CN111978307B - Detection reagent for fluorescent labeling amino compounds and application of detection reagent in protein fluorescent labeling - Google Patents

Detection reagent for fluorescent labeling amino compounds and application of detection reagent in protein fluorescent labeling Download PDF

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CN111978307B
CN111978307B CN202010780868.3A CN202010780868A CN111978307B CN 111978307 B CN111978307 B CN 111978307B CN 202010780868 A CN202010780868 A CN 202010780868A CN 111978307 B CN111978307 B CN 111978307B
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detection reagent
fluorescent labeling
protein
fluorescein
amino compounds
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CN111978307A (en
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李锦花
马明
冯睿
肖道清
石守江
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NINGBO ACADEMY OF SCIENCE AND TECHNOLOGY FOR INSPECTION AND QUARANTINE
Ningbo Customs Technology Center
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Ningbo Customs Technology Center
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Abstract

The invention belongs to the field of protein fluorescent labeling, and in particular relates to a detection reagent for fluorescent labeling of amino compounds and application of the detection reagent in protein fluorescent labeling, wherein the detection reagent is shown in a structural general formula (1), and R in the general formula (1) represents-OH and-OCH 3 、—OCH 2 CH 3 、—OCH(CH 3 ) 2 、—OC(CH 3 ) 3 . The detection reagent provided by the invention has the advantages of good stability of a marked product, high marking efficiency, high fluorescence quantum yield, short marking time and simple reaction conditions during marking.

Description

Detection reagent for fluorescent labeling amino compounds and application of detection reagent in protein fluorescent labeling
Technical Field
The invention belongs to the field of protein fluorescent labeling, and particularly relates to a detection reagent for fluorescent labeling of amino compounds and application of the detection reagent in protein fluorescent labeling.
Background
Proteins are important components of cells in the body and are the basis of all life. Protein and proteomics research is one of the most popular research directions in the scientific research field, and has an important role in medical science, life science and food safety detection. Detection of proteins is the basis for studying proteins. The detection signals of the protein are mainly ultraviolet absorption from the protein, and some proteins have weaker fluorescence, but the detection signals have weaker intensities, so that the detection sensitivity is lower, and for some trace proteins, effective detection is difficult to realize through the signals of the protein.
To compensate for this, proteins need to be stained or labeled to increase the sensitivity of detection. Common protein staining methods include coomassie brilliant blue staining, silver staining, eosin Y staining, nile red staining, fluorescent staining and the like, which are all realized by physical action between protein and a staining agent, and the sensitivity of the method is difficult to meet the requirement for detecting trace protein.
Today, protein labeling is a relatively sensitive protein detection method, such as radiolabeling, enzyme labeling, fluorescent labeling, etc., wherein fluorescent labeling is receiving a great deal of attention from researchers due to its advantages of high sensitivity, safety, reliability, etc. Fluorescent labeling, i.e., fluorescence derivatization, of proteins is to attach functional groups (e.g., amino, carboxyl, hydroxyl, etc.) of amino acids in the protein to fluorescent groups via covalent bonds, thereby causing fluorescence of the target protein. The effective fluorescent labeling of the protein is the basis of protein immunodetection, protein fluorescence polarization detection and protein chip detection technologies, and is also the premise of separating and detecting the protein by using separation technologies such as high performance liquid chromatography, capillary electrophoresis, gel electrophoresis and the like.
Common protein fluorescent labeling reagents are fluorescein isothiocyanate, phthalaldehyde, naphthaldehyde, tetramethylrhodamine isothiocyanate, dansyl chloride and the like. However, the existing protein fluorescent labeling reagents have the following disadvantages: 1. the stability of the marked product is poor; 2. the marking efficiency is low; 3. the fluorescence quantum yield is low; 4. it is necessary to use an organic solvent that can denature the protein, so that the time for fluorescent labeling of the protein is increased, and the reaction conditions are severe at the time of labeling.
