A chemiluminescent probe and its preparation method and application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a chemiluminescent probe and its preparation method and application. The structural formula of the chemiluminescent probe is wherein n≥1; and is an integer, and R is an aromatic ring connected to a dimethylamino group or an aromatic ring connected to a diethylamino group. The compound is combined with RT-QuIC and PMCA technology to detect protein markers of neurodegenerative diseases, which can eliminate the background interference generated by the autologous optical signal, significantly improve the signal-to-noise ratio, and the sensitivity is significantly better than that of detection using Thioflavin T as an indicator.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a chemiluminescent probe and a preparation method and application thereof. Background Art

[0002] Neurodegenerative diseases are a class of diseases caused by the loss of neurons and / or their myelin sheaths, usually manifesting as functional impairments, such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia or amyotrophic lateral sclerosis. In addition, neurodegenerative diseases gradually worsen over time, so early diagnosis is the key to preventing and treating such diseases.

[0003] At present, the commonly used diagnostic methods in clinical practice include behavioral scale assessment and imaging, but behavioral scale assessment is subject to many interference factors and is difficult to diagnose accurately; and imaging-based diagnosis requires reliance on expensive contrast agents. In addition, there are also studies on more accurate and lower-cost diagnostic methods based on biomarkers. For example, existing studies have shown that in the onset of neurodegenerative diseases, pathological protein markers of fibrosis will appear in the brain and body fluids, such as Abeta protein and tau protein closely associated with Alzheimer's disease, alpha-synuclein (α-synuclein) closely associated with Parkinson's disease, and TDP-43 protein closely associated with ALS. The above protein markers have been proven to be indicators for early diagnosis and typing of a variety of neurodegenerative diseases. However, the concentration of such markers in body fluids is low, often at the femtomolar or picomolar level, and existing protein detection methods such as ELISA, Simoa, and Meso Scale Discovery have limitations in detection.

[0004] Based on this, new in vitro detection technologies for this type of protein markers have been developed in recent years, such as real-time vibration-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) technology. Both technologies use the characteristics of this type of protein marker that are prone to misfolding, using a small amount of protein with misfolded conformation in the sample as a "seed" to induce a large number of protein monomers with natural conformation to misfold, and then amplify the misfolded protein markers through cyclic oscillation or ultrasound steps, and then detect the amplified protein markers, solving the problem of low concentrations of this type of protein in body fluids in the prior art. However, both of the above technologies rely on Thioflavin T (fluorescent dye) as an indicator, which has the disadvantages of high background interference and insufficient signal-to-noise ratio, and has limitations in the detection of low-concentration markers. Summary of the invention

[0005] The present invention develops a chemiluminescent probe suitable for detecting protein markers of neurodegenerative diseases using RT-QuIC and PMCA technologies. The chemiluminescent probe can emit light through self-oxidation after binding to the protein marker, thereby eliminating background interference; and the detection concentration of the chemiluminescent probe for the above protein marker can reach 1pg / mL, which is significantly lower than thioflavin.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a chemiluminescent probe, the chemical structure of which is shown below:

[0008] Wherein, n≥1; and is an integer, and R is a benzene ring connected to a dimethylamino group or a benzofuran connected to a diethylamino group.

[0009] More preferably, the chemiluminescent probe can be selected

[0010]

[0011] Another aspect of the present invention provides a method for preparing the chemiluminescent probe of the present invention, comprising: (1) reacting a compound represented by Formula I with a compound represented by Formula II to obtain a chemiluminescent probe represented by Formula III; (2) reacting a compound represented by Formula III with a compound represented by Formula IV to obtain a chemiluminescent probe represented by Formula V; (3) reacting a compound represented by Formula V with a compound represented by Formula VI to obtain the chemiluminescent probe of the present invention; wherein the structural formulas of the compounds represented by Formula I to Formula VI are respectively as follows:

[0012]

[0013]

[0014] In another aspect, the present invention provides a detection reagent or detection kit for protein detection, which includes the chemiluminescent probe of the present invention, and the protein is beta-amyloid protein, alpha-synuclein, tau protein, prion protein, amylin or transthyretin.

[0015] Preferably, the above detection reagent or detection kit further comprises dimethyl sulfoxide and a buffer.

[0016] More preferably, in the above detection reagent or detection kit, the volume percentage of dimethyl sulfoxide in the buffer system is 5%-100%.

[0017] Preferably, the above detection reagent or detection kit may further include an independently packaged RT-QuIC amplification reagent or an independently packaged PMCA amplification reagent.

[0018] In another aspect, the present invention provides a use of the chemiluminescent probe of the present invention in the preparation of a product for detecting neurodegenerative diseases, wherein the biomarker of the neurodegenerative diseases is α-synuclein.

[0019] In another aspect, the present invention provides a method for detecting α-synuclein for non-diagnostic purposes, comprising: (1) amplifying a sample to be detected and an α-synuclein monomer using an RT-QuIC or PMCA method to obtain an α-synuclein amplification product; (2) mixing the α-synuclein amplification product with the chemiluminescent probe of the present invention for reaction, and reading an optical signal after the reaction is completed; (3) qualitatively or quantitatively detecting the α-synuclein according to the optical signal.

[0020] Preferably, the above method for detecting α-synuclein satisfies one or more of the following conditions:

[0021] (a) In step (1), the sample to be tested is amplified using the RT-QuIC method to obtain an α-synuclein amplification product, and in the RT-QuIC method, the oscillation speed is 400 rpm-800 rpm;

[0022] (b) In step (1), the concentration of α-synuclein monomer is 0.1 mg / mL-2.5 mg / mL.

[0023] The beneficial effects of the present invention include at least:

[0024] (1) The chemiluminescent probe provided by the present invention is combined with RT-QuIC and PMCA technology to detect protein markers of neurodegenerative diseases. It can be activated by limiting factors such as enzymes and reactive oxygen species, and does not require external excitation light. The chemiluminescent probe protein marker can emit light by itself, which can eliminate the background interference generated by the autologous optical signal, significantly improve the signal-to-noise ratio, and realize highly sensitive in vitro detection of markers of neurodegenerative diseases in cerebrospinal fluid.

