FI13179Y1 - Device for detecting brain damage in a subject - Google Patents

Description

TECHNICAL FIELD The subject matter described herein relates generally to the field of diagnostic devices and detection methods. More particularly, but not exclusively, in some embodiments, the present invention relates to a device for detecting brain injury in a subject. BACKGROUND OF THE INVENTION Traumatic brain injury (TBI) is the leading cause of central nervous system disorder today, and in the United States alone, more than 2.8 million people suffer from TBI each year. According to the CDC, the highest incidence of TBI occurs in children aged 0-4 years, adolescents aged 15-19 years, and adults over 65 years of age. There are a total of more than 69 million TB1 cases worldwide each year. Although TBI affects a large population, it is still undertreated and has remained an unexplored pathological condition.

Traditionally, TBI has been acutely diagnosed and classified by neurological examinations such as the Glasgow Coma Scale (GCS). However, there are several important limitations to using GCS as a diagnostic tool. Recent studies have shown that the use of sedatives prevented accurate GCS assessment during the first 24 hours. Additional challenges to diagnosis are posed by the evolving nature of some brain injuries, which can lead to additional neurological injuries. In addition, neurological responses after TBI may vary over time for reasons unrelated to the injury. Still other challenges are the possible unconsciousness or inability of the injured subject to communicate. N Neurographic imaging techniques, such as X-ray, computed tomography, and magnetic resonance imaging, are used to provide information on the extent and location of the injury and are not affected by the above disadvantages. © 30 However, the sensitivity of computed tomography is low in diffuse brain injuries, and the availability and usefulness of magnetic resonance imaging is limited. Performing magnetic resonance imaging is also very impractical if the targets are physiologically unstable, and can lead to inaccurate diagnoses in war injuries where metal fragments are common. S 35 Mild to moderate TBI represents more than 90% of TBI injuries; this area of injury poses the greatest challenges for accurate acute diagnosis and prediction of outcomes.

Many mild cases of TBI are classified as subclinical brain injuries (SCI). It is widely acknowledged that approaches to diagnosing mild TBI are insufficient, indicating the need to significantly improve the diagnosis and classification of TBI, such as the use of biomarkers to complement functional and imaging-based assessments.

These biomarkers may be altered expression of genes, proteins, lipids, or glycan metabolites, or a combination of these changes following traumatic brain injury, reflecting the development of an initial injury (primary injury) and a secondary injury cascade (secondary injury). In particular, subclinical brain injury or SCI can be diagnosed by biomarker analysis.

Until recently, there have been no approved biomarkers for the diagnosis or prognosis of TBI.

The FDA has approved a marketing authorization for only one blood-based laboratory test to help assess whether or not a patient suspected of having a mild TBI needs a computed tomography scan.

Blood-brain barrier (BBB) may interfere with the assessment of brain biochemical changes in blood biomarkers in mild TBI, although the impaired integrity of BBB observed in severe TBI may increase blood levels of brain-derived proteins.

In addition, some potential biomarkers undergo proteolytic degradation in the blood and their levels may be affected by their clearance from the blood in the liver or kidneys.

As a result, reliable blood biomarkers have been very difficult to identify.

Metabolic biomarkers provide a picture of a patient's biochemical / physiological condition at a single time, which, when measured sequentially and repeatedly, may provide an indication of the progression of the initial disability condition or the treatments required.

The biomarker can be used as a screening indicator / method for early variations in cellular metabolism and tissue recovery associated with injury. = Most diagnostic methods require sample extraction, preparation and testing by healthcare professionals E using specific reagents and equipment and analytical protocols that o require special professional training.

Unfortunately, the ever-increasing burden on health care, the increase in general injuries and illnesses, and the queues for measuring equipment and the significant delays in treatment caused by remote testing are slowing down the uptake of technologies.

Early and timely detection of disease biomarkers can reduce long-term problems for the patient and mortality.

Because of the limited use of traditional diagnostic methods in low-resource environments due to large and expensive measurement equipment, the need for simple and inexpensive scroll test diagnostic equipment for timely and early biomarker diagnosis is immediate, especially in rural and developing countries.

In addition, the conditions on the battlefield are miserable and simple, but important treatment decisions must be made there without access to medical facilities.

Soldiers and military paramedics need durable, easy-to-use, military-level diagnostic equipment.

Significant barriers to rolling test equipment include manufacturing challenges, ease of use restrictions, and regulatory requirements.

Some of these problems have been solved by developing technology and recognizing the importance of side-by-side testing by governments and other regulators.

However, important considerations, including ease of use and accuracy, make side-testing unsuitable for many healthcare facilities and households, and especially for certain medical conditions such as brain injury.

Brief Description of the Invention The invention provides a novel device for detecting brain injury in a subject, said device comprising a detector, the detector comprising at least one glycan-based biomarker-binding / glycan-based biomarker binding reagent that selectively reacts with or binds to a glycan-based biomarker. said binding forming at least one data signal as measurement data, said at least one data signal having an intensity that correlates N to the level of said glycan-based biomarker in said sample, N said level being an indicator of the degree of brain injury to the subject.

In a preferred embodiment, said device comprises a sampling element configured to take a body fluid sample from the target and direct said sample to the detector.

E In a preferred embodiment, said device comprises a pump configured to transfer a sample from the collection point or sampling element to the detector.

In a preferred embodiment, said device comprises an analyzer S 35 configured to receive said at least one data signal from the detector and to generate visible and / or audible information indicative of brain injury in the subject.

In a preferred embodiment, said device comprises a transmitter configured to receive said at least one data signal from the detector and to transmit said at least one data signal to an analyzer.

In a preferred embodiment, said detector is configured to perform multiple measurements over time.

In a preferred embodiment, said device comprises a display configured to receive and display said visible and / or audible information generated by the analyzer as an indicator of a subject's brain injury.

In a preferred embodiment, said device comprises a memory configured to receive from the analyzer, retrievably store in the memory, and retrieve from the memory information indicative of a subject's brain injury.

In a preferred embodiment, said detector comprises an affinity pair member that is a counterpart of said glycan-based biomarker.

In a preferred embodiment, said body fluid is saliva and the device can be attached to or implanted in a tooth.

In a preferred embodiment, said body fluid is urine and said sampling element is a urine collection device.

In a preferred embodiment, said body fluid is a tear fluid and said sampling element is a tear fluid collection wire.

The invention provides a novel helmet comprising a device for detecting brain injury in a subject, said device comprising a detector, the detector comprising at least one glycan-based biomarker-binding / glycan-based biomarker-binding reagent that selectively reacts with a glycan-based biomarker. to body fluid-? in said sample, said reaction or said binding forming at least S30 as measurement data of one data signal, said at least one data signal having an intensity correlating with the level of said glycan-based biomarker in said sample, said level being an indicator of the degree of brain injury to said subject; body fluid is saliva. The invention provides a novel mouthguard comprising a device for detecting brain injury in a subject, said device comprising a detector, the detector comprising at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to the glycan-based biomarker. , said reaction or said binding forming measurement data of at least one data signal, said at least one data signal having an intensity correlating with the level of said glycan-based biomarker in said sample, said level being an indicator of the degree of brain injury to the subject where said body fluid is saliva.

The invention provides a novel adhesive patch comprising a device for detecting brain injury in a subject, said device comprising a detector, the detector comprising at least one glycan-based biomarker-binding / glycan-based biomarker-binding reagent that selectively reacts with or binds to a glycan-based biomarker. said reaction or said binding generating at least one data signal as measurement data, said at least one data signal having an intensity correlating with the level of said glycan-based biomarker in said sample, said level being an indicator of the degree of brain injury to said body fluid;

In a preferred embodiment, said body fluid is urine and said sampling element is a urine collection device.

In a preferred embodiment, said body fluid is a tear fluid and said sampling element is a tear fluid collection wire.

Brief Description of the Drawings Some embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

Referring specifically to the details of the drawings, it is emphasized that the details shown are exemplary and that S is shown to illustrate embodiments of the invention.

Accordingly, the description of the drawings will explain to those skilled in the art how embodiments of the invention may be applied. Fig. 1 illustrates a block diagram of an apparatus for detecting brain injury E in a subject according to an embodiment of the present invention. Figure 2 illustrates an apparatus for detecting brain injury in a subject S in accordance with an embodiment of the present invention.

Fig. 3 is a detailed description of a sampling element S35 of the device and a detector for detecting brain injury in a subject according to an embodiment of the present invention.

Figure 4 illustrates a block diagram of an apparatus for detecting brain injury in a subject in accordance with another embodiment of the present invention.

Figure 5 illustrates a snap-on device for detecting brain injury in a subject in accordance with another embodiment of the present invention.

Figure 6 illustrates a device mounted in a sports helmet for detecting brain injury in a subject according to an embodiment of the present invention.

Figure 7 illustrates a device mounted on a helmet for detecting brain injury in a subject according to an embodiment of the present invention.

Figure 8 illustrates a separate device mounted on a mouthguard for detecting brain injury in a subject according to an embodiment of the present invention.

Figure 9 illustrates a separate device mounted or implanted in a tooth for detecting brain injury in a subject in accordance with one embodiment of the present invention.

Figure 10 illustrates an apparatus arrangement for detecting brain injury in a subject and reporting and displaying its data in accordance with an embodiment of the present invention.

Figure 11 illustrates a measuring device for use in a battlefield for detecting brain injury in a subject according to an embodiment of the present invention.

Figure 12 shows an exemplary test strip for detecting several markers of brain injury a: sample pad; b: nitrocellulose; c: suction pad; d: ECA binding site; e: SNA-I binding site. The arrow shows the O direction.

> DETAILED DESCRIPTION OF THE INVENTION In some embodiments, the present invention relates to a diagnostic device, and more particularly, but not exclusively, to a device for continuous and repetitive detection of brain injury in a subject.

The principles and operation of the present invention can be better understood with reference to the figures and the accompanying explanations.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the application of the invention is not necessarily limited to the details set forth in the following description or examples.

The invention is applicable to other embodiments or can be used or implemented in various ways.

The present inventors have identified the need for a reliable tool for diagnosing brain injury based on a comprehensive view of the dynamics that may be characteristic of a variety of brain injury-related situations.

Measurement of a biomarker at one point after a major injury can be reliable when the level of the biomarker in a healthy, uninjured population is known.

An example of such a case is a situation where the level of the biomarker is measurable and known in healthy individuals or when it is zero or below the detection limit of the test.

Thus, an increase in the level of the biomarker above baseline can be interpreted as a positive result, indicating damage to the brain tissue.

However, this is not always the case because the level of biomarkers is high for other reasons, such as when baseline levels vary between individuals.

The baseline level of a biomarker may vary between individuals depending on variables such as age, weight, sex, physical condition, fat content, physical activity, and so on.

Thus, there are cases where a picture of a continuous trend is required to understand a patient’s true condition.

Repeated measurement may allow the creation of a biomarker level trend line as a function of time and allow monitoring and prediction.

The continued rise of a biomarker may indicate a progressive injury and, for example, indicate to the physician that it is not safe to repatriate the patient.

A decrease in the level of a biomarker may indicate that the person has suffered a brain injury, but his or her condition is improving.

Continuous steady state after the peak may in some cases indicate that the injury has healed and that the patient is safe to use.

The trend curve can represent the level of the biomarker quantitatively or relatively.

LÖ The level of relevant biomarkers may change over time after brain injury.

For example, some biomolecules may be released in a single burst of © 30 cell injury, resulting in their peak concentration shortly after injury E, followed by degradation and metabolism of circulating molecules and a gradual decrease in their concentration in the body fluids.

Alternatively, the initial injury may have caused progressive injuries that result in sustained leakage and increased release of biomarkers over time.

Depending on the type of injury and the biomarker being studied, the outcome may be either the peak of the biomarker (highest concentration).

a few days after the injury, followed by a gradual decrease or, in the worst case, a continuous increase in the concentration of the biomarker or a high level of its concentration. Repeated measurement, which provides a profile of the biomarker concentration as a function of time, provides valuable information about the status and progression of the condition compared to a single measurement at a single time point.

In addition, a number of biomolecules or biomolecules are constantly present in body fluids, the basic level of which varies from individual to individual depending on, for example, health status, medication, medication, previous disease, illness, disease, body composition, age, weight, sex, physical condition, physical condition, exercise, the use of chemicals, etc. Such molecules may also refer to brain tissue injury if their concentration in body fluids changes over time after the injury or if their structure changes after the brain injury. However, a single measurement of such a biomarker is not sufficient because the baseline / concentration is not known in an uninjured individual. However, a change in the concentration of such molecules over time may serve as a true biomarker of brain tissue destruction. Here, the constant trend describes the degree of space.

Thus, one aspect of the present invention is the design of diagnostic test devices that are fast and easy to use in the hands of special teams such as paramedics, first aid personnel, professional athletes, physicians, and soldiers. These can be achieved with the human and other mammalian device of the present invention, which is characterized in that it is non-invasive or invasive, is a small, wearable and portable device, has automatic sample handling, and has a simple interface. It can be obtained quickly, for example, and can be attached to the forehead or the mouthpiece, and it stores the level of the brain injury biomarker during the operation. = Another aspect of the present invention is the ability of a device to record readings and produce a time-dependent trend line to monitor the status and progression of brain tissue injury and to predict the outcome, i.e., the prediction, for medical professionals. This feature allows for more accurate detection of S brain tissue injury by taking into account the variation in the level of the basic bio-N marker between individuals and the different baselines / levels of injury. S 35 This means that, for example, when the concentration of the biomarker under study has dropped to a baseline level measured from an individual before head injury,

brain tissue injury is repaired. If a previous measurement is not available, brain tissue injury can be considered to have improved when the biomarker concentration has decreased and then remained stable for a sufficient period of time.

