CN107255720A - A kind of method that protein concentration is detected based on cobalt nano-particle - Google Patents
A kind of method that protein concentration is detected based on cobalt nano-particle Download PDFInfo
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Abstract
The present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle, including：(1) antibody of material to be detected is modified using cobalt nano-particle；(2) monoclonal antibody of testing sample is added to 96 orifice plates, constant temperature is stood overnight；(3) solution in (2) is discarded, 96 orifice plate twice is cleaned using the phosphate buffer of tween is added；Add the standard sample of different protein concentrations per hole to 96 orifice plates again, constant temperature is stood；(4) solution in (2) is discarded, 96 orifice plate twice is cleaned using the phosphate buffer of tween is added；Every hole into 96 orifice plates adds testing sample again, and constant temperature is stood；(5) solution in (3) and (4) is discarded, 96 orifice plate twice is cleaned using the phosphate buffer of tween is added；Every hole into 96 orifice plates adds cobalt nano-particle labelled antibody again, the step such as constant temperature is stood.With convenient and swift detection, analysis cost is low, do not need that large-sized analytic instrument, sensitivity be higher, testing result more accurately feature.
Description
Technical field
The invention belongs to technical field of analysis and detection, it is related to a kind of method that protein concentration is detected based on cobalt nano-particle.
Background technology
Existing chemiluminescence detection method of protein includes direct chemiluminescence, enzyme-catalyzed chemical luminescence and electrochemistry hair
Three kinds of light：(1) directly chemoluminescence method be possible to produce chemiluminescence signal molecule be marked directly on antibody, add phase
Reactant is closed, chemiluminescence signal is produced；(2) enzyme-catalyzed chemical luminescence utilizes enzyme labelled antibody, and substrate etc. produces chemiluminescence letter
Number；(3) electrochemical luminescence is to produce chemiluminescence signal using electrochemical reaction and related substrates.
But, there is following defect in above-mentioned prior art：(1) directly chemoluminescence method is directly produced using correlation molecule
Chemiluminescence signal, without reference to the signal amplification procedure such as catalytic reaction, sensitivity is not accurate enough；(2) enzyme-catalyzed chemical luminescence
Method is related to enzyme labelled antibody, it is necessary to consider the influence to testing result such as enzymatic activity, and detection requires higher；(3) Electrochemiluminescince
In relate to electrochemical reaction, detecting instrument is more more complicated than foregoing two kinds, and analysis cost is high.
The content of the invention
Cobalt nano-particle and chemiluminescence detection protein concentration are based on the technical problem to be solved in the present invention is to provide one kind
Highly sensitive method, with convenient and swift can detecting, analysis cost is low, do not need relatively large analytical instrument, sensitivity it is higher,
The characteristics of accuracy rate of testing result is high, be particularly suitable for use in large biological molecule, such as include protein, nucleic acid sample it is highly sensitive
Detection.
In order to solve the above-mentioned technical problem, the present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle,
It is characterised in that it includes following steps：
Step one, the antibody of detected sample is modified using cobalt nano-particle, cobalt nano-particle modified antibodies are obtained molten
Liquid；
In step 2, the experimental port that the monoclonal antibody of testing sample is added separately to 96 orifice plates, constant temperature was stood
At night, experimental port is divided into two groups；
Step 3, discards upper solution resulting in first group of experimental port in step 2, uses the phosphate for adding tween
Test hole in the orifice plate of buffer solution for cleaning 96；Again into 96 orifice plates in first group of each experimental port add same volume, no
With the standard sample of protein concentration, constant temperature is stood；
Step 4, discards upper solution resulting in second group of experimental port in step 2, uses the phosphate for adding tween
Test hole in the orifice plate of buffer solution for cleaning 96；Testing sample is added in second group of each experimental port into 96 orifice plates again, it is permanent
Temperature is stood；
Step 5, obtained by discarding respectively in first group of experimental port of step 3 and second group of experimental port of step 4
Upper solution, reuses the experimental port for adding the phosphate buffer of tween to clean 96 orifice plates；Each experiment into 96 orifice plates again
Hole adds gained cobalt nano-particle modified antibodies solution in step one, and constant temperature is stood；
Step 6, the upper solution obtained by discarding in first group, second group experimental port of step 5, using adding tween
Phosphate buffer cleans the experimental port of 96 orifice plates；Each experimental port into 96 orifice plates adds inorganic acid aqueous solution, shake again
Swing；
Step 7, each resulting solution for taking out same volume from first group, second group each experimental port of step 6, plus
Enter in the corresponding alkaline solution to haematine；
Step 8, the chemiluminescence signal value of the standard sample of different protein concentrations in first group of detection, and according to concentration
Quantitation curves are made with the relation of luminous signal value；
Its signal value is substituted into gained in step 8 by step 9, the luminous signal value of testing sample in second group of detection
In quantitation curves, the protein concentration in testing sample is drawn.
