TECHNICAL FIELD

The subject matter disclosed herein generally relates to the field of diagnostic devices and methods for detection. More particularly, but not exclusively the present invention, in some embodiments thereof, relates to a device for detecting of brain injury in a subject, and a method for diagnosing brain injury using said device.

BACKGROUND Traumatic brain injury (TBI) is the leading cause of central nervous system impairment in these days, with more than 2.8 million individuals suffering annually from TBI in the US alone. According to the CDC, the highest incidence of TBI occurs among children 0-4 years old, adolescents 15-19 years old, and adults over 65 years of age. In total, globally more than 69 million TBI cases occur annually. Despite the broad range of the population affected, TBI is still under-served and remains an unexplored pathological condition.

Traditionally, TBI has been acutely diagnosed and classified by neurological examinations, such as Glasgow Coma Scale (GCS). However, the use of the GCS as a diagnostic tool is subject to a number of important limitations. Recent research has provided evidence that the use of sedative drugs precluded accurate GCS assessment during the first 24 hours. Further challenges to diagnosis are presented by the evolving nature of some brain lesions, which can lead to further neurological impairment. In addition, neurological responses after TBI can vary over time for reasons unrelated to the injury. Still further challenges include the trauma subject's possible unconsciousness or inability to communicate.

Neuroimaging techniques, such as x-ray, CT scanning and MRI, are used to provide information on injury magnitude and location and are not influenced by the aforementioned disadvantages. However, CT scanning has low sensitivity to diffuse brain damage, and availability and utility of MRI is limited. MRI is also very impractical to perform if subjects are physiologically unstable and can lead to inaccurate diagnoses in military injuries in which metal fragments are common.

Mild and moderate TBI represent more than 90 % of TBI injuries; this injury range represents the greatest challenges to accurate acute diagnosis and outcome prediction. Many cases of mild TBI are classified as subclinical brain injury (SCI). The widespread recognition of inadequate approaches to diagnose mild TBI suggests the need for significant improvement in the diagnosis and classification of TBI, such as the use of biomarkers to supplement functional and imaging-based assessments. These biomarkers can be altered gene expression, protein, lipid or glycan metabolites, or a combination of these changes after traumatic brain injury, reflecting the initial insult (the primary injury) and the evolution of a cascade of secondary damage (the secondary injury). In particular, subclinical brain injury status or SCI could be diagnosed with a biomarker analysis.

Until recently, there have been no approved biomarkers for the diagnosis or prognosis of TBI. FDA has approved marketing permission for one blood-based laboratory test only which helps to assess whether a patient with suspected mild TBI needs CT scan or not. The blood-brain barrier (BBB) may hinder the assessment of biochemical changes in the brain by use of blood biomarkers in mild TBI, although impaired BBB integrity, as seen in severe TBI, can increase the levels of brain-derived proteins in the blood. Moreover, some potential biomarkers undergo proteolytic degradation in the blood, and their levels might be affected by clearance from blood via the liver or kidney. Consequently, reliable blood biomarkers have been extremely difficult to identify.

Metabolic biomarkers provide a picture of the biochemical/physiological condition of a patient at a single point in time, which, when taken sequentially and continuously, may provide insight into the progression of an initial injury state or needed treatments. Biomarker may be used as a screening indicator/method for early injury-associated fluctuations in cellular metabolism and tissue recovery.

Most diagnostic methodologies call for sample extraction, preparation and assaying, which is carried out by healthcare specialists using special reagents and equipment, as well as analytical protocols that require specific professional training. Unfortunately, the ever-growing strain on the healthcare system, the increased prevalence of common injuries and diseases, and the substantial delay in treatment caused by instrument-access queues and remote testing, stands in the way of utilizing technologies.

Early and timely detection of disease biomarkers can decrease long-term issue for the patient and mortality. Because conventional diagnostic methods have limited application in low-resource settings due to the use of bulky and expensive instrumentation, simple and low-cost point-of-care diagnostic devices for timely and early biomarker diagnosis is the need of the hour, especially in rural areas and developing nations. Additionally, battlefield settings are abject and crude, however, they need to make important medical decisions without access to medical facilities. Soldier and army paramedics need robust, easy to use, military grade diagnostic devices. Historic obstacles to point-of-care devices include manufacture challenges, ease-of-use limitations, and government regulations. Some of these obstacles have been reduced through advances in technology and recognition by governments and other regulatory bodies of the importance of point-of-care testing. However, important considerations, including ease-of-use and accuracy, still render point-of-care tests unsuitable for many healthcare facilities and domestic settings, and more so for particular medical conditions, such as brain injury.

BRIEF DESCRIPTION OF THE INVENTION

It is brought forward a new device for detecting of brain injury in a subject, said device comprising a detector, which detector comprises at least one glycan- based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject.

In a preferred embodiment, said device comprises a sampling element being configured to extract a sample of a bodily fluid from a subject and direct said sample to the detector.

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

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

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

In a preferred embodiment, said device comprises a display configured to receive and to display said displayable and/or audible information indicative of brain injury in the subject generated by the analyzer.

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

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

In a preferred embodiment, said bodily fluid is saliva, and the device is attachable to or implantable on/in a tooth. In a preferred embodiment, said bodily fluid is urine, and said sampling element is realized as a urine collection device.

In a preferred embodiment, said bodily fluid is tears, and said sampling element is realized as a tear collection wick.

It is brought forward a new helmet comprising a device for detecting of brain injury in a subject, said device comprising a detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is saliva.

It is brought forward a new mouth guard comprising a device for detecting of brain injury in a subject, said device comprising a detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is saliva.

It is brought forward a new adhesive patch comprising a device for detecting of brain injury in a subject, said device comprising a detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is sweat. In a preferred embodiment, said bodily fluid is urine, and said sampling element is realized as a urine collection device.

In a preferred embodiment, said bodily fluid is tears, and said sampling element is realized as a tear collection wick.

It is brought forward a new method for detecting brain injury in a subject, which method comprises: o contacting the subject with a device comprising a detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, o directing a sample of a bodily fluid to a detector of said device, o wherein said contacting and said directing is affected continuously and/or periodically.

In a preferred embodiment, said method comprises: o creating a trend line of said at least one glycan-based biomarker level in said sample as a function of time based on measurement data of said device, and o utilizing said created trend line for evaluating of brain injury in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. FIG. 1 illustrates a block diagram of a device for detecting of brain injury in a subject according to one embodiment of the present invention.

FIG. 2 illustrates a device for detecting of brain injury in a subject according to one embodiment of the present invention. FIG. 3 illustrates a detailed illustration of a sampling element and a detector of a device for detecting of brain injury in a subject according to one embodiment of the present invention.

FIG. 4 illustrates a block diagram of a device for detecting of brain injury in a subject according to another embodiment of the present invention. FIG. 5 illustrates a snap-on device for detecting of brain injury in a subject according to another embodiment of the present invention.

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

FIG. 7 illustrates a device for detecting of brain injury in a subject according to an embodiment of the present invention arranged in a protective helmet.

FIG. 8 illustrates a standalone device for detecting of brain injury in a subject according to an embodiment of the present invention arranged in a mouth guard.

FIG. 9 illustrates a standalone device for detecting of brain injury in a subject according to an embodiment of the present invention arranged or implanted in a tooth.

FIG. 10 illustrates a device arrangement for detecting and reporting and presenting data of brain injury in a subject according to an embodiment of the present invention.

FIG. 11 illustrates an instrument for use in battlefield for detecting of brain injury in a subject according to one embodiment of the present invention.

FIG. 12 illustrates an exemplary flow chart for evaluating a brain injury in a subject using the device of the present invention. Fig. 13 illustrates an exemplary test strip for detection of multiple markers from brain damage a: sample pad; b: nitrocellulose; c: wicking pad; d: ECA capture spot; e: SNA-l-capture spot. The arrow shows the direction of migration.

DETAILED DESCRIPTION

The present invention, in some embodiments thereof, relates to a diagnostic device and method for detecting head injury, and more particularly, but not exclusively, to a device for continuous and repeated detecting of brain injury in a subject.

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

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The present inventors have recognized the need for a reliable tool for diagnosing brain injury based on comprehensive view of the dynamics which may characterize various situations relating to brain injury.

A one-point measurement of the biomarker after a head injury may be reliable when the level of the biomarker in healthy, uninjured population, is known. An example of such a case is when in healthy individuals, the biomarker level is measurable and known, or it is zero, or when it is below the detection limit of the test. Thus, a rise in the level of the biomarker above a basal level can be interpreted as a positive result indicating an injury to the brain tissue. However, this is not always the case, since biomarkers level can be high for other reasons, for example when the basal level is varying among individuals. The basal level of a biomarker can vary among individuals depending on the variables such as age, weight, sex, physical condition, fat contents, physical activity, etc. Thus, there are cases when it is required to have a view of a continuous trend in order to comprehend the actual condition of the patient. Repeated measurement may allow creating a trend line of a biomarker level as a function of time and enable monitoring and making a prognosis. A continuous rise of the biomarker may indicate a progressing injury and may, for example, indicate to the doctor that it is not safe to release the patient. A decrease of the biomarker level may indicate that a person has suffered brain trauma but the condition is getting better. A continuous steady level after a peak may, in some cases, indicate that the injury is healed, and that it is safe to release the patient. The trend curve may present the biomarker level quantitatively or relatively.

The level of relevant biomarkers may change over time following a brain injury. For example, some biomolecules can be released in a single burst upon the cellular injury, resulting in a peak soon after the injury, and then followed by degradation and metabolism of the molecules in the circulation and gradual decrease of their concentration in the body fluids. Alternatively, the original injury may have set off progressive injuries resulting in continuous leakage and increasing release of biomarkers over time. Depending on the type of injury and the biomarker of interest, the outcome can be either peaking (highest concentration) of the biomarker in some days after the injury followed by gradual decrease, or in worse case, continuous increase of biomarker concentration, or a high-concentration level plateau of the biomarker. Repeated measurement, which gives a profile of the concentration of the biomarker as a function of time, provides valuable information of the status and progression of the condition compared to separate one time point measurement.

In addition, there are several biomolecules that are constantly present in the body fluids, or such biomolecules the basal level of which vary between individuals depending on, for example, health status, medication, drugs, prior sickness, illness, disease, composition of the body, age, weight, sex, physical condition, fat contents, recent exercise, use of chemicals, etc. Such molecules can also indicate brain tissue damage provided that their concentration in the body fluids changes over time after injury, or if their structure is modified following brain injury. A single measurement of such biomarker is not adequate, however, because of the unknown basal level/concentration in non-injured individual. However, a change in concentration of such molecules over time can serve as correct biomarker of brain tissue destruction. Here the continuous trend describes the state of the condition.

Hence, one of the provisions of the present invention is the design of diagnostic test devices and methods for the use in the hands of special groups such as paramedics, first responders, professional athletes, doctors, and soldiers are the fast response and ease to use. These can be achieved by a device for humans and other mammals according to the present invention, which device is characterized by or with: non-invasive or invasive sample collection; small, wearable and portable device; automatic sample processing and simple user interface; quick delivery of the analysis result; and which device can be incorporated e.g. in helmet or mouth guard and which records the brain injury biomarker level during a game.

