CN103760121B - A kind of detection method of determinating nitrite in blood - Google Patents
A kind of detection method of determinating nitrite in blood Download PDFInfo
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- CN103760121B CN103760121B CN201410054811.XA CN201410054811A CN103760121B CN 103760121 B CN103760121 B CN 103760121B CN 201410054811 A CN201410054811 A CN 201410054811A CN 103760121 B CN103760121 B CN 103760121B
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Abstract
The invention discloses a kind of quick determination method of determinating nitrite in blood.The absorbance of nitrite and the colored complex of Griess formation is determined using porous plate and ELIASA, will be eliminated the interference of blood matrix without nitrite blood as working curve Matrix Solution is made, improve sensitivity and the accuracy of detection.Method has the features such as degree of accuracy is high, detection sample size is big, detection sample size is few, detection efficiency is high.
Description
Technical field
The invention belongs to internal drug measurement techniques field, and in particular to a kind of detection method of blood nitrite.
Background technology
Nitric oxide（NO）It is that one kind plays a regulating and controlling effect bio signal point to angiocarpy, endocrine, nerve and immune system
Son, develops that NO in a kind of blood and body fluid is simple, accurate detection method is extremely important to its effect in clinical, medicine
's.The features such as NO has simple in construction, low molecule amount, highly lipophilic and free radical, it is extremely unstable in vivo, have and very short partly decline
Phase, only several seconds, easily form nitrite and nitrate.It is difficult directly to carry out NO detections, is used as metastable NO
Metabolite, nitrite（NO2 -）Turned into the focus of world medical circle at nearest 10 years, by determining in blood and body fluid
NO2 -Content carry out NO contents in antimer and as the important indicator or medical diagnosis on disease for reacting multinomial physical function.
The assay method of current nitrite detection nitrite have the chromatography of ions, AAS, fluorimetry,
High performance liquid chromatography etc..Nitrite in food one is that content is relatively high, and two be the basic interference without salt, the chromatography of ions
And photometry is all better suited detection method, and blood or body fluid nitrite are low in vivo, and sample size is few, and matrix is done
Disturb serious.Detection generally uses Griess detection kits at present, due to being limited and Matrix effects by detection sensitivity, generally
It is difficult to detect blood or body fluid nitrite, its sensitivity is typically only capable to reach 1 μ g/mg.Utilized before research group
High performance liquid chromatography (HPLC) is studied nitrate in blood urine and tissue and nitrite detection, is achieved good
Effect, applied for patent of invention（201310129291.X）, while also by nitrite and Griess reagent reactings, through cloud point
Extraction（CPE）Separated, be enriched with, direct visual colorimetric determination, progress blood urine nitrite is simple, quick detection, application
Patent of invention（201310274145.6、201310428467.1）.Further study show that, it can clinically determine with greater need for a kind of
Amount, accurate, convenient detection determinating nitrite in blood method, due to clinically blood nitrite detection have sample size it is few,
Matrix effects are big, the features such as sample size is big, otherwise further dilute if needing more sample size with existing photometry detection
Release, reduce detection sensitivity.The present invention substitutes general photometer using ELIASA, and one is that Detection wavelength is general, general enzyme mark
Instrument all has this wavelength；Two be that detection aequum is few, only needs 200 microlitres；Three be that detection limit is big, and 98 samples can be detected simultaneously,
The features such as detection efficiency is high.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide, a kind of simple and quick, sensitivity is high, energy is large quantities of
Amount, the method for quantitatively detecting determinating nitrite in blood.
The purpose of the present invention is achieved by the following technical programs.
Unless otherwise indicated, percentage of the present invention is percetage by weight.
A kind of detection method of determinating nitrite in blood, comprises the following steps：
（1）The making of nitrite standard working curve
1. the preparation of matrix blank solution：Fresh plus anti-coagulants blood of 10 mL without nitrite is taken, centrifuge is put into
Middle centrifugation, takes the mL of supernatant 5, and addition is taken off by the mL of 0.2mol/L sodium hydroxide solutions 1 and the g of zinc sulfate 1 albumen precipitation constituted
Toner, is well mixed 1min, centrifuges, and takes out supernatant, obtains almost colourless, transparent matrix blank solution.
