Devices and methods for detecting biomarkers associated with neurodegenerative diseases

Abstract

The present invention provides a device for the detection and / or quantification of neurotoxic amyloid-type protein aggregates, comprising a doxocycline derivative immobilized on a suitable surface, as well as electrochemical and immunochemical methods related to the use of such a device.

Description

Technical Field

[0001] Technical Field The present invention relates to the detection of biomarkers for the diagnosis of neurodegenerative diseases, in particular synucleinopathies such as Parkinson's disease and tauopathies such as Alzheimer's disease. More specifically, the present invention relates to a device for detecting said biomarkers, comprising a doxocycline derivative immobilized on a suitable surface, and to electrochemical and immunochemical methods related to the use of such a device.

Background Art

[0002] Background of the Invention The aging rate is increasing globally. According to the World Health Organization (WHO), the proportion of the population aged 60 years or older is expected to double worldwide between 2015 and 2050. Considering that the elderly are at high risk of suffering from neurodegenerative diseases, effective diagnosis and treatment of diseases are becoming increasingly important. Among several existing age-related neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease are common.

[0003] According to the World Health Organization (WHO), the prevalence of PD has doubled in the past 25 years and exceeded 10 million in 2020. However, these figures may be underestimated due to the lack of a globally standardized registration system and variability in the diagnostic criteria used in countries. Aging is considered the main risk factor for the disease, and with the extension of the average lifespan, it is predicted that diseases related to aging will increase significantly in the next few decades. However, the WHO also warns that the prevalence of PD is increasing at a faster rate than other NDs and predicts that the number of cases will double in the next few decades. On the other hand, the economic burden of symptomatic treatment is enormous not only for patients and their families but also for healthcare systems around the world. For example, in the USA, this figure was estimated to be 51 billion USD in 2017, and based on the prediction of the increasing incidence of the disease, there is a risk of burdening healthcare systems around the world in the next few decades. These figures have motivated the scientific community to study various mechanisms that can stop the neurogenic process of the disease. However, the effectiveness of neuroprotective treatments is affected by administration at a very early stage of the disease, which requires a diagnosis before the appearance of the motor symptoms typically associated with PD.

[0004] Despite significant efforts being made in quantifying alpha-synuclein (AS) aggregates, which are considered the main biomarker of Parkinson's disease, to date, no biochemical analysis of body fluids has been performed that can detect the early stages of the disease.

[0005] Details of existing techniques for diagnosing and / or monitoring PD mainly from cerebrospinal fluid (CSF) samples are as follows. - Conventional ELISA (enzyme-linked immunosorbent assay) In this immunoassay, the anti-synuclein antibody is immobilized on the polymer plaques. The main drawback of this technique is that while the aggregated species of AS are extremely heterogeneous, the antibody is sensitive to the conformation of the target antigen. Therefore, only some types of aggregates can be detected, and thus the existing species diversity cannot be embodied. This makes this technique non-specific and potentially affects sensitivity (Ganguly et al. (2021); Eusebi et al. (2016); Atik et al. (2016)).

[0006] - Amplification of α-synuclein seeds, also known as real-time quaking-induced conversion (RT-QuIC) or α-synuclein seed amplification assay (SAA). Based on the potential of this technique to distinguish people with Parkinson's disease from healthy controls, in a large-scale cross-sectional analysis (1123 participants) using SAA, it has been shown that this test can classify people with Parkinson's disease with high sensitivity and specificity, provide information on molecular heterogeneity, and detect individuals with prodromal symptoms before diagnosis (Siderowf et al. (2023). The main limitation of RT-QuIC is the availability of recombinant human α-synuclein, and its market cost complicates the large-scale application of this technique. Special equipment that is not available in low- to medium-complexity laboratories and personnel training for its implementation are also required. In addition, at present, there is no evidence suggesting that it may be miniaturized for application as a point-of-care (POC) system (Luan, M., et al. (2022); Poggiolini I, et al. (2022); Kuzkina, A, et al. (2021)).

[0007] - Nuclear imaging techniques: · Single-photon emission computed tomography (SPECT) This technique uses a radioactive ligand that binds to dopamine receptors to detect the emitted gamma rays. SPECT can provide information on the function of dopamineergic neurons typically associated with Parkinson's disease. · Positron Emission Tomography (PET) PET can also be used to evaluate dopamine activity in the brain by using a radioactive tracer that emits positrons and a radioactive ligand that binds to dopamine receptors or transporters. Nuclear imaging techniques have limited sensitivity and are not effective in detecting subtle changes in dopamine levels in the brain at the early stages of the disease, which can lead to false-negative results. In addition, the cost of this technology limits its use in low- or medium-complexity healthcare systems. Ultimately, the interpretation of the results becomes complex and requires nuclear medicine expertise. In conclusion, these techniques are only used to confirm the diagnosis of PD during the typical motor phase (Golan H, et al. (2022); Pavese, N, et al. (2011); Moore, R. Y., et al. (2008)).

[0008] - Immunomagnetic reduction by superconducting quantum interference device (IMR-SQUID). This technique uses antibody-functionalized magnetic nanoparticles and a highly sensitive SQUID magnetometer. Although this technique is promising because it can detect proteins at the femtomolar level, large-scale application will be complicated due to the high cost of the device and the difficulty of detecting different aggregate species presented by the antibody (Yang, SY., et al. (2016)). Jang et al. (2020) disclosed an electrochemical sensor for detecting AS oligomers for the early diagnosis of PD. The sensor contains a methylene blue aptamer adsorbed on the electrode, which desorbs when binding to the AS oligomers present in the sample, thus generating a detectable change in the electrical signal provided by the electrode. The electrode used in the sensor is a reduced graphene oxide electrode.

[0009] However, there is a need for alternative AS aggregate detection techniques with a wide range of specificities that use low-cost, readily available equipment to facilitate the detection of multiple types of aggregates that may be present in a sample. Regarding AD, it is a devastating neurodegenerative disease that gradually erodes memory, cognitive abilities, and behavior. To date, AD is the most common form of dementia, and it is more prevalent than vascular dementia, mixed dementia, Lewy body dementia (LBD), and frontotemporal dementia (FTD). Currently, AD accounts for 60 - 80% of all dementia cases. Similarly, the prevalence of AD is expected to increase over time. According to the U.S. Centers for Disease Control and Prevention (CDC), approximately 5.8 million Americans currently suffer from AD, and this number is predicted to increase with the aging of the population. In fact, beyond the age of 65, the risk of developing AD doubles every five years. Since the need for care continues to increase as the disease progresses, the prevalence of the disease is a significant burden on the healthcare system. This is further compounded by the fact that while the mortality rates for heart disease and cancer are decreasing, the mortality rate for Alzheimer's disease is increasing. The cost of caring for AD patients is also staggeringly high, estimated at over $500 billion per year, and this number is expected to increase with the aging of the population.

[0010] The key neuropathological feature of AD is the presence in the brain of deposits of the microtubule-associated protein Tau, called neurofibrillary tangles (NFTs), in various forms that appear years before the onset of clinical symptoms. Although the accumulation of NFTs was first described in AD, it is noteworthy that other neurodegenerative diseases such as frontotemporal dementia, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration are also associated with the presence of NFTs. In fact, some of the strongest evidence supporting Tau's involvement in neurodegenerative diseases comes from the identification of mutations in patients with frontotemporal dementia, highlighting the potential for Tau to be a causative factor in the development of these types of diseases. Like prions, tau pathology can spread from one area of the brain to another. There is increasing evidence that aggregated species of tau spread along neuroanatomically connected brain regions through a "prion-like" mechanism, transferring abnormal tau seeds from donor cells to recipient cells and generating new tau seeds. Therefore, early diagnosis of AD is essential to provide opportunities for pharmacological neuroprotective interventions to slow the progression of the disease. In the preclinical stage of Alzheimer's disease, there may be no obvious symptoms, but changes in the brain, such as the accumulation of amyloid-β (Aβ) and tau proteins, are already occurring. These changes can be detected through imaging and the detection and quantification of biomarkers.

