Marker and device for diagnosing overall health condition of individual

Abstract

The present invention provides a device for detecting an analyte in a liquid sample, the device comprising: a test element for testing whether the analyte exists in the liquid sample or the number of the analyte, the analyte being one or more of the following analyte: neopterin; the amino acid sequence is shown in the description, and the amino acid sequence is shown in the description. The invention relates to a preparation method of a graphene oxide-dopamine composite material. The invention also relates to a method for preparing the microalbuminuria. The microalbuminuria is prepared from the microalbuminuria.

Description

[0001] This invention patent application claims priority to the prior Chinese application, application number: 202311368227.7; application date: October 20, 2023, and its specification, abstract, claims and drawings are all incorporated herein by reference as part of the present invention. Technical Field

[0002] The present invention relates to the field of health status prediction or diagnosis, and in particular to a method and device for rapidly predicting an individual's overall health status by testing substances in urine samples using rapid diagnostic technology. Background Art

[0003] The following background technology introduction is merely an introduction to some background common sense and does not constitute any limitation to the present invention.

[0004] Currently, detection devices or methods for detecting the presence of an analyte in a sample are widely used in hospitals and at home. These rapid diagnostic devices, such as those used for pregnancy tests and drug abuse tests, include one or more test strips. These rapid diagnostic devices are very convenient, providing test results on the test strip within one minute, or at most ten minutes. With the increasing popularity of at-home testing, many of these rapid diagnostic tests can now be performed at home.

[0005] For specific disease diagnosis, the use of specific biomarkers in samples for diagnosis or prognosis is also common and is used in hospitals and at home. These specific biomarkers, such as the detection of one or more analytes, are often associated with specific diseases. For example, in infectious disease testing, such as COVID-19 testing, samples are tested for the presence of antibodies or antigens associated with the virus, thereby detecting infection. HIV testing, for example, tests for substances associated with HIV, such as HIV antibodies or the number of HIV nucleic acid copies, to determine whether an individual is infected with the virus. For another example, in cancer testing, specific biomarkers are used to detect cancer-related markers in individual samples, and the presence or level of these specific biomarkers can be used to determine whether an individual has cancer. Cancer biomarkers vary, such as small molecules in blood samples, autoimmune antibodies, nucleic acid sequences at the genetic level, or the presence or mutation of proteins. In cancer detection, as described in Chinese patent application No. 202110422182.1, a combination of specific nucleic acid sequences is used to detect whether an individual has liver cancer; as described in Chinese patent application No. 202310631059.X, specific proteins are used as specific markers to diagnose whether an individual has lung cancer; as described in Chinese patent application No. 2023100823544, small molecule chemicals in the blood or a combination of these compounds are used to diagnose whether an individual has lung cancer. In cardiovascular disease detection, specific markers associated with cardiovascular disease are used to test whether an individual is at risk of cardiovascular disease, such as troponin, myoglobin, natriuretic peptides, etc. to test whether an individual is at risk of heart failure or myocardial infarction. When testing markers for diabetes, the gold standard is to test the concentration of glucose in the blood to determine whether an individual has diabetes.

[0006] In addition, home self-testing is becoming increasingly common, and most self-testing products for COVID-19 have appeared. Testing for infectious diseases at home is becoming increasingly popular, which will also make it easier for individuals to test their own health at home, such as self-testing or testing for certain specific diseases. However, existing traditional tests or tests, including home self-tests (OTC), are all for diagnosing a specific disease, using specific markers or analogues to test in samples, and judging or predicting the diagnosis of a specific disease based on the specific quantity or the degree of change in quantity.

[0007] Such traditional tests are based on diagnosis of specific diseases. In fact, when an individual develops a specific disease, such as cancer, infectious diseases, cardiovascular disease, diabetes and other specific symptoms, such as the initial symptoms of a specific disease, testing will begin. Such tests are relatively late in time, which requires earlier testing of the individual's physical health status, so that the health status of the person being tested can be prompted in advance. Summary of the Invention

[0008] To overcome some of the shortcomings of traditional technologies, this invention provides a set of biomarkers. These markers do not diagnose specific diseases, but rather provide a more holistic assessment of an individual's health status at an early stage. The test results represent an individual's overall health status. This holistic health assessment can determine the risk of specific diseases in advance, allowing for early intervention or treatment to prevent the onset of specific diseases.

[0009] Therefore, in the first aspect of the present invention, the present invention provides markers for testing the overall health of an individual, wherein the markers are selected from one or more combinations of the following markers: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria. If any one marker is positive, it means that the overall health status of the individual is not very good. If multiple markers, such as two or more, are positive, the overall health status of the individual is not good, and there may be a possibility of the occurrence of a specific disease. It is necessary to further use specific markers to test or confirm the specific disease, or to intervene or treat it in advance. The positive and negative here are comparisons of these markers with pre-set thresholds. Generally, a value greater than the pre-set threshold is positive, and a value less than the pre-set threshold is negative.

[0010] In a second aspect, the present invention also provides the use of biomarkers for preparing reagents for diagnosing an individual's overall health status. The biomarkers are selected from one of the following markers to obtain multiple combinations: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria. These new uses of biomarkers are used to detect an individual's overall health status, rather than for diagnosing a specific disease. If any one marker is negative, it indicates that the individual's overall health status is good, such as being in a healthy state. If multiple markers, such as two or more, are negative, the individual's overall health status is good or very good, and there may be no possibility of developing a specific disease, or the possibility of developing a specific disease is very low. In this case, there is no need to further use specific markers to test or confirm a specific disease, and good living habits can still be maintained to maintain such good health. If one marker is positive, it indicates that the individual's overall health is not good. If the test results of multiple markers are positive, it means that the individual's health is very poor and they need to pay attention to their health or seek diagnosis for a specific disease. The diagnosis method is to use specific markers for specific diseases to conduct tests. It can be understood that the more positive the above markers are, the worsening of the overall health condition is. More positive results indicate worse health, and conversely, more negative results indicate increasing health.

[0011] In a third aspect, the present invention provides a device for detecting an analyte in a liquid sample, the device comprising: a test element configured to test the presence or quantity of an analyte in the liquid sample, wherein the analyte is one or more of the following: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria. In some embodiments, the test strip is a lateral flow test strip. In some embodiments, the test strip is capable of simultaneously testing four analytes in a sample, wherein the analytes include a combination of the following markers: neopterin; cystine, homocysteine, homocysteine; dopamine; and microalbuminuria. In some embodiments, the test strip can simultaneously test four analytes in a sample, consisting of the following: neopterin; cystine, homocysteine, homocysteine; dopamine; and microalbuminuria. Using such a device, individuals can perform self-tests at home to monitor their overall health or well-being, or changes in their overall health. For example, as individuals age, their health may change. Using this device, multiple tests can be performed to monitor changes in their overall health, such as whether they are experiencing negative or positive changes, such as becoming less healthy or becoming healthier. In some embodiments, for example, for individuals 60 years of age or older, testing is recommended twice a year, while for individuals 40 years of age or older, testing is recommended at least once a year. Testing can be performed at home or at a location deemed appropriate.

[0012] In some embodiments, the test strip includes a detection area and a labeling area. The labeling area includes a first receptor that specifically binds to an analyte or marker and is capable of flowing with the liquid sample. The detection area includes a second receptor immobilized thereon that specifically binds to the first receptor and is incapable of flowing with the liquid sample. In some embodiments, the first receptor comprises an antibody, an antibody fragment, a polypeptide, or a polypeptide fragment, wherein the antibody is a monoclonal antibody. The second receptor comprises an analog of the analyte. In some specific embodiments, the analog of the analyte comprises a linking reagent attached to the analyte. The linking reagent can be linked via any chemical bond, for example, one or more of the following: keyhole hyaluronidase (KLH), bovine globulin (BGG), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg white protein (HEL), ovalbumin (OVA), tetanus toxin (SWM), tetanus toxin (TT), methylated bovine serum albumin (mBSA), and rabbit serum albumin (RSA). These linking reagents are connected to the analyte or marker, making the marker substance immunogenic and capable of producing antibodies by injection into an animal. Such immunogenic markers can be called antigens. However, the marker of the present invention itself is not immunogenic and cannot produce antibodies in mammals.

[0013] A fourth aspect of the present invention provides a device for detecting analytes in a liquid sample, the device comprising: four test elements, each of which is used to test the presence or quantity of an analyte in the liquid sample, or to test the presence or absence of a marker or combination of markers that can predict or diagnose an individual's overall health status, wherein the analyte is one of the following: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria. In some embodiments, each test strip is used to detect one marker. In some embodiments, the test strip is a lateral flow test strip. In some embodiments, the test strip is capable of simultaneously testing four analytes in a sample, the analytes comprising the group consisting of: neopterin; cystine, homocysteine, homocysteine; dopamine; and microalbuminuria. In some embodiments, the test strip is capable of simultaneously testing four analytes in a sample, wherein the analytes consist of the following analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; and microalbuminuria.

