CN102279270B - Method for monitoring beta amyloid protein aggregation process by aggregation-induced emission - Google Patents

Abstract

The invention discloses a method for monitoring beta amyloid protein aggregation process by aggregation-induced emission, belonging to the technical field of biomedical research and clinical detection. With the method, the high affinity between sulfydryl on cysteine and maleimide is utilized to specially combine the beta amyloid protein on an aggregation-induced emission probe. The beta amyloid protein aggregation process is monitored on the basis of aggregation-induced emission enhancement phenomenon. The method has the advantages of high sensitivity, high selectivity, good stability, low manufacture cost and the like and is easy to control. The A beta42 and A beta40 contents can be monitored in a micromole, even a nano-mole level, the detection speed of the method is improved by 15 times if being compared with that of the detection method which utilizes probes, such as ANS (8-aniline-1-naphthalene sulfonic acid) and the like. The beta amyloid protein aggregation process can be qualitatively and quantitatively monitored, the method has a good application prospect if being applied to the pathologic diagnosis of latent patients suffering from senile dementia.

Description

A method for monitoring the aggregation process of β-amyloid protein using aggregation-induced luminescence

technical field

The invention relates to a monitoring method for the aggregation process of amyloid beta protein, in particular to a method for monitoring the aggregation process of amyloid beta protein by using an aggregation-induced luminescence method, and belongs to the technical field of biomedical research and clinical detection.

Background technique

Alzheimer's Disease (AD) is the most common disease causing senile dementia. Currently, AD is the fourth leading killer of the elderly after cardiovascular and cerebrovascular diseases, cancer, and stroke. The pathological feature of AD is that extracellular senescent plaques and intracellular neurofibrillary tangles can be observed in the brain of patients, which is due to the abnormal aggregation of β-amyloid (amyloid β-protein, Aβ) in and out of cells. Knots can form aggregates of short peptides that are toxic to neuronal cells. The most common Aβ in plasma, cerebrospinal fluid, and short peptide aggregates are Aβ 40 and Aβ 42 (Aβ 42 aggregates more easily and is more toxic), which are produced by amyloid precursors sequentially through β and γ-secretase Produced by site-specific digestion. The diagnosis of AD patients is currently mainly based on the partial loss of memory and behavior, and the pathological diagnosis of patients in the latent period is a challenging world-leading topic.

According to relevant medical data, among the 24 million senile dementia patients in the world, my country accounts for about 1/4, and the number is increasing at a rate of 1 million per year. Currently, the most commonly used techniques for the detection of asymptomatic AD patients are magnetic resonance imaging (magnetic resonance imaging, MRI), positron emission tomography (PET), optical imaging (optical imaging), surface-enhanced Raman ( surface enhanced Raman spectroscopy (SERS) spectroscopy, scanning electrical microscopy (SEM) technology and electrochemical methods, etc. The sensitivity of MRI is low, and it is difficult to effectively use it in clinical medicine; the price of PET is too high, and the available isotope-labeled PET probes are small, which greatly limits its wide application. Electrochemical methods require complex electrode preparation processes. There are also reports on the detection of β-amyloid aggregates by endogenous fluorescence method. However, the excitation and emission wavelengths of tryptophan, lysine, and phenylalanine that emit endogenous fluorescence are all in the ultraviolet region, so it is difficult to observe directly by imaging. , and the quantum yield is low, and the detection sensitivity is low. Fluorescent probes can be adsorbed or covalently bound to proteins, resulting in changes in their fluorescence properties (emission wavelength, fluorescence intensity, fluorescence polarization, etc.), which can then be used for qualitative and quantitative research on proteins. At present, the most commonly used fluorescent probes in the fluorescence detection of Aβ are Thioflavin (Thioflavin, ThT), Congo Red (Congo Red, CR) and 8-anilino-1-naphthalenesulfonic acid (8-Anilino-1- naphthalenesulfonic acid, ANS), etc. These probes are combined with proteins through hydrophobic interactions, causing changes in the fluorescence properties of fluorescent probes, so the specificity is poor; in addition, the detection repeatability of these probes is very poor; these probes cannot detect amyloid Some intermediates that occur during formation. In addition, β-amyloid protein has a low content in body fluids, and its aggregation speed is slow. Therefore, the design and synthesis of ultra-sensitive actuators and sensors that can monitor the aggregation process of β-amyloid protein have been developed for the early diagnosis of Alzheimer's patients. Medical kits can enable early detection and early treatment of many early patients, greatly reducing the physical pain and economic burden of patients. Roberti et al found that after modifying Aβ's sister protein α-synuclein (α-synuclein) on quantum dots (Qdot 605), a nucleation center that can promote the aggregation of α-synuclein is formed. This method can be quickly and sensitively studied The aggregation process of α-synuclein in living cells. Jovin et al. coupled maleimide (maleimide) to the fluorescent substance pyrene through a covalent bond, and mutated the inert alanine to cysteine ((Cysteine, Cys)) containing a hydroxyl group on α-synuclein, Taking advantage of the high affinity between maleimide and thiol, pyrene molecules were labeled on three mutants of α-synuclein. The aggregation of α-synuclein was detected by the emission peak generated by the formation of excimer after pyrene aggregation. Although pyrene molecules can form excimates, but because the fluorescence peak of excimates at 470 nm is small, it can only qualitatively indicate the formation of aggregates, but cannot accurately quantify the degree of aggregation.

