Protein-based biosensor suitable for MRI imaging

Abstract

The invention relates to the field of medicine. In particular, the present invention relates to a protein-based biosensor comprising a substrate binding protein that binds to an MRI contrast agent by a chelating agent. More specifically, the present invention relates to a glutamic acid biosensor comprising a glutamic acid binding protein bound to an MRI contrast agent, characterized in that the glutamic acid binding protein comprises SEQ ID NO: 10, preferably SEQ ID NO: 5, and a sequence encoding a fluorescent protein.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to a protein-based biosensor comprising a substrate-binding protein bound to a MRI contrast agent by a chelating agent. The present invention can be used to detect substrates that are difficult to access with optical imaging techniques. For example, it can be used to quantify neurotransmitters in the brain, thus facilitating the development of diagnostic methods for neurological disorders. BACKGROUND

[0002] Neurotransmitters are molecules that amplify, transmit and transduce signals in cells and play a key role in information transmission throughout the nervous system. Hundreds of such chemical substances have been discovered over the last century and are still being identified and studied to clarify their role on brain health. These substances have been observed to affect many functions, including mood, thought, memory, learning and movement. Therefore, disturbances in neurotransmitter homeostasis have been linked to a plethora of neurological and neurodegenerative diseases, including epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, depression and importantly stroke.

[0003] Stroke is the second leading cause of death worldwide and the leading cause of disability. In recent decades, improvements in stroke patient management, particularly in developed countries, have contributed to a decrease in mortality and morbidity. However, the incidence of brain stroke has not shown the same trend, with a continuous increase in developed countries. The World Stroke Organization recently published statistics showing that, in the last 20 years, stroke events have increased by 70% and stroke deaths by 43%, with an estimated global cost of over 721 billion dollars. In this context, finding targets that enable early diagnosis and preventive treatment in high-risk subjects is an important goal.

[0004] The endothelium has a key role in cerebrovascular diseases. The blood-brain barrier (BBB) is a highly selective, semi-permeable boundary of endothelial cells that prevents the non-selective passage of solutes in the circulating blood into the extracellular fluid of the central nervous system where neurons are located. It represents a biological and mechanical barrier between the brain and the vascular compartment.

[0005] The BBB endothelium is essential for the regulation of tone and vascular homeostasis. In healthy vascular endothelium, these functions are regulated by a variety of mechanisms, such as vasodilation, anti-oxidant, anti-inflammatory, inhibition of adhesion and leukocyte migration, inhibition of smooth muscle fibres, inhibition of platelet adhesion and aggregation, and anti-coagulant and fibrinolytic effects. Changes in these mechanisms usually reflect the presence of endothelial dysfunction. For example, atherosclerosis, which is associated with endothelial dysfunction, can be easily diagnosed by ultrasound or by several biomarkers of oxidative stress, adhesion molecule isoforms, inflammation, endothelial progenitor cells, etc. However, these markers of endothelial dysfunction are not associated with the development and severity of stroke.

[0006] More recently, it has been demonstrated that the BBB also exhibits other biological effects related to glutamate transport. Glutamate excitotoxicity refers to the process by which over-activation of glutamate receptors leads to neuronal damage and death. Glutamate is the major excitatory neurotransmitter in the brain and plays an important role in many physiological processes. However, when glutamate levels are too high, it can cause over-stimulation of neurons, leading to the production of reactive oxygen species (ROS) and calcium overload, which ultimately leads to cell death. Glutamate excitotoxicity is thought to play a role in a variety of neurological disorders, including stroke, multiple sclerosis, traumatic brain injury, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. In these cases, the release of excess glutamate leads to a surge of calcium ions into neurons, triggering a cascade of events that can ultimately lead to neuronal death.

[0007] The concentration of extracellular glutamate is tightly regulated to maintain physiological concentrations by sodium-dependent transporters, also known as excitatory amino acid transporters: EAATs. Following neuronal excitation, glutamate in the synaptic cleft is typically taken up by EAATs located on astrocytes, which bind and clear neurotransmitters for subsequent processing and recycling. Alternatively, when extracellular concentrations are elevated, sodium-dependent transporters located on the abluminal surface of brain capillaries bring glutamate from the extracellular space into endothelial cells. When glutamate accumulates in endothelial cells to concentrations that exceed plasma levels, it is released into the circulation via facilitated diffusion through the luminal side into the bloodstream. In this regard, endothelial regulation of glutamate in the central nervous system (CNS) can occur despite the presence of an unfavorable concentration gradient from the CNS to the plasma.

[0008] Based on the ability of astrocytes and endothelial cells to efficiently remove and metabolize glutamate, and because they are less susceptible to ischemic conditions than neurons, it can be hypothesized that both cellular components of the neurovascular unit should be able to protect neurons from glutamate excitotoxicity. However, experimental and clinical evidence suggests that these endogenous homeostatic mechanisms are not able to prevent the effects of glutamate following ischemic stroke.

[0009] Clinical studies have shown that glutamate is critical to neuronal damage following ischemic stroke. In this regard, ischemic stroke patients exhibit higher blood and cerebrospinal fluid (CSF) glutamate levels at admission than control subjects, indicating that glutamate concentrations above 200 μΜ in plasma are an important predictor of 48-hour progression of neuronal damage with a sensitivity of 85% and a specificity of 97%. In addition, high levels of glutamate in plasma persist for at least 24 hours in association with early neurological deterioration, while in patients with stable stroke, glutamate levels decrease to normal values in less than 6 hours from onset. All these clinical data have for the first time demonstrated the key role of glutamate in stroke pathophysiology and have provided a source of new strategies for the study of novel protective therapies based on the inhibition of glutamate toxicity. Therefore, controlling the increase of extracellular glutamate can provide neuroprotection by blocking the signal at the upstream converging point of multiple downstream death signaling cascades. Recently, it was demonstrated that a glutamate scavenger capable of reducing glutamate concentrations in blood after intravenous injection, effectively mitigated the excitotoxicity of glutamate in an ischemic stroke model, suggesting a potential neuroprotective effect of glutamate-reducing drugs.

[0010] Reducing extracellular excess glutamate in the brain is a key goal to eliminate excitotoxicity and reduce neuronal death and neurodegeneration in many neurological diseases.

[0011] Due to the importance of the homeostasis of glutamate and many other neurotransmitters in neurological and neurodegenerative disorders, a great effort has been made to develop neurotransmitter sensors that can be used to track and quantify neurotransmitters in vivo and in vitro. In particular, glutamate sensing has become the scope of a variety of basic research in biology. Several protein-based glutamate sensors have been developed and tested to measure glutamate concentrations at the cell surface.

