CN106645111A - Method for rapidly detecting concentration of antibiotic Zwittermicin A through electrochemical luminescence - Google Patents
Method for rapidly detecting concentration of antibiotic Zwittermicin A through electrochemical luminescence Download PDFInfo
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- CN106645111A CN106645111A CN201710114906.XA CN201710114906A CN106645111A CN 106645111 A CN106645111 A CN 106645111A CN 201710114906 A CN201710114906 A CN 201710114906A CN 106645111 A CN106645111 A CN 106645111A
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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
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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Abstract
The invention discloses a method for rapidly detecting the concentration of antibiotic Zwittermicin A through electrochemical luminescence. The method comprises the following steps: respectively preparing a tris bipyridine ruthenium solution, a Na2HPO4-NaOH buffer solution and a Zwittermicin A standard solution by taking redistilled water as a solvent; testing the prepared Zwittermicin A standard solution in a flow-injection chemiluminescence analyser through a cyclic voltammetric method; drawing a standard curve according to a test result; making a standard curve equation: Delta I is equal to 206.31x plus 26.677, wherein x is the concentration of the Zwittermicin A; changing the Zwittermicin A standard solution into a Zwittermicin A sample to be tested; repeating the testing process, so as to obtain the chemiluminescence intensity Is of the Zwittermicin A sample to be tested; working out brightening intensity Delta I; substituting the brightening intensity Delta I into the standard curve equation, so as to work out the concentration x of the Zwittermicin A sample. The method has the advantages that the method is simple to operate, the sensitivity is high, the repeatability is good, the linearity range is wide, the dosage of the sample to be tested is small, adopted instruments and equipment are simple, the detection cost is low, online routine determination can be performed, and the method can be easily applied to industrial or agricultural production.
Description
Technical field
The present invention is the on-line checking of antibiotic, belongs to biological technical field, and more particularly to a kind of electrochemical luminescence is quick
The method of detection antibiotic Zwittermicin A concentration.
Background technology
Zwittermicin A（ZwA）It is thuringiensis（English abbreviation Bt）And wax printing fabric（English contracting
Write Bc）A kind of antibiotic for producing during the fermentation, was separated first in 1994 by Siol-Suh et al., was abbreviated as
ZwA, its structural formula is referring to Figure of description, the molecular structural formula of Fig. 1 ZwA（Molecular weight 396D）.
Using for agricultural chemicals affects environmental ecology, and biological control is the developing direction in future.In biological control, although Bt bacterium
The application of insecticidal crystal protein can reduce the usage amount of agricultural chemicals, but insecticidal effect is slow.Research discovery in recent years, ZwA
There is synergy with the insecticidal effect of Bt bacterium insecticidal crystal proteins.That is ZwA is played very well to the insecticidal crystal protein that Bt bacterium produce
Synergistic effect, can play reduce Bt insecticidal crystal proteins usage amount, delay the generation of pest resistance, widen insecticidal spectrum.
At present, ZwA standard items are never had both at home and abroad, for this purpose, setting up extremely difficult to the exploitation of its detection method.
The ZwA detection methods having：(1) bioassay, i.e. ZwA have bacteriostasis to Erwinia herbicola, according to inhibition zone
Size judge ZwA contents；(2) ninhydrin colour developing spectrophotometer detection method, ninhydrin makes ZwA by coloured without discoloration, calculates
Content；(3) HPLC detection methods, chromatographic condition：Chromatographic column C18（10mm×25cm）, 210nm UV-detectors, 5mmol/L ammonium acetates
For mobile phase, flow velocity is for l.0mL/min, the retention time of ZwA is 2.27min.
The content of the invention
The invention aims to be directed in prior art detect the defects such as limit for height, selectivity difference, there is provided one kind operation
The method of lower, the inexpensive electrochemical luminescence quick detection antibiotic Zwittermicin A concentration of simpler, detection limit.
Concretely comprise the following steps：
(1) the terpyridyl ruthenium solution of 5mmol/L is prepared respectively as solvent with redistilled water（Ru(bpy)3 2+）、
60mmol/L and pH value are 8.5 Na2HPO4The Zwittermicin A standard liquids of-NaOH cushioning liquid and 1mg/mL, its
The Zwittermicin A standard liquids of middle 1mg/mL are used to draw calibration curve.
