CN112007620B - Preparation method of streptavidin magnetic microsphere - Google Patents
Preparation method of streptavidin magnetic microsphere Download PDFInfo
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- CN112007620B CN112007620B CN201910466538.4A CN201910466538A CN112007620B CN 112007620 B CN112007620 B CN 112007620B CN 201910466538 A CN201910466538 A CN 201910466538A CN 112007620 B CN112007620 B CN 112007620B
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- streptavidin
- carboxyl
- microspheres
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- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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Abstract
The invention discloses a preparation method of streptavidin magnetic microspheres, which comprises the following preparation steps: 1) Preparing carboxyl magnetic microspheres; 2) Preparing the carboxyl magnetic microsphere by activating and coupling streptavidin to obtain the streptavidin magnetic microsphere; thus, the streptavidin magnetic microsphere with good biological activity, high sensitivity and low non-specific adsorption for chemiluminescent immunoassay can be obtained.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a preparation method of streptavidin magnetic microspheres.
Background
Immunodiagnosis (immunodiagnosis) is the use of immunological theory, techniques and methods to diagnose various diseases and to determine immune status. The immunodiagnosis reagent has the most variety in the diagnosis kit, is widely applied to hospitals, blood stations and physical examination centers, and is mainly used for hepatitis detection, venereal disease detection, tumor detection, pregnancy detection and the like. Wherein the immunodiagnosis comprises radioimmunoassay, enzyme-linked immunoassay, chemiluminescence, etc.
The ELISA reagent has low cost, but has long reaction time and complicated steps, and is not beneficial to large-scale use. The radioimmunity has high sensitivity and strong specificity, but has the problems of radioactive rays, pollution and the like. The chemiluminescent reagent has the advantages of sensitivity, rapidness, stability, strong selectivity, good reproducibility, easy operation and flexible and various methods, and is clinically used in a large scale nowadays. Therefore, a preparation method of streptavidin magnetic microspheres for chemiluminescent immunoassay has the advantages of good biological activity, high sensitivity and low non-specific adsorption.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of streptavidin magnetic microspheres with good biological activity, high sensitivity and low non-specific adsorption for chemiluminescent immunoassay.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the preparation method of the streptavidin magnetic microsphere is characterized by comprising the following preparation steps:
1) Preparing carboxyl magnetic microspheres;
2) The carboxyl magnetic microsphere is prepared by activating and coupling streptavidin to obtain the streptavidin magnetic microsphere.
The preparation method of the streptavidin magnetic microsphere for chemiluminescent immunoassay provided by the invention has the advantages of good biological activity, high sensitivity and low non-specific adsorption.
On the basis of the technical scheme, the following improvement can be made:
as a preferred embodiment, the preparation of the carboxyl magnetic microsphere comprises the following steps:
1) By FeCl 3 ·6H 2 O is prepared into Fe 3 O 4 A magnetic core;
2) By Fe 3 O 4 Is magnetic core, siO 2 Is taken as a shell to prepare Fe 3 O 4 @SiO 2 Magnetic microspheres;
3) Adding MPS, which is modified polystyrene, and Fe 3 O 4 Is magnetic core, MPS and SiO 2 The polymer of (2) is taken as a shell to prepare Fe 3 O 4 @SiO 2 -MPS magnetic microspheres;
4) By Fe 3 O 4 @SiO 2 -MPS as magnetic core, GMA and MBA polymer as shell, GMA as glycidyl methacrylate, MBA as 2-mercaptobenzoic acid, and Fe 3 O 4 @SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S)；
5) Adding sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride, and reacting to obtain Fe 3 O 4 @SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S) -COOH, namely the carboxyl magnetic microsphere.
As a preferred scheme, the preparation step of the carboxyl magnetic microsphere further comprises the following preparation steps of separating the carboxyl magnetic microsphere through a magnetic separator, repeatedly washing the carboxyl magnetic microsphere for at least 5 times by purified water, storing the carboxyl magnetic microsphere in the purified water, checking the quality of the carboxyl magnetic microsphere to be qualified, and putting the carboxyl magnetic microsphere into the next step to prepare the streptavidin magnetic microsphere.
Preferably, the particle size of the carboxyl magnetic microsphere is 250-300nm.
As a preferred embodiment, the preparation of the streptavidin magnetic microsphere comprises the following steps:
1) Washing the carboxyl magnetic microspheres by an activation buffer solution;
2) Adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxy thiosuccinimide for 13-17min;
3) Adding streptavidin solution to react for 1.5-2.5h;
4) Blocking for 1.5-2.5 hours by using bovine serum albumin solution, washing and preserving to prepare the streptavidin magnetic microsphere.
As a preferred embodiment, the carbodiimide concentration in step 2) is 15-25mg/ml; the concentration of N-hydroxysulfosuccinimide is 15-25mg/ml.
As a preferred embodiment, the concentration of the bovine serum albumin solution in step 4) is between 0.5mg/ml and 1.0mg/ml.
As a preferred embodiment, the activation buffer solution comprises: 0.025% -0.075% polysorbate-20 and 0.05M-0.1M morpholinoethanesulfonic acid.
Preferably, the pH of the activation buffer is between 5.0 and 6.0.
As a preferred embodiment, streptavidin is 127AA core structure, 4 biotin molecules are combined per molecule SA, and the international specific activity is 12-15U/mg.
A method for preparing streptavidin magnetic microsphere includes preparing carboxyl magnetic microsphere with short magnetic response time, long suspension time, uniform particle size and high enough ligand content, and activating and coupling SA protein (streptavidin) to form streptavidin magnetic microsphere.
