Device and method for detecting biomarkers associated with neurodegenerative disease

Abstract

The present disclosure provides devices for the detection and / or quantification of neurotoxic amyloid aggregates comprising a doxycycline derivative immobilized on an appropriate surface, and also relates to electrochemical and immunochemical methods associated with such device applications.

Description

Field of the Invention

[0001] The present invention relates to a method for detecting biomarkers for diagnosing neurodegenerative diseases, in particular synuclein diseases, 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 doxycycline derivative immobilized on a suitable surface; the present invention also relates to electrochemical and immunochemical methods associated with the use of such a device. Background of the Invention

[0003] The proportion of the elderly population is growing worldwide. According to the World Health Organization (WHO), the percentage of the global population over 60 years old is expected to double between 2015 and 2050. Considering that the elderly are more susceptible to neurodegenerative diseases, effective diagnosis and treatment of said diseases are becoming increasingly important. Among the several existing age-related neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease are prevalent.

[0004] According to the World Health Organization (WHO), the incidence of PD has doubled over the past 25 years, reaching more than 10 million people in 2020. However, these figures are likely underestimates due to the lack of a standardized global registry and differences in the diagnostic criteria used in different countries. Aging is considered a major risk factor for the disease, and an increase in life expectancy portends a substantial increase in age-related diseases in the coming decades. However, the WHO also warns that the incidence of PD is increasing at a faster rate than other NDs and predicts that the number of cases will double in the coming 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 worldwide. In the United States, for example, in 2017, this figure was estimated at $51 billion, and projections based on the rising incidence of the disease in the coming decades could put a strain on healthcare systems worldwide. These figures have mobilized the scientific community to investigate different mechanisms that could block the neuroregenerative processes of the disease. However, the efficiency of any neuroprotective treatment depends on its administration at a very early stage of the disease, which must be diagnosed before the onset of motor symptoms typically associated with PD.

[0005] Despite the significant efforts devoted to quantifying α-synuclein (AS) aggregates, which are considered as the main biomarker for PD, to date, no biochemical analytes exist in biological fluids that can detect the first stages of the disease.

[0006] The existing technologies for diagnosing and / or monitoring PD mainly from cerebrospinal fluid (CSF) samples are described in detail as follows:

[0007] - Conventional ELISA (Enzyme-Linked Immunosorbent Assay):

[0008] This immunoassay involves the immobilization of anti-synuclein antibodies on polymer plaques. The main disadvantage of this technique is that the aggregated species of AS are very heterogeneous, and the antibodies are sensitive to the conformation of their intended antigen. Therefore, they are only able to detect certain types of aggregates and therefore fail to reflect the huge diversity of existing species. This makes the technique non-specific and may affect its sensitivity (Ganguly et al. (2021); Eusebi et al. (2016); Atik et al. (2016)).

[0009] -AS seed amplification is also called real-time shaking-induced conversion (RT-QuIC) or AS seed amplification assay (SAA):

[0010] Based on the potential of this technique to distinguish patients with Parkinson's disease from healthy controls, a large cross-sectional analysis (1123 participants) using SAA showed that the test classified patients with Parkinson's disease with high sensitivity and specificity, providing information about molecular heterogeneity and also allowing the detection of prodromal individuals before diagnosis (Siderowf et al. (2023)).

[0011] The main limitation of RT-QuIC is the availability of recombinant human AS, whose market cost complicates the large-scale application of this technology. It also requires specialized equipment that is not available in low- and medium-complexity laboratories, as well as human resource training required to implement this technology. In addition, to date, there is no evidence that it may be miniaturized for use as a point-of-care (POC) system. (Luan, M., et al. (2022); Poggiolini I, et al. (2022); Kuzkina, A, et al. (2021)).

[0012] - Nuclear imaging techniques:

[0013] Single Photon Emission Computed Tomography (SPECT)

[0014] The technique uses a radioligand that binds to dopamine receptors to detect emitted gamma rays. SPECT can provide information about the function of dopaminergic neurons that are typically associated with Parkinson's disease.

[0015] Positron emission tomography (PET)

[0016] PET uses a positron-emitting radioactive tracer and can also assess dopamine activity in the brain by using radioligands that bind to dopamine receptors or transporters.

[0017] Nuclear imaging techniques have limited sensitivity and are not effective in detecting subtle changes in dopamine levels in the brain early in the disease, which can lead to false-negative results. Additionally, the cost of the technology limits its availability in healthcare systems with low or moderate complexity. Finally, interpreting the results can be complex and requires expertise in nuclear medicine.

[0018] In summary, these techniques are often used for diagnostic confirmation of PD in motor phases (Golan H,

[0019] et al. (2022); Pavese, N, et al. (2011); Moore, RY et al. (2008)).

[0020] -Immunomagnetic reduction of superconducting quantum interference device (IMR-SQUID):

[0021] The technique uses antibody-functionalized magnetic nanoparticles and a highly sensitive SQUID magnetometer. Although the technique is promising, because it can detect proteins at the femtomolar level, its large-scale application is complicated by the high cost of the equipment and the difficulty that antibodies present in detecting different aggregation species (Yang, SY., et al. (2016)).

[0022] Jang et al. (2020) disclosed an electrochemical sensor for detecting AS oligomers for early PD diagnosis. The sensor comprises a methylene blue aptamer adsorbed on an electrode, which desorbs when it binds to AS oligomers present in the sample, thereby producing a detectable change in the electrical signal provided by the electrode. The electrode used in the sensor is a reduced graphene oxide electrode.

[0023] However, there is a need for alternative AS aggregate detection techniques with low operating costs, readily available equipment, and broad specificity to facilitate the detection of several types of aggregates that may be present in a sample.

[0024] Regarding AD, it is a devastating neurological condition that gradually erodes memory, cognitive abilities, and behavior. AD is by far the most common form of dementia; 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. Likewise, the incidence of AD is only expected to rise over time. According to the Centers for Disease Control and Prevention (CDC), approximately 5.8 million Americans currently suffer from AD, and this number is expected to increase as the population ages. In fact, after the age of 65, the risk of developing AD doubles every 5 years. The rising incidence of this disease places a heavy burden on the healthcare system, which is attributed to the increasing demand for care as the disease progresses. This fact is made even more challenging by the fact that the mortality rate of AD is increasing compared to heart disease and cancer, where the mortality rate is decreasing. The cost of caring for AD patients is also staggering, estimated to be over $500 billion per year, and this number is expected to increase as the population ages.

[0025] A key neuropathological feature of AD is the presence of various morphological deposits of the microtubule-associated protein tau (Tau), called neurofibrillary tangles (NFTs), in the brain, which appear many years before the onset of clinical symptoms. Although the accumulation of NFTs was first described in AD, it is noteworthy that other neurodegenerative disorders, such as frontotemporal dementia, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration, have also been associated with the presence of NFTs. In fact, some of the strongest evidence supporting the involvement of Tau in neurodegenerative diseases comes from the identification of mutations in patients with frontotemporal dementia, highlighting that Tau may be a causative factor in the development of these types of diseases.

[0026] Tau pathology can spread from one area of ​​the brain to another, similar to a prion. Accumulating evidence suggests that aggregated Tau species spread along neuroanatomically connected brain regions via a "prion-like" mechanism, transferring abnormal Tau seeds from donor to recipient cells, thereby generating new Tau seeds. Therefore, early diagnosis of AD is critical as it provides an opportunity for pharmacological neuroprotective intervention to slow the progression of the disease. In the preclinical stages of Alzheimer's disease, obvious symptoms may not be present, but changes have already occurred in the brain, such as the accumulation of amyloid beta (Aβ) and Tau protein. These changes can be detected and quantified by imaging and biomarker detection.

[0027] Ongoing research into AD biomarker detection is currently focused on developing reliable and sensitive assays for detecting biomarkers in body fluids, such as cerebrospinal fluid (CSF), plasma, and urine. In addition, concerted attempts are underway to improve imaging techniques to detect biomarkers other than Aβ aggregates in the brain via neuroimaging.

[0028] Tau aggregates contain a wide variety of species in a dynamic process until they are recruited into NFTs. Most of them are involved in cytotoxicity. In addition, increasing evidence suggests that Tau aggregates play a crucial role in spreading pathology from one neuron to another, triggering aggregation in healthy neurons. These characteristics make Tau aggregates widely regarded as a powerful biomarker, but their detection remains challenging due to the lack of antibodies that can detect all species. Typical immunoassays that rely solely on epitope / complement interactions are insufficient to identify aggregates of all conformations, often resulting in false positives or false negatives.

[0029] Although all amyloid aggregates, including those formed by Tau, have a cross-β structure, the compactness of this structure can both hide epitopes and expose new epitopes. Therefore, it has proven to be a real challenge to develop antibodies that can specifically recognize and bind to amyloid aggregates. In contrast, small molecules such as Thioflavin T (ThT), Congo Red (CR) and Doxycycline show strong and specific binding to amyloid aggregate structures (González-Lizárraga (2017), Medina (2021)).

