CN116574271B - Preparation method and application of fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification - Google Patents
Preparation method and application of fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification Download PDFInfo
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Abstract
The invention belongs to the technical field of immunodetection, and particularly relates to a preparation method and application of fluorescent dendrimers for immunodetection of low-abundance biomarker signal cascade amplification. Multiple Phycoerythrins (PE) are coupled using the thiol group of SA-PAMAM-SH. The invention amplifies the signal value of the biotin-marked capture molecule combined on the detected object in a cascade amplification mode, and utilizes the high stability of an avidin-biotin system and the SA-PAMAM marked by PE can be widely applied to the avidin-biotin system, thereby obviously improving the detection sensitivity, and particularly meeting the requirements of effective detection of low-abundance Alzheimer Disease (AD) markers such as blood phosphorylating Tau protein p-Tau-181, p-Tau-217, p-Tau-231, neurofilament light chain protein (NFL) and the like, and enabling the early diagnosis of AD to be possible.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a preparation method and application of a fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification.
Background
Alzheimer's Disease (AD) is a progressive neurodegenerative disease, the most common type of dementia, characterized by beta-amyloid (Abeta) plaque formation, intracellular Tau protein aggregation, neuronal and synaptic loss, and other pathologies. According to the degree of the disease, it can be divided into 3 stages, normal (NC), mild cognitive impairment (mild cognitive impatient, MCI), alzheimer's Disease (AD).
The first discovery of this neurodegenerative disease by Alzheimer in the early 20 th century was mainly manifested by deterioration of acquired cognitive abilities (including memory, observability, imagination, computing power, etc.), and decreased ability to live daily life, resulting in inability to independently live, learn, work. Wherein hypomnesis is the earliest and most prominent manifestation of decline in cognitive ability in Alzheimer's disease. The MCI phase is an early state of the AD phase, and patients in this phase are most likely to be transferred to AD phase patients. With the progress of aging of population, the incidence rate of AD increases year by year, bringing great economic burden to society. Early diagnosis of the patient in the MCI phase, delaying the progression of the disease, and thus reducing the pressure on the home and society is therefore of great interest.
Currently, there are still difficulties in diagnosing AD, and biomarkers are critical for accurate and early identification of AD and are a prerequisite for effective treatment of this disease. The current diagnosis method of Alzheimer's disease is mainly cognitive ability test and neuroimaging examination. The neuropsychological scale test is an evaluation of the cognitive field of AD patients, and mainly comprises seven fields of memory function, speech function, directional force, application capability, attention, perception and executive function. Imaging examinations are important means of diagnosing AD, including CT, MRI and PET. PET is a relatively new imaging method, including FDG-PET, which is a potent biomarker of brain metabolism reflecting synaptic activity, and amyloid-PET, which is a biomarker reflecting amyloid deposition in the cerebral cortex. FDG-PET and amyloid-PET are important biomarkers for early diagnosis of AD, and the method can assist doctors to carry out clinical diagnosis, but has higher cost and longer time, and is difficult to popularize in people with insignificant symptoms, low-income people and undeveloped areas. Biomarkers are of interest in recent years, which can establish early diagnosis, assess risk, assess disease stage, and monitor progression and treatment response, which is of great importance for the treatment of alzheimer's disease. Tau protein has more than 40 phosphorylation sites, surez-Calvet M et al (2020) compared the correlation of three Tau proteins P-Tau217, P-Tau181, P-Tau231 with AD, and found that P-Tau181 was significantly elevated in the MCI phase. P-tau217, P-tau181, P-tau231 are important indicators reflecting AD patients. Neurofibrillary tangles consisting of intra-neuronal hyperphosphorylated tau are one of the hallmark pathological features of Alzheimer's Disease (AD). Immunoassays for measuring threonine 181, 217, 231 phosphorylated tau (p-tau 181, p-tau217, p-tau 231) in cerebrospinal fluid (CSF) have been developed as biomarkers of neurofibrillary tangles to support clinical diagnosis of AD dementia for several years, and several studies have shown that plasma p-tau181, p-tau217 and p-tau231 are indicators of amyloid and tau pathology in the clinical AD lineage, which can distinguish AD dementia from other neurodegenerative diseases. Plasma p-tau217 is more effective as a marker of longitudinal changes in brain atrophy during early stages of AD. Numerous studies have shown that a convenient means of detection of blood AD biomarkers detects the progression of AD patients and evaluates the efficacy of treatment. However, due to the low levels of phosphorylated tau in blood, even as low as several tens of femtograms (fg) in the NC phase, only 1-2 picograms (pg) in the MCI phase and even tens to tens of pg in the AD phase, the need for accurate and efficient detection of these low abundance proteins is currently not met, and more sensitive immunodetection methods need to be developed.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the preparation and application of the fluorescent marker detection substance for detecting the cascade amplification of the low-abundance Alzheimer's disease signal, which is more sensitive, simple to operate, good in stability, clear in result, easy to judge and store and low in detection cost.