The preparation method of carboxyl magnetic microsphere with particle size of 300nm comprises the following steps of firstly using FeCl3.6H 2 Preparation of Fe from O 3 O 4 A magnetic core, then coating the magnetic core with SiO 2 Form Fe 3 O 4 @SiO 2 Adding MPS to react to form Fe 3 O 4 @SiO 2 -MPS, followed by coating GMA, MBA to form Fe 3 O 4 @SiO 2 -MPS@P (GMA-MBA), and finally adding sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride to form Fe 3 O 4 @SiO 2 -MPS@P (GMA-MBA) -COOH, i.e. carboxyl magnetic material used in the present inventionAnd (3) microspheres. And after the reaction is finished, removing the reaction device, separating the carboxyl magnetic microspheres by using a magnetic separator, repeatedly washing the carboxyl magnetic microspheres for at least 5 times by using purified water, finally storing the carboxyl magnetic microspheres in the purified water, and testing the mass concentration. And after the concentration of the carboxyl magnetic microspheres is calibrated, taking a carboxyl magnetic micro-racket electron microscope to see the coating effect of the polymer, the particle size uniformity degree, and testing whether residual magnetism exists or not. And testing the magnetic attraction time and the suspension time. And after all quality tests reach the standard, putting into the next step for use.
The carboxyl magnetic microsphere is activated by EDC/Sulfo-NHS water phase activation method. EDC is a water-soluble carbodiimide that is used as an activating reagent for carboxyl groups in amide synthesis and also for activating phosphate groups, cross-linking of proteins with nucleic acids and the preparation of immunoassays. The pH range is 4.0-6.0 when in use, and the coupling efficiency is improved by combining N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS). NHS is an additive in improving amidation and peptide coupling reactions for activating carbonyl groups upon amide bond formation. Sulfo-NHS is a derivative of NHS, and is more water soluble than NHS, and moreover, has a negative charge and does not readily cause polymerization of a linker (e.g., a protein). The reaction can select NHS or Sulfo-NHS, and the Sulfo-NHS has better effect. The preparation method comprises washing 10mg/mL carboxyl magnetic microsphere with MEST buffer (pH 5.0-6.0), adding half volume of 20mg/mL EDC in MEST and 20mg/mL Sulfo-NHS in MEST, activating for 15min, adding 1.0mg/mL streptavidin solution, reacting for two hours, blocking with 1% BSA (bovine serum albumin) solution for two hours, and washing and preserving to obtain streptavidin magnetic microsphere.
The streptavidin is a homotetrameric protein secreted by Streptomyces avermitilis (Streptomyces avidinii). The product has high affinity with biotin, the 127AA optimal core structure, 4 biotin molecules are combined per molecule SA, and the specific activity is 12-15U/mg. SA is more specific than Avidin (AV), and has a half-life of up to 6 hours after binding to biotin, whereas AV has a half-life of only 1 hour. Since SA contains no sugar groups and the isoelectric point is close to neutral, SA has a lower non-specific background than AV in detection applications. The molecular weight of the streptavidin used is about 60kDa, and the purity of the product is more than or equal to 95 percent.
The preparation method of the streptavidin magnetic microsphere comprises the following preparation steps:
1) Preparation method of 300nm carboxyl magnetic microsphere
Firstly, the particle size of the magnetic microsphere is selected, the particle size of the common immunomagnetic microsphere in the market is generally larger than or equal to 1 mu m, the magnetic microsphere with larger particle size has the common problems of short suspension time, adverse reaction and good suspension performance of the 300nm magnetic microsphere after test, and the suspension time is more than three hours. Secondly, siO is coated by a magnetic core 2 When the temperature is controlled, in the reaction process, the ultrasonic instrument is changed with water every 30-40min to prevent the reaction temperature from being higher than 50 ℃, the magnetic core oxidation is easy to be caused by the overhigh reaction temperature, the magnetism of the magnetic microsphere can be reduced by the magnetic core oxidation, and the magnetic microsphere with weaker magnetism can slowly run off in the magnetic attraction process, so that the use standard can not be met. In addition, the 300nm carboxyl magnetic microsphere is coated with two layers of polymers in the preparation process, so that a net structure can be formed, magnetic core leakage is reduced, and the magnetic microsphere is stronger in acid resistance, so that the magnetic microsphere can be used under an acid condition without damaging the structure of the magnetic microsphere. Finally, it is very important that the polymer added in the preparation process of the magnetic microsphere has various types, and is easy to form by-products which are difficult to clean, and the performance of the magnetic microsphere is directly affected, so that the adding amount, concentration and proportion of GMA (glycidyl methacrylate), MBA (2-mercaptobenzoic acid) and AIBN (azobisisobutyronitrile) are strictly controlled when the polymer layer is coated, such as 3g Fe 3 O 4 @SiO 2 The MPS magnetic microsphere reaction system is 9mL GMA, 9.0g MBA and 360mg AIBN, and the proportion is amplified when the preparation amount of the magnetic microsphere is increased. And the temperature of the step is also very critical, and the temperature is strictly controlled. After the deoxidation and stirring time is over, an oil bath switch and a cooling circulating water switch are turned on, the temperature is raised to 75 ℃, the consistency of the self-contained display temperature and the actual temperature of the instrument is required to be paid attention to, and if necessary, the instrument is required to be automatically detected and verified by a thermometer. Ensure the whole system to be heated uniformly and ensure the preparation of grainsThe magnetic microsphere has uniform diameter and no byproducts.