[0014] In some embodiments, each test strip includes a detection area and a labeling area, wherein the labeling area includes a first receptor that specifically binds to an analyte and is capable of flowing with the liquid sample, and the detection area includes a second receptor immobilized thereon that specifically binds to the first receptor and is incapable of flowing with the liquid sample. In some embodiments, the first receptor on each test strip specifically binds to one of four analytes, or the second receptor includes an analog of the analyte.

[0015] A fifth aspect of the present invention provides a device for detecting an analyte in a liquid sample, the device comprising: first, second, third, and fourth test elements, wherein the four test elements are located in a detection device, and the four test strips are capable of simultaneously contacting the liquid sample, wherein each test strip is capable of detecting one of the following four analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria. In some embodiments, the first test strip is used to detect neopterin, the second test strip is used to detect cystine, homocysteine, homocysteine; the third test strip is used to detect dopamine, and the fourth test strip is used to detect microalbuminuria. In some embodiments, the liquid sample comprises urine, saliva, or blood.

[0016] In a sixth aspect of the present invention, a method for testing an analyte in a fluid sample is provided, the method comprising: providing a liquid sample from an individual, detecting the content or quantity of a marker in the liquid sample, the marker comprising one or more of neopterin, cystine, homocysteine, homocysteine, dopamine, or microalbuminuria; when any marker exceeds a certain threshold, it can be determined that the individual's overall health is poor or unhealthy; when any marker is below a certain threshold, it can be determined that the individual's overall health is good or healthy.

[0017] In some approaches, when the levels or amounts of all four markers exceed a certain threshold, the individual's overall health can be judged to be very poor or very unhealthy. When the levels or amounts of all four markers are below a certain threshold, the individual's overall health can be judged to be very good or very healthy. The thresholds vary for each marker. If one marker exceeds a preset threshold, it indicates that the individual is unhealthy in some aspects or overall. Conversely, if one marker does not exceed the preset threshold, it indicates overall health or unhealthy in some aspects. In some approaches, these four markers can be used in combination to assess an individual's overall health. Using only one marker can only assess a specific aspect and not the overall picture. These substances have a normal range in healthy people. If these markers exceed the normal range, it indicates a problem with the individual's overall health and requires further evaluation. This evaluation can use other more accurate markers to test for specific diseases or to remind the individual to pay attention to their health. Therefore, if all four markers exceed their respective pre-set thresholds, it means that the individual's health is not very good, especially the overall health is not good, which indicates that there may be a specific disease. Of course, the emergence of this specific disease may not appear immediately, or may only appear within a certain period of time.

[0018] In some embodiments, the presence of a line in the test area of ​​any one of the test strips indicates a negative result for a particular marker, and the absence of a line in the test areas of all the test strips indicates a positive result for all markers. In some embodiments, the presence of a line in the test area indicates a negative result, and the absence of a line in the test area indicates a positive result. The absence of a line in the test result control area of ​​any or all of the test strips indicates an invalid test result.

[0019] In some embodiments, a testing device is provided, which includes four test strips, each of which tests a marker substance. The marker substance can test the overall health of an individual, but does not test a specific disease. The test strips of the device are allowed to contact a liquid sample at the same time, and the overall health of the individual is judged based on the results of the test strips. In some embodiments, the testing device includes an upper card and a lower card, and the lower card has a slot structure for carrying the test strips. Each test strip is set in the slot, wherein the sample application area of ​​the test strip is exposed for contacting the liquid sample. During the test, urine is collected in a container, and then the testing device is inserted into the urine, that is, the exposed sample application area is allowed to contact the urine. After waiting for a period of time, the number of lines that appear in the test area of ​​the test strip is observed to determine the test result. The overall health of the individual is judged based on the result.

[0020] In some embodiments, the sample is a urine sample, which is used to test markers in the urine sample. This marker includes one or a combined test of four of the aforementioned marker substances, which is used to evaluate, test or determine the overall health status of an individual.

[0021] In the seventh aspect of the present invention, the content of NPT in urine can be used to test whether the urine is adulterated. Generally, Neopterin (NPT) has a normal concentration in urine. If the urine is adulterated, for example, water is added to dilute the urine, so that the concentration of NPT is reduced or cannot be detected, it may be thought that the urine is adulterated. Therefore, if the NPT in the urine is lower than a preset threshold or cannot be detected, it means that the urine is adulterated. On the contrary, it means that the urine cup is not adulterated. At the same time, higher than the preset threshold or too high a concentration indicates that the individual has systemic health problems, such as the risk of developing cancer or infectious diseases in the later stage.

[0022] Beneficial effects

[0023] The device and method of the present invention can simultaneously detect four markers. These markers can indicate an individual's early health status. These early health conditions do not indicate the onset of a specific disease, but rather indicate the overall risk of disease. Furthermore, some markers can be used to test for urine adulteration. For example, NPT can be used to detect urine adulteration in addition to a comprehensive health assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of a testing device in a specific embodiment of the present invention.

[0025] Figure 2 is a schematic diagram of a testing device in a specific embodiment of the present invention.

[0026] Figure 3 Schematic diagram of the structure of a test strip in a specific embodiment of the present invention.

[0027] Figure 4 It is a structural schematic diagram of a testing device in a specific embodiment of the present invention.

[0028] FIG5 is a schematic diagram of a carrier containing a test element and a positive test structure in a specific embodiment of the present invention, wherein: Figure 5A This is a test chart showing all positive results. Figure 5B This is the test result chart showing that NPT and DOP are negative, while HCY and mALB are positive. Figure 5C This is a schematic diagram of the results in which mALB is negative and the other three indicators are positive.

[0029] Figure 6 Schematic diagram of a test card in a specific embodiment of the present invention showing negative test results for four markers.

[0030] FIG7 is a schematic diagram showing an invalid test result of a test card in a specific embodiment of the present invention, wherein: Figure 7A All test results are invalid. Figure 7B A negative result of HCY is considered valid, while other test results are considered invalid. Figure 7C All negative test results indicate that the individual is in good health. Figure 7D A test that returns all positive results indicates overall poor health and requires further testing or a doctor's appointment.

[0031] Figure 8 It is a schematic diagram of the principle structure of testing a marker substance in a specific embodiment of the present invention.

[0032] Figure 9 FIG. 1 is a top view of a test strip in a specific embodiment of the present invention.

[0033] Figure 10 The figure is a schematic structural diagram of a test strip in a specific embodiment of the present invention. Detailed description

[0034] The following is a further explanation of the structures involved in the present invention or the technical terms used. If not otherwise specified, they will be understood and interpreted according to the general terms commonly used in the art.

[0035] Detection

[0036] "Assay" means to test or examine a substance or material for the presence or absence of a substance or material, such as, but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolite, a drug or drug metabolite, an organic tissue or its metabolite, a nucleic acid, a protein, or a polymer. Additionally, "assay" means to test for the amount of a substance or material. Furthermore, "assay" also includes immunoassays, chemical assays, and enzyme assays.

[0037] Detection method

[0038] Any known method can be used to test for the markers of the present invention, which are present in liquid samples, such as urine. Testing methods include, for example, isotope analysis, a method developed by Refsum et al. in 1985, and chromatography. High-performance liquid chromatography (HPLC) is a relatively mature and widely used method, but its drawback is the wide variation in sample handling, chromatographic conditions, sample detection, and quantification, making standardization difficult. Fully automated high-performance liquid chromatography is also a method. HPLC can be categorized into various methods based on the derivatization method (pre-column or post-column) and detection method (fluorescence, electrochemical). HPLC can accurately measure one of the four markers of the present invention, or it can be used to test simultaneously. Testing methods also include immunological methods: these methods utilize specific antibodies against the four markers, such as monoclonal antibodies, to measure the four markers using fluorescence polarization or immunoassays. Immunological methods can employ fully automated chemiluminescence immunoassays, using instruments to detect the four markers. This method is rapid, simple to operate, and highly automated, reducing human error. It offers excellent accuracy and precision, making it suitable for use in most clinical laboratories. Of course, chromatography technology can also be used, that is, a disposable reagent strip that processes dry substances on a test strip (latteralflow) and performs chromatography in water, also known as a lateral flow test strip. Using this test strip, test results can be quickly obtained within 3-10 minutes, and it is more suitable for testing by non-professionals, such as being suitable for home self-testing, or belonging to OTC products, so that the results are fast and easy to operate. Of course, any other suitable testing method can be used to test one of the four markers provided by the present invention or test four at the same time, such as simultaneously testing the four markers in a sample, such as simultaneously testing the presence or absence of the four markers in saliva, urine, and blood, or the amount of the four markers, thereby testing whether the individual's body is healthy based on the level of the four expressed contents.