Another aspect of the present invention is to provide a device for use by hospitals and other medical professionals capable of storing a quantitative result for monitoring and prognostic use. This would allow paramedics and military assistance centers to obtain a more accurate diagnosis in outpatient settings and provide information to nurses in planning and monitoring the rehabilitation process for humans and other mammals. An aspect of the present invention is the ability of the device to provide more accurate diagnosis in about 12 to 24 hours, including invasive sampling, a small, wearable or bedside device, automatic sample processing and a simple interface, and rapid delivery of an analytical result.

Another aspect of the present invention is to design an apparatus for use in military first aid stations. The use of the device in a military environment requires a second level of durability compared to civilian use. The components must be designed for military use, the device must be completely watertight, it must be able to withstand shocks and damage, the measuring unit must be properly protected from dirt, it must be light and it must have its own energy source or battery. The result can be used to make a decision, for example, to remove a soldier from the battlefield.

The present invention provides means for diagnosing multi-injury / multi-organ injury subjects whose brain injuries can easily be overlooked in the presence of other major injuries. In addition, the method allows the detection of brain injury when the patient is unresponsive, such as in coma N or unconscious, because the device can measure the level of the biomarker without the active participation of the patient.

N Tracking means that the biomarker level is stored sequentially? at times and the level of the biomarker is presented as a function of time. The generated curve / trend line and its extrapolations are then used to diagnose and predict the outcome of the brain injury. Biomarker levels can be recorded using any measurements that give the absolute or relative amount / concentration of S biomarker associated with brain injury, preferably a glycan-based biomarker. At best, continuous monitoring can be performed with sensors that detect and record the level of the biomarker up to several times per second (real-time, on-line). The challenge with such a device is continuous sampling. The body fluid to be examined determines the design. As used herein, the term "brain injury" refers to the destruction or degeneration of brain cells due to one or more internal or external factors. Non-limiting examples of brain injuries include traumatic brain injury (TBI), acquired brain injury (ABI), subclinical brain injury (SCI), and neurodegenerative conditions and chronic traumatic encephalopathy (CTE). As used herein, the terms "brain injury" and "brain injury" are interchangeable unless otherwise indicated. In the context of embodiments of the present invention, the term "brain injury" is intended to encompass traumatic brain injury, concussion, axonal rupture damage, axonal lesion damage, non-neuronal membrane rupture, and brain tissue destruction.

As used herein, the term "traumatic brain injury" (TBI) refers to a brain injury caused by external physical trauma or sudden movement of the head, with or without physical contact with or impact with an external object. Non-limiting examples of cases leading to TBI include falls, vehicle collisions, sports collisions and battles, explosions, and pressure waves. The term includes both mild and severe TBI, including closed brain injuries, concussions or contusions, and piercing brain injuries.

As used herein, the term "acquired brain injury" (ABI) refers to brain damage that is not caused by an external brain injury or an inherited condition. ABI can occur after birth as a result of complications, disorder, or congenital disease, or it can result from, for example, stroke, surgery, brain tumor removal, infection, chemical and / or toxic poisoning, hypoxia, ischemia, substance abuse, or a combination thereof.

N The term "brain injury" also refers to subclinical brain injury and anoxic ischemic brain injury. The term "subclinical brain injury" (SCI) refers to a brain injury for which there is no clear clinical evidence of brain injury. The lack of clinical evidence = brain injury when the brain injury actually exists may be due to the degree of injury, the type of injury, the level of consciousness and / or medication, especially sedation and anesthesia. o As used herein, the term "subject" refers to any mammal, including animals and humans. Animals include, but are not limited to, pets, farm animals, working animals, racing animals, show animals and zoo animals. Non-limiting examples of typical human subjects suffering from or susceptible to brain injury, particularly TBlI, include infants, infants, children, and young adults, particularly men, the elderly, athletes, especially boxers, hockey players, soccer players, and American football. , cricket players, rugby players and skateboarders and soldiers. The terms "human subject" and "individual" are interchangeable. Typically, the subject is known to have or suspect a brain injury, such as TBI or ABI or CTE.

As used herein, the term "diagnosis" means the detection of an injury, disease, or disorder, collectively referred to as a medical condition, or the determination of the stage or degree of a medical condition. Usually, the diagnosis of a medical condition is based on an assessment of one or more factors (e.g., biomarkers) and / or symptoms indicative of the medical condition and / or its progression. That is, a diagnosis can be made based on the presence, absence, or amount of a factor that indicates the presence or absence of a medical condition. Each factor or symptom that is considered to be indicative of the diagnosis of a particular medical condition need not be related exclusively to a particular — medical condition, i.e., there may be differential diagnoses that can be inferred from the diagnostic factor or symptom. Similarly, there may be cases where a factor or symptom that indicates a particular medical condition occurs in an individual who does not have that disease.

The term "diagnosis" also encompasses the determination of the therapeutic effect of a drug treatment or the prediction of a response pattern to a drug treatment. Diagnostic methods may be used alone or in combination with other diagnostic methods and / or disease stage assays known in the medical art for a particular medical condition.

In embodiments of the present invention, the term "diagnosis" refers to determining whether a subject has brain damage, such as TBI or ABI N or CTE. The term is also intended to cover cases where the presence of brain damage is not definitively determined, but further diagnostic testing is warranted. N In such embodiments, the method does not in itself determine the presence or absence of brain damage in a subject, but may indicate that additional diagnostic tests are needed or that they would be useful. The methods may therefore be combined with one or more other diagnostic methods to definitively determine the presence or absence of brain damage in a subject. Examples of such other diagnostic methods include, but are not limited to, computed tomography and magnetic resonance imaging, and are well known to those skilled in the art. As used herein, "final assay" or "final diagnosis" refers to confirming the presence or absence of brain damage in a subject.

The final assay or final diagnosis may be the result of any method of the invention, which in some embodiments may include more than one diagnostic test.

As used herein, the term "comparison" refers to the evaluation of how the proportion, level, or cellular localization of one or more biomarkers in a patient-derived sample relates to the proportion, level, or localization of the corresponding one or more biomarkers in a standard or control sample. For example, "comparing" may refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a subject sample is the same, greater, less than, or different from the proportion, level, or localization of the corresponding one or more biomarkers in a standard or control sample.

More specifically, the term may refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a sample of a subject is the same, greater or less than or different from, or not corresponding to, the proportion, level, or cellular localization of a predetermined biomarker. the proportion, level, or cellular localization of levels of a defined biomarker, for example, in a subject with a subclinical brain injury (SCI) who does not have an SCI that responds to SCI treatment that does not respond to a particular SCI treatment. with or without any other disease or condition.

In a specific embodiment, the term "comparison" refers to evaluating whether the level of one or more biomarkers taken from a subject of the present invention is the same, greater or less, or different than other equivalent (or non-equivalent) levels of the same biomarkers in a control sample (e.g., pre- defined levels that correlate with uninfected individuals, standard SCI levels, etc.) The biomarkers and methods presented herein may be used not only for diagnostic purposes but also for prognosis or to predict the outcome of brain damage S or to monitor the survival or response of a subject to brain damage. = The biomarkers and methods presented herein can be used as clinical endpoints in clinical treatment studies of TBI or ABI or CTE, to achieve the outcome of brain damage, or to monitor the survival or response of a subject to brain damage.

S In some embodiments of the present invention, the diagnosis or prognosis of brain damage N may comprise determining the presence or absence of one or more of the present glycan-based biomarkers in a biological sample obtained from a subject for which potential brain damage is to be determined. Multiplexed tests can significantly improve diagnostic accuracy. Multiplexity can be based on measurement with specific lectins or antibodies. In a specific embodiment, the present invention provides methods for determining the risk of developing brain injury in a subject. The percentages, amounts or ranges of biomarkers are characteristic of different risk conditions, such as high, medium or low risk. The risk of developing brain injury is determined by measuring the relevant biomarkers and then either exposing them to a classification algorithm or comparing them to a reference quantity, ie a predetermined level of biomarkers or a spectrum associated with a particular level of risk.

In some embodiments, the present invention provides methods for determining the severity of a brain injury in a subject. Each degree or stage of brain injury is likely to have a typical biomarker level or a relative level of a set of biomarkers (spectrum). The severity of a brain injury is determined by measuring the relevant biomarkers and then either exposing them to a classification algorithm or comparing them to a reference quantity, ie a predetermined level or spectrum of biomarkers associated with a particular degree.

In embodiments of the present invention, the term "biomarker" as used herein refers to a class of biomolecules that are present in the body and increase or decrease in number following a brain injury, or that result from a physiological event, such as a brain injury, or whose structure / integrity changes as a result of the injury. , and which can be detected using a variety of molecular or biochemical methods under development or known. Biomolecules associated with brain injury can act as biomarkers for brain tissue injury. Accordingly, the term "biomarker" refers to a molecule that is detectable from a biological sample obtained from a subject and is an indicator of brain damage to the N target, and biomarkers of particular interest in embodiments of the present invention include glycan-based biomarkers having LOS glycosylation and glycans / sugars. / carbohydrate levels / concentration? There are differences between the S 30 and the healthy control of the individual with brain damage. E As used herein, the term "glycan-based biomarker" means o monosaccharides and polysaccharides, i.e., a polymer containing two or more monosaccharide residues, and a peptide of an alkoconjugate such as N glycopeptides and glycoproteins, glycolipid, or glycolipid. any fragment thereof. Glycan-based biomarkers can comprise either homopolymeric or heteropolymeric monosaccharide residues, and can be either linear or branched, and exist only as carbohydrates or conjugates with any other type of biomolecule. As used herein, the terms "glycan", "polysaccharide" and "carbohydrate" are interchangeable, unless otherwise indicated. In embodiments of the present invention, the term "glycan", as used in the term "glycan-based biomarker", is intended to encompass glycan carbohydrates, short (3 to 5 carbohydrate units), oligo- (5 to 20 units), di- and mono-glycans. , fragmented glycans, low molecular weight glycans, metabolically / enzymatically cleaved glycans and fully or partially hydrolyzed glycans. Glycan may be part of or associated with another molecule, molecular complex, or polymer of any type, but the term "glycan-based biomarker" is valid as long as the detection is substantially based on the glycan portion (s) of the structure.

In the context of the present invention, the body fluid sample (s) are intended to comprise saliva, sputum, saliva, mucus, phlegm, sweat, tear fluid, urine, expiratory air seal (EBC) and bronchoalveolar lavage samples, and any combination. As used herein, the term "saliva" is intended to encompass fluids and secretions available in the mouth, such as sputum, saliva, mucus, phlegm, submandibular gland secretion, parotid gland secretion, and salivary gland secretion.

It is stated herein that the device may be used or a portion of it may be used invasively to obtain a sample by intravenous, subcutaneous immersion — the device or a portion thereof in tissue.

N In some embodiments where the sampling is non-invasive or S-invasive, the samples may be from other body fluids, such as amniotic fluid, aqueous vitreous humor, bile, blood, plasma, T blood serum, breast milk, bronchoalveolar lavage fluid, , affected (earwax) milk fluid, food- E melt, endolymph and perilymph, exhaled air seal (ebc), inflammatory fluids, feces, female ejaculatory fluid, gastric acid, gastric S lymph fluid, mucus, mucus-like fluid and phlegm), pericardial fluid, peritoneal fluid, phlegm, S 35 pleural fluid, wet, bleeding, saliva, sebum (skin oil), semen, whey fluid, foreskin, saliva, sputum, sweat,

joint fluid, tear fluid, urine, vaginal discharge, vitreous humor, and vomiting, and any combination thereof.

In some embodiments of the present invention, the diagnosis or prognosis of brain damage may comprise determining the amount or relative amounts of one or more glycan-based biomarkers relative to, for example, each other, one or more other glycans, and / or a known standard. In some embodiments, the diagnosis or prognosis of brain injury may be based on the proportions of glycan-based biomarkers in various body fluids, such as the saliva / urine ratio or the blood / CSF ratio.

In some embodiments, glycan-based biomarkers can also be detected and / or quantified using lectins. Lectins are a well-known family of carbohydrate-binding proteins, i.e. macromolecules that are highly specific for certain glycans based on their sugar moiety structures and sequences. Lectins can be classified into separate groups according to their carbohydrate specificity, including, but not limited to, fucose-specific, mannose-specific, N-acetylglucosamine-specific, and galactose / N-acetylglucosamine-specific lectins. It should be noted that different sample types may have different profiles of lectin-binding glycan biomarkers. Accordingly, lectins capable of identifying brain-injured subjects can be used either singly or in any combination thereof.

In some embodiments, glycan-based biomarkers can also be detected and / or quantified using galectins, which is the most widely expressed class of lectins in all organisms. Galectins are a family of proteins defined by their binding specificity for β-galactoside sugars, such as N-acetylactosamine (Gal1-3GIcNAc or Gal1-4GIcNAc), which can bind to N proteins by either N-linked or O-linked glycosylation. They are also called AN S-type lectins because their stability and N-binding of carbohydrates are dependent on disulfide bonds. As used herein, the term "lectins" encompasses "ga- = lectins" unless otherwise indicated.

