CN102539420A - Method for detecting concentration of protein in liquid - Google Patents
Method for detecting concentration of protein in liquid Download PDFInfo
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- CN102539420A CN102539420A CN2012100302939A CN201210030293A CN102539420A CN 102539420 A CN102539420 A CN 102539420A CN 2012100302939 A CN2012100302939 A CN 2012100302939A CN 201210030293 A CN201210030293 A CN 201210030293A CN 102539420 A CN102539420 A CN 102539420A
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Abstract
The invention discloses a method for detecting concentration of protein in a liquid, and belongs to the technical field of food inspection and detection. The method comprises the following steps that: the protein in a liquid sample is combined with ferric porphyrin to form a compound with a peroxidase function, the compound catalyzes hydrogen peroxide (H2O2) to generate active oxygen, and the active oxygen oxidizes luminol to form an organic peroxide; the peroxide is instable, nitrogen is immediately obtained through decomposition, and excited state amino-o-phthalic acid is generated; and during conversion from an excited state to a ground state, the released energy is detected in a photon form, and the quantity of the protein in the liquid sample to be detected can be calculated by detecting the change of the number of photons before and after reaction. The invention has the advantages that: the method has the characteristics of high sensitivity, low interference, economy, practicability and the like, and can be widely applied to the field of food safety monitoring in medicine health and food manufacturers, farmer's markets, quality supervision and epidemic prevention departments and the like.
Description
Technical field
The invention belongs to Food Inspection, detection technique field.
Background technology
Protein and origin of life, existence and evolution cut are closed, and protein determination relates to various fields such as life science, Food Science.Protein determination mainly contains Kjeldahl, isoelectric point precipitation, ultraviolet absorption method, biuret method, coomassie brilliant blue staining method, capillary electrophoresis, ELISA method, direct potentiometric method, high performance liquid chromatography detection etc. at present.Three kinds of classical ways commonly used, i.e. Kjeldahl, biuret method and ultraviolet absorption method.The new determination method that also had just generally use in addition in a kind of nearly ten years, i.e. Coomassie brilliant blue method.Wherein the sensitivity of Coomassie brilliant blue method is the highest.Though the nitriding more complicated is more accurate, the protein of often measuring with nitriding is as the standard protein of additive method.
What be decided to be national standard is that Kjeldahl is the method for 19 th century later Dane John Kai Daer invention; Principle is very simple: protein contains the nitrogen element; Use the strong acid treatment sample; Let the nitrogen element in the protein discharge, measure the content of nitrogen, multiply by 6.38 and just can calculate Protein content.So what in fact Kjeldahl was surveyed is not protein content, but calculate protein content through surveying nitrogen content, if also have other compounds to contain nitrogen in the sample, the method is just inaccurate.Under normal conditions, the principal ingredient in the food has only protein to contain nitrogen, and other principal ingredients (carbohydrates, fat) are all nonnitrogenous, so Kjeldahl is a kind of method for determination of protein of measuring very accurately.If but there is the people in sample, to add nitrogenous other materials (like urea, melamine etc.) steathily, the Kjeldahl of just can out-tricking obtains false protein high-load, pretends to be former milk with converting buffalo milk.
Also there are different problems such as apparatus expensive, complicated operation, sensitivity be low in other protein concentration detection methods.
Peroxidase (PX) is one type of oxidoreducing enzyme that from plant, animal, microorganism, extracts.In the vital movement process, peroxidase mainly is oxide or superoxide and other toxin of oxygenolysis in the catalytic decomposition biosome.Though have common biologically active at the various peroxidase of occurring in nature, do not have the same or analogous structure and the mechanism of action.Wherein the protoheme peroxidase (HPX) of containing metal porphyrin ring is that a class formation is similar, and the enzyme that function is identical belongs to protoheme PX superfamily protein member, and ferriporphyrin is exactly one of them.
