Application of triphenylmethane compounds in detection of alzheimer's disease

Abstract

The application belongs to the technical field of disease detection, and particularly relates to application of triphenylmethane compounds in detection of Alzheimer's disease. The application is accidentally discovered that triphenylmethane compounds, especially crystal violet, can be inserted into misfolded beta-sheets of A beta oligomers, so as to cause change of spectrum. Macroscopically, oxidation and discoloration are caused, so that the triphenylmethane compounds can be used for monitoring and early screening of Alzheimer's disease patients. Compared with antigen-antibody preparation of antigen-antibody immunoreaction products, the product in the application has the defects of complicated antigen-antibody preparation and high cost, and the product in the application has low cost. Meanwhile, the main component of the application is a chemical reagent, has good stability, does not need refrigeration storage condition, is convenient to transport, and is convenient to use. Meanwhile, the technical scheme of the application effectively avoids the cleaning process of enzyme-labeled plates and chemiluminescence experiments, and does not need special equipment, so that the basic medical institutions can use the application, and therefore the application has good practical application value and application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease detection, and particularly relates to application of triphenylmethane compounds in detection of Alzheimer's disease. BACKGROUND

[0002] The number of Alzheimer's disease (AD) patients is increasing with the development of global aging. AD patients undoubtedly pose a huge burden on families and society, and the early clinical symptoms of AD are not specific, which is not conducive to clinical diagnosis. Extensive research has been conducted on the early biological markers of AD at home and abroad. The existing diagnostic methods include cerebrospinal fluid (CSF), blood, urine, neuropsychological assessment scale screening, and neuroimaging examination. CSF has high sensitivity and specificity for early diagnosis of AD, but it is an invasive examination, which is not easily accepted by the elderly and their families, and has poor clinical feasibility; at present, the neuropsychological assessment scale can only be used as an early AD screening tool; and the imaging markers need repeated research, dynamic observation and regular review to obtain more accurate results, which is high in cost and not suitable as a routine project. Blood and the above examination items are more easily obtained and accepted by patients and their families, and have small economic burden.

[0003] At present, blood biomarkers, including beta-amyloid, tau protein and its phosphorylated form, inflammatory factors, etc., detection methods include enzyme-linked immunoassay, colloidal gold immunochromatography, chemiluminescence, single molecule array, electrochemiluminescence, and immunomagnetic reduction technology. These detection methods are based on the specific binding of antigen and antibody, and the cost of antibody is high. Given that beta-amyloid, tau protein and its phosphorylated form, and inflammatory factors exist in the body of healthy or diseased individuals, biological differences between individuals, different stages of disease progression, and other factors that may affect the level of biomarkers (such as age, gender, lifestyle, and coexisting diseases) can cause significant variation in blood marker levels. This variability brings difficulties to the accurate interpretation and clinical application of markers. Improving the sensitivity and specificity of detection is also an important challenge in the field of AD blood marker detection. For neurodegenerative diseases such as AD, early diagnosis is crucial, but the concentration of many AD-related markers is extremely low in the early stages of the disease, which is beyond the detection limit of many traditional detection techniques, and the correlation of the above blood markers with AD is low, only 60-70%. Therefore, developing more sensitive and specific detection techniques is the key to achieving early diagnosis and effective monitoring. In addition, the methods and standards of AD blood marker detection differ in different laboratories and studies, which may affect the comparability and repeatability of the results. Therefore, overcoming the above challenges and improving the accuracy, sensitivity and reliability of AD blood marker detection are of great significance for achieving early diagnosis and individualized treatment of AD. SUMMARY