[0030] As for PD, there are several existing technologies for diagnosing and monitoring AD in low- and moderate-complexity laboratories, as described below:

[0031] - Immunohistochemistry

[0032] Immunohistochemistry is used to confirm the diagnosis of Alzheimer's disease after death by examining the presence and distribution of Tau aggregates in brain tissue samples obtained during autopsy. This technique involves staining thin sections of brain tissue with specific antibodies that bind to the Tau protein. The antibody is labeled with a color-producing molecule that enables the visualization of Tau aggregates under a microscope.

[0033] - Immunofluorescence

[0034] Immunofluorescence is a technique similar to immunohistochemistry, but it uses fluorescent dyes instead of color-producing molecules to label Tau. The technique involves incubating brain tissue samples with antibodies that specifically bind to Tau, which are conjugated to fluorescent dyes. It is important to note that although immunofluorescence is a valuable tool in Alzheimer's disease research, it is not routinely used in clinical diagnosis during autopsy.

[0035] - Conventional ELISA

[0036] Conventional ELISA is limited in its ability to detect Tau aggregate species due to its reliance on a single monoclonal capture antibody. This limitation reduces its ability to detect a diversity of Tau aggregate species. Commonly used commercial ELISA tests, such as BioLegend, Abcam, Thermofisher, and IBL International, are designed to detect total Tau protein and phosphorylated Tau as biomarkers. Unfortunately, these tests suffer from reduced sensitivity and specificity, leading to false positive or negative results (Mounsey (2018)). This highlights the need for better capture molecules to improve the accuracy of Tau aggregate detection.

[0037] -PET imaging

[0038] This technology is a powerful tool for visualizing and quantifying the accumulation of pathological proteins in the brains of patients with Alzheimer's disease (AD). Several radiotracers have been developed to target Tau aggregates in the brain, such as [ 18 [F]Tau PET tracers. As mentioned above for PD, this diagnostic technique is primarily available in high-complexity medical centers and research institutions due to the cost of the equipment and radiotracers. In addition, the interpretation of PET imaging is limited because it can only provide information about the distribution and density of pathological proteins, not about their specific composition or location in the brain. Furthermore, the interpretation of PET results can be challenging because there are no standardized methods for image analysis and quantification.

[0039] -Single-molecule fluorescence resonance energy transfer (smFRET)

[0040] This is a biophysical technique that can be used to measure intramolecular distances of proteins, including Tau. This technique has been used to study conformational changes in Tau in the presence or absence of aggregation-inducing agents, providing insights into the mechanism of Tau aggregation in AD. However, smFRET

[0041] It is a complex technology that requires specialized equipment and expertise and is typically only available through service providers or specialized research laboratories.

[0042] -RT-QuIC

[0043] This technology has been explored as a diagnostic tool for AD. It involves detecting misfolded proteins, such as Tau and β-amyloid, in CSF or other biological samples by inducing the conversion of normal proteins into abnormal aggregates, which can be detected by 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 in the experimental stage and has not yet been widely used in the clinic. It also requires specialized equipment and expertise, which limits its application in more sophisticated laboratories.

[0044] Clearly, there remains a need for alternative technologies to detect AD-related biomarkers with low operating costs, using readily available equipment and with broad specificity to facilitate the detection of several types of aggregates that may be present in a sample.

[0045] Furthermore, it is highly desirable to develop a general technology that is applicable to the early detection of biomarkers associated with neurodegenerative diseases. In particular, it would be very useful to obtain an apparatus and method for detecting biomarkers associated with diseases such as PD and AD.

[0046] Doxycycline is a well-known antibiotic in the tetracycline family that has been used to treat a variety of bacterial infections, such as bacterial pneumonia, cholera, syphilis, etc. SUMMARY OF THE INVENTION

[0048] According to a first aspect, the present invention provides a device for detecting neurotoxic amyloid aggregates, comprising a doxycycline derivative capable of binding to neurotoxic amyloid aggregates, wherein the doxycycline derivative is immobilized on a surface.

[0049] In one embodiment of this aspect of the invention, the device comprises a doxycycline derivative of formula (I):

[0050]

[0051] It is fixed on the surface, wherein R is selected from:

[0052] i- is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent of X, wherein n=0-10. and X is selected from Cl, Br and I;

[0053] ii - an alkyl or aryl linker with a sulfhydryl terminal group;

[0054] iii- Where n = 0-10;

[0055] iv- wherein n = 0-10; and

[0056] v- Where n=0-10.

[0057] In a preferred embodiment, R is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent for X, wherein n=0-10, and X is selected from Cl, Br and I.

[0058] In a particularly preferred embodiment of the invention, the device comprises a doxycycline derivative of formula (II),

[0059]

[0060] It is fixed to the surface.

[0061] In another embodiment of this aspect of the invention, the surface is selected from a polymer surface and a functionalized metal surface.

[0062] In a specific embodiment of the present invention, the surface is a polymer surface, wherein the polymer surface comprises a polymer selected from polystyrene, polystyrene / divinylbenzene copolymers and other synthetic or natural polymers, wherein the doxycycline derivative can be immobilized. Preferably, the polymer surface comprises polystyrene.

[0063] In a preferred embodiment of the present invention, the doxycycline derivative is immobilized on the polymer surface by covalently binding to a blocking agent adsorbed on the surface via a linker. Preferably, the blocking agent adsorbed on the polymer surface is bovine serum albumin.

[0064] In a particularly preferred embodiment, the device comprises a doxycycline derivative of formula (II) immobilized on a polymer surface via covalent bonding of glutaraldehyde to bovine serum albumin adsorbed on the surface.

[0065] In another specific 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.

[0066] In a particularly preferred embodiment, the device comprises a doxycycline derivative of formula (II) immobilized on a functionalized metal surface, wherein the functionalized metal surface is a functionalized gold surface, wherein the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and wherein the doxycycline derivative of formula (II) is covalently bound to the SAM of 3-mercaptopropionic acid.

[0067] Another aspect of the present invention provides an in vitro method for detecting and / or quantifying neurotoxic amyloid aggregates, comprising:

[0068] i-provides a device for detecting neurotoxic amyloid aggregates according to the first aspect of the present invention;

[0069] ii- contacting the device with a sample, wherein the presence and / or concentration of the neurotoxic amyloid aggregates is to be determined; and

[0070] iii- determining the presence and / or concentration of said neurotoxic amyloid aggregates by a detection technique.

[0071] In one embodiment of this aspect, the neurotoxic amyloid aggregates to be detected and / or quantified are α-synuclein (AS) aggregates.

[0072] In another embodiment of this aspect, the neurotoxic amyloid aggregates to be detected and / or quantified are tau protein (Tau) aggregates.

[0073] In one embodiment of this aspect of the invention, the detection technique is selected from the group consisting of an immunochemical assay and an electrochemical assay.

[0074] In a specific embodiment of this aspect of the invention, the detection technique of step iii- is an electrochemical assay. Preferably, the electrochemical assay uses a technique selected from cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for detecting neurotoxic amyloid aggregates. More preferably, the electrochemical assay uses cyclic voltammetry for detecting neurotoxic amyloid aggregates.

[0075] In another specific embodiment of this aspect of the invention, the detection technique of step iii- is an immunochemical assay, and step iii- comprises the following sub-steps:

[0076] a) contacting the device with antibodies suitable for binding to neurotoxic amyloid aggregates to be detected and / or quantified; and

[0077] b) Measuring bound antibody by a measurement technique.

[0078] In a preferred embodiment of this aspect of the invention, the neurotoxic amyloid aggregates to be detected and / or equivalent are AS aggregates, and the method comprises:

[0079] i-provides a device for detecting neurotoxic amyloid aggregates according to the first aspect of the present invention;

[0080] ii- contacting the device with a sample, wherein the presence and / or concentration of AS aggregates is to be determined; and

[0081] iii- determining the presence and / or concentration of said AS aggregates by an immunochemical assay, wherein the immunochemical assay further comprises:

[0082] a) contacting the device with an antibody suitable for binding AS aggregates; and

[0083] b) Measuring bound antibody by a measurement technique.

[0084] In another preferred embodiment of this aspect of the invention, the neurotoxic amyloid aggregates to be detected and / or quantified are Tau aggregates, and the method comprises:

[0085] i-provides a device for detecting neurotoxic amyloid aggregates according to the first aspect of the present invention;

[0086] ii- contacting the device with a sample, wherein the presence and / or concentration of Tau aggregates is to be determined; and

[0087] iii- determining the presence and / or concentration of Tau aggregates by an immunochemical assay, wherein the immunochemical assay further comprises:

[0088] a) contacting the device with an antibody suitable for binding to Tau aggregates; and

[0089] b) Measuring bound antibody by a measurement technique.

[0090] Another aspect of the present invention provides a method for preparing the device for detecting neurotoxic amyloid aggregates of the present invention, comprising:

[0091] i- providing a surface for immobilizing the doxycycline derivative; and

[0092] ii- contacting a surface capable of immobilizing a doxycycline derivative with a solution comprising a doxycycline derivative capable of binding to neurotoxic amyloid aggregates under certain conditions, such that the doxycycline derivative capable of binding to neurotoxic amyloid aggregates is immobilized on the surface.