Another provision of the present invention is the ability of the device to record the readings and produce a time-related trend line for monitoring the status and progress of the brain tissue damage, and to predict the outcome by medical professionals, i.e. to reach a prognosis. This feature enables the brain tissue damage to be detected more accurately, by taking into account the variability of the basal biomarker level between individuals and the different starting points/levels of damage. This means that, for example, when the concentration of the biomarker of interest has declined to the basic level measured from the individual before the hit to head, the brain tissue damage has resolved. If a prior measurement result is not available, the brain tissue damage can be considered being resolved once the concentration of the biomarker has declined followed by staying stable for an adequate period of time.

Another provision of the present invention is the design goal for the use of device in hospitals and other medical professionals is the ability to safe the quantitative result for monitoring and prognostic use. This would enable more accurate diagnosis in the point-of-care conditions also by paramedics and military first aid stations and give an information for caregivers to design and monitor rehabilitation process for humans and other mammals. The provision of the present invention is the ability of the device to enable more accurate diagnosis on-time in about 12-24 hours, also invasive sample collection, with small, wearable and portable device, or with a bedside device, to enable automatic sample processing and simple user interface, and to enable quick delivery of the analysis result.

Another provision of the present invention is the design goal of the use of the device in military first aid settings. Using the device in the field of a military setting requires another level of robustness compared to civilian use. The components need to be military grade, the device needs to be completely water proof, it needs to be able to take hits and damage, the measurement unit needs to be properly guarded from dirt, it needs to be light as well as have its own energy source or battery Result can be used to make decision, for example, about the extraction of the soldier from the battlefield.

The present invention provides means for diagnosing multi-trauma/poly-trauma subjects whose brain injuries may be easily overlooked in the presence of other more prominent injuries. In addition, the method enables to detect brain injury when the patient is unresponsive like in coma or loss of consciousness when the device can measure the level of biomarker without the patient’s active involvement.

Monitoring means that the biomarker level is recorded at successive time points and the level of biomarker is presented as a function of time. The composed curve/trend-line and its extrapolations are then used to diagnose and predict the outcome of the brain injury. The biomarker levels can be recorded using any measurements that give an absolute or relative quantity/concentration of the brain injury related biomarker, preferably a glycan-based biomarker. At its best, the continuous monitoring can be performed with sensors that detect and record the biomarker level even several times per second (real-time, on-line). The challenge in this kind of device is the continuous sample collection. The body fluid of the interest determines the design.

As used herein, the term "brain damage" refers to the destruction or degeneration of brain cells due to one or more internal or external factors. Non- limiting examples of brain damage 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 damage" and "brain injury" are interchangeable, unless otherwise indicated. In the context of embodiments of the present invention, the term “brain injury” is meant to encompass traumatic brain injury, concussion, shred axons damage, lesioned axons damage, neuronal cell body damage, non-neuronal membrane rupture, and brain tissue destruction.

As used herein, the term "traumatic brain injury" (TBI) refers to brain injury caused by external physical trauma, or by a sudden motion of the head, with or without a physical contact with or hit to an external object. Non-limiting examples of incidences resulting in TBI include falls, vehicle collisions, sports collisions, and combats, explosions, blasts. The term includes both mild and severe TBI including closed-head injuries, concussions or contusions and penetrating head injuries.

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

The term "brain injury" also refers to subclinical brain injury, and anoxic-ischemic brain injury. The term "subclinical brain injury" (SCI) refers to brain injury without overt clinical evidence of brain injury. A lack of clinical evidence of brain injury when brain injury actually exists could result from degree of injury, type of injury, level of consciousness and/or medications, particularly sedation and anesthesia.

As used herein, the term "subject" refers to any mammal, including animals and human subjects. Animals include, but are not limited to, pets, farm animals, working animals, sporting animals, show animals, and zoo animals. Non-limiting examples of typical human subjects suffering from or pre-disposed to brain damage, TBI in particular, include babies, infants, children and young adults, particularly male; elderly; athletes, particularly boxers, ice-hockey players, soccer players, football (American) players, cricket players, rugby players and skateboarders; and soldiers. The terms "human subject" and "individual" are interchangeable. Typically, the subject is known to have or suspected of having a brain injury, such as TBI or ABI or CTE.

As used herein, the term “diagnosis” means detecting an injury, a disease, or a disorder, jointly referred to as a medical condition, or determining the stage or degree of the medical condition. Usually, a diagnosis of a medical condition is based on the evaluation of one or more factors (e.g., biomarkers) and/or symptoms that are indicative of the disease and/or its progress. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the medical condition. Each factor or symptom that is considered to be indicative for the diagnosis of a particular medical condition does not need be exclusively related to the particular medical condition, i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular medical condition is present in an individual that does not have the particular disease. The term “diagnosis” also encompasses determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical arts for a particular medical condition.

In the context of embodiments of the present invention, the term "diagnosis" refers to the determination of whether or not a subject has a brain damage, such as TBI or ABI or CTE. The term is also meant to include instances where the presence of a brain damage is not finally determined but that further diagnostic testing is warranted. In such embodiments, the method is not by itself determinative of the presence or absence of a brain damage in the subject but can indicate that further diagnostic testing is needed or would be beneficial. The methods, therefore, can be combined with one or more other diagnostic methods for the final determination of the presence or absence of a brain damage in the subject. Examples of such other diagnostic methods include, but are not limited to, CT and MRI, and are well known to a person skilled in the art. As used herein, a "final determination" or "final diagnosis" refers to ascertaining the presence or absence of a brain damage in a subject. The final determination or final diagnosis can be the result of any of the methods of the invention which, in some embodiments, can include more than one diagnostic test.

As used herein, the term "comparing" refers to making an assessment of how the proportion, level or cellular localization of one or more biomarkers in a sample from a subject 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 sample from a subject is the same as, more or less than, or different from the proportion, level, or localization of the corresponding one or more biomarkers in 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 from a subject is the same as, more or less than, different from or otherwise corresponds (or not) to the proportion, level, or cellular localization of predefined biomarker levels that correspond to, for example, a subject having subclinical brain injury (SCI), not having SCI, is responding to treatment for SCI, is not responding to treatment for SCI, is/is not likely to respond to a particular SCI treatment, or having/not having another disease or condition. In a specific embodiment, the term "comparing" refers to assessing whether the level of one or more biomarkers of the present invention sampled from a subject is the same as, more or less than, different from other otherwise correspond (or not) to levels of the same biomarkers in a control sample (e.g., predefined levels that correlate to 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 predicting the outcome of the brain damage or monitoring the subject's survival from the brain damage or response to treatment. The biomarkers and methods presented herein may be used as a clinical end point in clinical trials for treating TBI or ABI or CTE, providing the outcome of the brain damage, or monitoring the subject's survival from the brain damage or response to treatment.

In some embodiments of the present invention, the diagnosis or prognosis of a brain damage may comprise determination of the presence or absence of one or more of the present glycan-based biomarkers in a biological sample obtained from a subject whose possible brain damage is to be determined. Multiplexed assays can provide substantially improved diagnostic precision. Multiplexity can be based on measuring with different specific lectins or antibodies. In a specific embodiment, the present invention provides methods for determining the risk of developing brain injury in a subject. Biomarker percentages, amounts or patterns are characteristic of various risk states, e.g., high, medium, or low. The risk of developing brain injury is determined by measuring the relevant biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount, i.e., a predefined level or pattern of biomarkers that is associated with the particular risk level.

In some embodiments, the present invention provides methods for determining the severity of brain injury in a subject. Each grade or stage of brain injury likely has a characteristic level of a biomarker or relative levels of a set of biomarkers (a pattern). The severity of brain injury is determined by measuring the relevant biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount, i.e., a predefined level or pattern of biomarkers that is associated with the particular stage.

In the context of embodiments of the present invention, the term “biomarker”, as used herein, refers to a class of biomolecules which occur in the body, and which increase or decrease in the amount following brain injury, or which emerge upon a physiological event, such as brain injury, or the structure/integrity of which changes upon injury, and which can be detected using various molecular or biochemical methods being developed or known in the art. Biomolecules that are associated with brain injury can serve as biomarkers of brain tissue injury. Accordingly, the term "biomarker" refers to a molecule that is detectable in a biological sample obtained from a subject and that is indicative of a brain damage in the subject, and biomarkers of particular interest in the context of embodiments of the present invention include glycan-based biomarkers, showing differences in glycosylation and glycan/sugar/carbohydrate level/concentration/profile/structure/integrity between a sample from an individual with a brain damage and a healthy control.

As used herein, the term "glycan-based biomarker" refers to monosaccharides and polysaccharides, i.e. a polymer comprising two or more monosaccharide residues, as well as to a carbohydrate portion of a glycoconjugate, such as glycopeptides and glycoproteins, glycolipid, a peptidoglycan, or a proteoglycan, and any fragment thereof. Glycan-based biomarkers may comprise either homo polymeric or hetero- polymeric monosaccharide residues, and they may be either linear or branched, and which exist/are presented as carbohydrates solely, or as conjugates with any other types of biomolecules As used herein, the terms "glycan", "polysaccharide" and "carbohydrate" are interchangeable, unless otherwise indicated. Glycan-based biomarkers include but are not limited to carbohydrates, sugars, glycans, monosaccharides and/or polysaccharides, glycoproteins and glycopolymers. In the context of embodiments of the present invention the term “glycan”, as used in the term “glycan-based biomarker”, is meant to encompass glycan carbohydrates, short- (3-5 units carbs), oligo- (5-20 units), di- and mono-glycan saccharides, fragmented glycans, low molecular weight glycans, metabolically/enzymatically cleaved glycans, and fully or partially hydrolyzed glycans. The glycan can be part of, or connected to, another molecule, molecular complex or polymer of any type, yet the term “glycan-based biomarker” applies as long as the detection is essentially based on the glycan moiety(ies) of the structure.

In the context of the present invention, a sample of bodily fluid(s) is meant to encompass samples of saliva, sputum, spit, mucus, phlegm, sweat, tears, urine, exhaled breath condensate (EBC), and broncho-alveolar lavage fluids, and any combination thereof. Herein thought, the term "saliva” is meant to encompass fluids and secretions that are accessible in the mouth, like sputum, spit, mucus, phlegm, submandibular gland secretion, parotid gland secretion, and salivary gland secretion.

It is noted herein that the device can be used, or part of it is used, invasively in order to obtain a sample intravenously, subcutaneously, by embedding the device or a part thereof in the tissue.

In some embodiments where the sampling is non-invasive or invasive, the samples can originate from other bodily fluids, such as amniotic fluid, aqueous humour, bile, blood, blood plasma, blood serum, breast milk, broncho-alveolar lavage, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph and perilymph, exhaled breath condensate (ebc), exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, phlegm, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, serous fluid, smegma, spit, sputum, sputum, sweat, synovial fluid, tears, urine, vaginal secretion, vitreous humour, and vomit, and any combination thereof.

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

In some embodiments, glycan-based biomarkers may also be detected and/or quantified with the use of lectins. Lectins are a well-known family of carbohydrate-binding proteins, i.e. macromolecules that are highly specific for given glycans on the basis of their sugar moiety structures and sequences. Lectins can be classified into distinct 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 is noted that different sample types may exhibit different profiles of lectin-binding glycan biomarkers. Accordingly, lectins capable of identifying subjects with brain injury may be used in either individually or in any combination thereof.

In some embodiments, glycan-based biomarkers may also be detected and/or quantified with the use of galectins, the most widely expressed class of lectins in all organisms. Galectins are a family of proteins defined by their binding specificity for b-galactoside sugars, such as N-acetyllactosamine (Gal1- 3GlcNAc or Gal1-4GlcNAc), which can be bound to proteins by either N-linked or O-linked glycosylation. They are also termed S-type lectins due to their dependency on disulfide bonds for stability and carbohydrate binding. As used herein, "galectins" are encompassed by the term "lectins", unless otherwise indicated.

