CN114252419A - Ciprofloxacin detection method using phycocyanin as fluorescent probe - Google Patents
Ciprofloxacin detection method using phycocyanin as fluorescent probe Download PDFInfo
- Publication number
- CN114252419A CN114252419A CN202111459365.7A CN202111459365A CN114252419A CN 114252419 A CN114252419 A CN 114252419A CN 202111459365 A CN202111459365 A CN 202111459365A CN 114252419 A CN114252419 A CN 114252419A
- Authority
- CN
- China
- Prior art keywords
- phycocyanin
- ciprofloxacin
- solution
- concentration
- buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 title claims abstract description 98
- 229960003405 ciprofloxacin Drugs 0.000 title claims abstract description 49
- 108010053210 Phycocyanin Proteins 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 28
- 239000007850 fluorescent dye Substances 0.000 title abstract description 12
- 238000000034 method Methods 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
- 229910052782 aluminium Inorganic materials 0.000 claims description 2
- 239000011888 foil Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 239000003242 anti bacterial agent Substances 0.000 abstract description 5
- 229940088710 antibiotic agent Drugs 0.000 abstract description 5
- 239000000575 pesticide Substances 0.000 abstract description 5
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 235000016425 Arthrospira platensis Nutrition 0.000 description 3
- 240000002900 Arthrospira platensis Species 0.000 description 3
- 239000000589 Siderophore Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000010453 quartz Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229940082787 spirulina Drugs 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- IXTLVPXCZJJUQB-VYJQSIGYSA-N 4-[[1-[[(2r)-1-[[(2s)-5-(diaminomethylideneamino)-1-[[(2r)-1-[[(2s)-5-[formyl(hydroxy)amino]-1-[[(3s,6s,9s,12s)-9-[3-[formyl(hydroxy)amino]propyl]-3,6-bis[(1r)-1-hydroxyethyl]-2,5,8,11-tetraoxo-1,4,7,10-tetrazacyclohexadec-12-yl]amino]-1-oxopentan-2-yl]am Chemical compound C1CCCNC(=O)[C@H]([C@H](O)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCN(O)C=O)NC(=O)[C@H]1NC(=O)[C@H](CCCN(O)C=O)NC(=O)[C@@H](CO)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](CO)NC(=O)C1N(C=2C(=CC(O)=C(O)C=2)C=C2NC(=O)CCC(O)=O)C2NCC1 IXTLVPXCZJJUQB-VYJQSIGYSA-N 0.000 description 2
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 2
- 102000008192 Lactoglobulins Human genes 0.000 description 2
- 108010060630 Lactoglobulins Proteins 0.000 description 2
- 229930186551 Pyoverdin Natural products 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- OATNQHYJXLHTEW-UHFFFAOYSA-N benzene-1,4-disulfonic acid Chemical compound OS(=O)(=O)C1=CC=C(S(O)(=O)=O)C=C1 OATNQHYJXLHTEW-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108010025281 pyoverdin Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 229940055764 triaz Drugs 0.000 description 2
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- -1 Thiom Chemical compound 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- DOFZAZXDOSGAJZ-UHFFFAOYSA-N disulfoton Chemical compound CCOP(=S)(OCC)SCCSCC DOFZAZXDOSGAJZ-UHFFFAOYSA-N 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000005558 fluorometry Methods 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-M methanethiolate Chemical compound [S-]C LSDPWZHWYPCBBB-UHFFFAOYSA-M 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000003306 quinoline derived antiinfective agent Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000000979 synthetic dye Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- AMFGTOFWMRQMEM-UHFFFAOYSA-N triazophos Chemical compound N1=C(OP(=S)(OCC)OCC)N=CN1C1=CC=CC=C1 AMFGTOFWMRQMEM-UHFFFAOYSA-N 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
Images
Classifications
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a ciprofloxacin detection method using phycocyanin as a fluorescent probe, and belongs to the technical field of detection. The invention takes the phycocyanin which is non-toxic, environment-friendly and easy to obtain as the fluorescent probe, establishes a rapid, low-cost and high-sensitivity detection method for detecting ciprofloxacin, has the detection limit of 5 mu mol/L, can be distinguished from other six antibiotics and six pesticides at the concentration of 50 mu mol/L, and has good selectivity.
Description
Technical Field
The invention relates to a ciprofloxacin detection method using phycocyanin as a fluorescent probe, and belongs to the technical field of detection.