[0004] In order to solve the problems in the prior art, the application provides the application of triphenylmethane compounds in detection of Alzheimer's disease. Through research, it is found that triphenylmethane compounds, especially crystal violet, can be inserted into the misfolded beta-sheet of A beta oligomer, thereby shortening the distance between the compound and a reactive oxygen species (ROS) molecule, so that the electron transfer oxidation reaction can occur within the oxidation reaction life time of the ROS free radical, and then the conjugate chemical bond in the molecular structure of the compound is oxidatively destroyed or the electron on the chemical bond is recombined, finally leading to a change in the spectrum: the absorbance at 590 nm is reduced, and the macroscopic oxidation discoloration is shown, so that the A beta oligomer and the ROS can be indirectly monitored by the change degree of the absorbance. The accumulation of ROS caused by the A beta oligomer damages the lipid membrane of neurons, and the accumulation of neurons leads to the occurrence and development of Alzheimer's disease, so that the A beta oligomer can be detected to indirectly determine whether Alzheimer's disease is suffered, and thus the triphenylmethane compounds can be used for monitoring and early screening of Alzheimer's disease patients. Based on the above research results, the application is completed.

[0005] In order to achieve the above technical purpose, the technical scheme of the application is as follows:

[0006] In a first aspect of the application, the application of triphenylmethane compounds in preparation of an Alzheimer's disease detection product is provided.

[0007] Further, the Alzheimer's disease detection product can be used for screening, diagnosis, monitoring or prognosis evaluation of Alzheimer's disease.

[0008] The diagnosis can be early diagnosis or early auxiliary diagnosis of Alzheimer's disease.

[0009] The Alzheimer's disease detection product can be a detection kit, a detection device or a detection equipment.

[0010] Further, the triphenylmethane compounds include but are not limited to ethyl violet, crystal violet, methyl violet B, Victoria blue B, malachite green, brilliant green, methyl green, bromophenol blue and rosolic acid; further, crystal violet, ethyl violet, methyl violet B and Victoria blue B; most preferably, crystal violet and ethyl violet.

[0011] In a second aspect of the application, an Alzheimer's disease detection kit is provided, and the detection kit at least contains triphenylmethane compounds.

[0012] Further, the detection kit can further comprise a matrix of the triphenylmethane compound, which includes but is not limited to purified water, distilled water, water for injection, tap water, alcohol solution, physiological saline, buffer, culture medium, animal serum, glycerol and chloroform; further purified water, distilled water, water for injection, tap water, alcohol solution, physiological saline and buffer; and further purified water and buffer.

[0013] The buffer includes but is not limited to acetic acid buffer, phosphoric acid buffer, boric acid buffer, citric acid buffer and Tris buffer, and the buffer concentration is 10 μM-100 mM.

[0014] In the present application, the concentration of the triphenylmethane compound is 5 μM-1 mM, preferably 10-500 μM, and more preferably 20-200 μM.

[0015] In a third aspect of the present application, a system for detecting Alzheimer's disease is provided, which comprises:

[0016] An acquisition module configured to acquire a mixture of a sample to be tested of a subject and a triphenylmethane compound or the Alzheimer's disease detection kit;

[0017] An analysis module configured to analyze and judge the disease condition of the subject according to the color, absorbance, absorption spectrum, fluorescence value or fluorescence spectrum of the mixture obtained by the acquisition module.

[0018] In the present application, the triphenylmethane compound includes but is not limited to ethyl violet, crystal violet, methyl violet B, Victoria blue B, malachite green, brilliant green, methyl green, bromophenol blue and rosolic acid; and further crystal violet, ethyl violet, methyl violet B and Victoria blue B; and most preferably crystal violet and ethyl violet.

[0019] The sample to be tested can be a body fluid, further a blood, cerebrospinal fluid, urine or saliva sample, and further a serum.

[0020] Specifically, the specific analysis and evaluation process of the analysis module includes: acquiring the absorbance value of the mixture of crystal violet detection solution (40 μM) and serum sample of the subject at 590 nm, and if the value is lower than the cutoff value, the subject is determined to have Alzheimer's disease, and if the value is higher than the cutoff value, the subject is determined to not have Alzheimer's disease. Further, the cutoff value is 0.32595.