2) Carboxyl magnetic microsphere activation mode selection
NHS is an additive in improving amidation and peptide coupling reactions for activating carbonyl groups upon amide bond formation. Sulfo-NHS is a sulfonated product of NHS, which is more water soluble than NHS, and additionally has a negative charge that does not readily cause polymerization of a linker (e.g., a protein). And not only NHS can be selected in the activation mode, but also organic activation and aqueous phase activation. The DMF (N, N-dimethylformamide) is selected as a solvent for organic activation, and is strictly dehydrated before use, and the DMF (N, N-dimethylformamide) is also operated to prevent moisture from entering as quickly as possible in the activation process, so that the intermediate product is hydrolyzed. By repeated experiments, the activation mode and the activation condition are compared, and an optimal activation coupling mode, namely a method of activating by EDC/Sulfo-NHS water phase, is found. Firstly, DMF (N, N-dimethylformamide), DMAC (dimethylacetamide) and other organic solvents with pungent smell and slight toxicity are avoided in the experimental process, so that the method is more friendly to the health of operators; secondly, the water phase activation energy greatly improves the amount of carboxyl magnetic microsphere coating protein, so that the immune magnetic microsphere loading is obviously improved; furthermore, the one-step water phase activation step is simple and easy to operate, the preparation of intermediate product NHS magnetic microspheres is avoided, and the immune magnetic microspheres are directly prepared; in addition, the phenomena of magnetic microsphere adhesion, agglomeration and the like can not occur in the activation process, and the physical and chemical properties of the magnetic microspheres are well maintained.
3) Selection of streptavidin
The Biotin-Avidin System (BAS) is a novel biological reaction amplification System, which can be combined with various markers which have been successfully studied at present. The high affinity firm binding between biotin and avidin and the multi-stage amplification effect make BAS (bovine serum albumin) immunolabeling and related tracer analysis more sensitive. It is widely used in qualitative and quantitative detection and positioning observation of trace antigen and antibody. Streptavidin (SA) is a protein secreted by Streptomyces streptomyces avidinii and has a molecular weight of 65kD. Streptavidin molecules consist of 4 identical peptide chains, each of which is capable of binding to a biotin and does not carry any sugar groups, so that, like avidin, a streptavidin molecule is also capable of binding to 4 biotin molecules, with an affinity constant (K) of 1015mol/L. The application range of streptavidin is wider than that of avidin. There are many types of streptavidin on the market today, with wild type and engineered ones. The wild streptavidin secreted by the streptomyces avermitilis (Streptomyces avidinii) is selected, and is subjected to purity test, concentration test and specific activity test before use, and is put into use after all the streptavidin meets the standards.
The invention takes 300nm carboxyl magnetic microsphere as raw material, and selects proper streptavidin for coupling to form the streptavidin magnetic microsphere. The magnetic response time of the streptavidin magnetic microsphere is less than or equal to 20s; the suspension time is more than or equal to 40min; the binding capacity of the free biotin is more than or equal to 1000pmol/mg; the binding capacity of Biotin-IgG is more than or equal to 20ug/mg; biotin-Probe binding capacity is not less than 450pmol/mg. In the chemiluminescence test, the standard curve has good linearity, R2 is more than or equal to 0.99, S1/S0 is more than 2.6, S7/S0 is more than 500, and CV value is less than or equal to 10% after the same sample is detected for 10 times.
Drawings
FIG. 1 is a diagram for a curve of a streptavidin magnetic microsphere MYO light-emitting cursor provided by an embodiment of the invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, the reagents used in the following examples were all commercially available from regular sources.
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
In order to achieve the aim of the invention, the invention provides a preparation method of streptavidin magnetic microspheres, which is characterized by comprising the following preparation steps:
1) Preparing carboxyl magnetic microspheres;
2) The carboxyl magnetic microsphere is prepared by activating and coupling streptavidin to obtain the streptavidin magnetic microsphere.
The preparation method of the streptavidin magnetic microsphere for chemiluminescent immunoassay provided by the invention has the advantages of good biological activity, high sensitivity and low non-specific adsorption.
On the basis of the technical scheme, the following improvement can be made:
in some embodiments, preparing the carboxyl magnetic microsphere comprises the steps of:
1) By FeCl 3 ·6H 2 O is prepared into Fe 3 O 4 A magnetic core;
2) By Fe 3 O 4 Is magnetic core, siO 2 Is taken as a shell to prepare Fe 3 O 4 @SiO 2 Magnetic microspheres;
3) Adding MPS, which is modified polystyrene, and Fe 3 O 4 Is magnetic core, MPS and SiO 2 The polymer of (2) is taken as a shell to prepare Fe 3 O 4 @SiO 2 -MPS magnetic microspheres;
4) By Fe 3 O 4 @SiO 2 -MPS as magnetic core, GMA and MBA polymer as shell, GMA as glycidyl methacrylate, MBA as 2-mercaptobenzoic acid, and Fe 3 O 4 @SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S)；
5) Adding sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride, and reacting to obtain Fe 3 O 4 @SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S) -COOH, namely the carboxyl magnetic microsphere.
In some embodiments, the preparation step 5) of the carboxyl magnetic microsphere further comprises the following preparation steps of separating the carboxyl magnetic microsphere through a magnetic separator, repeatedly washing the carboxyl magnetic microsphere for at least 5 times with purified water, storing the carboxyl magnetic microsphere in the purified water, checking the quality of the carboxyl magnetic microsphere to be qualified, and putting the carboxyl magnetic microsphere into the next step to prepare the streptavidin magnetic microsphere.