[0011] Ongoing research in biomarker detection for AD is currently focused on the development of reliable and sensitive assays for detecting biomarkers in body fluids such as cerebrospinal fluid (CSF), plasma, and urine. In addition, there are collaborative efforts to improve imaging techniques for detecting biomarkers other than Aβ aggregates in the brain through neuroimaging.

[0012] Tau aggregates contain a variety of species in a dynamic process until they are incorporated into NFTs. Most of them are involved in cytotoxicity. Furthermore, there is increasing evidence that tau aggregates play an important role in spreading lesions from neuron to neuron and triggering the aggregation process in healthy neurons. These features widely recognize tau aggregates as powerful biomarkers, but their detection remains difficult due to the absence of antibodies that can detect all species. Conventional immunological detection methods that rely only on epitope / paratope interactions are insufficient to recognize the diversity of all conformations of aggregates and often result in false positive or false negative results.

[0013] All amyloid aggregates, including those formed by Tau, share a cross-β structure, but the compactness of this structure can hide epitopes or expose new epitopes. As a result, the development of antibodies capable of specifically recognizing and binding amyloid aggregates has proven to be a real challenge. In contrast, small molecules such as Thioflavin T (ThT), Congo Red (CR), and Doxycycline bind strongly and specifically to the amyloid aggregate structure (Gonzalez-Lizarraga (2017), Medina (2021)). Regarding PD, there are several existing techniques for diagnosing and monitoring AD in both low-complexity and medium-complexity laboratories as follows.

[0014] - Immunohistochemistry Immunohistochemistry is used to confirm the diagnosis of Alzheimer's disease postmortem by examining the presence and distribution of Tau aggregates in brain tissue samples obtained from tissue. This technique stains thin sections of brain tissue with specific antibodies that bind to the Tau protein. The antibodies are labeled with chromogenic molecules, enabling the visualization of Tau aggregates under a microscope.

[0015] - Immunofluorescence Immunofluorescence is a technique similar to immunohistochemistry, but it uses a fluorescent dye instead of a chromogenic molecule when labeling Tau. This technique involves culturing brain tissue samples with an antibody that specifically binds to Tau conjugated to a fluorescent dye. While immunofluorescence is a valuable tool in Alzheimer's disease research, it is noteworthy that it is not routinely used for clinical diagnosis at autopsy. - Conventional ELISA Conventional ELISA is limited in its ability to detect Tau aggregation species due to its reliance on a single monoclonal capture antibody. This limitation reduces the ability to detect the various species of Tau aggregates. Commercially available ELISA tests commonly used, such as those from BioLegend, Abcam, Thermofisher, IBL International, etc., are designed to detect total Tau protein and phosphorylated Tau as biomarkers. Unfortunately, these tests suffer from decreased sensitivity and specificity, resulting in false positives or false negatives (Mounsey (2018)). This emphasizes the need for better capture molecules to improve the detection accuracy of Tau aggregates.

[0016] -PET imaging This technique is a powerful tool for visualizing and quantifying the accumulation of pathological proteins in the brains of patients with Alzheimer's disease (AD). 18 F] Several radioligands targeting Tau aggregates in the brain, such as TauPET tracers, have been developed. As discussed above for PD, this diagnostic technique is only available mainly at highly sophisticated medical centers and research institutions due to the high cost of the equipment and radioligands. In addition, there are limitations in the interpretation of PET images, which can only provide information about the distribution and density of pathological proteins, and not about specific compositions or locations within the brain. Moreover, the interpretation of PET results can be difficult due to the lack of standardized methods for image analysis and quantification. -Single molecule fluorescence resonance energy transfer (smFRET) This is a biophysical technique that can be used to measure intramolecular distances in proteins including Tau. This technique has been used to investigate conformational changes in Tau in the presence or absence of aggregation inducers, providing insights into the mechanisms underlying Tau aggregation in AD. However, smFRET is an advanced technique that requires special equipment and expertise and is usually only available through service providers or specialized research institutions.

[0017] -RT-QuIC This technique has been studied as a diagnostic tool for AD. It is involved in the detection of misfolded proteins such as Tau and beta-amyloid in cerebrospinal fluid (CSF) or other biological samples by converting normal proteins into abnormal aggregates that can be detected through changes in fluorescence or turbidity. RT-QuIC has shown promising results in detecting Tau aggregates in CSF samples from AD patients with high sensitivity and specificity. However, it is still at the experimental stage and not widely available for clinical use. Specialized equipment and expertise are also required, limiting its use to more advanced laboratories.

[0018] It is clear that there is still a need for alternative technologies that can detect several types of aggregates potentially present in samples with broad specificity, using easily accessible equipment at low operating costs and can detect AD-related biomarkers. Furthermore, it would be highly desirable to develop a versatile technology suitable for early detection of biomarkers related to neurodegenerative diseases. Specifically, it is extremely useful to obtain devices and methods for detecting biomarkers related to diseases such as PD and AD.

[0019] Doxycycline has long been known as a tetracycline antibiotic and has been used for the treatment of various bacterial infections such as bacterial pneumonia, cholera, and syphilis. SUMMARY OF THE INVENTION

[0020] SUMMARY OF THE INVENTION According to a first aspect, the present invention provides a device for detecting neurotoxic amyloid-type protein aggregates, comprising a doxycycline derivative capable of binding to neurotoxic amyloid-type protein aggregates, wherein the doxycycline derivative is immobilized on a surface. In an embodiment of this aspect of the present invention, the device has the formula (I) immobilized on a surface:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0021] In a preferred embodiment, R is a substituent selected from the group consisting of H, NH2, COOH, (CH2) n X, COOCH2(CH2) n X, where n is 0 to 10 and X is selected from Cl, Br, and I. In a particularly preferred embodiment of the present invention, the device includes a doxycycline derivative represented by formula (II) immobilized on the surface

Chemical formula

[0022] In another aspect of this side of the present invention, the surface is selected from a polymer surface and a functionalized metal surface. In certain embodiments of the present invention, the polymer surface comprises a polymer selected from the group consisting of polystyrene, polystyrene / divinylbenzene copolymer, and other synthetic or natural polymers to which a doxocycline derivative can be immobilized. Preferably, the polymer surface comprises polystyrene. In a preferred embodiment of the present invention, the doxocycline derivative is immobilized on the polymer surface by covalently bonding the doxocycline derivative to a blocking agent adsorbed on the surface via a linker. Preferably, the blocking agent adsorbed on the polymer surface is bovine serum albumin.

[0023] In a specifically preferred embodiment, the device comprises a doxocycline derivative represented by formula (II) that is immobilized on the polymer surface by covalently bonding to bovine serum albumin adsorbed on the surface via glutaraldehyde. In another particular embodiment of the present invention, the surface is a functionalized metal surface. Preferably, the functionalized metal surface is a functionalized gold surface. More preferably, the functionalized gold surface comprises a self-assembled monolayer (SAM) of mercapto acid. Most preferably, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid. In a specifically preferred embodiment, the device comprises a doxocycline derivative represented by formula (II) immobilized on a functionalized metal surface, where the functionalized metal surface is a functionalized gold surface, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and the doxocycline derivative represented by formula (II) is covalently bonded to the SAM of 3-mercaptopropionic acid.

[0024] Another aspect of the present invention is for detecting and / or quantifying neurotoxic amyloid-type protein aggregates i - providing a device for detecting neurotoxic amyloid-type protein aggregates according to a first aspect of the present invention, ii - contacting the device with a sample in which the presence and / or concentration of the neurotoxic amyloid-type protein aggregates is to be determined, and iii - determining the presence and / or concentration of said neurotoxic amyloid - type protein aggregates by a detection technique is to provide an in vitro method comprising the same. In one aspect of this aspect, the neurotoxic amyloid - type protein aggregates to be detected and / or quantified are alpha - synuclein (AS) aggregates.

[0025] In another aspect of this aspect, the neurotoxic amyloid - type protein aggregates to be detected and / or quantified are tau protein (Tau) aggregates. In an aspect of this aspect of the present invention, the detection technique is selected from the group consisting of immunochemical assays and electrochemical assays. In a specific aspect of this aspect of the present invention, the detection technique in step iii - is an electrochemical assay. Preferably, the electrochemical assay uses a technique selected from the group consisting of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to detect neurotoxic amyloid - type protein aggregates. More preferably, the electrochemical assay uses cyclic voltammetry to detect neurotoxic amyloid - type protein aggregates.