In the context of embodiments of the present invention, the term "lectins" E refers to macromolecules that bind glycans or glycan conjugates. o More broadly, this may include macromolecule binding proteins, S molecule recognition / specific binding proteins, attachment mediators, N complex affinity proteins, and complementary interaction proteins. A non-limiting example of this is an antibody capable of binding carbohydrates.

In the context of TBI, biomarkers are useful for detecting mild TBI cases and other TBI cases where other markers are vague or missing. Biomarkers are also useful for detecting cases in which the injured person cannot react (coma or unconsciousness) or communicate orally.

In some embodiments of the invention, the biomarker is a fragment of a larger molecule, such as glycan, formed by metabolic or enzymatic cleavage or hydrolysis caused by brain tissue damage and cell destruction.

Biomarkers are present in different ways in healthy subjects (normal control or non-brain subject) and in subjects with brain injury and are therefore useful in determining brain injury. In certain embodiments of the present invention, the biomarkers are measured in a sample taken from the subject using the methods described herein, and compared, for example, to predetermined levels of the biomarker and correlated with brain injury. In certain embodiments, the measurement (s) can then be compared to a relevant diagnostic amount (s), cut-off value (s), or multivariate model results that distinguish positive brain injury from negative brain injury. The diagnostic determinant (representative diagnostic representations) represents the measured amount of biomarker (s) above or below which a subject is classified to have a particular brain injury status. For example, if the expression of the biomarker (s) is higher than normal during brain injury, a measured amount higher than the diagnostic cut-off value (s) will result in (higher measured amounts will cause) a brain injury diagnosis. Alternatively, if the expression of the bio-N marker (s) is lower than normal during brain injury, a measured amount at or below the diagnostic cut-off value (s) will produce (lower measured amounts = produce) non-brain injury diagnosis. As is well known in the art, by adjusting the specific diagnostic limit (s) used in the assay (s), the sensitivity or specificity of the diagnostic assay can be increased according to the preference of the diagnostician. In certain embodiments, a particular diagnostic limit can be determined, for example, by measuring the number of biomarkers N in a statistically significant number of samples from different subjects suffering from ai-S 35 and by plotting the limit at the desired levels of specificity and sensitivity.

The device provided herein is configured for use in the repeated detection and assessment of brain integrity damage to brain tissue (neurons, non-neurons, glial cells, astrocytes, microglial cells, and other cells involved in brain function, structure, and integrity).

Brain tissue comprises both brain neurons and non-neurons.

Anatomically and morphologically, said injuries and lesions may be such as torn axons, lesioned axons, damage to the neuronal body, rupture of non-neuronal / neuronal membrane, and destruction of brain tissue.

Continuous monitoring of the glycan-based biomarker is possible - according to some embodiments of the present invention, by continuously or intermittently or repeatedly contacting a sample suspected of containing or expected to contain a glycan-based biomarker, the presence or increased levels of which in the sample suggest brain injury. a signal that correlates with the level of the glycan-based biomarker in the sample.

Detection involves contacting the sample with a detector / sensor, measuring the signal with the detector / sensor, and sending a signal to the processor.

The signal may be an electrical signal detected by an electrode, a magnetic signal detected by a magnetometer and converted to an electrical signal, any signal or signal modulation in the electromagnetic spectrum, including, but not limited to, UV radiation, visible light, infrared or radio frequency radiation. , detected by a photocell, an electromagnetic coil or an oscillator and converted into an electrical signal, a color change signal detected by the color detector and converted into an electrical signal, and the like.

The invention is not limited to one particular signal and signal detection mechanism, but N embodiments of the present invention focus on correlating and continuously monitoring the glycan-based biomarker level S of a sample.

N Continuous monitoring of a glycan-based biomarker can also be? provided by continuous or intermittent signal measurement or by continuous or intermittent signal transmission to the processor. By "intermittent" is meant that the detection operation (contacting, measuring, and transmitting) is initiated and terminated at predetermined times and at a predetermined cycle or frequency.

Thus, according to some embodiments S 35 of the present invention, there is provided an apparatus for repeatedly measuring brain injury at a subject comprising a sample extraction element or a vacuum-forced feed element configured to transport and contact a body fluid sample with a sensor adapted to generate a signal. , the intensity of which correlates with the level of at least one glycan-based biomarker in the sample in contact with the sensor, the level being an indicator of the degree of brain injury, wherein the sensor contains at least one glycan-based biomarker-responsive / glycan-based biomarker-responsive reagent to, react with or bind to the glycan-based biomarker in the sample.

In some embodiments, the sampling element further includes - a sample processing / manufacturing unit for pretreating and filtering or passing the resin and transferring it to the sensor free of debris.

In some embodiments, the glycan-based biomarker-reactive / glycan-based biomarker-binding reagent may be a biomarker-binding reagent that selectively binds the test biomarker by a bioaffinity binding reaction or a ligand binding reaction. In some embodiments, the reagent that reacts with the glycan-based biomarker / binds the glycan-based biomarker is a chemical reagent or series of chemical reagents that generate (s) a signal upon contact with the biomarker or the biomarker-initiated reaction cascade.

According to some embodiments of the present invention, the device provided herein is capable of monitoring brain injury biomarkers in real time. The term "real-time" as used herein refers to the ability of a device to obtain information regarding the level of a glycan-based biomarker in a sample extracted at the exact time the brain injury occurs and becomes apparent. In some embodiments, periodic monitoring is performed for a relatively short period of time, thus making the device such that the input data is processed within fractions of N seconds of the measurement event, with glycan-based = biomarker level information available for feedback almost immediately. Fig. 1 illustrates a block diagram of an apparatus for detecting brain injury E in a subject according to an embodiment of the present invention. The apparatus described comprises a sampling element 11, a detector 12, a pump 13, an analyzer S and a display 16. The sampling element 11 is configured to take a sample of the body fluid of the object S35 and direct said sample to the detector 12. The sampling element 11 may comprise a sample collector, a sample adsorption area or sample inlet. The sampling element 11 may also comprise a sample processing / pretreatment / preparation system by which the sample is pretreated and transferred to the detector 12 after debris removal by filtration and the use of resin.

Detector 12 is configured to receive said extracted sample of a patient's body fluid from sampling element 11. Detector 12 may comprise at least one glycan-based biomarker-binding / glycan-based biomarker binding reagent that selectively reacts with or binds to said glycan-based biomarker, wherein generating at least one data signal as measurement data, said at least one data signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury to the subject. Detector 12 is configured to transmit said at least one data signal to analyzer 15. Detector 12 is configured to perform multiple measurements over time to detect and monitor brain injury in a subject. The terms "detector" and "sensor" may be used interchangeably herein.

In the embodiment shown, the device for detecting brain injury to a subject comprises a pump 13 for transferring a sample from the collection point and the sampling element 11 to the detector 12. The pump 13 can be implemented e.g.

a microfluidic system equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, air flow, peristaltic tube, gravity, vacuum, electroosmotic flow, etc. A low suction vacuum can be advantageously used in the pump 13.

The analyzer 15 is configured to receive said at least data signal from the detector 12. The analyzer 15 is further configured to generate visible and / or audible information indicative of the subject's brain injury.

In the embodiment shown, the device for detecting brain injury to a subject comprises a display 16 for displaying said visible and / or audible information as an indicator of brain injury to the subject, which information is generated by an analyzer 15.

According to another aspect of the present invention, there is provided an S device for diagnosing brain injury that provides trend lines according to at least one N measured physical parameter correlated to the level of at least one glycan-S nip-based biomarker in a sample set.

The device of some embodiments of the present invention may include one or more components or one or more mechanisms for extracting at least one body fluid containing a glycan-based biomarker. This element of the device is also called the sampling element. According to some embodiments of the present invention, the device operates with a non-invasive sampling element, which makes the device a non-invasive diagnostic device for brain injury that can provide continuous or repeated monitoring of the brain injury status of a subject. The non-invasive sampling element may be in the form of a tubular pump element, as an absorbent pad with or without a protective liquid-permeable cover, or as one or more openings in the device that allow body fluid to enter and come into contact with the sensor. In some embodiments, the sampling element or portion of the sampling element is invasive, meaning that the device can be used, for example, intravenously, subcutaneously, or immersed in tissue.

Continuous monitoring of the biomarker in body fluid is most effective when the sensor is immersed in a continuous fluid flow or when the fluid flow is directed to the sensor. This allows the level of the biomarker to be recorded at very short intervals ("real time"). Alternatively, continuous monitoring of the biomarker in body fluid comprises separate, sequential off-line measurements of the biomarker level at specific time intervals and plotting the biomarker level as a function of time for diagnostic and prognostic purposes. Trend line presentation (e.g., drawing) and compilation can be done either on the device itself or on a separate data processing medium.

In some embodiments of the present invention, samples may be obtained by non-invasive means taken from, for example, saliva, urine, tear fluid, or sweat. The sample can be taken actively or passively. Active sampling means that the device or another person can take? sample without the subject's own participation. It allows sampling of a person who is speechless or in a coma or heavily intoxicated. Nevertheless, it should be noted that the use of embodiments of the present invention is not limited to samples extracted by non-invasive methods, which means that the apparatus and methods provided herein can be used to diagnose brain injury by sampling blood, plasma, spinal fluid and the corresponding S 35.

The devices of some embodiments of the present invention may be used by a variety of groups, such as paramedics, first aiders, professional athletes, physicians, and soldiers, whose work requires rapid response and ease of use. Such devices may include one or more of the following features: non-invasive sampling, small size, portability, wearability, automatic sample handling, simple interface, rapid availability of analysis results. Such devices can be incorporated into helmets, protective gear, dental implants, braces, and mouth guards (which can be used, for example, in the military and during sports performances).

The devices of some embodiments of the invention may be suitable for diagnosis by paramedics and military first aid personnel in a treatment facility or hospital having one or more of the following features: accessibility over several hours per day, potentially invasive sampling, small size, wearability, wearability , bedside care, automatic sample processing, simple user interface, fast availability of analysis results.

Figure 2 illustrates an apparatus for detecting brain injury in a subject in accordance with one embodiment of the present invention. The described device 20 comprises a sampling element 21, a detector 22, a cable 23 and an analyzer 25 on the display 26.

The sampling element 21 is configured to extract a body fluid sample from the target and direct said sample to the detector 22. The detector 22 is configured to receive said extracted target body fluid sample from the sampling element 21. The detector 22 may comprise at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent. N which selectively reacts with or binds to the glycan-based biomarker S in the sample, wherein said reaction or said binding forms at least one data signal as measurement data, said at least one da-? the level signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury to the subject. Detector 22 is configured to perform multiple measurements over time to detect and monitor a subject's brain injury. S In the illustrated embodiment, the device 20 for detecting brain injury N of the subject comprises a cable 23 having a data cable 27 connected to a suction cable 28 embedded in the S35 - said suction cable 28 is arranged to transmit suction from the collection point and the sampling element 21 to the detector 12. for example, a microfluidic system equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, air flow, peristaltic tube, gravity, vacuum, electroosmotic flow, etc.

Said data cable 27 is configured to transmit said at least one data signal generated by the detector 22 from the detector 22 to the analyzer 25. The detector 22 is configured to transmit said at least one data signal to the analyzer 25 via said data cable 27. The analyzer 25 is configured to receive said at least one data signal from the detector 22 via said data cable 27.

In the illustrated embodiment, the device 20 for detecting brain injury to a subject comprises an analyzer 25 having a display 26 configured to generate visible and / or audible information as an indicator of brain injury to the subject. Said display 26 of said analyzer 25 is configured to display said visible and / or audible information generated by the analyzer 25 as an indicator of a subject's brain injury.

According to various embodiments of the present invention, the entire device or sampling element or assembly of the sampling element and sensor may be integrated / embedded in a helmet, mouthpiece, protective device, dental implant, mouth guard, brace, garment, implanted on or under the skin, or implanted under the skin. In addition, the device or assembly of the sampling element and sensor may be an external unit / device that comes into direct contact with the organ / body fluid sample or to which the N sample travels by natural secretion or to which it is transferred using the aid O.

According to embodiments of the present invention, the device is an integrated device that continuously detects a glycan-based biomarker and is carried by the user so that it is in direct contact or attached to the body (organ or tissue) of the subject. Examples of such devices include, but are not limited to, an implant in a blood vessel, a device under or on the skin, or an implant in a tooth. Alternatively, the subject uses the device as part of a garment, accessories, or protective gear. Examples of such devices include, but are not limited to, a device that is part of a (recessed / integrated) mouth guard, helmet, or any other protective or wearable equipment.

Figure 3 is a detailed description of a sampling element and detector for detecting brain injury in a subject according to an embodiment of the present invention. The described device comprises a sampling element 21, a detector 22 and a cable 23 embedded in a data cable 27 connected to a suction cable 28.