[0012] Fluorescent sensors are molecules that emit light when in contact with a specific analyte. In the last decade, a new family of fluorescent sensors has been developed based on fluorescent proteins. These proteins fold in the presence of their targeted analyte, forming a new tertiary structure that in turn generates a fluorescent signal (usually achieved with the help of green fluorescent protein). In this regard, glutamate sensing has become the focus of many research groups, who have developed several glutamate-sensing proteins, such as IgLuSNFR or IgLu1-3. The main application of these sensors is the study of neuronal activity and synaptic function using in vivo fluorescence imaging. Although the results of these studies have a very high value in the field of neurological diseases, However, fluorescence imaging has limitations (it cannot visualize deep tissues, nor can it image through bone) Figure 1 This hinders the translation of fluorescent sensors to clinical applications.

[0013] Magnetic resonance imaging (MRI) is a non-invasive imaging technique widely used in clinical research of organs, tissues, and bones. Compared to other clinically used imaging techniques, such as positron emission tomography (PET) and X-ray-based computed tomography (CT), MRI offers higher resolution and is the preferred technique for diagnosing neurological diseases. The main drawback of MRI compared to PET is its lower sensitivity. PET, using radioactive tracers, can generate signals at extremely low concentrations (pM level), making it generally more suitable for studying cellular functions such as cell metabolism and oxygen consumption. This drawback can be partially mitigated by using contrast agents.

[0014] MRI contrast agents are currently used to increase the signal-to-noise ratio, providing images with a very high resolution (approximately 1 μm), which is unattainable by other imaging techniques. Among MRI contrast agents, gadolinium (Gd)-containing agents are the most widely used clinically, with nine types currently available on the market. Gadopiclenol is the latest Gd-containing contrast agent approved by the FDA in 2022. In 2017, the FDA raised significant concerns about the use of Gd-containing contrast agents: when gadolinium is physically bound to a linear chelating agent to prepare the contrast agent, Gd can accumulate in the brain. Studies have now demonstrated that cyclic chelating agents can stably bind Gd, and the safety of such contrast agents has been verified. The main advantage of commercially available Gd-containing contrast agents is that they enhance images of voxels containing Gd, making the corresponding areas brighter. This effect has many clinical applications, most commonly in detecting local lesions (tumors, abscesses, metastases, infarctions); vascular imaging (using angiography or venography); and calculating perfusion parameters. Other types of MRI contrast agents are based on nanoparticles and can also generate MRI signals. They are currently used in multiple clinical trials, with four already commercially available. The main application of Gd-containing contrast agents and nanoparticles is to visualize localized functional disorders in vivo through the abnormal aggregation of these contrast agents in target areas. However, recent research in this field has demonstrated that MRI has the ability to quantitatively measure different molecular concentrations, making it a practical analytical tool for diagnosis.

[0015] Because there is an unmet medical need to develop clinically applicable strategies for quantifying neurotransmitters to advance diagnostic methods for neurological disorders, this invention focuses on addressing this problem, and provides a protein-based biosensor suitable for MRI that uses glutamate as a proof-of-concept substrate. Summary of the Invention

[0016] Brief description of the invention This invention relates to protein-based biosensors comprising substrate-binding proteins that bind to MRI contrast agents via chelating agents.

[0017] The inventors of this invention recognized the need to develop clinically applicable strategies for detecting inaccessible substrates in tissues, particularly for the quantitative detection of neurotransmitters in the brain. They hypothesized that this problem could be addressed by generating protein-based biosensors comprising a substrate-binding protein that binds to an MRI contrast agent via a chelating agent. Upon contact with the substrate, this biosensor induces a conformational change, thereby altering the MRI signal generated by the contrast agent carried by that structure. Figure 2 ).

[0018] Using glutamate as a proof-of-concept substrate, the inventors constructed an MRI glutamate sensor by: using DOTA as a chelating agent, and labeling Gd onto the glutamate transporter (Glt1) via a stable amide bond between lysine residues and DOTA. Figure 6 This sensor is called the MRI glutamate sensor. The fluorescent glutamate sensor iGLuSnFR was used as a starting point because, in addition to containing the Glt1 domain, it also has a fluorescent protein reporter gene (GFP) that can be used for preliminary testing. They also generated two additional sensors using the same synthetic strategy: GOT-DOTA-Gd: A glutamate sensor in which the substrate-binding protein is glutamate oxaloacetate transaminase (GOT), an enzyme involved in glutamate metabolism.

[0019] BSA-DOTA-Gd: A bovine serum albumin (BSA) sensor that does not have a glutamate-specific binding domain but has a BSA-specific binding domain.

[0020] The protein sequences used to construct the three biosensors are summarized in Table 1.

[0021] Table 1: Protein sequences used to construct biosensors.

[0022]

[0023] To evaluate the sensing characteristics of the sensor, the inventors measured the MRI signals generated by the sensor in the presence and absence of a substrate. As expected, glutamate caused a shift in the MRI signal generated by the MRI glutamate sensor. Figure 8 and Figure 7 More precisely, it reduces MRI signal in a concentration-dependent manner. Figure 8 These results demonstrate that existing biosensors can be modified for use in MRI detection.

[0024] Similarly, glutamate also shifted the MRI signal of the GOT-DOTA-Gd sensor, although the effect was less than that observed with the MRI glutamate sensor. Figure 4 Importantly, BSA can also shift the MRI signal of the BSA-DOTA-Gd sensor. Other embodiments: This further demonstrates the applicability of the synthesis strategy to other substrates.

[0025] These results demonstrate that the synthetic strategy is applicable to different types of substrate-binding proteins; in particular, they suggest that substrate-specific transporters may be more suitable for synthesizing biosensors than other substrate-binding proteins. Compared to the sensor, the GOT sensor exhibits a weaker signal response than the glutamate sensor, possibly due to the latter undergoing a more significant conformational change in the presence of glutamate.

[0026] Therefore, a first embodiment of the present invention relates to a protein-based biosensor comprising a substrate-binding protein that binds to an MRI contrast agent via a chelating agent.

[0027] In a preferred embodiment, the protein-based biosensor comprises a neurotransmitter-binding protein that binds to an MRI contrast agent via a chelating agent.

[0028] In a preferred embodiment, the protein-based biosensor comprises a glutamate-binding protein that binds to an MRI contrast agent via a chelating agent.

[0029] In a preferred embodiment, the substrate-binding protein binds to the MRI contrast agent via a cyclic chelator.

[0030] In a preferred embodiment, the chelating agent is dodecanetetraacetic acid (DOTA).

[0031] In a preferred embodiment, the MRI contrast agent is a gadolinium-based MRI contrast agent.

[0032] In a preferred embodiment, the protein-based biosensor comprises a glutamate-binding protein (DOTA-Gd) that binds to gadotate, wherein the gadotate is composed of dodecanetetraacetic acid (DOTA, as a chelating agent) and gadolinium.

[0033] In a preferred embodiment, the protein-based biosensor is characterized by dodecanetetraacetic acid (DOTA) binding to glutamate-binding proteins via its amide bond with the lysine amino acid.

[0034] In a preferred embodiment, the characteristic sequence of the glutamate-binding protein is SEQ ID NO: 1.