[0039] sample

[0040] The detection method or detection device of the present invention can detect one or more combinations of the above four markers in biological samples. The biological samples here include biological fluids (such as case fluids or clinical samples). Liquid samples or liquid samples, or fluid samples or fluid samples, can be derived from solid or semi-solid samples, including excreta, biological tissues and food samples. Solid or semi-solid samples can be converted into liquid samples by any appropriate method, such as mixing, mashing, macerating, incubating, dissolving or utilizing enzymatic digestion of solid samples in a suitable solution (such as water, phosphate solution or other buffer solution). "Biological samples" include samples derived from animals, plants and food, such as urine, saliva, blood and its components, cerebrospinal fluid, vaginal secretions, sperm, feces, sweat, secretions, tissues, organs, tumors, cultures of tissues and organs, cell cultures and media derived from humans or animals. Preferably, the biological sample is urine, and preferably, the biological sample is saliva. Food samples include food-processed substances, final products, meat, cheese, wine, milk and drinking water. Plant samples include any plant, plant tissue, plant cell culture and medium. "Environmental samples" are derived from the environment (e.g., liquid samples from a lake or other body of water, sewage samples, soil samples, groundwater, seawater and wastewater samples). Environmental samples may also include sewage or other wastewater.

[0041] Any analyte can be detected using a suitable detection element or test element of the present invention, and preferably, the present invention is used to detect analytes in saliva or urine.

[0042] Downstream and upstream

[0043] The terms "downstream" and "upstream" are used to define the direction of liquid flow. Generally, liquid flows from the upstream to the downstream area. The downstream area receives liquid from the upstream area, and liquid can also flow along the upstream area to the downstream area. This is generally divided based on the direction of liquid flow. For example, in some materials that utilize capillary forces to promote liquid flow, liquid can flow in the direction opposite to gravity due to gravity. In this case, the upstream and downstream areas are still divided based on the direction of liquid flow. For example, in the detection device of the present invention, after the absorption element absorbs the fluid sample or sample, the fluid can flow from the liquid outflow outlet of the capillary channel into the detection chamber, contact the test element 300 above, and flow to the sample application area 304 of the test element 300. At this time, the liquid flows from the sample application area 304 to the absorption area 303 from upstream to downstream. During this flow, the liquid passes through the marking area 307 and then flows to the test area 306. The test area includes a detection area and a test result control area. The test area can be a polyester film, and the sample application area can be a glass fiber.

[0044] Gas connection or liquid connection

[0045] Gas connectivity or liquid connectivity means that liquid or gas can flow from one place to another, and the flow may pass through some physical structures to guide it. The so-called passing through physical structures generally refers to the liquid passing through the surface of these physical structures, or the internal space of these structures to passively or actively flow to another place. Passive flow is generally caused by external force, such as flow under capillary action. The flow here can also be the liquid or gas due to its own action (gravity or pressure), or it can be passive flow. The connectivity here does not necessarily mean that liquid or gas must exist. It only indicates the connection relationship or state between two objects in some cases. If there is liquid, it can flow from one object to another. This refers to the state of connection between two objects. On the contrary, if there is no liquid connectivity or gas connectivity between the two objects, if there is liquid in or on one object, the liquid cannot flow into or onto another object. Such a state is non-connected, a state of non-liquid or gas connectivity.

[0046] Test components

[0047] The so-called "test element" here refers to an element that can detect whether a sample or specimen contains an analyte of interest. This detection can be based on any technical principle, whether immunology, chemistry, electricity, optics, molecular biology, nucleic acid, physics, etc. The test element can be a lateral flow test strip, which can detect a variety of analytes. Of course, other suitable test elements can also be used in the present invention.

[0048] Various test elements can be combined and applied to the present invention. One form is a test strip. The test strip used to analyze the analyte in the sample (such as the four biomarkers of the present invention or metabolites indicating physical conditions) can be in various forms, such as immunoassay or chemical analysis. The test strip can adopt a non-competitive or competitive analysis mode. The test strip generally comprises a water-absorbing material with a sample addition area, a reagent area and a test area. The sample is added to the sample addition area and flows to the reagent area through capillary action. In the reagent area, if the analyte is present, the sample binds to the reagent. The sample then continues to flow to the detection area. Other reagents, such as molecules that specifically bind to the analyte, are fixed in the detection area. These reagents react with the analyte in the sample (if present) and bind the analyte to the area, or bind to a reagent in the reagent area. The marker used to display the detection signal is present in the reagent area or a separate labeling area.

[0049] In a typical non-competitive assay, if the sample contains the analyte or the concentration is above a pre-set threshold, a signal is generated, indicating a positive result; if the sample does not contain the analyte or the concentration is below the pre-set threshold, no signal is generated, indicating a negative result. In a competitive assay, if the analyte is absent from the sample or the concentration is below the pre-set threshold, a signal is generated, indicating a negative result; if the analyte is present or the concentration is above the pre-set threshold, no signal is generated, indicating a positive result.

[0050] The test element can be a test paper, which can be made of absorbent or non-absorbent materials. The test paper can include multiple materials for liquid sample delivery. One material of the test paper can be covered on another material, such as filter paper covered on a nitrocellulose membrane. One area of ​​the test paper can be made of one or more materials, while another area can be made of one or more different materials. The test paper can be adhered to a certain support or hard surface to increase the strength of the test paper.

[0051] The analyte is detected by a signal generating system, such as by using one or more enzymes that react specifically with the analyte, and by using the method for fixing the specific binding substance on the test paper as described above, to fix the combination of one or more signal generating systems to the analyte detection area of ​​the test paper. The substance that generates the signal can be in the sample addition area, the reagent area, or the detection area, or on the entire test paper. The substance can be filled with one or more materials of the test paper. A solution containing the signal substance is added to the surface of the test paper or one or more materials of the test paper are immersed in the solution containing the signal substance. The test paper to which the signal substance solution is added is dried.

[0052] The various zones of the test strip can be arranged as follows: a sample addition zone, a reagent zone, a test zone, a control zone, a zone for determining whether the sample is adulterated, and a liquid sample absorption zone. The control zone is located after the test zone. All zones can be arranged on a single strip of test strip made of only one material. Alternatively, different zones can be made of different materials. Each zone can be in direct contact with the liquid sample, or different zones can be arranged according to the direction of liquid sample flow, with the ends of each zone connected to the front ends of other zones and overlapping. The materials used can be highly absorbent materials such as filter paper, glass fiber, or nitrocellulose membrane. The test strip can also be in other forms.

[0053] The commonly used reagent strips are nitrocellulose membrane reagent strips, that is, the detection area includes a nitrocellulose membrane, and specific binding molecules are fixed on the nitrocellulose membrane to display the detection results; it can also be a cellulose acetate membrane or a nylon membrane, etc. For example, the following patents describe reagent strips or devices containing reagent strips: US 4857453; US 5073484; US 5119831; US ​​5185127; US 5275785; US 5416000; US 5504013; US 5602040; US 5622871; US ​​5654162; US 5656503; US 5686315; US 5766961; US ​​5770460; US 5916815; US 5976895; US 6248598; US 6140136; US 6187269; US 6187598; US 6228660; US 6235241; US The test strips and similar devices with test strips disclosed in the above patent documents can be applied to the test element or detection device of the present invention to detect the analyte, such as the analyte in the sample.

[0054] The test strips used in the present invention can be what are commonly known as lateral flow test strips. The specific structures and detection principles of these test strips are well known to those skilled in the art. Conventional test strips include a sample collection area or sample application area 301, a labeling area 302, a detection area 303, and a water absorption area 304. The sample collection area includes a sample receiving pad, the labeling area includes a labeling pad, and the water absorption area includes a water absorption pad. The detection area includes necessary chemical substances, such as immunoassays or enzyme chemical reagents, to detect the presence of the analyte. Commonly used test strips are nitrocellulose membrane test strips, i.e., the detection area includes a nitrocellulose membrane with specific binding molecules fixed to the membrane to display the test results. Alternatively, cellulose acetate membrane or nylon membrane may be used. Of course, a test result control area may also be included downstream of the detection area. Typically, the control area and the detection area appear as horizontal lines, representing a test line (T line) or a control line (C line). Such test strips are traditional test strips, but other types of test strips that utilize capillary action for detection may also be used. In addition, a general test strip contains dry chemical reagent components, such as fixed antibodies or other reagents. When the liquid encounters a liquid, the liquid flows along the strip due to capillary action. As it flows, the dry reagent components dissolve in the liquid, and then go to the next area for processing, where the dry reagent reacts, thereby performing the necessary detection. The flow of liquid is mainly carried out by capillary action. All of these can be applied to the detection device of the present invention, or be arranged in a detection chamber to contact a liquid sample, or be used to detect the presence or amount of an analyte in a liquid sample entering the detection chamber. The test element is generally arranged in the test chamber 105. When the test chamber has a fluid sample, the fluid sample contacts the test element and is tested or detected.