The sampling element 21 is configured to extract a body fluid sample from the target and direct said sample to the detector 22. The sampling element 21 may comprise a sample collector, a sample adsorption zone or a sample feed port. The sampling element 21 may further comprise one or more prefiltration units for sample processing, pretreatment and preparation. Said prefiltration units may perform one or more of the following functions: filtering the sample to remove cells, debris, precipitates, sediments, flocculations and / or particles based on their size / molecular weight, adjusting, maintaining or altering the composition of the sample carrier, diluting, adjusting the pH; ionic strength, temperature, viscosity, surface tension, modify the biomarker in the sample to make it detectable, modify the biomarker in the sample using chemical functionalization and / or derivatization, labeling, hydrolysis, degradation. The sampling element 21 may also comprise a sample processing / pretreatment / manufacturing system by which the sample is pretreated and transferred to the detector 22.

The detector 22 is configured to receive said extracted target body fluid sample from the sampling element 21. In the present embodiment, the detector 22 is physically connected to the sampling element 21 and in direct contact with the sampling element 21. It enables rapid and reliable detection of the biomarker and allows its real-time detection.

S The sampling element 21 may be a disposable and replaceable sampling element 21 arranged to be disposable. The sampling element = 21 can be arranged to be a replaceable sampling element 21. The sampling element 21 can be replaced by the user and can be attached to the detector 22 by snap-on fastening, snap-on fastening or short-thread fastening.

The suction cable 28 is arranged to transmit suction to transfer the sample S from the sampling point and the sampling element 21 to the detector 22. Said data-N cable 27 is configured to transmit said at least one data signal generated by the detector 22 from the detector 22 to the analyzer 25.

According to embodiments of the present invention, the device is an external device used to detect glycan-based biomarkers specific for brain injury.

The device is preferably hand-held and can be easily transported and used by lay people or professionals, in which body fluid used as a test sample is introduced into the device: by sweeping an organ or tissue with the device's sampling element / contacting the device with the tongue, skin, mucous membranes, eye, meninges or blood vessel, by passing body fluid through the sampling element of the device by natural means such as spitting or directing urine to the sampling element, wetting / immersing the device sampling element in a previously collected body fluid, or transferring - by means of a bed, or by pouring it from the tank / cup by means of a hose and a pump.

In embodiments where the body fluid sample is saliva or other sample from the oral cavity or upper respiratory tract, the sampling element may comprise: a perforated housing that allows saliva to penetrate the sensor, a sampling tube attached to the mouthpiece a sampling tube attached to a dental implant, such as that used in healthcare facilities, attached to the oral mucosa, e.g., with a magnet on one side of the cheek or a cheek clip, or a sampling unit connected to the comb implant.

In embodiments where the body fluid sample is sweat, the sampling element may comprise: a perforated housing or porous membrane that allows sweat to penetrate the sensor, an absorbent patch, and a microfluidic pump or capillary function that absorbs sweat into the device; , which passively absorb sweat inside the skin, from a self-adhesive swab or bandage, or from a swab of a sweat collection helmet. = In embodiments where the body fluid sample is blood, the sampling element may comprise an intravenous cannula that serves as a means for collecting blood E or directing a small amount of blood to the sensor of the device. o In embodiments, the body fluid sample is urine and the sampling element is a urinary catheter, which may or may not be integrated into the sensor.

N During sample transfer, it is also convenient or necessary to separate S 35 - some unwanted parts, such as cells, particles, contaminants, proteins and / or other non-essential components.

This can be done mechanically by forcing the liquid sample to flow through the filters, by chemical binding separation, or by utilizing fluid dynamics. Chemical separation separation is based on the adsorption of unwanted particles, molecules, on pretreated surfaces with well-defined chemistry while the sample flows past the surface. This surface may be the inner wall of the tube or an additional filler such as a filter, small beads, fibers or a porous material. The effect of adsorption can be increased by increasing the specific surface area exposed to the liquid. In addition, the separation may be based on electrical charges, gravity or centrifugal forces.

The sampling element may be shaped as a syringe tip adapter / adapter or stretchable and flexible to fit any external tip used for sampling, or it may be a helical tip, a piercing needle tip, a septum membrane, a butterfly needle adapter, and have any shape designed to be connected to an external source of liquid fluid.

In some embodiments, the purpose of the sampling portion is to provide additional reagents to the solution in addition to adding the sample.

The term "portal" as used in connection with some embodiments of the present invention refers to an element of a device designed to be an inlet and / or outlet for infusing or removing liquids and reagents in solution into and / or out of a sensor. In some embodiments, the device includes more than one portal for the passage of any one or more sample and / or standard analyte solutions and / or indicator formulation reagent and / or wash liquor, and any combination thereof. In such embodiments, the sampling element may have a plurality of inlet and outlet portals or may be connected to the inlet and outlet piping, or the sensor N may be connected to more than one portal.

AN In some embodiments, the device is provided with at least one N portal to which the container is attached. The container may be in the form of a piston / button and cylinder / tube combination (e.g., a syringe) with the button pulled back and the tube being the container. In some embodiments, the container may be pre-filled with E fluid used in the diagnostic process, and may be, for example, a standard analyte solution and / or indicator formulation reagent and / or wash fluid, and S any combination thereof.

Fig. 4 illustrates a block diagram of an apparatus for detecting brain injury in an S35 subject according to another embodiment of the present invention. The apparatus described comprises a sampling element 11, a detector 12, a pump 13, a transmitter 14, an analyzer 15 and a display 16. The sampling element 11 is configured to extract a body fluid sample from the target and direct said sample to the detector 12. The detector 12 is configured to receive said extracted target body fluid sample from the sampling element 11 Detector 12 may comprise at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to a glycan-based biomarker in a sample, wherein said reaction or said binding forms at least one data signal as measurement data, said at least one the data signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury to the subject. Detector 12 is configured to transmit said at least one data signal to transmitter 14. Detector 12 is configured to perform multiple measurements over time to detect and monitor a subject's brain injury. The terms "detector" and "sensor" may be used interchangeably herein.

Transmitter 14 is configured to receive said at least one data signal from detector 12 and transmit said at least one data signal to analyzer 15.

In another illustrated embodiment, the device for detecting brain injury comprises a pump 13 for transferring a sample from the collection site and the sampling element 11 to the detector 12. The pump 13 may be implemented with e.g. strip, airflow, peristaltic tube, gravity, vacuum, electrosmotic flow, etc. In the second embodiment shown, the analyzer 15 is configured to receive said at least one data signal from the detector 12. The analyzer 15 is further configured to generate visible and / or audible information, which is an indicator of a subject's brain injury. The device for detecting brain injury in a subject according to the second embodiment E comprises a display 16 for displaying said visible and / or audible information generated by the analyzer 15 as an indicator of brain injury to the subject. S 35 In an apparatus according to some embodiments of the present invention for detecting brain injury to a subject, the analyzer 15 may include a processor for transcoding said at least one data signal generated by the detector 12 into digitized display information that may be displayed or otherwise transmitted to the subject or caregiver. Alternatively, the processor may digitize a non-electrical signal, e.g., light, for display as digitized information. The processor may also store the data, analyze the data relative to the previously stored data, and / or send the information to another processor, monitor, and / or remote storage device.

Figure 5 illustrates a snap-on device for detecting brain injury in a subject in accordance with another embodiment of the present invention. The illustrated snap-on device 30 includes a detector unit 32, a cable 33, and a transmitter unit 34. The illustrated snap-on device 30 may be used to detect brain injury to an object wearing a helmet, e.g., a sports helmet, hard hat, or military helmet. Said snap-on device 30 can be snap-fitted to the helmet.

In the illustrated embodiment, the snap-on device 30 for detecting brain injury to a subject comprises a detector unit 32, wherein said detector unit 32 comprises a detector. Said detector unit 32 may thus comprise an assembly of a sampling element and a sensor. Alternatively, said detector unit 32 may comprise a detector in which said detector is configured to be in direct contact with the sample, making the sampling element unnecessary.

The detector of the detector unit 32 may comprise at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to a glycan-based biomarker in a sample, wherein said reaction or said binding forms at least one data signal as measurement data. S with a single data signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury to the subject. The detector of the detector unit 32 is configured to transmit said at least one data signal to the transmitter 34 via a cable 33. The detector of the detector unit E 32 is configured to perform multiple measurements over time to detect and monitor brain injury at the target. The transmitter 34 is configured S to receive said at least one data signal from the detector N of the detector unit 32 and to transmit to the analyzer digital information comprising S 35 - said at least one data signal.

In addition, the transmitter 34 may include a pump or suction for transferring the sample from the collection point to the detector unit 32. Said pump or suction can be realized, for example, by a microfluidic system equipped with pumps utilizing, for example, osmotic flow, capillary flow, side-flow strip, air flow, peristaltic tube, gravity, vacuum, electro-osmotic flow, or vacuum tank, etc. Correspondingly, in addition to transmitting said at least one data signal to the transmitter 34, the cable 33 is configured to transmit suction to transfer the sample from the collection point to the detector of the detector unit 32.

Figure 6 illustrates a device mounted in a sports helmet for detecting brain injury in a subject according to an embodiment of the present invention. The sports helmet 36 may be, for example, an American Football helmet 36. The sports helmet 36 described has a device for detecting brain injury at a subject, the device comprising a detector unit 32, a cable 33 and a transmitter unit 34. The sports helmet 36 comprises a detachable device for detecting brain injury without said device comprising a detector anatomically compatible with the mouthpiece, the detector comprising at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to said glycan-based biomarker in a body fluid sample; generating at least one data signal as measurement data, said at least one data signal having an intensity correlating with the level of said glycan-based biomarker in said sample, said level being an indicator the degree of brain injury to the subject, wherein said body fluid is saliva. Fig. 7 illustrates a device mounted in a helmet for detecting brain injury in a subject according to an embodiment of the present invention. The helmet 37 may be, for example, a helmet 37 used in cycling, ice skating, field sports or ice sports. The helmet 37 may also be a military helmet 37. The helmet 37 shown has a device for detecting brain injury in a subject, the device comprising a detector unit 32, a cable 33 and a transmitter unit 34. The helmet 37 described comprises a detachable device for detecting brain injury in a subject without the need to interrupt an ongoing N operation, said device comprising an S 35 detector anatomically compatible with the mouthpiece, the detector comprising at least one glycan-based biomarker-binding / glycan-based biomarker-binding reagent that selectively reacts with or binds to said glycan-based biomarker; as measurement data of at least one data signal, said at least one data signal having an intensity that correlates with the level of said glycan-based biomarker in said sample, said level being an indicator of brain injury to the subject; Figure 8 illustrates a separate device mounted on a mouthpiece for detecting brain injury in a subject according to an embodiment of the present invention.

The ski cover 41 can be, for example, a ski cover 41 used in field sports or martial arts, which measures the biomarker without the need to interrupt ongoing activities.

The described mouth guard 41 has a device 40 for detecting brain injury in a subject, the device comprising a detector-transmitter unit 42. The described mouth guard 41 comprises a dental guard, a jaw guard embedded in a device for detecting brain injury in a subject using the mouth guard, said device comprising a detector comprising at least one a glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to a glycan-based biomarker in a body fluid sample, said reaction or said binding forming at least one data signal as measurement data, said at least one data signal relaying the level of said glycan-based biomarker in said sample, said level being an indicator of the degree of brain injury in the subject at which said body fluid is saliva.

In the illustrated embodiment, the device 40 for detecting brain injury in a subject comprises a detector-transmitter unit 42, wherein said detector-transmitter unit 42 comprises a detector and a transmitter.

In said detector-transmitter unit S 42, said detector is configured to be brought into direct contact with the sample N. The detector of the detector-transmitter unit 42 may comprise at least one reagent that reacts with a glycan-based biomarker / binds to a glycan-based biomarker E that selectively reacts with or binds to a glycan-based biomarker in a body fluid sample, said reaction or S said binding forming at least one as measurement data, N said at least one data signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury to the subject.

The detector of the detector-transmitter unit 42 is configured to transmit said at least one data signal to the transmitter of the detector-transmitter unit 42. The detector of the detector-transmitter unit 42 is configured to perform multiple measurements over time to detect and monitor brain injury at the target. The transmitter of the detector transmitter unit 42 is configured to transmit to the analyzer digital information containing said at least one data signal.

Figure 9 illustrates a separate device mounted or implanted in a tooth for detecting brain injury in a subject in accordance with one embodiment of the present invention. The described device 50 for detecting brain injury in a subject is provided in a tooth 51 comprising a detector-transmitter unit 52. Said detector-transmitter unit 52 may, for example, be attached to or implanted in a tooth or tooth 51.

In the illustrated embodiment, the device 50 for detecting brain injury in a subject comprises a detector-transmitter unit 52, wherein said detector-transmitter unit 52 comprises a detector and a transmitter. In said detector-transmitter unit 52, said detector is configured to be brought into direct contact with the sample.

The detector of the detector-transmitter unit 52 may comprise at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to the glycan-based biomarker in a sample wherein said reaction or said binding forms at least one data signal as measurement data. the at least one data signal having an intensity that correlates with the level of the glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury in the subject. The detector of the detector-transmitter unit 52 is configured to transmit said at least one data signal to the transmitter of the detector-transmitter unit 52. S The detector of the detector-transmitter unit 52 is configured to perform multiple measurements over time to detect and monitor brain injury at the target. The transmitter of the detector unit 52 is configured to transmit to the analyzer digital information containing said at least one data signal. The terms "detector" and "sensor" may be used interchangeably herein.

o In one embodiment of the present invention, said body fluid is sweat and the device is designed as an adhesive patch.

In another embodiment of the present invention, said body fluid is urine and said sampling element is designed as a urine collection device.

