CN111624333A - Method for detecting loading capacity of streptavidin magnetic microsphere combined with free biotin - Google Patents
Method for detecting loading capacity of streptavidin magnetic microsphere combined with free biotin Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 101
- 229960002685 biotin Drugs 0.000 title claims abstract description 97
- 239000011616 biotin Substances 0.000 title claims abstract description 96
- 235000020958 biotin Nutrition 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 65
- 238000011068 loading method Methods 0.000 title claims abstract description 38
- 108010090804 Streptavidin Proteins 0.000 title claims abstract description 35
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- 238000004140 cleaning Methods 0.000 claims abstract description 7
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- 229930003756 Vitamin B7 Natural products 0.000 description 2
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- CBCKQZAAMUWICA-UHFFFAOYSA-N 1,4-phenylenediamine Chemical compound NC1=CC=C(N)C=C1 CBCKQZAAMUWICA-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
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- PFANGGLDSSGAFJ-UHFFFAOYSA-N [NH4+].[NH4+].[N+](=[N-])(C1SC2=C(N1CC)C=CC(=C2)S(=O)(=O)[O-])C2SC1=C(N2CC)C=CC(=C1)S(=O)(=O)[O-] Chemical compound [NH4+].[NH4+].[N+](=[N-])(C1SC2=C(N1CC)C=CC(=C2)S(=O)(=O)[O-])C2SC1=C(N2CC)C=CC(=C1)S(=O)(=O)[O-] PFANGGLDSSGAFJ-UHFFFAOYSA-N 0.000 description 1
- -1 antibodies Chemical class 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000002122 magnetic nanoparticle Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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Abstract
The invention discloses a method for detecting the loading capacity of free biotin combined with streptavidin magnetic microspheres, which comprises the following steps: pretreating a streptavidin magnetic nano microsphere solution, then respectively adding the pretreated streptavidin magnetic nano microsphere solution into a plurality of groups of biotin solutions for affinity adsorption to form a relatively stable complex, and cleaning a plurality of groups of complex products by using a cleaning solution; adding Biotin-labeled horseradish peroxide (Biotin-HRP) for adsorption; adding a color developing solution into the marked product to perform color developing reaction; measuring the OD (optical density) value of the solution after the color reaction; the method for detecting the loading capacity of the streptavidin magnetic nano-microspheres combined with the free biotin is obtained through calculation according to the obtained multiple groups of OD values, the loading capacity of the streptavidin magnetic nano-microspheres combined with the free biotin can be detected more accurately and rapidly, a basis is provided for the usage amount of the downstream streptavidin magnetic nano-microspheres, and the method is simple and convenient to operate, low in cost and high in sensitivity.
Description
Technical Field
The invention relates to the technical field of nano microsphere detection and molecular biology application, in particular to a method for detecting the loading capacity of streptavidin magnetic microsphere combined free biotin.
Background art:
biotin, also known as vitamin H, is a water-soluble vitamin belonging to the B group, is a sulfur-containing vitamin, and is also a coenzyme for the action of carboxy converting enzymes in the body, so it is also known as coenzyme R.
The streptavidin magnetic nano-microsphere is formed by covalently combining a superparamagnetic microsphere and high-purity streptavidin. The streptavidin-Biotin (SA-Biotin) system has extremely high binding affinity (Kd 10^ -15), and has wide application in the biological field. The streptavidin magnetic nano-microsphere can be simply, conveniently, quickly and effectively combined with a plurality of biotinylation complexes, such as: small molecules, peptides, proteins, antibodies, carbohydrates, lectins, oligonucleotides, DNA/RNA, and the like.
The streptavidin magnetic nano-microsphere is more and more widely applied, and usually, several indexes can reflect the loading capacity of the microsphere, such as free biotin loading capacity, biotinylated antibody loading capacity and biotinylated nucleic acid loading capacity. Free biotin can fully have an affinity effect with a binding site on streptavidin due to small molecular weight, so that the free biotin loading capacity can most visually reflect the binding capacity of the streptavidin magnetic nano-microspheres.