It is preferred that, in the step one, the method for material antibody to be detected is modified such as using cobalt nano-particle
Under：
(1) cobalt nano-particle solution is mixed with testing sample Anti-TNF-α liquid solution, shaken；
(2) gained supernatant in above-mentioned (1) is centrifuged off, the antibody after cobalt nano-particle modification is collected, then use phosphate
Buffer solution disperses again, obtains cobalt nano-particle modified antibodies solution；
It is preferred that, in above-mentioned (1) mass ratio of cobalt nano-particle and testing sample polyclonal antibody be 1~
1000:1；Powdered form cobalt nano-particle is dissolved in phosphate buffer；The particle diameter of cobalt nano-particle be 10nm or 20nm or
50nm or 100nm.
It is preferred that, in the step one, the method for material antibody to be detected is modified such as using cobalt nano-particle
Under：
(1) the polyethyleneimine polymer solution of positively charged is mixed with cobalt nano-particle solution, shaken；
(2) gained supernatant in above-mentioned (1) is centrifuged off, the cobalt nano-particle after modification is collected；Cobalt nanometer after modification
Particle phosphate buffer disperses again, the cobalt nano-particle solution after being modified；
(3) added in above-mentioned (2) in gained cobalt nano-particle solution in the Anti-TNF-α liquid solution of testing sample, profit
Cobalt nano-particle is modified on the surface of antibody with Electrostatic Absorption, then the antibody after cobalt nano-particle modification is collected by centrifugation.
It is preferred that, the macromolecule PEI of positively charged concentration is 10~500mg/mL in above-mentioned (1)；Above-mentioned (1)
The concentration of middle cobalt nano-particle solution is 0.1~100mg/mL；The pH of phosphate buffer is 5-9 in above-mentioned (2)；Above-mentioned (3)
The concentration of middle Anti-TNF-α liquid solution is 0.5~2mg/mL.
It is preferred that, the standard sample in the step 3 is concentration for 0,0.01,0.02,0.05,0.1,
0.25th, 0.5,1,2,5,10,20ng/mL creatine kinase isozyme solution.
It is preferred that, in the step 2, constant temperature is stood overnight at a temperature of 2~8 DEG C.
It is preferred that, in the step 3,37 DEG C of constant temperature stand 1~4h.
It is preferred that, in the step 4 or step 5,37 DEG C of constant temperature stand 0.5~2h.
It is preferred that, in the step 5, the inorganic acid is the sulfuric acid or nitric acid that concentration is 5~20mmol/L
Or hydrochloric acid；In the step 6, the concussion time is 5~20min.
The present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle, compared with existing design, its advantage
It is：(1) present invention by cobalt nano-particle by cobalt nano-particle modification by, in antibody surface, discharging cobalt ions, because of one
Tens nano cobalt granules can discharge a large amount of cobalt ions, and cobalt ions can make catalysis substrate haematine produce stronger chemiluminescence
Signal, the color signal or fluorescence signal in detection method compared with prior art, chemiluminescence signal of the invention makes detection spirit
Sensitivity is higher, and sensitivity detection limit can reach 0.01ng/mL.(2) luminous signal of according to standard sample of the present invention and its albumen
The relation of concentration is made after quantitation curves, subsequently only needs to determine the luminous signal of corresponding testing sample, you can quick and precisely
Go out the protein concentration of testing sample, detection method is convenient and swift, accuracy rate of testing result is high.(3) it is many used in the inventive method
It is general chemistry reagent to plant reagent, it is not necessary to which relatively large analytical instrument, simple to operate, analysis cost is low.