In another embodiment of the present invention, said body fluid is a tear fluid and said sampling element is designed as a tear fluid collection wire.

Sensors for detecting biomolecules, also called biosensors, may include biosensing elements and transducers. Bioassay elements specifically recognize and interact with analytes, typically by detecting changes in physicochemical properties (optical, thermal, electrical, and thermodynamic properties) that are generally converted to an electrical signal by a transducer.

The sensor comprises a recognition material, which may be an enzyme, an antibody, a microorganism, a tissue, an organelle, DNA and RNA, a ligand binding receptor, or any similar molecular affinity binding pair, such as lectin and glycan. Alternatively, it is the interaction between complementary substances and the interaction of ligands with 3D complementary 3D structures.

According to some embodiments of the present invention, the sensor may be based on the type of biosensing element used or the transducer method used. Based on the biosensing element, the sensor may be an enzyme sensor, an immunoassay, a nucleic acid probe sensor, or a cell, tissue, or organelle-based sensor. Depending on the converter method, the sensor may be a piezoelectric, optical or electrochemical sensor.

Some embodiments of the present invention contain carbon nanomaterials in the form of particles / dots, tubes / wires, and plates as biosensor substrates. Carbon nanomaterials in the form of nanotubes include single-walled (SWCNT) and multi-walled (MWCNT), graphenes include graphene (GR), graphene oxide (GO) and reduced graphene oxide (rGO). Carbon quantum N point sensor (OD) structures are also included in embodiments of the present invention. Thus, the device further comprises a sensor for converting / converting the presence and level of the biomarker into a quantifiable signal (e.g., an electrical signal) that can be processed in the processing unit for further analysis E. That is, the sensor is an element of the device that is modified to generate a signal whose intensity correlates with the level S of the glycan-based biomarker in the body fluid sample that is contacted with the sensor via the N sampling element. S 35 According to some embodiments of the invention, signal generation and measurements are based on at least one of the following: colorimetric measurements, light scattering measurements, thermal radiation measurements, fluorescence measurements, photoluminescence measurements, light polarization measurements, chemiluminescence measurements, electrochemiluminescence measurements, electrochemiluminescence measurements. In an alternative embodiment of the invention, the sensor receives a sample from the sampling element via a tube or sample transfer tool, such as a pipette.

Information, i.e. diagnostic / analytical results, can be obtained in a matter of seconds or minutes. According to some embodiments of the invention, the sensor and processor are capable of detecting and storing biomarker levels up to several times per second in real time, on-line.

The sensor may include a reaction cell to perform the following functions: providing diffuse reagents that can bind to or complex with a biomarker, where these diffuse reagents can be delivered either from a fluid-filled chamber containing the reagents or from a dried immobilized stain / spot containing the reagent; providing reagents (e.g., printed or spotted) to bind the biomarker or complex formed in a previous sample collection, and / or allowing the result to be detected by performing a reaction or event that allows the presence and amount of the biomarker to be detected and confirmed, and also optionally quantified.

The sensor may be in the form of a microfluidic cell and the biomarker is either labeled or otherwise altered in the cell characteristic signal. The signal is concentration dependent, which allows quantitative measurement of the biomarker.

Some biomarker tracking techniques are based on microfluidics, N which are considered simple, inexpensive, and fast. Microfluidics includes the use of polymer- and paper-based substrates in detection, colorimetric, LO fluorescence, chemiluminescence, electrochemiluminescence (ECL), magnetic, thermal radiation, radio frequency modulation, and electrochemical detection. The device according to some embodiments of the present invention may include microfluidic elements. The role of microfluidics in the development of biosensors has proven to be important in analytical studies of biological and chemical samples. The generic analytical operating model can be divided into three broad categories: the analytical policy on which the S 35 background is based, the analytical method, which is a concept for optimizing the conditions of the chosen analytical policy, and the analytical procedure, which includes all considerations from analytical to analytical. Microfluidic devices inherently have properties that make them advantageous for scroll testing applications (POCT): smaller amounts of test samples and reagents are required for analysis, which can also be processed accurately, test times are drastically reduced due to shortening of diffusion path lengths. ) and different process steps can be integrated and automated on the same platform. According to some embodiments of the present invention, a sample pretreatment and enrichment step may be included.

The microfluidic cell sensor may include a reaction and detection segment in a particular tube volume or surface area, for example, in the form of a layer, film, coated beads, fibers, needles, pore volume, or other three-dimensional matrix. Reactions triggered by the presence of a biomarker and a detectable / measurable outcome, an indicator of brain injury, occur in the reaction zone.

The sensor may comprise a spiral disk or a microfluidic centrifuge plate which may act as both a separation / filtration unit and a reaction cell.

As discussed above, the presence of one or more glycan-based biomarkers in a moving or stationary fluid sample is detected by a specific reaction triggered by the presence of the biomarker. Detection can be either quantitative in terms of mass per volume or relative between successive measurements.

The glycan-based biomarker can be adsorbed to the surface of the matrix functionalized with substances that bind / capture the biomarker from the sample. Such capture agents may be, for example, lectins, antibodies, or a synthetic functional mimetic thereof capable of binding a biomarker. Alternatively, non-specific binding of the biomarker, N, based on hydrophobic interactions, passive adsorption, van der Waals forces and / or hydrogen bonds, can also be used. © 30 In some embodiments, detection is based on label-free E techniques, e.g., measuring the change in electrical signal over the detection range / volume (e.g., electrochemical, conductivity, capacitance), or measuring changes in optical signal based on S sorption, e.g., N spectroscopic, absorbance, transmittance, reflectance and surface-S 35 lasmon resonance). Thus, quantitative or relative qualitative detection can be performed on a gold film by surface plasmon resonance, electrical detection of film capacitance or conductivity, electrochemical detection (PED, oxidation) or detection of optical resonance of a porous (nano) film.

In label-dependent detection, the term "label" refers to a substance used to trace, locate, or visualize a biomarker and make it detectable by visual techniques or any recognition technique.

The label is typically conjugated to a biomarker-specific molecule, such as an antibody or lectin, that specifically binds to a glycan-based biomarker.

The label can be a dye, a fluorescent molecule, an enzyme, a detectable nanoparticle, or a chelate.

These labels have a biomarker binding moiety to ensure specific binding of the biomarker.

The presence of such labels on / by the sensor generates a signal that is proportional to the amount of biomarker.

Fluorescence polarization detection is a label-dependent detection method; however, it is preferred because it does not require a separation or washing step, as detection is based on different levels of polarization of the emitted light depending on whether or not the biomarker is bound to the ligand.

In physical and analytical chemistry, colorimetry is a technique used to determine the concentration of colored compounds in solution.

Colorimetric analysis is a method for determining the concentration of a chemical element or chemical compound in a solution using a color reagent.

Testing the concentration of a solution can be performed by measuring its absorbance at a given wavelength of light.

In some embodiments of the invention, colorimetric techniques are used, but other color-based detection techniques may be used.

In some embodiments, the dye / dye / chromogen forms a color complex or changes color in the presence of a glycan-based biomarker (chemical glycan assays). According to some embodiments of the invention, the detection of glycan-based biomarkers is based on a reaction cascade initiated by the presence of the biomarker.

The reaction may or may not contain enzymes.

In some embodiments, an enzyme specific for the glycan-based bio-E marker initiates a conversion reaction in the presence of the biomarker.

The enzymatic reaction can be coupled to a dye / dye / chromogen that develops or changes color (enzymatic activity). Nor does such a detection mechanism require the immobilization of any element in the indicator formulation.

In another embodiment of the invention, the labeled reagent is a specific binding partner of the analyte. The labeled reagent, analyte (if present), and immobilized unlabeled specific binding reagent work together in a "layer reaction." This results in the labeled reagent binding to the second zone if analyte is present in the sample. These two binding reagents have specificities for different epitopes of the analyte.

The present invention also encompasses indicator formulations that may be based on soluble reagents for detecting a glycan-based biomarker.

For example, in an enzymatic glycan assay, the analyte in the sample (glycan) reacts with an enzyme that catalyzes the degradation of glycan. For example, hexokinase is an enzyme that phosphorylates hexoses (six-carbon sugars) to form hexose phosphate, which in turn can form a color donor with another reagent in the indicator formulation. This reagent is typically added to the formulation via a portal. Accordingly, galactose oxidase is an enzyme that catalyzes the oxidation of D-galactose, and glucose oxidase is an oxidoreductase that catalyzes the oxidation of glucose to hydrogen peroxide and D-alchono-β-lactone; these products of enzymatic reactions can be detected directly or indirectly.

For example, in an embodiment of "direct testing", a glycan-based biomarker reacts with a chromogen, e.g., a reducing sugar causes the chromogen to be reduced, thereby causing color development.

In some embodiments based on soluble and diffusible labels, the glycan-based biomarker reacts with an enzyme that is N specific for the moiety or molecular structure in the glycan-based biomarker. In some cases, this moiety may be galactose and the enzyme is galactose oxidase. In the presence of HRP and a chromogen such as Amplex® Red chromogen = (10H-phenoxazine-3,7-diol-10-acetyl, CAS 119171-73-2), the reaction results in the development of color (absorbance max @ 560 nm).

E In some embodiments based on soluble and diffusible labels, the glycan-based biomarker reacts directly with the chromogen / dye S. In some cases, the glycan biomarker may consist of a reducing sugar which in some cases reacts with 3-methyl-2-benzothiazolinone hydrazone (MBTH) to form a colored adduct.

Another detection technique relevant to some embodiments of the present invention involves inverse tuning of nanoparticle-based fluorescent probes that can be excited by low-energy photon (near-infrared). The signal is read at lower wavelengths, which means that autofluorescence is avoided.

In some embodiments of the present invention, the sensor is an antibody-based sensor, also known as an immunosensor, based on the specificity of the antibody-antigen response to cause a change in the sensor signal. Both polyclonal and monoclonal antibodies can be used in embodiments of the present invention. Antibodies can be immobilized directly on the surface of the sensor by covalent attachment via amino, carboxyl or aldehyde groups. Antibodies can also be attached to the surface of magnetic beads to perform immunomagnetic separation and detection.

In some embodiments, a combination of a lectin / galectin assay and an immunoassay can be used to detect, measure, and / or analyze the present biomarkers in a sample from a subject. For this purpose, both a capture reagent and a detection reagent are required. Said capture reagent may be lectin or galectin, while said detection reagent may be a detectable labeled antibody, or vice versa.

In immunological assays used to detect biomarkers in serum and biological fluids, antibodies bind non-covalently to antigenic epitopes through non-covalent interactions such as hydrogen bonds, electrostatic bonds, van der Waals forces, and hydrophobic interactions. These interactions are reversible and affect the strength of binding between antibodies and antigens. The present invention also contemplates conventional immunoassays, including, for example, layered immunoassays, such as enzyme-mediated immunoassays. egg sorbent test (ELISA) or fluorescence-based immunoassays, as well as other S 30 enzyme immunoassays. Antibodies are typically used in an ELISA. The specific antigenic epitope binds to pAbs immobilized on a solid surface. o Enzyme-linked sAb binds to other epitopes to form a layered s complex. Binding of antigen and pAb is detected using the activity of enzyme N, which converts the substrate to a colored product. S 35 As will be readily appreciated by those skilled in the art, more than one type of lectin / galectin and / or more than one type of

the substance can be used. In addition, several different reactions can be performed simultaneously or sequentially to detect different glycan-based biomarkers in a sample to be analyzed.

As described above, molecules suitable for use in detecting glycan-based biomarkers in a sample for analysis include, but are not limited to, lectins, galectins, antibodies, and competitive small molecules. Said detection molecules can be visualized or otherwise measured using, for example, conjugated color reagents, labels or dyes. Enzyme labels suitable for this purpose are those that, upon addition of a substrate, catalyze a reaction that results in a measurable change in color, luminescence, precipitate formation, or other measurable parameters. Non-limiting examples of such enzyme labels include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Photoluminescent labels, including fluorescent dyes (preferred), lanthanide chelates (for time-resolved fluorescence), and reverse photon excitation labels, can be used to detect said detection molecules.

In addition, detection can be based on bioluminescence and chemiluminescence (such as luciferin-based detection) or electrochemiluminescence (e.g. with ruthenium complexes). Biotin and its derivatives, which allow the binding and detection of labeled avidin or labeled streptavidin, as well as various radioactive isotopes can also be used for detection. Detection can also be performed using beads and particles, including, for example, latex particles, synthetic polymer particles, colloidal metals such as gold and silver particles, (para) magnetic beads, and fluorophore dyed particles.

In some embodiments, the bio-markers of the present invention can be detected by an electrochemical luminescence assay. Electrochemical luminescence detection uses labels that emit light when electrochemically stimulated. The background signals are minimal because the stimulation mechanism (electricity) is disconnected from the signal (light). The labels are stable, non-radioactive and provide a convenient alternative to coupling chemistry. o In addition, the sample can also be analyzed using a passive or active S biochip. Biochips generally comprise solid supports and generally have an N planar surface to which an capture reagent (also called an ad-sorbent or affinity reagent) is attached. Often, the surface of the biochip comprises several sites of known location, each of which is bound to a capture reagent.

Lectin biochips are biochips that have been engineered to capture glycans.

Many lectin biochips have been described in the art.

