CN105548058A - Method for detecting signal molecules in pseudomonas aeruginosa - Google Patents
Method for detecting signal molecules in pseudomonas aeruginosa Download PDFInfo
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- CN105548058A CN105548058A CN201610039697.2A CN201610039697A CN105548058A CN 105548058 A CN105548058 A CN 105548058A CN 201610039697 A CN201610039697 A CN 201610039697A CN 105548058 A CN105548058 A CN 105548058A
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- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 23
- 238000010521 absorption reaction Methods 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 230000011664 signaling Effects 0.000 claims description 91
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 26
- 239000012046 mixed solvent Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- PHSRRHGYXQCRPU-AWEZNQCLSA-N N-(3-oxododecanoyl)-L-homoserine lactone Chemical compound CCCCCCCCCC(=O)CC(=O)N[C@H]1CCOC1=O PHSRRHGYXQCRPU-AWEZNQCLSA-N 0.000 claims description 4
- 239000010949 copper Substances 0.000 description 25
- 239000002904 solvent Substances 0.000 description 6
- 150000001455 metallic ions Chemical class 0.000 description 4
- 238000011160 research Methods 0.000 description 3
- 230000007923 virulence factor Effects 0.000 description 3
- 239000000304 virulence factor Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for detecting signal molecules in pseudomonas aeruginosa. An ultraviolet-visible absorption spectrum study result shows that both the signal molecules in the pseudomonas aeruginosa and Cu<2+> do not have absorption peaks at about 280nm, and after the signal molecules are combined with Cu<2+>, a new absorption peak appears at about 280nm, and the strength of the absorption peak forms a good linear relationship with the concentration of the signal molecules. The method has the advantages of being simple and convenient to operate and low in cost; the signal molecules in the pseudomonas aeruginosa and Cu<2+> are coordinated to implement quantitative detection of the signal molecules in the pseudomonas aeruginosa in different solutions through an ultraviolet-visible absorption spectrum channel.
Description
Technical field
The invention belongs to chemical analysis test technical field, be specifically related to a kind of method detecting signaling molecule in pseudomonas aeruginosa, it is a kind of new detection method.
Background technology
Pseudomonas aeruginosa (Pseudomonaseaeruginosa, PA), belong to gram-Negative bacillus, extensively distribute in the skin, respiratory tract and enteron aisle etc. of water, air, soil and living organism, according to investigations, this bacterium is one of the main pathogenic fungi of inside-hospital infection, can cause skin disease, pneumonia, intestines problem, meningitis and septicemia etc., can cause death time serious after infection.Research shows, signaling molecule N-(3-oxododecanoyl) homoserinelactone (3OC of pseudomonas aeruginosa population effect (quorumsensing, QS) system
12-HSL) play an important role in the expression of regulation and control pseudomonas aeruginosa overwhelming majority virulence factor, and the release of the various severe infections that pseudomonas aeruginosa produces and its virulence factor is closely related.When pseudomonas aeruginosa number is lower, only produce the signaling molecule of foundation level, along with pseudomonas aeruginosa constantly growth in local, signaling molecule can reach certain concentration in local environment, when its concentration reaches threshold values, just activate the expression of relevant virulence factor in pseudomonas aeruginosa, strengthen the pathogenic of pseudomonas aeruginosa, cause life entity to infect.Therefore, the health of method to life entity developing quick and sensitive accurate detection pseudomonas aeruginosa is significant.
At present, in the context of detection of pseudomonas aeruginosa, need multiple step such as pure culture and biochemical identification, complex operation, sense cycle are long, sensitivity is low, and can not meet the demand detected fast.The concentration of signaling molecule is relevant to the number of pseudomonas aeruginosa, by the mensuration to signaling molecule concentration in pseudomonas aeruginosa, can reach the object of monitoring P. aeruginosa growth quantity.In pseudomonas aeruginosa, signaling molecule has multiple metal ion binding site (as O, N), and according to the combination of signaling molecule and metallic ion, thus the research detecting signaling molecule in pseudomonas aeruginosa have not been reported.