[0055] In some embodiments, the device of the present invention includes four test strips, each of which detects a different analyte, but these analytes are all detected in the same sample, such as urine. Therefore, during the test, all the test elements are brought into contact with the liquid sample at the same time, and four or five indicators can be detected at one time.

[0056] Healthy (welless) or unhealthy (no-welless)

[0057] The analytes or markers of the present invention are those that can provide early indications, indicators, predictions, forecasts, detections, or assessments of an individual's early health status. The individual herein refers to a human or mammal. Health status herein includes the individual's overall, comprehensive, or comprehensive health status, but does not indicate, indicate, forecast, forecast, detect, or assess specific or specific diseases in the individual. "Overall" herein can refer to 100% overall health status, or to overall or 80-95% overall health status, or to 60-79% overall health status, or even to over 50% overall health status. "Overall" herein also includes the collective manifestation or overall outcome of multiple factors influencing an individual's physical and psychological well-being. These factors include changes in the external environment, individual lifestyle habits, aging, and psychological changes. Individuals or a combination of these factors can influence an individual's health, such as physical and / or mental health.

[0058] The term health status in the present invention includes both healthy and unhealthy, and also refers to overall health or unhealth. Here, "healthy" and "unhealthy" are relative concepts, and the health or unhealth referred to in the present invention specifically refers to the overall, comprehensive or comprehensive health status of an individual, for example, overall unhealthy, overall healthy, or overall healthy and overall unhealthy.

[0059] At the same time, the definition of "unhealthy" in the present invention does not include or exclude individuals who already have specific diseases. This is merely a technical definition, but it does not preclude individuals with specific diseases from being tested using the device of the present invention to assess their overall health. The primary purpose is to assess the overall health level or condition of individuals who do not have specific diseases. That is, to conduct an early overall health assessment of individuals without specific diseases. This assessment method involves testing a liquid sample using one or a combination of the four specific markers screened by the present invention, and then comparing the test results with preset thresholds to obtain an overall health test result.

[0060] Individuals diagnosed with a "specific disease" are those whose disease can be confirmed or categorized using traditional methods. Traditional methods include diseases that can be confirmed using equipment, instruments, or test reagents, such as CT scans, ultrasound, X-rays, or other specific tests. Here, "specific disease" refers to a symptom with a clear medical name. For example, diseases generally have broad categories, as well as more specific subcategories within them. Examples of broad categories include cancer or malignant tumors, infectious diseases, cardiovascular disease, diabetes, and mental illnesses such as depression, schizophrenia, and mania. Subcategories within broad categories include, for example, cancer or malignant tumors, including all known types of cancer, such as lung cancer, intestinal cancer, pancreatic cancer, liver cancer, thyroid cancer, and breast cancer, as well as the various stages or subtypes of each specific cancer. Examples of infectious diseases include HIV, COVID-19, influenza, and other pre-existing viruses. Cardiovascular disease includes heart disease, such as acute or chronic myocardial infarction or hemangiomas, and diabetes includes type I and type II diabetes. All of the above belong to specific types of diseases, and the specific marker substances screened by the present invention can evaluate, predict, or test the overall health status of an individual before these specific diseases occur, indicating whether the individual is healthy or unhealthy as a whole. If unhealthy, it is predicted that a specific disease may occur or develop.

[0061] It is generally understood that in the "early stages" of a specific disease, an individual may have already developed unhealthy symptoms or an unhealthy state, or may be generally unhealthy, but without the appearance of specific disease symptoms. In this way, the markers of the present invention can be used to assess and test the overall health status at an early stage, prompting the individual to pay attention to their physical condition and take necessary measures to improve their overall health to avoid the occurrence of specific diseases, especially major diseases such as malignant tumors or cancer, infectious diseases, cardiovascular diseases, mental illnesses and other major diseases; or further confirmation of specific or specific diseases is required, such as testing cancer markers, such as tumor markers such as ACE, or certain specific tumors, such as lung cancer, intestinal cancer and other specific disease markers for confirmation. If any, early intervention or treatment can be carried out.

[0062] Understandably, this overall poor health doesn't guarantee the development of a specific illness; it simply indicates that the individual's overall health is suboptimal and requires personal attention. Failure to do so could lead to the development of a specific illness, particularly a serious one. This possibility is merely a hypothesis and doesn't guarantee the onset of a specific illness. If the overall, comprehensive health test result obtained in this way indicates poor health, it serves as a reminder for the individual to take steps to prioritize their health. Of course, if the individual taking the test actually takes steps to improve from overall poor health to overall, comprehensive health, they can avoid specific illnesses, particularly serious ones, later in life, or delay their onset, potentially extending their lifespan and improving their quality of life. If the test result indicates overall health, it indicates that the individual is in good health and should continue in their current state.

[0063] The "early stage" here refers to the period before an individual has developed a specific disease. This period can be 1 year, or 1-10 years, or even earlier. The specific diseases here include the definition of the specific diseases mentioned above, and of course, the time before a major disease occurs. On the one hand, as an individual ages naturally and gradually ages, their health status will change, for example, from healthy to unhealthy, from overall healthy to overall unhealthy, and at this time no specific disease appears. For example, among ordinary people aged 35-40, a certain percentage will show overall unhealthiness. If ordinary people are between 40-60 years old, a higher percentage will show overall unhealthiness. The present invention has discovered through screening that some specific markers can be used to evaluate, predict, test, detect, and confirm an individual's early health status, especially the overall health status. The early stage here can be divided based on age as a single factor. The above examples of early definition only use aging as a factor, but this does not exclude younger people, such as those under 35 years old. In some developing countries, the onset of specific diseases is becoming younger and younger. Any one or combination of the four markers screened by the present invention can also be used to conduct early overall health assessments of young people to obtain overall health results, thereby preventing specific diseases, especially the occurrence of major diseases. For example, the reagents of the present invention can be used for testing at the ages of 15, 25, and 30.

[0064] Another aspect is the long-term impact of adverse factors in the external environment, which can also cause the individual as a whole to go from healthy to unhealthy, which may eventually lead to the occurrence of specific diseases. Such adverse factors in the external environment include factors that are not beneficial to the health of the individual, such as people who receive ultraviolet rays and radiation for a long time, people who stay up late for a long time, or people with bad habits. These adverse factors can also lead to changes in the overall health status of the individual, which may eventually lead to the occurrence of specific diseases, especially the occurrence of major diseases. Such people can also use the reagents of the present invention, such as markers for testing, to test their overall health status, remind the health status of such personnel, take measures as soon as possible, and avoid specific diseases, especially the occurrence of major diseases.

[0065] It is understood that the development and progression of certain diseases is a lengthy process, taking, for example, 5-10 years. For example, the development or progression of cancer. Before a specific disease develops, the body's health may already be compromised. However, when these overall health issues arise, there is currently no effective method to assess overall health. The present invention addresses this issue by screening a panel of specific markers to assess an individual's overall health at an early stage, predicting their overall health and thereby preventing the onset of specific diseases, particularly serious illnesses. For infectious diseases, although the process from infection to symptom onset is relatively short, for example, 3-7 days, some people are susceptible to infection, while others are. Most susceptible individuals generally have poor overall health, while those who are less susceptible generally have better overall health. It is understood that the development of a specific disease in an individual is a reflection of a complex set of factors, such as changes in immunity, mental health, internal and external environmental factors, or other adverse factors, which can affect the individual, leading to poor or very poor overall health and, consequently, the onset of a specific disease. Therefore, from this perspective, assessing an individual's overall or comprehensive health is of significant benefit and significance.

[0066] After extensive research and screening, the team and inventors of the present invention have discovered that the following four markers can be tested in combination or individually to predict a person's overall health risk, overall health status, or comprehensive, systemic health. This prediction or test is typically an early warning. The term "comprehensive" here means predicting or testing whether an individual has a catch-all, comprehensive, or integrated health risk, but does not identify specific diseases. Furthermore, this overall disease risk does not indicate the presence of or direct association with a specific medical disease, but rather indicates an unhealthy condition or overall health. In some embodiments, these four markers are: neopterin (NPT); cystine, homocysteine, homocysteine ​​(HCY); dopamine (DOP); or microalbumin (mALB). These four markers can be tested in combination or individually, as described in detail below.