[0026] In another specific aspect of this aspect of the present invention, the detection technique in step iii - is an immunochemical assay, and step iii - comprises a) a sub - step of contacting the device with an antibody suitable for binding to the neurotoxic amyloid - type protein aggregates to be detected and / or quantified, and b) a sub - step of measuring the bound antibody by a measurement technique including the same. In a preferred aspect of this aspect of the present invention, the neurotoxic amyloid - type protein aggregates to be detected and / or quantified are AS aggregates, and the method comprises i - providing a device for detecting neurotoxic amyloid - type protein aggregates according to the first aspect of the present invention, ii - contacting the device with a sample in which the presence and / or concentration of the AS aggregates is determined, and iii - determining the presence and / or concentration of the AS aggregates by an immunoassay comprising, wherein the immunoassay further a) contacting the device with an antibody suitable for binding to the AS aggregates, and b) measuring the bound antibody by a measurement technique is included.

[0027] In another preferred embodiment of this aspect of the invention, the neurotoxic amyloid - type protein aggregates to be detected and / or quantified are Tau aggregates, and the method is i - providing a device for detecting neurotoxic amyloid - type protein aggregates according to the first aspect of the invention, ii - contacting the device with a sample in which the presence and / or concentration of the Tau aggregates is determined, and iii - determining the presence and / or concentration of the Tau aggregates by an immunoassay comprising, wherein the immunoassay a) contacting the device with an antibody suitable for binding to the Tau aggregates, and b) measuring the bound antibody by a measurement technique is further included.

[0028] Yet another aspect of the invention is to provide a method for preparing a device for detecting neurotoxic amyloid - type protein aggregates according to the invention, the method comprising i - providing a surface capable of immobilizing a doxycycline derivative, and ii - contacting the surface capable of immobilizing the doxycycline derivative with a solution containing a doxycycline derivative capable of binding to neurotoxic amyloid - type protein aggregates under conditions in which the doxycycline derivative is immobilized on the surface is included. In an aspect of this side of the present invention, the surface on which the doxycycline derivative can be immobilized is selected from the group consisting of a polymer surface and a functionalized metal surface.

[0029] In a specific aspect of this side of the present invention, the surface is a polymer surface, and the polymer plastic surface includes a polymer selected from the group consisting of polystyrene, a polystyrene / divinylbenzene copolymer, and other synthetic or natural polymers on which doxycycline and its derivatives can be immobilized. Preferably, the polymer plastic surface includes polystyrene.

[0030] In an aspect of this side of the present invention, the surface is a polymer surface, and step i of the method further includes the following sub-steps: a) contacting the polymer surface with a solution containing a blocking agent to cause adsorption of the blocking agent onto the polymer surface, thereby obtaining a blocked polymer surface, and b) contacting the blocked polymer surface with a solution containing a linker to obtain a polymer surface capable of immobilizing a doxycycline derivative further including. Preferably, the blocking agent is bovine serum albumin and the linker is glutaraldehyde.

[0031] In another specific aspect of this side of the present invention, the surface is a functionalized metal surface, and the method further includes, prior to step i, contacting the metal surface with a solution containing a functionalizing agent to obtain the functionalized metal surface. Preferably, the metal surface is a gold surface. Also preferably, the functionalizing agent is mercapto acid. More preferably, the functionalizing agent is 3-mercaptopropionic acid. Yet another aspect of the present invention is i - a polymer surface functionalized with or easily functionalizable with a doxycycline derivative according to the present invention, and ii - a detection reagent providing a kit for detecting and quantifying biomarkers related to PD. Yet another aspect of the present invention is to provide a point-of-care (POC) biosensor for detecting biomarkers related to PD, which comprises an electrode functionalized or easily functionalizable with a doxycycline derivative according to the present invention. The following figures form a part of this specification and are intended to further illustrate certain aspects of the present invention. **Brief Description of the Drawings**

[0032]

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[0033] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a device for detecting neurotoxic amyloid-type protein aggregates based on the binding ability of doxycycline to neurotoxic amyloid-type protein aggregates. As described above, AS aggregates (i.e., the polymeric form of AS) are considered the main biomarker for PD, while Tau aggregates are considered a promising biomarker for AD. Thus, the device of the present invention has the advantageous property of being useful for detecting both PD and AD biomarkers in biological samples. Correspondingly, the device of the present invention includes a doxycycline derivative immobilized on the surface.

[0034] Within the scope of the present disclosure, the doxycycline derivative is understood as a compound obtained by introducing minor modifications to doxycycline while maintaining the binding ability to neurotoxic amyloid-type protein aggregates such as AS aggregates and Tau aggregates. For example, such a doxycycline derivative has the formula (I) immobilized on the surface: CHEMICAL FORMULA In the formula, R is i-H, NH2, COOH, (CH2) n X, COOCH2(CH2) nA substituent selected from the group consisting of X, where n is from 0 to 10, and X is selected from Cl, Br, and I, An alkyl or aryl linker having a thiol terminal group, iii-

Chemical formula

Chemical formula

Chemical formula

[0035] For the doxycycline derivative represented by formula (I), the alkyl or aryl linker having a thiol terminal group refers to any suitable alkyl chain or aryl group that includes a thiol group at the terminal. When the linker is an alkyl linker, the linker may be any linear or branched alkyl hydrocarbon chain having a thiol group bonded to the carbon atom farthest from the bond of R to the aromatic ring represented by formula (I). When the linker is an aryl linker, the linker may be an aryl group having a thiol group directly bonded to its aromatic system, or an aryl group containing an alkyl substituent having a thiol group bonded to the carbon atom farthest from the bond of the alkyl substituent to the aromatic system of the aryl group.

[0036] In a preferred embodiment, R is a substituent selected from the group consisting of H, NH2, COOH, (CH2) n X, COOCH2(CH2) n A substituent selected from the group consisting of X, where n is from 0 to 10, and X is selected from Cl, Br, and I. In a particularly preferred embodiment, the doxycycline derivative has the formula (II): [Chemical formula] and is a compound represented by the formula (II). The compound represented by the formula (II) is also referred to herein as doxy-5. The device of the present invention requires a doxycycline derivative immobilized on a suitable surface. By "immobilized", those skilled in the art will understand that the compound should be bound to the surface in such a manner that it remains bound to the surface even after several washings. The manner in which the compound is immobilized on the surface depends on the nature of the surface.

[0037] In an embodiment of the present invention, the surface is a polymer surface. When using this type of surface, the doxycycline derivative is usually immobilized thereon either by an adsorption process, i.e., the compound binds to the surface by intermolecular forces, or by a blocking agent pre-adsorbed on the polymer surface to which the doxycycline derivative can covalently bind via a suitable linker.

[0038] The term "blocking agent" should be understood to refer to a protein that readily binds to the polymer surface of the device, such as a protein used to block the surface of an ELISA plate. Such blocking agents are known to those skilled in the art. However, the inventors have surprisingly found that the use of bovine serum albumin (BSA) as a blocking agent significantly improves the sensitivity of a detection device for neurotoxic amyloid-type protein aggregates. Therefore, when a blocking agent is adsorbed on the polymer surface of the device, the blocking agent adsorbed on the polymer surface of the device is preferably BSA. The linker through which the doxycycline derivative covalently attaches to the blocking agent is a bifunctional small molecule that contains two reactive moieties at opposite ends of a hydrocarbon chain, where one reactive moiety covalently attaches to the blocking agent adsorbed on the polymer surface, and the other reactive moiety covalently attaches to the doxycycline derivative, thus immobilizing the doxycycline derivative on the polymer surface. As will be appreciated by those skilled in the art, the linker selected depends on the binding agent and doxycycline derivative selected.