Embodiment
The present invention detects that the method for protein concentration can detect a variety of bioproteins based on cobalt nano-particle, only needs to meet
The antibody of testing protein sample is commercially available or prepared, the present embodiment bioprotein with creatine kinase isozyme (hereafter
Abbreviation CK-MB) exemplified by.
The testing sample A and content that 1-3 of the embodiment of the present invention is 0.04ng/mL using commercially available, CK-MB contents be
0.5ng/mL testing sample B, CK-MB is prepared using phosphate buffer (abbreviation PBS buffer solutions).
Embodiment 1
The present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle, comprises the following steps：
Step one, CK-MB antibody is modified using cobalt nano-particle, the present embodiment utilizes the big ratio table of cobalt nano-particle
The characteristics of area, surface can be high, it is easy to adsorb antibody, the effect of the step is exactly to allow antibody absorption on cobalt nano-particle surface,
Follow-up release cobalt ions, enhanced chemiluminescence signal improves detection sensitivity, and method is as follows：
(1) the PBS dissolving, cobalt nano-particle that concentration is 0.1mg/mL for the use of pH value being 7.2 by 500 μ L
Anti-TNF-α liquid solution of the solution with 50 μ L concentration for 1mg/mL CK-MB is mixed, and is placed in 2mL centrifuge tube, and concussion 2 is small
When；Wherein, cobalt nano-particle is powdered, and its particle diameter can be to make in 10nm or 20nm or 50nm or 100nm, the present embodiment
The cobalt nano-particle for being 50nm with particle diameter；The solvent of Anti-TNF-α liquid solution is PBS；Add the polyclonal of CK-MB
Antibody-solutions can recognize albumen to be detected；
(2) it is centrifuged off supernatant in above-mentioned (1), collects the antibody after cobalt nano-particle modification, then with 5000 μ L PBS
Buffer solution disperses again, obtains cobalt nano-particle modified antibodies solution, standby；
Step 2, the monoclonal antibody solution by 100 μ L concentration for 0.005mg/mL CK-MB is added separately to 96 holes
In 14 experimental ports of plate, constant temperature is stood overnight, and the temperature of constant temperature can be 2~8 DEG C, and the thermostat temperature in the present embodiment is 4
℃；It is divided to two groups to test respectively.This step is used to allow CK-MB monoclonal antibody to be adsorbed by physical absorption on 96 orifice plates
In subsequent captured thing to be detected.
Step 3, discards upper solution resulting in first group of experimental port in step 2, adds tween using 200 μ L
The experimental port that phosphate buffer (hereinafter referred PBST buffer solutions) is cleaned in 96 orifice plates twice；Again into 96 orifice plates first group
12 experimental ports in sequentially add 100 μ L concentration for 0,0.01,0.02,0.05,0.1,0.25,0.5,1,2,5,10,
20ng/mL solvent is the CK-MB solution of PBS, and 37 DEG C of constant temperature are stood, and the time that constant temperature is stood can be small for 1~4
When, constant temperature stands 2 hours in the present embodiment.
Step 4, discards upper solution resulting in second group of 2 experimental port in step 2, uses 200 μ L PBST
The experimental port cleaned in 96 orifice plates twice；Sequentially added again in 2 experimental ports into 96 orifice plates 100 μ L testing samples A and
100 μ L testing samples B, 37 DEG C of constant temperature are stood, and the time that constant temperature is stood can be 0.5~2 hour, and constant temperature is quiet in the present embodiment
Put 1 hour.
Step 5, the upper solution obtained by discarding respectively in step 3 and step 4 experimental port, uses 200 μ L PBST
Clean the experimental port of 96 orifice plates twice；Each experimental port into 96 orifice plates adds the step of 100 μ L concentration are 10 μ g/mL again
Gained cobalt nano-particle modified antibodies solution in one, 37 DEG C of constant temperature are stood, and the time that constant temperature is stood can be 0.5~2 hour,
Constant temperature stands 1 hour in the present embodiment.The effect of this step is to allow cobalt nano-particle modified antibodies and thing phase interaction to be detected
With, it is follow-up to discharge cobalt ions, chemiluminescence signal is produced, is detected.