[0093] In one embodiment of this aspect of the invention, the surface capable of immobilizing the doxycycline derivative is selected from the group consisting of a polymer surface and a functionalized metal surface.

[0094] In a specific embodiment of this aspect of the invention, the surface is a polymer surface, wherein the polymer plastic surface comprises a polymer selected from polystyrene, polystyrene / divinylbenzene copolymers and other synthetic or natural polymers to which doxycycline and its derivatives can be immobilized. Preferably, the polymer plastic surface comprises polystyrene.

[0095] In one embodiment of this aspect of the invention, the surface is a polymer surface and step i of the method further comprises the following sub-steps:

[0096] a) contacting the polymer surface with a solution comprising a blocking agent so as to allow the blocking agent to adsorb onto the polymer surface, thereby obtaining a blocked polymer surface; and

[0097] b) contacting the blocked polymer surface with a solution comprising a linker to obtain a polymer surface capable of immobilizing a doxycycline derivative.

[0098] Preferably, the blocking agent is bovine serum albumin and the linker is glutaraldehyde.

[0099] In another specific embodiment of this aspect of the invention, the surface is a functionalized metal surface, and the method further comprises a step prior to step i, wherein the metal surface is contacted with a solution comprising 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.

[0100] Another aspect of the present invention provides a kit for detecting and quantifying biomarkers associated with P1), comprising:

[0101] i- a polymer surface functionalized with a doxycycline derivative prepared according to the invention; and

[0102] ii-Detection reagents.

[0103] Yet another aspect of the present invention provides a point-of-care (POC) biosensor for detecting biomarkers associated with PD, comprising an electrode functionalized or to be functionalized with a doxycycline derivative according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The following drawings constitute an integral part of this specification and are included for the purpose of further illustrating certain aspects of the present invention.

[0106] Figure 1. CV characterization of each different stage of gold surface modification during the fabrication of the device of the present invention. Doxy-5 refers to the compound of formula (II) of the present invention.

[0107] Figure 2 Zoom in on the oxidation peaks of: A) cyclic voltammograms obtained at different AS concentrations using the gold-based device of the present invention; and B) cyclic voltammograms of control conditions without the addition of Doxy-5.

[0108] Figure 3 Evaluation of the concentration of the compound of formula (II) for good sensitization in the polystyrene-based device of the present invention. Doxy-5 refers to the compound of formula (II) of the present invention.

[0109] Figure 4 Characterization of the present invention for blocking non-specific binding sites in polystyrene-based devices using different treatments. Doxy-5 refers to a compound of formula (II) of the present invention.

[0110] Figure 5 Immunoassay of human CSF using the polystyrene-based device of the invention. ASm and ASf correspond to in vitro samples of monomeric and fibrillar AS, respectively, in PBS buffer. Samples F, B, C, G and E refer to CSF ​​obtained from patients without registered pathological conditions compatible with movement disorders.

[0111] Figure 6 Optimization of Doxy-5 fixation conditions in BSA using glutaraldehyde and Tau monomers (Tau m ) and preformed fibrils (PFF).

[0112] Figure 7 Effect of Doxy-5 fixation in BSA glutaraldehyde and replacement of Doxy-5 with BSA on Tau binding at different dilutions m or PFF interaction results (p≤0.05). Black bars show blank values ​​(OD<0.1) that were considered negative.

[0113] Figure 8 Comparative analysis of the binding efficiency of Doxy-5 and commercially available doxycycline hydrochloride (Doxy-Hy) to AS PFF (p<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0115] The present invention relates to a device for detecting neurotoxic amyloid protein aggregates based on the ability of doxycycline to bind to such protein aggregates.

[0116] As mentioned above, AS aggregates (ie, polymer forms of AS) are considered as the main biomarker for PD, while Tau aggregates are considered as promising biomarkers for AD. Therefore, the device of the present invention has advantageous properties for simultaneously detecting PD and AD biomarkers in biological samples.

[0117] Thus, the device of the present invention comprises a doxycycline derivative immobilized on a surface.

[0118] In the context of the present disclosure, doxycycline derivatives are understood to be compounds obtained by introducing minor modifications into doxycycline while maintaining its ability to bind to neurotoxic amyloid aggregates such as AS and Tau aggregates. For example, such doxycycline derivatives may be compounds of formula (I):

[0119]

[0120] It is fixed on the surface, wherein R is selected from:

[0121] i- is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent for X, wherein n=0-10, and X is selected from Cl, Br and I;

[0122] ii - an alkyl or aryl linker with a sulfhydryl terminal group;

[0123] iii- Where n = 0-10;

[0124] iv- wherein n = 0-10; and

[0125] v- Where n=0-10.

[0126] The alkyl or aryl linker with a terminal sulfhydryl group of the doxycycline derivative of formula (I) refers to any sufficient alkyl chain or aryl group including a sulfhydryl group at its terminal end.

[0127] In the case where the linker is an alkyl linker, the linker may be any alkyl hydrocarbon chain, which may be linear or branched, wherein the sulfhydryl group is bonded to the carbon atom farthest from the aromatic ring R of formula (I).

[0128] In case the linker is an aryl linker, the linker may be any aryl group having a sulfhydryl group directly bonded to its aromatic system or comprising an alkyl substituent having a sulfhydryl group bonded to a carbon atom which is furthest from the bond of such alkyl substituent to the aromatic system of the aryl group.

[0129] In a preferred embodiment, R is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent for X, wherein n=0-10, and X is selected from Cl, Br and I.

[0130] In a particularly preferred embodiment, the doxycycline derivative is a compound of formula (II):

[0131]

[0132] The compound of formula (II) is also referred to herein as Doxy-5.

[0133] The device of the present invention requires a doxycycline derivative that is immobilized on a sufficient surface. By "immobilized," it will be understood by those skilled in the art that the compound should be bound to the surface in a manner that allows it to remain bound to the surface even after several washes. The manner in which the compound is immobilized on the surface will depend on the characteristics of the surface.

[0134] In one embodiment of the invention, the surface is a polymer surface. When such a surface is used, the doxycycline derivative is usually immobilized thereon by an adsorption process, i.e. the compound is bound to the surface by intermolecular forces or by a blocking agent adsorbed on the polymer surface, wherein the doxycycline derivative may be covalently bound via a suitable linker.

[0135] The term "blocking agent" is understood to refer to proteins that are susceptible to binding to the polymer surface of a device, such as those used to block the surface of, for example, an ELISA plate. Such blocking agents are well known to those skilled in the art. However, the inventors surprisingly found that the sensitivity of a device for detecting neurotoxic amyloid aggregates was significantly improved using bovine serum albumin (BSA) as a blocking agent. Therefore, when the polymer surface comprises a blocking agent adsorbed thereon, the blocking agent adsorbed on the polymer surface of the device is preferably BSA.

[0136] The linker for covalently binding the doxycycline derivative to the blocking agent is a bifunctional small molecule comprising two reactive moieties at opposite ends of the hydrocarbon chain, so that one reactive moiety can be covalently bound to the blocking agent adsorbed on the polymer surface, and the other reactive moiety can be covalently bound to the doxycycline derivative, thereby immobilizing the doxycycline derivative on the polymer surface. The linker selected depends on the selected binding agent and doxycycline derivative, as can be understood by those skilled in the art.

[0137] When the polymer surface comprises a blocking agent adsorbed thereon, the doxycycline derivative is a doxycycline derivative that has been chemically modified to comprise a functional group capable of binding to the blocking agent adsorbed on the polymer surface. For example, the doxycycline derivative capable of binding to a linker attached to the blocking agent has formula (I) as defined above.

[0138] In a preferred embodiment, the device comprises a polymer surface to which bovine serum albumin has been adsorbed, and wherein the doxycycline derivative of formula (II) is covalently bound to the bovine serum albumin via glutaraldehyde as a linker.

[0139] In another preferred embodiment, no blocking agent is used and the device comprises a polymer surface to which the doxycycline derivative has been immobilized by direct adsorption without the use of a blocking agent.

[0140] The term "polymer surface" should be broadly understood to refer to any surface comprising a polymer to which a doxycycline derivative or blocking agent is susceptible to adsorption. For example, the polymer surface comprises a polymer selected from polystyrene, polystyrene / divinylbenzene copolymers and other synthetic or natural polymers, to which doxycycline and its derivatives can be immobilized. Preferably, the polymer surface comprises polystyrene.