The technical scheme adopted by the invention is as follows:
a method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascades, comprising the steps of:
(1) Preparation of SA-PAMAM dendrimer: coupling PAMAM dendrimer of G1.0 generation with carboxyl modified surface active group with streptavidin, activating PAMAM with N-hydroxysuccinimide solution and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution in an acidic environment, adding streptavidin, mixing and incubating in a dark place to obtain SA-PAMAM dendrimer;
(2) Preparation of SA-PAMAM-SH dendrimer: taking the SA-PAMAM dendrimer obtained in the step (1) as a center, mixing the SA-PAMAM dendrimer with PAMAM-PEG-SH dendrimer with surface active groups modified by mercapto groups, and adding a methanol solution of ethylenediamine for phthaleining reaction to generate a 0.5-generation SA-PAMAM-SH dendrimer product; then adding methyl acrylate methanol solution to perform addition reaction to generate 1.0 generation SA-PAMAM-SH dendrimer product; alternately repeating the two reaction steps to obtain SA-PAMAM-SH dendrimer products with algebraic sequentially increased numbers;
(3) Preparation of SA-PAMAM-Poly (PE) fluorescent dendrimer: and (3) activating the SA-PAMAM-SH dendrimer obtained in the step (2) by using N-maleimide in an acidic environment, and coupling phycoerythrin PE to obtain the SA-PAMAM-Poly (PE) fluorescent dendrimer dye.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the step of preparing the PAMAM dendrimer with the molecular weight of 2230.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the step of preparing the PAMAM-PEG-SH dendrimer with the molecular weight of 2000.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the following steps: taking 1mL of PAMAM solution with the concentration of 1mmol/L, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times; adding 900 mu L of MES buffer (pH 5.0), weighing NHS and EDC, and adding pure water to prepare 50mg/mL NHS and 10mg/mL EDC solution respectively; adding 10 mu L of NHS and EDC solution into PAMAM solution, mixing thoroughly, incubating at room temperature under dark shaking for 20min, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 2 times; adding 900. Mu.L of 1 XPBS buffer for resuspension, adding 100. Mu.L of SA solution, mixing uniformly, shaking at room temperature and light-shielding for incubation for 2h, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of methanol solution for resuspension, and standing for storage.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the following steps: mixing 1mL of PAMAM-PEG-SH solution with the SA-PAMAM solution in the step (1), adding 16mL of methanol solution of ethylenediamine according to the ratio of 1:8 of SA-PAMAM dendrimer to methanol solution of ethylenediamine, plugging a plug, magnetically stirring and mixing uniformly, incubating in a water bath for 2h, centrifugally washing a reaction product, and naturally drying to obtain a G0.5-generation SA-PAMAM-SH product; taking 1mL of 100.5-generation SA-PAMAM-SH dendrimer, adding 8mL of methyl acrylate methanol solution according to the ratio of 1:8 of the SA-PAMAM-SH dendrimer to the methyl acrylate methanol solution, adding a plug, magnetically stirring, incubating in a water bath for 2h, centrifugally washing a reaction product, and naturally drying to obtain the G1.0-generation SA-PAMAM-SH dendrimer; the reaction steps are alternately repeated to obtain the G2.0 generation SA-PAMAM-SH dendrimer.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the following steps:
labeling G2.0 generation SA-PAMAM-SH dendrimer with PE fluorescent dye, taking 1mL of SA-PAMAM-SH dendrimer solution, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times;
add 900 μl MES buffer: weighing a certain amount of N-maleimide, adding MES buffer solution to prepare 10mg/mL of N-maleimide solution, adding 50 mu L of N-maleimide solution into SA-PAMAM-SH solution, uniformly mixing, shaking at room temperature and light, incubating for 20min, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer solution for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 2 times;
after adding 900mL of MES buffer for resuspension, 100. Mu.L of PE fluorescent dye is added, the mixture is incubated for 30min under shaking at room temperature in a dark place, the supernatant is sucked off by centrifugation at 12000rpm for 20min, 1mL of 1 XPBS buffer is added for resuspension, the supernatant is sucked off by centrifugation at 12000rpm, and the mixture is subjected to resuspension at 1mL of 1 XPBS and then kept stand under dark place.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the steps that 1 XPBS buffer is 10mmol/L phosphate buffer, and pH7.4; the MES buffer is 0.05mol/L MES buffer with pH of 6.8.