Label-free electrochemical detection based on a nucleic acid aptamer can be used when binding to the immobilized aptamer can result in current attenuation (signal quenching) as measured by square wave voltammetry.

The advantage of this method is that it uses a reagent-free and label-free electrochemical biosensor based on a DNA aptamer.

The DNA aptamer can detect a specific antigen.

Gold nanoparticles (AUNPs) can serve as surfaces for the immobilization of proteins, but other surfaces with electrochemical properties can also be used to make electrochemical biosensors.

AUNP media can be combined with a multi-enzyme-labeled sAb-magnetic bead bioconjugate and used for ultra-sensitive detection.

Signal amplification can be achieved by using synthesized magnetic bioconjugate particle labels in conjunction with an sAb attached to 1 μm magnetic beads, or by using secondary particle amplification such as silver particle amplification.

In other embodiments of the invention, an organic electrochemical transistor based on poly (3,4-ethylenedioxythiophene) doped with poly (styrene sulfonate) (PEDOT-PSS) and pAb-labeled AuNP may be used.

However, Ab-labeled platinum nanoparticles and a hydrogen formation reaction can also be used in the electrochemiluminescence technique.

Recent advances in surface-assisted Raman spectroscopy (SERS) have led to the design of new nano probes called SERS tags.

SERS tags are combinations of metallic nanoparticles N and highly specific organic Raman reporter molecules.

AN SERS-active nano probes produce strong, characteristic Raman signals and can be used to detect biomarkers using = Raman laser spectrometry or SERS microscopy. © 30 Fluorescence-based detection of biomarker proteins E uses a simple quantification process, easy detection o of large sample volumes, and relatively easy labeling of biomolecules with fluorescent S tags.

Fluorescently labeled sAbs are used to quantify antigens in the samples.

In addition, signal amplification can be achieved by using fluorescent nanobeads instead of fluorescent molecules.

Fluorescent nanobeads are made by capturing fluorescent dyes inside polystyrene microbeads. These fluorescent beads are then conjugated to sAbs, which can form a layered biomolecular complex with the antigen and the immobilized pAb.

Since its first introduction in 1983, protein microchips have been widely accepted and innovated to detect biomarkers. Detection of biomarkers is based on the formation of a biomolecular complex between the immobilized pAb, the corresponding antigen and the sAb labeled with a fluorescent dye. Quantification of the antigen in the sample is performed by analyzing the intensity of the fluorescence signal. A protein microchip is preferred in that it requires a very small amount of sample and reagents. Although a very simple experimental protocol is used in protein microchips, the sensitivity of such a method depends in particular on the chemistry used to immobilize the proteins on the surface.

Semiconductor quantum point chemistry (QD) offers an alternative approach to detecting and quantifying biomarkers. ODs are generally labeled with sAb to detect certain antigens. Due to the photostability, adjustable nature, and sensitivity of fluorescence, ODs are superior to organic dyes for detecting biomarkers. Although the initial size of the OD is in the nanometer order, after bioconjugation, the QDs are found to form larger aggregates.

Enzyme-based biosensors are based on the use of enzymes specific for detectable biomolecules that catalyze the formation of a product that can be quantified by a transducer. Some of the enzymes used in biosensors are oxidases that react with dissolved oxygen to produce hydrogen peroxide.

N Nucleic acid-based biosensors integrate the natural and biomimetic forms of oligo- and polynucleotides and are based on the high sensitivity and selectivity of base-pairing affinity = complementary regions of nucleotide strands. Synthetic oligodeoxyribonucleotides (ODN) can also be used. Labels at the ends, such as thiols, disulfides, amines, or biotin, are added to immobilize the ODN on the surface of the transducer element. Some embodiments of the invention include a transducer element, optical biosensors including surface plasmon resonance (SPR) biosensors, adsorption biosensors, luminescence biosensors, fluorescence biosensors, and optical fiber biosensors. Fluorescence biosensors use fluorescent labels to produce a signal, and the intensity of the fluorescent signal is used to determine the amount detected. SPR is a direct optical detection technique that measures the change in refractive index due to biospecific interactions in the vicinity of a thin metal film surface.

Some embodiments of the invention include piezoelectric biosensors composed of mass-sensitive piezoelectric crystals with excitation electrons. Piezoelectric transducers allow the conversion event to be converted to a measurable signal, such as changes in resonant frequency.

Some embodiments of the invention include a quartz crystal microbalance sensor (QCM) embedded in a liquid medium that provides a direct response signal to characterize the binding event between the sensitive layer connected to the surface transducer and the detectable analyte.

Some embodiments of the invention involve the use of a microprojection, which is a type of mass-sensitive biosensor. This detection principle is based on the change in the mechanical response of the protrusion due to the molecular adsorption on the functionalized surface of the protrusion.

Other biosensors that may be used in some embodiments of the present invention include, but are not limited to, electrochemical biosensors, including amperometric, potentiometric, and impedance biosensors. Potentiometric biosensors use ion-selective electrodes (ISE) and ion-sensitive channel transistors (ISFETs) to measure the change in electrical potential due to the accumulation of ions caused by the enzyme reaction. Amperometric biosensors measure the change in electric current (typically in the region of nanoamperes and microamperes) due to the oxidation / reduction process of the electroactive species, for example by pulsed electrochemical detection (PED). The working electrodes are usually a precious metal or screen-printed layer covered with a bio-N identification element. Potentiometric biosensors measure the potential difference = formed over the ion-selective membrane separating the two solutions at virtually zero current. Impedance sensors are based on a combined measurement of the resistive and capacitive E properties of objects in response to a low-amplitude sinusoidal excitation signal. Impedance detection involves measuring the change in impedance S due to the binding of analytes to receptors immobilized on the surface of the electrodes (antibodies, DNA, proteins, etc.). The change in the conductivity of the S 35 medium is due to the change in the ion concentration of the medium caused by the activity of the labeled enzyme.

The calorimetric sensors that can be used in some embodiments of the invention are based on measuring the heat of a biochemical reaction in a sensor element. These biosensors consist of temperature sensors with immobilized biomolecules. When the analyte comes into contact with the biomolecules, the reaction temperature is measured. The total heat generated or absorbed is proportional to the molar enthalpy and the total number of molecules in the reaction. The change in temperature is proportional to the concentration of the analyte, i.e. the level of the glycan-based biomarker in the sample. In some embodiments of the present invention, the sensor is a unit that can directly measure the biomarker. Such units include HPLC, MS, Raman spectroscopy and FTIR spectroscopy sensors. Suitable methods for use in detecting or analyzing glycan-based biomarkers include, but are not limited to, Biacore methods, mass spectrometry, electrophoresis, nuclear magnetic resonance (NMR), chromatographic methods, or a combination thereof. Gas chromatography (GC) and liquid chromatography (LC) can be used to separate metabolites and determine their size, charge, affinity, and binding capacity. More specifically, the mass spectrometric method can be, for example, LC-MS, LC-MS / MS, MALDI-MS, MALDI-TOF, TANDEM-MS, FTMS, multi-reaction monitoring (MRM), quantitative MRM or label-free binding analysis. Examples of mass spectrometers include a time-of-flight mass spectrometer, a magnetic sector mass spectrometer, a quadrupole filter mass spectrometer, an ion trap mass spectrometer, an ion cyclotron resonance mass spectrometer, an electrostatic sector analyzer, hybrids or combinations thereof, and the like. In another embodiment, the mass spectrometry may be combined with other suitable method (s) known to those skilled in the art. In another embodiment, the mass spectrometric technique is N multi-reaction monitoring (MRM) or quantitative MRM. The electrophoretic = method may be, for example, capillary electrophoresis (CE) or isoelectric phosphor (IEF), and the chromatographic methods may be, for example, HPLC, chromatofocusing or ion exchange chromatography.

o In some additional embodiments, one or more different binding or substitution assays may be used to detect, measure, and / or analyze the present glycan-based bio-markers. For example, a competitive lectin / galectin assay format can be used in which the pre-labeled glycan competes with the glycan in the analyte for limited lectin / galectin binding sites. In some embodiments, the biomarkers of the present invention can be detected and / or measured by immunoassays in either competitive or layered assay format. Those skilled in the art will know how to perform such immunoassays. In addition, suitable antibodies for this purpose are commercially available. Other suitable antibodies can be produced by methods well known in the art. In some embodiments, the sensor may comprise a reaction cell or reaction zone separated from the unit that converts the signal to a readable form. For example, the reaction cell generates a light signal and the light is transferred to the photoelectric cell through a light guide such as an optical fiber.

In some embodiments, the sensor may comprise a reaction cell containing at least two blocks, one with a glycan-based biomarker co-reactive / glycan-based biomarker binding reagent and the other without such a biomarker-specific binding reagent. With such an arrangement, the noise level or the baseline level of the detector can be determined - a signal (electronic or optical) is detected from both blocks and the result of the baseline level is separated or subtracted from the result of the specifically binding block.

Figure 10 illustrates an apparatus arrangement for detecting brain injury in a subject and reporting and displaying its data in accordance with an embodiment of the present invention. The described device arrangement 60 comprises a detector 62, an analyzer 65, a display 66 and a memory 67.

The detector 62 of the apparatus 60 described in the present embodiment for detecting brain injury in a subject may comprise at least one glycan-based biomarker-responsive / glycan-based biomarker S-reagent that selectively reacts with or binds to a glycan-based biomarker in a sample, wherein said reaction or = accumulation forms at least one data signal as measurement data, said at least one data signal having an intensity that correlates with the level of the glycan-based biomarker E in the sample, the level being an indicator of the degree of brain injury to the subject. Detector 62 is configured to transmit digital information comprising said at least one data signal to analyzer 65. Detector 62 is configured to perform multiple measurements over time to detect trend S35 and to detect and monitor brain injury at the target.

The analyzer 65 of said described device arrangement 60 is configured to receive from said detector 62 said digital information comprising said at least one data signal. The analyzer 65 is further configured to generate visible and / or audible information as an indicator of a subject's brain injury. The analyzer 65 is configured to receive from the detector 62 said measurement data from a plurality of measurements performed over time. The analyzer 65 is further configured to analyze measurement data from a plurality of measurements performed over time and to generate visible and / or audible information as an indicator of a subject's brain injury.

The display 66 of the device arrangement 60 for detecting brain injury in the subject is configured to receive said visible and / or audible information generated by the analyzer 15 as an indicator of the subject's brain injury. The display 66 according to the present embodiment is configured to display said visible and / or audible information as an indicator of a subject's brain injury.

The memory 67 of the device arrangement 60 for detecting brain injury in the subject is configured to receive from the analyzer 15, retrievably store in the memory 67 and return from the memory 67 to the analyzer 15, for example, information which may include measurement data, digital information comprising said at least one data - nal and said visible and / or audible information as an indicator of a subject's brain injury. Said information may also include prior health information as an indicator of a subject's brain injury.

To be suitable for military use, a measuring instrument must be strong, N robust, resistant to drops, humidity and extreme temperatures, and capable of testing more than one marker or signal and giving a visual N or even an audible signal in an emergency. ? Figure 11 illustrates an exemplary measuring device 70 for use in an S 30 battlefield for detecting brain injury in a subject, reporting and displaying its data in accordance with an embodiment of the present invention. S The measuring device 70 comprises a sampling element 71 which is connected to the main unit 72 by a cable N or directly by a plug. If the unit is used for multiple patients, at least the pump unit is replaceable. The disposable part prevents cross-contamination between the objects to be tested. The sample is body fluid, preferably saliva.

Detector 73 comprises at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to the glycan-based biomarker in a sample wherein said reaction or said binding forms at least one data signal as measurement data, said at least one data signal correlates with the level of glycan-based biomarker in the sample, the level being an indicator of the degree of brain injury in the subject. The II sensor 73 is configured to perform multiple measurements over time to indicate a trend and to detect and monitor brain injury in a subject. Preferably, the glycan-reactive chemistry and reaction is performed at the other end of the cable. The sample is not fed to the main unit 72, which processes the signal and produces the final result.

The measuring device also comprises a transmitter 74 configured to receive said at least one data signal from the detector 73 and transmit said at least one data signal to the analyzer 75. The analyzer is configured to receive at least one data signal from the detector 72 and generate visible and / or audible information. which is an indicator of a subject's brain injury.

The measuring device 70 comprises a display 76, such as an OLED display, cursor buttons 77 and a measuring button 78, and a bar code reader 79 or an RFI reader 80 for reliable patient identification. According to a preferred embodiment, the patient ID is obtainable from the patient's wristband or tactile plate. N The device also includes a USB port 81 for charging and a rechargeable battery 82, such as an internal fixed rechargeable Li-ion battery 118650. The rechargeable battery is preferably sufficient for continuous use for seven days. = The measuring device is preferably a pocket-sized measuring device. The exemplary dimensions of the main unit, i.e. the device, without the sampling element are: length L 150 E mm, width W 85 mm and thickness D 22 mm. The main unit preferably comprises a durable shell which protects against falling and severe conditions. S The measuring device 70 comprises a user interface. It is started by pressing N on any of the buttons 77, 78. The device then requests a scan of the patient for identification. Patient data can be read, for example, from the RFI ID of the patient's RFI wristband label or the wristband's barcode. If reliable patient identification is not available, the sensor's own ID 74 can be read and the device operates in simple read mode (i.e., no trend or patient history is obtained).