[0039] When the polymer surface contains a blocking agent to which it is adsorbed, the doxycycline derivative is a chemically modified doxycycline derivative that contains a functional group capable of binding to a linker attached to the blocking agent adsorbed on the polymer surface. For example, a doxycycline derivative capable of binding to a linker attached to a blocking agent has formula (I) as defined above. In a preferred embodiment, the device comprises a polymer surface adsorbed with bovine serum albumin, and the doxycycline derivative represented by formula (II) covalently attaches to the bovine serum albumin via glutaraldehyde as a linker. In another preferred embodiment, no blocking agent is used, and the device comprises a polymer surface on which the doxycycline derivative is immobilized by direct adsorption without the need to use a blocking agent. The term "polymer surface" is broadly understood to refer to any surface that includes a polymer to which the doxycycline derivative or blocking agent can be readily adsorbed. For example, the polymer surface may include a polymer selected from the group consisting of polystyrene, polystyrene / divinylbenzene copolymer, and other synthetic or natural polymers to which doxycycline and its derivatives can be immobilized. Preferably, the polymer surface includes polystyrene.

[0040] In another aspect of the invention, the surface is a functionalized metal surface. The term "functionalized metal surface" should be understood to refer to a metal surface to which a functionalizing agent is attached. The function of the functionalizing agent is to function as a linker between the metal surface and the doxocycline derivatives by covalently binding to both the metal surface and the doxocycline derivatives. Preferably, the functionalized metal surface is a functionalized gold surface. More preferably, the functionalized metal surface is a functionalized gold surface to which mercapto acid is attached as the functionalizing agent, thus forming a self-assembled monolayer (SAM). Accordingly, in this preferred aspect, the functionalized gold surface comprises a SAM of linear mercapto acid. In such an arrangement, the linear mercapto acid molecules are regularly attached to the gold surface by their terminal thiol moieties, exposing a carboxylic acid moiety to which the doxocycline derivatives can covalently bind. Most preferably, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid.

[0041] When the surface is such a functionalized metal surface, the doxocycline derivative is a doxocycline derivative chemically modified to include a functional group capable of binding to the functionalizing agent of the functionalized metal surface. For example, a doxocycline derivative capable of binding to the functionalizing agent has the formula (I) as defined above. In a particularly preferred aspect, the device comprises a doxocycline derivative represented by formula (II) immobilized on a functionalized metal surface, the functionalized metal surface is a functionalized gold surface, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and the doxocycline derivative represented by formula (II) is covalently bound to the SAM of 3-mercaptopropionic acid.

[0042] The device according to the invention can be prepared by techniques readily available to those skilled in the art for immobilizing compounds on a suitable surface. Thus, another aspect of the invention is i - providing a surface capable of immobilizing a doxocycline derivative, and ii - contacting the surface capable of immobilizing a doxocycline derivative with a solution containing the doxocycline derivative under conditions such that the doxocycline derivative is immobilized on the surface To provide a method for preparing a device for detecting neurotoxic amyloid-type protein aggregates according to the present invention, which includes Providing a surface capable of immobilizing a doxycycline derivative includes, if necessary, modifying the corresponding surface to enable such immobilization.

[0043] In a specific embodiment, the surface is a polymer surface. In such an embodiment, the surface can immobilize the doxycycline derivative by an adsorption process without further modification. However, in an alternative embodiment of this aspect of the present invention, the polymer surface is pre-treated with a blocking agent that adsorbs thereto. Thus, in said embodiment, step i of the method is the following sub-steps: a) Contacting the polymer surface with a solution containing a blocking agent to cause adsorption of the blocking agent to the polymer surface, thereby obtaining a blocked polymer surface, and b) Contacting the blocked polymer surface with a solution containing a linker to obtain a polymer surface capable of immobilizing a doxycycline derivative including.

[0044] The polymer surface capable of immobilizing the doxycycline derivative thus provided includes a blocking agent adsorbed on the polymer surface and a linker covalently bonded thereto, providing a portion to which the doxycycline derivative can easily form a covalent bond. Optimizing the conditions under which adsorption of the blocking agent and reaction with the linker occur is within the knowledge of those skilled in the art. The blocking agent and the linker share the same characteristics as those described above herein. In a preferred embodiment, the blocking agent is BSA and the linker is glutaraldehyde.

[0045] In another specific embodiment, the surface is a functionalized metal surface. In such an embodiment, the metal surface must be functionalized so that a doxycycline derivative can be immobilized. Thus, in such an embodiment, the method further includes, prior to step i, contacting the metal surface with a solution containing a functionalizing agent to obtain the functionalized metal surface. Preferably, the metal surface is a gold surface. Also preferably, the functionalizing agent is a mercapto acid. More preferably, the functionalizing agent is 3-mercaptopropionic acid. Optimizing the conditions under which the functionalization of the metal surface is carried out is within the knowledge of those skilled in the art. Under the conditions in which the doxycycline derivative is immobilized on the surface, the step of contacting the surface capable of immobilizing the doxycycline derivative with a solution containing the doxycycline derivative also depends on the selected surface.

[0046] In an embodiment where the surface is a polymer surface, the step includes immersing the corresponding surface in a solution containing an appropriate concentration of the doxycycline derivative for an appropriate time for either the adsorption process or the reaction with the linker to be completed. In a specific embodiment where the polymer surface is pre-treated with a binder and a linker, the doxycycline derivative should also be a doxycycline derivative capable of binding to the linker that binds to the blocking agent, preferably a doxycycline derivative represented by formula (I), or most preferably a doxycycline derivative represented by formula (II). In embodiments where the surface is a functionalized metal surface, the solution containing the doxcycline derivative should also contain additional reagents necessary to form a covalent bond between the doxcycline derivative and the functionalized metal surface. The doxcycline derivative should preferably be a doxcycline derivative represented by formula (I), most preferably a doxcycline derivative represented by formula (II), and should also be a doxcycline derivative capable of binding to the functionalizing agent of the functionalized metal surface. In embodiments where the metal surface is a gold surface and the functionalizing agent is 3-mercaptopropionic acid, the solution containing the doxcycline derivative should contain the reagents necessary for the doxcycline derivative to effectively bind to the carboxylic acid moiety of 3-mercaptopropionic acid. For example, in such embodiments, the solution may also contain 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Optimizing the adsorption process or reaction conditions necessary to immobilize the doxcycline derivative on the selected surface is within the skill of the art.

[0047] The device of the present invention is intended to detect neurotoxic amyloid-type protein aggregates in a sample. As used herein, the term "neurotoxic amyloid-type protein aggregates" refers to protein aggregates associated with neurodegenerative diseases. Such aggregates are typically observed to accumulate in neurons and other parts of the nervous system during the progression of a neurodegenerative disease and can thus be used as biomarkers for diagnosing such diseases. For example, the device can be used to detect AS aggregates or Tau aggregates associated with synucleinopathies such as PD and tauopathies such as AD, respectively, in such samples. The signal emitted by the device for the detection of aggregates is related to its concentration in the sample.

[0048] Accordingly, another aspect of the present invention is i - providing a device for detecting neurotoxic amyloid-type protein aggregates according to the first aspect of the present invention, ii - contacting the device with a sample in which the presence and / or concentration of said aggregates is determined, and iii - determining the presence and / or concentration of said aggregates by a detection technique is to provide an in vitro method for detecting and / or quantifying neurotoxic amyloid - type protein aggregates, comprising.

[0049] The method utilizes the ability of a doxycycline derivative to bind to said aggregates. By contacting a device comprising a doxycycline derivative immobilized on a surface with a sample containing neurotoxic amyloid - type protein aggregates, said aggregates bind to the doxycycline derivative, enabling detection by an appropriate detection technique. The detection technique used to detect and / or quantify neurotoxic amyloid - type protein aggregates in the related method of the present invention may depend on the surface used to immobilize the doxycycline derivative. By way of example, the detection technique may be selected from the group consisting of immuno - chemical assays and electrochemical assays. In some embodiments, the detection technique includes a sandwich immunoassay.