According to the preparation method of the fluorescent dendrimer for immunodetection of the low-abundance biomarker signal cascade amplification, the mixing ratio of methyl acrylate to methanol solution in the methanol solution of methyl acrylate is 1:1.
The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification comprises the step of mixing ethylenediamine and methanol solution in a mixing ratio of 1:1 in the methanol solution of ethylenediamine.
The fluorescent dendrimer is used for detecting neural markers, including Alzheimer's disease, tumor markers and FPSA low-abundance biomarkers. .
The beneficial effects are that:
the SA-PAMAM-Poly (PE) fluorescent dendrimer prepared by the invention has a core at the center and a cavity at the inside, and has a large number of branching units. The volume, shape, functional groups and molecular weight can all be controlled at the molecular level. The fluorescent dye has high branching degree, regular and exquisite space structure, the high algebraic dendrimer is spherical, the nano-scale size is combined with a large amount of PE fluorescent dye on the surface, and the fluorescent signal intensity of protein detection can be greatly improved. Has good solubility and low viscosity.
The fluorescent detection substance for detecting signal cascade amplification is suitable for all flow detection kits, and has the advantages of simple operation, good stability, clear result, easy judgment and storage and low detection cost. Meanwhile, the method can be further extended to the immunodetection of a large number of low-abundance marker samples; as a general detection method, it will also be suitable for detection of a variety of product types. Therefore, the invention has wide application prospect.
Drawings
FIG. 1 is a flow chart of PAMAM dendrimer synthesis.
FIG. 2 is a schematic representation of PAMAM dendrimer synthesis.
FIG. 3 is a schematic diagram of the SA-PAMAM-SH dendrimer PE tag.
FIG. 4 shows concentration trends of different fluorescent substances for detecting the neural marker antigen.
FIG. 5 shows the concentration trend of different proportions of PE-bound fluorescent substances for detecting the neural marker antigen.
Detailed Description
The technical solution of the present invention will be clearly and completely described below, and the described embodiments are only preferred examples of the present invention and are not intended to limit the present invention, and various modifications and variations are possible based on the embodiments of the present invention. Any modification, equivalent replacement, improvement, etc. of the present invention should be included in the protection scope of the present invention.
Example 1
Referring to fig. 1-3, this embodiment provides a fluorescence detection system comprising the following components:
(1) Reaction buffer (T-AB): 20mmol/L Tris buffer, pH7.4.
(2) 10 Xwash (10 XWB): 100mmol/L phosphate buffer, pH6.4, containing 0.5% TWEEN 20 (Tween-20).
(3) Coupled microbead suspensions (Cap-Beads): the P-tau181, P-tau217 and P-tau231 monoclonal antibodies are respectively bound to three different fluorescent coding microbeads in a covalent crosslinking mode, and the three coupled microbeads are combined in equal quantity and diluted into a coupled microbead suspension with the concentration of 100 bds/mu L.
(4) Biotin-labeled Detection molecule (Detection): the biotin-labeled Tau monoclonal antibody is used to dilute the biotin-labeled antibody into a biotin-labeled detection molecule with a specified concentration.
(5) Stop Buffer: 20mmol/L phosphate buffer, pH7.4, containing 0.102% (v/v) ProClin.
The invention discloses a preparation method of fluorescent dendrimers for immunodetection of low-abundance biomarker signal cascade amplification, which comprises the following steps:
(1) preparation of SA-PAMAM dendrimer, 1mL of PAMAM solution with concentration of 1mmol/L is taken, and the molecular weight of the PAMAM dendrimer is 2230. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 3 times. The 1 XPBS buffer was 10mmol/L phosphate buffer, pH7.4. 900. Mu.L of MES buffer, 0.05mol/L MES buffer, pH5.0, was added. NHS and EDC were weighed and prepared as 50mg/mL NHS and 10mg/mL EDC solutions with purified water, respectively. To the PAMAM solution, 10. Mu.L of NHS and EDC solutions were added, respectively, and after mixing, the mixture was incubated at room temperature for 20min with shaking in the absence of light. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and 1mL of phosphate buffer, pH6.0 was added and resuspended, and after centrifugation at 12000rpm for 20min, the supernatant was aspirated and repeated 2 times. 900. Mu.L of phosphate buffer (pH 6.0) pH6.0 was added for resuspension, 100. Mu.L of SA solution was added, and after mixing, incubation was performed at room temperature under shaking in the dark for 2h. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and after resuspension with 1mL of methanol solution, the mixture was allowed to stand in the dark.