[0035] A second embodiment of the present invention relates to a nanocarrier comprising any of the above-described protein-based sensors, characterized in that its surface is modified with ligands capable of binding to the blood-brain barrier.

[0036] In a preferred embodiment, the nanocarrier is characterized in that the ligand is selected from the list including antibodies, peptides, aptamers, or small molecules having affinity for at least one receptor at the blood-brain barrier, said receptor being selected from the list including VCAM-1, E-selectin, PECAM-1, CD44, ICAM-1, transferrin receptor, LDL-associated protein, leptin receptor, insulin-like growth factor receptor, diphtheria toxin receptor, neonatal Fc receptor, or nicotinic acetylcholine receptor.

[0037] In a preferred embodiment, the nanocarrier is characterized in that the nanocarrier is a nanoparticle selected from the group consisting of polymer nanoparticles, lipid nanoparticles, or inorganic nanoparticles.

[0038] A third embodiment of the invention relates to the use of any of the above-described methods for quantifying or monitoring substrates or neurotransmitters in vivo based on protein biosensors or nanocarriers, preferably in the brain or blood-brain barrier.

[0039] In a preferred embodiment, protein-based biosensors or nanocarriers are used to diagnose neurological or neurodegenerative diseases.

[0040] One of the main applications of the proposed MRI biosensor is to measure glutamate transport across the brain barrier (BBB). Since the endothelial cells at the BBB are the final barrier preventing excess glutamate from entering the bloodstream from the brain parenchyma, the BBB's ability to transport glutamate into the bloodstream or retain it in the brain parenchyma can serve as an excellent target for identifying patients at risk for neurological disorders. The inventors considered several strategies for delivering the sensor to the BBB using nanoparticles; for example, using BBB-targeting nanoparticles to facilitate drug delivery to the brain. Because the glutamate sensor has been designed to measure glutamate at the BBB, rather than in the brain parenchyma, the nanoparticles used for diagnostics do not need to cross the BBB, avoiding one of the major barriers to brain delivery and also avoiding interaction with neural cells. They designed a nanoparticle in which the sensor is encapsulated inside the particle, and the outer surface of the particle is modified with molecules that are BBB-targetingly active.

[0041] The nanoparticle composition plays a key role in two important features of brain imaging: (i) protecting the sensor from degradation and rapid removal of the bleeding flow after intravenous injection, and (ii) delivering the sensor to the BBB.

[0042] In this regard, the first point is achieved by encapsulating the sensor within nanoparticles. Nanoparticles typically consist of one or a combination of the following categories: - Polymers: Biocompatible and biodegradable polymers, such as polyesters (PLA, PLGA, PLA-PLGA, PCL); polysaccharides; polyethylene glycol, polyglycerol, polystyrene; -Lipids and oils; -Inorganic nanoparticles (silicon, iron, gold, silver); The second point regarding active brain targeting is achieved by modifying the surface of nanoparticles with targeting molecules. The targeting component is an antibody, peptide, aptamer, or small molecule with affinity for a receptor at the brain border (BBB). Cellular receptors used for brain targeting include: VCAM-1, E-selectin, PECAM-1, CD44, ICAM-1, transferrin receptor, LDL-associated protein, leptin receptor, insulin-like growth factor receptor, diphtheria toxin receptor, neonatal Fc receptor, and nicotinic acetylcholine receptor.

[0043] A fourth embodiment of the present invention relates to an in vitro detection method for determining the presence of a substrate or neurotransmitter in a biological sample obtained from a subject, the method comprising: a. Contact any of the above protein-based biosensors with a biological sample obtained from a subject suspected of containing the substrate or neurotransmitter, and b. If an MRI signal is detected to be deviated from the MRI signal when there is no substrate or neurotransmitter, it indicates that there is a substrate or neurotransmitter in the sample.

[0044] In the context of this invention, the following terms are defined: The term "comprising" refers to anything that includes, but is not limited to, the content following the word "comprising". Therefore, the use of the term "comprising" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0045] "Consisting of" means "composed of" and includes only what follows the phrase. Therefore, "consisting of" indicates that the listed elements are required and that no other elements exist.

[0046] Figure 2 The fifth aspect of the present invention relates to a glutamate biosensor (hereinafter referred to as the glutamate biosensor of the fifth aspect of the present invention), comprising a glutamate-binding protein that binds to an MRI contrast agent, characterized in that the glutamate-binding protein comprises SEQ ID NO: 10 and a sequence encoding a fluorescent protein, or both thereof. Preferably, SEQ ID NO: 10 is SEQ ID NO: 5 or SEQ ID NO: 9, more preferably SEQ ID NO: 5.

[0047] In a preferred embodiment, the glutamate-binding protein comprises SEQ ID NO: 10 and SEQ ID NO: 6, or both. Preferably, SEQ ID NO: 10 is SEQ ID NO: 5 or SEQ ID NO: 9, more preferably SEQ ID NO: 5.

[0048] In a more preferred embodiment, the glutamate-binding protein comprises SEQ ID NO: 10 and SEQ ID NO: 6, or both, separated by a sequence comprising 108 to 128 amino acids. Preferably, SEQ ID NO: 10 is SEQ ID NO: 5 or SEQ ID NO: 9, more preferably SEQ ID NO: 5.

[0049] In one or even more preferred embodiments, the glutamate-binding protein comprises or consists of SEQ ID NO: 10 and SEQ ID NO: 6, separated by a sequence comprising SEQ ID NO: 7. Preferably, SEQ ID NO: 10 is SEQ ID NO: 5 or SEQ ID NO: 9, more preferably SEQ ID NO: 5.

[0050] In a most preferred embodiment, the glutamate-binding protein comprises or consists of SEQ ID NO: 11. Preferably, SEQ ID NO: 11 is SEQ ID NO: 8 or SEQ ID NO: 13, more preferably SEQ ID NO: 8.

[0051] In one, or even the most preferred, embodiment, the glutamate-binding protein comprises or consists of SEQ ID NO: 12. Preferably, SEQ ID NO: 12 is SEQ ID NO: 4 or SEQ ID NO: 14, more preferably SEQ ID NO: 4.

[0052] It should be noted that the terms "comprising" or "consisting of" in this invention are also intended to cover sequences that are highly identical to and have comparable biological properties to those sequences mentioned in the above embodiments. This includes sequences that have more than 95% sequence identity with the aforementioned sequences. In particular, this includes SEQ ID NO: 9, which has more than 95% identity with SEQ ID NO: 5 and corresponds to a known variant of the fluorescent glutamate sensor used as a starting point in this invention (see [link to original document]). Figure 1 ).

[0053] In another preferred embodiment, the MRI contrast agent comprises a chelating agent that binds to a chemical element suitable for MRI.

[0054] In a more preferred embodiment, the chemical element suitable for MRI is selected from gadolinium, manganese, cobalt, dysprosium, and / or chromium. Preferably, the chemical element suitable for MRI is gadolinium.