[0021] The one or more technical solutions have the following beneficial technical effects:

[0022] The technical scheme discloses application of triphenylmethane compounds in detection of Alzheimer's disease, research shows that the triphenylmethane compounds can effectively distinguish healthy people from Alzheimer's disease patients, compared with an antigen-antibody preparation based on an antigen-antibody immune reaction product, the product has the defects of complicated preparation and high cost, the product in the application has low cost, meanwhile, the main component of the product is a chemical reagent, the product has good stability, does not need refrigeration storage conditions, is convenient to transport, and is convenient to use; meanwhile, the technical scheme effectively avoids the cleaning process required by an enzyme-labeled plate and a chemiluminescence experiment, and does not need special equipment, so that the product can be used by basic medical institutions, and therefore, the product has good practical application value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a screening result diagram of 40 μM methyl violet B detection liquid (columns 1-2), 40 μM nile blue detection liquid (columns 3-4), 200 μM victoria blue B detection liquid (columns 5-6), 200 μM methyl violet B detection liquid (columns 7-8), 40 μM ethyl violet detection liquid (columns 9-10) and 40 μM thymol blue detection liquid (columns 11-12) in the application, wherein every two columns are one kind of detection liquid, the left column of every two columns is a negative sample, and the right column of every two columns is a positive sample.

[0024] Figure 2 Figure 2 is a screening result diagram of 40 μM crystal violet (row 1), 200 μM victoria blue B (row 2), 40 μM ethyl violet (row 3), 200 μM methyl violet B (row 4), 200 μM crystal violet (row 5), 100 μM methyl green (row 6), 40 μM malachite green (row 7) and 200 μM brilliant green detection liquid (row 8) in the application, wherein every row is one kind of detection liquid, the left 6 holes in every row are negative samples, and the right 6 holes in every row are positive samples.

[0025] Figure 3 Figure 3 is an absorption spectrum of 40 μM crystal violet detection liquid in the application.

[0026] Figure 4 Figure 4 is an absorption spectrum of 200 μM victoria blue B detection liquid in the application.

[0027] Figure 5 Figure 5 is an absorption spectrum of 40 μM ethyl violet detection liquid in the application.

[0028] Figure 6 Figure 6 is an absorption spectrum of 200 μM methyl violet B detection liquid in the application.

[0029] Figure 7 Figure 7 is an absorption spectrum of 200 μM crystal violet detection liquid in the application.

[0030] Figure 8Absorption spectrum of 100 μΜ methylene green detection solution in the present application.

[0031] Figure 9 Absorption spectrum of 40 μΜ malachite green detection solution in the present application.

[0032] Figure 10 Absorption spectrum of 200 μΜ brilliant green detection solution in the present application.

[0033] Figure 11 AUC-ROC curve of 40 μΜ crystal violet detection solution in the present application. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed description is illustrative only, and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0036] As mentioned above, there are many difficulties in early screening and diagnosis of Alzheimer's disease. The inventors have found that β-amyloid protein (Aβ) is generated by cleavage of amyloid precursor protein (APP) by β- and γ-protease, forming Aβ 16 , Aβ 40 , Aβ 42 and other peptide segments. These peptide chains contain hydrophobic amino acid residues and can be induced to misfold by metal ions (such as Cu 2+ , Fe 2+ , Zn 2+ ) or active molecules (such as H2O2), forming soluble oligomers or insoluble fiber aggregates. Among them, soluble Aβ oligomers dominate in the early stage of Alzheimer's disease (AD) and have stronger neurotoxicity, and gradually transform into insoluble plaques (senile plaques) in the later stage.

[0037] Metal ion homeostasis imbalance is an important inducer of AD. The content of Cu 2+ , Fe 2+ , Zn 2+ and other metal ions in amyloid plaques is significantly increased. Fe 2+ produces ROS through Fenton reaction, triggering oxidative stress and "ferroptosis"; Zn 2+promote Aβ aggregation and enhance toxicity; Cu 2+ then inhibit protein degradation, leading to Aβ accumulation. Aβ-metal complexes further exacerbate aggregation and oxidative damage. In addition, Aβ oligomers initiate a vicious cycle by promoting Tau protein hyperphosphorylation, forming neurofibrillary tangles, and inducing neuroinflammation.