(2) Preparing SA-PAMAM-SH dendrimer, taking 1mL of PAMAM-PEG-SH solution, mixing the PAMAM-PEG-SH dendrimer with the molecular weight of 2000 with the SA-PAMAM solution in (1), adding the mixture into a three-necked bottle, adding 16mL of ethylenediamine methanol solution into the three-necked bottle according to the ratio of 1:8 of the SA-PAMAM dendrimer to the ethylenediamine methanol solution by using a dropping funnel, adding the mixture ratio of ethylenediamine and the ethylenediamine methanol solution into the ethylenediamine methanol solution by using a 1:1, adding a plug, magnetically stirring the mixture, incubating the mixture for 2h in a water bath, centrifugally washing the reaction product, and naturally drying the reaction product to obtain the G0.5 generation product. 1mL of the G0.5 generation product is taken and added into a three-necked bottle, 8mL of methyl acrylate methanol solution is added into the three-necked bottle by a dropping funnel according to the ratio of 1:8 of dendrimer to methyl acrylate methanol solution, the mixing ratio of methyl acrylate to methyl acrylate methanol solution is 1:1, a plug is plugged and magnetically stirred, water bath incubation is carried out for 2h, the reaction product is centrifugally washed and naturally dried, and the G1.0 generation product is obtained. The two reaction steps are alternately repeated to obtain the G2.0 generation product.
(3) A preparation method of SA-PAMAM-Poly (PE) fluorescent dendrimer comprises the step of labeling G2.0 generation SA-PAMAM-SH dendrimer by PE fluorescent dye. Taking 1mL of SA-PAMAM-SH dendrimer solution, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeatedly washing for 3 times.
mu.L MES buffer was added. 1mg of N-maleimide was weighed and 100. Mu.LMES buffer was added to prepare a 10mg/mL N-maleimide solution. 50 mu L of N-maleimide solution is added into the SA-PAMAM-SH solution, and after uniform mixing, the mixture is incubated for 20min at room temperature under light-proof shaking. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 2 times. After adding 900mL of MES buffer for resuspension, 100. Mu.L of PE fluorescent dye is added, and the mixture is incubated for 30min at room temperature under light-proof shaking. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and after 1mL of 1 XPBS was added for resuspension, the mixture was allowed to stand in the dark.
Example 2
The embodiment provides a preparation method of SA-PAMAM-Poly (PE) fluorescent dendrimers combined by SA-PAMAM-SH dendrimers of different generations and PE fluorescent dyes, which comprises the following steps:
(1) Preparation of SA-PAMAM dendrimer, 1mL of PAMAM solution with concentration of 1mmol/L was centrifuged at 12000rpm for 20min, the supernatant was aspirated, and after 1mL of 1 XPBS buffer was added for resuspension, the supernatant was aspirated and repeated 3 times at 12000 rpm. The 1 XPBS buffer was 10mmol/L phosphate buffer, pH7.4. 900. Mu.L of MES buffer, 0.05mol/L MES buffer, pH5.0, was added. A certain amount of NHS and EDC were weighed and prepared into 50mg/mL of NHS and 10mg/mL of EDC solution with pure water, respectively. To the PAMAM solution, 10 μl of NHS and EDC solution was added, and after mixing, incubation was performed at room temperature with shaking for 20min. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 2 times. Add 900. Mu.L 1 XPBS buffer for resuspension and 100. Mu.L SA solution, mix upside down and incubate for 2h with shaking at room temperature. Centrifuge at 12000rpm for 20min, suck the supernatant, add 1mL1 XPBS buffer to resuspend, centrifuge at 12000rpm for 20min, suck the supernatant, add 1mL methanol solution to resuspend, and then keep stand for storage.
(2) Preparing SA-PAMAM-SH dendrimer, mixing 1mL of PAMAM-PEG-SH solution with the SA-PAMAM solution in the step (1), adding into a three-necked bottle, adding 16mL of ethylenediamine methanol solution with a dropping funnel according to the ratio of 1:8 of the dendrimer to the ethylenediamine methanol solution, adding a plug, magnetically stirring, incubating for 2h in a water bath, centrifugally washing the reaction product, and naturally drying to obtain the G0.5 generation product. 1mL of the G0.5 generation product is taken and added into a three-necked bottle, 8mL of methyl acrylate methanol solution is added into the three-necked bottle by a dropping funnel according to the ratio of 1:8 of dendrimer to methyl acrylate methanol solution, the mixing ratio of methyl acrylate to methyl acrylate methanol solution is 1:1, a plug is plugged and magnetically stirred, water bath incubation is carried out for 2h, the reaction product is centrifugally washed and naturally dried, and the G1.0 generation product is obtained. The two reaction steps are alternately repeated to obtain SA-PAMAM-SH dendrimer products G0.5-G5.0 with algebraic sequentially increased.