Disclosure of Invention
The invention provides a detection reagent for fluorescent labeling of amino compounds, which has the advantages of good stability of labeled products, high labeling efficiency, high fluorescence quantum yield, short labeling time and simple reaction conditions during labeling.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a detection reagent for fluorescent labeling amino compounds is shown in a structural general formula (1),
in the general formula (1), R represents-OH, -OCH 3 、—OCH 2 CH 3 、—OCH(CH 3 ) 2 、—OC(CH 3 ) 3 ；
As preferable:
when R represents-OCH 3 、—OH、—OCH 2 CH 3 、—OCH(CH 3 ) 2 、—OC(CH 3 ) 3 When the fluorescent detection reagent product is used, the yield of the fluorescent detection reagent product can reach 92%, 68%, 86%, 80% and 73%.
Preferably, the detection reagent includes a compound represented by the following formula (2):
preferably, the detection reagent includes a compound represented by the following formula (3):
preferably, the detection reagent includes a compound represented by the following formula (4):
preferably, the detection reagent includes a compound represented by the following formula (5):
preferably, the detection reagent includes a compound represented by the following formula (6):
the yield of the formula (2) can reach 92%, and the yield of the formula (3) is 68%; the yield of formula (4) was 86%, the yield of formula (5) was 80%, and the yield of formula (6) was 73%.
The detection reagent provided by the invention can be applied to protein fluorescent labeling.
The beneficial effects of the invention are as follows:
the invention designs and synthesizes a protein fluorescent labeling reagent by taking fluorescein with higher fluorescence quantum yield as a fluorophore and N-hydroxysuccinimide active ester as a reaction group through simple 2-step reaction, establishes a method for successfully labeling protein by the synthesized fluorescent reagent, and designs a kit for fluorescent labeling of protein. The designed protein fluorescent labeling reagent has the advantages of simple synthetic route, mild synthetic condition, higher yield, higher protein labeling efficiency, higher labeling speed compared with the existing fluorescent labeling reagent, capability of being labeled in aqueous solution and at room temperature, no protein denaturation and the like.
Drawings
FIG. 1 is a scheme showing the synthesis of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein from example 1;
FIG. 2 is a scheme showing the synthesis of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -ethoxycarbonyl) fluorescein from example 3;
FIG. 3 is a fluorescence spectrum of hydrolysis and derivatives of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein in test example 1;
FIG. 4 is a schematic illustration of fluorescence intensity measurements;
FIG. 5 is a table of fluorescence quantum yield statistics for fluorescently labeled products;
FIG. 6 is a table of fluorescence quantum yield statistics in different media;
FIG. 7 is a schematic illustration of the amount of fluorescent label used;
FIG. 8 is an electrophoresis spectrum of an antigen-antibody complex;
fig. 9 is a standard curve.
Detailed Description
Example 1
A detection reagent for fluorescent labeling amino compounds has the following structural formula:
the preparation method of the detection reagent comprises the following steps:
(1) Synthesis of methyl fluorescein:
(1.1) fluorescein (3.32 g,10 mmol) was dissolved in 10ml methanol, added dropwise to 2.5ml concentrated sulfuric acid, heated under reflux for 14 hours, and then usedAnd (5) drying the molecular sieve. After the above reaction was stopped, 1g of ice, 10g of NaHCO3 powder was added, filtered, and washed with a small amount of water, and the obtained red solid was put into 50ml of NaHCO3 aqueous solution having a mass fraction of 2%, filtered again, and washed again with water. The water washing process was repeated three times.
After the completion of the washing with water (1.2), a 1% acetic acid solution was added, followed by filtration and washing with water.
(1.3) the solid obtained by the reaction (red solid) was washed with water and then dried to obtain 3.4g (98%) of fluorescein methyl ester as a red solid, i.e., fluorescein methyl ester.
(2) Synthesis of 6-oxo-maleic acid monoesterified-9- (2' -methoxycarbonyl) fluorescein:
(2.1) the methyl fluorescein ester (2.7 g, about 7.8 mmol) prepared above was dissolved in 10ml of anhydrous DMF, and 0.9g (about 9 mmol) of maleic anhydride was added thereto, and the reaction was stopped by refluxing under heating (about 75 to 80 ℃ C.) for 10 hours. About 100ml NaHCO 2% by mass was added to the product 3 After the reaction was completed (10 min), the system was adjusted to acidity with 0.1M hydrochloric acid, and filtered. The obtained reddish brown solid is washed 3 times by acetic acid with the mass fraction of 1%, and is dried in vacuum at 40 ℃ for more than 24 hours.