How to measure the loading capacity of the bound free biotin needs a simple, convenient, efficient and sensitive method for determining, and few methods for detecting the loading capacity of the streptavidin magnetic nanospheres bound with the biotin can be found in the market, and no specific detection method is disclosed. Foreign manufacturers measure the free biotin loading index through biotin-labeled fluorescent molecules, and measure the adsorption amount of the microspheres to the biotinylated fluorescent molecules through a fluorometer, but the biotinylated fluorescent molecules and detection equipment in the method are high in price. The invention provides a detection method which is simple and easy to implement, has lower cost and relatively lower reagent and equipment prices.
Disclosure of Invention
The invention aims to provide a method for detecting the loading capacity of free biotin combined with streptavidin magnetic microspheres, and aims to overcome the defects that the method for detecting the loading capacity of the free biotin combined with the streptavidin magnetic nanoparticles in the prior art is complex and has low accuracy of detection data.
A method for detecting the loading capacity of free biotin combined with streptavidin magnetic microspheres comprises the following steps:
pretreating the magnetic nano microsphere solution, and then respectively adding the pretreated magnetic nano microsphere solution into a plurality of groups of biotin solutions for complexing to obtain a plurality of groups of mixed solutions;
respectively cleaning the multiple groups of mixed liquor and carrying out a labeling reaction;
adding a color developing solution into the marked product to perform color developing reaction;
measuring the OD value of the solution after the color reaction;
and calculating the biotin binding capacity according to the obtained multiple groups of OD values.
Further, the method for pretreating the magnetic nano microsphere solution comprises the following steps:
carrying out magnetic separation on the magnetic nano microsphere solution, and removing the supernatant;
the magnetic nanospheres were resuspended in PBST (phosphate tween buffer), and the supernatant removed after at least two magnetic separations.
Further, the method for complexing by respectively adding the pretreated magnetic nano microsphere solution into a plurality of groups of biotin solutions comprises the following steps:
dividing the solution into a plurality of groups of biotin solutions with different concentrations according to the content of biotin,
adding a magnetic nano microsphere solution into a biotin solution to obtain a plurality of groups of mixed solutions;
and carrying out constant-temperature oscillation complexation on the mixed solution, wherein the oscillation time is 30-70min, the temperature is set to be 20-40 ℃, and the rotation speed during oscillation is 800-.
Further, the Biotin solution comprises a D-Biotin (D-Biotin) working solution.
Further, the method for respectively cleaning the multiple groups of mixed liquor and carrying out the labeling reaction comprises the following steps:
adding PBST into the mixed solution, and oscillating at constant temperature;
after shaking, carrying out magnetic attraction separation and removing supernatant;
adding a marking solution, and oscillating at constant temperature to carry out marking reaction.
Further, the labeling solution comprises one or more of Biotin-HRP and Biotin-AP (Biotin-labeled alkaline phosphatase).
Further, the method for adding the color developing solution into the marked product to carry out color developing reaction comprises the following steps:
PBST is respectively added into the products for washing, constant temperature oscillation is carried out, and the supernatant is removed after magnetic attraction separation;
adding color development liquid, mixing uniformly, and performing light-proof color development;
and adding the color developing solution again after the color development to terminate the reaction.
Further, the color development liquid comprises one or more of TMB color development liquid, OPD, ABTS, p-NPP.
Further, the method for measuring the OD value comprises the following steps:
measuring the value of OD450 by a microplate reader when developing with TMB;
the value of OD492 was measured by a microplate reader when developing with OPD;
measuring the value of OD405 by a microplate reader when developing using ABTS;
the value of OD405 was measured by a microplate reader when developing with p-NPP.
Further, the method for calculating the biotin binding capacity according to the obtained multiple groups of OD values comprises the following steps:
drawing a scatter plot line graph according to the content value and OD value of biotin;
calculating by combining a scattered point line graph to obtain an OD value of a biotin binding capacity saturation point;
and calculating the biotin loading capacity of the streptavidin magnetic microspheres according to the OD value.
The invention has the advantages that: the method for detecting the loading capacity of the streptavidin magnetic nano-microspheres combined with the free biotin limits parameters such as specific detection conditions, can detect the loading capacity of the streptavidin magnetic nano-microspheres combined with the free biotin more accurately and quickly, provides basis for the usage amount of the downstream streptavidin magnetic nano-microspheres, and is simple and convenient to operate, high in sensitivity and relatively low in cost.
Drawings
FIG. 1 is a schematic flow chart of the method of the present invention.