Step 6, the upper solution obtained by discarding in step 5 experimental port cleans three 96 holes using 200 μ L PBST
Plate；Each experimental port into 96 orifice plates adds the dust technology that 10 μ L concentration are 10mmol/L again, concussion, and the concussion time can be with
For 5~20min, the concussion time is 10min in the present embodiment.It can be dissolved and released using inorganic acids such as dust technology, hydrochloric acid, sulfuric acid
Put the cobalt ions in cobalt nano-particle.
Step 7, it is each from first group, second group each experimental port of step 6 to take out 10 μ L resulting solutions, add to Soviet Union
In another name for alkaline solution.Haematine alkaline solution obtains mixed liquor using following solution mixing system in the present invention：The anhydrous second of 2.5mL
The concentration of alcohol configuration is 0.005mol/L hematoxylin solutions, and the concentration of 2.5mL water configuration is 2.58mol/L H2O2,0.2mL
The KOH solution that the concentration of water configuration is 1mol/L.Under alkaline reaction environment, cobalt ions catalysis haematine produces stronger change
Luminous signal is learned, cobalt ions is catalyst, and one tens nanometers of cobalt nano-particle contains up to ten thousand cobalt ions, realizes signal
The effect of amplification.
Step 8,12 concentration knowns in first group are detected using BioTek multi-function microplate readers (Synergy H1)
The chemiluminescence signal value of CK-MB standard samples, and quantitation curves are made according to the relation of its concentration and luminous signal value；See
Table 1, Fig. 1.
Step 9, testing sample A and to be measured in second group is detected using BioTek multi-function microplate readers (Synergy H1)
Sample B luminous signal value, its signal value is substituted into the quantitation curves obtained by step 8, CK-MB in testing sample is drawn
Concentration.
As shown in figure 1, the quantitation curves equation of the embodiment of the present invention 1 is：Y=118674.4x+14252.7, wherein, R2
=0.999；The luminous signal value for measuring testing sample A, B is respectively 19305.7,69105.6, by above-mentioned quantitation curves equation
It is 0.043 ng/mL, 0.46ng/mL respectively to draw the CK-MB concentration in testing sample A, B, with its concentration known 0.04ng/
ML, 0.5ng/mL are compared, and relative error is 7.5%, 8%.
Embodiment 2
The present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle, the embodiment of the present invention 2 and embodiment
1 method is essentially identical, except that the concentration of cobalt nano-particle solution, comprises the following steps：
Step one, CK-MB antibody is modified using cobalt nano-particle, the present embodiment utilizes the big ratio table of cobalt nano-particle
Area directly adsorbs antibody, because cobalt nano-particle specific surface area is big, and surface can be high, it is easy to which the absorption of protein, method is as follows：
(1) by 500 μ L are dissolved using PBS, cobalt nano-particle solution and the 50 μ L concentration that concentration is 100mg/mL be
1mg/mL CK-MB Anti-TNF-α liquid solution mixing, is placed in 2mL centrifuge tube, shakes 2 hours；Wherein, cobalt nanometer
Grain is powdered, and its particle diameter can be using the cobalt that particle diameter is 50nm in 10nm or 20nm or 50nm or 100nm, the present embodiment
Nano particle.
(2) it is centrifuged off supernatant in above-mentioned (1), collects the antibody after cobalt nano-particle modification, then with 5000 μ L PBS
Buffer solution disperses again, obtains cobalt nano-particle modified antibodies solution, standby；
Step 2, the monoclonal antibody solution by 100 μ L concentration for 0.005mg/mL CK-MB is added separately to 96 holes
In 14 experimental ports of plate, 4 DEG C of constant temperature are stood overnight, and are divided to two groups to test respectively.
Step 3, discards upper solution resulting in first group of 12 experimental port in step 2, uses 200 μ L PBST
The experimental port cleaned in 96 orifice plates twice；Sequentially added again in 12 experimental ports into 96 orifice plates 100 μ L concentration for 0,
0.01st, 0.02,0.05,0.1,0.25,0.5,1,2,5,10,20ng/mL CK-MB solution, 37 DEG C of constant temperature stand 2 hours.