Summary of the invention
The objective of the invention is: a kind of liquid protein concentration detection method is provided; It utilizes the principle that has the peroxidase function after ferriporphyrin and the protein bound; Prepare a kind of protein detection reagents, set up a kind of detection method of protein in the fluid sample being carried out quantitative measurement.Through the variation of photon number before and after the detection reaction, can calculate the amount of protein in the testing liquid sample.
Detection method of the present invention is:
1, detecting principle is:
Protein combines the back to form compound in the fluid sample with ferriporphyrin, has the peroxidase function, catalyzing hydrogen peroxide (H
2O
2) produce active oxygen, the oxidation luminol, luminol forms an organic peroxide.This superoxide is very unstable, decomposites nitrogen immediately, generates the aminophthalic acid of excited state.During excited state to ground state transformed, the energy of release through the variation of photon number before and after the detection reaction, can calculate the amount of protein in the testing liquid sample with this detection of form of photon.
2, the reagent composition is:
One, with TMB be the experimental procedure of substrate:
Experiment reagent:
1, ferriporphyrin reagent: secondary heme (or secondary heme six peptides): 4-20 μ M/L.(I)
2, pH 7.4PBS:KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, NaCL 8.0g, KCL 0.2g adds water to 1L.(II)
3, TMB (tetramethyl benzidine) uses liquid: TMB (10mg/mL absolute ethyl alcohol) 0.5mL, substrate buffer solution (pH5.5) 10mL, 0.75%H
2O
232 μ L.(III)
4, stop buffer: distilled water 178.3mL dropwise adds the concentrated sulphuric acid (98%) 21.7mL.(IV)
Experimental technique:
The protein sample of ferriporphyrin reagent (I) with variable concentrations diluted and mixing with PBS (II); Every hole adds 100 μ L (I) and mixed liquid of protein on 96 orifice plates; The concentration of (I) is in 4-20 μ M/L (cause consumption different because of testing difference, the common experimental scope is concentration for this reason) in every hole at this moment, and both every hole includes 0.2-1 μ g secondary heme (or 0.4-2 μ g secondary heme six peptides); Every then hole 100 μ L add TMB and use liquid (III), and the final concentration of (I) is 2-10 μ M/L at this moment.Behind the reaction beginning 10min, every hole adds 50 μ L stop buffers (IV), and ELIASA detects the 450nm light absorption value then.
Two, with the luminol be the experimental procedure of substrate:
Experiment reagent:
1, ferriporphyrin reagent: secondary heme (or secondary heme six peptides): 4-20 μ M/L.(I)
2, pH 7.4PBS:KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, NaCL 8.0g, KCL 0.2g adds water to 1L.(II)
3, luminol uses liquid: take by weighing quantitative luminol and be added among the PBS, put into 10 seconds of ultrasonic generator then, add 1 triethylamine hydrotropy after the taking-up, can use after the filtration.Luminol dilutes 100 times with PBS during use, adds oxydol to final concentration 100mM.(V)
Experimental technique:
The protein sample of ferriporphyrin reagent (I) with variable concentrations diluted and mixing with PBS (II); Every hole adds 100 μ L (I) and mixed liquid of protein on 96 orifice plates; The concentration of (I) is in 4-20 μ M/L (cause consumption different because of testing difference, the common experimental scope is concentration for this reason) in every hole at this moment, and both every hole includes 0.2-1 μ g secondary heme (or 0.4-2 μ g secondary heme six peptides); Every then hole 100 μ L add luminol and use liquid (V), and the final concentration of (I) is 2-10 μ M/L at this moment.Add the back and detect values of chemiluminescence with ELIASA at once.
3, operating process
1. fluid sample is joined in the test tube that ferriporphyrin reagent is housed following 10 minutes of room temperature;
2. to 1. adding hydrogen peroxide agent and luminol reagent respectively in the test tube, liquid in the test tube is moved in the luminous detection appearance, room temperature was reacted preceding photon counting in following 1 minute;
3. room temperature is reacted the back photon counting in the time of following 5 minutes, calculates the variation of reaction front and back photon number, and quantitative measurement goes out Protein content in the testing liquid sample.