[0055] It should be noted that the inventors of this invention have used the chelating agent DOTA as a proof of concept. However, other macrocyclic chelating agents with similar structures and the ability to form stable complexes with metal ions can also be used in this invention. These include 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraphosphonic acid (NOTP), ethylenediaminetetraacetic acid (EDTA), hydrazine nicotinamide (HYNIC), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), and 1,4,7-triazacyclononane-1,4-bis(methylenephosphonic acid) (NOTB).

[0056] Therefore, in another more preferred embodiment, the chelating agent is selected from: dodecanetetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraphosphonic acid (NOTP), ethylenediaminetetraacetic acid (EDTA), hydrazine nicotinamide (HYNIC), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), and 1,4,7-triazacyclononane-1,4-bis(methylenephosphonic acid) (NOTB). Preferably, the chelating agent is DOTA.

[0057] Preferably, the glutamate-binding protein is composed of SEQ ID NO: 4, the MRI contrast agent contains a chelating agent that binds to a chemical element suitable for MRI, the chemical element suitable for MRI being gadolinium, and the chelating agent being DOTA.

[0058] In another preferred embodiment, the MRI contrast agent or chelating agent binds to the glutamate-binding protein by forming an amide bond with the lysine amino acid. Preferably, at least 50%, at least 90%, at least 93%, at least 95%, at least 97%, at least 99%, or 100% of the lysine amino acid in the glutamate-binding protein is bound to the MRI contrast agent or chelating agent.

[0059] Preferably, the glutamate-binding protein is composed of SEQ ID NO: 4, the MRI contrast agent comprises a chelating agent that binds to a chemical element suitable for MRI; the chemical element suitable for MRI is gadolinium, and the chelating agent is DOTA; the MRI contrast agent or chelating agent binds to the glutamate-binding protein by forming an amide bond with a lysine amino acid, and at least 90%, at least 93%, at least 95%, at least 97%, at least 99%, or at least 100% of the lysine amino acids of the glutamate-binding protein are bound to the MRI contrast agent or chelating agent.

[0060] The sixth aspect of the present invention relates to a glutamate biosensor (hereinafter referred to as the glutamate biosensor of the sixth aspect of the present invention), comprising a glutamate-binding protein that binds to an MRI contrast agent, characterized in that the glutamate-binding protein comprises SEQ ID NO: 2. Preferably, the glutamate-binding protein comprises SEQ ID NO: 2.

[0061] In another preferred embodiment, the MRI contrast agent comprises a chelating agent that binds to a chemical element suitable for MRI.

[0062] The chelating agent and the chemical elements suitable for MRI are the same as those described in the fifth aspect. Therefore, all relevant embodiments mentioned in the description of the first aspect are applicable here.

[0063] It should be noted that the terms "comprising" or "consisting of" in this invention are also intended to cover sequences that are highly identical to and have comparable biological properties with the sequences mentioned in the above embodiments. This includes sequences that exhibit more than 95% sequence identity with SEQ ID NO: 2.

[0064] The seventh aspect of the present invention relates to a method for obtaining a glutamate biosensor (hereinafter referred to as the method of the seventh aspect of the present invention), wherein the method comprises: a. Provides glutamate-binding proteins and chelating agents that bind to chemical elements suitable for MRI, and b. Reacting the glutamate-binding protein with the chelating agent of step (a), wherein the reaction is carried out under conditions suitable for the formation of a covalent bond between the glutamate-binding protein and the chelating agent; The glutamate-binding protein is characterized in that it contains SEQ ID NO: 5 and a sequence encoding a fluorescent protein.

[0065] The glutamate-binding protein and the MRI-suitable chemical elements are the same as those described in the fifth aspect. Therefore, all relevant embodiments mentioned in the description of the first aspect are applicable here.

[0066] It should be noted that the inventors of this invention have used the chelating agent DOTA as a proof of concept. However, other chelating agents containing suitable chemical groups can also be used in this invention, which can react with glutamate-binding proteins under conditions where an amide bond is formed between the terminal amine group of a lysine residue suitable for glutamate-binding proteins and the chelating agent. This includes dodecanetetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), hydrazinenicotinamide (HYNIC), satoreotidetrizoxetan (NODAGA), 2-[11-(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-yl]acetic acid (CB-TE2A) and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), or their salts, solvates, stereoisomers or esters.

[0067] Therefore, in a preferred embodiment, the chelating agent comprises a chemical group suitable for reacting with an amine group.

[0068] In a more preferred embodiment, the chelating agent is selected from: dodecanetetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), hydrazinenicotinamide (HYNIC), satoreotidetrizoxetan (NODAGA), 2-[11-(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-yl]acetic acid (CB-TE2A), and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), or salts, solvates, stereoisomers, or esters thereof. Preferably, the chelating agent is DOTA or a salt, solvate, stereoisomer, or ester thereof.

[0069] In the context of this invention, the term "salt" must be understood to mean any form of compound used in this invention, wherein the compound is in ionic form or charged and coupled with a counter ion (cation or anion) or in solution. This definition also includes quaternary ammonium salts and complexes of active molecules with other molecules and ions, particularly complexes formed through ionic interactions.

[0070] In the context of this invention, the term "solvent" should be understood to mean any form of the compound of this invention, wherein the compound is bonded to another molecule (typically a polar solvent) by a non-covalent bond, particularly including hydrates and alcohols, such as methanols.

[0071] In the context of this invention, the term "stereoisomer" should be understood as a type of isomer in which atoms are connected in the same order but have different spatial arrangements. This difference in spatial arrangement can arise from double bonds, rings, or chiral centers in the molecule. Stereoisomers can be further classified into two main types: Conformational isomers: Stereoisomers that can transform into each other by rotating around a single bond. They have the same connectivity but different spatial orientations of their atoms.

[0072] Configurational isomers: Stereoisomers that cannot be converted into each other without breaking the bonds.

[0073] In the context of this invention, the term "ester" should be understood as a compound derived from an acid (organic or inorganic) wherein at least one -OH (hydroxyl) group is substituted with -O-alkyl (alkoxy) groups.

[0074] In another preferred embodiment, step (b) includes reacting the glutamate-binding protein with the chelating agent of step (a), wherein the reaction is carried out under suitable conditions to promote the formation of an amide bond between the terminal amino group of the lysine residue of the glutamate-binding protein and the chelating agent.

[0075] In one or even more preferred embodiment, the glutamate-binding protein is composed of SEQ ID NO: 4; the chemical element suitable for MRI is gadolinium; the chelating agent is DOTA or its salt, solvate, stereoisomer or ester; and step (b) includes reacting the glutamate-binding protein with the chelating agent of step (a), wherein the reaction is carried out under suitable conditions to promote the formation of an amide bond between the terminal amino group of the lysine residue of the glutamate-binding protein and the chelating agent.