Step 4, discards upper solution resulting in second group of 2 experimental port in step 2, uses 200 μ L PBST
The experimental port cleaned in 96 orifice plates twice；Sequentially added again in 2 experimental ports into 96 orifice plates 100 μ L testing samples A and
100 μ L testing samples B, 37 DEG C of constant temperature stand 1 hour.
Step 5, the upper solution obtained by discarding respectively in step 3 and step 4 experimental port, uses 200 μ L PBST
Clean the experimental port of 96 orifice plates twice；Each experimental port into 96 orifice plates adds the step of 100 μ L concentration are 10 μ g/mL again
Gained cobalt nano-particle modified antibodies solution in one, 37 DEG C of constant temperature stand 1 hour.
Step 6, the upper solution obtained by discarding in step 5 experimental port cleans three 96 holes using 200 μ L PBST
Plate；Each experimental port into 96 orifice plates adds the dust technology that 10 μ L concentration are 10mmol/L again, shakes 10min.
Step 7, it is each from first group, second group each experimental port of step 6 to take out 10 μ L resulting solutions, add to Soviet Union
Another name for alkaline solution.
Step 8,12 concentration knowns in first group are detected using BioTek multi-function microplate readers (Synergy H1)
The chemiluminescence signal value of CK-MB standard samples, and quantitation curves are made according to the relation of its concentration and luminous signal value；See
Table 1, Fig. 2.
Step 9, testing sample A and to be measured in second group is detected using BioTek multi-function microplate readers (Synergy H1)
Sample B luminous signal value, its signal value is substituted into the quantitation curves obtained by step 8, CK-MB in testing sample is drawn
Concentration.
As shown in Fig. 2 the quantitation curves equation of the embodiment of the present invention 2 is：Y=122670.6x+11874.2, wherein, R2
=0.999；The luminous signal value for measuring testing sample A, B is respectively 17208.2,70025.3, by above-mentioned quantitation curves equation
It is that 0.043 ng/mL, 0.47ng/mL, i.e. relative error are 7.5% respectively to draw the CK-MB concentration in testing sample A, B,
6%.
Embodiment 3
The present invention provides a kind of method that protein concentration is detected based on cobalt nano-particle, the embodiment of the present invention 2 and embodiment
1 put is sent out essentially identical, except that the antibody method of material to be detected is modified using cobalt nano-particle, including following step
Suddenly：
Step one, the antibody of material to be detected is modified using cobalt nano-particle, the present embodiment will be anti-using Electrostatic Absorption method
Body modification is on cobalt nano-particle surface, because cobalt nano-particle specific surface area is big, and surface can be high, it is easy to which coated high molecular, method is such as
Under：
(1) by polyethyleneimine polymer solution (abbreviation PEI) and 500 of the 500 μ L concentration for 100mg/mL positively charged
μ L are dissolved using PBS solvents, concentration mixes for 10mg/mL cobalt nano-particle solution, is placed in 2mL centrifuge tube, room temperature
Shake 2h；Wherein, the concentration of the polyethyleneimine polymer solution (abbreviation PEI) of positively charged is 10~500mg/mL；Cobalt nanometer
The concentration of particle solution can be 0.1~100mg/mL；Cobalt nanometer is powdered, and its particle diameter can be 10nm or 20nm or 50
The cobalt nano-particle that particle diameter is 50nm is used in nm or 100nm, the present embodiment；This step uses physical absorption, cobalt nano-particle
Specific surface area is big, and surface can be high, it is easy to coated high molecular；
(2) supernatant obtained by being centrifuged off in above-mentioned (1), collects the cobalt nano-particle after modification；Cobalt after modification
Nano particle is disperseed again with 500 μ L PBS, the cobalt nano-particle solution after being modified；Wherein, the pH value of PBS
Can be 5~9, the present embodiment uses the PBS buffer solutions that pH is 7.2；
(3) many grams of the CK-MB that 50 μ L concentration are 1mg/mL are added in gained cobalt nano-particle solution in above-mentioned (2)
Grand antibody-solutions, are adsorbed antibody on cobalt nano-particle surface using Electrostatic Absorption, then are collected by centrifugation after cobalt nano-particle modification
Antibody, then disperseed again with 5000 μ L PBSs, obtain cobalt nano-particle modified antibodies solution, it is standby；Wherein, it is many
The concentration range of clonal antibody solution is that concentration is 1mg/mL in 0.5~2mg/mL, the present embodiment.