[0067] Generally, people develop specific diseases or illnesses, such as cancer or tumors, infectious diseases, diabetes, or physiological depression. These only indicate a broad category of diseases. If the levels of any of the four markers above are abnormal, it indicates a health risk and requires further testing to clarify. For example, specific markers for tumors, infectious diseases, or diabetes can be tested. For example, markers directly associated with tumors or cancer, direct targets of infectious diseases such as antigens or antibodies, or direct markers for diabetes or other markers can be selected. The term "further" here does not mean that symptoms of a specific disease will immediately appear, but only indicates a risk of developing a specific disease. The term "specific" here means that there is a clear direct correlation with a specific disease. Using these direct markers, if they are at a certain level, it indicates a specific disease, such as cancer, cardiovascular disease, infectious disease, or diabetes. While the four markers mentioned above cannot be directly linked to a specific disease, they can be used to indicate health risks and overall or systemic health, especially for early detection of specific diseases.

[0068] Neopterin and its analogs

[0069] In some embodiments, the inventors of the present invention have discovered that abnormally elevated levels of neopterin (NTP) (chemical formula: C9H11N5O4, CAS Registry Number: 2009-64-5, molecular weight: 253.2147, as shown in the molecular formula below) in the human body indicate an individual's overall health risk or overall poor health. Without early intervention, the individual is at risk for specific diseases, particularly tumors, cancers, and / or infectious diseases. This suggests that further testing for tumor-related markers, antibodies or antigens associated with infectious diseases, or other specific markers related to tumors, cancers, or infectious diseases is warranted. As previously explained, elevated NTP levels do not necessarily indicate that the individual has already developed a malignant tumor, cancer, or / and infectious disease; they simply indicate a risk of developing a malignant tumor, cancer, or / and infectious disease, a risk that is absent without intervention. If the result is positive, timely intervention can restore the individual's overall health and turn the NPT negative, significantly reducing the risk of developing a malignant tumor, cancer, or / and infectious disease. Human urine typically contains a stable or constant concentration of NPT. An abnormally elevated NPT indicates overall poor health and an increased risk of developing a disease, such as cancer or a tumor, or an infectious disease, such as an acute infection.

[0070]

[0071]

[0072] Neopterin and 7,8-dihydropterin are the main pterins found in urine, with neopterin accounting for 25-45% of the total pterins ( et al., 1992, Lindsay et al., 2014). 7,8-Dihydropterin is the main component of the rest. Both are produced by macrophages during immune system activation and inflammatory responses and are used as clinical markers of monocyte / macrophage activation. However, the present invention can use both substances as markers of overall health. When testing, antibodies can be selected to recognize the main common functional groups of the two substances. Of course, it is also possible to choose to bind to or recognize neopterin while not recognizing 7,8-Dihydropterin. Therefore, 7,8-Dihydropterin can also be used as a marker of the present invention to predict whether an individual is healthy and their health status.

[0073] In healthy adults, the average urinary neopterin concentration is approximately 125 μmol / mol creatinine. Urine concentrations are often standardized to creatinine to compensate for urine dilution. The upper limits of normal neopterin / mol creatinine for women are: 208 (18-25 years), 209 (26-35 years), 239 (36-45 years), 229 (46-55 years), 249 (56-65 years), and 251 (over 65 years). The upper limits of normal for men are slightly lower: 195 (18-25 years), 182 (26-35 years), 176 (36-45 years), 197 (46-55 years), 218 (56-65 years), and 229 (over 65 years). These upper limits include 97.5% of healthy controls. Creatinine concentrations vary widely depending on the subject's hydration level but range around 12 mmol / L. Peaks occur late at night and early in the morning. Therefore, the new guanine concentration detected in the first morning urine is approximately 1500 nmol / L (379 ng / mL) (Wachter et al., 1989, Lindsay et al., 2014). The above concentrations can be selected as a baseline level or threshold for healthy people, and individuals above this threshold or baseline level may have unhealthy symptoms. Of course, higher concentrations or abnormally higher concentrations above the baseline level indicate poor overall health or unhealthy conditions, and are particularly in need of treatment and intervention to reduce the risk of tumors or cancers and / or infectious diseases.

[0074] Neopterin and Adulteration

[0075] In some cases, NPT or 7,8-dihydropterin of the present invention can also be used as a marker for urine adulteration. Traditionally, urine adulteration is determined by testing urine for specific gravity, pH, and other factors. While urine adulteration is considered unusual, it does occur frequently in real-world testing. For example, in job recruitment, applicants are often required to undergo drug abuse tests. However, candidates who do not wish to receive a positive result may adulterate their urine, for example by diluting the urine. For example, by adding a large amount of water to the collected urine, the concentration of the analyte in the urine is reduced, rendering it undetectable. Furthermore, simply replacing urine with another solution (so-called artificial urine) can also lead to inaccurate test results. While NPT or 7,8-dihydropterin maintains a normal concentration range in a person's urine, if NPT is found to be below normal or undetectable during testing, this indicates possible urine adulteration. This indicator can be used to test for urine adulteration, which is much simpler than conventional tests such as specific gravity and pH, providing a new approach to urine adulteration. While an abnormally elevated NPT concentration indicates the urine is not adulterated, it also suggests an individual's overall health may be poor, with an increased risk of cancer or acute infectious diseases later in life. If the possibility of artificial injection or addition of NPT to urine is ruled out, urine adulteration can be considered a possibility.

[0076] "Drugs of abuse" (DOA) are the use of drugs for non-medical purposes (usually for narcotic effects). Abuse of these drugs can lead to physical and mental impairment, dependence, addiction, and / or death. Examples of drugs of abuse include: depressants (downers, goofballs, barbs, blue devils, yellow syrup, etc.). jackets, methaqualone); tricyclic antidepressants (TCAs, i.e. imipramine, amitriptyline and doxepin); phencyclidine (PCP); tetrahydrocannabinol (THC, pot, dope, hash, weed, etc.); anxiolytics and sedatives and hypnotic drugs. Anxiolytics are a class of drugs mainly used to relieve anxiety, tension, fear, stabilize mood, and have hypnotic and sedative effects, including benzodiazepines BZO (benzodiazepines), atypical BZs, fused diazepam NB23Cs, benzodiazepines, BZ receptor ligands, open-ring BZs, diphenylmethane derivatives, piperazine carboxylates, piperidine carboxylates, quinazolinones, thiazine and thiazole derivatives, other heterocyclics, imidazole-type sedatives / analgesics (such as oxycodone OXY, methadone MTD), propylene glycol derivatives - carbamates, aliphatic compounds, anthracene derivatives, etc. The detection device of the present invention can also be used to detect medications that are medically useful but prone to overdose, such as tricyclic antidepressants (imipramine or its analogs) and acetaminophen. These drugs break down into various small molecules after being absorbed by the body. These small molecules are present in body fluids such as blood, urine, saliva, and sweat, or in some body fluids.

[0077] Homocysteine

[0078] In some embodiments, the present invention occurs, for example, that cystine, homocysteine, and homocysteine ​​(abbreviated as "HCY") (homocysteine, chemical formula C4H9NO2S) are markers associated with cardiovascular disease. If the content of the substance in the human body is higher than a certain level, it indicates that the overall health of the body is at risk, and it is also a marker for early prediction of cardiovascular disease. Although it is known here that the marker is a marker associated with cardiovascular disease, the inventors have also found that the marker is also a marker related to overall health. If the marker is higher than the normal level, the test result is positive, indicating that the individual's overall and comprehensive health status is not good. Similarly, a positive result does not mean that the individual must have suffered from cardiovascular disease, but only means that if no intervention is performed, there is a higher possibility of suffering from cardiovascular disease. If the HCY is lower than or equal to the normal level and the test result is negative, the individual's overall health is good and the risk of suffering from cardiovascular disease is reduced. That is, the marker has a new function and can be used to test, measure, predict, and diagnose an individual's overall health status, whether it is good or bad, good or bad. From the previous definition of the term, it can be seen that the overall health status and specific diseases are different in definition, and the diagnostic significance and scope they represent are also different.

[0079] Trace amount of white egg

[0080] In some approaches, for diabetes, we don't choose glucose as a test because glucose is a marker directly associated with diabetes, or HbA1C is also a more commonly used marker of diabetes. Instead, we choose microalbumin (abbreviated as "mALB") to predict diabetes. This is a new discovery. Generally, in a healthy state, after being filtered by the kidneys, trace amounts of protein are present in the blood. However, if it appears in the urine, it is called urinary protein, which has also been shown to be a marker for early diabetes. The present invention has discovered a new marker for testing an individual's overall health. This marker can be used to test an individual's overall health, specifically microalbumin in urine. If this marker exceeds normal levels, it indicates that the individual's overall health is not good, or is poor, alerting the individual to the need for attention and intervention. If not, the risk of developing specific diseases in the future will increase, particularly the risk of diabetes. Conversely, if intervention is implemented and the mALB level is restored to a normal, healthy level, the individual's overall health will be restored to a healthy and good state, and the risk of developing specific diseases in the future will be reduced, such as the risk of diabetes.