[0025] (2) The chemiluminescent probe provided by the present invention is combined with RT-QuIC and PMCA technology to detect protein markers of neurodegenerative diseases, and the sensitivity is higher than that of detection using thioflavin T (fluorescent dye) as an indicator; for example, for the detection of α-synuclein, the sensitivity of thioflavin T is 20pg / mL (refer to the document "O'Hara, DM, Kalia, SK, & Kalia, LV (2020). Methods for detecting toxic α-synuclein species as a biomarker for Parkinson's disease. Critical Reviews in Clinical Laboratory Sciences, 57(5), 291–307."), while the sensitivity of detection based on the chemiluminescent probe of the present invention can be as low as 1pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the detection method flow chart;

[0027] Figure 2 The comparison of signal enhancement times of probe 1 and Thioflavin T before and after binding to different model protein fibers in the present invention;

[0028] Figure 3 The optical signals of the probe 2 in the present invention when it is respectively combined with different model protein fibers;

[0029] Figure 4 The optical signal after the probe 1 of the present invention is combined with protein aggregates (protein fibers) of different concentrations;

[0030] Figure 5 The optical signal after the probe 2 of the present invention is combined with protein aggregates (protein fibers) of different concentrations;

[0031] Figure 6 is the optical signal intensity before and after Thioflavin T binds to α-synuclein in the present invention;

[0032] Figure 7 The RT-QUIC method based on the probe 1 of the present invention is used to detect the condition of Parkinson's cerebrospinal fluid samples;

[0033] Figure 8 The RT-QUIC method based on Thioflavin T is used to detect the condition of Parkinson's cerebrospinal fluid samples;

[0034] Fig. 9 The RT-QUIC method based on the chemiluminescent probe of the present invention is used to detect the condition of Parkinson's cerebrospinal fluid samples;

[0035] Fig.10 The effect of different DMSO / PBS% on the luminescence of probe 1;

[0036] Fig.11 The effect of different DMSO / PBS% on the luminescence of probe 2;

[0037] Fig.12 This is the effect of the oscillation speed on the luminescence of probe 1 in the RT-QUIC amplification reaction;

[0038] Fig.13 The effect of different concentrations of α-synuclein monomers on the luminescence of probe 1 and probe 2. DETAILED DESCRIPTION

[0039] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0040] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0041] The present invention provides a chemiluminescent probe, the chemical structure of which is shown below:

[0042] Wherein, n≥1; and is an integer, and R is a benzene ring connected to a dimethylamino group or a benzofuran connected to a diethylamino group.

[0043] It should be noted that n≥1, and can be 1-30, 1-20 or 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; it should also be noted that the key group contained in the chemiluminescent probe of the present invention is IPO

[0044] (imidazo[1,2-a]pyrazin-3(7H)-one), which functions to produce a chemiluminescent reaction; the key skeleton is a molecular rotor skeleton, specifically, with conjugated double bonds of different lengths as the axis, one end connected to IPO, and the other end connected to a targeting protein group (R group), and the function of the R group is to amplify the signal; specifically, its principle is: when the chemiluminescent probe is not bound to a marker, its energy is dissipated in the form of heat energy, and only a weak light signal is generated; when the chemiluminescent probe is bound to a marker, its bond energy rotation is inhibited, and the energy is dissipated in the form of a significantly enhanced light signal.

[0045] In some examples, the chemical structure of the chemiluminescent probe described above can be as follows:

[0046]

[0047] It should be noted that, as described above, the two ends of the conjugated double bond of the chemiluminescent probe in the present invention are IPO groups, which are responsible for generating chemiluminescent reactions, while the other end of the conjugated double bond is connected to an R group that can target a protein group, which is responsible for generating an enhanced light signal after binding to a protein marker. In the example of the present invention, the R group can be preferably selected from And the R group is selected When the length of the conjugated double bond is n = 1, that is, the above-mentioned chemiluminescent probe R Group Selection When the length of the conjugated double bond is n = 2, that is, the above-mentioned chemiluminescent probe When the above two R groups are preferred, after they are combined with α-synuclein amplified by RT-QuIC and PMCA technology, the enhancement factor of the luminescence signal is better than that of other R groups.

[0049] The present invention also provides a method for preparing the chemiluminescent probe of the present invention, comprising: (1) reacting a compound represented by Formula I with a compound represented by Formula II to obtain a compound represented by Formula III; (2) reacting a compound represented by Formula III with a compound represented by Formula IV to obtain a compound represented by Formula V; (3) reacting a compound represented by Formula V with a compound represented by Formula VI to obtain the chemiluminescent probe of the present invention; wherein the structural formulas of the compounds represented by Formula I to Formula VI are respectively as follows:

[0050]

[0051]

[0052] It should be noted that it should be understood that R in the above formula I, formula III and formula V is consistent with R in the present invention.

[0053] An embodiment of the present invention also provides a detection reagent or detection kit for protein detection, which includes the chemiluminescent probe of the present invention, and the protein is a protein that is easily misfolded, such as beta-amyloid protein, α-synuclein, tau protein, prion protein, amylin or transthyretin protein.

[0054] It should be noted that after the chemiluminescent probe in the present invention is combined with the above-mentioned protein, the chemiluminescent signal will be enhanced, thereby realizing the detection of the above-mentioned protein; in addition, the above-mentioned protein can be in the form of an oligomer or a protein aggregate.

[0055] In some examples, the detection reagent or detection kit further includes dimethyl sulfoxide and a buffer.

[0056] It should be noted that the buffer in the present invention includes PBS buffer, pipes buffer, tris buffer, water or other conventional buffers, preferably PBS buffer, which has better detection effect.

[0057] In some examples, in the above detection reagent or detection kit, the volume percentage of dimethyl sulfoxide (DMSO) in the buffer system is 5%-100%.

[0058] It should be noted that when the detection is carried out, the compound of the present invention is dissolved in a solvent and then combines with the aggregated form of the protein, and then emits light to achieve the purpose of protein detection; wherein, the volume ratio of DMSO in the buffer solution will affect the luminescence efficiency, and 5%-100% is selected in the present invention, such as 7%-18%, 8%-16%-10%-15% or 12%-14%, etc.; or 7%, 10%, 15% or 18%, etc.; in addition, the buffer system refers to all reagents such as DMSO, buffer solution and probe solution, that is, the volume percentage of dimethyl sulfoxide (DMSO) in the buffer system is 5%, which means that in 100mL of the buffer system, the volume of DMSO is 5mL, and the volume of other reagents (including buffer solution and probe solution, etc.) is 95mL, and so on.

[0059] In some examples, the above detection reagent or detection kit may further include an independently packaged RT-QuIC amplification reagent or an independently packaged PMCA amplification reagent.