The measurement is started by pressing the measurement button 78. The results and the previous trend are displayed when the analysis is complete. You can scroll through the trend with the cursor buttons 77 (left / right). The device may display previous readings to monitor improvement or deterioration. Patients can also be scrolled with the cursor buttons 77 (up / down) when the device is not in use. The display 76 preferably displays one or more of the following: patient information, date and time, analyte level as a function of function, analyte level as a function of time, analyte concentration decrease or increase with an arrow, and battery charge level.

According to a preferred embodiment, the measuring device is adapted o to take a saliva sample at intervals, o to detect two or more reagents that react with the glycan-based biomarker / that bind selectively bind to the glycan-based biomarker or that to determine the level of two or more glycan-based biomarkers in saliva, o to represent the level of two or more glycan-based biomarkers as a function of time.

Preferably, the measuring device is also adapted to trigger a visual alarm indicating a change in the level of two or more glycan-based biomarkers as a function of time, i.e., worsening of a brain injury in a subject.

In one embodiment of the present invention, there is provided a method N for diagnosing a brain injury in a subject, said method N comprising: contacting the subject with a device 20, 30, 36, 37, 40, 50, 60 or measuring device 70 according to the present invention; 30 o directing a body fluid sample to a detector 12, 22, 62, 72 of said device 20, 30, 36, 37, E 40, 50, 60 or said measuring device 70, wherein said contacting and said control are performed continuously and / or periodically. According to one embodiment of the present invention, measuring said level of an S 35 glycan-based biomarker in said sample of a particular patient is repeated over time to form a quantitative or relative trend line of the biomarker level as a function of time and allowing this patient to be tracked and predicted based on said generated trend line.

Patient-specific measurement results can be stored in memory and retrieved from said memory.

Patient-specific measurement results can be used to understand the patient's condition, i.e., whether the person has had brain damage, whether the condition is healing, whether the injury has healed, and whether it is safe to repatriate the patient.

Healthcare professionals can retrieve and view patient-specific trend lines at the biomarker level (s) as a function of time to monitor the condition and progression of brain tissue injury and to predict the outcome.

In diagnosing a patient's condition, said patient-specific measurement results can be utilized with certain biomolecules, the basal level of which may vary between individuals.

However, the ever-changing trend in the concentration of such molecules over time may serve as a true biomarker of brain tissue destruction.

The continued rise of the biomarker may indicate progression of the injury and indicate to the physician that it is not safe to repatriate the patient.

A decrease in the biomarker level may indicate that the person has suffered a brain injury, but that the condition is improving.

A continuous steady state after the peak may indicate that the injury has healed and that it is safe to repatriate the patient.

An example of a device for detecting brain injury according to the present embodiment is a microfluidic device characterized by a micrometer range in which the surface area / volume ratio increases, where surface forces become dominant and affect the operation of the microfluidic devices.

Mixing liquids in microfluidic devices is challenging due to limited convection and diffusion dependence.

Typically, POCT devices according to embodiments of the present invention are expected to be low cost, portable, user friendly, have lower power requirements, and contain fewer accessories required to operate.

The most power consuming and N most expensive components, which also increase the size of the POCT, are = usually the converter and the pump components with the valve.

Reducing the size of the pump system or even eliminating the need for pumps and valves would be of great benefit in some embodiments of the present invention.

Microfluidic substrates can be classified based on fluid propulsion principles, such as pressure-controlled flow.

Another example of the device S35 according to the present embodiment for detecting brain injury of a subject is a capillary flow device, also known as a test strip. - In a capillary flow device - the sample fluid - moves by capillary forces without added pressure; such a device may be used in some embodiments of the present invention. The flow of the liquid sample is regulated by the moisture and specific size of the microstructured substrate. All necessary chemicals are added to the test strip or test strip. Typically, the test results are optical signals and are usually performed as a color change in the detection range.

Another example of a device for detecting brain injury in a subject is an added side pressure device that can be used in some embodiments of the present invention. These are pressure-driven devices in which external pumps or built-in micropumps are used to transport liquids through the substrate. This medium allows various fluid operations such as mixing, ejection, dosing and separation.

Another example of a device for detecting brain injury in a subject is an added transverse pressure device that can be used in some embodiments of the present invention. The increased transverse pressure is based on the transverse pressure to move or stop the fluid flow. The microchannels and containers are made of soft elastomers that can be compressed or narrowed by external pressure caused by a fluid flowing in adjacent channels, such as compressed air. The substrate material can be made from soft elastomers such as polydimethylsiloxane (PDMS). In some embodiments of the present invention, fluid webs may be used in these substrates.

In some embodiments of the present invention, tear microfluidics involving the formation and processing of micrometer-sized emulsion droplets in a micro-sized device may be used. Droplet microfluidics make the use of traditional micropumps unnecessary. The controlled and rapid mixing of the liquids N in the droplet reactors results in shorter reaction times compared to continuous flow microfluidic devices. Droplet microfluidics can also store all reagents in droplets and eliminate the need for fluid coupling to external reagent tanks. Scaling the droplet microfluidic device to a larger size does not increase the size or complexity of the device. In digital microfluidic devices, unlike droplet microfluidic devices, it is possible to align each droplet separately in the electrode array without microchannels, and the droplets can be moved based on the electrowetting-on-dielectric principle. In some N embodiments, the device is a fully integrated "digital" lab-on-a-chip droplet-S 35 microfluidic device for performing body fluid testing.

Centrifugal pumping of various fluids, including body fluids, aqueous solutions, solvents, and surfactants, can be used in the present invention. Surface acoustic waves (SAW) traveling over the surface of the substrate carrying a drop of liquid in the direction of wave propagation may be used in the apparatus according to some embodiments of the present invention to form, transport, mix and disperse the drop of liquid. The SAW device enables enhanced mixing and centrifugation within individual droplets on a micro scale.

The device presented here is very well suited for use as a pendulum testing device (POCT). Diagnostic techniques can be divided into laboratory-based test equipment and POCT platforms. POCT is a procedure in which samples are analyzed at or near a subject, assuming that test results are available immediately or in a short period of time to assist caregivers in making an immediate diagnosis and clinical procedures. Some of the features of POCT are rapid processing, rapid transmission of results to guide clinical decisions, rapid completion of testing, and follow-up in the same clinical testing environment. Prompt processing of results is important because test results can influence clinical decisions, such as the classification of a patient's urgency of care, referral, and the decision to repatriate a patient. Fast can mean seconds, minutes, or a few hours while the patient is still present. A rapid test result without a mechanism to report the results quickly to care providers and convert the results into a treatment plan is unlikely to have a significant effect on public health outcomes. The POCT can solve this problem by making testing convenient for both patients and care providers by completing the diagnosis process "in the same clinical testing environment" and allowing the treatment plan to be implemented on the same day. Early diagnosis based on POCT N may also provide an opportunity for the caregiver to begin the treatment process earlier and thus increase the chances of preventing or ameliorating the medical condition. z Some embodiments of the present invention encompass a POCT that includes a lab-on-a-chip device, total analysis microsystems, and liquid stunts / side flow strips (LF). These devices may have integrated or separate N reading and display elements. S 35 POCT trays can range in size from a small chip to a desktop system that can be placed in physicians' offices, hospitals, or ambulances, and ideally can only be used with little training. POCT systems should, in principle, require as little user intervention as possible, and should have the required reagents and, preferably, automated processing steps integrated into the system. According to one embodiment of the present invention, there is provided a kit for the continuous diagnosis of a brain injury in a subject that can be diagnosed by a professional or lay person at any location and facility. As will be apparent to those skilled in the art, a lectin chip kit may be used in the invention. In one embodiment, a kit is used for biotinylated samples containing glycans for direct detection on a chip via a Cy3-equivalent dye-conjugated biotin-streptavidin complex. In some embodiments, a layer-type method is used to detect antibodies captured on the chip by antibodies. In some embodiments, a kit for detecting a biomarker comprises HRP protein and fluorescent light can be used to detect the biomarker in body fluid and to indicate the amount of biomarker as a percentage. This can be included in a portable application that indicates the severity of brain damage on a scale that includes, without limitation, the values: none, mild, moderate, and severe. In another embodiment, an analog yes / no answer is obtained. These examples do not exclude other possible embodiments.

In some embodiments, the present invention provides the use of at least one antibody in a kit or device for detecting brain damage, wherein the antibody may be a polyclonal or N monoclonal antibody or fragment thereof of any species, either enzymatically cleaved or S recombinant DNA. and wherein the N antibody recognizes and binds glycan, glycoprotein, peptidoglycan, proteoglycan, glycolipid, protein, small molecule, lectin, or other species of antibody (generally "antigens"). . E All embodiments, details, advantages, and the like of the present invention also apply to the apparatus used in the various S aspects and embodiments of the present invention. Also, all embodiments, details, advantages, and the like of the present methods N apply to the present kit S 35 and vice versa. In particular, one or more compounds, compositions, or reagents described as suitable for carrying out the present methods may be included in the present kit. Likewise, all the points referred to in connection with the packaging of the accessories also apply to the present methods.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.

Non-limiting examples of the advantages of the present glycan-based biomarkers are that they are brain tissue specific, are able to cross the blood-brain barrier into the bloodstream within minutes of injury, and can be detected by a blood or other body fluid bypass test. In addition, the number of biomarkers may either increase or decrease after injury, but still correlate with the severity of the injury. Preferably, the present biomarkers may correlate with the extent of injury, survival, and / or neurological outcome, or may be indicators of the extent of neuronal and glial cell destruction, axonal, and vascular damage. The present biomarkers can significantly increase the current range of brain damage diagnostics.

It is expected that during the period of validity of the utility model evolving from this utility model application, many relevant, substantially continuous brain injury diagnosis devices will be developed and the scope of the term 'saliva-based brain injury device' is intended to cover all such new technologies.

As used herein, the term "about" means + 10%.

The terms "comprising", "comprising", "comprising", "including", "are" and their conjugates mean "including, but not limited to".

The term "consisting of" means "including and not limited to".

N The term "consisting essentially" means that the composition, method S or structure may contain additional ingredients, steps and / or components, but only if the additional components, steps and / or components do not significantly alter the protection requirements? the basic properties and novel properties of the composition, method or structure of the invention. E As used herein, the terms "substantially absent" and / or "substantially absent" in reference to a particular substance refer to a composition in which this substance S is completely absent or which contains less than about 5, 1, 0.5 or 0.1% of this N substance in the composition. according to total weight or total volume. Alternatively, the terms "substantially absent" and / or "substantially absent" in connection with a process, method, property or characteristic refer to a process

a composition, structure or product that is completely devoid of a particular process / method step or a particular property or characteristic, or a process / method in which a particular process / method step is performed in an amount of less than about 5, 1, 0.5 or 0.1% compared to to a particular standard process / method, or to a feature or characteristic characterized in that it has less than about 5, 1, 0.5, or 0.1 percent of the feature or characteristic relative to a particular standard. The term "exemplary" is used herein to mean "which serves as an example, instance, or description." None of the embodiments described as "exemplary" are to be construed as preferred or advantageous over other embodiments and / or to exclude features embodied in other embodiments.

The words "optionally" or "alternatively" are used herein to mean "provided in some embodiments and not provided in other embodiments." Any particular embodiment of the invention may include a number of "optional" features, unless such features are inconsistent with each other.

As used herein, singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" may include multiple compounds, including mixtures thereof.

Through this utility model application, various embodiments of the invention may be presented in a range. It is to be understood that the specification is presented in a range of variations only for convenience and brevity and should not be construed as inflexibly limiting the scope of the invention. Thus, the description of the range should be considered to describe in particular all possible ranges within the range as well as the individual numerical values in that range. For example, a description of a range such as 1 to 6 should be considered to also describe specific ranges within the range, such as 1-3.1-4 1-5, 2-4 2-6, 3-6, etc., and individual numbers in that = range, for example 1, 2, 3, 4, 5, and 6. This is true regardless of the length of the range © 30.

E Whenever a numeric range is shown here, it is intended to contain o any referenced number (fraction or integral) in the range indicated. The terms "vary / vary between" having the first assigned number of the range and the second assigned number, and "vary / varying somewhere" having the range S35 from the first assigned number to the second assigned number,

are used interchangeably herein and are intended to include the first and second assigned numbers and all fractions and integrals therebetween.

As used herein, the terms "process" and "method" refer to ways, means, techniques, and methods for performing a particular task, including, but not limited to, methods, tools, techniques, and procedures in the chemical, materials, mechanical, computational, and digital fields. actors are familiar with or easily able to develop from known practices, tools, techniques and methods in these fields.

As used herein, the term "treating" includes ameliorating, substantially preventing, slowing or reversing the progression of a medical condition, substantially ameliorating the clinical or aesthetic symptoms of a medical condition, or substantially preventing the onset of clinical or aesthetic symptoms of a medical condition.

It is to be understood that certain features of the invention, which are, for clarity, described in connection with separate embodiments, may also be provided in combination in a single embodiment.

Conversely, various aspects of the invention, which are described in connection with one embodiment for brevity, may also be provided separately or as any suitable subcombination, or suitably in any of the other embodiments of the invention described.

Certain features described in connection with the various embodiments should not be construed as essential features of these embodiments unless the functionality of the embodiment requires such elements.

It is to be understood that the above description and the accompanying figures are intended only to illustrate the best mode known to the inventors for making and using the invention.

It will be clear to a person skilled in the art that the concept of the invention can be implemented in different ways.