(3) The preparation method of the SA-PAMAM-Poly (PE) dendrimer fluorescent dye is characterized by comprising the following steps: the G2.0 generation SA-PAMAM-SH dendrimer is marked by PE fluorescent dye. Taking 1mL of SA-PAMAM-SH dendrimer solution, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times.
mu.L MES buffer was added. 1mg of N-maleimide was weighed and 100. Mu.LMES buffer was added to prepare a 10mg/mL N-maleimide solution. 50 mu L of N-maleimide solution is added into the SA-PAMAM-SH solution, and after the mixture is mixed evenly in a reversed manner, the mixture is incubated for 20min at room temperature in a dark place. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 2 times.
After adding 900mL of MES buffer for resuspension, 100. Mu.L of PE fluorescent dye is added, and the mixture is incubated for 30min at room temperature under light-proof shaking. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and after 1mL of 1 XPBS was added for resuspension, the mixture was allowed to stand in the dark.
Example 3
The embodiment provides a preparation method of SA-PAMAM-Poly (PE) fluorescent dendrimer combined by the SA-PAMAM-SH dendrimer of the same generation and PE fluorescent dyes with different addition amounts, which comprises the following steps:
(1) Preparation of SA-PAMAM dendrimer, 1mL of PAMAM solution with concentration of 1mmol/L was centrifuged at 12000rpm for 20min, the supernatant was aspirated, and after 1mL of 1 XPBS buffer was added for resuspension, the supernatant was aspirated and repeated 3 times at 12000 rpm. The 1 XPBS buffer was 10mmol/L phosphate buffer, pH7.4. 900. Mu.L of MES buffer, 0.05mol/L MES buffer, pH6.8, was added. A solution of 50mg/mL NHS and 10mg/mL EDC was prepared by weighing a certain amount of NHS and EDC, respectively, with pure water. To the PAMAM solution, 10 μl of NHS and EDC solution was added, and after mixing, incubation was performed at room temperature with shaking for 20min. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 2 times. Add 900. Mu.L 1 XPBS buffer for resuspension and 100. Mu.L SA solution, mix upside down and incubate for 2h at room temperature in the dark. Centrifuge at 12000rpm for 20min, suck the supernatant, add 1mL1 XPBS buffer to resuspend, centrifuge at 12000rpm for 20min, suck the supernatant, add 1mL methanol solution to resuspend, and then keep stand for storage.
(2) Preparing SA-PAMAM-SH dendrimer, mixing 1mL of PAMAM-PEG-SH solution with the SA-PAMAM solution in the step (1), adding into a three-necked bottle, adding 16mL of ethylenediamine methanol solution with a dropping funnel according to the ratio of 1:8 of the dendrimer to the ethylenediamine methanol solution, adding a plug, magnetically stirring, incubating for 2h in a water bath, centrifugally washing the reaction product, and naturally drying to obtain the G0.5 generation product. 1mL of the G0.5 generation product is taken and added into a three-necked bottle, 8mL of methyl acrylate methanol solution is added into the three-necked bottle by a dropping funnel according to the ratio of 1:8 of dendrimer to methyl acrylate methanol solution, the mixing ratio of methyl acrylate to methyl acrylate methanol solution is 1:1, a plug is plugged, magnetic stirring and uniform mixing are carried out, water bath incubation is carried out for 2h, the reaction product is centrifugally washed, and natural blow-drying is carried out, thus obtaining the G1.0 generation product. The two reaction steps are alternately repeated to obtain the G2.0 generation product.
(3) The preparation method of the SA-PAMAM-POLY (PE) dendrimer fluorescent dye is characterized by comprising the following steps: the PE fluorescent dye is used for marking the G2.0 generation of other SA-PAMAM-SH dendrimer. Taking 1mL of SA-PAMAM-SH dendrimer solution, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times.
mu.L MES buffer was added. 1mg of N-maleimide was weighed and 100. Mu.LMES buffer was added to prepare a 10mg/mL N-maleimide solution. 50 mu L of N-maleimide solution is added into the SA-PAMAM-SH solution, and after the mixture is mixed evenly in a reversed manner, the mixture is incubated for 20min at room temperature in a dark place. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and then 1mL of 1 XPBS buffer was added for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and the procedure was repeated 2 times.
SA-PAMAM-SH dendrimers and PE fluorescent dye were mixed according to Table 1 and incubated for 30min at room temperature under shaking in the absence of light. After centrifugation at 12000rpm for 20min, the supernatant was aspirated, and after 1mL of 1 XPBS buffer was added to each tube for resuspension, centrifugation at 12000rpm for 20min, the supernatant was aspirated, and after 0.1mL of 1 XPBS was added to each tube for resuspension, the tube was left to stand in a dark place.