Aggregation-induced emission enhancement (AIEE) compounds are a research hotspot in fluorescent probes in recent years. Due to its special molecular structure, its fluorescence is weak or even non-luminous in organic solvents. If the polarity of the solvent environment where it exists increases, or the compound exists in the form of aggregates or solids, some substituents in the molecule that can rotate freely The rotation is blocked, which strongly inhibits the non-radiative inactivation process of fluorescent molecules, resulting in greatly enhanced fluorescence of AIEE-type compounds. If an AIEE-type compound molecule is specifically labeled on Aβ42 or Aβ40, the aggregation of Aβ42 or Aβ40 molecules will inevitably lead to the aggregation of the AIEE-type compound, and the fluorescence will increase. It can qualitatively or quantitatively monitor the aggregation process of β-amyloid protein, and it will be used in the pathological diagnosis of senile dementia patients in the latent stage. It has a good application prospect, and there are no related literature reports.

Contents of the invention

The purpose of the present invention is to provide a new aggregation-induced luminescence method that can monitor the aggregation process of β-amyloid protein with high sensitivity and high selectivity, so as to provide beneficial information for early diagnosis of diseases such as Alzheimer's disease.

In order to achieve the purpose of the present invention, the present invention utilizes the high affinity between maleimide and the sulfhydryl group on cysteine to specifically bind β-amyloid protein to the aggregation-induced fluorescent probe, based on the phenomenon of aggregation-induced luminescence enhancement , highly sensitive, highly selective monitoring of amyloid beta aggregation process, so as to qualitatively or quantitatively monitor changes in amyloid beta in the human body.

Specific technical plan: First, synthesize a fluorescent probe with an aggregation-induced luminescence enhancement effect, use a covalent bond to couple maleimide to the aggregation-induced fluorescent probe molecule, and place the second position on the β-amyloid 42 molecule Glycine is mutated into cysteine, using the high affinity between maleimide and sulfhydryl on cysteine, specifically binding β-amyloid to the aggregation-inducing fluorescent probe, and using aggregation-induced luminescence enhancement A method to monitor the aggregation process of β-amyloid protein.

Specifically, it is realized through the following steps:

1) Dissolve the β-amyloid protein in which the 2nd glycine on the Aβ42 molecule is mutated into cysteine in phosphate buffer solution;

2) Then add a fluorescent probe with aggregation-induced luminescence enhancement effect with the following structure:

Fluorescent probe 1

or

fluorescent probe 2

or

Fluorescent probe 3 ;

3) Add the β-amyloid sample to be tested into the solution prepared in step (2), monitor its fluorescence intensity, and monitor the aggregation process of β-amyloid by using aggregation-induced luminescence enhancement.

Described phosphate buffer solution is phosphate 0.2 M, pH 7.4 buffer solution.

The present invention establishes a new aggregation-induced luminescence method for monitoring the aggregation process of β-amyloid protein, selects three kinds of fluorescent probe molecules with aggregation-induced luminescence effect coupled with maleimide groups, and injects the first one on the Aβ42 molecule The glycine at position 2 is mutated into cysteine, and the high affinity between maleimide and the sulfhydryl group on cysteine is used to specifically bind β-amyloid to the aggregation-inducing fluorescent probe. The aggregation of β-amyloid protein can increase the hydrophobicity of the environment where the aggregation-induced fluorescent probe molecules marked on it increase, and the fluorescence is enhanced, which can monitor the aggregation process of β-amyloid protein with high sensitivity and high selectivity. Has the following advantages:

1) The aggregation-induced luminescence method for monitoring β-amyloid protein has a very low fluorescence background, and the sensitivity is greatly improved;

2) Mutate the 2nd glycine on the Aβ42 (β-amyloid 42) molecule to cysteine-β-amyloid 42, the mutation position can effectively label the fluorescent probe on the β-amyloid The Aβ42 molecule does not change the aggregation behavior of the Aβ42 molecule. Utilizing the high affinity between maleimide and the sulfhydryl group on cysteine, the selectivity of the monitoring method is improved;

3) The preparation method is simple, easy to control, low in production cost, simple and efficient; the monitoring method has good stability and good reproducibility;

4) It has a wide range of applications. It can be connected to other proteins on the aggregation-induced fluorescent probe or use the phenomenon of aggregation-induced luminescence enhancement to study the structural changes of protein molecules or DNA molecules.