The invention has the beneficial effects as follows: have characteristics such as highly sensitive, interference is low and economical and practical, can be widely used in departments such as medical and health and food production enterprise, the market of farm produce, quality supervision, health and epidemic prevention food security is carried out the monitoring field.
Description of drawings
Fig. 1 is that secondary heme six peptides combine the back to measure luminol chemiluminescence value result with variable concentrations bovine serum albumin(BSA) (BSA)
(A group is secondary heme six peptide final concentrations 2.5 μ M/L, and BCDEF is respectively that to have mixed final concentration in 2.5 μ M/L secondary hemes, six peptide solutions be 62.5,125,250, the BSA of 500,750 μ g/ml).
Fig. 2 is that secondary heme six peptides combine the back to measure luminol chemiluminescence value result with variable concentrations HSA
(A group is secondary heme six peptide final concentrations 2.5 μ M/L, and BCDEF is respectively that to have mixed final concentration in 2.5 μ M/L secondary hemes, six peptide solutions be 62.5,125,250, the HSA of 500,750 μ g/ml).
Fig. 3 is that secondary heme combines back TMB reaction solution result with variable concentrations human serum albumins (HSA)
(A group is secondary heme final concentration 10 μ M/L, and BCDEF is respectively that to have mixed final concentration in the 10 μ M/L secondary heme solution be 100,200,300, the HSA of 400,500 μ g/ml).
Fig. 4 is that secondary heme combines the back to measure luminol chemiluminescence value result with different protein samples
(the A group is secondary heme final concentration 2 μ M/L, and BCDE is respectively 62.5,125 in the secondary heme solution of 2 μ M/L, having added final concentration; The BSA of 500,750 μ g/ml, FG are two kinds of milk dilute samples; Its standard protein final concentration is respectively 375,375 μ g/ml).
Bring FG two sample measured values into the typical curve protein concentration record that converts and be respectively 365.62,354.82 μ g/ml, the two standard protein concentration is 375 μ g/ml, so correction coefficient=375*2/ (365.62+354.82)=1.041.
Fig. 5 be secondary heme with dilution after human serum combines TMB reaction solution result afterwards
(A group is secondary heme final concentration 10 μ M/L, and BCDEF is respectively and has mixed in the 10 μ M/L secondary heme solution that to have mixed final concentration in the secondary heme solution be 100,200; 300; The HAS of 400,500 μ g/ml, GH have mixed two kinds of standard serums in the secondary heme solution; The albumin final concentration is respectively 400,162.5 μ g/ml).
Fig. 6 secondary heme combines the back to measure luminol chemiluminescence value result with different protein samples
(the A group is secondary heme final concentration 2 μ M/L, and BCDE is for to have added the BSA that final concentration is respectively 62.5,125,500,750 μ g/ml in the secondary heme solution of 2 μ M/L, and F sour milk dilute sample, its standard protein final concentration are 375 μ g/ml).
Fig. 7 is that secondary heme combines the back to measure luminol chemiluminescence value result with different protein samples
(the A group is secondary heme final concentration 2 μ M/L, and BCDE is for to have added the BSA that final concentration is respectively 62.5,125,500,750 μ g/ml in the secondary heme solution of 2 μ M/L, and F milk beverage dilute sample, its standard protein final concentration are 125 μ g/ml).
Fig. 8 secondary heme combines the back to measure luminol chemiluminescence value result with different protein samples
(A group is secondary heme final concentration 5 μ M/L, and BCDEF has been for having added the BSA that final concentration is respectively 62.5,125,250,500,750 μ g/ml in the secondary heme solution of 5 μ M/L, and GH is respectively 125,500 urea for having added final concentration).
Fig. 9 secondary heme combines TMB reaction solution result with different protein samples
(the A group is secondary heme final concentration 5 μ M/L, and BCDE is respectively 62.5,125 in the secondary heme solution of 5 μ M/L, having added final concentration; 187.5; The BSA of 250 μ g/ml, FGH is respectively 62.5,125 for having added the protein final concentration; The almond milk of 250 μ g/ml, I is for having added 250 μ g/ml melamine groups).