FIG. 2 is a schematic diagram of complexation in the process of the present invention.
FIG. 3 is a schematic view of a dotted broken line curve in example 1 of the present invention.
FIG. 4 is a schematic view of a dotted broken line curve in example 2 of the present invention.
FIG. 5 is a schematic view of a dotted broken line curve in example 3 of the present invention.
Fig. 6 is a schematic view of a dotted broken line curve in embodiment 4 of the present invention.
FIG. 7 is a schematic view of a dotted broken line curve in example 5 of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Preparation of reagents required for detection:
(1)1×PBS
weighing NaCl 8.0g, KCl 0.2g and KH2PO40.24g，Na2HPO4Adding 1.44g of the mixture into a 1L blue bottle, measuring 950mL of ultrapure water by using a measuring cylinder, adding dropwise 1M NaOH to adjust the pH value to 7.4 after the solid powder is completely dissolved (can be dissolved by ultrasonic waves), transferring the liquid in the blue bottle into the 1L measuring cylinder, fixing the volume to 1L, and transferring the liquid into the 1L blue bottle for later use after suction filtration;
(2)1×PBST
adding 2mL of Tween-20 into the blue-mouth bottle filled with 1 XPBS, fully mixing, and storing in a refrigerator at 4 ℃;
(3)0.448mg/mL (2mM) D-Biotin solution
Taking 4.48mg D-Biotin to a 15mL EP tube, adding 10mL DMSO, and shaking to dissolve for later use;
(4)100ng/100 mu L D-Biotin solution
Adding 10 mu L of 0.448mg/mL D-Biotin solution into 4.470mL 1 xPBST, and mixing uniformly for later use;
(5) 1% BSA solution
0.15g BSA was taken in a 15mL centrifuge tube and dissolved in 15mL 1 XPBST. Mixing uniformly for later use;
(6) biotinylated enzyme solution
6.1, Biotin-HRP solution: taking 1 mu L of Biotin-HRP to a 15mL centrifuge tube, adding 10mL of 1% BSA (bovine serum albumin) to dilute by 10000 times, and uniformly mixing for later use;
6.2, Biotin-AP solution: take 1. mu.L of Biotin-AP to a 15mL centrifuge tube, add 10mL of 1% BSA to dilute 10000 times, and mix well for use.
(7) Color developing liquid
TMB color development liquid: commercial single-component TMB color developing solution
OPD color development liquid: taking 6.1ml of 0.1mol/L citric acid (2.1g/100ml) and 0.2mol/L Na2HPO4.12H2O (7.163g/100ml)6.4ml, 12.5ml of distilled water was added thereto, 10mg of OPD was dissolved therein, and after dissolution, 240. mu.L of 30% H2O was added immediately before use.
ABTS color development liquid: commercial ABTS single-component color developing liquid
p-NPP developing solution: commercial p-NPP developing liquid
(8) Reaction terminating liquid
TMB reaction stop solution: taking 5.45mL of 98% sulfuric acid solution, dropwise adding the sulfuric acid solution into a beaker filled with 35mL of ultrapure water, slowly dropwise adding concentrated sulfuric acid (the concentrated sulfuric acid releases a large amount of heat when meeting water, and is required to be slowly dropwise added), stirring while dropwise adding, and fixing the volume to 50mL after dropwise adding is finished;
OPD reaction termination solution: taking 5.45mL of 98% sulfuric acid solution, dropwise adding the sulfuric acid solution into a beaker filled with 35mL of ultrapure water, slowly dropwise adding concentrated sulfuric acid (the concentrated sulfuric acid releases a large amount of heat when meeting water, and is required to be slowly dropwise added), stirring while dropwise adding, and fixing the volume to 50mL after dropwise adding is finished;
ABTS reaction stop solution: 1.0g SDS (sodium dodecyl sulfate) was weighed into a beaker, 100mL ultrapure water was added, and the mixture was stirred until it was completely clear.
p-NPP reaction stop solution: 12.0g NaOH was weighed into a beaker, 80mL of ultrapure water was added, stirred until completely dissolved, and the volume was 100 mL.
(9) D-Biotin working solution with different gradient concentrations
D-Biotin working solution with different concentration gradients is obtained by carrying out gradient dilution on 100ng/100 mu L D-Biotin solution by 1 XPBST, and the prepared gradient concentration (ng/100 mu L) is 0, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50 and 60.