Background
Chemical contaminants in the environment pose a great threat to humans and animals. Ciprofloxacin is a fluoroquinolone antibiotic, can be used for resisting various gram-negative bacteria, certain gram-positive bacteria and certain viruses due to the broad-spectrum action, is widely used for treating infections of urinary tracts, respiratory tracts and the like, and is highly absorbed by human tissues and body fluids. Ciprofloxacin (CIP) is often released into municipal sewage through the excreta of patients, and further drug residues may appear in drinking water. Long-term exposure of humans to ciprofloxacin can produce allergic reactions and toxic effects on cells, organs and organisms.
In view of this, a large number of analytical methods for ciprofloxacin have been reported. Some of these techniques include high performance liquid chromatography, high performance thin layer chromatography, gas chromatography, and the like. These methods based on large analytical devices have the disadvantages of high cost, cumbersome procedures, use of large amounts of organic solvents, etc. The rapid detection method based on the mechanisms of immunoreaction, aptamer, chromogenic reaction, chemical bonding and the like, such as a colorimetric method and a fluorescence method, has the advantages of rapid reaction, simple principle, visual observation and the like, and is always the key point for research and development of rapid detection products. Among them, the fluorescence measurement method has a promising prospect in the aspect of detecting chemical pollutants such as antibiotics due to higher sensitivity, quick response and non-invasiveness to a certain extent. The development of novel fluorescent probes has always been the most important research content of fluorescent rapid detection methods.
Phycocyanin is a deep blue water-soluble protein isolated from spirulina, and is a complex of fluorescent dye and protein. Phycocyanin is a pure natural fluorescent material, can be used as a food additive, has the advantages of safety, low price, small environmental pollution, good stability and high brightness compared with chemical synthetic dyes. At present, no literature reports a method for detecting ciprofloxacin by using phycocyanin as a fluorescent probe.
In the research, the applicant finds that phycocyanin reacts specifically with ciprofloxacin, and the fluorescence emission intensity of phycocyanin is reduced. Based on the method, a detection method for detecting ciprofloxacin by using phycocyanin as a fluorescent probe is developed, and the method can be used for developing a rapid detection kit for ciprofloxacin.
In chemical sensing for fluorescent applications, there are a variety of fluorescent dyes and proteins that are simply used. In detecting ciprofloxacin based on fluorometry, different proteins and organic fluorescent molecules have also been used. For example, Mehreban and his team used beta-lactoglobulin to determine ciprofloxacin. Pawar et al reported the use of the fluorescent siderophore pyoverdin, a non-protein pigment extracted from a soil isolate, Pseudomonas sp, for ciprofloxacin detection in aqueous media.
In summary, it is important to detect ciprofloxacin with different fluorophores. However, most fluorophore agents still present problems, since most of them are artificially synthesized, they are hazardous and present carcinogenic problems. Furthermore, the synthesis of such materials requires a large number of steps, a long time, and also a complicated technique in the extraction process.
Disclosure of Invention
In order to solve the problems, the invention provides a novel method for detecting ciprofloxacin, phycocyanin is used as a fluorescent probe, and a rapid, low-cost, high-sensitivity and good-selectivity method is established for detecting ciprofloxacin.
The invention aims to provide a method for detecting ciprofloxacin, which comprises the following steps:
dispersing phycocyanin in ultrapure water to obtain a phycocyanin probe solution; diluting a series of ciprofloxacin samples by using a buffer solution to obtain corresponding sample solutions, mixing the sample solutions with a phycocyanin solution, uniformly mixing, standing and culturing to obtain a mixed solution; measuring the fluorescence intensity value of the mixed solution; and then constructing by using the concentration of the ciprofloxacin and the fluorescence intensity value of the corresponding mixed solution to obtain a linear detection model.
In one embodiment of the invention, the concentration of the phycocyanin solution is 1-5 mg/mL; specifically, 2mg/mL can be selected.
In one embodiment of the invention, the buffer is 0.01M PBS buffer, pH 6.2.
In one embodiment of the invention, the volume ratio of sample to buffer is 1: (3-5); specifically, the selection is 1: 3.5.
in one embodiment of the invention, the volume ratio of the sample solution to the phycocyanin solution is (6-10): 1; specifically, the selection is 9: 1.
in one embodiment of the invention, the concentration of phycocyanin in the mixed solution is 0.1-0.5 mg/mL; specifically, the concentration of the surfactant may be 0.2 mg/mL.