Step 2, the monoclonal antibody solution by 100 μ L concentration for 0.005mg/mL CK-MB is added separately to 96 holes
In 14 experimental ports of plate, 4 DEG C of constant temperature are stood overnight, and are divided to two groups to test respectively.
Step 3, discards upper solution resulting in first group of 12 experimental port in step 2, uses 200 μ L PBST
The experimental port cleaned in 96 orifice plates twice；Sequentially added again in 12 experimental ports into 96 orifice plates 100 μ L concentration for 0,
0.01st, 0.02,0.05,0.1,0.25,0.5,1,2,5,10,20ng/mL CK-MB solution, 37 DEG C of constant temperature stand 2 hours.
Step 4, discards upper solution resulting in second group of 2 experimental port in step 2, uses 200 μ L PBST
The experimental port cleaned in 96 orifice plates twice；Sequentially added again in 2 experimental ports into 96 orifice plates 100 μ L testing samples A and
100 μ L testing samples B, 37 DEG C of constant temperature stand 1 hour.
Step 5, the upper solution obtained by discarding respectively in step 3 and step 4 experimental port, uses 200 μ L PBST
Clean the experimental port of 96 orifice plates twice；Each experimental port into 96 orifice plates adds the step of 100 μ L concentration are 10 μ g/mL again
Gained cobalt nano-particle modified antibodies solution in one, 37 DEG C of constant temperature stand 1 hour.
Step 6, the upper solution obtained by discarding in step 5 experimental port cleans three 96 holes using 200 μ L PBST
Plate；Each experimental port into 96 orifice plates adds the dust technology that 10 μ L concentration are 10mmol/L again, shakes 10min.
Step 7, it is each from first group, second group each experimental port of step 6 to take out 10 μ L resulting solutions, add to Soviet Union
Another name for alkaline solution.
Step 8,12 concentration knowns in first group are detected using BioTek multi-function microplate readers (Synergy H1)
The chemiluminescence signal value of CK-MB standard samples, and quantitation curves are made according to the relation of its concentration and luminous signal value；See
Table 1, Fig. 3.
Step 9, testing sample A and to be measured in second group is detected using BioTek multi-function microplate readers (Synergy H1)
Sample B luminous signal value, its signal value is substituted into the quantitation curves obtained by step 8, CK-MB in testing sample is drawn
Concentration.
As shown in figure 3, the quantitation curves equation of the embodiment of the present invention 3 is：Y=114272.3x+15065.7, wherein, R2
=0.999；The luminous signal value for measuring testing sample A, B is respectively 19856.8,75684.5, by above-mentioned quantitation curves equation
It is that 0.042 ng/mL, 0.53ng/mL, i.e. relative error are 5%, 6% respectively to draw the CK-MB concentration in testing sample A, B.
Comparative example
The direct chemoluminescence method detection CK-MB specifications of certain company of the invention write detection sensitivity for less than
0.625ng/mL, measures testing sample A, B in 1-3 of the embodiment of the present invention using this method, because sample concentration is too low, exceeds
The detection range of available reagent box, it is impossible to accurate detection.
Table 1
As shown in table 1, Fig. 1-3,1-3 of the embodiment of the present invention compares with comparative example to be drawn, is demonstrated the present invention and is based on cobalt
The feasibility of the method for nano particle detection protein concentration, can be used for detecting a variety of bioproteins, for different biological eggs
In vain, detection sensitivity is up to 0.01ng/mL, and this method is simple to operate, detection sensitivity is high, accuracy is very high, no longer separately lifts
Example.