(2) add in the detection cell of Flow Injection Analysis/Chemiluminescence instrument 150 L steps (1) prepare cushioning liquid,
The Zwittermicin A standard liquids that the terpyridyl ruthenium solution and 20 L steps (1) that 100 L steps (1) are prepared is prepared, make
The Zwittermicin A standard liquids that step (1) is prepared are tested with cyclic voltammetry, set initial voltage as 0.2V,
High potential is 1.25V, and electronegative potential is 0.2V, and sweep speed is 0.1V/s, and quiescent time is 4s, and the high pressure of photomultiplier is
800V, blank sample is that the chemiluminescence intensity of cushioning liquid and terpyridyl ruthenium solution is designated as Io, Zwittermicin A samples
Chemiluminescence intensity be designated as Is, intensity of adding lustre to is designated as △ I, △ I=Is-Io, photomultiplier and examination used in the test process
Agent is carried out under 30 DEG C of constant temperatures.
(3) according to the result tested the Zwittermicin A standard liquids that step (1) is prepared in step (2),
Calibration curve is drawn, and makes calibration curve equation：△ I=206.31x+26.677, wherein x are dense for Zwittermicin A's
Degree, its unit is mg/mL, and its span is 0.001 ~ 0.1, R2=0.9947。
(4) concentration mensuration of Zwittermicin A samples：The operating process of repeat step (2), by Zwittermicin
A standard liquids are changed to Zwittermicin A samples to be measured, test its chemiluminescence intensity Is, calculate the intensity △ I that adds lustre to, generation
Enter the calibration curve equation that step (3) is made, that is, calculate concentration x of Zwittermicin A samples.
The inventive method is simple to operate, and sensitivity is high, reproducible, range of linearity width, and detected amount of samples is few, and institute
Simple using instrument and equipment, testing cost is low, can routinely be determined online, it is easy to be applied to industry or agricultural production
In.
Description of the drawings
Fig. 1 is the structural formula of Zwittermicin A in the present invention.
Fig. 2 is the Zwittermicin A calibration curves drawn in the embodiment of the present invention.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Test in following embodiments
Method, if no special instructions, is conventional method.Experiment material used in following embodiments, if no special instructions, is often
Rule biochemical reagents company is commercially available.
Embodiment：
Instrument used is IFFM-E type Flow Injection Analysis/Chemiluminescence instrument in the present embodiment（The limited public affairs of the auspicious electronics advanced in years in Xi'an
Department）.Sampling, note sample, experimental data collection and process during analysis, by IFFM-E types flowing note under Windows XP systems
Penetrate chemiluminescent analyzer to complete.
In the present embodiment, antibiotic ZwA sterlings：≤ 85%, laboratory oneself separation and Extraction；Antibiotic activity detection is indicated
Bacterium：Erwinia herbicola LS005, bacterial classification is provided by Plant Protection institute, Chinese Academy of Agricultral Sciences.
Concretely comprise the following steps：
(1) the terpyridyl ruthenium solution of 5mmol/L is prepared respectively as solvent with redistilled water（Ru(bpy)3 2+）、
The Na of the pH 8.5 of 60mmol/L2HPO4The Zwittermicin A standard liquids of-NaOH cushioning liquid and 1mg/mL, wherein
The Zwittermicin A standard liquids of 1mg/mL are used to draw calibration curve.
(2) in IFFM-E type Flow Injection Analysis/Chemiluminescence instrument（Xi'an Rui Mai Electronics Co., Ltd.s）Detection cell in plus
Enter the cushioning liquid of 150 L steps (1) preparation, the terpyridyl ruthenium solution that 100 L steps (1) are prepared and 20 L steps (1) to match somebody with somebody
The Zwittermicin A standard liquids of system, it is molten to the Zwittermicin A standards that step (1) is prepared using cyclic voltammetry
Liquid is tested, and sets initial voltage as 0.2V, and high potential is 1.25V, and electronegative potential is 0.2V, and sweep speed is 0.1V/s, quiet
Only the time is 4s, and the high pressure of photomultiplier is 800V, and blank sample is the chemiluminescence of cushioning liquid and terpyridyl ruthenium solution
Intensity is designated as Io, and the chemiluminescence intensity of Zwittermicin A samples is designated as Is, and intensity of adding lustre to is designated as △ I, △ I=Is-Io,
Photomultiplier and agents useful for same are carried out under 30 DEG C of constant temperatures in the test process.
(3) according to the result tested the Zwittermicin A standard liquids that step (1) is prepared in step (2),
Calibration curve is drawn, and makes calibration curve equation：△ I=206.31x+26.677, wherein x are dense for Zwittermicin A's
Degree, its unit is mg/mL, and its span is 0.001 ~ 0.1, R2=0.9947。
(4) concentration mensuration of Zwittermicin A samples：The operating process of repeat step (2), by Zwittermicin
A standard liquids are changed to Zwittermicin A samples to be measured, test its chemiluminescence intensity Is, calculate the intensity △ I=5 that adds lustre to,
The calibration curve equation that step (3) is made is substituted into, that is, calculates concentration x of Zwittermicin A samples.