[0038] Aβ oligomers coupled with metal ions produce a peroxidase-like effect, catalyzing the generation of short-lived (about 10 -9 seconds) ROS (such as ·OH, H2O2, ONOO - , HOCl). The inventors have unexpectedly found that triphenylmethane compounds, especially crystal violet, can insert into the β-sheet structure of Aβ oligomers, undergo a fading reaction (absorbance decrease at 590 nm) under ROS oxidation, thereby indirectly reflecting the levels of Aβ oligomers and ROS. This mechanism can be used for early screening of AD, by monitoring soluble Aβ oligomers and their oxidative damage to assess disease progression.

[0039] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application.

[0040] Example 1

[0041] (1) Preparation of 1 mM crystal violet solution

[0042] Weigh 0.0408 g of crystal violet and dissolve it in 100 mL of purified water, mix well to prepare a 1 mM crystal violet solution.

[0043] (2) Preparation of 40 μM crystal violet solution

[0044] Measure 10 mL of 1 mM crystal violet solution and add 240 mL of purified water, mix well to prepare a 40 μM crystal violet solution.

[0045] (3) Preparation of 20 μM crystal violet solution

[0046] Measure 10 mL of 1 mM crystal violet solution and add 490 mL of purified water, mix well to prepare a 20 μM crystal violet solution.

[0047] Example 2

[0048] (1) Preparation of 1 mM bromophenol blue solution

[0049] Weigh 0.0670 g of bromophenol blue and dissolve it in 100 mL of purified water, mix well to prepare a 1 mM bromophenol blue solution.

[0050] (2) Preparation of 100 μM bromophenol blue solution

[0051] Take 10 mL of 1 mM bromophenol blue solution, add 90 mL of purified water, mix well, and prepare 100 μM bromophenol blue solution.

[0052] Example 3

[0053] (1) Preparation of 1 mM Victoria Blue B solution

[0054] Take 0.0506 g of Victoria Blue B, add 100 mL of purified water, mix well, and prepare 1 mM Victoria Blue B solution.

[0055] (2) Preparation of 200 μM Victoria Blue B solution

[0056] Take 10 mL of 1 mM Victoria Blue B solution, add 40 mL of purified water, mix well, and prepare 200 μM Victoria Blue B solution.

[0057] Example 4

[0058] (1) Preparation of 1 mM Methyl Violet B solution

[0059] Take 0.0358 g of Methyl Violet B, add 100 mL of purified water, mix well, and prepare 1 mM Methyl Violet B solution.

[0060] (2) Preparation of 200 μM Methyl Violet B solution

[0061] Take 10 mL of 1 mM Methyl Violet B solution, add 40 mL of purified water, mix well, and prepare 200 μM Methyl Violet B solution.

[0062] (3) Preparation of 40 μM Methyl Violet B solution

[0063] Take 10 mL of 1 mM Methyl Violet B solution, add 240 mL of purified water, mix well, and prepare 40 μM Methyl Violet B solution.

[0064] Example 5

[0065] (1) Preparation of 1 mM Ethyl Violet solution

[0066] Take 0.0492 g of Ethyl Violet, add 100 mL of purified water, mix well, and prepare 1 mM Ethyl Violet solution.

[0067] (2) Preparation of 40 μM Ethyl Violet solution

[0068] Take 10 mL of 1 mM Ethyl Violet solution, add 240 mL of purified water, mix well, and prepare 40 μM Ethyl Violet solution.

[0069] Example 6

[0070] (1) Preparation of 1 mM Thymolphthalein solution

[0071] Take 0.0467g thymol blue, add 100mL purified water, mix well, and prepare 1mM thymol blue solution.

[0072] (2) Preparation of 40μM thymol blue solution

[0073] Take 10mL 1mM thymol blue solution, add 240mL purified water, mix well, and prepare 40μM thymol blue solution.