5) Through the phenomenon of aggregation-induced luminescence enhancement, micromolar or even nanomolar Aβ42 and Aβ40 contents can be monitored, and the detection speed is 15 times higher than the monitoring method using ANS (8-aniline-1-naphthalenesulfonic acid) and other probes .

Detailed ways

For a better description of the present invention, examples are as follows:

Example 1 The aggregation process of β-amyloid protein 42 is monitored by fluorescent probe 1 coupled with maleimide, which is achieved by the following steps:

1. Synthesis of fluorescent probe 1 coupled with maleimide, which is water-soluble. The synthetic route is shown below.

2. Purchase Aβ42 whose glycine at position 2 is mutated into cysteine, and dissolve it in phosphate buffer solution (0.2 M, pH 7.4).

3. Add the maleimide-coupled fluorescent probe 1 in the above step 2, and use the specific covalent bond formed between the maleimide and the sulfhydryl group on cysteine to convert the amyloid-beta protein specificity binding to aggregation-inducing fluorescent probes.

4. Use aggregation-induced luminescence enhancement to monitor the aggregation process of β-amyloid protein: put the prepared probes into a mixture containing 50 nM Aβ42 and 400 times complement C3, complement C4, IGA, IGM, IGG, IGD, IGE, fibroblast Cell growth factor, epidermal growth factor, platelet growth factor, 200 times serum albumin, β-lipoprotein, α-1 acid glycoprotein, α-1 antitrypsin, alpha-fetoprotein, haptoglobin, ceruloplasmin, α -2 macroglobulin, α-2 lipoprotein, transferrin, 100 times the α-1 globulin, α-2 globulin, β-globulin, γ-globulin samples, found that β-amyloid aggregates , the aggregation of β-amyloid protein causes the hydrophobicity of the environment where the aggregation-induced fluorescent probe molecule 1 exists on which the label is increased, and the fluorescence is enhanced. Aggregation-induced luminescence enhancement was used to monitor the aggregation process of β-amyloid protein, with a linear range of 4-100 nM (R=0.9958), and a detection limit of 0.5 nM (3σ), with good sensitivity. Moreover, none of the above-mentioned interfering substances interferes with the determination of β-amyloid protein (the relative standard deviation is controlled within 5%), which has good selectivity. 30 minutes to complete the test.

Synthetic route of fluorescent probe 1 coupled with maleimide

Compounds 1 and 2 were reacted under the conditions of NaH and Ph ₃ P to generate intermediate product 3, intermediate product 3 was reacted with maleic anhydride under CHCl ₃ and NaOAc to generate compound 4, and compound 4 and BBr ₃ were reacted to generate compound 5, Compound 5 was reacted with dibromoethane in a sodium hydroxide environment to generate intermediate 6, and intermediate 6 was reacted with triethylamine in a mixed solvent of tetrahydrofuran and water to generate target product 7.

Example 2 The aggregation process of β-amyloid protein 42 is monitored by fluorescent probe 2 coupled with lymide, which is achieved by the following steps:

1. Synthesis of fluorescent probe 2 coupled with maleimide, which is water-soluble. The synthetic route is shown below.

2. Purchase Aβ42 whose glycine at position 2 is mutated into cysteine, and dissolve it in phosphate buffer solution (0.2 M, pH 7.4).

3. Add the maleimide-coupled fluorescent probe 2 in the above step 2, and use the specific covalent bond formed between the maleimide and the sulfhydryl group on cysteine to convert the β-amyloid protein specificity binding to aggregation-inducing fluorescent probes.