In the context of embodiments of the present invention, the term “lectins” refers to macromolecules binding glycans or glycan conjugates. In an extended concept this can comprise macromolecules binding proteins, molecular recognition/specific binding proteins, mediators of attachment, complex affinity proteins, and complementary interaction proteins. A non-limiting example of such is an antibody that is capable of binding carbohydrates.

In the context of TBI, biomarkers are useful for detecting cases of mild TBI and any other TBI cases in which other signs are vague or absent. Biomarkers are also useful for detecting cases in which the injured person cannot respond (coma, or unconsciousness) or cannot communicate verbally.

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

The biomarkers are differentially present in unaffected subjects (normal control or non-brain injury) and subjects with brain injury, and, therefore, are useful in aiding in the determination of brain injury status. In certain embodiments of the present invention, the biomarkers are measured in a sample taken from a subject using the methods described herein and compared, for example, to predefined biomarker levels and correlated to brain injury status. In particular embodiments, the measurement(s) may then be compared with a relevant diagnostic amount(s), cut-off(s), or multivariate model scores that distinguish a positive brain injury status from a negative brain injury status. The diagnostic amount(s) represents a measured amount of a biomarker(s) above which or below which a subject is classified as having a particular brain injury status. For example, if the biomarker(s) is/are up-regulated compared to normal during brain injury, then a measured amount(s) above the diagnostic cut-offs(s) provides a diagnosis of brain injury. Alternatively, if the biomarker(s) is/are down-regulated during brain injury, then a measured amount(s) at or below the diagnostic cut-offs(s) provides a diagnosis of non- brain injury. As is well understood in the art, by adjusting the particular diagnostic cut-off(s) used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. In particular embodiments, the particular diagnostic cut-off can be determined, for example, by measuring the amount of biomarkers in a statistically significant number of samples from subjects with the different brain injury statuses, and drawing the cut-off to suit the desired levels of specificity and sensitivity.

The device provided herein is configured to be used for repeated detection of damage occurred to the brain tissue (neuronal, non-neuronal, glial cells, astrocytes, microglia, and any other cells related to function, structure and integrity of the brain) and assessment of the brain integrity. The brain tissue comprises both neuronal and non-neuronal cells of the brain. In anatomical and morphological terms said injuries and damage can be such as shred axons, lesioned axons, neuronal cell body damage, non-neuronal/neuronal membrane rupture and brain tissue destruction.

The continuity of monitoring a glycan-based biomarker is made possible, according to some embodiments of the present invention, by continuously or periodically or repeatedly contacting a sample suspected or expected to contain the glycan-based biomarker, which presence or increased levels thereof in the sample is indicative of brain injury, while measuring a signal that correlates to the level of the glycan-based biomarker in the sample. Detecting comprises contacting the sample with the detector/sensor, measuring the signal by the detector/sensor, and transmitting the signal to a processor. The signal can be an electric signal detected by an electrode, a magnetic signal detected by a magnetometer and converted into an electric signal, any signal or signal modulation on the electromagnetic spectrum, including but not limited to UV radiation, visible light, infrared or radio-frequency radiation, detected by a photoelectric cell, electromagnetic coil or oscillator and converted into an electric signal, a color change signal detected by a color detector device and converted into an electric signal, and the likes. The invention is not limited to one particular signal and signal detection mechanism, as whereas embodiments of the present invention are drown to the correlation of glycan-based biomarker level in the sample, and the continuous monitoring thereof. The continuity of monitoring the glycan-based biomarker can also be afforded by continuous or periodical measurements of the signal, or by continuous or periodical transmittal of the signal to the processor. By “periodical”, it is meant that the detecting operation (contacting, measuring, and transmitting) is commenced and stopped at predetermined time points and at a predetermined cycle or frequency.

Thus, according to an aspect of some embodiments of the present invention, there is provided a device for repeated measurement of brain injury in a subject, which includes a sample extraction element or passing forced by vacuum that is configured to convey and contact a sample of a bodily fluid with a sensor, a sensor adapted to generate a signal having an intensity that correlates to a level of at least one glycan-based biomarker in the sample in contact with the sensor, wherein the level is indicative of a degree of brain injury, whereas the sensor includes at least one glycan-based biomarker responsive/binding reagent for selectively respond to, react with, or bind to the glycan-based biomarker in the sample.

In some embodiments, the sampling element further includes a sample processing/preparation unit, via which the sample is pre-treated and filtered or passed through a resin and moved to the sensor clean of debris. In some embodiments the glycan-based biomarker responsive/binding reagent can be biomarker-binding reagent which selectively binds the biomarker of interest by bioaffinity-binding reaction or ligand binding reaction. In some embodiments the glycan-based biomarker responsive/binding reagent is a chemical reagent or a series of chemical reagents that generate a signal upon contacting the biomarker, or upon a reaction cascade set off by the biomarker.

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 the device to provide information pertaining to the level of a glycan-based biomarker in a sample extracted from a subject at the actual time during which the brain injury event occurs and transpires. In some embodiments, the periodic monitoring is effected at a relatively short time cycle, thereby rendering the device such that the input data is processed within fractions of a second from the measuring event, so that information pertaining to the level of the glycan-based biomarker is made available virtually immediately as feedback.

FIG. 1 illustrates a block diagram of a device for detecting of brain injury in a subject according to one embodiment of the present invention. The illustrated device comprises a sampling element 11 , a detector 12, a pump 13, an analyzer 15 and a display 16.

The sampling element 11 is configured to extract a sample of a bodily fluid from a subject and direct said sample to the detector 12. The sampling element 11 may comprise a sample collector, sample adsorbing area or sample inlet. The sampling element 11 may also comprise a sample processing/pre treatment/preparation system, via which the sample is pre-treated and moved to the detector 12 following debris removal by filtration and the use of resin.

The detector 12 is configured to receive said extracted sample of a bodily fluid of a subject from the sampling element 11 . The detector 12 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector 12 is configured to forward said at least one data signal to the analyzer 15. The detector 12 is configured to carry out multiple measurements over time for detecting and monitoring of brain injury in a subject. The terms "detector" and "sensor" may be used interchangeably herein.

In the presented embodiment, the device for detecting of brain injury in a subject comprises a pump 13 to transfer the sample from the collection site and from the sampling element 11 to the detector 12. The pump 13 can be realized, for example, in a microfluidic system, equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, airflow, peristaltic tube, gravitation, vacuum, electroosmotic flow, etc. The pump 13 may preferably use low suction vacuum.

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 displayable and/or audible information indicative of brain injury in the subject.

In the presented embodiment, the device for detecting of brain injury in a subject comprises a display 16 for displaying said displayable and/or audible information indicative of brain injury in the subject generated by the analyzer 15.

According to another aspect of the present invention, there is provided a device for diagnosis of brain injuries, which provides trend lines according to at least one measured physical parameter, which is correlated to the level of at least one glycan-based biomarker in a series of samples.

The device, according to some embodiments of the present invention, may include one or more section or one or more mechanism for extracting at least one bodily fluid that includes a glycan-based biomarker. This element of the device is also referred to as the sampling element. According to some embodiments of the present invention, the device operates with a non-invasive sampling element, rendering the device a non-invasive brain injury diagnostic device, which can provide a continuous or repeated monitoring of a subject’s brain injury condition. A non-invasive sampling element may be present in a form of a tube-and-pump element, an absorbent pad with or without a protective fluid- permeable shield, or a single or a plurality of openings in the device that allow the bodily fluid to enter and come in contact with the sensor. In some embodiments the sampling element, or a part of the sampling element, is invasive, meaning that the device can be used, for example, intravenously, subcutaneously, or embedded in the tissue.

Continuous monitoring of a biomarker in a bodily fluid is most effective when the sensor is immersed in continuous flow of the fluid, or when a flow of the fluid is directed to the sensor. This enables recording of the biomarker level at very short intervals (“real-time”). Alternatively, continuous monitoring of a biomarker in a bodily fluid comprises discrete, successive off-line measurements of the biomarker level at certain time intervals and plotting of the biomarker level as a function of time for diagnostic and prognostic purposes. Presenting (e.g., plotting) and compiling of the trend line can be made either by the device itself or by a separate data processing means.

In the context of some embodiments of the present invention, samples may be obtained by non-invasive means taken for example from saliva, urine, tears or sweat. Sample can be taken actively or passively. Active sample taking means that the sample can be retrieved, without the subject’s own activity, by the device or by another person. This enables sampling from an unconscious subject or subject who is in coma or heavily intoxicated subject. Nonetheless, it is noted herein that use of the provisions of the present invention are not limited to samples extracted by non-invasive methods, meaning that the device and methods provided herein can be used to diagnose brain injury by sampling blood, plasma, spinal fluid and the like.

Devices according to some embodiments of the present invention may be used by variety of groups such as paramedics, first responders, professional athletes, doctors, and soldiers in a need for fast response and ease of use. Such devices may include one or more of the following features: non-invasive sample collection; small size; portable; wearable; automatic sample processing; simple user interface; quick delivery of the analysis results. Such devices may be incorporated in helmet, protection gear, dental implant, braces, and mouth guard (which can be used for example in military and during sports events)

Devices according to some embodiments of the invention may be suitable for diagnosis in the point-of-care by paramedics and military first aid staff, or in hospitals with one or more of the following characteristics: accessibility during many hours during the day; possibly invasive sample collection; small size; portable; wearable; bedside; automatic sample processing; simple user interface; quick delivery of the analysis results.

FIG. 2 illustrates a device for detecting of brain injury in a subject according to one embodiment of the present invention. The illustrated device 20 comprises a sampling element 21 , a detector 22, a cable 23 and an analyzer 25 with a display 26.

The sampling element 21 is configured to extract a sample of a bodily fluid from a subject and direct said sample to the detector 22. The detector 22 is configured to receive said extracted sample of a bodily fluid of a subject from the sampling element 21. The detector 22 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector 22 is configured to carry out multiple measurements over time for detecting and monitoring of brain injury in a subject.

In the presented embodiment, the device 20 for detecting of brain injury in a subject comprises a cable 23, which has a data cable 27 combined with a suction cable 28 embedded in said cable 23. Said suction cable 28 is arranged to forward the suction for transferring the sample from the collection site and from the sampling element 21 to the detector 12. Said suction can be realized, for example, in a microfluidic system, equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, airflow, peristaltic tube, gravitation, vacuum, electroosmotic flow, etc.

Said data cable 27 is configured to forward 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 forward 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 presented embodiment, the device 20 for detecting of brain injury in a subject comprises an analyzer 25 with a display 26, said analyzer 25 configured to generate displayable and/or audible information indicative of brain injury in the subject. Said display 26 of said analyzer 25 is configured for displaying said displayable and/or audible information indicative of brain injury in the subject generated by the analyzer 25.

According to various embodiments of the present invention, the entire device, or the sampling element, or an assembly of the sampling element and the sensor, can be integrated/embedded in a helmet, mouthpiece, protection gear, dental implant, mouth guard, brace, article of clothing, implanted on tooth or on the skin surface, or implanted under the skin, or in the blood vessel, etc. In addition, the device, or an assembly of the sampling element and the sensor, can be an external unit/device which is contacted directly with the organ/body fluid sample, or to which a sample is passed by natural excretion, or by transferring using an auxiliary piece of equipment.