In some embodiments, the carboxyl magnetic microspheres have a particle size of 250-300nm.
In some embodiments, preparing the streptavidin magnetic microsphere comprises the steps of:
1) Washing the carboxyl magnetic microspheres by an activation buffer solution;
2) Adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxy thiosuccinimide for 13-17min;
3) Adding streptavidin solution to react for 1.5-2.5h;
4) Blocking for 1.5-2.5 hours by using bovine serum albumin solution, washing and preserving to prepare the streptavidin magnetic microsphere.
In some embodiments, the carbodiimide in step 2) is at a concentration of 15-25mg/ml; the concentration of N-hydroxysulfosuccinimide is 15-25mg/ml.
In some embodiments, the concentration of the bovine serum albumin solution in step 4) is 0.5mg/ml to 1.0mg/ml.
In some embodiments, the activation buffer is 0.05M to 0.1M morpholinoethanesulfonic acid containing 0.025% to 0.075% polysorbate-20.
In some embodiments, the pH of the activation buffer is between 5.0 and 6.0.
In some embodiments, streptavidin is 127AA core structure, binding 4 biotin molecules per molecule SA, international specific activity of 12-15U/mg.
The preparation method of the 300nm carboxyl magnetic microsphere comprises the following preparation steps:
(1)Fe 3 O 4 preparation of magnetic core
50g FeCl was weighed out 3 ·6H 2 O is dissolved in 1000mL of ethylene glycol;
respectively adding 1g of sodium citrate and 55g of sodium acetate into the solution in sequence, and continuously stirring until the sodium citrate and the sodium acetate are dissolved to obtain a precursor solution;
subpackaging the precursor solution into 250mL hydrothermal reaction kettles;
placing the installed reaction kettle into a baking oven, setting the reaction temperature to be 200 ℃ and the reaction time to be 20 hours;
collecting Fe after the reaction 3 O 4 Repeatedly washing the magnetic microspheres with ethanol and purified water for more than ten times;
washing Fe 3 O 4 The magnetic microspheres were stored in purified water and tested for mass concentration.
(2)Fe 3 O 4 @SiO 2 Is prepared from
A5L round bottom flask was charged with 2500mL ethanol, 700mL purified water, 30mL ammonia, and 6g Fe, respectively 3 O 4 Magnetic microspheres;
a mechanical stirring device is arranged on an ultrasonic cleaning instrument, a switch is turned on, the ultrasonic time is set to be 6.5 hours, the reaction solution is subjected to ultrasonic treatment while being stirred, and the stirring speed is 200rpm;
9mL of TEOS (tetraethyl orthosilicate) solution is diluted in 100mL of ethanol and then added into a 100mL constant pressure dropping funnel;
after ultrasonic treatment for 30min, a constant pressure funnel is arranged on the round bottom flask, TEOS (tetraethyl orthosilicate)/ethanol mixed solution is dripped into a reaction system, and ultrasonic stirring reaction is continuously maintained for 6h;
in the reaction process, changing water for the ultrasonic instrument at intervals to prevent the temperature from being too high;
dismantling the device after the reaction is finished, and collecting Fe by using a magnetic separator 3 O 4 @SiO 2 The magnetic microsphere is washed by ethanol and purified water for more than 10 times, and finally Fe is added 3 O 4 @SiO 2 The magnetic microspheres were stored in purified water and tested for mass concentration.
(3)Fe 3 O 4 @SiO 2 Preparation of MPS
1000mL of ethanol, 500mL of purified water, 10mL of ammonia water, and Fe were each added to a 2L round bottom flask 3 O 4 @SiO 2 Magnetic microspheres;
ultrasound the reaction solution for 30min;
installing a mechanical stirring device on the experiment table, and setting the stirring speed to be 200rpm;
9mL of MPS (modified polystyrene) is diluted in 100mL of ethanol and added into a 100mL constant pressure dropping funnel;
installing a constant pressure funnel on a round bottom flask, dripping an MPS (modified polystyrene)/ethanol mixed solution into a reaction system, and stirring for reaction for 20 hours;
after the reaction is finished, the reaction device is removed, and a magnetic separator is used for collecting Fe 3 O 4 @SiO 2 -MPS magnetic microspheres, washed 5 times with ethanol, finally Fe 3 O 4 @SiO 2 -MPS magnetic microspheres were stored in ethanol and tested for mass concentration.
(4)Fe 3 O 4 @SiO 2 Preparation of MPS@P (GMA-MBA)
3g of Fe 3 O 4 @SiO 2 -MPS magnetic microspheres were dispersed with ethanol in a centrifuge tube and washed 3 times, finally in 50mL ethanol;
adding 3L of purified water and Fe3O4@SiO2-MPS magnetic microsphere dispersion liquid, 300mg of sodium dodecyl benzene sulfonate into a 5L round bottom flask respectively, performing ultrasonic treatment for 10min, installing an oil bath reaction device, setting the stirring speed to 300rpm, and stirring for 1h;
9mLGMA (glycidyl methacrylate), 9.0g MBA (2-mercaptobenzoic acid) and 360mg AIBN (azodiisobutyronitrile) are added into the reaction system, stirring is continued for 1h, and simultaneously nitrogen is introduced into the flask to remove oxygen;
after the deoxidation and stirring time is over, an oil bath switch and a cooling circulating water switch are turned on, the temperature is raised to 75 ℃, and the protection and stirring of nitrogen are continued to be carried out for 16 hours;
after the reaction is finished, the reaction device is removed, and a magnetic separator is used for separating Fe 3 O 4 @SiO 2 -mps@p (GMA-MBA) magnetic microspheres, followed by repeated washing with ethanol and purified water more than 10 times, finally Fe 3 O 4 @SiO 2 -mps@p (GMA-MBA) magnetic microspheres were stored in purified water and tested for mass concentration.