4. Use the aggregation-induced luminescence enhancement method to monitor the aggregation process of β-amyloid protein: put the above-prepared probes into 30 nM Aβ42 and 300 times complement C3, complement C4, IGA, IGM, IGG, IGD, IGE, Fibroblast growth factor, epidermal growth factor, platelet growth factor, 150 times serum albumin, β-lipoprotein, α-1 acid glycoprotein, α-1 antitrypsin, alpha-fetoprotein, haptoglobin, ceruloplasmin , α-2 macroglobulin, α-2 lipoprotein, transferrin, 50 times more α-1 globulin, α-2 globulin, β-globulin, γ-globulin samples, found β-amyloid protein Aggregation occurs, and the aggregation of β-amyloid protein causes an increase in the hydrophobicity of the environment where the aggregation-induced fluorescent probe molecule 2 labeled on it increases, and the fluorescence increases. The aggregation-induced luminescence enhancement method was used to monitor the aggregation process of β-amyloid protein, with a linear range of 5-90 nM (R=0.9975) and a detection limit of 1 nM (3σ), with good sensitivity. Moreover, none of the above-mentioned interfering substances interferes with the determination of β-amyloid protein (the relative standard deviation is controlled within 5%), which has good selectivity. 30 minutes to complete the test.

 

 

Synthetic route of fluorescent probe 2 coupled with maleimide

Compound 1 reacted with Ph ₃ P under NaH condition to generate intermediate 2, intermediate 2 reacted with compound 3 to generate product 4, product 4 reacted with AgNO ₃ to generate product 5, and product 5 reacted with cis-butyl in CHCl ₃ and HAc/NaOAc environment Alkenic anhydrides react to give the desired product 6.

Example 3 The aggregation process of β-amyloid protein 42 is monitored by fluorescent probe 3 coupled with maleimide, which is achieved by the following steps:

1. Synthesis of fluorescent probe 3 coupled with maleimide, which is water-soluble. The synthetic route is shown below.

2. Purchase Aβ42 whose glycine at position 2 is mutated into cysteine, and dissolve it in phosphate buffer solution (0.2 M, pH 7.4).

3. Add the maleimide-coupled fluorescent probe 3 in the above step 2, and use the specific covalent bond formed between the maleimide and the sulfhydryl group on cysteine to convert the amyloid-β specificity binding to aggregation-inducing fluorescent probes.

4. Use the aggregation-induced luminescence enhancement method to monitor the aggregation process of β-amyloid protein: put the above-prepared probes into a medium containing 50 nM Aβ42 and 500 times complement C3, complement C4, IGA, IGM, IGG, IGD, IGE, fibroblast Cell growth factor, epidermal growth factor, platelet growth factor, 200 times serum albumin, β-lipoprotein, α-1 acid glycoprotein, α-1 antitrypsin, alpha-fetoprotein, haptoglobin, ceruloplasmin, α -2 macroglobulin, α-2 lipoprotein, transferrin, 150 times more α-1 globulin, α-2 globulin, β-globulin, γ-globulin samples, found that β-amyloid aggregates , the aggregation of β-amyloid causes the hydrophobicity of the environment where the aggregation-induced fluorescent probe molecule 3 present on the label increases, and the fluorescence increases. The aggregation process of β-amyloid was monitored by aggregation-induced luminescence enhancement method with a range of 10-150 nM (R=0.9982) and a detection limit of 2 nM (3σ). Has better sensitivity. Moreover, none of the above-mentioned interfering substances interferes with the determination of β-amyloid protein (the relative standard deviation is controlled within 5%), which has good selectivity. 30 minutes to complete the test.

Synthetic route of fluorescent probe 3 coupled with maleimide

Compound 1 reacts with ethylene glycol in p-toluenesulfonic acid environment to generate compound 2, compound 2 reacts with compound 3 under the catalysis of Zn/ _TiCl4 to generate product 4, and product 4 reacts with butene under the conditions of chloroform and NaOAC The dianhydride reacts to generate compound 5, which is transformed into compound 6 in the environment of p-toluenesulfonic acid, and the compound 6 reacts with the reactant 7 to generate the target product 8.

Based on the fluorescent probe aggregation-induced luminescence enhancement phenomenon, the invention monitors the aggregation process of amyloid beta protein with high sensitivity and high selectivity, and achieves better results.

Claims (2)

1. A method for monitoring amyloid-beta aggregation process by aggregation-induced luminescence, characterized in that, it is realized through the following steps: 1) Dissolve the β-amyloid protein in which the 2nd glycine on the Aβ42 molecule is mutated into cysteine in phosphate buffer solution; 2) Then add a fluorescent probe with aggregation-induced luminescence enhancement effect with the following structure: or

; 3) Add the β-amyloid sample to be tested into the solution prepared in step (2), monitor its fluorescence intensity, and monitor the aggregation process of β-amyloid by using aggregation-induced luminescence enhancement. 2. A method of utilizing aggregation-induced luminescence to monitor amyloid beta aggregation process as claimed in claim 1, wherein the phosphate buffer solution is 0.2 M, pH 7.4 buffer solution. the