In one embodiment of the invention, the range of ciprofloxacin concentrations is from 0 to 600 μmol/L.
In one embodiment of the invention, the time for standing for incubation is 10-20 minutes; specifically, 15 minutes can be selected. The temperature for placing and culturing is room temperature (20-30 ℃); specifically, 25 ℃ can be selected.
In one embodiment of the present invention, the time for mixing is 1 minute and the time for culturing is 15 minutes.
In one embodiment of the invention, the phycocyanin solution is stored at 4-6 ℃ until use. The phycocyanin solution was stored in plastic tubes wrapped in aluminum foil.
In one embodiment of the present invention, the phycocyanin is 40kDa phycocyanin powder.
A ciprofloxacin detection method specifically comprises the following steps:
s1: preparing phycocyanin stock solution from phycocyanin powder (40kDa) purified from blue Spirulina using ultrapure water, storing in refrigerator until use;
s2: diluting a sample to be detected by using a PBS buffer solution;
s3: the diluted sample was further mixed with a phycocyanin solution.
S4: after mixing, violently oscillating in a vortex, then placing and culturing, transferring to a quartz reaction cup for measurement after culturing;
s5: the fluorescence intensity was measured and the concentration of ciprofloxacin in the solution was calculated using a standard curve method.
The invention also provides application of the detection method in the field of environmental protection.
The invention has the advantages and effects that:
the invention uses the non-toxic, environment-friendly and easily obtained phycocyanin obtained from blue algae as a fluorescent probe, establishes a rapid, low-cost and high-sensitivity detection method for detecting ciprofloxacin, has the detection limit of 5 mu mol/L, can be distinguished from other six antibiotics and six pesticides at the concentration of 50 mu mol/L, and has good selectivity. The phycocyanin is natural and safe, and the method can also be safely processed.
Drawings
FIG. 1 is a graph showing the change in fluorescence intensity of phycocyanin (. lamda.em. 532nm) with ciprofloxacin (0-120. mu. mol/L) at various concentrations.
FIG. 2 is a standard graph of fluorescence intensity over a range of ciprofloxacin concentrations from 5 to 50. mu. mol/L.
FIG. 3 shows fluorescence intensities of phycocyanin in the presence of ciprofloxacin and in the presence of six equal concentrations (50. mu. mol/L) of antibiotics (Pen G, Cap, strep. S, Kana, TTC, Vanco) (FIG. 3A); fluorescence intensity of phycocyanin in the presence of ciprofloxacin with six pesticides (Azin-methyl, Par-ethyl, Fenith, Disulf, Thiom, Triaz) at six equal concentrations (50. mu. mol/L) (FIG. 3B).
Detailed description of the preferred embodiments
Phycocyanin powder (40kDa) referred to below was purchased from mitsubishi gmbh of beijing, zhejiang.
Example 1
For ciprofloxacin detection, 100. mu.L of various ciprofloxacin concentrations (0-600. mu. mol/L) were diluted with 350. mu.L of PBS pH6.2, and then 50. mu.L of phycocyanin (2mg/mL) was added. The final concentration of ciprofloxacin ranged from 0 to 120 μmol/L (0,5,8,13,20,29,37,50,80 and 120 μmol/L). All solutions were shaken for 1 minute in a vortex shaker. Incubation was performed for 15 minutes at room temperature, the solution was transferred to a quartz cell, fluorescence measurement was performed using a fluorescence spectrophotometer, and a standard curve was plotted (baseline correction was performed using ultrapure water in all experiments).
As shown in fig. 1, the fluorescence intensity decreased as the ciprofloxacin concentration increased. As shown in FIG. 2, the fluorescence intensity and the ciprofloxacin concentration have a good linear relationship in the concentration range of 0-50 nM, and the regression equation can be expressed as Y-2544X +230777 (R)20.9995), Y represents the fluorescence intensity, and X represents the ciprofloxacin concentration.
Mu.l of the aqueous solution to be tested was diluted with 350. mu.L of PBS pH6.2, then 50. mu.L of a 2mg/mL phycocyanin aqueous solution was added, shaken in a vortex shaker for 1 minute, further incubated for 14 minutes, and then the solution was transferred to a quartz cell for fluorescence measurement, and then the ciprofloxacin concentration was calculated from a standard curve.