[0141] In another embodiment of the present invention, the surface is a functionalized metal surface. The term "functionalized metal surface" is understood to refer to a metal surface to which a functionalizing agent has been bound. The function of the functionalizing agent is to act as a linker between the doxycycline derivatives by covalently binding the metal surface and the doxycycline derivative. Preferably, the functionalized metal surface is a functionalized gold surface. More preferably, the functionalized metal surface is a functionalized gold surface that is bound to a mercapto acid as a functionalizing agent, thereby forming a self-assembled monolayer (SAM). Therefore, in this preferred embodiment, the functionalized gold surface comprises a SAM of linear mercapto acids. In this type of arrangement, the linear mercapto acid molecules are bound to the gold surface in an orderly manner through their terminal sulfhydryl moieties, thereby exposing the carboxylic acid moieties to which the doxycycline derivatives are covalently bound. Most preferably, the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid.

[0142] When the surface is such a functionalized metal surface, the doxycycline derivative is a doxycycline derivative that has been chemically modified to include a functional group of a functionalizing agent capable of binding to the functionalized metal surface. For example, the doxycycline derivative capable of binding to the functionalizing agent has formula (I) as defined above.

[0143] In a particularly preferred embodiment, the device comprises a doxycycline derivative of formula (II) immobilized on a functionalized metal surface, wherein the functionalized metal surface is a functionalized gold surface, wherein the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and wherein the doxycycline derivative of formula (II) is covalently bound to the SAM of 3-mercaptopropionic acid.

[0144] The device of the present invention can be prepared by readily available techniques used by those skilled in the art to immobilize compounds on a sufficient surface. Therefore, another aspect of the present invention provides a method for preparing the device of the present invention for detecting neurotoxic amyloid aggregates, comprising:

[0145] i- providing a surface for immobilizing the doxycycline derivative; and

[0146] ii- contacting a surface capable of immobilizing a doxycycline derivative with a solution comprising a doxycycline derivative under conditions such that the doxycycline derivative is immobilized on the surface.

[0147] If necessary, providing a surface for immobilizing a doxycycline derivative comprises modifying the corresponding surface so that it is capable of such immobilization.

[0148] In a specific embodiment, the surface is a polymer surface. In such embodiments, the surface is capable of immobilizing the doxycycline derivative by an adsorption process without any additional modification.

[0149] However, in an alternative embodiment of this aspect of the invention, the polymer surface is treated in advance with a blocking agent adsorbed thereon. Therefore, in said embodiment, step i of the method comprises the following sub-steps:

[0150] a) contacting the polymer surface with a solution comprising a blocking agent so as to allow the blocking agent to adsorb onto the polymer surface, thereby obtaining a blocked polymer surface; and

[0151] b) contacting the blocked polymer surface with a solution comprising a linker to obtain a polymer surface capable of immobilizing a doxycycline derivative.

[0152] The polymer surface capable of immobilizing a doxycycline derivative provided thereby comprises a blocking agent adsorbed on the polymer surface, and a linker covalently bound thereto provides a moiety to which the doxycycline derivative can be easily covalently bound. It is within the knowledge of those skilled in the art to optimize the conditions under which the blocking agent adsorption and reaction with the linker occur.

[0153] The blocking agent and linker have the same characteristics as described above in this specification. In a preferred embodiment, the blocking agent is BSA and the linker is glutaraldehyde.

[0154] In another specific embodiment, the surface is a functionalized metal surface. In such embodiments, the metal surface must be functionalized in order to be able to immobilize the doxycycline derivative. Therefore, in such embodiments, the method further comprises a step before step i, wherein the metal surface is contacted with a solution comprising 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. It is within the knowledge of those skilled in the art to optimize the conditions under which metal surface functionalization occurs.

[0155] The step of contacting the surface capable of immobilizing the doxycycline derivative with a solution comprising the doxycycline derivative under conditions such that the doxycycline derivative is immobilized on the surface also depends on the surface selected.

[0156] In the embodiment where the surface is a polymer surface, the step comprises immersing the corresponding surface in a solution containing an appropriate concentration of a doxycycline derivative for a sufficient time to complete the adsorption process or reaction with the linker. In the specific embodiment where the polymer surface is previously treated with a binding agent and a linker, the doxycycline derivative should also be a doxycycline derivative capable of binding to a linker bound to a blocking agent, preferably a doxycycline derivative of formula (I), or most preferably a doxycycline derivative of formula (II).

[0157] In embodiments where the surface is a functionalized metal surface, the solution comprising the doxycycline derivative should also include any additional reagents necessary to form a covalent bond between the doxycycline derivative and the functionalized metal surface. The doxycycline derivative should also be a doxycycline derivative capable of binding to the functionalizing agent of the functionalized metal surface, preferably a doxycycline derivative of formula (I), or most preferably a doxycycline derivative of formula (II). In embodiments where the metal surface is a gold surface and the functionalizing agent is 3-mercaptopropionic acid, the solution comprising the doxycycline derivative should also include reagents necessary to effectively bind to the carboxylic acid portion of 3-mercaptopropionic acid. For example, in such embodiments, the solution may also include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0158] The reaction conditions necessary to optimize the adsorption process or immobilize the doxycycline derivative on a selected surface are well within the skill of one skilled in the art.

[0159] It is contemplated that the device of the present invention is used to detect neurotoxic amyloid aggregates in a sample. The term "neurotoxic amyloid aggregates" as used herein refers to protein aggregates associated with neurodegenerative diseases. Such aggregates are those typically observed to accumulate in neurons and other parts of the nervous system during the progression of certain neurodegenerative diseases, 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 synuclein diseases such as PD and Tau pathologies such as AD in such samples, respectively. The signal emitted by the device when the aggregates are detected is related to its concentration in the sample. The signal emitted by the device when detecting the aggregates is related to its concentration in the sample. Therefore, another aspect of the present invention provides an in vitro method for detecting and / or quantifying neurotoxic amyloid aggregates, comprising:

[0160] i- providing a device for detecting and / or quantifying neurotoxic amyloid aggregates according to the present invention;

[0161] ii- contacting the device with a sample in which the aggregates to be detected are present; and

[0162] iii- determining the presence and / or concentration of said aggregates by a detection technique.

[0163] The method exploits the ability of doxycycline derivatives to bind to said aggregates. By contacting a device comprising a doxycycline derivative immobilized on a surface with a sample comprising neurotoxic amyloid aggregates, said aggregates bind to the doxycycline derivative, making it possible to detect them by appropriate detection techniques.

[0164] The detection technology used to detect and / or quantify neurotoxic amyloid aggregates in the methods related to the present invention can depend on the surface used to immobilize the doxycycline derivative. For example, the detection technology can be selected from immunochemical assays and electrochemical assays. In some embodiments, the detection technology comprises a sandwich immunoassay.

[0165] In a specific embodiment, the detection technique is an immunochemical assay, which requires a surface that is a polymer surface, preferably a polystyrene surface. Such an immunochemical assay comprises contacting the device with neurotoxic amyloid aggregates from a sample bound to an immobilized doxycycline derivative as obtained in step ii- of the method, with antibodies having a specific affinity for proteins present in the bound aggregates, which can then be detected by the detection device itself or by another antibody.

[0166] For example, if the neurotoxic amyloid aggregates present in the sample are AS aggregates, they can be detected by anti-AS antibodies for binding to immobilized doxycycline. Then, the binding of the anti-AS antibody to the bound AS can be detected using a HRP-connected secondary antibody. Similarly, if the neurotoxic amyloid aggregates present in the sample are Tau aggregates, their binding to immobilized doxycycline can be detected by anti-Tau antibodies. Then, the binding of the anti-Tau antibody to the bound Tau can be detected by a HRP-connected secondary antibody. This type of immunochemical assay is completely within the technical scope of those skilled in the art. However, a general method is provided, such as the method described herein, in which it can be applied to a method for detecting and / or quantifying a series of neurotoxic amyloid aggregates associated with neurodegenerative diseases, only by changing the antibodies used to detect the specific proteins involved in the formation of the binding aggregates, which obviously cannot be obtained from the prior art in an obvious manner.

[0167] It will be appreciated by those skilled in the art that, as described above, the above detection techniques are applicable when the surface of the device is a polymer surface, regardless of whether the doxycycline derivative is immobilized on the polymer surface by direct adsorption or via a blocking agent-linker bond.

[0168] 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 embodiments, the metal surface is used as an electrode in an apparatus (e.g., a potentiostat) for performing electrochemical measurements. Preferably, the electrochemical assay uses a technique selected from CV and EIS for detecting and / or quantifying neurotoxic amyloid aggregates. More preferably, the electrochemical assay uses CV for detecting and / or quantifying neurotoxic amyloid aggregates.

[0169] In this embodiment of the invention, the specificity of each protein ((AS) aggregates and (Tau) aggregates) can then be assessed by using specific antibodies, the binding of which can also be assessed electrochemically. That is, antibodies with selective affinity for a particular protein (AS or Tau) can bind to the corresponding aggregates bound to the doxycycline derivative, thereby changing the detected electrochemical signal. However, a positive signal in this sensor already indicates the presence of some aggregates, so it can be a point-of-care (POC) assay result, which already gives a positive diagnosis.