[0060] It should be noted that, as described above, the above two chemiluminescent probes can significantly enhance the optical signal, especially the chemiluminescence enhancement of α-synuclein, so they can be prepared into a detection product for diagnosing and detecting α-synuclein, and the form of the detection product can be a form known in the art, such as a detection reagent or a detection kit. In addition, since the chemiluminescent probe in the present invention is directed to a protein marker after RT-QuIC amplification or PMCA amplification, when preparing a detection reagent or a detection kit, RT-QuIC amplification or PMCA amplification reagent is required at the same time, and both reagents are reagents known in the art, such as buffer, etc.

[0061] It should also be noted that there are protein fibers / aggregates in the samples to be tested (such as cerebrospinal fluid samples from patients), but the concentration is very low, so they need to be amplified by specific methods (RT-QuIC amplification or PMCA amplification) before detection; in addition, since fibers / aggregates can turn protein monomers into toxic fibers / aggregates like viruses, protein monomers are generally added during amplification; at the same time, only fibers / aggregates can bind to Thioflavin T or probes to emit light.

[0062] The embodiment of the present invention also provides a use of the compound of the present invention in preparing a product for detecting neurodegenerative diseases, wherein the biomarker of neurodegenerative diseases is α-synuclein.

[0063] It should be noted that the present invention can detect neurodegenerative disease protein markers through compounds to achieve the diagnosis of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia or amyotrophic lateral sclerosis, etc. In addition, the optical signal enhancement after the chemiluminescent probe in the present invention binds to α-synuclein is stronger than that of other protein markers. Therefore, the chemiluminescent probe in the present invention can be used for the detection of α-synuclein, and the sensitivity can reach 1pg / mL.

[0064] The embodiment of the present invention also provides a method for detecting α-synuclein for non-diagnostic purposes, which comprises: (1) using RT-QuIC or PMCA method to amplify the sample to be detected and the α-synuclein monomer to obtain an α-synuclein amplification product; (2) mixing the α-synuclein amplification product with the chemiluminescent probe of the present invention for reaction, and reading the optical signal after the reaction is completed; (3) qualitatively or quantitatively detecting the α-synuclein according to the optical signal.

[0065] It should be noted that the schematic diagram of the detection method of α-synuclein based on RT-QuIC amplification is as follows Figure 1As shown. In addition, the chemiluminescent probe in the present invention will react immediately after mixing with the amplification product, generating a significantly enhanced signal, so it must be detected immediately; the signal of the probe will decay over time, and its decay parameters (half-life, etc.) are directly related to the protein conformation, which can provide more information for the disease; the signal from the time of adding the sample to 3-5 hours is most conducive to fitting and calculating the decay parameters, and the signal is too low after that, which is difficult to use for calculation; detecting once every X minutes will make the signal record more standardized and more conducive to fitting and calculation; after adding the probe, it must be detected immediately, and it is recommended that the detection time last for 3-5 hours and be measured once every 1-10 minutes.

[0066] It should also be noted that the mixing time of the chemiluminescent probe and the α-synuclein amplification product in the present invention is 12 hours to 160 hours after the start of the RT-QUIC or PMCA reaction, such as 20 hours, 50 hours, 80 hours, 100 hours, 120 hours or 140 hours.

[0067] In some examples, in step (1) of the above method for detecting α-synuclein, the sample to be detected is amplified using the RT-QuIC method to obtain an α-synuclein amplification product. In the RT-QuIC method, the oscillation speed is 400 rpm-800 rpm, such as 500 rpm, 600 rpm or 700 rpm.

[0068] In some examples, in step (1) of the above method for detecting α-synuclein, the concentration of α-synuclein monomer is 0.1 mg / mL-2.5 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL or 2 mg / mL.

[0069] It should also be noted that during RT-QUIC amplification, if the temperature is too low, it is difficult for the reaction to occur, and if the temperature is too high, the protein will undergo irreversible conformational destruction; if the shaking is too slow, it is difficult for the reaction to occur, and if the shaking is too fast, the instrument and the well plate will not be able to withstand it. Therefore, the amplification temperature in the present invention can be 30°C-42°C, such as 32°C, 35°C, 37°C or 40°C, and the shaking speed can be 400rpm-800rpm, such as 500rpm, 600rpm or 700rpm, etc. The interval between each shaking is 1 minute to 5 minutes, such as 2 minutes, 3 minutes or 4 minutes, etc.

[0070] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0071] 1. Sample preparation

[0072] Example 1

[0073] In the embodiment of the present invention, probe 1 (ADLumin-1) was prepared according to the following collective route:

[0074]

[0075] The specific preparation method of probe 1 is as follows:

[0076] To a solution of TMP (2,2,6,6-tetramethylpiperidine, 0.25 mmol, 1.0 equiv) in anhydrous THF (1.0 mL) at 0°C was added n-BuLi (0.25 mmol, 1.0 equiv) to obtain mixture 1; the mixture 1 was stirred at 0°C for 5 minutes, and then a solution of bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methane (compound 1b) (0.30 mmol, 1.2 equiv) in anhydrous THF (0.60 mL) was added. Mixture 2 was obtained; Mixture 2 was stirred at 0°C for 15 minutes to react to obtain a reaction solution; then, the reaction solution was cooled to -78°C, and an anhydrous THF (0.30 ml) solution containing aldehyde (Compound 1a) (0.25 mmol, 1.0 equivalent) was added to obtain Mixture 3; Mixture 3 was stirred at -78°C for another 4 hours to obtain Mixture 4; then, Mixture 4 was concentrated under reduced pressure, and the 1,2-disubstituted boric acid vinyl ester product was purified by flash silica chromatography to obtain a yellow solid (Compound 1c);

[0077] Under argon protection, compound 1c (106.0 mg, 0.39 mmol) and 5-bromo-2-aminopyrazine (compound 1d) (57.0 mg, 0.33 mmol) were dissolved in a mixture of 1,4-dioxane (1,4-dioxane, 1,4-dioxane) (6.0 mL) and 1.0 M sodium bicarbonate aqueous solution (400.0 μL), and the resulting mixture was degassed under vacuum to obtain mixture 5; then tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) (0.0033 mmol, 3.8 mg) was added to the degassed mixture, and the mixture was heated at 75°C for 6 hours and then cooled; after cooling, the mixture was diluted with ethyl acetate (20 mL), washed with water and brine, dried over sodium sulfite and evaporated; the resulting residue was purified by flash silica gel chromatography (silica gel, eluent: chloroform / methanol = 50 / 1) to obtain a yellow solid, named 1e;

[0078] Under argon protection, compound 1e (50.0 mg, 0.18 mmol) and methylglyoxal-1,1-dimethyl acetal (compound 1f) (30.0 mg, 0.25 mmol) were dissolved in a mixture of 20% aqueous hydrochloric acid solution (200.0 μL), pure water (200.0 μL) and ethanol (3.0 mL), and the resulting mixture was stirred and heated at 70°C for 12 hours, and then cooled; after cooling, the reaction mixture was evaporated; and the residue was then purified by flash silica chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain probe 1 (ADLumin-1) as a yellow solid.