Claims (1)
1. a kind of method of electrochemical luminescence quick detection antibiotic Zwittermicin A concentration, it is characterised in that concrete steps
For：
(1) with redistilled water as solvent, terpyridyl ruthenium solution, 60mmol/L and the pH value that 5mmol/L is prepared respectively is
8.5 Na2HPO4The Zwittermicin A standard liquids of-NaOH cushioning liquid and 1mg/mL, wherein 1mg/mL's
Zwittermicin A standard liquids are used to draw calibration curve；
(2) cushioning liquid, 100 L for adding 150 L steps (1) to prepare in the detection cell of Flow Injection Analysis/Chemiluminescence instrument
The Zwittermicin A standard liquids that the terpyridyl ruthenium solution and 20 L steps (1) that step (1) is prepared is prepared, using circulation
Voltammetry is tested the Zwittermicin A standard liquids that step (1) is prepared, and sets initial voltage as 0.2V, high potential
For 1.25V, electronegative potential is 0.2V, and sweep speed is 0.1V/s, and quiescent time is 4s, and the high pressure of photomultiplier is 800V, empty
White sample is that the chemiluminescence intensity of cushioning liquid and terpyridyl ruthenium solution is designated as Io, and the chemistry of Zwittermicin A samples is sent out
Luminous intensity is designated as Is, and intensity of adding lustre to is designated as △ I, △ I=Is-Io, and photomultiplier and agents useful for same are 30 in the test process
Carry out under DEG C constant temperature；
(3) according to the result tested the Zwittermicin A standard liquids that step (1) is prepared in step (2), draw
Calibration curve, and make calibration curve equation：△ I=206.31x+26.677, wherein x are the concentration of Zwittermicin A, its
Unit is mg/mL, and its span is 0.001 ~ 0.1, R2=0.9947；
(4) concentration mensuration of Zwittermicin A samples：The operating process of repeat step (2), by Zwittermicin A marks
Quasi- solution is changed to Zwittermicin A samples to be measured, tests its chemiluminescence intensity Is, calculates the intensity △ I that adds lustre to, and substitutes into
The calibration curve equation that step (3) is made, that is, calculate concentration x of Zwittermicin A samples.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0871887B1 (en) * | 1995-06-07 | 2007-03-28 | BioVeris Corporation | Electrochemiluminescent enzyme immunoassay |
| CN101539525A (en) * | 2009-04-21 | 2009-09-23 | 中国科学院长春应用化学研究所 | Method for measuring formaldehyde in aqueous solution by electrochemical luminescence of terpyridyl ruthenium |
| CN101949885A (en) * | 2010-08-12 | 2011-01-19 | 东南大学 | Capillary electrophoresis-electrochemiluminescence detection method for spectinomycin |
| CN102249952A (en) * | 2011-05-06 | 2011-11-23 | 桂林理工大学 | Method for separating and purifying water-soluble antibiotic Zwittermicin A in Bacillus thuringiensis |
| CN103091305A (en) * | 2013-01-14 | 2013-05-08 | 大连理工大学 | Method for detecting quinolone antibiotic by means of electrochemical luminescence |
-
2017
- 2017-03-01 CN CN201710114906.XA patent/CN106645111A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0871887B1 (en) * | 1995-06-07 | 2007-03-28 | BioVeris Corporation | Electrochemiluminescent enzyme immunoassay |
| CN101539525A (en) * | 2009-04-21 | 2009-09-23 | 中国科学院长春应用化学研究所 | Method for measuring formaldehyde in aqueous solution by electrochemical luminescence of terpyridyl ruthenium |
| CN101949885A (en) * | 2010-08-12 | 2011-01-19 | 东南大学 | Capillary electrophoresis-electrochemiluminescence detection method for spectinomycin |
| CN102249952A (en) * | 2011-05-06 | 2011-11-23 | 桂林理工大学 | Method for separating and purifying water-soluble antibiotic Zwittermicin A in Bacillus thuringiensis |
| CN103091305A (en) * | 2013-01-14 | 2013-05-08 | 大连理工大学 | Method for detecting quinolone antibiotic by means of electrochemical luminescence |
Non-Patent Citations (1)
| Title |
|---|
| 陶晟辰: "三联吡啶合钌电化学发光在药物分析中的应用", 《中国优秀硕士学术论文全文数据库 医药卫生科技辑》 * |
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Application publication date: 20170510 |