[0076] The eighth aspect of the present invention relates to a protein-based glutamate biosensor (hereinafter referred to as the glutamate biosensor of the eighth aspect of the present invention) that can be obtained by any method of the sixth aspect of the present invention.

[0077] The ninth aspect of the present invention relates to a nanocarrier comprising any glutamate sensor of the fifth, sixth or eighth aspect of the present invention, characterized in that the nanocarrier is functionalized with ligands capable of binding to the blood-brain barrier.

[0078] In a preferred embodiment, the ligand is selected from a list including antibodies, peptides, aptamers, or small molecules having affinity for at least one receptor at the blood-brain barrier, wherein the receptor is selected from a list including VCAM-1, E-selectin, PECAM-1, CD44, ICAM-1, transferrin receptor, LDL-associated protein, leptin receptor, insulin-like growth factor receptor, diphtheria toxin receptor, neonatal Fc receptor, or nicotinic acetylcholine receptor.

[0079] In another preferred embodiment, the nanocarrier is a nanoparticle selected from the group consisting of polymer nanoparticles, lipid nanoparticles, or inorganic nanoparticles.

[0080] The tenth aspect of the present invention relates to the use of any glutamate biosensor of the fifth, sixth or eighth aspect of the present invention, or any nanocarrier of the ninth aspect of the present invention, in a method for quantifying or monitoring a substrate or neurotransmitter in vivo, preferably in the brain or blood-brain barrier.

[0081] In a preferred embodiment, a glutamate biosensor or nanocarrier is used in a method for diagnosing neurological or neurodegenerative diseases.

[0082] The eleventh aspect of the present invention relates to an in vitro detection method for determining the presence of glutamate in a biological sample obtained from a subject, the method comprising: a. To contact any glutamate biosensor of the fifth, sixth, or eighth aspect of this invention, or any nanocarrier of the ninth aspect of this invention, with a biological sample obtained from a subject suspected of containing a substrate or neurotransmitter, and b. If the MRI signal is detected to be shifted compared to the MRI signal without glutamate, it indicates that the substrate or glutamate is present in the sample. Attached Figure Description

[0083] Figure 2 A schematic diagram of the synthesis of the glutamate MRI sensor. The starting point is the Glt1 protein domain in the iGluSnFr protein. Upon binding to glutamate, Glt1 folds and induces MRI signal enhancement, which is generated by Gd-DOTA linked to 27 lysine residues in the protein.

[0084] Figure 3The structures of a fluorescent glutamate sensor (iGluSnFr) and a synthetic MRI sensor comprising SEQ ID NO: 1 are described. The first labeled region in the protein is a histidine tag, the second labeled region is the glutamate recognition subunit (Glt1) (SEQ ID NO: 5), and the third labeled region is the GFP subunit (SEQ ID NO: 7). The amino acid K* represents a lysosome modified with DOTA. The complete sequence corresponds to SEQ ID NO: 4.

[0085] Figure 4 The fluorescence signals generated by the sensor in the presence or absence of 250 μM glutamate at different sensor concentrations were investigated.

[0086] Figure 5 The fluorescence signal (0.43 nM) generated by the sensor under different concentrations of glutamate (glu) and arginine (arg).

[0087] Figure 6 Comparison of T1 relaxation rate profiles of bovine serum albumin (BSA) and Gd reaction in the presence and absence of DOTA. The slope of BSA-DOTA-Gd is the highest.

[0088] Figure 7 Comparison of T1 relaxation rate maps of MRI sensors in the presence (+glu) and absence (-glu) 1mM glutamate.

[0089] Figure 8 The T1 signal of the MRI glutamate sensor was measured as glutamate concentration increased. Measurements were performed in triplicate. Standard deviation is expressed as an error bar.

[0090] Figure 1 Left: T1 contrast of the sensor, GOT, and albumin labeled with DOTA-Gd, expressed as the percentage change in T1 value measured without glutamate and after the addition of 250 μM glutamate (relative to the signal without glutamate). Right: Representative MRI image of the sensor. Detailed Implementation

[0091] The present invention is illustrated by the following embodiments, but is not intended to limit its scope of protection.

[0092] Example 1: Materials and Methods Example 1.1. Materials The plasmid pRSET.GltI253-cpGFP.L1LV / L2NP in the bacterial agar slant was provided by Loren Looger (Addgene plasmid #41733; RRID: Addgene_41733). *E. coli* BL21(DE3)pLysS competent cells, PureYield plasmid MiniPrep, enzymes EcoRI and NdeI, DNALadder, and BlueJuice 10X were purchased from Promega. Luria-Bertani (LB) agar and SYBR were purchased from Invitrogen. LB medium, ampicillin, isopropyl-β-D-1-thiogalactoside (IPTG), lysozyme (from eggs), imidazole, gadolinium(III) chloride, and poly(lactic-co-glycolic acid) (PLGA) (Resomer) were purchased from Sigma-Aldrich. Protease inhibitors (whole tablets) and nickel resin (whole His-Tag purified resin) were purchased from Roche. Ultrafiltration tubes were purchased from Millipore, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS) was purchased from Chematech, and dialysis bags (Float-A-Lyzer G2) were purchased from Spectra / Por.

[0093] Example 1.2. Synthesis of Fluorescent Sensor A fluorescent sensor was synthesized and purified using plasmids from *E. coli* bacterial cultures as starting materials, following a standard protein synthesis protocol. The plasmid containing iGLuSnFR (Addgene, plasmid #41733) was transformed into *E. coli* BL21(DE3)pLysS competent cells, inoculated onto LB agar containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C with shaking at 220 rpm. To select recombinant bacteria, their plasmids were isolated using the commercial kit "PureYield Plasmid Miniprep" and digested with EcoRI and Ndel. Agarose gel electrophoresis was performed on the digestion products using SYBR as a contrast agent, DNA ladder bands as fragment length markers, and Bluejuice 10X as loading buffer. Correctly transformed bacteria will exhibit banding patterns of 1673 bp and 2759 bp.

[0094] The transformed bacteria were cultured overnight at 37°C with shaking in LB medium containing 100 μg / mL ampicillin. This pre-culture was then inoculated at a 1:50 ratio into LB medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking at 220 rpm until the OD600 reached between 0.4 and 0.6. Once the optical density reached the target range, sensor synthesis was induced by adding 1 mM IPTG, and the cells were cultured overnight at 37°C with shaking at 220 rpm. Finally, the cells were centrifuged, and the pellet was collected.

[0095] The precipitate was resuspended in 10 mL of phosphate-buffered saline containing 10 mg of lysozyme (from eggs) and a protease inhibitor (whole tablets). The mixture was sonicated for 5 min at 0.5 sec and 80% amplitude. Then, the mixture was centrifuged at 15000 g and 4 °C for 30 min, and the supernatant was collected. The supernatant was passed through a 0.22 μm microfiltration filter and purified using nickel resin (complete His-Tag purification resin). The glutamate fluorescent sensor was eluted using the same phosphate-buffered saline with 250 mM imidazole. The sensor was concentrated at 15000 g using a 10 kDa filter, and the medium was replaced with storage buffer (50 mM HEPES; 200 mM NaCl and pH 7.5). Finally, glycerol was added to a final volume of 10%, and the purified fluorescent sensor was stored at -80 °C.