In the ciprofloxacin detection method using phycocyanin as the probe, compared with other protein probes, the method for detecting ciprofloxacin has the following advantages:
the detection limit of the fluorescent siderophore (pyoverdin) is 7.13 mu M, the wavelength is in a blue light region (458-455 nm), and the fluorescent siderophore is not beneficial to visual detection relative to red light emitted by phycocyanin. Produced by pathogenic strains, while phycocyanin is isolated from spirulina.
The wavelength of beta-lactoglobulin is also around the blue light region (450nm), which is not beneficial to visual detection. The acquisition of hemocyanin is more complicated than that of phycocyanin.
Example 2
This method was used for the detection of ciprofloxacin in an actual sample (pond water). Water samples of ponds were collected from reservoirs in the campus of university in south of the river and filtered through a 0.22 μm pore filter, the detection procedure was the same as in example 1, and the results are shown in Table 1. The recovery rate and the Relative Standard Deviation (RSD) result are in an acceptable range, and the method has great prospect in detecting ciprofloxacin in real environmental water samples.
TABLE 1 results of actual sample testing
Example 3
To examine the selectivity of this fluorescent detection method for ciprofloxacin, the fluorescence intensity of phycocyanin in the presence of ciprofloxacin was compared to six antibiotics: tetracycline (TTC), penicillin g (pen g), chloramphenicol (Cap), streptomycin sulfate (strep.s), kanamycin (Kana), and vancomycin (Vanco); and six pesticides: comparison was made between Azin-methyl, parathion (Par-ethyl), fenuth, disulfoton (Disulf), thiomethoxide (Thiom) and triazophos (Triaz). As shown in FIG. 3, at an equivalent concentration (50. mu. mol/L) of each drug or pesticide, ciprofloxacin could be easily distinguished between them.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for detecting ciprofloxacin, which comprises the following steps:
dispersing phycocyanin in water to obtain phycocyanin solution; diluting a series of ciprofloxacin samples by using a buffer solution to obtain corresponding sample solutions, mixing the sample solutions with a phycocyanin solution, uniformly mixing, standing and culturing to obtain a mixed solution; measuring the fluorescence intensity value of the mixed solution; and then constructing by using the concentration of the ciprofloxacin and the fluorescence intensity value of the corresponding mixed solution to obtain a linear detection model.
2. The method of claim 1, wherein the concentration of the phycocyanin solution is 1-5 mg/mL.
3. The method of claim 1, wherein the buffer is 0.01M PBS buffer, pH 6.2.
4. The method of claim 1, wherein the volume ratio of sample to buffer is 1: (3-5).
5. The method of claim 1, wherein the volume ratio of the sample solution to the phycocyanin solution is (6-10): 1.
6. The method of claim 1, wherein the concentration of phycocyanin in the mixture is 0.1-0.5 mg/mL.
7. The method of claim 1, wherein the range of ciprofloxacin concentrations is from 0 to 600 μmol/L.
8. The method of claim 1, wherein the time for the standing culture is 10 to 20 minutes; specifically, 15 minutes can be selected.
9. The method according to any one of claims 1 to 8, wherein the phycocyanin solution is stored in plastic tubes wrapped in aluminum foil and kept at 4 to 6 ℃ until use.
10. Use of the process according to any one of claims 1 to 9 in the field of environmental protection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111459365.7A CN114252419A (en) | 2021-12-02 | 2021-12-02 | Ciprofloxacin detection method using phycocyanin as fluorescent probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111459365.7A CN114252419A (en) | 2021-12-02 | 2021-12-02 | Ciprofloxacin detection method using phycocyanin as fluorescent probe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114252419A true CN114252419A (en) | 2022-03-29 |
Family
ID=80793782
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111459365.7A Pending CN114252419A (en) | 2021-12-02 | 2021-12-02 | Ciprofloxacin detection method using phycocyanin as fluorescent probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114252419A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS626691A (en) * | 1985-07-02 | 1987-01-13 | Dainippon Ink & Chem Inc | Phycocyanin pigment a from blue-green alga, production and use thereof |
| CN101915844A (en) * | 2010-08-03 | 2010-12-15 | 中国农业大学 | Method and special quantum dot fluorescent immunoassay kit for detecting quinolone compounds |
| CN101980019A (en) * | 2010-09-17 | 2011-02-23 | 颜世敢 | Method for preparing allophycocyanin-marked fluorescent antinuclear antibody |