According to embodiments of the present invention, the device is an integrated device that detects a glycan-based biomarker continuously and which is carried by the user so that it is directly contacted or attached to the body (organ or tissue) of the subject. Examples of such devices include, but are not limited to, an implant in the blood vessel, a device under the skin or on the skin, or an implant on tooth. Alternatively, the subject is wearing the device as part of an article of clothing, equipment, or protective gear. Examples of such devices include, but are not limited to, a device being part of (embedded in/integrated to) a mouth guard, a helmet, or any other protective or wearable equipment. FIG. 3 illustrates a detailed illustration of a sampling element and a detector of a device for detecting of brain injury in a subject according to one embodiment of the present invention. The illustrated device comprises a sampling element 21 , a detector 22 and a cable 23 having embedded a data cable 27 combined with a suction cable 28.

The sampling element 21 is configured to extract a sample of a bodily fluid from a subject and direct said sample to the detector 22. The sampling element 21 may comprise a sample collector, sample adsorbing area or sample inlet. The sampling element 21 may further include one or more pre-filter units for processing, pre-treatment and preparation of the sample. Said pre-filter units may perform one or more of the following functions: filter the sample to exclude cells, debris, precipitates, sediments, flocculation, and/or particles based on their size/molecular weight; adjust, maintain or change the composition of the carrier of the sample, dilute, adjust pH, ionic strength, temperature, viscosity, surface tension; modify the biomarker in the sample in order to render it detectable, modify the biomarker in the sample using chemical functionalization, and/or derivatization, labelling, hydrolysis, degradation. The sampling element 21 may also comprise a sample processing/pre-treatment/preparation system, via which the sample is pre-treated and moved to the detector 22.

The detector 22 is configured to receive said extracted sample of a bodily fluid of a subject from the sampling element 21. In the present embodiment, the detector 22 is physically coupled to the sampling element 21 and in direct contact with the sampling element 21 . This enables a fast and reliable detection of the biomarker, as well as enabling real-time detection thereof.

The sampling element 21 may be a disposable and replaceable sampling element 21 arranged for one time-use. The sampling element 21 may be arranged as a changeable sampling element 21 . The sampling element 21 may be changed by the user and may be fastened to the detector 22 by a click fastening, a snap fastening or a short thread fastening.

The suction cable 28 is arranged to forward the suction for transferring the sample from the collection site and from the sampling element 21 to the detector 22. Said data cable 27 is configured to forward 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 for detecting glycan-based biomarkers specific to brain injury. The device is preferably a hand-held device that can be easily transported and used by laymen or professionals, wherein the body fluid used as the test sample is introduced to the device by: sweeping/contacting the sampling element of the device directly with the organ or tissue, such as mouth, tongue, skin, mucous membranes, eye, meningitis, or blood vessel; passing body fluid through the sampling element of the device by natural means, such as by spitting or by passing urine into the sampling element; wetting/immersing the sampling element of the device in a bodily fluid which has been collected previously; or transferring bodily fluid to the sampling element of the device by means of an auxiliary equipment, such as by a pipette, or by pouring from a container/cup, by a tubing and pump.

In embodiments where the sample of bodily fluid is saliva or other oral cavity sample or upper respiratory tract sample, the sampling element may comprise: a perforated encasement allowing saliva to penetrate and reach the sensor; a sampling tube that is attached to the mouth guard, such as used in contact sports; a sampling tube attached to the tooth piece/implant, such as used in medical care facilities; a sampling tube attached to the oral mucosa, e.g., with a magnet on the other side of the cheek, or with a cheek clip; or a sampling unit connected to a tooth implant.

In embodiments where the sample of bodily fluid is sweat, the sampling element may comprise: a perforated encasement or a porous film allowing sweat to penetrate and reach the sensor; an absorbing patch and microfluidic pump or capillary action sucking it to the device; a hollow or porous non-invasive microneedles with passive sweat suction inside the skin; a sweat collection from self-adhering or wrapped patch; or a sweat collection helmet padding. In embodiments where the sample of bodily fluid is blood, the sampling element may comprise an intravenous cannula that serves as a port to collect blood or to direct a small flow of blood to the sensor in the device.

In embodiments, the sample of bodily fluid is urine, and the sampling element is a urine catheter, which may or may not be integrated with the sensor.

During the transfer of the sample, it is also convenient or necessary to separate some unwanted parts thereof, such as cells, particles, contaminants, proteins and/or other non-relevant components. This can be done mechanically, by forcing the flow of the fluid sample through filters, chemical binding separation, or by utilizing fluid dynamics. Chemical binding separation would be based on adsorption of unwanted particles, molecules onto pre-treated surfaces with well- defined chemistry while the sample flows by the surface. This surface can be inside wall of the tube, or additional filling such as filter, small beads, fibres, or porous material. The effect of adsorption can be increased by increasing the specific surface area which is exposed to the liquid. Moreover, the separation can be based on electrical charges, gravity, or centrifugal forces.

The sampling element can be shaped as a syringe tip fitting/adaptor, or be stretchable and elastic for fitting any tip of the external source of the sample, or be a screw threaded tip, a piercing needle tip, a septum membrane, a butterfly needle adaptor, and have any shape designed to connect to an external source of a liquid sample.

In some embodiments, the purpose of the sampling member, in addition to introducing the sample, is to deliver additional reagents in solution.

The term “portal”, as used in the context of some embodiments of the present invention, refers to an element of the device, which is designed as an inlet and/or outlet for infusing or retracting liquids and reagents in solution into and/or out of the probe. In some embodiments, the device includes more than one portal, for letting any one or more of a sample and/or a standard analyte solution and/or an indicator formulation reagent and/or a washing liquid, and any combination thereof. In such embodiments the sampling member can have a multiple inlets and outlets portals or be connected to a manifold of inlets and outlets, or the probe can be in communication with more than one portal.

In some embodiments, the device is equipped with at least one portal to which a reservoir is attached. The reservoir may be in the form of a piston/plunger and cylinder/barrel) combination (e.g., a syringe), wherein the plunger is retracted and the barrel is the reservoir. In some embodiments, the reservoir can be pre filled with a liquid that is used in the diagnosis process, and can be, for example, a standard analyte solution and/or an indicator formulation reagent and/or a washing liquid, and any combination thereof.

FIG. 4 illustrates a block diagram of a device for detecting of brain injury in a subject according to another embodiment of the present invention. The illustrated device 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 sample of a bodily fluid from a subject and direct said sample to the detector 12. The detector 12 is configured to receive said extracted sample of a bodily fluid of a subject from the sampling element 11. The detector 12 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector 12 is configured to forward said at least one data signal to the transmitter 14. The detector 12 is configured to carry out multiple measurements over time for detecting and monitoring of brain injury in a subject. The terms "detector" and "sensor" may be used interchangeably herein.

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

In the presented another embodiment, the device for detecting of brain injury in a subject comprises a pump 13 to transfer the sample from the collection site and from the sampling element 11 to the detector 12. The pump 13 can be realized, for example, in a microfluidic system, equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, airflow, peristaltic tube, gravitation, vacuum, electroosmotic flow, etc.

In the presented another embodiment, 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 displayable and/or audible information indicative of brain injury in the subject. The device for detecting of brain injury in a subject according to the presented another embodiment comprises a display 16 for displaying said displayable and/or audible information indicative of brain injury in the subject generated by the analyzer 15.

In the device for detecting of brain injury in a subject according to some embodiments of the present invention The the analyzer 15 may include a processor for transcoding said at least one data signal generated by the detector 12 into digitized presentable information that can be displayed or otherwise transmitted to the subject or the caretaker. Alternatively, the processor may digitize a non-electric signal, e.g. light, into presentable digitized information. The processor may also store the data, analyze the data with respect to previously stored data therein, and/or transmit the information to another processor, a display, and/or a remote storing device.

FIG. 5 illustrates a snap-on device for detecting of brain injury in a subject according to another embodiment of the present invention. The illustrated snap- on device 30 comprises a detector unit 32, a cable 33 and a transmitter unit 34. The illustrated snap-on device 30 may be used for detecting of brain injury in a subject wearing a helmet, e.g. a sports helmet, a protective helmet or a military helmet. Said snap-on device 30 can be attached Snap-on to a helmet.

In the presented embodiment, the snap-on device 30 for detecting of brain injury in 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, wherein said detector is configured to be brought in direct contact with the sample, rendering a sampling element unnecessary.

The detector of the detector unit 32 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector of the detector unit 32 is configured to forward said at least one data signal to the transmitter 34 via the cable 33. The detector of the detector unit 32 is configured to carry out multiple measurements over time for detecting and monitoring of brain injury in a subject. The transmitter 34 is configured to receive said at least one data signal from the detector of the detector unit 32 and to transmit digital information comprising said at least one data signal to an analyzer.

Furthermore, the transmitter 34 may comprise a pump or suction to transfer the sample from the collection site to the detector of the detector unit 32. Said pump or suction may be realized, for example, in a microfluidic system, equipped with pumps utilizing, for example, osmotic flow, capillary flow, lateral flow strip, airflow, peristaltic tube, gravitation, vacuum, electroosmotic flow or a stored vacuum punched upon a sample-taking, etc. Respectively, in addition to forwarding said at least one data signal to the transmitter 34, the cable 33 is configured to forward the suction for transferring the sample from the collection site to the detector of the detector unit 32.

FIG. 6 illustrates a device for detecting of brain injury in a subject according to an embodiment of the present invention arranged in a sports helmet. The sports helmet 36 may e.g. be an American football helmet 36. The illustrated sports helmet 36 has a device for detecting of brain injury in a subject comprising a detector unit 32, a cable 33 and a transmitter unit 34. The illustrated sports helmet 36 comprises a detachable device for detecting of brain injury in a subject without the need to stop ongoing activity, said device comprising a mouthpiece anatomically fitting, detector, which detector comprises at least one glycan- based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is saliva.

FIG. 7 illustrates a device for detecting of brain injury in a subject according to an embodiment of the present invention arranged in a protective helmet. The protective helmet 37 may e.g. be a protective helmet 37 used for cycling, skating, field sports or ice sports. The protective helmet 37 may also be a military helmet 37. The illustrated protective helmet 37 has a device for detecting of brain injury in a subject comprising a detector unit 32, a cable 33 and a transmitter unit 34. The illustrated protective helmet 37 comprises a detachable device for detecting of brain injury in a subject without the need to stop ongoing activity, said device comprising a mouthpiece anatomically fitting, detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan-based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is saliva.

FIG. 8 illustrates a standalone device for detecting of brain injury in a subject according to an embodiment of the present invention arranged in a mouth guard. The mouth guard 41 may e.g. be a mouth guard 41 used for court sports or combat sports measuring the biomarker without the need to stop activity. The illustrated mouth guard 41 has a device 40 for detecting of brain injury in a subject comprising a detector-transmitter unit 42. The illustrated mouth guard 41 comprises a teeth protection, jaw protection embedded with a device for detecting of brain injury in a subject wearing it in his/her mouth, said device comprising a detector, which detector comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan- based biomarker in said sample, said level being indicative of a degree of brain injury in the subject, wherein said bodily fluid is saliva.

In the presented embodiment, the device 40 for detecting of 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 42 said detector is configured to be brought in direct contact with the sample.