Based on the problems referred to above, the present invention is devoted to the method developing signaling molecule in easy, quick and sensitive detection pseudomonas aeruginosa.By uv-visible absorption spectra, utilize copper ion solution, realize the quantitative detection to signaling molecule in pseudomonas aeruginosa.The present invention not only method is easy, and all has outstanding advantages in sensitivity, response time and biologic applications etc.
Summary of the invention
The object of this invention is to provide a kind of method detecting signaling molecule in pseudomonas aeruginosa.
Technical scheme of the present invention is as follows:
1. one kind is detected signaling molecule N-(3-oxododecanoyl) homoserinelactone (3OC in pseudomonas aeruginosa
12-HSL) new method, signaling molecule and Cu in pseudomonas aeruginosa
2+after coordination combines, there is following structural formula:
2. detect a method for signaling molecule in pseudomonas aeruginosa, comprise the following steps:
At DMSO and H
2o volume ratio is in the mixed solvent of 4:1, adds the CuCl that initial concentration is 1.0mM
22H
2o, then, the initial concentration adding different amount is successively signaling molecule in the pseudomonas aeruginosa of 1.0mM, makes Cu in solution
2+concentration be 5.0x10
-4m, in pseudomonas aeruginosa, the concentration of signaling molecule is respectively 1.0 μMs, 2.0 μMs, 5.0 μMs, 10.0 μMs, 20.0 μMs, 50.0 μMs, 100 μMs, 200 μMs, 300 μMs, 400 μMs, 500 μMs, do not add signaling molecule in contrast, leave standstill 30min system and reach balance, with the uv-visible absorption spectra under uv-visible absorption spectra instrument test variable concentrations pseudomonas aeruginosa signaling molecule condition, detect signaling molecule in pseudomonas aeruginosa in the mode that 277nm and 280nm place absorption peak strength in uv-visible absorption spectra obviously changes; Described DMSO and H
2the pH value of O mixed solvent is by dripping the pH to 6.5 of hydrochloric acid and NaOH adjustment detection system.
A kind of above-mentioned method detecting signaling molecule in pseudomonas aeruginosa, is applied to Cu
2+cH
3oH/H
2in O solution, the detection of signaling molecule in pseudomonas aeruginosa, detects signaling molecule in pseudomonas aeruginosa in the mode that 277nm place absorption peak strength in uv-visible absorption spectra obviously changes.
A kind of above-mentioned method detecting signaling molecule in pseudomonas aeruginosa, is applied to Cu
2+dMF/H
2in O solution, the detection of signaling molecule in pseudomonas aeruginosa, detects signaling molecule in pseudomonas aeruginosa in the mode that 280nm place absorption peak strength in uv-visible absorption spectra obviously changes.
In the present invention, above-mentioned Cu
2+dMSO/H
2o, CH
3oH/H
2o and DMF/H
2signaling molecule in pseudomonas aeruginosa can be quantitatively detected, this Cu in O solution
2+solution this there is no ultraviolet-ray visible absorbing peak in about 280nm place, after being combined with signaling molecule, the ultraviolet-ray visible absorbing peak located at about 280nm significantly strengthens.
Beneficial effect of the present invention:
1. the new method detecting signaling molecule in pseudomonas aeruginosa of the present invention, has broad prospect of application in life science and analysis field.
2. new method of the present invention is obvious to the detected artifacts of signaling molecule in pseudomonas aeruginosa, is convenient to the quantitative judge of signaling molecule in pseudomonas aeruginosa.
3. in detection pseudomonas aeruginosa of the present invention, the new method of signaling molecule is easy and simple to handle, quick, cost is low.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 1 in pseudomonas aeruginosa signaling molecule to the uv-visible absorption spectra figure of different metal Ion response.
Fig. 2 is Cu in the embodiment of the present invention 2
2+with the uv-visible absorption spectra figure under signaling molecule condition in variable concentrations pseudomonas aeruginosa.
In figure, solvent used is DMSO/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m; Nethermost curve is not for add signaling molecule 3OC
12the absorption curve of-HSL, curve is signaling molecule 3OC from the bottom up
12the concentration of-HSL increases successively.
Fig. 3 is the graph of a relation of signaling molecule concentration in the absorption intensity at 277nm place in the embodiment of the present invention 2 and pseudomonas aeruginosa.