As shown in fig. 1 to 3, a method for detecting the loading of free biotin bound to streptavidin magnetic microspheres includes the following steps:
the method comprises the following steps: pretreating 50-3000nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple-group gradient biotin solution for complexing;
the method for pretreating the magnetic nano microsphere solution comprises the following steps:
carrying out magnetic separation on the magnetic nano microsphere solution, and removing the supernatant;
carrying out resuspension on the magnetic nano-microspheres through PBST, carrying out magnetic separation at least twice, and then removing supernatant;
the method comprises the following specific steps:
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
the method for adding the pretreated magnetic nano microsphere solution into a plurality of groups of biotin solutions for complexing comprises the following steps:
adding the D-Biotin working solution into the magnetic nano microsphere solution to obtain a mixed solution;
carrying out constant-temperature oscillation on the mixed solution to realize complexation, wherein the oscillation time is 30-70min, the temperature is set to be 20-40 ℃, and the rotation speed during oscillation is 800-;
the method comprises the following specific steps:
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
step two: cleaning and complexing multiple groups of products, and respectively carrying out Biotin-HRP combination;
the method for washing and complexing multiple groups of products and labeling biotin respectively comprises the following steps:
adding PBST into the complexed product, and oscillating at constant temperature;
after shaking, carrying out magnetic attraction separation and removing supernatant;
adding Biotin-HRP working solution, and vibrating at constant temperature to realize combination;
the method comprises the following specific steps:
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
step three: adding a color developing solution into the combined product to perform color developing reaction;
the method for adding the color developing solution into the combined product to carry out color developing reaction comprises the following steps:
respectively adding the combined products into PBST for washing, oscillating at constant temperature, and removing supernatant after magnetic separation;
adding TMB color development liquid, mixing uniformly, and then performing light-shielding color development;
adding a reaction termination solution to terminate the reaction after color development;
the color developing solution comprises one of TMB (tetramethylbenzidine) color developing solution, OPD (p-phenylenediamine), ABTS (2,2' -diazobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) or derivatives with similar functional structures, and when Biotin-AP is used as the marking solution, the color developing solution is p-NPP (nitrophenylphosphate) or derivatives with similar functional structures;
the method comprises the following specific steps:
after the reaction was completed, the supernatant was magnetically separated. Adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator, mixing uniformly for 30 seconds at 1100r/min, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
step four: measuring the OD value of the solution after the color reaction;
measuring the value of OD450 by a microplate reader when developing with TMB;
the value of OD492 was measured by a microplate reader when developing with OPD;
measuring the value of OD405 by a microplate reader when developing using ABTS;
when developing with p-NPP, the value of OD405 was measured by a microplate reader;
keeping the recorded numerical value after the detection is finished;
step five: calculating the biotin binding capacity according to the obtained multiple groups of OD values;
the method for calculating the biotin binding capacity according to the obtained multiple groups of OD values comprises the following steps:
drawing a scatter plot according to the content value of biotin and the value of OD 450;
calculating by combining a scatter plot line graph to obtain an OD450 value of a biotin binding capacity saturation point;
calculating the amount of free biotin according to the OD450 value;
the method comprises the following steps:
acquiring a biotin binding capacity saturation point on a scattered point line graph, and recording a corresponding abscissa numerical value of the saturation point;
substituting the OD450 value of the biotin binding capacity saturation point into an equation system to obtain a biotin structure comprises the following specific steps:
measuring an OD450 value by using an enzyme-labeling instrument, and calculating the binding amount of free biotin bound to each mg of magnetic nano-microspheres;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
the dosage of each hole of the magnetic nano microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point of the present example is 35, which is calculated
B＝35/244.3×1000×40＝5370.66pmol/mg。
The process of the invention is further illustrated below with reference to examples:
example 1
The method comprises the following steps: pretreating 50nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple-group gradient biotin solution for complexing;
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
magnetically separating the supernatant after the reaction is finished, adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator at 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
performing OD450 detection on the microplate reader and keeping a recorded numerical value;
as shown in fig. 3, the amount of bound free biotin per mg of magnetic nanospheres was calculated;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
each holeThe dosage of the magnetic nano-microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point in this example is 38, calculated
B＝38/244.3×1000×40＝6221.86pmol/mg。
Example 2
The method comprises the following steps: pretreating 300nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple groups of gradient biotin solutions for complexing;