(3) Synthesis of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein:
(3.1) 6-oxo-maleic acid monoesterified-9- (2' -methoxycarbonyl) fluorescein 0.88g (2 mmol), N-hydroxysuccinimide 0.276g (2.4 mmol), dicyclohexylcarbodiimide 0.5g (2.4 mmol) were taken in 3ml anhydrous DMF and stirred under closed conditions at room temperature (20 ℃ C.) overnight. The reaction was frozen at-20 ℃ for 3h, the white precipitate was removed by rapid filtration, rinsed with a small amount of anhydrous acetonitrile, added with anhydrous diethyl ether and petroleum ether, vigorously stirred under nitrogen protection for 15min, the precipitate was collected, rinsed with diethyl ether, dried in vacuo at 40 ℃ for 24h to give a red orange 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein powder.
The synthetic route is shown in fig. 1.
Example 2
A detection reagent for fluorescent labeling amino compounds has the following structural formula:
the preparation method is substantially the same as that of the detection reagent in example 1, except that the synthesis is directly performed from step (2) without performing step (1).
Example 3
A detection reagent for fluorescent labeling amino compounds has the following structural formula:
the preparation method is the same as that of the detection reagent in example 1, except that ethanol is selected as the solvent in step (1.1).
Example 4
A detection reagent for fluorescent labeling amino compounds has the following structural formula:
the preparation method was the same as that of the detection reagent in example 1, except that isopropanol was selected as the solvent in step (1.1).
Example 5
A detection reagent for fluorescent labeling amino compounds has the following structural formula:
the preparation method was the same as that of the detection reagent in example 1, except that t-butanol was selected as the solvent in step (1.1).
Test example 1
To test the properties of the test reagents provided by example 1, the following tests were performed:
1. preparation of reagent hydrolysis products: take l ml 5mmol l -1 Is placed in a 10ml volumetric flask, and a proper amount of pH 8.5H is added 3 BO 3 -Na 2 B 4 O 7 The buffer solution was then diluted to the scale, shaken well and hydrolyzed at room temperature for 20 minutes.
2. Reagent and amino group combinationPreparation of derivative products of the materials: the appropriate excess of methylamine or glycine solution and 1ml of 5mmol L were taken -1 In a 10ml volumetric flask, 1.5ml of pH 8.5H was added 3 BO 3 -Na 2 B 4 O 7 Buffer, then diluted to scale, shaken well and reacted at room temperature for 20 minutes. The complete reaction of the reagent was known by chromatographic detection.
3. Determination of fluorescence Spectroscopy
The hydrolysate or derivative prepared above is diluted with water to the desired concentration. The fluorescence photometry was performed on a Hitachi F4600 fluorescence spectrophotometer, a 1cm×1cm quartz cuvette, and the slit of the fluorescence spectrophotometer was adjusted depending on the measurement object.
The fluorescence spectrum of the hydrolysis and derivative of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein is shown in figure 3.
In fig. 3: 1: excitation spectrum diagram of 6-oxygen- (N-hydroxysuccinimide maleic monoester) -9- (2' -methoxycarbonyl) fluorescein hydrolysate; 1': an emission spectrum diagram of a 6-oxo- (N-hydroxysuccinimide maleic acid monoester) -9- (2' -methoxycarbonyl) fluorescein hydrolysate; 2: excitation spectrum diagram of 6-oxygen- (N-hydroxysuccinimide maleic monoester) -9- (2' -methoxycarbonyl) fluorescein labeled methylamine; 2': an emission spectrum diagram of 6-oxygen- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein labeled methylamine; 3: excitation spectrum diagram of 6-oxygen- (N-hydroxysuccinimide maleic acid monoester) -9- (2' -methoxycarbonyl) fluorescein anhydrous acetonitrile solution; 3': emission spectrum of 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein in anhydrous acetonitrile.
4. Determination of fluorescence stability
The concentration of the reagent was 5X 10 -8 mol·l -1 . The cuvette containing the sample was irradiated with a 200W incandescent bulb (general company, china) at 10cm and cooled with water at room temperature. Fluorescence intensity measurement was performed after irradiation for a certain period of time. The fluorescence intensity is shown in FIG. 4.