(5)Fe 3 O 4 @SiO 2 Preparation of-MPS@P (GMA-MBA) -COOH
Taking a beaker, adding 20g of sodium carbonate, 3g of sodium bicarbonate and 40g of 6-aminocaproic acid respectively, adding 1200mL of purified water, magnetically stirring until the solution is dissolved, then adding 1.9g of sodium borohydride for continuous dissolution, finally controlling the pH of the solution to be about 10.6, and transferring the solution into a 2L round-bottomed flask;
fe is added to 3 O 4 @SiO 2 -mps@p (GMA-MBA) magnetic microspheres are added into the reaction system and sonicated for 20min;
installing an oil bath reaction and mechanical stirring device, setting the stirring speed to be 200rpm, introducing nitrogen into the flask, and deoxidizing for 30min;
after the deoxidization is finished, opening an oil bath switch and a cooling circulating water switch, heating to 70 ℃, continuing to introduce nitrogen for protection and stirring, and reacting for 16 hours;
after the reaction is finished, the reaction device is removed, and a magnetic separator is used for separating Fe 3 O 4 @SiO 2 -MPS@P (GMA-MBA) -COOH magnetic microspheres, followed by repeated washing with purified water for 5 more times, finally Fe 3 O 4 @SiO 2 -mps@p (GMA-MBA) -COOH magnetic microspheres were stored in purified water and tested for mass concentration.
(II) carboxyl 300nm magnetic microsphere activation coupling streptavidin experiment
Reagent:
washing buffer: MEST buffer, TBST buffer (0.05M TBS+0.15M NaCl+0.05%Tween (Polysorbate-20), pH 7.5)
Activation buffer: MEST buffer (0.1M MES (morpholinoethanesulfonic acid) +0.05% Tween (Polysorbate-20), pH5.0
Coupling buffer: MEST buffer (0.1M MES (morpholinoethanesulfonic acid) +0.05% Tween (Polysorbate-20), pH5.0
Sealing liquid: TBST buffer containing 10mg/mL BSA (bovine serum Albumin), i.e. TBST buffer containing 1% BSA
Preservation solution: PBS (phosphate buffered saline) buffer containing 0.02% sodium azide
The steps are as follows:
(1) Taking 15mg of 300nm carboxyl magnetic microspheres in a 2mL EP tube, and washing 3 times by using MEST buffer (1.5 mL);
(2) Preparing 1mL each of 20mg/mL EDC (carbodiimide) in MEST (activating buffer solution) and 20mg/mL Sulfo-NHS (N-hydroxysulfosuccinimide) in MEST (activating buffer solution);
(3) Adding 750 mu L of EDC and Sulfo-NHS into a magnetic microsphere centrifuge tube respectively, uniformly mixing, and placing the mixture in a vertical mixer for reaction and activation for 15min;
(4) A1 mg/mL solution of streptavidin was prepared using a MEST buffer.
(5) After the activation of the magnetic microspheres is finished, magnetically sucking the supernatant, adding 1.5mL of prepared antibody solution, uniformly mixing, and placing on a vertical mixer for mixing reaction for 2 hours;
(6) 10mg/mL BSA blocking solution was prepared: weighing 16mg BSA, adding into 1.6mL TBST buffer, and uniformly mixing for later use;
(7)Fe 3 O 4 @SiO 2 -finishing the reaction of MPS@P (GMA-MBA) -COOH magnetic microsphere grafting SA, magnetically sucking the supernatant, adding 1.5mL BSA blocking solution, uniformly mixing, and placing on a vertical mixer for mixing reaction and blocking for 1h;
(8) After the end of the blocking reaction, magnetically removing the supernatant, repeatedly washing the magnetic microspheres (10 times or more) with TBST buffer;
(9) Repeated washing 3 times with 1.5mL SA storage buffer (SA storage buffer);
(10) The supernatant was magnetically aspirated, and 1.5. 1.5mL SA storage buffer (SA storage buffer) was added accurately to a constant volume to maintain a concentration of 10mg/mL.
(III) streptavidin magnetic microsphere binding biotinylated oligonucleotide ability test experiment.
Reagent:
1 XPBST (0.2% Tween-20): 100mL of 10 XPBS (phosphate buffered saline) was diluted to 1L with ultrapure water, and 2mL of Tween-20 was added thereto and mixed well. The mixture was filtered through a 0.22 μm filter and stored at 4 ℃.
Biotin-Probe (Biotin-labeled Probe) and NTC-Probe (unlabeled Biotin Probe): both oligonucleotide probes were submitted to Shanghai chemical synthesis and dry probe powders were prepared according to the instructions to give stock solutions at 100. Mu.M each, which were diluted 50-fold to 2 pmol/. Mu.L with 1 XPBST when used.