[0079] The hydrogen spectrum data of the prepared compound 1c are as follows:

[0080] 1 H NMR (500MHz, CDCl3): δ7.32(d,J=8.8Hz,2H),7.20–7.14(m,1H),6.70–6.59(m,4H),5.55(d,J=17.5Hz,1H),2.97(s,6H),1.29(s,12H). 13 C NMR(125MHz, CDCl3): δ150.93,150.53,136.86,128.20,126.51,125.09,112.28,83.12,40.43,24.89.ESI-MS(m / z):300.1[M+H] + .

[0081] The hydrogen spectrum data of the prepared 1e is as follows:

[0082] 1 H NMR (500MHz, DMSO-d6): δ7.93(s,1H),7.87(s,1H),7.32(d,J=8.7Hz,2H),7.10(dd,J=15.2,10.9Hz,1H),6. 81(dd,J=15.4,11.0Hz,1H),6.69(d,J=8.8Hz,2H),6.59(d,J=15.5Hz,1H),6.53–6.50(m,3H),2.92(s,6H). 13 C NMR (125MHz, DMSO-d6): δ154.43,149.92,140.65,139.02,132.97,132.18,128 .81,127.42,126.80,125.21,124.78,112.32,40.03.ESI-MS(m / z):267.1[M+H] + .

[0083] The hydrogen spectrum data of probe 1 are as follows:

[0084] 1 H NMR (500MHz, CD3OD): δ9.03(s,1H),8.39(s,1H),7.76–7.64(m,4H),7.55(t,J =15Hz,1H),7.23(t,J=15Hz,1H),6.95–6.87(m,2H),3.29(s,6H),2.52(s,3H). 13 C NMR (125MHz, CD3OD): δ143.14,140.61,139.91,139.03,135.88,135.47,135.14,131.73, 129.69,128.56,127.26,124.46,122.22,113.97,47.2,10.17.ESI-MS(m / z):321.1[M+H] + .HR-MS(m / z):321.1710[M+H] + .

[0085] Example 2

[0086] In the present embodiment, probe 2 (ADLumin-2) was prepared according to the following collective route:

[0087]

[0088] To a solution of TMP (2,2,6,6-tetramethylpiperidine, 0.25 mmol, 1.0 equiv) in anhydrous THF (1.0 mL) at 0°C was added n-BuLi (0.25 mmol, 1.0 equiv) to obtain mixture 1; mixture 1 was stirred at 0°C for 5 minutes, and then a solution of bis-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methane (compound 2b) (0.30 mmol, 1.2 equiv) in anhydrous THF (0.60 mL) was added. Mixture 2 was obtained; Mixture 2 was stirred at 0°C for 15 minutes to react to obtain a reaction solution; then, the reaction solution was cooled to -78°C, and an anhydrous THF (0.30 ml) solution containing aldehyde (compound 2a) (0.25 mmol, 1.0 equivalent) was added to obtain mixture 3; Mixture 3 was stirred at -78°C for another 4 hours to obtain mixture 4; then, mixture 4 was concentrated under reduced pressure, and the 1,2-disubstituted boronic acid vinyl ester product was purified by flash silica chromatography to obtain a yellow solid (compound 2c);

[0089] Under argon protection, compound 2c (106.0 mg, 0.39 mmol) and 5-bromo-2-aminopyrazine (compound 2d) (57.0 mg, 0.33 mmol) were dissolved in a mixture of 1,4-dioxane (6.0 mL) and 1.0 M sodium bicarbonate aqueous solution (400.0 μL), and the resulting mixture was degassed under vacuum to obtain mixture 5; then tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) (0.0033 mmol, 3.8 mg) was added to the degassed mixture, and the mixture was heated at 90°C for 6 hours and then cooled; after cooling, the mixture was diluted with ethyl acetate (20 mL), washed with water and brine, dried over sodium sulfite and evaporated; the resulting residue was purified by flash silica gel chromatography (silica gel, eluent: chloroform / methanol = 50 / 1) to obtain a yellow solid, named 2e;

[0090] Under argon protection, compound 2e (50.0 mg, 0.18 mmol) and methylglyoxal-1,1-dimethyl acetal (compound 2f) (30.0 mg, 0.25 mmol) were dissolved in a mixture of 20% aqueous hydrochloric acid solution (200.0 μL), pure water (200.0 μL) and ethanol (3.0 mL), and the resulting mixture was stirred and heated at 70°C for 12 hours, and then cooled; after cooling, the reaction mixture was evaporated; and the residue was then purified by flash silica chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain probe 2 (ADLumin-2) as a yellow solid.

[0091] The hydrogen spectrum of the prepared compound 2c is as follows:

[0092] 1 H NMR (500MHz, CDCl3) δ7.32(d,J=8.7Hz,1H),7.19(d,J=18.1Hz,1H),6.72(d,J=1.5Hz,1H),6.65(dd,J=8.7, 2.2Hz,1H),6.57(s,1H),6.09(d,J=18.1Hz,1H),3.40(q,J=7.1Hz,4H),1.31(s,12H),1.19(t,J=7.1Hz,6H); 13 C NMR (125MHz, CDCl3)158.0,153.0,147.4,136.9,121.7,121.7,118.2,109.8,108.0,93.9,83.4,45.1,25.0,12.7.

[0093] The hydrogen spectrum data of the prepared 2e is as follows:

[0094] 1H NMR(500MHz, CDCl3)δ8.00(d,J=6.1Hz,2H),7.36–7.28(m,2H),7.07(d,J=15.7Hz,1H),6.75(s,1H), 6.65(d,J=8.1Hz,1H),6.57(s,1H),4.60(br,2H,-NH2),3.41(q,J=7.0Hz,4H),1.20(t,J=7.0Hz,6H); 13 C NMR (125MHz, CDCl3)157.6,152.9,152.7,147.0,141.4,132.4,122.5,121.2,118.6,117.8,109.7,106.8,93.9,45.1,29.8,12.7.