2. the NO that concentration is 1.0 μ g/mL is taken respectively2 - 20th, 40,60,80,100 μ L standard liquids are in microtest tube,
The Matrix Solution 1. prepared with step is diluted to 500 μ L, adds the μ L of GriessA reagents 25, is well mixed；Added after 1min
The μ L of GriessB reagents 25, are well mixed.Take 200 μ L to mix nitrite ion in ELISA Plate, determined at ELIASA wavelength 490nm
Absorbance, makes working curve, obtains the test limit and equation of linear regression of nitrite；
（2）Sample is determined
The detection of blood：Take 5 mL fresh plus anti-coagulants blood, be put into centrifuge and centrifuge, take the mL of supernatant 2, add
The albumen precipitation decolorising agent being made up of 0.2mol/L sodium hydroxide solutions 0.4mL and zinc sulfate 0.4g, vortex mixed is uniform
1min, centrifuges, takes in 500 μ L of supernatant liquid microtest tubes, adds the μ L of GriessA reagents 25, is well mixed；Added after 1min
The μ L of GriessB reagents 25, are well mixed.Take 200 μ L to mix nitrite ion in ELISA Plate, determined at ELIASA wavelength 490nm
Absorbance, and compare step（1）The equation of linear regression of gained, calculates the content of sample nitrite.
Wherein, step（1）（2）Described in GriessA reagents be dissolved in 5mL hydrochloric acid for 50mg p-aminobenzene sulfonic acid
（0.5mol/L）；GriessB reagents are that 4mg naphthalidines are dissolved in 4mL methanol（50%）；
Described centrifugal condition is 5~10min of centrifugation time, 3000~6000r/min of centrifugation rate.
Relative to prior art, the present invention has following remarkable advantage:
1st, general photometer is substituted using ELIASA, Detection wavelength is set at the 490nm that nearly all ELIASA is all matched somebody with somebody,
Detect that aequum is few, only need 200 microlitres, 98 samples can be detected simultaneously, detection efficiency is high.
2nd, the matrix blank made by the use of treated blank plasma as working curve, is overcome and makees matrix with distilled water
The detection error that blank is brought.
3rd, by taking off plasma protein processing the effect of decolouring is reached while, matrix blank solution is almost colourless, reduces and surveys
Determine blank value, improve detection sensitivity.
Embodiment
The present invention is further described with reference to embodiment, but protection scope of the present invention is not limited to this.
Embodiment 1
1st, the making of nitrite standard working curve
1. the preparation of matrix blank solution：Fresh plus anti-coagulants blood of 10 mL without nitrite is taken, centrifuge is put into
In, 6000r/min centrifugation 8min take the mL of supernatant 5 into clean centrifuge tube, added by 0.2mol/L sodium hydroxide solutions 1
The albumen precipitation decolorising agent of the mL and g of zinc sulfate 1 compositions, is well mixed, 5000r/min centrifugation 10min separation, takes out supernatant,
Obtain almost colourless, transparent matrix blank solution.
2. the NO that concentration is 1.0 μ g/mL is taken respectively2 - 20th, 40,60,80,100 μ L standard liquids are used in microtest tube
The matrix blank solution that 1. step prepares is diluted to 500 μ L, adds the μ L of GriessA reagents 25, is well mixed；Added after 1min
The μ L of GriessB reagents 25, are well mixed.Take 200 μ L to mix nitrite ion in ELISA Plate, determined at ELIASA wavelength 490nm
Absorbance, obtains equation of linear regression y=0.1875x-0.0007, coefficient R2=0.9960；
（2）Sample is determined
The detection of blood：Take 5 mL fresh plus anti-coagulants blood, be put into centrifuge, 6000r/min centrifugation 8min take
Clear liquid, adds the protein precipitant being made up of 0.2mol/L sodium hydroxide solutions 0.4mL and zinc sulfate 0.4g, is well mixed,
5000r/min centrifugation 10min separation, takes out supernatant, takes the μ L of supernatant 500 into microtest tube, add GriessA reagents 25
μ L, are well mixed；The μ L of GriessB reagents 25 are added after 1min, are well mixed.200 μ L are taken to mix nitrite ion in ELISA Plate,
It is 0.008 that absorbance is determined at ELIASA wavelength 490nm, and compares step（1）The equation of linear regression of gained, calculates sample
The content of nitrite is 0.046 μ g/mL.
Embodiment 2
1. make nitrite standard working curve by embodiment 1.