[0050] In a specific embodiment, the detection technique is an immuno - chemical assay and requires that the surface be a polymer surface, preferably a polystyrene surface. Such an immuno - chemical assay involves contacting neurotoxic amyloid - type protein aggregates from the sample that bind to the immobilized doxycycline derivative obtained in step ii - of the method with an antibody having a specific affinity for the protein present in the bound aggregates, and then detecting, by a detection device, either itself or further by another antibody. For example, if the neurotoxic amyloid-type protein aggregates present in the sample are AS aggregates, their binding to the immobilized doxycycline may be detected by an anti-AS antibody. The binding of the anti-AS antibody to the bound AS may be detected using an HRP-conjugated secondary antibody. Similarly, if the neurotoxic amyloid-type protein aggregates present in the sample are Tau aggregates, their binding to the immobilized doxycycline may be detected by an anti-Tau antibody. The binding of the anti-Tau antibody to the bound Tau may be detected by an HRP-conjugated secondary antibody. Such immunoassays are within the skill of those in the art. However, the provision of the versatile method as described herein, which may be applied to the detection and / or quantification of a series of neurotoxic amyloid-type protein aggregates related to neurodegeneration by simply changing the antibody used to detect the specific protein involved in the formation of the bound aggregates, clearly cannot be derived in an obvious manner from the prior art.

[0051] Those skilled in the art will understand that the detection technique described above is applicable when the surface of the device is a polymer surface, and also regardless of whether the doxycycline derivative is immobilized on the polymer surface by direct adsorption as described above or via a blocking agent-linker bond. In another specific embodiment, the detection technique is an electrochemical assay, which requires the surface to be a functionalized metal surface, preferably a functionalized gold surface. In such an embodiment, the metal surface functions as an electrode in an apparatus for performing electrochemical measurements such as a potentiostat. Preferably, the electrochemical assay uses a technique selected from the group consisting of CV and EIS to detect and / or quantify neurotoxic amyloid-type protein aggregates. More preferably, the electrochemical assay uses CV for the detection and / or quantification of neurotoxic amyloid-type protein aggregates.

[0052] In this aspect of the invention, the specificity for each protein ((AS) aggregates and (Tau) aggregates) may then be evaluated by the use of a specific antibody, and its binding may also be evaluated electrochemically. That is, an antibody having a selective affinity for a specific protein (either AS or Tau) binds to the corresponding aggregate that binds to the doxycycline derivative and can thus modify the electrochemical signal detected. However, since the positive signal in this sensor already indicates the presence of aggregates, it may be a point-of-care (POC) determination for a patient and already gives a positive diagnosis.

[0053] As mentioned above, the formation of neurotoxic amyloid-type protein aggregates that can be detected and / or quantified by the method of this aspect of the invention is observed in several neurodegenerative diseases. In one aspect, the method is for the detection and / or quantification of AS aggregates associated with synucleinopathies such as PD, dementia with Lewy bodies (DLB), multiple system atrophy (MSA). In another aspect, the method is for the detection and / or quantification of Tau aggregates associated with tauopathies such as AD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD). Accordingly, the method of the invention may be used for the early diagnosis of such neurodegenerative diseases. For such practical use, the sample for determining the presence of neurotoxic amyloid-type protein aggregates is a sample from a subject in whom the onset of one of the diseases is suspected.

[0054] As used herein, the term "diagnosis" means detecting a disease or disorder or determining the stage or extent of a disease or disorder. Diagnostic methods may be used independently or in combination with other diagnostic and / or staging methods known in the art for a particular disease or disorder. Additional diagnostic markers may be combined with the markers described herein to predict the presence or stage of a disease. For example, clinical factors relevant to the diagnosis of neurodegenerative diseases include, but are not limited to, the patient's medical history, physical examination, and other biomarkers. As used herein, the terms "biological sample" and "sample" may be used interchangeably and refer to a sample obtained from a patient or subject. In some cases, the biological sample may be a tissue, cell, or body fluid (e.g., blood, serum, synovial fluid, sputum, lung fluid, mucus, tears, lymph fluid, synovial fluid, cerebrospinal fluid (CSF), feces, saliva, amniotic fluid, umbilical cord blood of amniotic fluid, urine, vaginal fluid, semen). Preferably, such a sample is a cerebrospinal fluid (CSF) sample.

[0055] Throughout, when used, the term subject means an individual, e.g., an adult subject. Preferably, the subject is an animal, e.g., a mammal such as a primate, and more preferably a human. Non-human primates of the order Primates are also subjects. Subject animals include cats, dogs, reptiles, amphibians, livestock (such as cows, horses, pigs, sheep, goats, etc.), and laboratory animals (such as ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses are contemplated herein.

[0056] Another aspect of the invention is a polymer surface functionalized or easily functionalizable with an i-doxycycline derivative, and ii-a detection reagent and providing a kit for detecting and / or quantifying neurotoxic amyloid-type protein aggregates. The term "polymer surface functionalized with or amenable to functionalization with a doxycycline derivative" is understood to refer to a polymer surface on which a doxycycline derivative is already immobilized or can be readily immobilized upon contact with a solution containing a doxycycline derivative, as described above for the devices of the present invention.

[0057] The detection reagent includes reagents known to those skilled in the art for performing immunochemical detection and / or quantification of neurotoxic amyloid-type protein aggregates according to the in vitro methods described above. Such detection reagents may include, for example, secondary antibodies, staining reagents, and the like. In a specific embodiment, the polymer surface of the kit is prepared to be functionalized with a doxycycline derivative, and the detection reagent also contains a doxycycline derivative. In a preferred embodiment, the kit according to this aspect of the present invention contains a doxycycline derivative represented by formula (I) immobilized on the polymer surface or contained within the detection reagent. More preferably, the doxycycline derivative is a doxycycline derivative represented by formula (II). The kit according to this aspect of the present invention may be operated by laboratory technicians and in a laboratory of moderate complexity.

[0058] Another aspect of the present invention is to provide a point-of-care (POC) biosensor for detecting biomarkers related to PD, comprising an electrode that (i) contains a doxycycline derivative immobilized thereon or (ii) is capable of immobilizing a doxycycline derivative thereon.

[0059] The term "electrode (i) containing a doxcycline derivative immobilized thereon or (ii) capable of immobilizing a doxcycline derivative thereon" is understood to refer to a functionalized metal surface on which a doxcycline derivative is already immobilized or can be easily immobilized upon contact with a solution containing a doxcycline derivative, as described above for the device of the present invention. Preferably, the POC biosensor comprises an electrode on which a doxcycline derivative is immobilized, more preferably a doxcycline derivative represented by formula (I), and most preferably a doxcycline derivative represented by formula (II). The present invention lies in the fact that it can selectively detect neurotoxic aggregate proteins such as AS and Tau over their monomeric forms, is easy to perform, and has the advantage that it does not require particularly expensive devices or sophisticated devices. Therefore, it shows great potential as a POC device for the early detection of biomarkers associated with diseases such as PD and AD, enabling early diagnosis of the diseases and thus obtaining a much better prognosis.

[0060] Example The present invention will be further illustrated by the following examples, which are not intended to limit its scope. Instead, the examples described below should be understood only as exemplary embodiments for better practicing the present invention (taking into practice).

[0061] Example 1 Organic synthesis of the compound represented by formula (II) General experimental procedures NMR spectra were recorded at 500 MHz ( 1 H) or 125.7 MHz ( 13 C), or at 300 MHz ( 1 H) or 75.6 MHz ( 13 C). Chemical shifts (δ, in ppm) were referenced to internal standards ( 1 Me4Si (δ: 0.0) in CDCl3 for 13For the case of C, it was referenced against CDCl3 (δ: 77.0)) or against the residual solvent peak. The data multiplicity is indicated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad); the coupling constant (J) is given in Hertz (Hz). 1 H and 13 The assignment of the 13C NMR spectrum was assisted by 2D 1 H-COSY or NOESY, and 2D 1 H- 13 CHSQC. High-resolution mass spectra (HRMS) were obtained using electrospray ionization (ESI) technique and Q-TOF detection. Analytical thin-layer chromatography (TLC) was carried out on silica gel 60 F254 aluminum support plates (layer thickness 0.2 mm) and on silica gel 60 RP F254S aluminum support plates. Spots were visualized by exposure to UV light and by charring with Ce / Mo staining. Column chromatography was carried out on silica gel 60 (230 - 400 mesh), or in the case of reverse phase, octadecyl-functionalized silica gel was employed as the stationary phase. The chromatographic solvents or stepwise solvent polarity gradients used were specified for each individual compound. The specific rotation was measured at room temperature in a 1 dm cell in the indicated solvent at the sodium D line. Unless otherwise stated, all commercially available compounds were used as received from the supplier without further purification.