| CN107741414A (en) * | 2017-12-04 | 2018-02-27 | 上海海洋大学 | A kind of method using long wavelength's allophycocyanin fluoroscopic examination oxidation resistance |
| CN111690045A (en) * | 2020-07-15 | 2020-09-22 | 中国科学院水生生物研究所 | Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method |
-
2021
- 2021-12-02 CN CN202111459365.7A patent/CN114252419A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS626691A (en) * | 1985-07-02 | 1987-01-13 | Dainippon Ink & Chem Inc | Phycocyanin pigment a from blue-green alga, production and use thereof |
| CN101915844A (en) * | 2010-08-03 | 2010-12-15 | 中国农业大学 | Method and special quantum dot fluorescent immunoassay kit for detecting quinolone compounds |
| CN101980019A (en) * | 2010-09-17 | 2011-02-23 | 颜世敢 | Method for preparing allophycocyanin-marked fluorescent antinuclear antibody |
| CN107741414A (en) * | 2017-12-04 | 2018-02-27 | 上海海洋大学 | A kind of method using long wavelength's allophycocyanin fluoroscopic examination oxidation resistance |
| CN111690045A (en) * | 2020-07-15 | 2020-09-22 | 中国科学院水生生物研究所 | Artificial binding protein capable of specifically recognizing allophycocyanin and acquisition method |
Non-Patent Citations (1)
| Title |
|---|
| TAO LU等: "《Evaluation of the taxonomic and functional variation of freshwater plankton communities induced by trace amounts of the antibiotic ciprofloxacin》", 《ENVIRONMENTAL INTERNATIONAL》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pathak et al. | Detection of reactive oxygen species (ROS) in cyanobacteria using the oxidant-sensing probe 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) | |
| Axelrod et al. | Bioluminescent bioreporter pad biosensor for monitoring water toxicity | |
| Wu et al. | Nanoparticles assembled by aptamers and crystal violet for arsenic (III) detection in aqueous solution based on a resonance Rayleigh scattering spectral assay | |
| He et al. | Highly specific bacteriophage-affinity strategy for rapid separation and sensitive detection of viable Pseudomonas aeruginosa | |
| Yu et al. | CdTe/CdS quantum dot-labeled fluorescent immunochromatography test strips for rapid detection of Escherichia coli O157: H7 | |
| Tarasova et al. | Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances | |
| Yu et al. | A novel near-infrared fluorescent sensor for zero background nitrite detection via the “covalent-assembly” principle | |
| Yang et al. | Insights into the binding interactions of autochthonous dissolved organic matter released from Microcystis aeruginosa with pyrene using spectroscopy | |
| Son et al. | A BODIPY-functionalized bimetallic probe for sensitive and selective color-fluorometric chemosensing of Hg 2+ | |
| CN107121415A (en) | The method of the label-free fluorescence quick detection mercury ion of single step | |
| Potekhina et al. | Biodiversity of mycelial fungi in fresh water in the territory of the park" mari chodra" of the Russian federation | |
| Damirchi et al. | A comparison between digital camera and spectrophotometer for sensitive and selective kinetic determination of brilliant green in wastewaters | |
| Arita et al. | Fluorometric detection of nitric oxide with diaminofluoresceins (DAFs): applications and limitations for plant NO research | |
| Masner et al. | Rapid in situ toxicity testing with luminescent bacteria Photorhabdus luminescens and Vibrio fischeri adapted to a small portable luminometer | |
| Panhwar et al. | A novel approach for real-time enumeration of Escherichia coli ATCC 47076 in water through high multi-functional engineered nano-dispersible electrode | |
| Deb et al. | Development of an on-site lateral flow immune assay based on mango leaf derived colloidal silver nanoparticles for rapid detection of Staphylococcus aureus in milk | |
| CN114252419A (en) | Ciprofloxacin detection method using phycocyanin as fluorescent probe | |
| Sun et al. | Using a safe and effective fixative to improve the immunofluorescence staining of bacteria | |
| Kiersztyn et al. | Coomassie blue G250 for visualization of active bacteria from lake environment and culture | |
| AU2018266138B2 (en) | Apparatus and method for detecting microbial contamination | |
| CN107941773B (en) | endotoxin detection method based on fluorescent molecules | |
| Chen et al. | A Novel Fluorescence Tool for Monitoring Agricultural Industry Chain Based on AIEgens | |
| Gu et al. | Sensitive determination of proteins with naphthol green B by resonance light scattering technique | |
| Peng et al. | Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ | |
| Rohtla et al. | Biosensor for the detection of cyanobacterial toxin microcystin-LR |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220329 |