The steps are as follows:
(1) Taking 4 EP pipes with 1.5mL inlets, dividing the EP pipes into two groups A and B, carrying out parallel experiments on each group of the EP pipes, and adding 20 mu L (200 mu g) of magnetic microspheres to be detected into the 4 EP pipes;
(2) 200. Mu.L of 1 XPBST was added to each tube of magnetic microspheres and vortexed. Magnetically separating, removing the supernatant, and repeatedly cleaning for 2 times;
(3) mu.L of 100. Mu.M (100 pmol/. Mu.L) Biotin-Probe was taken, 245. Mu.L of 1 XPBST was added thereto, and the mixture was homogenized, at which time OD260 was about 0.8 and labeled S1. An NTC-Probe stock solution was prepared in the same manner, at which time OD260 was about 0.8, labeled S2;
note that: "2 pmol/. Mu.L" represents only the concentration labeled by the synthesis company, and the actual measurement should be converted according to the actual A260 value; the stock solution of Probe is adjusted to OD260 of about 0.8 by 1 XPBST;
(4) 100 mu L S1 is added to each of the group A2 magnetic microspheres, 100 mu L S2 is added to each of the group B2 magnetic microspheres, and the mixture is uniformly mixed. Placing the EP pipe on a vertical mixer, and carrying out rotary mixing reaction for 2 hours at room temperature (all representing the range of 22-28 ℃);
(5) After the reaction, putting the 4-tube magnetic microsphere and the residual S1 and S2 after sample addition into a centrifugal machine together for centrifugation at 12,000g for 5min (4 ℃), taking the supernatant after centrifugation, and measuring the ssDNAA260 value of each tube by using Onedrop2000 (Nucleic Acid module, ssDNA mode) (repeatedly measuring three times for averaging);
(6) The measurement results were recorded and the amount of single stranded oligonucleotide bound to the magnetic microspheres was calculated as follows:
1mg magnetic microsphere bound to 2 Probe probes:
Biotin-Probe binding amount (pmo 1): 5 x [ S1- (a1+a2)/2 ] 4.54 x 100
NTC-Probe binding amount (pmo 1): 5 x [ S2- (b1+b2)/2 ] 4.54 x 100
Amount of Biotin-Probe bound per mg magnetic microsphere (pmo 1): biotin-Probe binding amount-NTC-Probe binding amount.
The calculation results are shown in the following table:
table C.1 calculation of the amount of Single-stranded oligonucleotides bound by magnetic microspheres
The results show that: the streptavidin magnetic microsphere provided by the invention has good capability of combining with Biotin-Probe, and has low nonspecific adsorption.
(IV) streptavidin magnetic microsphere binding biotin IgG ability test experiment.
Reagent:
1×PBST(0.2％Tween20)：
100mL of 10 XPBS was diluted to 1L with ultrapure water, and 2mL of Tween-20 was added thereto and mixed well. The mixture was filtered through a 0.22 μm filter and stored at 4 ℃.
5% bsa solution:
0.6g of BSA was weighed out and dissolved in 12mL of 1 XPBST. Mixing well and storing at 4 ℃. The shelf life is 24 hours.
TMB (tetramethylbenzidine) reaction termination solution:
2M H 2 SO 4 54.5mL of 98% sulfuric acid solution was measured, and the solution was added dropwise to 350mL of ultrapure water with stirring. After cooling to room temperature, ultrapure water is continuously added to fix the volume to 500mL.
Biotin (Biotin) -rabbit IgG working fluid (3. Mu.g/mL):
biotin (Biotin) -rabbit IgG was diluted to 3. Mu.g/mL with 1 XPBST. Stored at 4 ℃ for 24 hours (approximately 4mL per batch of magnetic microspheres tested).
Biotin-rabbit IgG standard yeast stock (100 ng/mL):
the Biotin-rabbit IgG working solution (3. Mu.g/mL) was diluted 30-fold, 100. Mu.L of the Biotin-rabbit IgG working solution was taken, and 1 XPBST was added to a total volume of 3m L. Preserving at 4deg.C for 24 hr.
HRP (horseradish peroxidase) -goat anti-rabbit IgG:
HRP (horseradish peroxidase) -goat anti-rabbit IgG was taken and diluted 10000-fold with 5% bsa solution. Preserving at 4deg.C for 24 hr.