[0081] Dopamine

[0082] Compared with physical diseases or health conditions, mental health is increasingly valued. The inventors have found that for some mental health conditions, the following four substances are commonly used markers of mental health, such as dopamine (abbreviated as "DOP") (dopamine is a compound obtained by hydroxylation of phenylethylamine, and its chemical name is 3,4-dihydroxyphenylethylamine. Its molecular formula is C8H11NO2, and its relative molecular mass is 153.18 g / mol), serotonin; cortisol; and epinephrine.

[0083] The present invention has discovered that using dopamine as a marker can indicate early risks of mental health problems. Compared to several other substances, dopamine is a more effective indicator of mental health and can also be used to assess an individual's overall, comprehensive, or integrated health. Compared to other substances, dopamine is a more robust indicator of an individual's overall health. For example, anxiety, increased sodium intake, mercury poisoning, primary aldosteronism, post-traumatic stress disorder, and psychological stress can all elevate dopamine levels. Furthermore, compared to other substances, high dopamine levels are also associated with hyperactivity, inattention, mood swings, poor gastrointestinal function, psychosis, and sleep disorders. Therefore, dopamine can be used as a marker to assess an individual's overall health, allowing for early assessment of their overall health and preventing the development of specific diseases in the future. Numerous studies have shown that long-term mental ill-health can lead to physical harm, which in turn can lead to the development of specific diseases, such as depression, cancer, or one or more of the following: cardiovascular disease, diabetes, and so on.

[0084] Chronic abuse of illegal drugs such as methamphetamine can increase dopamine levels above a certain level: even if the addict is only sober for a few days, dopamine levels will remain high. Therefore, this is a sign of long-term abuse, such as methamphetamine, similar to ETG and alcohol abuse. Therefore, when using dopamine testing, it is necessary to rule out drug abuse. If methamphetamine is abused for a long time, dopamine levels will increase. However, if drug abuse is ruled out and dopamine levels are still higher than normal, it means that the individual is under long-term stress and is generally unhealthy. This unhealthy state may lead to the development of specific diseases. This specific disease may be a neurological or psychiatric disease, or it may lead to other physical diseases such as cancer and infectious diseases.

[0085] Four markers combined

[0086] Abnormalities in the four markers above indicate elevated levels compared to healthy individuals. These increases are defined by a standard or threshold. If these levels do not exceed a certain threshold, the individual is considered very healthy. Conversely, if they exceed a certain threshold, the individual's overall health is poor or very poor. Of course, if all four markers are elevated above a threshold, this indicates that the individual's overall health is very poor, potentially indicating a high risk of developing a specific disease or a combination of specific diseases. If only three markers are elevated above the normal threshold, this indicates poor overall health and a high risk of developing a specific disease. Furthermore, if only two or one marker is elevated above the normal threshold, this indicates poor overall health and a high risk of developing a specific disease. Conversely, if all four markers are within the healthy threshold, this indicates that the individual is generally very healthy and has a very low risk of developing a specific disease. Health status can be ranked according to the degree of health: very healthy, very healthy, averagely healthy, unhealthy, very unhealthy, and extremely unhealthy. If all four markers are at normal levels, the individual is very healthy. If all four are above normal, the individual is very unhealthy. If one or two of the four markers are above normal, the individual is generally healthy or unhealthy, but attention should be paid to the risk of specific diseases brought about by the elevation of one or two other markers, and the individual should be reminded to improve their functional behavior. For example, if microalbumin levels are abnormally elevated while the other three markers are at normal levels, the individual is generally healthy, but intervention or improvement is needed to avoid the development of diabetes, such as paying attention to dietary habits. Health status here is merely a relative concept.

[0087] Threshold (cut off) setting

[0088] In some approaches, the threshold values ​​for healthy individuals may vary across countries, living environments, and ages. However, clinical testing or screening can be used to determine average healthy threshold levels. Markers above these threshold levels indicate overall poor health. Of course, higher concentrations may indicate a higher degree of poor health and a greater risk of developing specific diseases. In some approaches, the threshold value for NPT in urine may be around 200 ng / mL, the threshold for HCY in urine may be 2000 ng / mL, the threshold for mALB in urine may be 40 ug / mL, and the threshold for DOP in urine may be 500 ng / mL. It should be understood that these threshold values ​​are merely estimates and may be adjusted based on clinical needs. Generally, the concentrations of markers that represent normal healthy levels for different populations, regions, and ages fall within a range, and threshold values ​​can be selected based on different objectives. In some approaches, the threshold values ​​for each marker may be different. In some embodiments, the threshold for NPT testing urine adulteration may be the same as or different from the threshold for testing overall health status, and may be adjusted according to different circumstances. Any adjusted value is also within the scope of protection of the present invention.

[0089] After verifying samples of four different markers, we found that each individual marker was able to effectively distinguish between healthy and unhealthy people. The AUC value of NPT was above 0.75, the AUC value of HCY was above 0.70, the AUC value of mALB was above 0.72, and the AUC value of DOP was above 0.75. The specific testing method was to use the reagent strip method and pre-set specific thresholds for testing (specific data and process omitted).

[0090] The specific validation method involves collecting samples from confirmed healthy and unhealthy populations (those without other specific diseases, particularly cancer, infectious diseases, diabetes, psychiatric conditions, cardiovascular diseases, and others) at different stages of their development. The number of unhealthy individuals who subsequently developed specific diseases is further grouped and categorized, and then tested to see if good discrimination can be achieved. The AUC values ​​for the markers are then calculated. Urine samples from these populations are obtained from a sample center using existing, stored samples collected over multiple years. These samples are associated with specific individuals (without specific information about the individuals, but with numbers). Samples stored at different times are tested and analyzed for specificity, sensitivity, and predictive performance of the four markers of the present invention. When the four markers are tested in combination, they can better distinguish between overall health and unhealthy states with high sensitivity and specificity. The AUC for the combined four markers can reach above 0.8. Of course, the AUC values ​​here are based on a limited sample size and may vary depending on the sample size. However, our experiments have demonstrated that the four markers described above can distinguish between general health and unhealthy states, allowing for early intervention and improvement to prevent the onset of specific diseases.

[0091] In some approaches, it's generally understood that physical health encompasses both physical and physiological health risks, as well as mental health risks, and that these two interact and influence each other. It's widely known that psychological factors play a significant role in human health and the development of certain diseases. Chronic psychological stress can easily lead to certain illnesses. Conversely, maintaining a positive mindset can enhance the body's resistance and reduce the risk of disease. The present invention has discovered a direct correlation between dopamine and the development of cancer, infectious diseases, cardiovascular disease, and diabetes. Therefore, in a preferred embodiment of the present invention, simultaneous testing of these four markers in an individual's fluid sample allows for a more comprehensive assessment of an individual's overall health. Currently, no test kit can simultaneously assess both physical and mental health, using urine samples simultaneously. The present invention combines the early prediction of physical and mental health risks with the early prediction of psychological health risks, enabling systemic testing, prediction, and monitoring of early health risks. Since the sample is primarily urine, it's easily accessible; this test can be performed at home, allowing users to monitor their overall health status at any time.

[0092] In some embodiments, all four markers are tested using a lateral flow test strip. A single test strip can be used to simultaneously test the levels of the four markers in urine. Alternatively, two test strips can be used, each testing two markers. Alternatively, four test strips can be used, each testing one marker. By allowing these different combinations or forms of test strips to simultaneously contact a liquid sample, such as urine, the same sample can be tested for a combination of multiple markers. In some embodiments, the test strip includes a sample application pad, a labeling pad, a test pad, and an absorbent pad. The labeling pad contains antibodies that specifically bind to these markers, which are conjugated to a label. Analogs of these markers are immobilized on the test pad. When the sample contains the markers, the marker analogs compete with the antibody-marker complex on the test pad for binding. The test result is determined by whether a color appears on the test pad. If a color appears, it indicates a negative result, meaning the sample is below a preset threshold. If no line appears, it indicates a positive result, meaning the sample is above the preset threshold.