In fig. 4: stability of fluorescence intensity (fluorescein (■), fluorescein isothiocyanate (.: fluorescein isothiocyanate derived methylamine (+); 6-oxo- (N-hydroxysuccinimide maleate) -9- (2 '-methoxycarbonyl) fluorescein (×); 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein derived methylamine (.: c.)).
5. Determination of Quantum yield
The invention uses 0.1mol L of fluorescein -1 The NaOH solution is the standard, under which conditions the fluorescence quantum yield of fluorescein is 0.90. The calculation is performed according to the following formula:
Φu＝Φs·Fu·As/Fs·Au,
wherein Φu, Φs are respectively the fluorescence quantum yields of the substance to be detected and the reference substance; au and As are respectively the fluorescence peak areas of the substance to be detected and the reference substance; fu, fs are the absorbance of the test and reference substances, respectively, at that wavelength.
The fluorescence quantum yield results at different pH are shown in FIG. 5.
6. 6-oxo- (N-succinimidyl maleate) -9- (2' -methoxycarbonyl) fluorescein labeled protein
6.1 instruments
Beckman-Coulter P/ACE MDQ capillary electrophoresis apparatus, and 488 argon ion laser induced fluorescence detector of Beckman-Coulter company, excitation wavelength/emission wavelength: 488nm/520nm. The capillary tube was a 60.2cm (effective length 50 cm) by 75 μm (inner diameter) quartz capillary tube (Hebei Yongnian fiber works). Separation temperature: 25 ℃; separation voltage: 28kV; sample injection mode: pressure injection (0.5 psi,34.5 mbar); sample introduction time: 5s; the sample loading was about 25nL.
The new capillary was rinsed with methanol, water, 0.1mol/L HCl, water, 0.1mol/L NaOH, 5, 10,5, 30,5min, respectively. The two runs were rinsed with 0.1mol/L HCl, water, 0.1mol/L LNaOH and running buffer for 2min each.
The pH was determined by a Mettler-Toledo SK-40pH meter (Metrele-tolidol, shanghai).
6.2 reagents
Rabbit anti-bovine serum albumin polyclonal antibodies were purchased from beijing boaosen biotechnology limited.
Bovine serum albumin was purchased from Shanghai Boaoko biotechnology Co.
Sephadex G-25 was prepared by Amersham, and the new Sephadex was used after swelling in boiling water for 2 hours and filling the column (20 cm. Times.1.5 cm) and equilibration with a physiological buffer solution of phosphoric acid (pH 7.4). SSMF was synthesized by the owner. All other reagents used in the experiments were analytically pure. The water used in the experiments was ultrapure water (Millipore, bedford, mass., USA) prepared by Milli-Q system.
H 3 BO 3 -Na 2 B 4 O 7 Buffer solution is prepared from 0.05mol/L Na 2 B 4 O 7 Solution and 0.2mol/L H 3 BO 3 The solution mixture was adjusted to the desired pH on a pH meter. Boric acid buffer solution was prepared from an amount of boric acid and adjusted to the desired pH with 1mol/L NaOH. The running buffer was filtered through a 0.22 μm filter before use.
The Phosphate Buffered Saline (PBS) consists of 8.0g/L NaCl,0.2g/L KCl,2.88g/L Na 2 HPO 4 ,0.2g/L KH 2 PO 4 Formulated to pH 7.4 with 0.1mol/L HCl.
6.3 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein labeled BSA
Bovine Serum Albumin (BSA) with H at pH 8.5 3 BO 3 -Na 2 B 4 O 7 The buffer solution was prepared as a 10mg/mL solution and was refrigerated in a refrigerator at 4 ℃.
Into a 0.5mL centrifuge tube containing 15. Mu.L of a 20 mmol/L6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein solution, 100. Mu.L of 10mg/mL BSA was added, diluted to 200. Mu.L with water, mixed and shaken well, and reacted at room temperature for 45min. The reaction mixture was then passed through a sephadex column equilibrated with PBS in advance to remove excess small molecule material, and the first eluted bright yellow band (i.e., 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein labeled BSA) was collected and diluted to 2.0mL with PBS solution, stored in a low temperature refrigerator at-30℃and diluted to the desired concentration with PBS when used. The concentration of labeled BSA was calculated as the concentration of BSA before unlabeling.