The steps are as follows:
(1) 50uL of the magnetic microspheres to be detected (10 mg/mL) were weighed by a pipette and placed in a 1.5mL EP tube, 900uL of 1 XPBST was added and mixed well, the magnetic microspheres were washed, magnetically separated, and the supernatant was removed. Repeating washing for 3 times, adding 500uL of 1 XPBST, mixing, and diluting for 10 times (i.e. 1 mg/mL);
(2) Three 2.0mL EP tubes were taken, 100. Mu.L (i.e., 100. Mu.g) of magnetic microspheres were added to each tube (run in parallel), magnetically separated, and the supernatant removed;
(3) The prepared Biotin-rabbit IgG working fluid was labeled S. Each tube of magnetic microspheres was added with 1.5mL of Biotin-rabbit IgG working solution, and the EP tube was placed on a vertical mixer and mixed by rotation at room temperature (both representing 22-28 ℃ C.) for 2h. The supernatants were collected into new EP tubes, labeled F1, F2, F3, respectively. Finally, S is diluted 150 times by 1 XPBST (S concentration can be directly replaced by initial addition concentration of 3 ug/ml), and F1, F2 and F3 are diluted 120 times for standby. Namely 10 is diluted to 1.5ml or 1.2ml;
(4) 5 pieces of Streptavidin Coated Plate (streptavidin-coated plate), 96-well plate strips, 200. Mu.L of 1 XPBST per well, were taken and washed in a horizontal shaker for 3min. The washing liquid is removed, and the plate holes from which the residual liquid is removed are tapped. Repeating the washing for 2 times according to the step;
(5) Biotin-rabbit IgG working solution was diluted to 35ng/mL, 30ng/mL, 25ng/mL, 20ng/mL, 15ng/mL, 10ng/mL, 5ng/mL, 0ng/mL with 1 XPBST for standard curve preparation (Biotin-rabbit IgG concentration is on the abscissa, OD450 values measured at the corresponding concentrations are on the ordinate);
TABLE C.2
(6) Subjecting the diluted samples (200 uL or more) of S, F and F2 in C.3 to 17,000g centrifugation for 5min (4 ℃);
(7) According to the loading layout of Table C.3, the solution prepared in 5.6 steps was added to Streptavidin Coated Plate (streptavidin coated plate) from a 4 step spot check, 96-well microplate strips, 100uL per well;
TABLE C.3
(8) The ELISA plate was placed on a horizontal shaker and incubated for 2h at room temperature. (alternatively, the reaction mixture may be left at 4℃overnight);
(9) After completion of the reaction, 200. Mu.L of 1 XPBST was added to each well, and the mixture was placed on a shaking table at room temperature and washed for 3 minutes. Removing supernatant, and repeatedly washing for 2 times;
(10) 200 mu L of 5% BSA is added into each hole, the mixture is placed on a shaking table at room temperature, and the mixture is sealed for 90min, and the supernatant is removed;
(11) Adding 100 mu L of HRP-goat anti-rabbit IgG working solution into each hole, placing the mixture on a shaking table at room temperature, mixing and incubating for 2 hours, and removing supernatant;
(12) 200. Mu.L of 1 XPBST was added to each well, and the mixture was placed on a shaking table at room temperature and washed for 3 minutes. Removing supernatant, and repeatedly washing for 4 times;
(13) Adding 100 mu L of TMB color development liquid into each hole, and developing color for 5min in dark; after the reaction was completed, 50. Mu.L of TMB reaction termination solution (2M concentrated sulfuric acid) was added to each well; measuring the OD450 value in an enzyme-labeled instrument, and calculating the binding capacity of Biotin-rabbit IgG of each milligram of magnetic microsphere;
the calculation method comprises the following steps:
(14) Taking the concentration (ng/mL) of Biotin-rabbit IgG as an abscissa, taking the OD450 value (average value of No. 1 strip, no. 2 strip and very No. 3 strip) measured under the corresponding concentration as an ordinate to make an XY scatter diagram, and calculating a linear equation of the standard curve;
(15) Calculating the OD450 mean value of each diluted sample, calculating the concentration of the diluted sample according to a standard curve equation, and calculating the concentrations C (S) and C (F) (ng/mL) of the stock solution sample by multiplying the concentrations of the diluted samples respectively;
remarks: c (F) represents: average value of OD450 calculation results of C (F1), C (F2) and C (F3);
(16) The amount of Biotin-rabbit IgG bound per 100 μg of magnetic microsphere was (ng): (C (S) -C (F))1.5;
remarks: 1.5 represents the volume of C.3.3 added Biotin-rabbit IgG working fluid;
(17) The amount of Biotin-rabbit IgG bound per mg of magnetic microspheres was (μg): (C (S) -C (F))1.5 x 10/1000, i.e., (C (S) -C (F))0.015;
the calculation results are shown in the following table:
TABLE C.4
| Magnetic microsphere designation | Biotin-IgG binding Capacity (ug/mg) |
| SA300nm20191004 | 23.81 |
The results show that: the 300nm streptavidin magnetic microsphere has good capability of combining with Biotin-IgG, and can reach more than or equal to 20ug/mg.
And (V) application of streptavidin magnetic beads in a Myoglobin (MYO) chemiluminescence detection system.
Reagent:
wash Buffer (washing solution): TBST (washing) buffer (20 mM Tris (triisopropylethanesulfonyl) and 30mM NaCl and 0.1% Tween 20)
Enzyme-labeled antibody dilution: PBST containing 5% BSA, ALP-MYO antibody 1:2000 was diluted and 1ul was taken and diluted to 2ml.
Biotinylated antibody dilution: PBS contained 0.1% Tween20, 10ug/ml, and 10ul of antibody was diluted to 1ml.
Antigen dilution: pbs contained 0.1% tween20, starting from 1000ng for dilution, plus blank for 10 wells, 8 wells after use.
Free biotin: 2 μg/ml diluted with 1 x PBS containing 0.1% Tween 20. (8 ul of 1mg/ml of D-Biotin was diluted to 4 ml)
Substrate solution: shenzhen Meinate
Streptavidin magnetic microsphere: after the biotinylated antibody is coupled, it is blocked with free organisms.