The detector of the detector-transmitter unit 42 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector of the detector-transmitter unit 42 is configured to forward 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 carry out multiple measurements over time for detecting and monitoring of brain injury in a subject. The transmitter of the detector-transmitter unit 42 is configured to transmit digital information comprising said at least one data signal to an analyzer.

FIG. 9 illustrates a standalone device for detecting of brain injury in a subject according to an embodiment of the present invention arranged or implanted in a tooth. The illustrated device 50 for detecting of brain injury in a subject is arranged in a tooth 51 comprising a detector-transmitter unit 52. Said detector- transmitter unit 52 may e.g. be attached to or implanted on or in a tooth 51 . In the presented embodiment, the device 50 for detecting of 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 in direct contact with the sample.

The detector of the detector-transmitter unit 52 may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector of the detector-transmitter unit 52 is configured to forward said at least one data signal to the transmitter of the detector- transmitter unit 52. The detector of the detector-transmitter unit 52 is configured to carry out multiple measurements over time for detecting and monitoring of brain injury in a subject. The transmitter of the detector-transmitter unit 52 is configured to transmit digital information comprising said at least one data signal to an analyzer. The terms "detector" and "sensor" may be used interchangeably herein.

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

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

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

Sensors for detecting biomolecules, also referred to as biosensor, may include bio-recognition elements and a transducers. A bio-recognition elements specifically identify and interacts with analytes, typically by detecting changes in physicochemical properties (optical, thermal, electrical, and thermodynamic properties) that are usually converted into an electrical signal by a transducer. The sensor comprises a sensing material, which may be an enzyme, an antibody, a microorganism, a tissue, an organelle, DNA, and RNA a receptor which binds a ligand, or any correspondent molecular affinity binding pair such as lectin and glycan. Alternatively, interaction between complementary substances and ligands interaction with 3D complementary 3D structures. The sensor, according some embodiments of the present invention, may be based on the type of bio-recognition element or the transducing method used. Based on the bio-recognition element, the sensor can be an enzyme sensor, immunosensor, nucleic acid probe sensor, or cell-, tissue-, or organelle-based sensor. Based on the transducing method, the sensor can be piezoelectric, optical, or electrochemical sensor.

Some embodiments of the present invention include carbon nanomaterials in the form of particle/dots, tube/wires, and sheets as biosensor platforms. Carbon nanomaterials as nanotubes include single-walled (SWCNT), and multiple- walled (MWCNT); graphenes include graphene (GR), graphene oxide (GO), and reduced graphene oxide (rGO). Carbon quantum-dots (QD) sensors constructions are also encompassed within embodiments of the present invention.

Hence, the device further comprises a sensor for converting/transducing the presence and level of the biomarker into a quantifiable signal (e.g., an electrical signal) that can be processed in a processing unit for further analysis. In other words, the sensor is an element in the device that is adapted to generate a signal having an intensity that correlates to the level of a glycan-based biomarker in a sample of a bodily fluid, which is brought in contact with the sensor, via the sampling element.

According to some embodiments of the invention, the generation and measurements of the signal, are based on at least one of colorimetric measurements, light scattering measurements, thermal radiation measure ments, fluorescence measurements, photoluminescence measurements, light polarization measurements, chemiluminescence measurements, electrochemi luminescence (ECL) measurements, magnetic field measurements, and electrochemical measurements. In an alternative embodiment of the invention, the sensor receives the sample from the sampling element via a tube or a sample transfer tool, such as, for example, a pipette.

The information, i.e. diagnostic/analytical results, may be obtained within a matter of seconds or minutes. According to some embodiments of the invention, the sensor and processor are capable of detecting and recording biomarker levels even several times per second real-time, on-line.

The sensor may contain a reaction cell, for performing the following functions: providing diffuse reagents that can bind or form a complex with the biomarker, wherein these diffuse reagents can be provided either from a prefilled chamber with liquid containing the reagents, or from a dried patch/smear thereof; providing immobilized/bound (e.g., printed or dotted) reagents to capture the biomarker or a complex formed in the preceding sample collection; and/or enabling detection of the outcome, by performing a reaction or an event upon which the presence and amount of the biomarker can be detected and verified, and also optionally quantified.

The sensor can be in the form of a microfluidic cell, and the biomarker is either labelled or otherwise cause the change in the signal characterizing the cell. The signal is concentration dependent, thereby enabling a quantitative measurement of the biomarker.

Some biomarker monitoring technologies rely on microfluidics that are considered simple, low-cost, and rapid. Microfluidic includes using polymer and paper-based platforms, colorimetric, fluorescence, chemiluminescence, electrochemiluminescence (ECL), magnetic, thermal radiation, radiofrequency modulation and electrochemical detection. A device, according to some embodiments of the present invention, may include microfluidic elements. The application of microfluidics for the development of biosensors has proven to play a significant role in analytical investigations of biological and chemical samples. A generic analytical procedure can be broken down into three broad categories: the analytical principle on which the measurement is based, the analytical method that is the concept for optimizing the conditions of the analytical principle chosen, and the analytical procedure that includes all considerations from the analyte to the analytical result. Microfluidic devices inherently possess characteristics that make them advantageous for point-of-care testing (POCT) applications: smaller quantities of test samples and reagents required for analysis that can also be precisely handled, drastic reductions in test times due to reductions in diffusion path lengths in microfluidic devices, performing multiple test types simultaneously (multiplexing), and the capability to integrate and automate various process steps on the same platform. According to some embodiments of the present invention, sample pretreatment and enrichment step may be included.

The microfluidic cell sensor may include a reaction and detection segment on/in volume or area inside a tube or on a surface, for example in the form of a layer, a film, coated beads, fibers, needles, porous volume, or other three dimensional matrix. The reactions that are triggered by the presence of the biomarker, and the detectable/measurable outcome that are indicative of brain injury, is taking place in the reaction area.

The sensor may comprise a spiral disc, or a microfluidic centrifugal platform, which can serve as a separation/filtering unit as well as a reaction cell.

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

The glycan-based biomarker can adsorb to the surface of a matrix which is functionalized with agents that bind/capture the biomarker from the sample. Such capturing agents can be, for example, lectins, antibodies, or a synthetic functional mimic thereof which is capable of binding the biomarker. Alternatively, non-specific binding of biomarker can also be utilized, i.e. binding that is based on hydrophobic interactions, passive adsorption, van der Waals forces and/or hydrogen bonds. In some embodiments the detection is based on label free techniques, e.g., by measuring the change of electrical signal over detection area/volume (e.g. electro-chemical, conductivity, capacitance) or sorption-based changes in optical signal (e.g. spectroscopic, absorbance, transmittance, reflectance, and surface plasmon resonance). Hence, quantitative or relative-qualitative detection may be effected by a gold film in a surface plasmon resonance setting, by an electric detection of capacitance or conductivity of a film, an electrochemical detection (PED; oxidation), or by a porous (nano) film with optical resonance detection.

In label dependent detection, the term “label” refers to an agent that is used for tracing, localization, or visualization the biomarker and making it detectable by visual inspection or by any detection technology. The label is typically conjugated with a biomarker-specific molecule such as antibody or lectin that specifically binds the glycan-based biomarker. The label can be a dye, a fluorescent molecule, an enzyme, a detectable nanoparticle, or a chelate. These labels possess a biomarker binding moiety to ensure specific binding of the biomarker. The presence of such labels in/on the sensor generates a signal relative to the amount of the biomarker. Fluorescent polarization detection is a label dependent detection method, however, it is advantageous as it does not require separation or a washing step because the detection is based on different polarization planes of emitted light depending on whether the biomarker is bound to the ligand or not.

In physical and analytical chemistry, colorimetry or colourimetry is a technique used to determine the concentration of colored compounds in solution. Colorimetric analysis is a method of determining the concentration of a chemical element or chemical compound in a solution with the aid of a color reagent. Testing of the concentration of a solution can be done by measuring its absorbance of a specific wavelength of light. In some embodiments of the invention colorimetry techniques are used however other color based detection techniques may be used as well. In some embodiments, a dye/colorant/chromogen forms a colored complex or changes its 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 which is initiated by the presence of the biomarker. The reaction may or may not include enzymes. In some embodiments, an enzyme specific for the glycan-based biomarker starts a conversion reaction in the presence of the biomarker. The enzymatic reaction may be coupled to a dye/colorant/chromogen which develops color or change it color (enzymatic activity). Such detection mechanism also does not require immobilization of any element in the indicator formulation.

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

The present invention also encompasses indicator formulations which can be based on soluble reagents for glycan-based biomarker detection.

For example, in an enzymatic glycan assay embodiment, the analyte (a glycan) in the sample reacts with an enzyme that catalyzes the glycan’s decomposition. For example, a hexokinase is an enzyme that phosphorylates hexoses (six- carbon sugars), forming hexose phosphate, which in turn can with another reagent in the indicator formulation to form a substance that gives-off a color. This reagent is typically added thereto via a portal. Similarly, galactose oxidase is an enzyme that catalyzes the oxidation of D-galactose, and glucose oxidase is an oxido-reductase that catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-5-lactone; these products of the enzymatic reactions can be detected directly or indirectly.

For example, in a “direct assay” embodiment, the glycan-based biomarker reacts with a chromogen, e.g. a reducing sugar generates reduction of a chromogen thereby affording color development. In some embodiments based on soluble and diffusible labels, the glycan-based biomarker reacts with an enzyme that is specific for a moiety or a molecular structure present in the glycan-based biomarker. In some cases this moiety can be galactose and the enzyme is galactose oxidase. In the presence of HRP and a chromogen such as Amplex Red (10H-phenoxazine-3,7-diol, 10-acetyl, CAS 119171-73-2) the reaction results in color development (abs max @ 560 nm.

In some embodiments based on soluble and diffusible labels, the glycan-based biomarker reacts directly with a chromogen/dye. In some cases the glycan biomarker can consist of a reducing sugar which in some cases reacts with 3- methyl-2-benzothiazolinonehydrazone (MBTH), developing a colored adduct.

Another detection technology relevant in some embodiments of the present invention involves upconversion nanoparticles-based fluorescent probes, which can be excited with low energy light photon (near infrared). The reading of the signal occurs in lower wavelengths, which means that auto-fluorescence is circumvented.

In some embodiments of the present invention, the sensor is an antibody-based sensor, also known as immunosensor, which is based on the specificity of an antibody-antigen reaction to produce a change in the transducer signal. Both polyclonal and monoclonal antibodies can be used in the context of embodiments of the present invention. Antibodies can be directly immobilized on the surface of the transducer by covalent bonding through 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 may be employed for detecting, measuring and/or analyzing the present biomarkers in a sample taken from a subject. For this purpose, both a capture reagent and a detection reagent are required. Said capture reagent may be a lectin or a galectin, while said detection reagent may be a detectably labelled antibody, or vice versa. In immunoassays used for the detection of biomarkers in serum and biological fluids antibodies bind non-covalently to the antigen 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 the binding between antibodies and antigens.

The present invention also contemplates traditional immunoassays including, for example, sandwich immunoassays such as Enzyme-Linked Immunosorbent Assay (ELISA) or fluorescence-based immunoassays, as well as other enzyme immunoassays. ELISA typically employs antibodies. A specific epitope of antigens bind to the pABs immobilized on the solid surface. The enzyme linked sAB binds to the other epitopes to form a sandwich like complex. The binding of the antigen and pAB is detected using an activity of enzyme which changes the substrate into a coloured product.