Claims (10)
1. a kind of method that protein concentration is detected based on cobalt nano-particle, it is characterised in that comprise the following steps：
Step one, the antibody of detected sample is modified using cobalt nano-particle, cobalt nano-particle modified antibodies solution is obtained；
In step 2, the experimental port that the monoclonal antibody of testing sample is added separately to 96 orifice plates, constant temperature is stood overnight, will be real
Verify and be divided into two groups；
Step 3, discards upper solution resulting in first group of experimental port in step 2, uses the phosphate-buffered for adding tween
The test hole that liquid is cleaned in 96 orifice plates；Add same volume, different eggs in first group of each experimental port into 96 orifice plates again
The standard sample of white concentration, constant temperature is stood；
Step 4, discards upper solution resulting in second group of experimental port in step 2, uses the phosphate-buffered for adding tween
The test hole that liquid is cleaned in 96 orifice plates；Testing sample is added in second group of each experimental port into 96 orifice plates again, constant temperature is quiet
Put；
Step 5, the upper strata obtained by discarding respectively in first group of experimental port of step 3 and second group of experimental port of step 4 is molten
Liquid, reuses the experimental port for adding the phosphate buffer of tween to clean 96 orifice plates；Each experimental port into 96 orifice plates is added again
Gained cobalt nano-particle modified antibodies solution in step one, constant temperature is stood；
Step 6, the upper solution obtained by discarding in first group, second group experimental port of step 5, uses the phosphate for adding tween
The experimental port of the orifice plate of buffer solution for cleaning 96；Each experimental port into 96 orifice plates adds inorganic acid aqueous solution, concussion again；
Step 7, each resulting solution for taking out same volume from first group, second group each experimental port of step 6, is added relative
Should be into haematine alkaline solution；
Step 8, the chemiluminescence signal value of the standard sample of different protein concentrations in first group of detection, and according to concentration and hair
The relation of optical signal value makes quantitation curves；
Step 9, detection second group in testing sample luminous signal value, by its signal value substitute into step 8 in gained quantify
In curve, the protein concentration in testing sample is drawn.
2. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that described
In step one, the method for modifying material antibody to be detected using cobalt nano-particle is as follows：
(1) cobalt nano-particle solution is mixed with testing sample Anti-TNF-α liquid solution, shaken；
(2) gained supernatant in above-mentioned (1) is centrifuged off, the antibody after cobalt nano-particle modification is collected, then use phosphate-buffered
Liquid disperses again, obtains cobalt nano-particle modified antibodies solution.
3. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 2, it is characterised in that above-mentioned
(1) mass ratio of cobalt nano-particle and testing sample polyclonal antibody is 1~1000 in:1；Powdered form cobalt nano-particle is dissolved in
In phosphate buffer；The particle diameter of cobalt nano-particle is 10nm or 20nm or 50nm or 100nm.
4. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that described
In step one, the method for modifying material antibody to be detected using cobalt nano-particle is as follows：
(1) the polyethyleneimine polymer solution of positively charged is mixed with cobalt nano-particle solution, shaken；
(2) gained supernatant in above-mentioned (1) is centrifuged off, the cobalt nano-particle after modification is collected；Cobalt nano-particle after modification
Disperseed again with phosphate buffer, the cobalt nano-particle solution after being modified；
(3) added in above-mentioned (2) in gained cobalt nano-particle solution in the Anti-TNF-α liquid solution of testing sample, utilize electrostatic
Absorption modifies cobalt nano-particle on the surface of antibody, then the antibody after cobalt nano-particle modification is collected by centrifugation.
5. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 4, it is characterised in that above-mentioned
(1) the macromolecule PEI of positively charged concentration is 10~500mg/mL in；The concentration of cobalt nano-particle solution is 0.1 in above-mentioned (1)
~100mg/mL；The pH of phosphate buffer is 5-9 in above-mentioned (2)；The concentration of Anti-TNF-α liquid solution is 0.5 in above-mentioned (3)
~2mg/mL.
6. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that described
Standard sample in step 3 is concentration for 0,0.01,0.02,0.05,0.1,0.25,0.5,1,2,5,10,20ng/mL flesh
Acid kinase isodynamic enzyme solution.
7. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that institute
State in step 2, constant temperature is stood overnight at a temperature of 2~8 DEG C.
8. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that described
In step 3,37 DEG C of constant temperature stand 1~4h.
9. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that described
In step 4 or step 5,37 DEG C of constant temperature stand 0.5~2h.
10. a kind of method that protein concentration is detected based on cobalt nano-particle according to claim 1, it is characterised in that institute
State in step 5, the inorganic acid is the sulfuric acid or nitric acid or hydrochloric acid that concentration is 5~20mmol/L；In the step 6, concussion
Time is 5~20min.
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