[0062] Doxycycline free form (D-1) from doxycycline hydrochloride

Chemical Structure

[0063] Synthesis of methyl doxycycline iodide (D-2)

Chem.

[0064] Synthesis of 4-demethyldoxycycline D-3

Chem.

[0065] Synthesis of 4-Dimethylamino-9-nitrodoxycycline D-4

Chemical Structure

[0066] Synthesis of 9-amino-4-demethyldoxycycline doxy-5 (compound represented by formula (II))

Chemical formula

[0067] Example 2 - Generation of AS Aggregates as Biomarker Analytes 2.1. Expression and Purification of Human AS Human AS was expressed in E. coli (BL21) using the pT7-7 plasmid containing the gene encoding the protein. Purification was carried out by selective precipitation with (NH4)2SO4 and anion exchange chromatography. The purity of the protein was confirmed by electrophoresis (SDS-PAGE) under denaturing conditions. The AS solution was prepared in 20 mM HEPES, 150 mM NaCl buffer (pH 7.4). The samples were filtered and centrifuged at 12000 xg for 30 minutes to remove pre-formed aggregates. The concentration of AS was measured by spectrophotometry using the molar extinction coefficient.

[0068] 2.2. Preparation of aggregated AS species Aggregated AS species were obtained by incubating an AS solution (70 μM) for 24 h with a temperature-controlled orbital stirrer (600 rpm, 37 °C) as previously reported (Avila C.L. et al., (2014)).

[0069] 2.3. Kinetic measurement of amyloid aggregation Kinetic measurements were performed by Congo Red (CR) and Thioflavin T (ThT) techniques that specifically bind to amyloid fibers. Free CR exhibits an absorption maximum at 490 nm, but upon binding to amyloid fibers, the absorption peak shifts to 520 nm, showing an increase in the intensity of the absorption band. When ThT binds to amyloid fibers, it changes from having an excitation maximum at 415 nm to having a fluorescence emission wavelength at 480 nm and a much stronger maximum at 450 nm, and is optimized as described in Avila C.L., et al. (2014) based on the teachings of LeVine (1999).

[0070] Example 3 - Immobilization of a doxocycline derivative (doxy-5) on a conductive surface for electrochemical detection of AS aggregates A comparative study of various crosslinking agents for the immobilization of dox-5 on a gold conductive surface was carried out. 3-Mercaptopropionic acid (MPA) was used to form a self-assembled monolayer (SAM) on the substrate, thus facilitating its functionalization. Gold was selected because it is inert to the target molecule. In addition, the use of other agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to generate or activate the moieties present is necessary for the formation of covalent bonds with dox-5. The concentration and incubation time were optimized at each modification stage of the gold surface. First, cyclic voltammetry (CV) measurements were performed to clarify the characterization of each immobilization stage on the gold substrate. The measurements were carried out in a three-electrode configuration cell using a platinum electrode as the counter electrode, a silver / silver chloride as the reference electrode, and a modified gold electrode as the working electrode. Fe 3+ / Fe 2+ The electrolyte [Fe(CN)6]K4 containing the redox couple was used in all characterization assays. With this technique, the optimal polarization voltage value for electrochemical impedance spectroscopy (EIS) measurements, i.e., the voltage at which the biosensor exhibits the highest sensitivity and specificity in the interaction between the AS aggregate species and dox-5 immobilized on the conductive electrode, was determined. CV and EIS measurements were also performed using phosphate buffered saline (PBS) at pH 7.4 at each immobilization stage (Figure 1).

[0071] For the electrochemical measurements, a Solartron® 1287 potentiostat and a Solarton® 1250 frequency response analyzer were used. For data processing, the software provided by the manufacturer (CorrWare®, CorrView®, ZPlot®, ZView®) was used. The measurements were carried out at room temperature. The biosensor was calibrated for the increase in the concentration of toxic AS species. The gold biosensor modified with doxy-5 prepared according to Example 3.1 was used to assay the electrochemical measurements of AS at different concentrations (0.1 and 100 ng / mL). An alternating voltage of 10 mV overlapping at different potentials (0 V and -0.7 V) was used to perform impedance measurements in the frequency range of 0.1 - 65000 Hz. CV measurements were performed in the potential range of -0.6 V to 0.6 V and at a scan rate of 30 mV / seg. The CV assay first shows that there is no interference by the monomer. The response obtained for the monomer is much lower than the response obtained for the AS fiber. In fact, at a monomer concentration of 100 ng / mL, the response is lower than the response obtained for the fiber at a concentration of 0.1 ng / mL. Second, a distinct electrochemical response of the sensor was observed in the presence of the AS fiber. An increase in the oxidation peak of the voltammogram was observed from a low concentration of 100 pg / mL to a concentration exceeding 100 ng / mL (Figure 2). CV has been proven to be a selective technique because the presence of the AS fiber is recognized more selectively than the monomer variant.

[0072] Example 4 - Immuno-chemical detection of AS aggregates and immobilization of doxy-5 on the polymer surface by direct adsorption The design is based on an immunoassay by a conventional sandwich system that captures the fibrillar species of AS by dox-5 and subsequently detects it with two antibodies, an anti-human AS antibody and an antibody conjugated to peroxidase. Briefly, the compound represented by formula (II) is incubated in a polystyrene microplate and adsorbed there. After several washings, the system is incubated with a primary anti-human AS rabbit antibody to remove the unadsorbed molecules. After washing, the system is incubated with an anti-rabbit secondary antibody conjugated to peroxidase. The conjugated antibody specifically reacts with the primary antibody bound to the immunocaptured AS. The unbound conjugated antibody is removed by washing, and the presence of peroxidase is revealed by the addition of tetramethylbenzidine (TMB) as a chromogenic substrate. After incubation, blue color develops, and its intensity depends on the concentration of dox-5 and the affinity for AS species. The addition of sulfuric acid stops the enzyme reaction, and a color change to yellow occurs, which can be quantified by spectrophotometry at 450 nm. The effectiveness of this detection platform can be affected by various parameters such as the concentrations of the compound represented by formula (II) and the antibodies, the use and their concentrations of various proteins to block non-specific sites, and the temperature and incubation time at each stage. To obtain the best analytical performance for this platform, an assay was performed to optimize the above parameters (Figure 3).

[0073] The specificity of commercially available primary antibodies (Ab138501, Ab209538, Ab27766, Abcam, Sc-7011-R, Santa Cruz) against various AS species (monomers, oligomers, fibrils) was demonstrated. Each antibody showed picomolar affinity for each molecular AS form. The highest sensitivity was observed for the polyclonal antibody (Sc-7011-R, Santa Cruz) and one of the monoclonal antibodies (Ab209538). The anti-AS antibodies did not show a significant difference in the dosage values between fibrillar AS and monomeric AS. This is crucial in the design of this enzyme immunoassay where the bioreceptor bears the specificity for fibrillar AS rather than monomeric AS. The concentrations of the primary and secondary antibodies were also optimized (Ab32460, Invitrogen, Ab6721, Abcam).

[0074] The immobilization of the compound represented by formula (II) was analyzed using Microlon® polystyrene plates (96 wells, high binding, Greiner Bio-One). The concentration of the compound represented by formula (II) was characterized in combination with the use of different proteins and their concentrations to block non-specific sites. The results of analyzing various blocking agents such as bovine serum albumin (BSA), lysozyme, and casein at concentrations of 0.05% - 1% confirmed that lysozyme at a concentration of 0.1% was the best option (Figure 4). The protocol was defined as follows.

[0075] A 96-well polystyrene plate was protected from light with 20 μg / mL of the compound represented by formula (II) in 20 mM sodium acetate pH 5 (50 μL / well) and incubated overnight at 4 °C for sensitization. The plate was washed with PBST (10 mM PBS pH 7.4 + 0.01% Tween-80) and blocked with 150 μL / well of 0.1% lysozyme solution in 10 mM PBS pH 7.4 at 37 °C for 1 h. After washing, 50 μL of a sample with the target analyte prepared in 10 mM PBS pH 7.4 was added to each well and the plate was incubated at 37 °C for 2 h. The plate was then washed again, and 50 μL of anti-AS antibody diluted to 1 μg / ml with 10% fetal bovine serum (BFS in 10 mM PBS pH 7.4) was added to each well, and then the plate was incubated for 1.5 h. The plate was washed once more, and 50 μL / well of peroxidase-conjugated antibody diluted to 1 μg / ml with 10% fetal bovine serum was incubated at 37 °C for 1 h. The plate was washed and 50 μL / well of substrate (TMB) was added and incubated at room temperature for 15 min. The reaction was stopped with 25 μL of 1 M H2SO4 solution. Immediately thereafter, the absorbance was measured with a TECAN Spark microplate reader.