As is readily understood by those skilled in the art, more than one type of lectins/galectins and/or more than one type of antibodies may be used. Furthermore, multiple different reactions may be carried out simultaneously or sequentially for detecting different glycan-based biomarkers in a sample to be analyzed.

In accordance with the above, molecules suitable for use in detecting glycan- based biomarkers in a sample to be analyzed include, but are not limited to, lectins, galectins, antibodies, and competitive small molecules. Said detection molecules may be visualized, or made otherwise measurable, using for instance conjugated color reagents, labels, or dyes. Enzyme labels suitable for this purpose include those that upon addition of a substrate catalyze a reaction leading to a measurable change in color, in luminescence, in production of a precipitate, or other measurable parameters. Non-limiting examples of such enzyme labels include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Photoluminescent labels, including fluorescent dyes (prompt), lanthanide chelates (for time- resolved fluorescence), and photon upconversion labels may be used for detecting said detection molecules. Furthermore, the detection may be based on bioluminescence and chemiluminescence (as e.g. in luciferin-based detection), or on electrochemiluminescence (with e.g. ruthenium complexes). Also biotin and its derivatives, which enable binding and detection by labeled avidin or labeled streptavidin, as well as various radioactive isotopes may be used for the detection. The detection may also be carried out 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 biomarkers of the present invention may be detected by means of an electrochemical-luminescent assay. Electrochemiluminescence detection uses labels that emit light when electrochemically stimulated. Background signals are minimal because the stimulation mechanism (electricity) is decoupled from the signal (light). Labels are stable, non- radioactive and offer a choice of convenient coupling chemistries.

Furthermore, a sample may also be analyzed by means of a passive or active biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there. Lectin biochips are biochips adapted for the capture of glycans. Many lectin biochips are described in the art.

Label-free electrochemical detection based on the nucleic acid aptamer may be used where binding with the immobilized aptamer may result in the decrease in current (signal-off), which is measured by square wave voltammetry. The advantage of this method is that it uses a reagentless and label-free electrochemical biosensor based on a DNA aptamer. The DNA aptamer may detect specific antigen.

Gold nanoparticles (AuNPs) may serve as the surfaces for the immobilization of proteins, however, other surfaces which have electrochemical properties also may be used to obtain electrochemical biosensors. AuNP platforms can be combined with a multiple-enzyme labeled sAB-magnetic bead bioconjugate and be used for the ultrasensitive detection. The signal amplification can be achieved by using synthesized magnetic bioconjugate particles labels along with the sAB attached to 1 pm magnetic beads, or secondary particle amplification like silver particle amplification.

In other embodiments of the invention, an organic electrochemical transistor based on poly(styrenesulfonate) doped poly(3,4-ethylenedioxythiophene) (PEDOT-PSS) and AuNP labeled with pAb may be used. However, platinum nanoparticles labeled with Ab and the hydrogen evolution reaction in an electrochemiluminescence technique may be used as well.

Recent advances in Surface enhanced Raman spectroscopy (SERS) have led to the design of novel nanoprobes which are called as SERS tags. The SERS tags are the combination of metallic nanoparticles and highly specific organic Raman reporter molecules. The SERS-active nanoprobes produce strong, characteristic Raman signals and can be used to detect biomarkers using laser Raman spectrometry or SERS microscopy.

The fluorescence-based detection of biomarker proteins uses a simple quantification process, easy detection of large-scale samples, and comparatively easy labelling of biomolecules with fluorescent tags. The fluorescent labelled sAB’s are used for the quantification of the antigens in the samples. Moreover, the signal amplification may be achieved by using fluorescent nanobeads instead of fluorescent molecules. The fluorescent nanobeads are prepared by entrapping fluorescent dyes inside the microbeads composed polystyrene. Then these fluorescent beads are conjugated with the sABs, which can form a sandwich type biomolecular complex with the antigen and immobilized pAB.

After their induction in 1983, the protein microarrays have been widely accepted and innovated for the detection of biomarkers. The detection of biomarkers is based on the formation of the biomolecular complex between the immobilized pAB, respective antigen and the sAB labelled with the fluorescent dye. The quantification of the antigen in the sample is performed by analysing the fluorescence signal intensity. The protein microarray is advantageous in the sense that it requires a very small amount of the sample and reagents. Though protein microarrays use a very simple experimental protocol, the sensitivity of such a method depends on the use of the particular chemistry for the immobilization of proteins on the surface.

Semiconductor quantum dot (QD) chemistry provides an alternative approach for detection and quantification of biomarkers. The QD are usually tagged with sAB for the detection of particular antigens. Because of the photostability, tunable nature, and sensitivity of fluorescence, the QDs are preferred over organic dyes for detection of biomarkers. Though the initial size of the QD is in nanometer, after bio-conjugation the QDs are found to form the aggregates of larger sizes.

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

Nucleic acid-based biosensors integrate a natural and biomimetic form of oligo- and polynucleotides and rely on base pair affinity between complementary sections of nucleotide strands for their high sensitivity and selectivity. Synthetic oligodeoxyribonucleotides (ODNs) may be used as well. End labels, such as thiols, disulfides, amines, or biotin, are incorporated to immobilize ODNs to the surface of a transducer element.

Some embodiments of the invention include transducer element optical biosensors include surface plasmon resonance (SPR) biosensors, adsorption and reflection biosensors, luminescence biosensors, fluorescence biosensors, and optical fiber biosensors. Fluorescence biosensors use fluorescent labels for signal generation, and the intensity of the fluorescent signal is used to determine the amount being detected. SPR is a direct optical-sensing technique that measures the refractive index change due to bio-specific interactions occurring in the vicinity of a thin metal film surface. Some embodiments of the invention include piezoelectric biosensors consisting mass-sensitive piezoelectric crystals with excitation electrodes. The piezoelectric transducers allow a binding event to be converted into a measurable signal, such as resonant frequency changes.

Some embodiments of the invention include quartz crystal microbalance (QCM), adapted to the liquid medium, which gives a direct response signal to characterize the binding event between a sensitive layer grafted onto the surface transducer and an analyte to be detected.

Some embodiments of the invention include microcantilevers which is a type of mass-sensitive biosensor. The principle of this detection is based on the change in the mechanical response of a cantilever due to molecular adsorption on the functionalized cantilever surface.

Additional biosensors that can be used in some embodiments of the present invention include, without limitation, electrochemical biosensors including amperometric, potentiometric, and impedance biosensors. Potentiometric biosensors use ionselective electrodes (ISEs) and ion-sensitive field-effect transistors (ISFETs) to measure the change in electric potential due to the accumulation of ions resulting from an enzyme reaction. Amperometric biosensors measure the change in electrical current (typically, in the nanoampere to microampere range) due to the oxidation/reduction process of an electroactive species, for example, pulse electrochemical detection (PED). The working electrodes are usually a noble metal or a screen-printed layer covered by a bio-recognition element. Potentiometric biosensors measure the potential difference generated across an ion-selective membrane separating two solutions at virtually zero current flow. Impedance biosensors are based on the combined measurement of the resistive and capacitive properties of the targets in response to a small-amplitude sinusoidal excitation signal. Impedance detection involves measuring the change in impedance caused by the binding of analytes to receptors (antibodies, DNA, proteins, etc.) immobilized on the surface of electrodes. Change in the conductivity of the medium caused by change in the ionic concentration of a medium caused by activity of enzyme used as labels.

Calorimetric sensors that may be used in some embodiments of the invention are based on measuring the heat of a biochemical reaction at the sensing element. These biosensors consist of temperature sensors with immobilized biomolecules. Once the analyte comes into contact with the biomolecules, the reaction temperature is measured. The total heat produced 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 analyte concentration, namely the level of the glycan-based biomarker in the sample.

In some embodiments of the present invention, the sensor is a unit that can measure the biomarker directly. Such units include HPLC, MS, Raman- spectroscopy, FTIR-spectroscopy detectors. Suitable methods for use in detecting or analyzing glycan-based biomarkers include, but are not limited to, Biocore studies, mass spectrometry, electrophoresis, nuclear magnetic resonance (NMR), chromatographic methods or a combination thereof. Gas chromatography (GC) and liquid chromatography (LC) can be used for separation of metabolites and define size, charge, affinity, binding capacity. Specifically, the mass spectrometric method can be, for example, LC-MS, LC- MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS, FTMS, multiple reaction monitoring (MRM), quantitative MRM, or Label-free binding analysis. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, hybrids or combinations of the foregoing, and the like. In yet another embodiment, mass spectrometry can be combined with another appropriate method(s) as may be contemplated by one of ordinary skill in the art. In another embodiment, the mass spectrometric technique is multiple reaction monitoring (MRM) or quantitative MRM. The electrophoretic method can be, for example, capillary electrophoresis (CE) or isoelectric focusing (IEF), and the chromatographic methods can be, for example, HPLC, chromatofocusing, or ion exchange chromatography. In some further embodiments, one or more different kinds of binding or replacement assays may be used for detecting, measuring and/or analyzing the present glycan-based biomarkers. For instance, a competitive lectin/galectin mode may be employed, wherein a pre-labelled glycan competes with a glycan from a sample to be analyzed for a limited number of binding sites offered by the lectin/galectin.

In some embodiments, the biomarkers of the present invention can be detected and/or measured by immunoassays, either in a competitive or sandwich mode. Those skilled in the art know how to carry out such immunoassays. Furthermore, antibodies suitable for this purpose are available commercially. Further suitable antibodies may be produced by methods well known in the art.

In some embodiments, the sensor may comprise a reaction cell or area, which is 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 a photoelectric cell via a light conductor, such as an optical fiber.

In some embodiments, the sensor may comprise a reaction cell that includes at least two sections, one with a glycan-based biomarker responsive/binding reagent, and another without such biomarker-specific binding reagent. Such a setup allows determining a noise level or a baseline of the detector - the signal (electrical or optical) is detected from both sections and the baseline result is divided or subtracted from the specific binding section.

FIG. 10 illustrates a device arrangement for detecting and reporting and presenting data of brain injury in a subject according to an embodiment of the present invention. The illustrated device arrangement 60 comprises a detector 62, an analyzer 65, a display 66 and a memory 67.

In the presented embodiment, the a detector 62 of the illustrated device arrangement 60 for detecting of brain injury in a subject may comprise an at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector 62 is configured to forward digital information comprising said at least one data signal to the analyzer 65. The detector 62 is configured to carry out multiple measurements over time for indicating a trend and detecting and monitoring of brain injury in a subject.

The analyzer 65 of said illustrated device arrangement 60 is configured to receive said digital information comprising said at least one data signal from the detector 62. The analyzer 65 is further configured to generate displayable and/or audible information indicative of brain injury in the subject. The analyzer 65 is configured to receive said measurement data from multiple measurements carried over time from the detector 62. The analyzer 65 is further configured to analyze the measurement data from multiple measurements carried over time and to generate displayable and/or audible information indicative of brain injury in the subject.

The display 66 of the device arrangement 60 for detecting of brain injury in a subject according to the presented embodiment is configured to receive said displayable and/or audible information indicative of brain injury in the subject generated by the analyzer 15. The display 66 according to the presented embodiment is configured to display said displayable and/or audible information indicative of brain injury in the subject.

The memory 67 of the device arrangement 60 for detecting of brain injury in a subject according to the presented embodiment is configured to receive from the analyzer 15, to retrievably store to the memory 67 and to retrieve from the memory 67 e.g. to the analyzer 15 information, which information may include measurement data, digital information comprising said at least one data signal and said displayable and/or audible information indicative of brain injury in the subject. Said information may also comprise historical information indicative of brain injury in the subject.