[0170] As described above, the formation of neurotoxic amyloid aggregates that can be detected and / or quantified by the method of this aspect of the invention has been observed for several neurodegenerative diseases. In one embodiment, the method is used to detect and / or quantify AS aggregates, which are associated with synuclein diseases, such as PD, Lewy body dementia (DLB) and multiple system atrophy (MSA). In another embodiment, the method is used to detect and / or quantify Tau aggregates, which are associated with tauopathies, such as AD, progressive supranuclear palsy (PSP), cortical basal degeneration (CBD), chronic traumatic encephalopathy (CTE) and Pick's disease (PiD).

[0171] Accordingly, the method of the invention can be used for early diagnosis of such neurodegenerative diseases.For such use, the sample in which the presence of neurotoxic amyloid aggregates is to be determined is a sample from an individual suspected of having one of said diseases.

[0172] As used herein, the term diagnosis refers to detecting a disease or disorder, or determining the stage or extent of a disease or disorder. Diagnostic methods can be used independently or in combination with other diagnostic and / or staging methods known in the art for a particular disease or disorder. Other diagnostic markers can be combined with the markers described herein to predict the presence or absence of a disease or the stage of a disease. For example, clinical factors associated with the diagnosis of a neurodegenerative disease include, but are not limited to, the patient's medical history, physical examination, and other biomarkers.

[0173] As used herein, the terms biological sample and specimen are used interchangeably and refer to a sample obtained from a patient or subject. In some cases, a biological sample can be a tissue, cell, or body fluid (e.g., blood, serum, synovial fluid, sputum, lung fluid, mucus, tears, lymph, synovial fluid, cerebrospinal fluid (CSF), feces, saliva, amniotic fluid, amniotic fluid cord blood, urine, vaginal fluid, and semen). Preferably, this sample is a cerebrospinal fluid (CSF) sample.

[0174] As used throughout, a subject refers to an individual, such as an adult subject. Preferably, the subject is an animal, for example, a mammal such as a primate, more preferably a human. Non-human primates are also research subjects. The term subject includes cats, dogs, reptiles, amphibians, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.) and experimental animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, veterinary use is contemplated herein.

[0175] Another aspect of the present invention provides a kit for detecting and / or quantifying neurotoxic amyloid aggregates, comprising:

[0176] i- a polymer surface functionalized or to be functionalized with a doxycycline derivative; and

[0177] ii-Detection reagents.

[0178] The term "polymer surface functionalized or intended to be functionalized with a doxycycline derivative" is to be understood as a polymer surface as described above for the device of the invention, which comprises a doxycycline derivative already immobilized thereon or is capable of readily immobilizing a doxycycline derivative upon contact with a solution comprising it.

[0179] The detection reagent comprises a reagent known to those skilled in the art for immunochemical detection and / or quantification of neurotoxic amyloid aggregates according to the above-mentioned in vitro method. Such detection reagents may include, for example, secondary antibodies, staining reagents, etc. In a specific embodiment, the polymer surface of the kit is susceptible to functionalization with a doxycycline derivative, and the detection reagent also comprises a doxycycline derivative.

[0180] In a preferred embodiment, the kit of this aspect of the invention comprises a doxycycline derivative of formula (I) which is immobilized on the polymer surface or contained in the detection reagent. More preferably, the doxycycline derivative is a doxycycline derivative of formula (II).

[0181] The kit of this aspect of the invention can be handled by a laboratory technician and in a laboratory of moderate sophistication.

[0182] Yet another aspect of the present invention provides a point-of-care (POC) biosensor for detecting biomarkers associated with PD, comprising an electrode, wherein the electrode: (i) comprises a doxycycline derivative immobilized thereon, or (ii) is capable of immobilizing a doxycycline derivative thereon.

[0183] The term "electrode: (i) comprising a doxycycline derivative immobilized thereon, or (ii) capable of immobilizing thereon a doxycycline derivative" is understood to mean a functionalized metal surface as described above for the device of the present invention, which comprises a doxycycline derivative capable of readily immobilizing upon contact with a solution comprising it. Preferably, the POC biosensor comprises a doxycycline derivative immobilized thereon, more preferably a doxycycline derivative of formula (I), most preferably a doxycycline derivative of formula (II).

[0184] The main advantage of the present invention is that it is selective for detecting neurotoxic aggregated forms of proteins such as AS and Tau over their monomeric forms, while being easy to implement and not requiring particularly expensive or complex equipment. Therefore, it shows great potential as a POC device for early detection of biomarkers associated with diseases such as PD and AD, which can lead to early diagnosis of the diseases, thereby providing better prognosis for them. Example

[0185] The present invention is further illustrated by the following examples, which are not intended to limit its scope. On the contrary, in order to better implement the present invention, the examples described below should be understood as exemplary embodiments only.

[0186] Example 1 - Organic Synthesis of Compounds of Formula (II)

[0187] General experimental methods

[0188] At 500MHz( 1 H) or 125.7MHz( 13 C) or at 300MHz( 1 H) or 75.6MHz( 13 C) Record NMR spectra. Chemical shifts (δ, in ppm) referenced to internal standards (for 1 H in CDCl3 Me4Si (δ0.0) and for 13 C is CDCI3 (δ: 77.0) or reference residual solvent peak. Data diversity is expressed as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak); coupling constants (J) are specified in Hertz (Hz). 1 H and 13 Assignment of C NMR spectra by 2D 1 H-COSY or NOESY and 2D 1 H- 13 C HSQC assisted. High resolution mass spectra (HRMS) were obtained using electrospray ionization (ESI) technology and Q-TOF detection. Analytical thin layer chromatography (TLC) was performed on silica gel 60F254 aluminum support plates (layer thickness 0.2 mm) and silica gel 60RP F254S aluminum support plates. The spots can be developed by exposure to UV light and carbonization with Ce / Mo staining. Column chromatography was performed with silica gel 60 (230-400 mesh), or for reverse phase, octadecyl functionalized silica gel was used as the stationary phase. The chromatographic solvent or stepwise solvent polarity gradient used was specified for each individual compound. Optical rotation was measured at the sodium D line in a 1 dm cell in the solvent at room temperature. Unless otherwise specified, all commercially available compounds were obtained from the supplier and used without further purification.

[0189] Doxycycline free form from doxycycline hydrochloride (D-1)

[0190]

[0191] Doxycycline hydrochloride (2.0 g, [α] D 20= -113.5 (c 1, 10 mM HCL in MeOH) was dissolved in distilled water (6 mL) and 1 M NaOH aqueous solution was added dropwise to reach pH ≈ 5. At this point, a white solid was formed. The solid was filtered and dissolved in methanol (20 mL). The solution was stirred for 20 min until a new white precipitate appeared. The solid was filtered and dried to obtain doxycycline free form (D-1, 1.3 g, 75%); [α] D 20 = +250.7 (c 1, THF), 1 H NMR ((CD3)2CO, 500MHz) δ: 7.53 (t, 1H, J 7，8 =J 8，9 =8.1Hz, H-8), 7.02(d, 1H, J 8，9 =8.1Hz, H-9), 6.82(d, 1H, J 7，8 =8.1Hz, H-7), 4.21(brt, 1H, J 5a，5 ≈J 4a，5 ≈4.0Hz, H-5), 3.59(brd, 1H, J 4a，4 =9.6Hz,H-4),2.93(dq,1H,J 6，Me =6.7, J 5a，6 =12.8Hz, H-6), 2.74(dd, 1H, J 5a，5 =4.0, J 5a，6 =12.8Hz, H-5a), 2.63(dd, 1H, J 4a，4 =9.6, J 4a，5 =4.0Hz, H-4a), 2.53(s, 6H, N(CH3)2), 1.61(d, 3H, J 6，Me =6.7 Hz, CH3); 13 C NMR ((CD3)2CO, 125.7MHz) δ: 194.7 (C-1, 3, 11), 174.9, 174.5 (C-12, CONH2), 163.4 (C-10), 148.7 (C-6a), 137.6 (C-8), 117.4, 116.8, 116.4 (C- 7, 9, 10a), 106.4 (C-11a), 92.0 (C-2), 76.0 (C-12a), 69.9 (C-5), 66.2 (C -4), 48.9(C-4a), 48.2(C-5a), 42.6(N(CH3)2), 38.5(C-6), 16.7(CH3).

[0192] This method was also applied to doxycycline hydrate, yielding 69% pure D-1.

[0193] Synthesis of methyl iodide doxycycline salt (D-2)

[0194]

[0195] To a solution of D-1 (2.0 g, 4.5 mmol) in dry THF (40 mL) was added iodomethane (2.5 mL, 40 mmol) dropwise at room temperature under Ar atmosphere. The reaction system was stirred at 45 ° C for 24 h, and the solvent was removed by evaporation under reduced pressure. The obtained solid was washed with anhydrous CH2Cl2 (15 mL) and dried to give D-2 (2.6 g, 98%). [α] D 20 +31.2 (c 1.0, THF); 1 H NMR ((CD3)2CO, 200MHz) δ: 7.55 (t, 1H, J 7，8 =J 8，9 =8.0Hz, H-8), 6.97(d, 1H, J 8，9 ≈8.0Hz, H-7), 6.86 (d, 1H, J 7，8 ≈8.1Hz, H-9), 5.44 (s, 1H, OH), 3.89 (brt, 1H, H-5), 3.69 (s, 9H, N (CH3)3), 3.53 (brd, 1H, H-4), 2.97-2.60 (m, 3H, H-4a, 5a, 6), 1.56 (d, 3H, J 6，Me =6.4Hz, CH3); HRMS(ESI)m / z[M] + Calculated value C 23 H 27 N2O5459.1762, measured value 459.1762.