Embodiment
Embodiment 1
(1) experiment reagent:
1, substrate buffer solution: 0.2M Na
2HPO
425.7ml 0.1M citric acid 24.3ml adds water to 50ml.
2, TMB (tetramethyl benzidine) uses liquid: TMB (10mg/ml absolute ethyl alcohol), 0.5ml substrate buffer solution (PH5.5) 10ml, 0.75%H
2O
232 μ l.
3, PH 7.4PBS:KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, NaCl 8.0g, KCl 0.2g adds water to 1L.
4, stop buffer: distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.
5, luminol uses liquid: take by weighing quantitative luminol and be added among the PBS, put into 10 seconds of ultrasonic generator then, add 1 triethylamine hydrotropy after the taking-up, can use after the filtration.Luminol dilutes 100 times with PBS during use, adds oxydol to final concentration 100mM.
(2) experimental technique:
1, with TMB be the experimental procedure of substrate:
The protein sample of secondary heme (or secondary heme six peptides) with variable concentrations diluted and mixing with PBS; Every hole 100 μ l secondary heme and mixed liquid of protein; Each concentration protein sample is done in parallel adding 96 orifice plates in 4 holes; The concentration of secondary heme (or secondary heme six peptides) (causes consumption different because of testing difference at 4-20 μ M/L in every hole at this moment; The common experimental scope is concentration for this reason), both every hole includes the every then hole 100 μ l of 0.2-1 μ g secondary heme (or 0.4-2 μ g secondary heme six peptides) and adds TMB use liquid, and the final concentration of secondary heme (or secondary heme six peptides) is 2-10 μ M/L.Behind the reaction beginning 10min, every hole adds 50 μ l stop buffers, and ELIASA detects the 450NM light absorption value then.
2, with the luminol be the experimental procedure of substrate:
The protein sample of secondary heme and variable concentrations is diluted and mixing with PBS; Every hole 100 μ l secondary heme six peptides (or secondary heme) and mixed liquid of protein; Each concentration protein sample is made 4 parallel holes and is added in 96 orifice plates; The concentration of secondary heme (or secondary heme six peptides) is at 4-20 μ M/L in this moment every hole; Both every hole includes the every then hole 100 μ l of 0.2-1 μ g secondary heme (or 0.4-2 μ g secondary heme six peptides) and adds luminol use liquid, and making the final concentration of secondary heme (or secondary heme six peptides) is 2-10 μ M/L.Add the back and detect values of chemiluminescence with ELIASA at once.
3, protein detection method:
Utilize above-mentioned two kinds of methods; When detecting protein sample protein sample to be detected is mixed back and TMB or luminol reflection detection luminous value with the same secondary heme of the albumen of concentration known (or secondary heme six peptides) at every turn; Utilize the detected value and the protein concentration of known protein solution to set up typical curve; Bring sample result into typical curve again; Protein concentration after the conversion multiply by the protein concentration that correction coefficient promptly obtains this sample (because the material of interference measurement is different in the different protein samples, so measure the correction coefficient that difference such as serum, milk should add a standard greatly during the class sample).Deviation (%)=[(detection-standard)/standard] * 100.
4, standard curve determination:
Like Fig. 1,2, shown in 3.
(1), measure the milk sample correction coefficient:
As shown in Figure 4, to bring FG two sample measured values into the typical curve protein concentration record that converts and be respectively 365.62,354.82 μ g/ml, the two standard protein concentration is 375 μ g/ml, so correction coefficient=375*2/ (365.62+354.82)=1.041.
(2), blood serum sample correction coefficient:
As shown in Figure 5; Bring three kinds of sample determination values of GHI into the typical curve protein concentration record that converts and be respectively 362.5,130.40 μ g/ml, the two standard protein concentration is respectively 400; 162.5 μ g/ml, thus correction coefficient=(400/362.5+162.5/130.4)/2=1.17.