TABLE 1 addition ratio of SA-PAMAM-SH to PE fluorescent dye
Experimental example 1
(1) The purpose of the experiment is as follows: is used for detecting the low-abundance marker of the Alzheimer disease blood. The fluorescence detection system described in example 1 was tested for differences from the SA-PE detection system.
(2) Experiment design: samples of Alzheimer's disease plasma (20 each) were collected from a medical university affiliated hospital in Xuzhou, and assayed simultaneously with the SA-PE assay (control) using the fluorescence assay (experimental) described in example 1, and compared for concentration values.
(3) The experimental process comprises the following steps:
dissolving antigen CAL1 and nerve marker antigen diluent with purified water, standing at room temperature for 10 min, and mixing uniformly. The neural marker antigen is diluted in a gradient with a neural marker antigen diluent.
A96-well plate was used, 25. Mu.L of reaction buffer (T-AB) and labeled Detection antibody (Detection) were added to each well, 25. Mu.L of a well-mixed conjugate bead suspension was added to each well, and 25. Mu.L of a sample was added to each well, and well-mixed. The reaction was continued for 1.5 hours at 37℃with shaking (shaking: 700 rpm).
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process, reversely buckling on a low-dust water absorbing paper to absorb residual liquid), adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, absorbing residual liquid by using the low-dust water absorbing paper, and taking the 96-well plate off the magnetic frame.
50. Mu.L of SA-PAMAM-POLY (PE) dendrimer fluorescent dye was added to each well of the experimental group; 50. Mu.L of SA-PE (1.25. Mu.g/mL) was added to each well of the control group, the sealing film was attached, and the mixture was homogenized. The reaction was carried out at 37℃for 0.5 hour with shaking (shaking: 700 rpm)
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process), sucking out the residual liquid by using low-dust paper, adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, sucking out the residual liquid by using low-dust paper, and taking the 96-well plate out of the magnetic frame.
120. Mu.L of the termination detecting solution was added thereto, and the mixture was shaken on a shaker at 700 rpm. The result is read on a multifunctional flow dot matrix instrument Luminex System 200, and the operation of the machine is detailed in the instruction book.
(4) Experimental results: the results of the two fluorescence detection systems for detecting the Alzheimer's disease low-value sample are shown in Table 2.
TABLE 2 mean value comparison (Unit: pg/mL) of three detection results of two fluorescence detection systems for detecting Alzheimer's disease low-value sample
(5) Conclusion of experiment: the SA-PAMAM-POLY (PE) fluorescent detection system detects Alzheimer's disease low-value samples, and compared with the SA-PE fluorescent detection system, the SA-PAMAM-POLY (PE) fluorescent detection system detects, the sensitivity is higher, the low-value stability is better (CV value is lower), and the established reference range of each index and the sample concentration lower than the lowest detection line can be detected.
Experiment 2
(1) The purpose of the experiment is as follows: the different generations of other SA-PAMAM-SH dendrimers described in example 2 were tested in combination with PE fluorescent dyes for the most suitable generation of other SA-PAMAM-POLY (PE) dendrimer fluorescent dyes.
(2) Experiment design: the biomarker antigens were detected in experimental groups 1-10 using the different generation fluorescent substances G0.5-G5.0 described in example 2, and the detected concentration values were compared.
(3) The experimental process comprises the following steps: dissolving antigen CAL1 again with purified water, standing at room temperature for 10 min, and mixing well for use. The neural marker antigen is diluted in a gradient with a neural marker antigen diluent.
A96-well plate was used, 25. Mu.L of reaction buffer (TMAB) and labeled Detection antibody (Detection) were added to each well, 25. Mu.L of well-mixed conjugate bead suspension was added to each well, and 25. Mu.L of sample was added to each well, and well-mixed. The reaction was continued for 1.5 hours at 37℃with shaking (shaking: 700 rpm).
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process, reversely buckling on a low-dust water absorbing paper to absorb residual liquid), adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, absorbing residual liquid by using the low-dust water absorbing paper, and taking the 96-well plate off the magnetic frame.
50. Mu.L of SA-PAMAM-POLY (PE) dendrimer fluorescent dye was added to each well of experimental groups 1-10; and (5) pasting a sealing film, uniformly mixing, pasting the sealing film, and uniformly mixing. The reaction was carried out at 37℃for 0.5 hour with shaking (shaking: 700 rpm)
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process), sucking out the residual liquid by using low-dust paper, adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, sucking out the residual liquid by using low-dust paper, and taking the 96-well plate out of the magnetic frame.