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
magnetically separating the supernatant after the reaction is finished, adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator at 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
performing OD450 detection on the microplate reader and keeping a recorded numerical value;
as shown in fig. 4, the amount of bound free biotin per mg of magnetic nanospheres was calculated;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
the dosage of each hole of the magnetic nano microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point of the present example is 35, which is calculated
B＝35/244.3×1000×40＝5370.66pmol/mg。
Example 3
The method comprises the following steps: pretreating 1000nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple groups of gradient biotin solutions for complexing;
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
magnetically separating the supernatant after the reaction is finished, adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator at 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
performing OD450 detection on the microplate reader and keeping a recorded numerical value;
as shown in fig. 5, the amount of bound free biotin per mg of magnetic nanospheres was calculated;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
the dosage of each hole of the magnetic nano microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point of the present example is 29, which is calculated
B＝29/244.3×1000×40＝4748.26pmol/mg。
Example 4
The method comprises the following steps: pretreating 2000nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple groups of gradient biotin solutions for complexing;
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
magnetically separating the supernatant after the reaction is finished, adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator at 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
performing OD450 detection on the microplate reader and keeping a recorded numerical value;
as shown in fig. 6, the amount of bound free biotin per mg of magnetic nanospheres was calculated;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
the dosage of each hole of the magnetic nano microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point in this example is 22, which is calculated
B＝25/244.3×1000×40＝3602.13pmol/mg。
Example 5
The method comprises the following steps: pretreating 3000nm magnetic nano microsphere solution, and respectively adding into the prepared 0-60ng/100uL multiple-group gradient biotin solution for complexing;
(1.1) taking 1 EP tube with the volume of 1.5mL, adding 75 mu L (0.75mg) of SA magnetic nano microspheres which are uniformly mixed in a vortex mode, carrying out magnetic separation on a magnetic frame, and removing supernate;
(1.2) Add 1mL of 1 XPBST to EP tube, resuspend magnetic nanospheres, and magnetically separate out the supernatant. Repeating the steps once, and then adding 1.5mL of 1 xPBST heavy suspension magnetic nano microspheres, wherein the concentration of the microspheres is 0.5 mug/muL;
(1.3) taking a 96-hole enzyme label plate, and sequentially adding 50 mu L of the uniformly mixed SA magnetic nano microspheres at the hole positions. Placing the 96-hole plate on a 96-hole enzyme label plate magnetic separator, performing magnetic separation, and removing supernatant;
sequentially adding D-Biotin working solution with gradient concentration into an enzyme label plate, adding 100 mu L of the D-Biotin working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out treatment at 37 ℃ for 50min at 1100 r/min;
after the reaction is finished, magnetically separating out the supernatant, adding 100 mu L of 1 xPBST into each hole, placing the 96-hole plate on a constant-temperature oscillator, 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, carrying out supernatant clarification, and then sucking and removing the supernatant; this step was repeated 1 more time;
adding 100 mu L of Biotin-HRP working solution into each hole, placing a 96-hole plate on a constant temperature shaking instrument, and carrying out heating at 37 ℃ for 50min at 1100 r/min;
magnetically separating the supernatant after the reaction is finished, adding 100 mu L of 1 xPBST washing magnetic nano microspheres into each hole, placing a 96-hole plate on a constant-temperature oscillator at 1100r/min, uniformly mixing for 30 seconds, then placing the 96-hole plate on a magnetic frame for magnetic separation, and after the supernatant is clarified, sucking and removing the supernatant; this step was repeated 3 more times;
adding 200 μ L of TMB color developing solution into each well with a row gun, blowing, mixing, and developing at 37 deg.C in dark for 10 min. After the reaction is completed, 50 mu L of TMB reaction termination solution is added into each hole, and the mixture is blown and uniformly mixed;
performing OD450 detection on the microplate reader and keeping a recorded numerical value;
as shown in fig. 7, the amount of bound free biotin per mg of magnetic nanospheres was calculated;
(5.1) taking the addition of the biotin as an abscissa and taking a corresponding OD450 value as an ordinate to make a scattered point line graph, wherein the curve gradually decreases and then tends to be stable, and the first point on the stable line is a saturation inhibition point and is marked as a point B;
(5.2) calculating the free biotin binding amount per mg of magnetic nanospheres:
the dosage of each hole of the magnetic nano microsphere is 50uL, and the concentration is 0.5mg/mL, namely 0.025 mg; abscissa m corresponding to saturation suppression point(B)The microsphere free biotin loading was calculated as follows:
B(nmol/0.025mg)＝m(B)/244.3
converting a unit to nmol/mg, the formula can be modified as follows:
B(pmol/mg)＝m(B)/244.3×1000×40。
244.3-molecular weight for D-Biotin;
the horizontal coordinate value of the saturation suppression point of the present example is 15, which is calculated
B＝15/244.3×1000×40＝2456.00pmol/mg。
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.