2. sample treatment and measurement result
The detection of blood：Take 5 mL fresh plus anti-coagulants blood, be put into 8000r/min in centrifuge and centrifuge 5min separation,
The mL of supernatant 2 is taken, the albumen precipitation decolorising agent being made up of 0.2mol/L sodium hydroxide solutions 0.4mL and zinc sulfate 0.4g is added,
It is well mixed, 6000r/min centrifugation 6min separation, supernatant is taken out, acetonitrile 1mL, 6000r/min centrifugation 5min separation is added,
Take in the μ L microtest tubes of supernatant 500, add the μ L of GriessA reagents 25, be well mixed；The μ of GriessB reagents 25 is added after 1min
L, is well mixed.200 μ L are taken to mix nitrite ion in ELISA Plate, it is 0.011 that absorbance is determined at ELIASA wavelength 490nm,
And compare step（1）The equation of linear regression of gained, the content for calculating sample nitrite is 0.062 μ g/mL.
Claims (3)
1. a kind of detection method of determinating nitrite in blood, comprises the following steps：
（1）The making of nitrite standard working curve
1. the preparation of matrix blank solution：10mL is taken without nitrite fresh plus anti-coagulants blood, be put into centrifuge from
The heart, takes supernatant 5mL, adds the protein decolouring agent being made up of 0.2mol/L sodium hydroxide solutions 1mL and zinc sulfate 1.0g, mixing
Uniformly, centrifuge, take out supernatant, obtain almost colourless, transparent matrix blank solution；
2. the NO that concentration is 1.0 μ g/mL is taken respectively2 - 20th, 40,60,80,100 μ L standard liquids use step in microtest tube
1. the matrix blank solution prepared is diluted to 500 μ L, adds the μ L of GriessA reagents 25, is well mixed；Added after 1min
The μ L of GriessB reagents 25, are well mixed, and take 200 μ L to mix nitrite ion in ELISA Plate, are determined at ELIASA wavelength 490nm
Absorbance, makes working curve, obtains the test limit and equation of linear regression of nitrite；
（2）Sample is determined
The detection of blood：Take that 5mL is fresh plus anti-coagulants blood, be put into centrifuge and centrifuge, take supernatant 2mL, add by
The albumen precipitation decolorising agent of 0.2mol/L sodium hydroxide solutions 0.4mL and zinc sulfate 0.4g compositions, the uniform 1min of vortex mixed, from
The heart is separated, and is taken out in the μ L microtest tubes of supernatant 500, is added the μ L of GriessA reagents 25, is well mixed；Added after 1min
The μ L of GriessB reagents 25, are well mixed, and take 200 μ L to mix nitrite ion in ELISA Plate, are determined at ELIASA wavelength 490nm
Absorbance, and compare step（1）The equation of linear regression of gained, calculates the content of sample nitrite.
2. detection method according to claim 1, it is characterised in that：Described GriessA reagents are 50mg p-aminophenyls
Sulfonic acid is dissolved in the hydrochloric acid solution that 5mL contents are 0.5mol/L；GriessB reagents are that to be dissolved in 4mL concentration be 50% to 4mg naphthalidines
In methanol solution.
3. the detection method described in claim 1, it is characterised in that：Centrifugal condition described in sample detection for centrifugation time 5 ~
10min, 3000 ~ 6000r/min of centrifugation rate.
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| CN105277542B (en) * | 2015-02-05 | 2018-02-06 | 温州医科大学 | A kind of water nitrite field fast detection method for eliminating reagent blank influence |
| CN106596209B (en) * | 2015-10-14 | 2020-05-12 | 杭州量康科技有限公司 | Dry blood sample pretreatment and detection method |
| CN106323894B (en) * | 2016-08-01 | 2019-02-05 | 广西中烟工业有限责任公司 | A kind of nitrite method in silver nanoparticle auxiliary cloud point extraction measurement tobacco |
| CN108562574A (en) * | 2018-01-19 | 2018-09-21 | 常州大学 | Hyaluronic acid high throughput micro-scale rapid detection method |
| CN109060694B (en) * | 2018-10-15 | 2021-01-22 | 广州中医药大学(广州中医药研究院) | Colorimetric detection method for hydroxyl free radicals and application thereof |
| CN109507410B (en) * | 2018-11-20 | 2022-03-18 | 湖北工业大学 | Method and portable device for rapidly detecting nitrate in urine |
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| CN103235093A (en) * | 2013-04-15 | 2013-08-07 | 云南健牛生物科技有限公司 | Detection method of nitrate and nitrite in blood, urine and tissue |
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| CN103235093A (en) * | 2013-04-15 | 2013-08-07 | 云南健牛生物科技有限公司 | Detection method of nitrate and nitrite in blood, urine and tissue |
Non-Patent Citations (2)
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| 尿一氧化氮代谢产物亚硝酸盐的比色测定法;胡明昌等;《临床检验杂志》;19950530;第13卷(第3期);第129页 * |
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