[0093] Antibody

[0094] The antibody used in the present invention can be any antibody that can specifically bind to the analyte in the sample, in particular, can specifically bind to the four markers of the present invention for testing the overall health of an individual: neopterin (NPT); cystine, homocysteine, homocysteine ​​(HCY); dopamine (DOP); or microalbumin. The antibody that can be used as a binding agent can be any antibody known to those skilled in the art. "Antibodies" can be immunoglobulin molecules and antigen-binding portions of immunoglobulin molecules, that is, molecules containing an antigen-binding site that specifically binds to an analyte, analyte analog, or ligand ("immunoreaction"). This term also includes derivatives of antibodies that retain binding ability, as well as any protein containing a binding domain that is homologous or largely homologous to the binding domain of an immunoglobulin. These proteins may be derived from natural substances or may be partially or completely synthesized. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human immunoglobulin classes: IgG, IgM, IgA, IgD, IgG, and IgE. An "antibody fragment" is a derivative of an antibody or a portion of an antibody that is less than full-length. An antibody fragment retains at least one significant site of the binding ability of the full-length antibody. Examples of antibody fragments include Fab, Fab', F(ab'), 2, scFv, Fv, dsFv dimer, and Fd fragment, but not limited to the above. Antibody fragments can be generated by any means. For example, antibody fragments can be generated by enzymatic or chemical cleavage of an intact antibody, or by recombination from genes encoding partial antibody sequences. In other words, antibody fragments can be partially or completely recombinantly produced. Antibody fragments can be any single-chain antibody fragment. In other words, antibody fragments can contain multiple peptide chains linked to each other, for example, by disulfide bonds. Antibody fragments can also be any multimolecular complex. A functional antibody fragment typically contains at least about 50 amino acids, while more antibody fragments typically contain at least about 200 amino acids. Single-chain Fvs (scFvs) are recombinant antibody fragments that consist only of a variable light chain (V L ) and variable heavy chain (V H ) are covalently bound to each other via polypeptide chains. L and V H One of the two variable domains has an amino-terminal region. The polypeptide chain length and composition are variable, and the length can bridge the two variable domains without significantly affecting the atomic arrangement. The polypeptide chain is typically composed primarily of stretches of glycine and serine residues, with some glutamic acid and lysine residues interspersed to enhance solubility. "Dimer" refers to a dimer of a single-chain Fv. The monomers of the dimer typically contain shorter peptide chains than most single-chain Fvs, and they exhibit a propensity to form dimers.

[0095] The "Fv" segment consists of a V H and a V L The term "dsFv" herein refers to a fragment comprising a stable V H -V L Fv that eliminates the intermolecular disulfide bonds of the heavy chains. A "F(ab')" fragment is an antibody fragment that is essentially the same as the fragment obtained by digesting an immunoglobulin (usually IgG) with pepsin at pH 4.0-4.5. This fragment can also be recombinantly synthesized. A "Fab'" fragment is an antibody fragment that is essentially the same as the fragment obtained by reducing the disulfide bonds connecting the two heavy chains of the F(ab') fragment. A Fab' fragment can also be recombinantly synthesized. A "Fab" fragment is an antibody fragment that is essentially the same as the fragment obtained by digesting an immunoglobulin (usually IgG) with papain. A Fab fragment can also be recombinantly synthesized. The heavy chain fragment on the Fab fragment is the Fd fragment.

[0096] In the present invention, the antibody can specifically bind to the analyte in the liquid sample, such as urine, and the analyte here is the four marker substances mentioned above. In some ways, the antibody also carries a marker substance, which is a substance that indicates the amount of the antibody, and these marker substances are substances that can be read by equipment or by the naked eye. The marker substance can be a colored example, such as gold particles, latex particles, fluorescent substances, etc. These colored particles can be directly read and observed by the naked eye, or they can be read by taking pictures. Other marker substances, such as fluorescent substances or others, can be read by equipment and instruments. In some ways, the antibody that specifically binds to the four markers of the present invention carries a marker substance, which is processed and sprayed on the marker pad and exists in a dry state. After the marker pad is wetted by the liquid sample, it can dissolve in the liquid sample and flow to the downstream of the test strip as the liquid sample flows. When a sample contains any of the four markers mentioned above, antibodies can bind to the marker to form complexes. For example, complexes such as marker-anti-NPT antibody-NPT (in urine), marker-anti-HCY antibody-HCY, marker-anti-DOP antibody-DOP, and marker-anti-mALB antibody-mALB (where the antibodies are monoclonal antibodies) form. These complexes flow into the test area and compete with immobilized analyte analogs on the test area. Generally, higher analyte concentrations in the sample, exceeding a preset threshold, result in no lines appearing in the test area. Lines appear when the concentration falls below the threshold.

[0097] Similar substances to the analyte (marker substance)

[0098] The analogous substance of the analyte can be a substance similar to the analyte, but it can form a consortium with the antibody, and the antibody can also form a consortium with the analyte. The analyte here is the four marker substances of the present invention. In some embodiments, the analogous substance of the analyte shows the ability to compete with the analyte for the antibody on the labeled area, or shows the same ability to bind to the antibody, or the two have different binding abilities with the antibody. The binding ability between the analogous substance of the analyte and the antibody can be stronger or weaker than the binding force between the analyte and the antibody. The binding ability between the analogous substance of the analyte and the antibody can be greater than or lower than the binding force between the analyte and the antibody. This binding ability can be selected or optimized according to the needs of the test according to existing known technologies. For example, binding molecules, such as members of binding sites or binding pairs, can be obtained by arbitrarily or non-arbitrarily mutating.

[0099] The analog of the analyte can be a fragment of the analyte that retains some sites of the analyte. The marker substance can be attached to these analogs via linkers. These linkers include a binding site that is not present on the analyte, the analog of the analyte, and the marker substance. For example, these sites can be recognized by some antibodies but not by the analyte, the analog of the analyte, and the marker substance. Alternatively, these linkers can be recognized by antibodies but not by sites on other linkers. These linkers include, but are not limited to, proteins, natural or synthetic polypeptides, or some carbohydrates, such as hemocyanin (KLH), bovine hemoglobin (BGG), bovine serum albumin (BSA), bovine thyroprotein (BTG), ovalbumin (OVA), sperm whale myosin (SWM), tetanus toxoid (TT), methylated bovine serum albumin (mBSA), and rabbit serum albumin (RRSA).

[0100] In some embodiments of the present invention, the analog of the marker substance can be an antigen, while the marker itself is a hapten. To make the marker capable of eliciting an immune response, the marker needs to be modified to become an antigen. This modification involves chemically linking molecules to make it immunogenic. These molecules can be any of the proteins in the aforementioned linkers. In some embodiments, the analogs of these markers of the present invention are fixed to the test area and cannot move with the flow of the liquid. These analogs compete with the analyte for binding to the antibody carrying the marker substance. Thus, if there is no colored line on the test area, it indicates a positive test result. If a colored line appears, it indicates a negative test result. That is, a positive result indicates that the marker substance in the sample exceeds the pre-screened threshold, and a negative result indicates that the marker substance in the sample is below the preset threshold.

[0101] Liquid flow

[0102] Liquid flow typically refers to the movement from one location to another. Generally, most natural liquid flows rely on gravity to flow from higher to lower locations. This flow also relies on an external force, that is, flow under external gravity, which can be called natural gravity flow. In addition to gravity, liquid flow can also overcome gravity and move from lower to higher locations. For example, this can occur through the extraction of liquid, the compression of liquid, or the flow of liquid from the bottom to the top due to pressure, or the flow of liquid overcoming its own gravity due to pressure.

[0103] In some embodiments, providing Figure 1The four test strips shown each detect a marker. For example, one test strip is used to detect NPT, another test strip is used to test HCY, and the other test strips are used to detect DOP and mALB separately. The specific method uses a competitive method similar to drug abuse to perform the test. If no lines appear on the test line T, it means that these substances exceed a preset threshold value (cut-off), indicating that the early health status of the whole body is not good. On the contrary, if lines appear on the test line T, it means that these substances are below the preset threshold value, indicating that the whole body is in good health. During the test, the test strips can be tested individually, or the four test strips can be tested on the same sample at the same time.

[0104] In some methods, analogs of four analytes are fixed to the T-line, while the labeling pad is labeled with antibodies that specifically bind to the analytes in the sample. The labeling material can be gold particles, latex particles, or a water-soluble dye. The antibodies on the labeling pad can flow with the liquid. The principles of testing for other markers are similar. During the test, if any one of the NPT, HCY, DOP, and mALB in the sample, such as in a urine sample, exceeds the preset threshold, it indicates that the person being tested has a comprehensive health risk, but the specific disease cannot be determined. If all four markers are positive, the overall health risk is relatively high, and further professional testing is required to determine whether there is a possibility of a specific disease.

[0105] Figure 2 There is another way, that is, the test strips of the four markers are connected together by a guide element. When the sample is applied to the guide element, the sample flows to the four test strips respectively to complete the simultaneous testing of the four markers in the fluid sample.