[0095] The hydrogen spectrum data of the prepared probe 2 is as follows:

[0096] 1 H NMR (500MHz, CD3OD) δ9.04(s,1H),8.54(s,1H),7.93(s,1H),7.88(d,J=8.4Hz,1H),7.74(d,J=15 .6Hz,1H),7.52(d,J=15.6Hz,2H),7.14(s,1H),3.76(m,4H),2.54(s,3H),1.19(t,J=7.2Hz,6H); 13 C NMR (125MHz, CD3OD)158.4,156.2,135.5,132.3,128.5,125.9,124.3,121.7,118.6,114.9,108.3,107.1,55.3,10.8,9.8; HRMS-ESI(m / z)[M+H + ]:calcdfor C 21 H 23 N4O2363.1816, found 363.1813.

[0097] 2. Sample testing

[0098] In the following tests, the preparation method of the probe solution (300 μM) used is as follows: first, probe 1 and probe 2 prepared in the above examples and Thioflavin T (Sigma-Aldrich, the same below) are dissolved in DMSO. During the test, a small amount of the probe solution is then drawn and added to the sample to be tested (usually a buffer solution) so that the final volume ratio of DMSO in the sample solution to be tested is 5%.

[0099] (I) Comparison of chemiluminescent probes and Thioflavin T in detecting common markers of neurodegenerative diseases

[0100] (1) Take the protein marker powder (protein marker monomer) in Table 1, add 0.01M PBS buffer (Sigma-Aldrich, the same below) with pH=7.4 respectively, and then shake (900 rpm) and incubate in a constant temperature shaker (ThermoMixer) at 37°C (to promote the transformation of the protein structure from monomer form to aggregate form) for 72 hours to obtain different protein marker aggregate solutions. The concentration of protein marker aggregates in the protein marker aggregate solutions is shown in Table 1 below.

[0101] Table 1 Information on different protein markers and aggregate concentrations

[0102]

[0103]

[0104] (2) Sample loading: Take a black flat-bottom 96-well plate and add each beta-amyloid protein aggregate solution in Table 1 to 18 wells, 95 μL per well; add 5 μL of probe 1 solution to 3 wells, add 5 μL of probe 2 solution to 3 wells, and add 5 μL of thioflavin T solution to the other 3 wells, mix well, so that the final concentration of the protein marker aggregation solution is 70 μM, and the final concentrations of probe 1, probe 2 and thioflavin T are all 15 μM; in addition, add PBS buffer to other blank wells, and then add probe 1 solution, probe 2 solution and thioflavin T solution (3 wells for each solution) respectively, as blank controls to calibrate the test data;

[0105] (3) Detection: Place the well plate in an imager (IVIS Lumina III, the same below), set the luminescence mode to a closed excitation light source, collect the emission spectrum full-spectrum signal, and read the chemiluminescence signal; then change the luminescence mode to fluorescence, set the excitation wavelength to 450 nm, and the emission wavelength to 480 nm; read the optical signal;

[0106] (4) Result analysis: quantify the signal intensity of each well, calculate the signal-to-noise ratio, and find the average value;

[0107] The other proteins in Table 1 were tested according to the above method, and the results are as follows:

[0108] First, the detection results of probe 1 and Thioflavin T are shown in Table 2 below. The signal ratio in Table 2 is the value divided by the signal enhancement factor of probe 1 (optical signal enhancement factor of probe 1 = optical signal of (probe 1 + protein) / optical signal of probe 1 itself) and the signal enhancement factor of Thioflavin T (optical signal enhancement factor of Thioflavin T = optical signal of (Thioflavin T + protein) / optical signal of Thioflavin T itself). Each group was repeated 3 times; the median of the ratio was plotted as shown in Figure 2 shown.

[0109] Table 2 Signal ratio of the optical signal enhancement factor of probe 1 divided by the optical signal enhancement factor of Thioflavin T

[0110]

[0111]

[0112] From the above table 2 and Figure 2 It can be seen that after binding to the above six protein aggregates, the optical signals of probe 1 are greater than those of Thioflavin T, especially Amylin protein, Transthyretin protein and Aβ protein, followed by α-Synuclein protein and Tau protein. Prion has little difference, and probe 1 is slightly stronger than Thioflavin T.

[0113] Second, the optical signal values ​​of probe 2 before and after binding to different protein aggregates are shown in Table 3, and the average optical signal values ​​are plotted as Figure 3 shown.

[0114] Table 3 Optical signal values ​​of probe 2 after binding to different protein aggregates

[0115]

[0116] From the above table 3 and Figure 3 It can be seen that the optical signal value of probe 2 is significantly enhanced after binding to different protein aggregates, especially Tau protein, Transthyretin protein, α-Synuclein protein and Aβ protein; followed by Prion protein and Amylin protein; indicating that probe 2 can detect the above protein aggregates.

[0117] (II) Sensitivity and detection limit test of α-synuclein by chemiluminescent probe and Thioflavin T

[0118] (1) Preparation of an in vitro model of Parkinson's disease markers: α-synuclein powder was added to 0.01 M PBS buffer (pH = 7.4), and incubated in a constant temperature shaker at 37° C. for 72 hours; then, the protein marker aggregate samples were diluted with PBS buffer, and the final concentrations of the protein marker aggregates in the protein marker aggregate samples were set to 70 fM, 350 fM, and 700 fM, respectively;

[0119] (2) Sample loading: Take a black flat-bottom 96-well plate and add different concentrations of protein marker aggregate samples to 18 wells, 95 μL per well, of which 5 μL of probe 1 solution is added to 3 wells, 5 μL of probe 2 solution is added to 3 wells, and thioflavin T solution is added to the other 3 wells. Mix well so that the final concentrations of the protein marker aggregates are 70 fM, 350 fM and 700 fM, respectively. The final concentrations of probe 1, probe 2 and thioflavin T are 15 μM, respectively. Each sample is tested in 3 parallel groups. In addition, PBS buffer is added to the other 9 wells, and then probe 1 solution (15 μM), probe 2 solution (15 μM) and thioflavin T solution (15 μM) are added respectively as blank controls to calibrate the test data.

[0120] (3) Detection: Place the well plate in the imager, set the luminescence mode to a closed excitation light source, collect the full spectrum signal of the emission spectrum, and read the chemiluminescence signal; then change the luminescence mode to fluorescence, set the excitation wavelength to 450 nm and the emission wavelength to 480 nm; read the optical signal.

[0121] (4) Result analysis: Quantify the signal intensity of each well, with the protein aggregate concentration as the horizontal axis and the signal intensity as the vertical axis.