Thus, the embodiments S of the invention described above may be modified or modified without departing from the scope of the invention, as will be appreciated by those skilled in the art in light of the above teachings.

It is therefore to be understood that the invention and its embodiments are not limited to the examples described above, but may vary within the scope of the protection claims and their equivalents. o For various embodiments and aspects of the present invention, S described above and claimed in the appended claims, N is the experimental and / or calculated support found in the following examples.

S 35 Examples

Reference is made here to the following examples, which, together with the above description, illustrate some embodiments of the invention in a non-limiting manner.

Gold sols for use in the immunoassay can be prepared from commercially available colloidal gold and an antibody preparation.

For example, colloidal gold G 2 O (particle size 20 nm, supplied by Janssen Life Sciences Products) is adjusted to pH 7 with 0.1 M K 2 CO 2 filtered through a 0.22 μm filter and added to 20 ml of clean beaker.

To the gold sol is added 200 μl of antibody prepared in 2 mM borax buffer pH 9 to a concentration of 1 mg / ml and filtered through a 0.22 μm filter, and the mixture is stirred continuously for two minutes.

The pH of the antibody-gold sol mixture is adjusted to 90.1 M with KxCO 2 and 2 ml of 10% (w / v) BSA is added.

The antibody gold is purified in a series of three centrifugation steps at 12,000 g for 30 minutes and 4 ° C, and only the loose part of the pellet is resuspended for later use.

The final pellet is resuspended in 1% (w / v) BSA in a solution of 20 mM Tris, 150 mM NaCl, pH 8.2. Dye sols can be prepared from commercially available hydrophobic dyes such as Foron Blue SRP and Resolin Blue BBLS.

For example, 50 g of dye is dispersed in 1 distilled water by stirring with a magnetic stirrer for 2 to 3 minutes.

Fractionation of the dye dispersion can be performed in a first centrifugation step of 1,500 g for 10 minutes at room temperature to remove larger sol particles as a solid pellet while maintaining the supernatant suspension for further centrifugation.

The suspension is centrifuged at 3,000 g for 10 minutes at room temperature, the supernatant is discarded and the pellet is resuspended in 500 ml of distilled water.

This procedure is repeated three more times so that the final N pellet is resuspended in 100 ml of distilled water. ? The spectra of the dye sols prepared as described above can be measured to give maximum lambda values of about 657 E nm for Foron Blue and 690 nm for Resolin Blue.

The absorbance at a maximum lambda value of 1 cm in a measuring cuvette is used as an arbitrarily selected measure of the dye sol concentration.

N Latex particles (polymer) for immunoassays are commercially available.

They can be based on a variety of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid, and polyacrolein. The monomers used are generally insoluble in water and emulsify in an aqueous surfactant to form monomer moles, which are then induced to polymerize by adding an initiator to the emulsion. This produces substantially spherical polymer particles.

Colored latex particles can be prepared either by incorporating a suitable dye such as anthraquinone (yellow) into the emulsion prior to polymerization or by dyeing the preformed particles. In the latter case, the dye should be dissolved in a water-immiscible solvent such as chloroform, which is then added to the aqueous suspension of latex particles. The particles absorb the non-aqueous solvent and dye and can then be dried. The maximum diameter of such latex particles is preferably less than about 0.5 microns.

Colored latex particles can be sensitized with a protein, and in particular an antibody or lectin, to provide selective binding reagents, as described above. For example, polystyrene beads with a diameter of about 0.3 microns (supplied by Polymer Laboratories) can be sensitized with an anti-glycan-based biomarker antibody by the method described below: dilute 0.5 ml (12.5 mg solids) of the suspension to 1 ml 0, 1 M borate buffer, pH 8.5, in an Eppendorf tube. These particles are washed four times in borate buffer, each wash consisting of centrifugation at 13,000 rpm for 3 minutes in an MSE microcentrifuge at room temperature. The final pellet is resuspended in 1 ml of borate buffer, mixed with 300 μg of anti-glycan-based biomarker antibody, and the suspension is vortexed for 16-20 hours at room temperature. The antibody-latex suspension N is centrifuged for 5 minutes at 13,000 rpm, the supernatant is discarded and the pellet is resuspended in 1.5 ml of borate buffer containing 0.5 mg of N bovine serum albumin. After rotating the suspension for 30 minutes at room temperature, the suspension is washed three times in 5 x 30 mg / ml BSA in phosphate buffered saline, pH 7.2, centrifuging at 13,000 rpm for 5 minutes. The pellet is resuspended in 5 mg / ml BSA / 5% (w / v) glycerol in phosphate buffered saline, pH 7 2, S and stored at 4 ° C until use. N The protein can be coupled to a dye sol in a process involving S 35 passive adsorption. The protein may be, for example, a lectin or an antibody preparation such as an anti-glycan-based biomarker antibody prepared in phosphate buffered saline, pH 7.4, at a concentration of 2 mg / ml. A reaction mixture is prepared containing 100 ul of antibody solution, 2 ml of dye salt, 2 ml of 0.1 M phosphate buffer, pH 5.8, and 15.9 ml of distilled water. After gentle stirring of this solution, the preparation is left at room temperature for 15 minutes. Excess binding sites can be blocked by the addition of, for example, bovine serum albumin: 4 ml of 150 mg / ml BSA in 5 mM NaCl, pH 7.4 is added to the reaction mixture, and after 15 minutes incubation at room temperature, the solution is centrifuged at 3,000 g for 10 minutes and the pellet is resuspended in 10 ml of 0.25% (w / v) dextran / 0.5% (w / v) lactose / 0.04 M phosphate buffer. This antibody-dye sol conjugate is best stored lyophilized.

In an exemplary embodiment of the present invention, a model for testing fetuin and asialofetuin is provided. Fetuin is a rich glycoprotein in fetal serum and asialofetuin is its acylated form. Lectins or lectin that bind selectively to the glycan moiety of fetuin or asialofetuin are permanently immobilized on a solid matrix. The fetuin or asialofetuin in solution is contacted with the lectin and then a binding reaction takes place. The reaction chamber is then washed and the labeled conjugate is added. The conjugate binds to fetuin or asialofetuin, which is bound to the surface in the previous step.

Alternatively, fetuin or asialofetuin is first contacted with a labeled conjugate to form a complex. The complex is then contacted with immobilized lectin (s). The conjugate comprises a fetuin-specific or asialofetuin-specific antibody coupled to a detectable label. The detectable label is N one of those shown in the text, preferably a colloidal / particulate substance, which enables N visual detection.

N Evidence that a lectin-based glycan detection method = can detect multiple markers simultaneously © 30 Below is an example of simultaneous detection of multiple biomarkers in a single assay. The lectins used in the example are listed in Table 1 and the test strip is shown in Figure 12. The experiments were performed as follows: S Strip preparation and assembly: Sample pads were blocked and after strips the strips were assembled: nitrocellulose membrane ) and an absorption pad (lower part of the strip)

was fixed to the base plate with 2 mm overlaps and the wide base plate was cut into 5 mm wide individual test strips. Functionalization of the reaction zone: The two capturing lectins were pipetted separately onto the nitrocellulose membrane (middle of the strip) and dried to permanently adsorb the lectins onto the membrane. Sample Analysis: A sample from a TBI patient was pipetted onto a sample pad and allowed to pass through the membrane. A second lectin (tracer) conjugated to the gold particles was added and migrated. The strips were washed by dipping the end of the conjugate pad in a well containing wash buffer and rinsing excess and unbound gold conjugate out of the reaction area with migration buffer. Two dots appeared in the reaction zone, indicating that two different glycans were present in the sample and could be detected in a single assay. The lectins used in the example are listed in Table 1. Table 1.

Lectin Full name Carbohydrate Function - example- abbreviation specificity in the cat ECA Erythrina Capture cristagalli GalB4GIcNAc nigra | nigra | tracer N The specificities of lectins are clearly different, the most significant difference being the specificity of O SNA-I for sialic acid, whereas ECA does not have this specificity; sialic acid actually inhibits ECA binding, and ECA binds primarily galactic = 20-toose glucosamine. © Confirmation by mass spectrometry analysis: Glycans were isolated and E: purified from saliva samples from TBI patients and healthy controls. The released ® N-glycans were analyzed by mass spectrometry and the m / z values of each peak in the spectrum were converted to the glycan composition by fitting the peak to the glycan mass spectrometer databases. The relative abundance of each glycan in Sample S was calculated from the peak height (area) and the relative abundance was compared between TBI patients and healthy controls.

At least two significant types were found to be predominant in TBI samples compared to healthy samples: 1) hybrid type N-glycans with an x-galactose-NANA end, and 2) acidic N-glycan with an x-glucosamine galactose end. These results are consistent with the lectin specifics of the side-flow example.

Summary: Mass spectrometry showed the presence of two glycan species in the sample, and the side flow example showed the ability to detect them simultaneously.

Although the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will occur. Accordingly, the invention is intended to cover all such alternatives, modifications, and variations as fall within the spirit and broad scope of the appended claims.

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Claims (18)

Security requirements A device for detecting brain injury in a subject, said device (20), (30), (36), (37), (40), (50), (60) comprising a detector (12), (22), (62), said detector comprising at least one glycan-based biomarker-reactive / glycan-based biomarker-binding reagent that selectively reacts with or binds to the glycan-based biomarker in a body fluid sample, said reaction or said binding constituting at least one data signal as measuring data, said at least one correlating the level of said glycan-based biomarker in said sample, said level being an indicator of the degree of brain injury in a subject, characterized in that said detector (12), (22), (62) is configured to perform multiple measurements over time. Apparatus according to claim 1, wherein said apparatus (20), (30), (36), (37), (40), (50), (60) comprises: - a sampling element (11), (21) which is configured to extract a body fluid sample from the target and direct said sample to the detector (12), (22), (62). Apparatus according to claim 1 or 2, wherein said apparatus (20), (30), (36), (37), (40), (50), (60) comprises: - a pump (13) configured transfer the sample from the collection point or sampling element (11), (21) to the detector (12), (22), (62). Apparatus according to any one of claims 1 to 3, wherein said apparatus (20), (30), (36), (37), (40), (50), (60) comprises: - an analyzer (15), (25) ), (65) configured to receive said at least one data signal from the detector (12), (22), (62) and to generate visible and / or audible information indicative of N subject brain injury. OF The device of claim 4, wherein said device (20), (30), = (36), (37), (40), (50), (60) comprises: © 30 - a transmitter (14) that is configured to receive said at least one data signal from the detector (12), (22), (62) and to provide said at least one data signal to the analyzer (15), (25), (65). S The device according to claim 4 or 5, wherein said device (20), N (30), (36), (37), (40), (50), (60) comprises: S 35 - a display (16), (26), (66) configured to receive and display said visible and / or audible information generated by the analyzer (15), (25), (65) as an indicator of subject brain injury. Apparatus according to any one of claims 4 to 6, wherein said apparatus comprises: - a memory (67) configured to receive from the analyzer (15), retrievably store in the memory (67) and retrieve information from the memory (67) indicative of brain injury to the subject. The device of any one of claims 1 to 1, wherein said detector (12), (22), (62) comprises a member of an affinity pair that is a counterpart of said glycan-based biomarker. The device of any one of claims 1 to 8, wherein said body fluid is saliva and the device (40), (50) is attachable to or implantable in the tooth / tooth (51). The device of any one of claims 1 to 9, wherein said body fluid is urine and said sampling element is a urine collection device. The device of any one of claims 1 to 9, wherein said body fluid is a tear fluid and said sampling element is a tear fluid collection wire. A helmet (36), (37) comprising a device according to any one of claims 1 to 9, wherein said body fluid is saliva. A mouth guard (41) comprising a device according to any one of claims 1 to 9, wherein said body fluid is saliva. An adhesive patch comprising a device according to any one of claims 1 to 9, wherein said body fluid is sweat. OF A measuring device (70) for use in a battlefield, the measuring device comprising a N 0 detector (73) comprising at least one glycan-based biomarker / glycan-based biomarker-binding reagent that selectively reacts with or binds to a glycan-based biomarker. in a body fluid sample, said reaction or said binding forming measurement data of at least one data signal, said at least one data signal having an intensity correlating with the level of said glycan-based biomarker in said sample, said level being S 35 - an indicator of the target o a sampling element (71) configured to extract a body fluid sample from the target and direct said sample to a detector (73); o a transmitter (74) configured to receive said at least one data signal from the detector (73) and transmit said at least one data signal; a level signal to the analyzer (75), the analyzer configured to receive said at least one data signal from the detector (73) and generate visible and / or audible information indicative of the subject's brain injury, and a display (76) configured to receive and display the analyzer said visible and / or audible information as an indicator of a subject's brain injury. The measuring device of claim 15, wherein the body fluid is saliva. The measuring device of claim 16, wherein the measuring device is adapted to o sample saliva at intervals, o to detect two or more glycan-based biomarker-binding / glycan-based biomarker-binding reagents that selectively react with the two glycan-based biomarkers, or or the level of two or more glycan-based biomarkers in saliva, and to represent the level of two or more glycan-based biomarkers as a function of time. The measuring device of claim 17, wherein the measuring device N is adapted to trigger a visual alarm indicating a change in the level of two S or more glycan-based biomarkers as a function of time. OF LÖ <Q o O I a a 00 <[<] + OF OF O N 5