[0196] Synthesis of 4-Desdimethylaminodoxycycline D-3

[0197]

[0198] To a solution of D-2 (1 g, 1.7 mmol) in 50% (v / v) aqueous acetic acid (30 ml) was added zinc powder (0.6 g, 9.2 mmol) and the mixture was stirred at room temperature for 20 min. The suspension was filtered through a pad of celite. The filtrate was diluted with water (100 mL) containing concentrated HCl (1 mL) and the mixture was stirred in an ice bath for 1 h. The solid formed was filtered and dried in vacuo. This amorphous solid was characterized as D-3 (0.48 g, 70%); [α] D 2 0-50.8 (c 1.0, acetone); 1H NMR ((CD3)2CO, 500MHz) δ: 7.51 (t, 1H, J 7，8 =J 8，9 =8.0Hz, H-8), 6.96 (d, 1H, J 8，9 =8.0Hz, H-9), 6.84(d, 1H, J 7，8 =8.0Hz, H-7), 4.41(d, 1H, J 5，OH =8.5Hz, OH), 3.79 (br q, 1H, J 4a，5 =9.5, J 5a，5 =7.7, J 5，OH =8.5Hz, H-5), 3.06(dd, 1H, J 4a，4 =5.5, J 4，4， =18.6Hz, H-4), 2.97(dd, 1H, J 4a，4， =2.9, J 4，4’ =18.6Hz, H-4′), 2.79(m, 1H, J 6，Me =6.8, J 5a，6 =12.5Hz, H-6), 2.51(dd, 1H, J 5a，5 =7.7, J 5a，6 =12.5Hz, H-5a), 2.47(ddd, 1H, J 4a，4 =5.5, J 4a，4’ =2.9, J 4a，5 =9.5Hz, H-4a), 1.57(d, 3H, J 6，Me =6.8 Hz, CH3); 13 C NMR ((CD3)2CO, 125.7MHz) δ: 195.9, 194.6, 193.2 (C-1, 3, 11), 176.0, 174.9 (C-12, CONH2), 163.1(C-10), 149.1(C-6a), 137.4(C-8), 116.8, 116.7, 116. 6(C-7, 9, 10a), 107.4(C-11a), 99.7(C-2), 75.7(C-12a), 69.6(C-5), 44.4(C -4a), 47.6(C-5a), 39.5(C-6), 30.6(C-4), 16.4(CH3); HRMS(ESI)m / z[M+Na] + Calculated value C 20 H 19 NNaO8424.0998, measured value 424.1003.

[0199] Synthesis of 4-Desdimethylamino-9-nitro-doxycycline D-4

[0200]

[0201] To D-3 (0.5 g, 1.2 mmol) was slowly added 97% H2SO4 (4 mL) which had been previously cooled in an ice bath. To this solution was added KNO3 (0.16 g, 1.6 mmol) and the mixture was stirred at 0°C for 2 h. The reaction was diluted with cold methanol (5 mL) and a precipitate formed when water (35 mL) was added. The light brown solid was filtered, dried in vacuo, and then dissolved in acetone (4 mL). A black precipitate appeared when dichloromethane (15 mL) was added. The mixture was treated with activated charcoal while stirring for 20 min and then filtered through a pad of celite. The solid was decanted and slowly diluted with hexane (70 mL) to induce precipitation of D-4 (0.35 g, 65%) as a yellow solid; [α] D 20 -6.5 (c1.0, acetone); 1 H NMR ((CD3)2CO, 200MHz) δ: 8.15 (d, 1H, J 7，8 =8.6Hz, H-8), 7.17(d, 1H, J 7，8 =8.6Hz, H-7), 3.83(dd, 1H, J 4a，5 =11.4, J 5a，5 =7.8Hz, H-5), 3.00-2.91 (m, 3H, H-4, H-4′and H-6), 2.61 (dd, 1H, J 5a，6 =12.5, J 5a，5 =7.8Hz, H-5a), 2.47(ddd, 1H, J 4a，4 =5.3, J 4a，4′ =3.3, J 4a，5 =11.4Hz, H-4a), 1.60(d, 3H, J 6，Me =6.8 Hz, CH3); 13 C NMR ((CD3)2CO, 50.3MHz) δ: 165.5 (C-12, CONH2), 155.1 (C-7), 137.4 (C--6a), 132.1 (C-9), 118.5, 118.2 (C-9, 10a) 116.5 (C-8), 107. 5(C-11a), 94.6(C-2), 75.6(C-12a), 69.2(C-5), 46.6(C-5a), 44.2(C-4a), 38.8(C-6), 33.4(C-4), 16.2(CH3); HRMS(ESI)m / z[M+Na] + Calculated value C 20 H 18 N2NaO10 469.0854, measured value 469.0854.

[0202] Synthesis of 9-amino-4-desdimethylaminodoxycycline Doxy-5 (compound of formula (II))

[0203]

[0204] To a solution of D-4 (0.2 g, 0.5 mmol) in methanol (6 mL) containing 0.1% concentrated HCl was added 10% Pd / C (30 mg), and the mixture was treated with hydrogen at 44 psi at room temperature for 20 h. The mixture was filtered through a pad of celite and the residue was washed with methanol. The filtrate and washings were pooled and concentrated. The resulting residue was dissolved in ethanol (2 mL) and ethyl acetate (30 mL) was added dropwise to induce precipitation to give Doxy-5 (125 mg, 60%) as a light gray solid; [α] D 20 -30.0 (c 0.5, MeOH); 1 H NMR (CD3OD, 500MHz) δ: 7.61 (d, 1H, J 7，8 =8.3Hz, H-8), 7.10(d, 1H, J 7，8 =8.3Hz, H-7), 3.67(dd, 1H, J 4a，5 =10.8, J 5a，5 =8.0Hz, H-5), 3.05(dd, 1H, J 4a，4 =5.5, J 4，4’ =18.6Hz, H-4), 2.92(dd, 1H, J 4a，4’ =2.4, J 4，4’ =18.6Hz, H-4′), 2.78(m, 1H, J 6，Me =6.9, J 5a，6 =12.4Hz, H-6), 2.44(dd, 1H, J 5a，5 =8.0, J 5a，6 =12.4Hz, H-5a), 2.32(ddd, 1H, J 4a，4 =5.5, J 4a，4’ =2.4, J 4a，5 =10.8Hz, H-4a), 1.55(d, 3H, J 6，Me =6.9 Hz, CH3); 13C NMR (CD3OD, 125.7MHz) δ: 196.5, 194.5x 2(C-1,3,11),177.2,175.04(CONH2,C-12),155.8(C-10),150.4(C-6a),130.9(C-8),118.9,118.1(C-9,10a)117.3(C-7),108. 0(C-11a), 99.5(C-2), 76.0(C-12a), 69.8(C-5), 47.7(C-5a), 44.9(C-4a), 40.0(C-6), 31.3(C-4), 16.2(CH3); HRMS(ESI)m / z[M] + Calculated value C 20 H 21 N2O8417.1292; measured value 417.1295.

[0205] Example 2 - Generation of AS Aggregates as Biomarker Analytes

[0206] 2.1. Expression and purification of human AS

[0207] Human AS was expressed in E. coli (BL21) using the pT7-7 plasmid containing the coding gene of the protein. Purification was performed by selective precipitation of (NH4)2SO4 and anion exchange chromatography. The purity of the protein was confirmed by electrophoresis (SDS-PAGE) under denaturing conditions. AS solution was prepared with HEPES20mM, 150mM NaCl buffer at pH 7.4. The sample was filtered and centrifuged at 12000xg for 30 minutes to remove any aggregates formed. The concentration of AS was determined by spectrophotometry using its molar extinction coefficient.

[0208] 2.2. Preparation of aggregated AS species

[0209] Aggregated AS species ( AS ) were obtained by incubating AS solution (70 μM) in a controlled temperature orbital stirrer (600 rpm, 37° C.) during 24 h as described above. CL, et al. (2014).

[0210] 2.3. Determination of amyloid aggregation kinetics

[0211] Kinetic assays were performed using the Congo red (CR) and Thioflavin T (ThT) techniques that specifically bind to amyloid fibers. Free CR exhibits a maximum absorption at 490 nm, while when it binds to amyloid fibers, the peak shifts to 520 nm and also exhibits an increase in the intensity of the absorption band. When ThT binds to amyloid fibers, it changes from an excitation maximum at 415 nm with a fluorescence emission wavelength of 480 nm to a more intense maximum at 450 nm, based on the teachings of LeVine (1999), as Optimization as described in CL et al. (2014).