[0074] Example 7

[0075] (1) Preparation of 1mM Nile blue solution

[0076] Take 0.0733g Nile blue, add 100mL purified water, mix well, and prepare 1mM Nile blue solution.

[0077] (2) Preparation of 40μM Nile blue solution

[0078] Take 10mL 1mM Nile blue solution, add 240mL purified water, mix well, and prepare 40μM Nile blue solution.

[0079] Example 8

[0080] (1) Preparation of 1mM Brilliant green solution

[0081] Take 0.0483g Brilliant green, add 100mL purified water, mix well, and prepare 1mM Brilliant green solution.

[0082] (2) Preparation of 200μM Brilliant green solution

[0083] Take 10mL 1mM Brilliant green solution, add 40mL purified water, mix well, and prepare 200μM Brilliant green solution.

[0084] Example 9

[0085] (1) Preparation of 1mM Methyl green solution

[0086] Take 0.0458g Methyl green, add 100mL purified water, mix well, and prepare 1mM Methyl green solution.

[0087] (2) Preparation of 100μM Methyl green solution

[0088] Take 10mL 1mM Methyl green solution, add 90mL purified water, mix well, and prepare 100μM Methyl green solution.

[0089] Example 10

[0090] (1) Preparation of 1mM Malachite green solution

[0091] Take 0.0927g malachite green, add 100mL purified water, mix well, and prepare 1mM malachite green solution.

[0092] (2) Preparation of 40μM malachite green solution

[0093] Take 10mL 1mM malachite green solution, add 240mL purified water, mix well, and prepare 40μM malachite green solution.

[0094] Effect verification

[0095] (1) Probe screening

[0096] Using 200μM Victoria blue B, 40μM and 200μM methyl violet B, 40μM ethyl violet, 40μM thymol blue, 40μM nile blue, 40μM and 20μM crystal violet, 200μM brilliant green, 100μM methyl green, 40μM malachite green and 100μM bromophenol blue, the detection was carried out by using positive and negative samples, and the clinical sample was serum, which was from Qilu Hospital of Shandong University and Weifang People's Hospital. In the present application, the positive sample is the serum sample of patients definitely diagnosed as Alzheimer's disease, and the negative sample is the serum sample of healthy subjects.

[0097] Experimental process: dissolve various compounds in water for injection to prepare the above corresponding concentrations; take 100μL of the detection solution in the microwell, add 20μL of the clinical sample, and the detection results can be distinguished by color change, absorbance, absorption spectrum or fluorescence value.

[0098] From the color, 40μM and 20μM crystal violet detection solution, 40μM ethyl violet detection solution, 200μM Victoria blue B, 40μM and 200μM methyl violet B detection solution have the best effect, and the color distinction degree of positive and negative samples is the best, and the results are shown in Figures 1 to 2 .

[0099] Figures 3 to 10 The absorbance detection results of different compound detection solutions are shown in the following table, wherein, except that the scanning wavelength of the methyl green detection solution is 400-700nm, the scanning wavelengths of the remaining compound detection solutions are all 500-700nm. From the absorption spectrum, 40μM and 200μM crystal violet detection solution ( Figure 3 and Figure 7 ), Victoria blue B detection solution ( Figure 4 ), 40μM ethyl violet detection solution ( Figure 5 ), 200μM methyl violet B detection solution ( Figure 6 ) and 200μM brilliant green detection solution ( Figure 10 ) can effectively distinguish positive samples and negative samples. While 100μM methyl green detection solution (Figure 8 ) and 40 μM malachite green detection solution (B) Figure 9 ) cannot effectively distinguish between positive and negative samples.

[0100] In view of the small difference in color change between positive and negative samples of bromophenol blue detection solution, the fluorescence value was detected by a microplate reader with an excitation wavelength of 595 nm and an emission wavelength of 615 nm. The fluorescence value is shown in Table 1. From the results, the fluorescence value of the negative sample is higher than that of the positive sample, which can distinguish between positive and negative samples.