Table C.5 antigen preparation method
Experimental procedure
1) Preparing 1ml of biotinylated antibody with the concentration of 10ug/ml, and diluting 10ul of the antibody to 1ml;
2) Taking 25 mu l (10 mg/ml) of 2-tube streptavidin magnetic microspheres and magnetically removing supernatant (1-tube control magnetic beads, 1-tube SA300 nm), washing once, and adding 500 mu l of biotinylated antibody of 10ug/ml each and incubating for 1h at room temperature;
3) After 3 washes, 100. Mu.l each, 2. Mu.g/ml free biotin was added and incubated for 1h at room temperature; after 3 washes, 500. Mu.l of PBST was added to each and diluted to 0.5mg/ml;
4) Taking 2 chemiluminescent plates, adding 50 mu L/hole of streptavidin magnetic microspheres into a 96-well plate, adding 50 mu L/hole (3-10) of a test standard substance, and adding 100 mu L of enzyme-labeled antibody into each hole; sufficiently vibrating the heavy strepavidin magnetic microspheres, incubating for 15min in a 37 ℃ incubator, magnetically separating, sucking supernatant by a pipettor, and taking down the 96-well plate from the magnetic separator;
5) Adding 200 mu L of Washing buffer into each hole, fully vibrating and re-suspending the streptavidin magnetic microspheres, magnetically separating, sucking the supernatant by a pipette, and taking down the 96-well plate from the magnetic separator, wherein the step is repeated for 2 times and is washed for 3 times;
6) Adding 150 mu L of substrate solution into each hole, fully vibrating and re-suspending the streptavidin magnetic microspheres, and incubating for 5min in a dark place;
7) The 96-well plate was placed into a chemiluminescent reader for reading, spot-on-screen FIG. 2.0- -password with user name login- -quick measurement- -shock (low intensity 5S, all wells) - -starting measurement- -Ctr1-C- - -Ctrl-V to U disk.
Experimental results:
table C.6 Experimental results of streptavidin magnetic beads in Myoglobin (MYO) chemiluminescent detection System
Table C.7 Experimental results of streptavidin magnetic beads in Myoglobin (MYO) chemiluminescent detection System
| Degree of separation | Magnetic microsphere (separation degree) | 300nm streptavidin magnetic microsphere (degree of separation) |
| S1/S0 | 8.761 | 11.030 |
| S2/S0 | 19.517 | 12.913 |
| S3/S0 | 45.167 | 27.895 |
| S4/S0 | 102.075 | 61.386 |
| S5/S0 | 235.894 | 154.772 |
| S6/S0 | 547.873 | 330.983 |
| S7/S0 | 972.698 | 632.695 |
From the experimental results and fig. 1, it is shown that: the streptavidin magnetic microsphere has good linearity of standard curve in chemiluminescence test, R2 is more than or equal to 0.99, S1/S0 is more than 2.6, and S7/S0 is more than 500.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements could be made by those skilled in the art without departing from the inventive concept, which fall within the scope of the present invention.
Claims (3)
1. The preparation method of the streptavidin magnetic microsphere is characterized by comprising the following preparation steps:
1) Preparing carboxyl magnetic microspheres;
2) Preparing the carboxyl magnetic microsphere by activating and coupling streptavidin to obtain the streptavidin magnetic microsphere;
the preparation method of the carboxyl magnetic microsphere comprises the following steps:
1.1 By FeCl 3 •6H 2 O is prepared into Fe 3 O 4 A magnetic core;
1.2 In Fe (B) 3 O 4 Is magnetic core, siO 2 Is taken as a shell to prepare Fe 3 O 4 @ SiO 2 Magnetic microspheres;
1.3 MPS is added and is modified polystyrene, fe is used 3 O 4 Is magnetic core, MPS and SiO 2 The polymer of (2) is taken as a shell to prepare Fe 3 O 4 @ SiO 2 -MPS magnetic microspheres;
1.4 In Fe (B) 3 O 4 @ SiO 2 -MPS as magnetic core, GMA and MBA polymer as shell, GMA as glycidyl methacrylate, MBA as 2-mercaptobenzoic acid, and Fe 3 O 4 @ SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S)；
1.5 Sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride are added to react to prepare Fe 3 O 4 @ SiO 2 -MPS@P(C 7 H 10 O 3 -C 7 H 6 O 2 S) -COOH, namely the carboxyl magnetic microsphere;
in the reaction process of the step 1.2), water is changed for the ultrasonic instrument every 30-40min to prevent the reaction temperature from being higher than 50 ℃;
the particle size of the carboxyl magnetic microsphere is 250-300nm;
the preparation method of the streptavidin magnetic microsphere comprises the following steps:
2.1 Washing the carboxyl magnetic microspheres by an activation buffer solution;
2.2 Adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxysulfosuccinimide for 13-17min;
2.3 Adding streptavidin solution to react for 1.5-2.5h;
2.4 Sealing for 1.5-2.5h with bovine serum albumin solution, washing and preserving to prepare streptavidin magnetic microspheres;
the concentration of carbodiimide in step 2.2) is 15-25mg/ml; the concentration of the N-hydroxy thiosuccinimide is 15-25mg/ml;
the concentration of the bovine serum albumin solution in step 2.4) is 0.5mg/ml to 1.0 mg/ml;
the activation buffer solution in step 2.1) comprises: 0.025% -0.075% polysorbate-20 and 0.05M-0.1M morpholinoethanesulfonic acid.
2. The method for preparing streptavidin magnetic microspheres according to claim 1, wherein the step 1.5) of preparing the carboxyl magnetic microspheres is followed by a step of separating the carboxyl magnetic microspheres by a magnetic separator, repeatedly washing the carboxyl magnetic microspheres with purified water at least 5 times or more, storing the carboxyl magnetic microspheres in the purified water, checking the quality of the carboxyl magnetic microspheres, and putting the carboxyl magnetic microspheres into a next step to prepare the streptavidin magnetic microspheres.
3. The method for preparing streptavidin magnetic microspheres according to claim 1, wherein the pH value of the activation buffer solution is 5.0-6.0.
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