[0212] Example 3 - Immobilization of a Doxycycline Derivative (Doxy-5) on a Conductive Surface for Electrochemical Detection of AS Aggregates

[0213] A comparative study of different cross-linking agents for the immobilization of Doxy-5 on gold conductive surfaces was performed. 3-Mercaptopropionic acid (MPA) was used, which is able to form a self-assembled monolayer (SAM) on the substrate, thus facilitating its functionalization. Gold was chosen because it is inert towards the molecules of interest. In addition, the application of other reagents, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), is essential to form covalent bonds with Doxy-5, in order to generate or activate the moieties present. At each stage of modification of the gold surface, the concentration and incubation time were optimized.

[0214] Cyclic voltammetry (CV) measurements were first performed to characterize each immobilization stage on the gold substrate. The measurements were performed in a cell with a three-pole configuration using a platinum electrode as the counter electrode, a silver / silver chloride reference electrode, and a modified gold electrode as the working electrode. A Fe-containing 3+ / Fe 2+ The electrolyte of the redox couple [Fe(CN)6]K4 was used. Using this technique, the optimal polarization voltage value for electrochemical impedance spectroscopy (EIS) measurements was determined, i.e., the voltage at which the biosensor exhibited its highest sensitivity and specificity in the interaction between AS aggregated species and Doxy-5 immobilized on the conductive electrode. At each immobilization stage, CV and EIS measurements were also performed using phosphate buffer (PBS) at pH 7.4 ( Figure 1 ).

[0215] For electrochemical measurements, use 1287 Potentiostat and 1250 frequency response analyzer. For data processing, the software provided by the manufacturer ( y ). The measurements were performed at room temperature. The biosensor was calibrated against increasing concentrations of toxic AS species.

[0216] Electrochemical measurements of AS at different concentrations (0.1 and 100 ng / mL) were determined using a Doxy-5 modified gold biosensor prepared according to Example 3.1. Impedance measurements were performed using overlapping 10 mV alternating voltages at different potentials (0 V and -0.7 V) in the frequency range of 0.1-65000 Hz. CV measurements were performed at a potential range of -0.6 V to 0.6 V and a scan rate of 30 mV / seg. CV analysis showed that, first, there was no interference from the monomer. The response obtained for the monomer was much lower than that obtained for the AS fiber. In fact, at a monomer concentration of 100 ng / mL, the response was lower than that obtained for the fiber at a concentration of 0.1 ng / mL. Secondly, in the presence of AS fibers, a significant electrochemical response of the sensor was observed. The oxidation peak of the voltammogram was observed to increase from a concentration as low as 100 pg / mL to a concentration above 100 ng / mL ( Figure 2 ).

[0217] CV has been shown to be a selective technique, as the presence of AS fibrils can be selectively recognized over monomeric variants.

[0218] Example 4 - Immobilization of Doxy-5 on polymer surfaces by direct adsorption and immunochemical detection of AS aggregates

[0219] This design is based on an immunoassay using a conventional sandwich system, which involves Doxy-5 capturing the fibril species of AS, which is then detected with two antibodies: an anti-human AS antibody and an antibody conjugated to peroxidase. In brief, the compound of formula (II) is incubated in a polystyrene microplate, where it is adsorbed. After several rinses, the system is incubated with a primary anti-human AS rabbit antibody to eliminate any unadsorbed molecules. After rinsing, the system is then incubated with a secondary anti-rabbit antibody conjugated to peroxidase. The conjugated antibody reacts specifically with the primary antibody bound to the immune-captured AS. Unbound conjugated antibodies are eliminated by rinsing, and the presence of peroxidase is displayed by adding tetramethylbenzidine (TMB) as a chromogenic substrate. After incubation, a blue color will appear, the intensity of which depends on the concentration and affinity of Doxy-5 to the AS species. The enzymatic reaction is stopped by adding sulfuric acid, resulting in a color shift to yellow, which can be quantified by spectrometry at 450nm.

[0220] The effectiveness of this detection platform may be affected by different parameters, such as the concentration of the compound of formula (II) and the antibody, the application of different proteins and their concentrations to block non-specific sites, and the temperature and incubation time at each stage. In order to obtain the best analytical performance of this platform, an analysis was performed to optimize the parameters ( Figure 3 ).

[0221] The specificity of commercially available primary antibodies (Ab 138501, Ab209538, Ab27766, Abcam and Sc-7011-R, SantaCruz) to different AS species (monomer, oligomer, fibril) was verified. Each antibody showed picomolar affinity for each molecular AS form. The highest sensitivity was observed for polyclonal antibodies (Sc-7011-R, Santa Cruz) and one of the monoclonal antibodies (Ab209538). Anti-AS antibodies did not show significant differences in the dosage values ​​of fibrils and monomeric AS. This plays a decisive role in the design of this enzyme immunoassay, in which biological receptors are responsible for the specificity of fibrils to AS exceeding the specificity of monomeric AS. The concentrations of primary and secondary antibodies have also been optimized (Ab 32460, Invitrogen and Ab 6721, Abcam).

[0222] exist The immobilization of the compound of formula (II) was analyzed on polystyrene plates (96-well, high binding, Greiner Bio-One). The concentration of the compound of formula (II) was characterized in conjunction with the use of different proteins and their concentrations in blocking non-specific sites. After testing different blocking agents, such as bovine serum albumin (BSA), lysozyme, and casein in a concentration range of 0.05% to 1%, it was determined that lysozyme was the best choice, with a concentration of 0.1% ( Figure 4 ).

[0223] The scheme is defined as follows:

[0224] The 96-well polystyrene plate was sensitized by incubation overnight at 4°C and protected from light with 20 μg / mL of the compound of formula (II) in 20 mM sodium acetate at pH 5 (50 μL / well). The plate was rinsed with PBST (PBS 10 mM pH 7.4 + Tween-80 0.01%) and blocked for 1 h at 37°C with 150 μL / well of 0.1% lysozyme solution in PBS 10 mM pH 7.4. After rinsing, 50 μL of a sample containing the analyte of interest 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 rinsed again, and 50 μL of an anti-AS antibody diluted to 1 μg / ml in 10% fetal bovine serum (BFS in 10 mM PBS pH 7.4) was added to each well, after which the plate was incubated for 1.5 h. The plate was rinsed again and incubated with 50 μL / well of antibody conjugated to 1 μg / ml peroxidase diluted in 10% fetal bovine serum for 1 h at 37°C. The plate was rinsed and incubated with 50 μL / well of substrate (TMB) for 15 minutes at room temperature. The reaction was stopped with 25 μL of 1M H2SO4 solution. Immediately thereafter, the absorbance of the development was measured using a TECAN Spark microplate reader.

[0225] The results were confirmed by at least 3 independent experiments, each performed in triplicate.

[0226] The immunoassay focuses on the action of peroxidase on a substrate (TMB) to generate a colored compound that is proportional to the concentration of AS analyte captured by Doxy-5. Absorbance readings at 450 nm were taken using a TECAN Spark microplate reader.

[0227] As described in Example 3.2 above, monomeric and fibrillar AS standard solutions at a wide concentration range were incubated in wells previously sensitized with Doxy-5. The system exhibited nanomolar affinity for fibrillar AS with no cross-reactivity with native AS included as a negative control. Using the protocol described in Example 3.2, concentrations exceeding 30 ng / mL showed the best differential reactivity between AS aggregates and monomeric species.

[0228] Additionally, the application of this platform to CSF ​​samples from patients without enrolled pathological conditions compatible with movement disorders was explored. All samples were centrifuged at 2000 g for 10 min to eliminate cells and debris and then stored in aliquots at -80 °C until analysis. The values ​​displayed by the assayed biofluids were in order of magnitude consistent with the absorbance readings of the AS samples used as negative controls ( Figure 5 ).

[0229] Example 5 - Immobilization of Doxy-5 on polymer surfaces via indirect binding to BSA and immunochemical detection of Tau aggregates

[0230] The sensitization process of the wells was carried out as follows: First, the binding sites of the microplate were blocked with albumin. Thereafter, the compound of formula (II) was covalently linked to albumin using glutaraldehyde as a linker.