[0122] The optical signals after probe 1 binds to protein marker aggregates of different concentrations are shown in Table 4 below. The linear relationship between the average optical signal and the concentration is analyzed. Figure 4 shown.

[0123] Table 4 Optical signals after probe 1 binds to protein marker aggregates at different concentrations

[0124]

[0125] There was no obvious optical signal enhancement after probe 2 was combined with protein marker aggregates at concentrations of 70fM, 350fM and 700fM; therefore, different concentrations of protein marker aggregates (70nM, 700nM and 7000nM) were set to combine with probe 2, and the optical signal was detected after combination according to the above method. The results are shown in Table 5 below. The linear relationship between the average optical signal and the concentration was analyzed. The results are as follows Figure 5 shown.

[0126] Table 5 Optical signals after probe 2 binds to protein marker aggregates at different concentrations

[0127]

[0128] It can be seen from Tables 4 and 5 above that there is no obvious optical signal enhancement after probe 2 binds to 700fM protein marker aggregates, and the optical signal after probe 1 binds to 700fM protein marker aggregates is not very different from the optical signal after probe 2 binds to 70nM protein marker aggregates, indicating that probe 2 is not as sensitive as probe 1 in detecting α-synuclein aggregates.

[0129] In addition, the optical signals of single Thioflavin T and Thioflavin T and protein aggregates (protein aggregate concentration is 70 fM) are shown in Table 6, and the graphs are as follows: Figure 6 shown.

[0130] Table 6 Optical signals before and after Thioflavin T binds to protein aggregates

[0131] Sample Group Thioflavin t Thioflavin t+1pg / ml (70fM) alpha synuclein aggregates Signal value 1 6900000 7100000 Signal value 2 7410000 7130000 Signal value 3 7710000 7940000

[0132] From the above table 6 and Figure 6 It can be seen that after Thioflavin T binds to protein aggregates, the optical signal is basically not enhanced, so α-synuclein aggregates at this concentration cannot be well detected using Thioflavin T.

[0133] From the above, it can be seen that for the detection of α-synuclein aggregates, the sensitivity of probe 1 is better than that of probe 2 and thioflavin T.

[0134] (III) RT-QUIC testing of cerebrospinal fluid samples

[0135] (1) Sample collection and pretreatment: 3 healthy cerebrospinal fluid samples were collected. About 10 ml of cerebrospinal fluid was collected after lumbar puncture, and the samples were immediately centrifuged at 1300 g for 5 min at 4°C and stored in a -80°C refrigerator. α-synuclein powder (monomer) was added to 0.01 M PBS buffer (pH = 7.4) (Sigma-Aldrich), and incubated in a constant temperature shaker at 37°C for 72 hours to obtain an α-synuclein aggregate solution, which was then divided and frozen in a -80°C refrigerator. Before use, the α-synuclein aggregates were diluted with PBS buffer (Sigma-Aldrich) and added to the healthy cerebrospinal fluid samples to make the final concentration of α-synuclein aggregates 35 pM to prepare 3 simulated Parkinson's disease samples, namely simulated Parkinson's disease sample 1, simulated Parkinson's disease sample 2, and simulated Parkinson's disease sample 3.

[0136] (2) Prepare buffer solution: add 1.357 g potassium dihydrogen phosphate, 8.086 g disodium hydrogen phosphate and 9.927 g sodium chloride to 800 mL deionized water, and adjust the solution to pH 8.0 using hydrochloric acid or sodium hydroxide; add 0.015 g sodium dodecyl sulfate, add deionized water until the volume is 1 L, and stir evenly; filter with a 0.22 μm filter, and dispense into small bottles, avoiding direct sunlight and high temperature;

[0137] (3) preparing a substrate solution: dissolving α-synuclein in the buffer prepared in step (2) above to a concentration of 0.1 mg / mL, and filtering with a 100KD filter membrane to obtain a substrate solution containing α-synuclein monomers;

[0138] (4) Sample loading: Take a black flat-bottom 96-well plate, add 85 μL of the substrate solution containing α-synuclein monomers prepared in (3) above to each well, take 15 μL of the samples to be tested (simulated Parkinson's disease sample 1, simulated Parkinson's disease sample 2, simulated Parkinson's disease sample 3) and continue to add them to the single wells, and set up 3 replicate wells respectively; according to the same method, set α-synuclein aggregates of known concentration (1000 pg / ml) as the positive control, obtain cerebrospinal fluid samples of 3 healthy people (prepared according to the above method) as the negative control (healthy control 1, healthy control 2, healthy control 3), and RT-QUIC buffer (Sigma-Aldrich) as the blank control; after the sample loading is completed, seal the 96-well plate with a transparent sealing film;

[0139] (5) RT-QUIC amplification reaction: Place the sealed 96-well plate into a microplate reader (BioTek Synergy H1) and run the microplate reader with the following program: temperature 42°C, 1 min oscillation (600 rpm dual track), rest 1 min, oscillation cycle, total running time set to 80 h, read once every 30 minutes;

[0140] (6) Preparation of detection reagents: Weigh powdered probe 1 and thioflavin T, dissolve them in dimethyl sulfoxide solution to make 750 μM probe 1 solution and 750 μM thioflavin T solution; use a dispenser to draw 2 μL of probe 1 solution into each well of a 96-well plate, and quickly place the plate in an ELISA reader for signal reading; at the same time, add thioflavin T solution to other wells, and quickly place the plate in an ELISA reader for signal reading as a comparison;

[0141] (7) Chemiluminescence detection: Set the microplate reader program to the chemiluminescence mode, the operating temperature to 37°C, the shaking time to 10 seconds, and the detection immediately. Set the total running time to 180 minutes, and read once every 5 minutes.

[0142] (8) Result interpretation: Data were normalized to the maximum response signal of the microplate reader, and a curve of the sample chemiluminescence signal changing over time was plotted as a reference for disease classification; the mean of the maximum response signal of the negative control + 3SD was used as the threshold for determining a positive result; when more than 2 of the 3 parallel samples were positive, the sample was considered positive; when 0 were positive, the sample was considered negative; when only 1 was positive, the sample was tested again; if ≤ 1 sample was positive, the result was considered negative.

