comprising a mobile software application that displays basal biomarker concentrations, health category scores, and actionable insights.

181. The system of claim 176, further comprising a device having a microprocessor and a sensing element, the device being capable of measuring biomarker concentrations, storing and modifying weighting factors manually or based on an artificial intelligence algorithm, and calculating a final health category score.

182. The system of claim 176, further comprising means for communicating and transmitting biomarker concentrations, weighting factors, and general health category scores between the measuring device and a remote device running a software application and / or a remote database.

183. The system of claim 176, further comprising means for sharing the quantitative health category scores with a coach, medical advisor, or other person for further actionable insights or consultation.

184. An electrochemical sensor device for detecting and measuring the concentration of an analyte in saliva, comprising: A saliva sample collection mechanism, comprising a handle and a wicking swab; Saliva sample collection reservoir; means for calculating a general health, fitness, or wellness score based in part on the analyte concentration; a device that displays and transmits analyte concentrations and health, fitness and wellness scores to an external device; and Software that displays said content with self-directed actionable insights and links to external professionals or others for additional insights; The saliva sample collection mechanism and reservoir are disposable after each use.

185. A collection device according to any one of claims 56-72, which is devoid of any electronic devices and / or batteries and / or other power sources.

186. The collection device of any one of claims 56-72, being free of any electronics and / or batteries and / or other power source on a portion thereof configured to be received in a cavity of a body from which a biological sample is to be collected.

187. The cartridge base of any one of claims 116-121 , which does not have a battery.

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