[0231] To this end, a 96-well polystyrene microplate was incubated with 150 μL / well 1% bovine serum albumin (BSA) at 4°C in a humidified chamber overnight. Then, after three cycles of washing with PBST, 50 μL / well glutaraldehyde at different concentrations (0.1%, 0.5% and 1%) was added to the wells, and the plate was placed in a fume hood during 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 plate was then incubated at 37°C in the dark for 30 minutes. Thereafter, the wells were washed again with PBST, and 50 μL / well of 1M TRIS was added as a glutaraldehyde inactivator. The microplate was then incubated at 37°C for 30 minutes. After this incubation, the wells were subsequently washed with PBST, and 50 μL / well of Tau samples (monomer (Tau m ) and preformed fibers (PFF)), and the microplate was incubated at 37°C for 2 hours. Tau m The concentration was 100ng / ml (5ng / well), and the dilution of PFF was 1 / 10000. Then, the wells were washed three times with PBST, and 50μL / well of 1 / 2000 anti-Tau antibody (ab80579, abcam) diluted in PBS-FBS 10% was added. The microplate was then incubated at 37°C for 1.5 hours. The microwells were then washed five times with PBST, and 50μL / well of HRP-linked secondary antibody (ab6789, abcam) (1 / 75000) was added. The microplate was then incubated at 37°C for 1 hour, after which excess secondary antibody was removed by washing five times with PBST. Finally, 50μL / well of TMB substrate was added and incubated in the dark at room temperature for 10 minutes. To stop the reaction, 25 μL / well of 2N H2SO4 was added and the microplate was immediately read on a microplate reader (Tecan Spark, Austria) at a wavelength of 450 nm. All different conditions demonstrated with glutaraldehyde showed similar results, and 0.1%-10' was selected for further assays because it contained the lowest concentration. Doxy-5 was able to distinguish Tau under these conditions. m With PFF, and thus with Tau m Compared with PBS (working buffer), it showed higher signal ( Figure 6 ).

[0232] In addition, we evaluated whether PFFs bind to Doxy-5 or to the remaining reactive sites from glutaraldehyde-treated BSA. To this end, after adding glutaraldehyde to the above protocol, Doxy-5 was replaced with 0.1% BSA. Then, different dilutions of Tau (1 / 2500, 1 / 5000, 1 / 10000) were added. m or PFF samples and the assay was continued as described. Using BSA instead of Doxy-5, the signal for PFF was significantly lower than that obtained for Doxy-5 ( Figure 7 ).

[0233] Example 6 - Comparative Binding Effectiveness of Doxycycline and Doxy-5 Against α-Synuclein Aggregates

[0234] Doxycycline hydrochloride and Doxy-5 were immobilized on polystyrene microplates and contacted with AS aggregates (PPF) using an AS aggregate concentration of 20 ng / mL according to the protocol described in Example 4. The binding of AS aggregates to each molecule was also determined as described in Example 4.

[0235] Figure 8 Doxy-5 was shown to be more effective than doxycycline HCl in binding to amyloid-aggregated AS species. Although a positive signal was observed with doxycycline HCl, the signal obtained with Doxy-5 was significantly higher, suggesting that Doxy-5 has a higher affinity for AS-PFF than doxycycline HCl. The results indicate that Doxy-5 is a superior capture element compared to conventional doxycycline HCl, confirming higher selectivity for target analytes. These findings highlight the importance of chemically modifying this tetracycline to develop devices with higher sensitivity. Removal of the bulky dimethylamine from the C4 position of doxycycline results in an enhanced affinity of the molecule for amyloid-aggregated AS species.

[0236] References

[0237] Atik A et al. Alpha-Synuclein as a Biomarker for Parkinson's Disease. Brain Path0l. 2016 May;26(3):410-8. doi:10.1111 / bpa.12370.

[0238] C.L. et al., "Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing a-Synuclein Oligomeric Species Toxicity," Journal of Biological Chemistry, Vol. 289, No. 20, 2014, pp. 13838-13850, ISSN 0021-9258, https: / / doi.org / 10.1074 / jbc.Ml 13.544288.

[0239] Eusebi P et al., "Diagnostic utility of CSF a-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.

[0240] 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.

[0241] Golan H, et al., "Nuclear imaging in Parkinson's disease: The past, the present, and the future." Journal of the Neurological Sciences. Vol. 436. 2022. 120220. ISSN 0022-510X. https: / / doi.org / 10.1016 / j.jns.2022.120220.

[0242] González-Lizárraga, 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.

[0243] Jang et al. α-Synuclein Oligomer Detection with Aptamer Switch on Reduced Graphene Oxide Electrode. Nanomaterials 2020, 10, 832; doi:10.3390 / nano10050832.

[0244] 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.

[0245] Luan, M et al. (2022), Diagnostic Value of Salivary Real-Time Quaking-Induced Conversion in Parkinson′s Disease and Multiple System Atrophy. MovDisord. https: / / doi.org / 10.1002 / mds.28976.

[0246] 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).

[0247] Moore, R.Y. et al. Extrastriatal monoamine neuron function in Parkinson’s disease: an 18F-dopa PET study. Neurobiol Dis 29, 381-390 (2008).

[0248] 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).

[0249] Pavese, N. et al. Progression of monoaminergic dysfunction in Parkinson’s disease: a longitudinal 18F-dopa PET study. Neuroiinage 56, 1463-1468 (2011).

[0250] 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.

[0251] Siderowf, A. et al. Assessment of heterogeneity among participants in the Parkinson′s Progression Markers Initiative cohort using α-synucIein 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.

[0252] 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 aggregates, comprising a doxycycline derivative capable of binding to neurotoxic amyloid aggregates, wherein the doxycycline derivative is immobilized on a surface.

2. The device of claim 1, wherein the device comprises a doxycycline derivative of formula (I): It is fixed on the surface, wherein R is selected from: i- is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent for X, wherein n=0-10, and X is selected from Cl, Br and I; ii - an alkyl or aryl linker with a sulfhydryl terminal group; iii- Where n = 0-10; iv- wherein n = 0-10; and v- Where n=0-10.

3. The device of claim 2, wherein R is selected from H, NH2, COOH, (CH2) n X、COOCH2(CH2) n A substituent for X, wherein n=0-10, and X is selected from Cl, Br and I.

4. The device of any one of claims 1 to 3, wherein the device comprises a doxycycline derivative of formula (II):

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

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

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

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

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

10. The device of claim 1, wherein the device comprises a doxycycline derivative of formula (II) It is immobilized on a polymer surface, wherein the polymer surface comprises polystyrene.

11. The device of claim 10, wherein the device comprises a doxycycline derivative of formula (II) immobilized on the polymer surface by covalent binding to bovine serum albumin adsorbed on the surface via glutaraldehyde as a linker.

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

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

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

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

16. The device of claim 1, wherein the device comprises a doxycycline derivative of formula (II) It is immobilized on a functionalized metal surface, wherein the functionalized metal surface is a functionalized gold surface, wherein the functionalized gold surface comprises a SAM of 3-mercaptopropionic acid, and wherein the doxycycline derivative of formula (II) is covalently bound to the SAM of 3-mercaptopropionic acid.

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

18. The method of claim 17, wherein the neurotoxic amyloid aggregates to be detected and / or quantified are alpha-synuclein (AS) aggregates.

19. The method of claim 17, wherein the neurotoxic amyloid protein aggregates to be detected and / or quantified are tau protein (Tau) aggregates.

20. The method of any one of claims 17-19, wherein the detection technique is selected from the group consisting of an immunochemical assay and an electrochemical assay.

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

22. The method of claim 21, wherein the electrochemical assay uses a method selected from cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for detecting neurotoxic amyloid aggregates.

23. The method of claim 22, wherein the electrochemical assay uses cyclic voltammetry for detecting neurotoxic amyloid aggregates.

24. The method of claim 20, wherein the detection technique is an immunochemical assay.

25. The method of claim 24, wherein step iii- comprises the following sub-steps: a) contacting the device with antibodies suitable for binding to neurotoxic amyloid aggregates to be detected and / or quantified; and b) Measuring bound antibody by a measurement technique.

26. The method of claim 25, wherein the neurotoxic amyloid aggregates to be detected and / or quantified are AS aggregates, and step iii- comprises the following sub-steps: a) contacting the device with an antibody suitable for binding AS aggregates; and b) Measuring bound antibody by a measurement technique.

27. The method of claim 25, wherein the neurotoxic amyloid aggregates to be detected and / or quantified are Tau aggregates, and step iii- comprises the following sub-steps: a) contacting the device with an antibody suitable for binding to Tau aggregates; and b) Measuring bound antibody by a measurement technique.

28. The method of any one of claims 24 to 27, wherein the means for detecting neurotoxic amyloid aggregates comprises a doxycycline derivative immobilized on a polymer surface by covalent binding via a linker to a blocking agent adsorbed on said surface.

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

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

31. A method for preparing a device for detecting neurotoxic amyloid aggregates according to any one of claims 1 to 16, comprising: i- providing a surface capable of immobilizing a doxycycline derivative; and ii- contacting a surface capable of immobilizing a doxycycline derivative with a solution comprising a doxycycline derivative capable of binding to neurotoxic amyloid aggregates under certain conditions, such that the doxycycline derivative capable of binding to neurotoxic amyloid aggregates is immobilized on the surface.

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

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

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

35. The method of any one of claims 31 to 33, wherein step i of the method further comprises the following sub-steps: a) contacting the polymer surface with a solution comprising a blocking agent so as to allow the blocking agent to adsorb onto the polymer surface, thereby obtaining a blocked polymer surface; and b) contacting the blocked polymer surface with a solution comprising a linker to obtain a polymer surface capable of immobilizing a doxycycline derivative.

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

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

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

39. The method of claim 38, wherein the functionalizing agent is a mercapto acid.

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

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