120. Mu.L of the termination detecting solution was added thereto, and the mixture was shaken on a shaker at 700 rpm. The result is read on a multifunctional flow dot matrix instrument Luminex System 200, and the operation of the machine is detailed in the instruction book.
(4) Experimental results: the results of the detection of the neural marker antigen by the fluorescent substances of different generations G0.5-G5.0 are shown in Table 3.
(5) Conclusion of experiment: referring to FIG. 4, the fluorescent substances of the generation G3.0-4.0 have the best effect of detecting the neural marker antigen.
TABLE 3 results of detection of the neural marker antigens by different generations of fluorescent substances (concentration pg/mL, signal value: MFI)
Experiment 3
(1) The purpose of the experiment is as follows: the effect of the different proportions of PE fluorochromes described in example 3 on binding to SA-PAMAM-SH dendrimer fluorochromes was tested.
(2) Experiment design: the different proportions of PE fluorochromes described in example 3 were used in combination with SA-PAMAM-SH dendrimer fluorochromes, and were set as test groups 1-10 for detection of the biomarker antigens, and the concentration values were compared.
(3) The experimental process comprises the following steps: re-dissolving the freeze-dried antigen with purified water, standing for 10 minutes at room temperature, and uniformly mixing for later use. The neural marker antigen is diluted in a gradient with a neural marker calibrator diluent.
A96-well plate was used, 25. Mu.L of reaction buffer (TMAB) and labeled Detection antibody (Detection) were added to each well, 25. Mu.L of well-mixed conjugate bead suspension was added to each well, and 25. Mu.L of sample was added to each well, and well-mixed. The reaction was continued for 1.5 hours at 37℃with shaking (shaking: 700 rpm).
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process, reversely buckling on a low-dust water absorbing paper to absorb residual liquid), adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, absorbing residual liquid by using the low-dust water absorbing paper, and taking the 96-well plate off the magnetic frame.
50 μl of PE-conjugated SA-PAMAM-POLY (PE) dendrimer fluorochromes of different concentrations were added to each well of experimental groups 1-10; and (5) pasting a sealing film, uniformly mixing, pasting the sealing film, and uniformly mixing. The reaction was carried out at 37℃for 0.5 hour with shaking (shaking: 700 rpm)
Directly adding 150 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the liquid (the 96-well plate is always placed on a magnetic frame in the process), sucking out the residual liquid by using low-dust paper, adding 200 mu L of 1 Xwashing liquid, magnetically separating for 2-3 min, pouring out the washing liquid, sucking out the residual liquid by using low-dust paper, and taking the 96-well plate out of the magnetic frame.
120. Mu.L of the termination detecting solution was added thereto, and the mixture was shaken on a shaker at 700 rpm. The result is read on a multifunctional flow dot matrix instrument Luminex System 200, and the operation of the machine is detailed in the instruction book.
(4) Experimental results: the results of detecting the neural marker antigen by the SA-PAMAM-POLY (PE) dendrimer fluorescent dye combined by PE with different concentrations are shown in Table 4.
TABLE 4 results of detection of neural marker antigens by PE-conjugated fluorescent substances in different proportions (concentration pg/mL, signal value: MFI)
(5) Conclusion of experiment: referring to FIG. 5, the PE and SA-PAMAM-SH addition ratio described in example 3 gave the best combination effect.
Claims (10)
1. The preparation method of the fluorescent dendrimer for immunodetection of low-abundance biomarker signal cascade amplification is characterized by comprising the following steps of:
(1) Preparation of SA-PAMAM dendrimer: coupling PAMAM dendrimer with a surface active group of carboxyl modified G1.0 generation with SA, activating PAMAM with N-hydroxysuccinimide NHS solution and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC solution in an acidic environment, adding the activated PAMAM dendrimer into SA solution, mixing and incubating to obtain SA-PAMAM dendrimer;
(2) Preparation of SA-PAMAM-SH dendrimer: mixing the SA-PAMAM dendrimer obtained in the step (1) serving as a center with PAMAM-PEG-SH dendrimer with surface active groups modified by mercapto groups, and then adding a methanol solution of ethylenediamine to perform a phthalamination reaction to generate a G0.5 generation SA-PAMAM-SH dendrimer product; then adding methyl acrylate methanol solution to perform addition reaction to generate SA-PAMAM-SH dendrimer product of G1.0 generation; alternately repeating the two reaction steps to obtain SA-PAMAM-SH dendrimer products with algebraic sequentially increased numbers;
(3) Preparation of SA-PAMAM-POLY (PE) fluorescent dendrimer: and (3) activating the SA-PAMAM-SH dendrimer obtained in the step (2) by using N-maleimide in an acidic environment, and coupling PE to obtain the SA-PAMAM-Poly (PE) fluorescent dendrimer dye.
2. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 1, wherein the method is characterized in that: the molecular weight of the PAMAM dendrimer is 2230.
3. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 1, wherein the method is characterized in that: the molecular weight of the PAMAM-PEG-SH dendrimer is 2000.
4. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 1, wherein the method is characterized in that: the preparation of the SA-PAMAM dendrimer comprises the following steps: taking 1mL of PAMAM solution with the concentration of 1mmol/L, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times; adding 900 mu L of MES buffer solution, weighing NHS and EDC, and preparing 50mg/mL of NHS and 10mg/mL of EDC solution by pure water respectively; adding 10 mu L of NHS and EDC solution into PAMAM solution respectively, mixing thoroughly, incubating at room temperature under dark shaking for 20min, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 2 times; adding 900. Mu.L of 1 XPBS buffer for resuspension, adding 100. Mu.L of SA solution, reversing and mixing uniformly, performing shaking incubation at room temperature for 2h under dark conditions, centrifuging at 12000rpm for 20min, sucking off the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking off the supernatant, adding 1mL of methanol solution for resuspension, and standing at dark conditions.
5. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 1, wherein the method is characterized in that: the preparation of the SA-PAMAM-SH dendrimer comprises the following steps: mixing 1mL of PAMAM-PEG-SH solution with the SA-PAMAM solution in the step (1), adding 16mL of methanol solution of ethylenediamine according to the ratio of 1:8 of SA-PAMAM dendrimer to methanol solution of ethylenediamine, plugging a plug, magnetically stirring and mixing uniformly, incubating in a water bath for 2h, centrifugally washing a reaction product, and naturally drying to obtain a G0.5-generation SA-PAMAM-SH product; taking 1mL of 100.5-generation SA-PAMAM-SH dendrimer, adding 8mL of methyl acrylate methanol solution according to the ratio of 1:8 of the SA-PAMAM-SH dendrimer and the methyl acrylate methanol solution, plugging a plug, magnetically stirring and mixing uniformly, incubating in a water bath for 2h, centrifugally washing a reaction product, and naturally drying to obtain the G1.0-generation SA-PAMAM-SH dendrimer; the reaction steps are alternately repeated to obtain the G2.0 generation SA-PAMAM-SH dendrimer.
6. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 1, wherein the method is characterized in that: the preparation of the SA-PAMAM-Poly (PE) fluorescent dendrimer comprises the following steps:
labeling G2.0 generation SA-PAMAM-SH dendrimer with PE, taking 1mL of SA-PAMAM-SH dendrimer solution, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 3 times;
add 900 μl MES buffer: weighing a certain amount of N-maleimide, adding MES buffer solution to prepare 10mg/mL of N-maleimide solution, adding 50 mu L of N-maleimide solution into SA-PAMAM-SH solution, uniformly mixing, shaking at room temperature and light, incubating for 20min, centrifuging at 12000rpm for 20min, sucking the supernatant, adding 1mL of 1 XPBS buffer solution for resuspension, centrifuging at 12000rpm for 20min, sucking the supernatant, and repeating for 2 times;
after adding 900mL of MES buffer for resuspension, 100. Mu.L of PE was added, the mixture was incubated at room temperature under light-shielding conditions for 30min, centrifuged at 12000rpm for 20min, the supernatant was sucked off, and after adding 1mL of 1 XPBS buffer for resuspension, the supernatant was sucked off, and after adding 1mL of 1 XPBS for resuspension, the mixture was kept at rest under light-shielding conditions.
7. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signal cascade amplification according to claim 4 or 6, wherein the method is characterized in that: the 1 XPBS buffer is 10mmol/L phosphate buffer, and the pH value is 7.4; the MES buffer solution is 0.05mol/L MES buffer solution, and the pH value is 6.8.
8. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 5, wherein the method is characterized by: in the methyl acrylate methanol solution, the mixing ratio of the methyl acrylate to the methanol solution is 1:1.
9. The method for preparing fluorescent dendrimers for immunodetection of low abundance biomarker signaling cascade according to claim 5, wherein the method is characterized by: in the methyl alcohol solution of the ethylenediamine, the mixing ratio of the ethylenediamine to the methyl alcohol solution is 1:1.
10. Use of fluorescent dendrimer SA-PAMAM-Poly (PE) according to any one of claims 1 to 6, characterized in that: the fluorescent dendrimer is used for detecting a nerve marker or a tumor marker.
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