(3), sour milk sample correction coefficient:
As shown in Figure 6, to bring G sample determination value into the typical curve protein concentration record that converts and be respectively 309.68 μ g/ml, standard protein concentration is 375 μ g/ml, so correction coefficient=375/309.68=1.21.
(4), fruit milk (milk beverage) sample correction coefficient:
As shown in Figure 7, to bring G sample determination value into the typical curve protein concentration record that converts and be respectively 187.32 μ g/ml, standard protein concentration is 125 μ g/ml, so correction coefficient=125/187.32=0.667.
Table one, 5 kinds of milk sample protein contents (dilution back μ g/ml) testing result and and deviation
Table two, 5 kinds of sour milk sample protein content (dilution back μ g/ml) testing result and and deviation
Table three, 5 kinds of fruits suckle sample protein content (dilution back μ g/ml) testing result and and deviation
Table four, 6 parts of blood serum sample protein contents (dilution back μ g/ml) testing result and and deviation
As shown in Figure 8; The result shows that mixing with BSA afterwards for secondary heme is that the peroxidase activity of substrate all has humidification with the luminol; This humidification and BSA concentration are approximate proportional; Live and do not see that obvious increase, the urea that contains in the interpret sample can not disturb combining of secondary heme and albumen and mixed enzyme behind the urea.
As shown in Figure 9; Can find by this result; The enzyme activity that mixes the back secondary heme with almond milk or melamine does not have obvious raising, in almond milk even also, is suppressed, and explains and utilizes this method can't detect protein content in the almond milk; When detecting the liquid milk protein content, might not receive the interference of melamine.
Claims (2)
1. liquid protein concentration detection method, its method is:
Reagent is formed
With TMB is the experimental procedure of substrate
Experiment reagent
A, ferriporphyrin reagent, 4-20 μ M/L;
B, pH 7.4PBS:KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, NaCL 8.0g, KCL 0.2g adds water to 1L;
C, tetramethyl benzidine use liquid, TMB0.5mL, substrate buffer solution 10mL, 0.75%H
2O
232 μ L;
D, stop buffer, distilled water 178.3mL dropwise adds concentrated sulphuric acid 21.7mL;
Experimental technique
The protein sample of ferriporphyrin reagent and variable concentrations is diluted and mixing with PBS; Every hole adds 100 μ L and mixed liquid of protein on 96 orifice plates; The concentration of ferriporphyrin reagent is at 4-20 μ M/L in this moment every hole; Both every hole includes 0.2-1 μ g secondary heme or 0.4-2 μ g secondary heme six peptides, and every then hole 100 μ L add TMB and use liquid, and this moment, the final concentration of ferriporphyrin reagent was 2-10 μ M/L; Behind the reaction beginning 10min, every hole adds 50 μ L stop buffers, and ELIASA detects the 450nm light absorption value then;
Operating process
1. fluid sample is joined in the test tube that ferriporphyrin reagent is housed following 10 minutes of room temperature;
2. to 1. adding hydrogen peroxide agent and luminol reagent respectively in the test tube, liquid in the test tube is moved in the luminous detection appearance, room temperature was reacted preceding photon counting in following 1 minute;
3. room temperature is reacted the back photon counting in the time of following 5 minutes, calculates the variation of reaction front and back photon number, and quantitative measurement goes out Protein content in the testing liquid sample.