[0143] Among them, the optical signals of probe 1 combined with healthy controls and Parkinson's disease samples are shown in Table 7 below, wherein the optical signals of probe 1 combined with healthy controls and Parkinson's disease are the maximum values ​​of the optical signals (the probe is a consumable reagent, and the reaction starts as soon as it is added, and then the signal will gradually weaken); In addition, the RT-QUIC kinetic signals of the samples to be tested and the healthy controls based on the probe 1 of the present invention and amplified using RT-QUIC are shown in Table 7. Figure 7 shown.

[0144] Table 7 Optical signals after probe 1 combined with healthy controls and simulated Parkinson's disease samples

[0145] Grouping Probe + healthy control (au) Probe + simulated Parkinson sample (au) Probe 1 + Sample 1 382 23046 Probe 1 + Sample 2 386 20471 Probe 1 + Sample 3 337 14775

[0146] It can be seen from Table 7 above that there is no obvious optical signal when the probe 1 of the present invention is combined with the healthy control, but it is significantly enhanced after being combined with the simulated Parkinson's sample, indicating that the probe 1 of the present invention can detect α-synuclein in the cerebrospinal fluid samples of Parkinson's patients.

[0147] In addition, the optical signals of Thioflavin T combined with healthy controls and simulated Parkinson's disease samples are shown in Table 8 below, where the optical signals of Thioflavin T combined with healthy controls and Parkinson's disease are the maximum values ​​of the optical signals (the highest value of the RT-QUIC experiment, 44h); In addition, the RT-QUIC kinetic signals of the samples to be tested and the healthy controls based on Thioflavin T and amplified using RT-QUIC are shown in Table 8 below. Figure 8 shown.

[0148] Table 8 Optical signals after combination of Thioflavin T and healthy controls and simulated Parkinson's disease samples

[0149] Grouping Thioflavin T+ healthy control (au) Thioflavin T+ simulated Parkinson's sample (au) Thioflavin T+ Sample 1 5614 8687 Thioflavin T+ Sample 2 5515 9934 Thioflavin T+ Sample 3 5901 8836

[0150] It can be seen from Table 8 above that the optical signal after thioflavin T combined with the simulated Parkinson's sample is higher than that combined with the healthy control, indicating that thioflavin T can detect α-synuclein in cerebrospinal fluid samples of Parkinson's patients, but the signal enhancement effect is weaker than that of probe 1.

[0151] The optical signal enhancement factors of Parkinson's disease patients labeled with probe 1 and thioflavin T respectively and the optical signal enhancement factors of healthy controls are shown in Table 9 below; wherein, the optical signal enhancement factor of probe 1 = (optical signal of probe 1 + simulated Parkinson's sample) / (optical signal of probe 1 + healthy control); the optical signal enhancement factor of thioflavin T = (optical signal of thioflavin T + simulated Parkinson's sample) / (optical signal of thioflavin T + healthy control).

[0152] Table 9 Signal enhancement in Parkinson's disease patients and healthy controls

[0153] Parkinson's disease patients / healthy controls Optical signal enhancement factor of probe 1 Thioflavin T optical signal enhancement factor Parkinson's disease 1 / healthy control 1 60.33 1.55 Parkinson's disease 2 / healthy control 2 53.03 1.80 Parkinson's disease 3 / healthy control 3 43.84 1.50

[0154] It can be seen from Table 2 above that the signal enhancement factor based on the probe 1 of the present invention is more than 40 times that based on the signal enhancement of thioflavin T.

[0155] (IV) PMCA testing of cerebrospinal fluid samples

[0156] (1) Sample collection and pretreatment: For healthy cerebrospinal fluid samples, about 10 ml of cerebrospinal fluid was collected after lumbar puncture, and the samples were immediately centrifuged at 1300 g for 5 min at 4°C and stored in a -80°C refrigerator; α-synuclein powder (monomer) was added to 0.01 M PBS buffer (pH = 7.4) (Sigma-Aldrich), and incubated in a constant temperature shaker at 37°C for 72 hours to obtain an α-synuclein aggregate solution, which was then divided and frozen in a -80°C refrigerator; before use, the α-synuclein aggregates were diluted to 3500 fM with PBS buffer (Sigma-Aldrich), and 4 μl was taken and added to 196 μl of healthy cerebrospinal fluid sample to make the final concentration of α-synuclein aggregates 70 fM to prepare a simulated Parkinson's sample;

[0157] (2) Prepare buffer solution: add 30.237 g of 1,4-piperazine diethanesulfonic acid and 29.220 g of sodium chloride to 800 mL of deionized water, adjust the solution to pH 6.5 with hydrochloric acid or sodium hydroxide, add deionized water until the volume is 1 L, and stir evenly; filter with a 0.22 μm filter, and dispense into small bottles, avoiding direct sunlight and high temperature;

[0158] (3) Preparation of substrate solution: Dissolve α-synuclein in the buffer prepared in step (2) above to a concentration of 1 mg / mL, and filter with a 100KD filter membrane to obtain a substrate solution containing α-synuclein monomers;

[0159] (4) Sampling: Take a 0.5 mL polyethylene centrifuge tube, add 200 μL of the substrate solution containing α-synuclein monomers prepared in step (3) above to each tube, add 40 μL of the sample to be tested (the simulated Parkinson's disease sample prepared above) to the substrate solution, and set up a probe group 1; in the same way, set the sample obtained from the healthy population as the negative control and the buffer solution as the blank control; at the same time, prepare the same sample but do not perform the amplification reaction as a control; after the sample loading is completed, seal the centrifuge tube with a sealing film;

[0160] (5) PMCA amplification reaction: Place the probe set 1 of the sample to be detected in an ultrasonic instrument and perform cyclic ultrasonication at 37°C, first ultrasonicating for 1 minute, and then incubating for 29 minutes; repeat the above operation for a total of 144 cycles, and the reaction time is a total of 72 hours; after the reaction is completed, transfer the sample to a black 96-well plate, with 100 μL of sample in each well;

[0161] (6) Preparation of detection reagents: Weigh the probe 1 prepared above, dissolve it in dimethyl sulfoxide solution to 315 μM, use a dispenser to draw 12 μL into each well of a 96-well plate, and quickly place it in an ELISA reader for signal reading;

[0162] (7) Chemiluminescence detection: Place the well plate in an imager (IVIS imager), set the luminescence mode to a closed excitation light source, collect the full spectrum signal of the emission spectrum, and read the chemiluminescence signal; then change the luminescence mode to fluorescence, set the excitation wavelength to 450 nm and the emission wavelength to 480 nm; and read the optical signal.