With H at pH 8.5 3 BO 3 -Na 2 B 4 O 7 As a medium for the derivatization reaction, 100. Mu.L of 10mg/mL BSA was derivatized, and SSMF concentration was examined at 5X 10 -4 mol/L-3×10 -3 In the mol/L range. As shown in FIG. 4, when the SSMF concentration is less than 1.5X10 -3 At the time of mol/L, the peak area of SSMF-BSA becomes larger rapidly with the increase of SSMF concentration, and the SSMF concentration is larger than 1.5X10 -3 At mol/L, the peak area of SSMF-BSA is not substantially increased, so 1.5X10 - 3 mol/L is used as the optimal reagent dosage.
100. Mu.L of 10mg/mL BSA was reacted with SSMF under the above conditions for 15 to 75 minutes, and the change in the peak area of SSMF-BSA was measured. The experiment finds that: after 45min the peak area of the SSMF-labelled BSA reached a maximum and stable, so the time of the derivatization reaction was chosen to be 45min.
6.4 capillary electrophoresis competitive immunoassay
6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein-BSA (Ag) * ) AntiBSA (Ab), unlabeled BSA, were diluted to a certain concentration with PBS. Into 200. Mu.L tubes, 10. Mu.L of Ag was added, respectively * (0.02 mg/mL), 15. Mu.L Ab (0.1 mg/mL) and a certain amount of unlabeled BSA were formulated into 30. Mu.L with an appropriate amount of PBS, sonicated for 1min, incubated at room temperature (25 ℃) for 10min, and then injected into the CE-LIF system for analysis.
The fluorescent antigen-antibody complex is obtained by reacting SSMF-BSA with anti-BSA. As shown in FIG. 6, when no antibody was added, SSMF-BSA was unimodal (curve 1 in FIG. 6), and the migration time was about 4.7min. After addition of the antibody, a new peak (curves 2-4 in FIG. 5) appears on the electropherogram, i.e., the peak of the SSMF-labeled fluorescent antigen-antibody complex, which has a shorter migration time than SSMF-BSA, by about 3.7min. As the amount of antibody added increases, the peak height of the complex gradually increases, while the peak height of the SSMF-BSA gradually decreases. These fully confirm the formation of antigen-antibody complexes, and in subsequent experiments we selected an SSMF-BSA concentration of 6.8. Mu.g/mL, with an amount of antibody added of 50. Mu.g/mL.
6.5 regression equation, linear Range and detection Limit
Different amounts of unlabeled BSA were added, respectively, as a ratio of the peak area of free SSMF-BSA to the peak area of BSA antigen-antibody complex (A F /A C ) Linear regression analysis was performed on the added BSA concentration, see fig. 7. The linear range is 5-200 μg/mL, and the linear regression equation is Y=0.1045X+3.9201 (Y: A) F /A C The method comprises the steps of carrying out a first treatment on the surface of the X is BSA concentration, μg/mL; the correlation coefficient is 0.9983). The relative standard deviations of time and peak area ratio were 1.54% and 3.35%, respectively. The detection limit was 0.1. Mu.g/mL.
Test example 2
7.1 instruments and reagents
The pH value was determined by SK-40pH meter (Metrele-tolidol, switzerland)
SSMF, homemade;
acetonitrile and methanol are both chromatographic purity and purchased from the technology of dima; boric acid and borax are analytically pure and purchased from Chinese medicine groups.
The ultra-pure water is ultra-pure water treated by Milli-Q.
7.2 preparation of Anhydrous methanol
Taking 100ml of methanol in a large flask, adding 5g of magnesium strips, heating and refluxing, adding 400ml of methanol after magnesium is completely dissolved, refluxing for 5-6h, and distilling the methanol at 80-100 ℃ to obtain anhydrous methanol. The water content of the distilled methanol can be measured by the karl fischer method if necessary.
7.3 preparation of Anhydrous acetonitrile
Taking 500ml of acetonitrile, adding 100g of anhydrous calcium chloride, drying for 24 hours, filtering, adding 2.5-5g of phosphorus pentoxide, heating, refluxing for 10 hours, and distilling at normal pressure. Repeating the operation for 3 times to obtain the anhydrous acetonitrile. The water content of the distilled methanol can be measured by the karl fischer method if necessary.