Claims (10)
1. A method for detecting the loading capacity of free biotin combined with streptavidin magnetic microspheres is characterized by comprising the following steps:
pretreating the magnetic nano microsphere solution, and then respectively adding the pretreated magnetic nano microsphere solution into a plurality of groups of biotin solutions for complexing to obtain a plurality of groups of mixed solutions;
respectively cleaning the multiple groups of mixed liquor and carrying out a labeling reaction;
adding a color developing solution into the marked product to perform color developing reaction;
measuring the OD value of the solution after the color reaction;
and calculating the biotin binding capacity according to the obtained multiple groups of OD values.
2. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the method for pretreating the magnetic nano microsphere solution comprises the following steps:
carrying out magnetic separation on the magnetic nano microsphere solution, and removing the supernatant;
resuspending the magnetic nanospheres by PBST, performing magnetic separation at least twice, and removing the supernatant.
3. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the method for complexing by respectively adding pretreated magnetic nano microsphere solution into multiple groups of biotin solutions comprises the following steps:
dividing the solution into a plurality of groups of biotin solutions with different concentrations according to the content of biotin,
adding a magnetic nano microsphere solution into a biotin solution to obtain a plurality of groups of mixed solutions;
and carrying out constant-temperature oscillation complexation on the mixed solution, wherein the oscillation time is 30-70min, the temperature is set to be 20-40 ℃, and the rotation speed during oscillation is 800-.
4. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the Biotin solution comprises a D-Biotin (D-Biotin) working solution.
5. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the method for respectively cleaning and carrying out the labeling reaction on the multiple groups of mixed liquor comprises the following steps:
adding PBST into the mixed solution, and oscillating at constant temperature;
after shaking, carrying out magnetic attraction separation and removing supernatant;
adding a marking solution, and oscillating at constant temperature to carry out marking reaction.
6. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 5, wherein the method comprises the following steps: the marking solution comprises one or more of Biotin-HRP and Biotin-AP.
7. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the method for carrying out color reaction by adding color developing solution into the marked product comprises the following steps:
PBST is respectively added into the products for washing, constant temperature oscillation is carried out, and the supernatant is removed after magnetic attraction separation;
adding color development liquid, mixing uniformly, and performing light-proof color development;
and adding the color developing solution again after the color development to terminate the reaction.
8. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the color development liquid comprises one or more of TMB color development liquid, OPD, ABTS and p-NPP.
9. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 8, wherein the method comprises the following steps: the method for measuring the OD value comprises the following steps:
measuring the value of OD450 by a microplate reader when developing with TMB;
the value of OD492 was measured by a microplate reader when developing with OPD;
measuring the value of OD405 by a microplate reader when developing using ABTS;
the value of OD405 was measured by a microplate reader when developing with p-NPP.
10. The method for detecting the loading amount of free biotin combined with streptavidin magnetic microspheres according to claim 1, wherein the method comprises the following steps: the method for calculating the biotin binding capacity according to the obtained multiple groups of OD values comprises the following steps:
drawing a scatter plot line graph according to the content value and OD value of biotin;
calculating by combining a scattered point line graph to obtain an OD value of a biotin binding capacity saturation point;
and calculating the biotin loading capacity of the streptavidin magnetic microspheres according to the OD value.
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| CN114324857A (en) * | 2021-12-31 | 2022-04-12 | 北京普赞生物技术有限公司 | Microsphere for amplifying ELISA kit signal and preparation method thereof |
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