[0076] The results were confirmed in at least three independent experiments (three times each). The focus of the immunoassay is to utilize the colored compound generated by the action of peroxidase on a substrate (TMB) that is proportional to the concentration of the AS analyte captured by Doxy-5. The measured absorbance at 450 nm was obtained with a TECAN Spark microplate reader.

[0077] Standard solutions of monomeric and fibrillar AS over a wide concentration range were incubated in wells pre-sensitized with Doxy-5 as shown in Example 3.2 above. The system showed nanomolar-level affinity for fibrillar AS without cross-reactivity with native AS included as a negative control. At concentrations above 30 ng / mL, using the protocol described in Example 3.2, the best differential reactivity was shown between the aggregated and monomeric species of AS.

[0078] In addition, the use of this platform was investigated in CSF samples from patients without a medical condition consistent with motor impairment. All samples were centrifuged at 2000 g for 10 minutes to remove cells and debris, aliquoted, and stored at -80 °C until analysis. The biological fluids tested showed values in the order of absorbance measurements of the AS samples used as negative controls (Figure 5).

[0079] Example 5 - Immobilization of Doxy-5 on the Polymer Surface by Indirect Binding to BSA and Immunochemical Detection of Tau Aggregates Well sensitization was performed as follows: First, the binding sites of the microplate were blocked with albumin. Then, the compound represented by formula (II) was covalently bound to albumin using glutaraldehyde as a linker. For this purpose, 96-well polystyrene microplates were incubated overnight at 4 °C in a humid chamber with 150 μL / well of 1% bovine serum albumin (BSA). Next, after washing three times with PBST, 50 μL / well of glutaraldehyde at different concentrations (0.1%, 0.5%, and 1%) was added to the wells, and the plates were placed under a hood for the specified time (10 minutes for each concentration, or 5 minutes for 0.5% and 1%). The wells were then washed three times with PBS, and 50 μL / well of Doxy-5 (40 μg / ml) was added. The plates were then incubated at 37 °C for 30 minutes while protecting from light. Thereafter, the wells were washed again with PBST, and 50 μL / well of 1M TRIS was added as a glutaraldehyde inactivator. The microplates were then incubated at 37 °C for 30 minutes. After this incubation, the wells were washed with PBST, and 50 μL / well of Tau sample (monomer (Tau m ) and preformed fibrils (PFF)) was added, and the microplates were incubated at 37 °C for 2 hours. Tau mThe concentration of [[ID=]] was 100 ng / ml (5 ng / well), and PFF was demonstrated at a 1 / 10000 dilution. Subsequently, the wells were washed three times with PBST, and 50 μL / well of a 1 / 2000 solution of anti-Tau antibody (ab80579, abcam) diluted with 10% PBS-FBS was added. The microplate was then incubated at 37 °C for 1.5 hours. Subsequently, the wells were washed five times with PBST, and 50 μL / well of HRP-conjugated secondary antibody (ab6789, abcam) (1 / 75000) was added. Next, the microplate was incubated at 37 °C for 1 hour and then washed five times with PBST to remove excess secondary antibody. Finally, 50 μL / well of TMB substrate was added, covered from light, and incubated at room temperature for 10 minutes. To stop the reaction, 25 μL / well of 2N H2SO4 was added, and the microplate was immediately read at a wavelength of 450 nm with a microplate reader (Tecan Spark, Austria). All the various conditions demonstrated with glutaraldehyde showed similar results, and 0.1%-10’ containing the lowest concentration was selected for further assays. Doxy-5 m could distinguish Tau m from PFF and showed a higher signal for PFF compared to Tau

[0080] and PBS (working buffer) (Figure 6). m In addition, it was evaluated whether PFF binds to doxy-5 or to the remaining reactive sites of BSA treated with glutaraldehyde. For this purpose, after adding glutaraldehyde in the above protocol, doxy-5 was replaced with 0.1% BSA. Then samples of Tau

[0081] Example 6 - Comparison of the binding effects of doxycycline and doxy-5 to alpha-synuclein aggregates Both doxycycline hyclate and doxy-5 were immobilized on polystyrene microplates and contacted with AS aggregates (PPF) using a concentration of 20 ng / mL of AS aggregates according to the protocol described in Example 4. The binding of the AS aggregates to each molecule was also determined as described in Example 4. Figure 8 shows that doxy-5 is more effective than doxycycline hyclate in binding to amyloid aggregation species of AS. A positive signal is observed with doxycycline hyclate, but the signal obtained with doxy-5 is significantly higher, suggesting that doxy-5 binds to AS PFF with higher affinity than doxycycline hyclate. The results indicate that doxy-5 is an excellent capture element compared to conventional doxycycline hyclate and has higher selectivity for the target analyte. These findings highlight the importance of this chemical modification of tetracycline for developing devices with enhanced sensitivity. The removal of the bulky dimethylamine from the C4 position of doxycycline resulted in an enhanced affinity of the molecule for amyloid aggregation species of AS.

[0082] References Atik A, et al. Alpha-Synuclein as a Biomarker for Parkinson's Disease. Brain Pathol. 2016 May;26(3):410-8. doi: 10.1111 / bpa.12370. Avila C.L., et al. Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity, Journal of Biological Chemistry, Volume 289, Issue 20, 2014, Pages 13838-13850, ISSN 0021-9258, https: / / doi.org / 10.1074 / jbc.M113.544288. Eusebi P, et al. Diagnostic utility of CSF α-synuclein species in Parkinson's disease: protocol for a systematic review and meta-analysis. BMJ Open 2016; 6:e011113. doi: 10.1136 / bmjopen-2016-011113. Ganguly U, et al. Alpha-Synuclein as a Biomarker of Parkinson’s Disease: Good, but Not Good Enough. Frontiers in Aging Neuroscience. 2021; 13. DOI=10.3389 / fnagi.2021.702639. Golan H, et al. Nuclear imaging in Parkinson's disease: The past, the present, and the future. Journal of the Neurological Sciences. Volume 436. 2022. 120220.ISSN 0022-510X. https: / / doi.org / 10.1016 / j.jns.2022.120220 Gonzalez-Lizarraga, F., et al. Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species. Sci Rep 7, 41755 (2017). https: / / doi.org / 10.1038 / srep41755. Jang et al. α-Synuclein Oligomer Detection with Aptamer Switch on Reduced Graphene Oxide Electrode. Nanomaterials 2020, 10, 832; doi:10.3390 / nano10050832. Kuzkina, A., et al. Diagnostic value of skin RT-QuIC in Parkinson’s disease: a two-laboratory study. npj Parkinsons Dis. 7, 99 (2021). https: / / doi.org / 10.1038 / s41531-021-00242-2 Luan, M, et al. (2022), Diagnostic Value of Salivary Real-Time Quaking-Induced Conversion in Parkinson's Disease and Multiple System Atrophy. Mov Disord. https: / / doi.org / 10.1002 / mds.28976 Medina, L. et al. Doxycycline Interferes With Tau Aggregation and Reduces Its Neuronal Toxicity. Frontiers in aging neuroscience 13, 635760, doi:10.3389 / fnagi.2021.635760 (2021) Moore, R. Y., et al. Extrastriatal monoamine neuron function in Parkinson’s disease: an 18F-dopa PET study. Neurobiol Dis 29, 381-390 (2008). Mounsey, A. L. & Zeitler, M. R. Cerebrospinal Fluid Biomarkers for Detection of Alzheimer Disease in Patients with Mild Cognitive Impairment. Am Fam Physician 97, 714-715 (2018). Pavese, N., et al. Progression of monoaminergic dysfunction in Parkinson’s disease: a longitudinal 18F-dopa PET study. Neuroimage 56, 1463-1468 (2011). Poggiolini I, et al. Diagnostic value of cerebrospinal fluid alpha-synuclein seed quantification in synucleinopathies, Brain, Volume 145, Issue 2, February 2022, Pages 584-595, https: / / doi.org / 10.1093 / brain / awab431 Siderowf, A. et al. Assessment of heterogeneity among participants in the Parkinson's Progression Markers Initiative cohort using α-synuclein seed amplification: a cross-sectional study. The Lancet Neurology, Volume 22, Issue 5 (2023), Pages 407-417, https: / / doi.org / 10.1016 / S1474-4422(23)00109-6. Yang, SY., et al. Development of an ultra-high sensitive immunoassay with plasma biomarker for differentiating Parkinson disease dementia from Parkinson disease using antibody functionalized magnetic nanoparticles. J Nanobiotechnol 14, 41 (2016). https: / / doi.org / 10.1186 / s12951-016-0198-5.