2. liquid protein concentration detection method, its method is:
Reagent is formed
With the luminol is the experimental procedure of substrate
Experiment reagent
A, ferriporphyrin reagent, 4-20 μ M/L;
B, pH 7.4PBS, KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, NaCL 8.0g, KCL 0.2g adds water to 1L;
C, luminol use liquid, take by weighing quantitative luminol and are added among the PBS, put into 10 seconds of ultrasonic generator then, add 1 triethylamine hydrotropy after the taking-up, can use after the filtration; Luminol dilutes 100 times with PBS during use, adds oxydol to final concentration 100mM;
Experimental technique
The protein sample of ferriporphyrin reagent and variable concentrations is diluted and mixing with PBS; Every hole adds 100 μ L ferriporphyrin reagent and mixed liquid of protein on 96 orifice plates; The concentration of ferriporphyrin reagent is at 4-20 μ M/L in this moment every hole; Both every hole includes 0.2-1 μ g secondary heme or 0.4-2 μ g secondary heme six peptides, and every then hole 100 μ L add luminol and use liquid, and this moment, the final concentration of ferriporphyrin reagent was 2-10 μ M/L; Add the back and detect values of chemiluminescence with ELIASA at once;
Operating process
1. fluid sample is joined in the test tube that ferriporphyrin reagent is housed following 10 minutes of room temperature;
2. to 1. adding hydrogen peroxide agent and luminol reagent respectively in the test tube, liquid in the test tube is moved in the luminous detection appearance, room temperature was reacted preceding photon counting in following 1 minute;
3. room temperature is reacted the back photon counting in the time of following 5 minutes, calculates the variation of reaction front and back photon number, and quantitative measurement goes out Protein content in the testing liquid sample.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359897A (en) * | 2014-10-24 | 2015-02-18 | 福建工程学院 | Method for rapidly detecting melamine based on nanogold chemiluminiscence |
| CN105911005A (en) * | 2016-06-06 | 2016-08-31 | 长春理工大学 | Hydrogen peroxide photometric method adopting DhHP-6 mimic enzyme |
| CN107024584A (en) * | 2017-06-07 | 2017-08-08 | 嘉兴朝云帆生物科技有限公司 | Pass through the biological test paper detector of network calibration standard curve and its bearing calibration |
| CN110441250A (en) * | 2019-06-13 | 2019-11-12 | 吉林大学 | A kind of preparation method that double enzymes are total to fixed copper nanometer floral material and the application in glucose detection |
| CN111579548A (en) * | 2020-05-20 | 2020-08-25 | 重庆师范大学 | Luminol-gallium nano assembly and preparation method and application thereof |
| CN112858269A (en) * | 2020-12-25 | 2021-05-28 | 广东绿康医疗器械有限公司 | Preparation method and application method of detection reagent for nasopharyngeal mucosa cell free heme |
| CN115112742A (en) * | 2022-07-01 | 2022-09-27 | 山东先声生物制药有限公司 | Method for correcting molecular weight isoelectric point by using physicochemical reference substance |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359897A (en) * | 2014-10-24 | 2015-02-18 | 福建工程学院 | Method for rapidly detecting melamine based on nanogold chemiluminiscence |
| CN105911005A (en) * | 2016-06-06 | 2016-08-31 | 长春理工大学 | Hydrogen peroxide photometric method adopting DhHP-6 mimic enzyme |
| CN107024584A (en) * | 2017-06-07 | 2017-08-08 | 嘉兴朝云帆生物科技有限公司 | Pass through the biological test paper detector of network calibration standard curve and its bearing calibration |
| CN110441250A (en) * | 2019-06-13 | 2019-11-12 | 吉林大学 | A kind of preparation method that double enzymes are total to fixed copper nanometer floral material and the application in glucose detection |
| CN111579548A (en) * | 2020-05-20 | 2020-08-25 | 重庆师范大学 | Luminol-gallium nano assembly and preparation method and application thereof |
| CN111579548B (en) * | 2020-05-20 | 2022-03-18 | 重庆师范大学 | Luminol-gallium nano assembly and preparation method and application thereof |
| CN112858269A (en) * | 2020-12-25 | 2021-05-28 | 广东绿康医疗器械有限公司 | Preparation method and application method of detection reagent for nasopharyngeal mucosa cell free heme |
| CN115112742A (en) * | 2022-07-01 | 2022-09-27 | 山东先声生物制药有限公司 | Method for correcting molecular weight isoelectric point by using physicochemical reference substance |
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