To be suitable for military use an instrument must be strong, robust, capable of withstanding falls, moisture and extreme temperatures with the ability to test more than one marker or signal and to give a visual or even audio signal in case of danger.

FIG. 11 illustrates an exemplary instrument 70 suitable for use in battlefield for detecting, reporting and presenting data of brain injury in a subject according to an embodiment of the present invention.

The instrument 70 comprises a sampling element 71 which is cable connected or connected directly with a socket to a main unit 72. The sampling unit comprises a detector 73, serial number, memory and needed sensors 74, and a pumping unit 75. Use of a bubble pump is the easiest method for pumping. If the unit is used for several patients at least the pumping unit is replaceable. Disposable part prevents cross-contamination between tested subjects. The sample is body fluid, preferably saliva.

The detector 73 comprises at least one glycan-based biomarker responsive/binding reagent for selectively reacting with or binding to a glycan- based biomarker in the sample, wherein said reacting or said binding generates an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of the glycan-based biomarker in the sample, the level is indicative of a degree of brain injury in the subject. The detector 73 is configured to carry out multiple measurements over time for indicating a trend and detecting and monitoring of brain injury in a subject. Preferably, the glycan-responsive chemistry and reaction is taken place in the in the sampling in the other end of the cable. The sample is not carried to the main unit 72 which processes the signal and produces the output.

The instrument comprises also a transmitter 74 configured to receive said at least one data signal from the detector 73 and to transmit said at least one data signal to an analyzer 75. The analyzer is configured to receive at least one data signal from the detector 72 and to generate displayable and or audible information indicative of brain injury in the subject.

The instrument 70 comprises a display 76 such as an oled display, cursor buttons 77 and a measurement button 78, and a barcode reader 79 or a RFI reader 80 for reliable patient identification. According to a preferable embodiment, the patient ID is obtainable from a patient wristband or badge. The device comprises also USB connection 81 for charging and a rechargeable battery 82 such as internal fixed Lion cell 118650 rechargeable battery. The rechargeable battery is preferably sufficient for 7 days continuous operation.

The instrument is preferable a pocket size instrument. Exemplary dimensions of the main unit, i.e. the device excluding the sampling element are 150 mm, 85 mm and 22 mm for length L, width W, and thickness, D, respectively. The main unit comprises preferably a resistant shell for protecting against falls and harsh conditions.

The instrument 70 comprises a user interface. It wakes by pressing any of the buttons 77,78. Then the instrument asks patient scan for identification. Patient data can be read e.g. from patient RFI tag of RFI wristband sticker or from a wristband barcode. If no reliable patent identification is available, sensor own ID 74 can be read, and the device operates in a simple readout mode (i.e. no trend or patient history).

The measurement starts by pressing the measurement button 78. Results and previous trend are shown when analysis is ready. The trend can be browsed using the cursor buttons 77 (left/right). The device can show previous readings to indicate improvements or worsening of the situation. Patients can also be browsed using the cursor buttons 77 (up/down) when the device is not in operation. The display 76 shows preferably one or more of: patient information, date and time, analyte level as a number, analyte level as a function of time, decrease or increase of the analyte concentration as an arrow, and charge level of the battery.

According to a preferable embodiment the instrument is adapted to o sample saliva at time intervals, o detect two or more glycan-based biomarkers responsive/binding reagent for selectively reacting with or binding to a glycan-based biomarker in the sample of saliva o determine level of the two or more glycan-based biomarkers in the saliva, o display the level of the two or more glycan-based biomarkers as function of time.

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

In one embodiment or the present invention there is provided a method for diagnosing brain injury in a subject, said method comprising: o contacting the subject with a device 20, 30, 36, 37, 40, 50, 60, or an instrument 70 according to the present invention, o directing a sample of a bodily fluid to a detector 12, 22, 62, 72 of said device 20, 30, 36, 37, 40, 50, 60, 70 wherein said contacting and said directing is affected continuously and/or periodically.

In one embodiment or the present invention there is provided a method for detecting brain injury in a subject, said method comprising: o contacting the subject with a device 20, 30, 36, 37, 40, 50, 60, or an instrument 70 according to the present invention, o directing a sample of a bodily fluid to a detector 12, 22, 62, 72 of said device 20, 30, 36, 37, 40, 50, 60, or of said instrument 70, wherein said contacting and said directing is effected continuously and/or periodically.

An exemplary non-limiting flow chart of the method of the present invention is shown in figure 12. The method comprises the following actions

Action 80: Contact a body fluid sample obtained from a subject with a device or an instrument comprising a detector comprising at least one glycan-based biomarker responsive/binding reagent or selectively reacting with or binding to a glycan-based biomarker in a sample of a bodily fluid, said reacting or said binding generating an at least one data signal as measurement data, said at least one data signal having an intensity that correlates to a level of said glycan- based biomarker in said sample, said level being indicative of a degree of brain injury in the subject. Action 81 : Direct the sample to the detector.

Action 82: analyze intensity of the data signal

Action 83 Generate a displayable and/or audible information indicative to brain injury in the subject based on the intensity.

According to the method the contacting and directing is effected continuously and/or periodically.

According to a preferable embodiment the method comprises also the following actions

Action 84: Create a trendline of the at least one glycan-based biomarker level in said sample as a function of time, and

Action 85: Utilize the trendline for evaluating of brain injury in the subject.

According to an aspect of embodiments of the present invention, the measurement of said level of the glycan-based biomarker in said sample of a specific patient is repeated over time, this creating a trend line of a quantitative or a relative biomarker level as a function of time and enable monitoring and making a prognosis for this patient based on said created trend line. The patient- specific measurement results may be stored to a memory and retrieved from said memory. The patient-specific measurement results may be used to comprehend the condition of the patient, i.e. whether a person has suffered brain trauma, whether the condition is getting better, whether the injury is healed, and whether it is safe to release the patient.

The patient-specific trend lines of biomarker level(s) as a function of time may be retrieved and viewed by medical professionals for monitoring the status and progress of the brain tissue damage, and for predicting the outcome. In diagnosing the condition of the patient utilizing said patient-specific measurement results, with certain biomolecules the basal level of which vary between individuals. However, a continuously changing trend in concentration of such molecules over time can serve as correct biomarker of brain tissue destruction. A continuous rise of the biomarker may indicate a progressing injury and may indicate to the doctor that it is not safe to release the patient. A decrease of the biomarker level may indicate that a person has suffered brain trauma, but the condition is getting better. A continuous steady level after a peak may indicate that the injury is healed, and that it is safe to release the patient.

One example of a device for detecting of brain injury in a subject according to the presented embodiment is a microfluidic device, which microfluidic device is characterized by a micrometer scale with increased surface area-to-volume ratio, wherein surface forces become dominant and influence the operation of microfluidic devices. It becomes challenging to mix fluids in microfluidic devices due to limited convection and reliance on diffusion. Typically, POCT devices such as according to embodiments of the present invention are expected to have low cost, portability, user-friendliness, reduced power requirements, and reduced accessories required for their operation. The most power-consuming and expensive components that also increase the footprint of a POCT device are usually the transducer and the pumping components with valves. Size reduction in the pumping system or even eliminating a need for pumps and valves in some embodiments of the present invention would be of great advantage. Microfluidic platforms can be classified based on liquid propulsion principles such as pressure-driven flow.

Another example of a device for detecting of brain injury in a subject according to the presented embodiment is a capillary flow device, also known as test strip. In a capillary flow device, the sample liquid is driven by capillary forces without any applied pressure; such device can be used in some embodiments of the present invention. The liquid sample flow is controlled by the wettability and feature size of the microstructured substrate. All of the required chemicals are pre-applied on or within the test strip. Typically, the test results are optical signals and are usually implemented as a color change in the detection area.

Another example of a device for detecting of brain injury in a subject is an applied lateral pressure device can be used in some embodiments of the present invention. These are pressure-driven devices in which external pumps or built- in micropumps that are applied to drive fluids through the platform. This platform allows a variety of fluidic operations such as mixing, ejection, metering, and separation.

Another example of a device for detecting of brain injury in a subject is an applied transverse pressure device that can be used in some embodiments of the present invention. The applied transverse pressure is based on transverse pressure to propel or stop the fluid flow. The microchannels and reservoirs are made of soft elastomers that can be squeezed or pinched off by external pressure exerted by fluid such as compressed air flowing in adjacent channels. Material of platforms may be produced from soft elastomers such as polydimethylsiloxane (PDMS). In some embodiments of the present invention, fluidic networks on these platforms can be used.

Droplet microfluidics, which involves the generation and manipulation of micrometer-sized emulsion droplets on a microfabricated device, may be in use in some embodiments of the present invention. Droplet microfluidics makes the need for conventional micropumps redundant. A controlled and rapid mixing of fluids in the droplet reactors results in decreased reaction times when compared to continuous-flow microfluidic devices. Droplet microfluidics can also store all the reagents in droplets and eliminate the need for fluidic coupling to external reagent reservoirs. Any scaling up of droplet microfluidic device does not increase device size or complexity. In digital microfluidic devices, unlike the droplet microfluidic devices, it is possible to address each droplet separately in an array of electrodes without any microchannels, and the droplets can be moved based on the electrowetting-ondielectric principle. In some embodiments, the device is a fully integrated droplet-based “digital” microfluidic lab-on-a-chip for performing an assay on bodily fluids.

Centrifugal pumping of a variety of liquids, including bodily fluids, aqueous solutions, solvents, and surfactants, is applicable in the context of the present invention.

Surface acoustic waves (SAWs) traveling over the surface of substrate to propel a liquid droplet in the wave propagation direction, may be used in the device, according to some embodiments of the present invention, to generate, propel, mix, and break up liquid droplets. A SAW device enables enhanced mixing and centrifugation within individual droplets on a microscale.

The device presented herein is highly suitable for performing as a point-of-care testing (POCT) device. Diagnostic technologies can be divided into lab-based testing devices and POCT platforms. POCT is a procedure wherein samples are assayed at or near the subject with the assumption that test results will be available instantly or in a short timeframe to assist caregivers with immediate diagnosis and/or clinical intervention”. Some of the features of POCT include rapid turnaround, communication of results to guide clinical decisions, completion of testing, and follow-up action in the same clinical encounter. A rapid turnaround of results is important for the test results to influence clinical decisions such as triage, referral, and decision to discharge the patient. Rapid can refer to seconds, minutes, or a few hours while the patient is still present at the site. A rapid test result that does not also have a mechanism for rapid reporting of the result to the care providers and its translation to a treatment plan is highly unlikely to have any significant impact on public health outcomes. POCT can overcome this problem by making it convenient for both patients and care providers by completing the diagnostic process “in the same clinical encounter” and allowing a treatment plan to be implemented on the same day. An early diagnosis based on POCT can also enable the caretaker to start the treatment process earlier and thereby increase chances of preventing or curing the medical condition.

Some embodiments of the present invention encompass POCT which include lab-on-a-chip device, total analysis microsystems, and fluidic cartridges/lateral flow (LF) strips. These devices may have an integrated or dedicated readout and display elements.

The footprint of POCT platforms can range from a small chip to a table top system that might be placed in doctors’ offices, hospitals, or ambulances and ideally is capable of being operated by minimally trained users. POCT systems should essentially require minimal operator intervention, have required reagents, and preferably have automated processing steps that are integrated within the system.