[0106] Figure 3 Another embodiment is a test strip having four test lines, each test line corresponding to a marker substance of the present invention. The marker pad has a mixture of four different antibodies and is also provided with color particles. When performing the test, urine is applied to the sample pad or the sample front of the test strip is inserted into the urine to see whether the four test lines show color or the depth of the color. If color appears, it indicates a negative result, indicating that the individual's overall health is good. If no color appears, it indicates that one or more markers are positive, indicating that the overall health is poor or not good.

[0107] Figure 4 This is another specific implementation method, in which two marker substances are detected on two test strips respectively, and then connected together through a guide pad. Two indicators can be tested simultaneously on each test strip. Of course, 4 marker tests are also possible.

[0108] Figure 5A -7 is a specific embodiment. The test strips are loaded into a test card, and the sample pad extends from the test card. Each test strip is used to detect a marker substance. When performing the test, urine is collected in a urine cup, and the test card is then inserted into the urine. The test results are read after approximately 10 minutes. If any one indicator is positive, it is considered positive (no T line appears), indicating that the systemic health status is not good in the early stage. Conversely, if all indicators are negative (T lines appear), it indicates good health. If the C line does not appear, the test result is invalid.

[0109] Figure 8 This is a diagram explaining the basic principle of the present invention, using NPT as an example to illustrate how to set up the test strip of the present invention. Of course, the other three indicators can also be detected using the same design principle as NPT, or other similar principles.

[0110] Example 1 Preparation of the Immunoassay Lateral Flow Detection Device Provided by the Present Invention

[0111] The lateral flow detection device for detecting four markers by immunoassay prepared in this embodiment is as follows Figure 9 and Figure 10 As shown, the test strip comprises a sample area 101, a labeling area 102, a detection area 103 and a water absorption area 104 in the order of the liquid flow direction from upstream to downstream; wherein the water absorption area is prepared by using a general water absorption filter paper as a water absorption pad; the sample area 101 adopts a sample application pad, and the material of the sample application pad is glass fiber, so that the liquid sample contacts the glass fiber and flows to the labeling pad 102 by capillary force; the labeling area is made into a labeling pad, including labeling particles (such as gold particles, fluorescent particles, latex particles or dyes, or other colored labels) The antibody coupled with the marker substance is then sprayed onto a polyester film through a spraying device to form a marker pad. The marker substance on the marker pad can flow with the flow of the liquid; the detection area uses a nitrocellulose membrane, and the antigen of the detection line is dissolved with a buffer solution PBS. Then, a film dot device is used to draw lines on the nitrocellulose membrane, leaving a distance of 3-8 mm between different antibodies. The nitrocellulose membrane is then placed in an oven to dry for later use. The antigens or other binding substances processed on the membrane are generally immobile. The detection area includes a detection area 206 and a detection result control area 607.

[0112] After completing the preparation of the water absorption area 104, the sample area 101, the marking area 102, and the detection area 103 respectively, they are assembled so that one end of the sample application pad is superimposed on one end of the marking pad, the other end of the marking pad is superimposed on the nitrocellulose membrane, and the nitrocellulose membrane at one end of the control line is superimposed by the water absorption filter paper. In this way, the entire test paper strip is formed and then assembled in the test card, wherein the sample pad extends from the test card for contacting the liquid sample, and the degree window corresponding to the nitrocellulose membrane is used to read the test results.

[0113] All patents and publications cited in this specification are intended to indicate that they are state of the art and that the present invention may be used. All patents and publications cited herein are incorporated by reference in their entirety, as if each publication were specifically incorporated by reference. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations, unless otherwise specified. For example, in each instance, the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The term "a" or "an" herein simply means "one" and does not exclude the inclusion of only one or more. The terms and expressions used herein are intended to be descriptive, not limiting, and are not intended to exclude any equivalent features. However, it is understood that any suitable changes or modifications may be made within the scope of the present invention and the appended claims. It is understood that the embodiments described herein are preferred embodiments and features, and that modifications and variations can be made by persons of ordinary skill in the art based on the spirit of the present invention. Such modifications and variations are considered to be within the scope of the present invention and the scope of the independent and appended claims.

Claims

1. A device for detecting an analyte in a liquid sample, comprising: A test element for testing the presence or quantity of an analyte in a liquid sample, wherein the analyte is one or more of the following: neopterin; homocysteine; dopamine; or microalbuminuria.

2. The device according to claim 1, wherein The test strip is a lateral flow test strip.

3. The device according to claim 2, wherein The test strip can simultaneously test four analytes in a sample, wherein the analytes include the following group: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria.

4. The device according to claim 2, wherein The test strip can simultaneously test four analytes in a sample, wherein the analytes are composed of the following analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; and Microalbuminuria.

5. The device according to any one of claims 1 to 4, wherein: The test strip includes a detection area and a labeling area. The labeling area includes a first receptor that can specifically bind to the analyte and can flow with the liquid sample. The test area includes a second receptor that cannot flow with the liquid.

6. The device according to claim 5, wherein The second receptor competes with the analyte for binding to the first receptor.

7. The device according to claim 6, wherein the first receptor comprises an antibody, an antibody fragment, a polypeptide, or a polypeptide fragment, wherein the antibody is a monoclonal antibody.

8. The device according to claim 5, wherein the second receptor is an analog of the analyte or an antigen of the analyte.

9. The device according to claim 8, wherein the analog of the analyte comprises a linker molecule, and the molecule is selected from one of the following molecules: keyhole hyaluronidase (KLH), bovine globulin (BGG), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg albumin (HEL), ovalbumin (OVA), whale thyroglobulin (SWM), tetanus toxin (TT), methylated bovine serum albumin (mBSA) and rabbit serum albumin (RSA).

10. The device according to claim 5, wherein the first receptor further comprises a labeling substance, and the labeling substance comprises gold particles, latex particles, water-soluble dyes or fluorescent substances.

11. The device according to claim 1, wherein the liquid sample is a saliva, urine or blood sample.

12. The apparatus according to claim 1, comprising: Four test elements, each of which is used to test the presence or quantity of an analyte in a liquid sample, wherein the analytes are the following four analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria.

13. The device according to claim 1, wherein The device includes a test strip that can simultaneously test a liquid sample for four analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria.

14. The device according to claim 1, wherein The device includes two test strips, each of which tests two different analytes, wherein the analytes are selected from a combination of two of the following analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria.

15. The device according to claim 1, wherein The test strip can simultaneously test four analytes in a sample, wherein the analytes include the following group: neopterin; cystine, homocysteine, homocysteine; dopamine; or microalbuminuria.

16. The device according to claim 1, wherein The test strip can simultaneously test four analytes in a sample, wherein the analytes are composed of the following analytes: neopterin; cystine, homocysteine, homocysteine; dopamine; and Microalbuminuria.

17. Use of a biomarker for the preparation of a reagent for predicting the overall health of an individual, wherein the biomarker is selected from one or more combinations of the following markers: neopterin; cystine, homocysteine ​​or homocysteine; dopamine; and microalbuminuria.

18. The use according to claim 17, wherein The individual does not suffer from a particular disease.

19. The use according to claim 18, wherein The specific diseases include one or more of cancer, infectious diseases, cardiovascular diseases, and diabetes.

20. The use according to claim 17, wherein The reagents include immune reagents, and the immune reagents include antibodies that specifically bind to biomarkers, and analogs of biomarkers.

21. The use according to claim 20, wherein The antibody that specifically binds to the biomarker is also coupled with a labeling substance, which includes gold particles, latex particles or colored dyes.

22. The use according to claim 21, wherein The antibody specifically binding to the biomarker is processed on the label pad and can move with the flow of liquid, and the similar substance is fixed on the film and cannot move with the flow of liquid.

23. The use according to claim 22, wherein The film is a nitrocellulose film.

24. A method for testing urine for adulteration, the method comprising: Test the amount of neopterin (NPT) in urine and determine whether the urine is adulterated based on the amount or concentration.

25. The method according to claim 24, wherein when the tested NPT is lower than a preset threshold or is 0, it indicates that the urine is adulterated.

26. The method according to claim 24, wherein when the NPT value is higher than a preset threshold, it indicates that the individual is generally unhealthy, has a risk of developing a specific disease, or that the urine is not adulterated.

27. The method according to claim 24, wherein a test strip is used to test whether urine is adulterated, wherein the test strip has a labeling area and a testing area, wherein the labeling area is treated with an antibody that specifically binds to NPT, and the testing area is immobilized with a substance similar to NPT.

28. The method according to claim 27, wherein when no lines appear in the test area on the test strip, it indicates that the NPT is higher than a preset threshold value, indicating that the urine is not adulterated.

29. The method according to claim 27, wherein when a line appears in the test area on the test strip, it indicates that the NPT is lower than a preset threshold value, indicating that the urine is adulterated.

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