[0096] Example 1.3. MRI Sensor Synthesis First, the fluorescent sensor (2 mg, 0.03 μmol) was dissolved in 400 μL of carbonate buffer (50 mM, pH 9), followed by the addition of 3 μmol (100 equivalents) of DOTA-NHS dissolved in 100 μL of DMSO. The reaction mixture was stirred at 4 °C for 5 hours. Then, the mixture was transferred to 1 mL of acetate buffer (50 mM, pH 5), and 1.65 μmol of gadolinium(III) was added. The mixture was incubated overnight at 4 °C with continuous mixing. Finally, unbound gadolinium(III) and DOTA were removed, and the buffer was replaced with PBS via dialysis using a Float-A-Lyzer G2 (10 kDa pore size) filter.

[0097] GOT-DOTA-Gd was synthesized from GOT (0.5 mg, 0.03 μmol). Initially, GOT (0.5 mg, 0.03 μmol) was dissolved in 400 μL of carbonate buffer (50 mM, pH 9), followed by the addition of 3 μmol (100 equivalents) of DOTA-NHS dissolved in 100 μL of DMSO. The reaction mixture was stirred at 4 °C for 5 hours. Then, the mixture was transferred to 1 mL of acetate buffer (50 mM, pH 5), and 1.65 μmol of gadolinium(III) was added. The mixture was then continuously incubated overnight at 4 °C. Finally, unbound gadolinium(III) and DOTA were removed, and the buffer was replaced with PBS by dialysis through 10 kDa wells using a Float-A-Lyzer G2.

[0098] BSA-DOTA-Gd was prepared by first dissolving BSA (2 mg, 0.03 μmol) in 400 μL carbonate buffer (50 mM, pH 9), then adding 3 μmol (100 equivalents) of DOTA-NHS dissolved in 100 μL DMSO. The reaction mixture was stirred at 4 °C for 5 hours. The mixture was then transferred to 1 mL acetate buffer (50 mM, pH 5), and 1.65 μmol of gadolinium(III) was added. The mixture was then incubated overnight at 4 °C. Finally, unbound gadolinium(III) and DOTA were removed, and the buffer was replaced with PBS by dialysis through 10 kDa wells using a Float-A-Lyzer G2.

[0099] Example 1.4. MRI Characterization All studies were conducted using a Bruker Biospec 9.4 T MR scanner (equipped with a 12 cm wide Bruker BioSpin horizontal aperture magnet and an actively shielded gradient coil with a gradient intensity of 440 mT). m -1 The data acquisition is performed on an external device. For external data acquisition, an RF resonator is used as the transmitter-receiver (orthogonal volume coil with a diameter of 7 cm).

[0100] T1 relaxation rate (R1) or T2 relaxation rate (R2), as a measure of MRI contrast, was evaluated at 9.4T in a multi-well agar (1.6% w / v) mold following procedures described elsewhere. The sensor was first diluted in water and placed in an Eppendorf tube, which was then inserted into the mold. The mold was then sealed with liquid agar and cooled to room temperature. T2-weighted images (T2-wi) were obtained using a multi-spin echo (MSME) sequence with an echo time (ET) of 11.32 ms, a repetition time (RT) of 3 s, 16 echoes (echo spacing ES of 11.32 ms), a spectral bandwidth (SB) of 50 kHz, a flip angle (FA) of 90°, 14 slices (1 mm thick), and one mean. The field of view (FOV) of T2-wi was 7.5 × 7.5 cm. 2 (Outside-of-field signal suppressed by saturation band), matrix size 300×300, planar resolution 250μm / pixel, no fat suppression was used. T1-weighted images (T1-wi) were acquired using a Rapid Relaxation Enhanced Variable Acquisition (RARE-VTR) sequence, with ET=17.48ms, RT=1s, NA=1, repetitions (NR)=1, SW=50kHz, and rarity factor (RF)=4; 6 sets of T1 experiments, RT=900, 2000, 3500, 5000, 7000, and 11000ms, and with the same T2 geometry.

[0101] Example 2, Results Example 2.1. Fluorescence characterization of the existing iGluSnFR glutamate sensor The inventors of this invention hypothesize that linking gadolinium (Gd) to a glutamate-binding protein will produce a new protein that undergoes a conformational change upon contact with glutamate, thereby shifting the MRI signal provided by Gd bound to its structure. Figure 2 ).

[0102] To verify this hypothesis, the inventors prepared an MRI glutamate sensor by using DOTA as a chelating agent and labeling the glutamate transport protein (Glt1) with Gd. Figure 3 The fluorescent glutamate sensor iGluSnFR was used as the starting material because, in addition to containing the Glt1 domain, it also carries a fluorescent protein reporter group (GFP), which facilitates preliminary testing.

[0103] Following standard procedures, the fluorescent glutamate sensor iGluSnFR was synthesized and isolated via histidine tagging. The inventors tested the sensor's glutamate detection capability by measuring changes in fluorescence after incubation with glutamate. Figure 4 They also tested changes in fluorescence in the presence of other amino acids to assess the selective binding of the sensor to glutamate. Figure 3As expected, fluorescence intensity in the presence of glutamate occurred in a sensor-dependent manner. Figure 4 ) and increases in a glutamate-dependent manner ( Figure 1 ).

[0104] Example 2.2. Synthesis of an MRI Glutamate Sensor Based on the Existing iGluSnFR Glutamate Sensor A novel MRI glutamate sensor was synthesized by reacting 52 lysine residues on the iGluSnFR sensor with the chelating agent DOTA to achieve coupling via the formation of a stable amide bond between lysine and DOTA. Subsequently, Gd was linked to the sensor via the DOTA chelating agent. Figure 2 and Figure 5 ).

[0105] First, the reaction of lysine with DOTA-NHS was tested in a model protein (albumin in this case) to optimize the reaction conditions. Albumin was first reacted with DOTA-NHS, and then Gd was labeled via DOTA-lysine residues. A control was set up, with DOTA-free albumin directly labeled with Gd. Measurement of MRI T1 relaxation rate revealed a higher signal in DOTA-labeled albumin, indicating that Gd-labeled proteins require a chelating agent (…). Figure 6 Subsequently, using the same reaction as BSA, DOTA-Gd was linked to the lysine of iGluSnFR to generate an MRI glutamate sensor.