Claims

1. A device for detecting neurotoxic amyloid-type protein aggregates, comprising a doxcycline derivative capable of binding to neurotoxic amyloid-type protein aggregates, wherein the doxcycline derivative is immobilized on the surface.

2. The device according to claim 1, comprising a doxcycline derivative represented by formula (I) immobilized on the surface: 【Chemical 1】 wherein R is i-H, NH ２ , COOH, (CH ２ ) ｎ X, COOCH ２ (CH ２ ) ｎ a substituent selected from the group consisting of X, wherein n is from 0 to 10, and X is selected from Cl, Br and I, an alkyl or aryl linker having an ii-thiol terminal group, iii - 【Chemical 2】 wherein n is from 0 to 10, iv - 【Chemical 3】 wherein n is from 0 to 10, and v - 【Chemical Formula 4】 wherein n is from 0 to 10, selected from the group consisting of and represented by

3. R is H, NH ２ , COOH, (CH ２ ) ｎ X, COOCH ２ (CH ２ ) ｎ X is a substituent selected from the group consisting of, wherein n is from 0 to 10, and X is selected from Cl, Br and I, the device according to claim 2.

4. The device according to any one of claims 1 to 3, comprising a doxcycline derivative represented by formula (II): 【Chemical Formula 5】

5. The device according to any one of claims 1 to 4, wherein the surface is a polymer surface.

6. The device according to claim 5, wherein the polymer surface comprises a polymer selected from the group consisting of polystyrene, a polystyrene / divinylbenzene copolymer, and other synthetic or natural polymers on which doxcycline and its derivatives can be immobilized.

7. The device according to claim 6, wherein the polymer surface comprises polystyrene.

8. The device according to any one of claims 1 to 7, wherein the doxcycline derivative is immobilized on the polymer surface by covalently bonding via a linker to a blocking agent adsorbed on the surface.

9. The device according to claim 8, wherein the blocking agent is bovine serum albumin (BSA) and the linker is glutaraldehyde.

10. The device according to claim 1, comprising a doxcycline derivative represented by formula (II) immobilized on the polymer surface, wherein the polymer surface comprises polystyrene.

11. 【Chemical Formula 6】 The device according to claim 10, comprising a doxcycline derivative represented by formula (II) immobilized on the polymer surface by covalently bonding via glutaraldehyde as a linker to bovine serum albumin adsorbed on the surface.

12. The device according to any one of claims 1 to 4, wherein the surface is a functionalized metal surface.

13. The device according to claim 12, wherein the functionalized metal surface is a functionalized gold surface.

14. ​ ​ The device according to claim 13, wherein the functionalized gold surface comprises a self-assembled monolayer (SAM) of mercapto acid.

15. The device according to claim 14, wherein the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid.

16. The device comprises a doxocycline derivative represented by formula (II) immobilized on a functionalized metal surface 【Chemical Formula 7】 wherein the functionalized metal surface is a functionalized gold surface, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and the doxocycline derivative represented by formula (II) is covalently bonded to the SAM of 3-mercaptopropionic acid. The device according to claim 1.

17. i - providing a device for detecting a neurotoxic amyloid-type protein aggregate according to any one of claims 1 to 16, ii - contacting the device with a sample in which the presence of the aggregate is to be determined, and iii - determining the presence and / or concentration of the aggregate by a detection technique An in vitro method for detecting and / or quantifying a neurotoxic amyloid-type protein aggregate, comprising:

18. The method according to claim 17, wherein the neurotoxic amyloid-type protein aggregate to be detected and / or quantified is an alpha-synuclein (AS) aggregate.

19. The method according to claim 17, wherein the neurotoxic amyloid-type protein aggregate to be detected and / or quantified is a tau protein (Tau) aggregate.

20. The method according to any one of claims 17 to 19, wherein the detection technique is selected from the group consisting of immunoassay and electrochemical assay.

21. The method according to claim 20, wherein the detection technique is an electrochemical assay.

22. The method according to claim 21, wherein the electrochemical assay uses a technique selected from the group consisting of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to detect a neurotoxic amyloid-type protein aggregate.

23. The method according to claim 22, wherein the electrochemical assay uses cyclic voltammetry to detect a neurotoxic amyloid-type protein aggregate.

24. The method according to claim 20, wherein the detection technique is an immunoassay.

25. Step iii - is a) a sub-step of contacting the device with an antibody suitable for binding to the neurotoxic amyloid-type protein aggregates to be detected and / or quantified, and b) a sub-step of measuring the bound antibody by a measurement technique The method according to claim 24, comprising:

26. The neurotoxic amyloid-type protein aggregates to be detected and / or quantified are AS aggregates, and step iii- is a) a sub-step of contacting the device with an antibody suitable for binding to AS aggregates, and b) a sub-step of measuring the bound antibody by a measurement technique The method according to claim 25, comprising:

27. The neurotoxic amyloid-type protein aggregates to be detected and / or quantified are Tau aggregates, and step iii- is a) a sub-step of contacting the device with an antibody suitable for binding to Tau aggregates, and b) a sub-step of measuring the bound antibody by a measurement technique The method according to claim 25, comprising:

28. The method according to any one of claims 24 to 27, wherein the device for detecting neurotoxic amyloid-type protein aggregates is immobilized on the polymer surface by covalently bonding a doxycycline derivative to a blocking agent adsorbed on the surface via a linker.

29. The method according to claim 28, wherein the blocking agent is bovine serum albumin (BSA) and the linker is glutaraldehyde.

30. The method according to any one of claims 28 to 29, wherein the doxycycline derivative is a compound represented by formula (II).

31. i - providing a surface on which the doxycycline derivative can be immobilized, and ii - contacting the surface on which the doxycycline derivative can be immobilized with a solution containing a doxycycline derivative capable of binding to neurotoxic amyloid-type protein aggregates under conditions in which the doxycycline derivative capable of binding to neurotoxic amyloid-type protein aggregates is immobilized on the surface The method for preparing a device for detecting neurotoxic amyloid-type protein aggregates according to any one of claims 1 to 16, comprising:

32. The method according to claim 31, wherein the surface is a polymer surface.

33. The method according to claim 32, wherein the polymer surface comprises a polymer selected from the group consisting of polystyrene, a polystyrene / divinylbenzene copolymer, and other synthetic or natural polymers to which doxcycline and its derivatives can be immobilized.

34. The method according to claim 33, wherein the polymer surface comprises polystyrene.

35. Step i of the method comprises the following sub-steps: a) contacting the polymer surface with a solution containing a blocking agent to cause adsorption of the blocking agent to the polymer surface, thereby obtaining a blocked polymer surface; b) contacting the blocked polymer surface with a solution containing a linker to obtain a polymer surface capable of immobilizing a doxcycline derivative The method according to any one of claims 31 to 33, further comprising.

36. The method according to claim 35, wherein the blocking agent is bovine serum albumin and the linker is glutaraldehyde.

37. The method according to claim 31, wherein the surface is a functionalized metal surface and the method further comprises, prior to step i, contacting the metal surface with a solution containing a functionalizing agent to obtain the functionalized metal surface.

38. The method according to claim 37, wherein the metal surface is a gold surface.

39. The method according to claim 38, wherein the functionalizing agent is mercapto acid.

40. The method according to claim 39, wherein the functionalizing agent is 3-mercaptopropionic acid.

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