[0101] Table 1 Clinical sample-bromophenol blue fluorescence value detection

[0102]

[0103] (2) Determination of positive judgment value of product

[0104] The samples were detected by crystal violet detection solution. 48 negative samples and 48 positive samples (samples from Shandong University Qilu Hospital and Weifang People's Hospital) were taken. 100 μL of 40 μM crystal violet detection solution was added to the 96-well plate, 20 μL of clinical sample was added, and the microplate reader was used for detection. The absorbance value at 590 nm was used to distinguish between positive and negative samples.

[0105] The detection results of positive samples are shown in Table 2.

[0106] Table 2 Detection results of positive samples based on crystal violet detection

[0107]

[0108] The detection results of negative samples are shown in Table 3.

[0109] Table 3 Detection results of negative samples based on crystal violet detection

[0110]

[0111] Through receiver operating characteristic curve (ROC) analysis, the results are as follows Figure 11The AUC value is 0.916, the AUC value is greater than 0.9, and the accuracy is high, and the classification ability is high. The cutoff value is 0.32595. In the present application, the cutoff value is obtained by calculation based on the conventional ROC curve method. Briefly, by drawing an ROC curve, the horizontal axis is 1-specificity (false positive rate), and the vertical axis is sensitivity (true positive rate). Each point on the curve corresponds to a potential cutoff value. By calculating the Youden Index of each point on the curve, the Youden Index calculation formula is: Youden Index = Sensitivity + Specificity - 1, and the cutoff value corresponding to the maximum Youden Index is 0.32595, that is, the optimal cutoff value.

[0112] (3) Verification of the crystal violet detection solution of the clinical sample

[0113] The established cutoff value is verified, 80 negative samples and 64 positive samples (the samples are derived from Qilu Hospital of Shandong University, Weifang People's Hospital, Xiamen Xianyue Hospital and company physical examination samples), and the detection method is consistent with the method of positive judgment value. The results of the negative samples and the positive samples are shown in Table 4 and Table 5.

[0114] Table 4 Verification results of negative samples based on crystal violet detection

[0115]

[0116] Table 5 Verification results of positive samples based on crystal violet detection

[0117]

[0118] After detection, the sensitivity is 98.44%, and the specificity is 97.5%, indicating that crystal violet can be used for the diagnosis and detection of Alzheimer's disease patients.

[0119] The remaining matters of the present application are known technologies.

[0120] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a triphenylmethane compound in the preparation of an Alzheimer's disease detection product, characterized in that, the triphenylmethane compound is crystal violet; when the absorbance value of the mixture of the crystal violet detection solution and the serum sample of the subject at 590 nm is lower than the cutoff value, the subject is determined to have Alzheimer's disease, and when the absorbance value is higher than the cutoff value, the subject is determined not to have Alzheimer's disease.

2. Use according to claim 1, wherein The cutoff value is 0.32595.

3. The use according to claim 1, wherein the compound is ###0002### The Alzheimer's disease detection product is used for screening, diagnosis, monitoring or prognosis evaluation of Alzheimer's disease.

4. Use according to claim 3, wherein the compound is ###0002### The diagnosis is early diagnosis or early auxiliary diagnosis of Alzheimer's disease.

5. The use according to claim 1, wherein The Alzheimer's disease detection product is a detection kit.

6. A system for detecting Alzheimer's disease, characterized by, The system comprises: an acquisition module configured to acquire a mixture of a serum sample of a subject and a detection solution of a triphenylmethane compound, and when the absorbance value of the obtained mixture at 590 nm is lower than the cutoff value, the subject is determined to have Alzheimer's disease, and when the absorbance value is higher than the cutoff value, the subject is determined not to have Alzheimer's disease, and the triphenylmethane compound is crystal violet; an analysis module configured to analyze and determine the disease condition of the subject according to the absorbance of the mixture obtained by the acquisition module.

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