[0106] Example 2.3. In vitro evaluation of the sensing properties of an MRI glutamate sensor To investigate the detection performance of the MRI glutamate sensor, the inventors analyzed the variation of the MRI signal generated by the sensor with its own concentration. For example... Figure 7 As shown, the signal slope and signal intensity are improved in the presence of glutamate, indicating that the MRI signal generated by the sensor in the presence of glutamate is different from the MRI signal generated in the absence of glutamate.

[0107] The MRI detection performance was also evaluated by incubating the sensor with different concentrations of glutamate. Figure 7 Increased glutamate concentration was associated with decreased MRI signal, which is due to glutamate-induced conformational changes in GltI.

[0108] The glutamate sensing ability of the MRI glutamate sensor was then compared with that of other proteins labeled with DOTA-Gd:BSA-DOTA-Gd and GOT-DOTA-Gd. BSA-DOTA-Gd was used as a negative control because BSA has no affinity for glutamate. On the other hand, glutamate oxaloacetate transaminase (GOT) is an enzyme involved in glutamate metabolism. GOT-DOTA-Gd was used to assess whether a reliable glutamate sensor could be produced when the same synthetic strategy was applied to another glutamate-binding protein. The inventors compared the T1 signal of the MRI sensor in the presence and absence of 250 μM glutamate with the signals provided by BSA-DOTA-Gd and GOT-DOTA-Gd under the same conditions. The results were consistent with expectations; minimal changes in the MRI signal were detected when the control sensor (BSA-DOTA-Gd) was incubated with glutamate, while the signal changes of the MRI glutamate sensor (containing the Glt1 domain) and the GOT-DOTA-Gd sensor were more significant. SEQUENCE LISTING: It is worth noting that the MRI glutamate sensor (containing the Glt1 domain) outperforms the GOT-DOTA-Gd sensor in detection performance.

[0109] In summary, the above analysis shows that (i) the synthetic glutamate sensor containing a glutamate transporter (GLTL-DOTA-Gd) can generate a strong glutamate-dependent MRI signal, (ii) the synthetic glutamate sensor prepared with a glutamate-binding protein (GOT-DOTA-Gd) which is not a glutamate-specific transporter can also generate a glutamate-induced MRI signal, but the signal intensity is lower than that generated when using a glutamate transporter, and (iii) the protein sensor prepared with a protein that is not specific to glutamate (BSA-DOTA-Gd) does not generate a glutamate-dependent MRI signal. This indicates that a glutamate-binding protein is essential for the generation of this signal.

[0110] AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDVLLAGERAKAKKPDNWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI SEQ ID NO: 1 DRWGS AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDVLLAGERAKAKKPDNWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDVLLAGERAKAKKPDNWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL SEQ ID NO: 2 MWLTEAQKFGVAFTFGGFLFFLFGIFTFFDRALLALGNILFLIGVFLIIGSQKTYIFFTRPNKRRGSLFFLVGAFLILLKWTFLGFIIESLGIIGLFGDFFGVIVQFLRSMPIIGPILSHPAIAPIVDKLAGVRVLPV SEQ ID NO: 3 MKWVTFISLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFK ADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDCGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLECCD KPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA SEQ ID NO ： 4 MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGS AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNE EQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDVLLAGERAKAKKPDNWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNE ​ LVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE ​ ​ EYNFNNPLNMNFELSDEMKALFKEPNDKALK SEQ ID NO ： 5 ​ ​ ​ ​ SEQ ID NO ： 6 LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL SEQ ID NO ： 7 LVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE SEQ ID NO ： 8 AAGSTLDKIAKNGVIVVGHRESSVPFSYYDNQQKVVGYSQDYSNAIVEAVKKKLNKPDLQVKLIPITSQ NRIPLLQNGTFDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNE EQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDVLLAGERAKAKKPDNWEIVGKPQSQEAYGCMLRKDDPQFKKLMD DTIAQVQTSGEAEKWFDKWFKNPI LVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNY KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL SEQ ID NO ： 9 AAGSTLDKIAKNGVIVVGHRDSSVPFSYYDSQHKVVGYSQEYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTYDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDSLLAGERAKAKKPDDWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI SEQ ID NO ： 10 AAGSTLDKIAKNGVIVVGHRX 1 (E / D)SSVPFSYYDX 2 (N / S)QX 3 (Q / H)KVVGYSQX 4 (D / E)YSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTX 5 (F / Y)DFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDX 6 (V / S)LLAGERAKAKKPD* 7 (N / D)WEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPI where X 1 is E or D, X 2 is N or S, X 3 is Q or H, X 4 is D or E, X 5 is F or Y, X 6 is V or S, and X 7 is N or D.

[0111] SEQ ID NO: 11 AAGSTLDKIAKNGVIVVGHRX 1 (E / D)SSVPFSYYDX 2 (N / S)QX 3 (Q / H)KVVGYSQX 4 (D / E)YSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTX 5 (F / Y)DFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDX 6 (V / S)LLAGERAKAKKPDX 7(N / D)WEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPILVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED GNILGHKL Wherein, X 1 is E or D, X 2 is N or S, X 3 is Q or H, X 4 is D or E, X 5 is F or Y, X 6 is V or S, and X 7 is N or D.

[0112] SEQ ID NO: 12 MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSAAGSTLDKIAKNGVIVVGHRX 1 (E / D)SSVPFSYYDX 2 (N / S)QX 3 (Q / H)KVVGYSQX 4 (D / E)YSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTX 5 (F / Y)DFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDX 6 (V / S)LLAGERAKAKKPDX 7 (N / D)WEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPILVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNNPLNMNFELSDEMKALFKEPNDKALK Wherein, X 1 is E or D, X 2 is N or S, X 3 is Q or H, X 4 is D or E, X 5 is F or Y, X 6 is V or S, and X 7 is N or D.

[0113] SEQ ID NO: 13 AAGSTLDKIAKNGVIVVGHRDSSVPFSYYDSQHKVVGYSQEYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTYDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSFRTLESGRAVAFMMDDSLLAGERAKAKKPDDWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAEKWFDKWFKNPILVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSV SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNY KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL SEQ ID NO: 14 MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSAAGSTLDKIAKNGVIVVGHRDSSVPFSYYDSQHKVVGYSQEYSNAIVEAVKKKLNKPDLQVKLIPITSQNRIPLLQNGTYDFECGSTTNNVERQKQAAFSDTIFVVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDSF RTLESGRAVAFMMDDSLAGERAKKPDDWEIVGKPQSQEAYGCMLRKDDPQFKKLMDDTIAQVQTSGEAAEKWFDKWFKNPILVSHNVYIMADKQRNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL EYNFNNPLNMNFELSDEMKALFKEPNDKALK

Claims

1. A glutamate biosensor comprising a glutamate binding protein bound to a magnetic resonance imaging (MRI) contrast agent, characterized in that, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and a sequence encoding a fluorescent protein.

2. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO:

6.

3. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO: 6, separated by a sequence comprising SEQ ID NO:

7.

4. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO: 6, separated by a sequence comprising SEQ ID NO:

7.

5. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 11, preferably SEQ ID NO:

8.

6. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 12, preferably SEQ ID NO:

4.

7. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein consists of SEQ ID NO:

12.

8. The glutamate biosensor according to the preceding claim, wherein, The glutamate binding protein consists of SEQ ID NO:

4.

9. The glutamate biosensor according to any one of the preceding claims, wherein, The MRI contrast agent comprises a chelator bound to a chemical element suitable for MRI.

10. The glutamate biosensor according to the preceding claim, wherein, The chemical element suitable for MRI is selected from the list consisting of gadolinium, manganese, cobalt, dysprosium and / or chromium.

11. The method of any one of claims 9 or 10, wherein, The chemical element suitable for MRI is gadolinium.

12. The glutamate biosensor according to any one of claims 9-11, wherein, The chelator is selected from the group consisting of dodecanetetraoetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraphosphonic acid (NOTP), ethylenediaminetetraacetic acid (EDTA), hydrazinonicotinamide (HYNIC), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and 1,4,7-triazacyclononane-1,4-bis(methylene phosphonic acid) (NOTB).

13. The glutamate biosensor according to the preceding claim, wherein, The chelator is DOTA.

14. The glutamate biosensor according to any one of the preceding claims, wherein, The glutamate binding protein consists of SEQ ID NO: 4, wherein the MRI contrast agent comprises a chelator bound to a chemical element suitable for MRI, the chemical element suitable for MRI is gadolinium and the chelator is DOTA.

15. The glutamate biosensor according to any one of the preceding claims, wherein, The MRI contrast agent or chelator is bound to the glutamate binding protein via an amide bond it forms with a lysine amino acid.

16. The glutamate biosensor according to the preceding claim, wherein, At least 50%, at least 90%, at least 93%, at least 95%, at least 97%, at least 99% or 100% of the lysine amino acids in the glutamate binding protein are bound to the MRI contrast agent or chelator.

17. The glutamate biosensor according to any one of the preceding claims, wherein, The glutamate binding protein consists of SEQ ID NO: 4, the MRI contrast agent comprises a chelator bound to a chemical element suitable for MRI, the chemical element suitable for MRI is gadolinium, and the chelator is DOTA; the MRI contrast agent or chelator is bound to the glutamate binding protein via an amide bond formed with a lysine amino acid; at least 90%, at least 93%, at least 95%, at least 97%, at least 99% or 100% of the lysine amino acids in the glutamate binding protein are bound to the MRI contrast agent or chelator.

18. Nanocarrier comprising the glutamate sensor according to any one of claims 1 to 17, characterized in that, The nanocarrier is functionally modified with a ligand that can bind to the blood brain barrier.

19. Nanocarrier according to the preceding claim, characterized in that, The ligand is selected from the list consisting of: an antibody, a peptide, an aptamer or a small molecule having affinity for at least one receptor of the blood brain barrier; the receptor is selected from the list consisting of: VCAM-1, E-Selectin, PECAM-1, CD44, ICAM-1, transferrin receptor, LDL related protein, leptin receptor, insulin-like growth factor receptor, diphtheria toxin receptor, neonatal Fc receptor or nicotinic acetylcholine receptor.

20. The nanocarrier of any one of claims 18 or 19, wherein, The nanocarrier is a nanoparticle selected from the group consisting of: a polymeric nanoparticle, a lipid nanoparticle or an inorganic nanoparticle.

21. The glutamate biosensor of claims 1 to 17, or the nanocarrier of claims 18 to 20, for use in a method of quantifying or monitoring a substrate or a neurotransmitter in vivo, preferably in the brain or the blood brain barrier.

22. The use according to claim 21, the glutamate biosensor of claims 1 to 17 or the nanocarrier of claims 18 to 20, for use in a method of diagnosing a neurological or neurodegenerative disease.

23. An in vitro method for determining the presence of glutamate in a biological sample obtained from a subject, comprising: a. contacting the glutamate biosensor of claims 1-17 or the nanocarrier of claims 18-20, with a biological sample obtained from a subject suspected of containing a substrate or a neurotransmitter, and b. wherein, the presence of a shift in the MRI signal compared to the MRI signal obtained in the absence of glutamate indicates the presence of a substrate or glutamate in the sample.

24. A method for obtaining a glutamate biosensor, wherein, The method comprises: a. providing a glutamate binding protein and a chelator bound to a chemical element suitable for MRI, and b. reacting the glutamate binding protein with the chelator of step (a), wherein the reaction is performed under conditions suitable for the formation of a covalent bond between the glutamate binding protein and the chelator, wherein the glutamate binding protein is characterized in that it comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and a sequence encoding a fluorescent protein.

25. The method of the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO:

6.

26. The method of the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO: 6, which is separated by a sequence comprising 108 to 128 amino acids.

27. The method of the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 10 (preferably SEQ ID NO: 5) and SEQ ID NO: 6, which is separated by a sequence comprising SEQ ID NO:

7.

28. The method of the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 11, preferably SEQ ID NO:

8.

29. The method of the preceding claim, wherein, The glutamate binding protein comprises SEQ ID NO: 12, preferably SEQ ID NO:

4.

30. The method of the preceding claim, wherein, The glutamate binding protein consists of SEQ ID NO:

12.

31. The method of the preceding claim, wherein, The glutamate binding protein consists of SEQ ID NO:

4.

32. The method of any one of claims 24-31, wherein, The chemical element suitable for MRI is selected from the list consisting of gadolinium, manganese, cobalt, dysprosium and / or chromium.

33. The method of the preceding claim, wherein, The chemical element suitable for MRI is gadolinium.

34. The method of any one of claims 24-33, wherein, The chelator comprises a chemical group suitable for reacting with an amine group.

35. The method of the preceding claim, wherein, The chelator is selected from the group consisting of dodecanetetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane (TETA), hydrazinonicotinamide (HYNIC), satoreotide trizoxetan (NODAGA), 2-[11-(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl]acetic acid (CB-TE2A) and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), or a salt, solvate, stereoisomer or ester thereof.

36. The method of the preceding claim, wherein, The chelator is DOTA, or a salt, solvate, stereoisomer or ester thereof.

37. The method of any one of claims 24 to 36, wherein, Step (b) comprises reacting the glutamate binding protein with the chelator of step (a), wherein the reaction is performed under conditions suitable for forming an amide bond between a terminal amine group of a lysine residue of the glutamate binding protein and the chelator.

38. The method of any one of claims 24 to 37, wherein, The glutamate binding protein consists of SEQ ID NO: 4, wherein the chemical element suitable for MRI is gadolinium and the chelator is DOTA, or a salt, solvate, stereoisomer or ester thereof; wherein step (b) comprises reacting the glutamate binding protein with the chelator of step (a), the reaction being performed under conditions suitable for forming an amide bond between a terminal amine group of a lysine residue of the glutamate binding protein and the chelator.

Images