[0163] The optical signals of probe 1 combined with healthy controls and Parkinson's disease are shown in Table 10 below, where the optical signals of probe 1 combined with healthy controls and Parkinson's disease are the maximum values ​​of the optical signals (the probe is a consumable reagent, and the reaction starts as soon as it is added, and then the signal gradually weakens); the average values ​​are plotted as shown in Fig. 9 As shown ( Fig. 9 In the figure, CSF (i.e., probe 1) + healthy controls, CSF + 70fM fibers (i.e., probe 1) + simulated Parkinson's disease samples).

[0164] Table 10 Optical signals of probe 1 binding to cerebrospinal fluid of healthy controls and Parkinson's disease

[0165] Grouping Probe + healthy control (au) Probe + simulated Parkinson sample (au) Probe 1-1 20850 71960000 Probe 1-2 23730 70800000 Probe 1-3 9555 71360000

[0166] It can be seen from Table 10 above that the optical signal of probe 1 combined with the simulated Parkinson's sample is significantly higher than that of the healthy control, indicating that probe 1 can detect α-synuclein in cerebrospinal fluid.

[0167] 3. Cost Comparison

[0168] The present invention compares the costs of detecting α-synuclein by the prior art and the present invention, as shown in Table 11 below.

[0169] Table 11 Cost estimates for different detection methods

[0170]

[0171]

[0172] It can be seen from Table 11 above that the sensitivity of the detection method in the present invention is significantly better than that of the prior art, and the detection cost of a single sample is also lower than that of the prior art.

[0173] 4. Other amplification parameter tests

[0174] (I) DMSO volume ratio

[0175] In this test, probe 1 and probe 2 were weighed and dissolved in dimethyl sulfoxide (DMSO) solution to 750 μM, and then PBS buffer was added to make the final volume ratio of DMSO in the buffer solution 1%, 5%, 10%, 50% and 100% (for example, 1% means that the volume of DMSO is 1 mL and the volume of buffer is 99 mL); 2 μL was drawn by a pistol and added to each well of a 96-well plate, and the plate was quickly placed in an ELISA reader for signal reading, and 3 parallel groups were set up. The results showed that too low an amount (1%) would not allow the probe to undergo a chemiluminescent reaction, and too high an amount (20%) would destroy the protein conformation. The luminescence conditions of 5%, 10%, 50% and 100% are shown in Tables 12 and 13, and the average values ​​are plotted as shown in Tables 12 and 13. Fig.10 and Fig.11 As shown, the results show that both can emit optical signals.

[0176] Table 12 Optical signals of probe 1 at different DMSO percentages

[0177]

[0178] Table 13 Optical signals of probe 2 at different DMSO percentages

[0179]

[0180] 2.2 Environment of protein aggregation reaction

[0181] Probe 1 was tested according to the method for cerebrospinal fluid sample detection in the above sample detection (III), wherein the oscillation speed in the RT-QUIC amplification reaction was set to 400 rpm, 500 rpm, 600 rpm, 700 rpm or 800 rpm respectively; the test results are shown in Tables 14 and Fig.12As shown, the results show that protein amplification can be achieved at an oscillation speed of 400rpm-800rpm.

[0182] Table 14 Effect of different rotation speeds on optical signals

[0183] Speed / rpm 400 500 600 700 800 Optical signal (au) 17200 21700 25400 37700 49800

[0184] (III) Effects of different α-synuclein monomers on the luminescence of probe 1

[0185] Probe 1 was tested according to the method for cerebrospinal fluid sample detection in the above sample detection (III), wherein the concentration of α-synuclein monomer was 0.1 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL or 2.5 mg / mL, and its luminescence was shown in Tables 15 and Fig.13 As shown, the results showed that the concentration of α-synuclein monomers in the range of 0.1 mg / mL-2.5 mg / mL had an optical signal enhancement effect, but when the concentration was greater than 2 mg / mL, the optical signal did not change much.

[0186] Table 15 Effect of different α-synuclein monomer concentrations on luminescence values

[0187] Concentration mg / ml 0.1 1 1.5 2 2.5 Optical signal (au) 51600 95900 103000 145000 154000

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A chemiluminescent probe, the chemical structure of which is shown below: in, n≥1; and is an integer, and R is a benzene ring connected to a dimethylamino group or a benzofuran connected to a diethylamino group.

2. The chemiluminescent probe according to claim 1, characterized in that Its chemical structure is shown below:

3. The method for preparing the chemiluminescent probe according to claim 1 or 2, characterized in that: include: (1) reacting the compound represented by formula I and the compound represented by formula II to obtain the compound represented by formula III; (2) reacting the compound represented by formula III with the compound represented by formula IV to obtain the compound represented by formula V; (3) reacting the compound represented by formula V with the compound represented by formula VI to obtain the chemiluminescent probe according to any one of claims 1 to 3; wherein the structural formulas of the compounds represented by formula I to formula VI are respectively as follows:

4. A detection reagent or detection kit for protein detection, characterized in that: The chemiluminescent probe according to claim 1 or 2, wherein the protein is beta-amyloid protein, alpha-synuclein, tau protein, prion protein, amylin or transthyretin protein.

5. The detection reagent or detection kit according to claim 4, characterized in that: The detection reagent or detection kit further comprises dimethyl sulfoxide and a buffer.

6. The detection reagent or detection kit according to claim 6, characterized in that: The volume percentage of dimethyl sulfoxide in the buffer system is 5%-100%.

7. The detection reagent or detection kit according to any one of claims 4 to 6, characterized in that: Also included are individually packaged RT-QuIC amplification reagents or individually packaged PMCA amplification reagents.

8. Use of the chemiluminescent probe according to claim 1 or 2 in the preparation of a product for detecting neurodegenerative diseases, wherein the biomarker of neurodegenerative diseases is α-synuclein.

9. A method for detecting α-synuclein for non-diagnostic purposes, characterized in that: include: (1) Amplifying a sample to be tested and an α-synuclein monomer using the RT-QuIC or PMCA method to obtain an α-synuclein amplification product; (2) Mixing the α-synuclein amplification product with the chemiluminescent probe according to claim 1 or 2, and reading an optical signal after the reaction is completed; (3) Qualitatively or quantitatively detecting α-synuclein based on the optical signal.

10. The method according to claim 9, characterized in that The method meets one or more of the following conditions: (a) In step (1), the sample to be tested is amplified using the RT-QuIC method to obtain an α-synuclein amplification product, and in the RT-QuIC method, the oscillation speed is 400 rpm-800 rpm; (b) In step (1), the concentration of α-synuclein monomer is 0.1 mg / mL-2.5 mg / mL.

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