According to an aspect of embodiments of the present invention, there is provided a kit for continuous diagnosis of brain injury in a subject, which can be carried out by a professional or a layman at any location and facility.

As is apparent to a skilled person, a lectin array kit can be used. In one embodiment, the kit is used for biotinylated samples containing glycans for direct detection on the array via a Cy3 equivalent dye-conjugated Biotin-Streptavidin complex. In some embodiments, a sandwich-based method is used for antibody detection of glycans captured on the array.

In some embodiments, the biomarker detection kit comprises HRP protein and a fluorescent light may be employed in order to detect the biomarker in a body fluid and to indicate the quantity of the biomarker in percentage. This may be incorporated into a portable application that indicates the severity of brain damage on a scale comprising, but not limited to, none, mild, moderate and severe. In another embodiment, an analogous yes/no reply is received. These examples do not exclude other possible embodiments.

In some embodiments, the present invention provides use of at least one antibody in a kit or in a device to detect brain damage, where the antibody may be a polyclonal or a monoclonal antibody of any species, or a fragment thereof, either enzymatically cleaved or recombinantly produced, or a humanized antibody, and where the antibody recognizes and binds glycan, glycoprotein, peptidoglycan, proteoglycan, glycolipid, protein, small molecule, lectin, or antibody of another species (generally 'antigens'). All embodiments, details, advantages, and the like of the present device also apply to a device for use in different aspects and embodiments of the present invention. Also, all embodiments, details, advantages, and the like of the present methods apply to the present kit, and vice versa. In particular, one or more compounds, compositions, or reagents disclosed as suitable for carrying out the present methods may be comprised in the present kit. Likewise, anything disclosed with reference to the kit, apply to the present methods as well.

Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. Non-limiting examples of advantages associated with the present glycan-based biomarkers include that they are brain-tissue specific, able to cross the blood- brain barrier into the bloodstream within minutes of injury, and can be detected using a point-of-care blood test or other body fluids. Furthermore, the biomarkers may either increase or decrease following the injury, but nevertheless they are in correlation with the severity of the injury. Preferably, the present biomarkers may correlate with injury magnitude, survivability, and/or neurologic outcome, or they may be indicative of the extent of neuronal and glial cell loss, axonal, and vascular damage. The present biomarkers can significantly add to the current diagnostic palette for brain damage. It is expected that during the life of a patent maturing from this application many relevant continuous brain injury diagnosis devices will be developed and the scope of the term saliva-based brain injury diagnosis device is intended to include all such new technologies a priori.

As used herein the term “about” refers to ± 10 %. The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of means “including and limited to”.

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the phrases "substantially devoid of and/or "essentially devoid of in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1 , 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases "substantially devoid of and/or "essentially devoid of in the context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process/method step, or a certain property or a certain characteristic, or a process/method wherein the certain process/method step is effected at less than about 5, 1 , 0.5 or 0.1 percent compared to a given standard process/method, or property or a characteristic characterized by less than about 5, 1 , 0.5 or 0.1 percent of the property or characteristic, compared to a given standard.

The term “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The words “optionally” or “alternatively” are used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

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

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the terms “process” and "method" refer to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, material, mechanical, computational and digital arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments unless the embodiment is inoperative without those elements.

It is to be understood that the above description and the accompanying Figures are only intended to teach the best way known to the inventors to make and use the invention. It will be apparent to a person skilled in the art that the inventive concept can be implemented in various ways. The above-described embodiments of the invention may thus be modified or varied, without departing from the invention, as 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 claims and their equivalents.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental and/or calculated support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Gold sols may be prepared for use in immunoassay from commercially available colloidal gold, and an antibody preparation. For example, colloidal gold G20 (20 nm particle size, supplied by Janssen Life Sciences Products) is adjusted to pH 7 with 0.22 pm filtered 0.1 M K2CO3, and 20 ml is added to a clean glass beaker. 200 pi of antibody, prepared in 2 mM borax buffer pH 9 at 1 mg/ml, and 0.22 pm filtered, is added to the gold sol, and the mixture stirred continuously for two minutes. 0.1 M K2CO3 is used to adjust the pH of the antibody gold sol mixture to 9, and 2 ml of 10 % (w/v) BSA is added.

The antibody-gold is purified in a series of three centrifugation steps at 12000 g, 30 minutes, and 4 °C, with only the loose part of the pellet being resuspended for further use. The final pellet is resuspended in 1 % (w/v) BSA in 20 mM Tris, 150 mM NaCI pH 8.2.

Dye sols may be prepared from commercially available hydrophobic dyestuffs such as Foron Blue SRP and Resolin Blue BBLS. For example, fifty grams of dye is dispersed in 1 liter of distilled water by mixing on a magnetic stirrer for 2- 3 minutes. Fractionation of the dye dispersion can be performed by an initial centrifugation step at 1500 g for 10 minutes at room temperature to remove larger sol particles as a solid pellet, with the supernatant suspension being retained for further centrifugation.

The suspension is centrifuged at 3000 g for 10 minutes at room temperature, the supernatant being discarded, and the pellet resuspended in 500 ml distilled water. This procedure is repeated a further three times, with the final pellet being resuspended in 100 ml distilled water.

The spectra of dye sols prepared as described above can be measured, giving lambda-max values of approximately 657 nm for Foron Blue, and 690 nm for Resolin Blue. The absorbance at lambda-max, for 1 cm path length, is used as an arbitrary measure of the dye sol concentration.

Latex (polymer) particles for use in immunoassays are commercially available. These can be based on a range of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid and polyacrolein. The monomers used are normally water-insoluble and are emulsified in aqueous surfactant so that monomer micellae are formed, which are then induced to polymerize by the addition of initiator to the emulsion. Substantially spherical polymer particles are produced.

Colored latex particles can be produced either by incorporating a suitable dye, such as anthraquinone (yellow), in the emulsion before polymerization, or by coloring the pre-formed particles. In the latter route, the dye should be dissolved in a water-immiscible solvent, such a chloroform, which is then added to an aqueous suspension of the latex particles. The particles take up the non- aqueous solvent and the dye and can then be dried. Preferably such latex particles have a maximum dimension of less than about 0.5 micron.

Colored latex particles may be sensitized with protein, and in particular antibody or lectin, to provide selective binding reagents as described in the foregoing. For example, polystyrene beads of about 0.3 micron diameter, (supplied by Polymer Laboratories) may be sensitized with an anti-glycan-based biomarker antibody, in the process described below: 0.5 ml (12.5 mg solids) of suspension is diluted with 1 ml of 0.1 M borate buffer pH 8.5 in an Eppendorf vial. These particles are washed four times in borate buffer, each wash consisting of centrifugation for 3 minutes at 13000 rpm in an MSE microcentrifuge at room temperature. The final pellet is resuspended in 1 ml borate buffer, mixed with 300 pg of anti-glycan-based biomarker antibody, and the suspension is rotated end-over-end for 16-20 hours at room temperature. The antibody-latex suspension is centrifuged for 5 minutes at 13000 rpm, the supernatant is discarded, and the pellet resuspended in 1 .5 ml borate buffer containing 0.5 mg bovine serum albumin. Following rotation end-over-end for 30 minutes at room temperature, the suspension is washed three times in 5 mg/ml BSA in phosphate buffered saline pH7.2, by centrifugation at 13000 rpm for 5 minutes. The pellet is resuspended in 5 mg/ml BSA/5 % (w/v) glycerol in phosphate buffered saline pH 7.2 and stored at 4 °C until used.

Protein may be coupled to dye sol in a process involving passive adsorption. The protein may, for example, be a lectin or an antibody preparation such as anti-glycan-based biomarker antibody prepared in phosphate buffered saline pH 7.4 at 2 mg/ml. A reaction mixture is prepared which contains 100 pi antibody solution, 2 ml dye sol, 2 ml 0.1 M phosphate buffer pH 5.8- and 15.9-ml distilled water. After gentle mixing of this solution, the preparation is left for fifteen minutes at room temperature. Excess binding sites may be blocked by the addition of, for example, bovine serum albumin: 4 ml of 150 mg/ml BSA in 5 mM NaCI pH 7.4 is added to the reaction mixture, and after 15 minutes incubation at room temperature, the solution is centrifuged at 3000 g for 10 minutes, and the pellet resuspended in 10 ml of 0.25 % (w/v dextran/0.5 % w/v lactose in 0.04 M phosphate buffer). This antibody-dye sol conjugate is best stored in a freeze- dried form.

In an exemplary embodiment of the present invention is a model of assaying fetuin and asialofetuin. Fetuin is an abundant glycoprotein in fetal serum, and asialofetuin is its asialylated form. Lectins or a lectin that selectively binds to the glycan part of fetuin or asialofetuin is permanently immobilized on solid matrix. Fetuin or asialofetuin in solution is brought into contact with the lectin and the binding reaction is subsequently taking place. Thereafter, the reaction compartment is washed and a labeled conjugate is added. The conjugate binds to the fetuin or asialofetuin that was captured on the surface in the preceding phase.

Alternatively, fetuin or asialofetuin is first contacted with the labeled conjugate to form a complex. Thereafter the complex is brought into contact with the immobilized lectin(s). The conjugate comprises a fetu in-specific or asialofetuin- specific antibody which is coupled to a detectable label. The detectable label is one of those presented in the text, preferably a colloidal/particulate matter which enables visual detection.

Evidence on the ability to detect multiple markers simultaneously with a lectin- based qlvcan detection method

An example on simultaneous detection of multiple biomarkers in one assay is provided below. Lectins used in the example are listed in Table 1 , and a test strip is shown in Fig 13. The experiments were performed as follows:

Preparation and assembly of the strips: Sample pads were blocked, and after drying, the strips were assembled: nitrocellulose membrane (central part of the stirp), conjugate pad (upper part of the strip), and wicking pad (lower part strip) were attached on backing card with a 2 mm overlap and the broad sheet was cut in 5 mm wide individual assay strips.

Functionalization of the reaction area: Two capture lectins were pipetted spatially separated on the nitrocellulose membrane (central part of the strip) and dried to permanently adsorb the lectins on the membrane.

Sample analysis: A sample from a TBI-patient was pipetted on the sample pad and was allowed to migrate through the membrane. A gold-particle-conjugated second lectin (tracer) was added and migrated. The strips were washed by dipping the conjugate pad end in a well containing washing buffer and the migrating buffer flushed the excess and unbound gold-conjugate away from the reaction area. Two dots appeared in the reaction area indicating that two different glycans were present in the sample and they could be detected in one assay. Lectins used in the example are listed in Table 1 . Table 1.

The specificities of the lectins are clearly different, the most remarkable difference being the specificity of SNA-I to sialic acid whereas ECA doesn’t have this specificity; sialic acid will actually prevent ECA binding and it is preferentially binding galactose-glucoseamine.

Confirmation by mass spectrometry analysis: Glycans were isolated and purified from saliva samples of 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 was translated into glycan composition by matching the peak value to glycan mass spectrometry databases. The relative abundance of each glycan in the sample was calculated from the peak height (area) and the relative abundancies were compared between the TBI patients and healthy controls.

At least two prominent types were found to be dominant in the TBI-samples compared to healthy samples: 1) Hybrid-type N-glycans with x- Galactose - NANA terminal and 2) Acidic N-glycan x- Glucoseamine - Galactose terminal. These results match with the lectin specificities of the lateral flow example.

Summary : Mass spectrometry proved the presence of the two glycans species in the sample, and it was proved in the lateral flow example the ability to detect them simultaneously.

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