7.4 preparation of buffer solution
H 3 BO 3 -Na 2 B 4 O 7 Buffer solution is prepared from 0.05mol/L Na 2 B 4 O 7 Solution and 0.2mol/L H 3 BO 3 The solution mixture was adjusted to the desired pH on a pH meter.
7.5 selection of SSMF physical states
N-hydroxysuccinimide active ester is a reagent that can undergo a rapid nucleophilic reaction with an amino compound and also a reagent that can undergo a nucleophilic reaction with water, and thus, if a long-term storage of N-hydroxysuccinimide active ester reagent cartridge is desired without hydrolysis, water blocking is the most important factor. Therefore, in order to better isolate the influence of water, two measures are adopted, namely, after the preparation of the SSMF is finished, a vacuum drying method is adopted to ensure that the moisture in the SSMF is completely vacuumized, and the SSMF is packaged in a sealed brown reagent bottle with a silica gel plug, so that the infiltration of water vapor can be avoided, and the brown color of the reagent bottle can be used to ensure that the reagent cannot be photobleached.
7.6 selection of solvent
SSMF is soluble in methanol or acetonitrile, however, since methanol contains a nucleophilic group, it has been reported that N-hydroxysuccinimide active ester is also capable of nucleophilic reaction with hydroxyl groups, and therefore we choose anhydrous acetonitrile as solvent.
7.7 selection of buffer solution
In the previous studies, the SSMF-derived fatty amines, amino acids, proteins all occurred in alkaline environment, and the buffer solution system generally used was boric acid-borax system, at 50mm concentration and ph 8.5, so the buffer solution was chosen as the buffer solution for the kit.
7.8 methods of Forming and Using the kits
From the foregoing, it can be seen that the kit consists essentially of three parts: a-reagent (SSMF crystals), B-solvent (anhydrous acetonitrile) and C-reaction system (boric acid-borax buffer solution, pH 8.5, 50 mM).
The using method comprises the following steps: adding proper amount of B into the bottle A, and shaking until no insoluble matters exist. The sample to be tested is a self-made sample, dissolved by a proper amount of C, and the solution in the bottle A is added into the object to be tested.
7.9 stability of the kit
A batch of kits are prepared, and are respectively placed in a refrigerator at 4 ℃ for 1 month, 3 months, 6 months and 10 months, and then the amino acid and the protein are marked and tested, so that the results show that the kits still have higher reactivity after being placed for 10 months, and the peak area of the derivatives is not obviously changed. The kit is stable for a long time.
If mass production is possible in the future, SSMF and anhydrous acetonitrile can be stored in a more sealed ampoule, with a longer shelf life being expected.
SSMF means 6-oxo- (N-hydroxysuccinimide maleate) -9- (2' -methoxycarbonyl) fluorescein.

Claims (6)

1. A detection reagent for fluorescent labeling amino compounds is characterized in that the detection reagent is shown in a structural general formula (1),
in the general formula (1), R represents-OH, -OCH 3 、—OCH 2 CH 3 、—OCH(CH 3 ) 2 、—OC(CH 3 ) 3 。
2. The detection reagent for fluorescent-labeled amino compounds according to claim 1, characterized by the following formula (2):
3. the detection reagent for fluorescent-labeled amino compounds according to claim 1, characterized by the following formula (3):
4. the detection reagent for fluorescent-labeled amino compounds according to claim 1, characterized by the following formula (4):
5. the detection reagent for fluorescent-labeled amino compounds according to claim 1, characterized by the following formula (5):
6. the detection reagent for fluorescent-labeled amino compounds according to claim 1, characterized by the following formula (6):
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN101059507A (en) * 2006-04-18 2007-10-24 中国科学院大连化学物理研究所 Use of BODIPY analog fluorescent reagent in biological large molecule marking
CN106645745A (en) * 2016-10-19 2017-05-10 山东大学齐鲁医院 Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101059507A (en) * 2006-04-18 2007-10-24 中国科学院大连化学物理研究所 Use of BODIPY analog fluorescent reagent in biological large molecule marking
CN106645745A (en) * 2016-10-19 2017-05-10 山东大学齐鲁医院 Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof

Non-Patent Citations (1)

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Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells;Flebus, Luca;International Journal of Pharmaceutics;第478卷(第1期);278-287 *

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