In figure, solvent used is DMSO/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m.
Fig. 4 is Cu in the embodiment of the present invention 3
2+with the uv-visible absorption spectra figure under signaling molecule condition in variable concentrations pseudomonas aeruginosa.
In figure, solvent used is CH
3oH/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m; Nethermost curve is not for add signaling molecule 3OC
12the absorption curve of-HSL, curve is signaling molecule 3OC from the bottom up
12the concentration of-HSL increases successively.
Fig. 5 is the graph of a relation of signaling molecule concentration in the absorption intensity at 277nm place in the embodiment of the present invention 3 and pseudomonas aeruginosa.
In figure, solvent used is CH
3oH/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m.
Fig. 6 is Cu in the embodiment of the present invention 4
2+with the uv-visible absorption spectra figure under signaling molecule condition in variable concentrations pseudomonas aeruginosa.
In figure, solvent used is DMF/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m; Nethermost curve is not for add signaling molecule 3OC
12the absorption curve of-HSL, curve is signaling molecule 3OC from the bottom up
12the concentration of-HSL increases successively.
Fig. 7 is the graph of a relation of signaling molecule concentration in the absorption intensity at 280nm place in the embodiment of the present invention 4 and pseudomonas aeruginosa.
In figure, solvent used is DMF/H
2o (4/1, v/v), Cu
2+concentration be 5.0 × 10
-4m.
Specific embodiments
Below by specific embodiment, the present invention will be further described, but the invention is not restricted to embodiment.
Raw materials used in embodiment, be conventional commercial products if no special instructions.
Signaling molecule N-(3-oxododecanoyl) homoserinelactone (3OC in a kind of detection pseudomonas aeruginosa
12-HSL) method, signaling molecule and Cu in pseudomonas aeruginosa
2+after coordination combines, there is following structural formula:
Embodiment 1
In pseudomonas aeruginosa, signaling molecule is to the response research of metallic ion
At DMSO and H
2o volume ratio is (pH=6.5) in the mixed solvent of 4:1, is that in the pseudomonas aeruginosa of 20 μMs, signaling molecule is the CuCl of 200 μMs respectively with initial concentration by initial concentration
22H
2o, CdCl
2, NiCl
2and ZnCl
2mixing, configuration detection sample, makes the concentration of signaling molecule in pseudomonas aeruginosa in sample be 10 μMs, Cu
2+, Cd
2+, Ni
2+and Zn
2+concentration be 100 μMs, do not add metallic ion in contrast, leave standstill 30min system reach balance.
With in uv-visible absorption spectra instrument test pseudomonas aeruginosa, signaling molecule is to the response condition of metallic ion, and result as shown in Figure 1.As shown in Figure 1, Cd is added
2+, Ni
2+and Zn
2+after, in pseudomonas aeruginosa, the uv-visible absorption spectra of signaling molecule does not change substantially; Only when adding Cu
2+after, there is new absorption peak under 277nm wavelength, to illustrate in pseudomonas aeruginosa signaling molecule can with Cu
2+coordination combines.
Embodiment 2
A kind ofly detect the method for signaling molecule in pseudomonas aeruginosa to comprise step as follows:
At DMSO and H
2o volume ratio is (pH=6.5) in the mixed solvent of 4:1, adds the CuCl that initial concentration is 1.0mM
22H
2o, then, the initial concentration adding different amount is successively signaling molecule in the pseudomonas aeruginosa of 1.0mM, makes Cu in solution
2+concentration be 5.0x10
-4m, in pseudomonas aeruginosa, the concentration of signaling molecule is respectively 1.0 μMs, 2.0 μMs, 5.0 μMs, 10.0 μMs, 20.0 μMs, 50.0 μMs, 100 μMs, 200 μMs, 300 μMs, 400 μMs, 500 μMs, do not add signaling molecule in pseudomonas aeruginosa in contrast, to leave standstill 30min system and reach balance.
Test the uv-visible absorption spectra in different pseudomonas aeruginosa under signaling molecule condition with uv-visible absorption spectra instrument, result as shown in Figure 2.As shown in Figure 2, along with the concentration of signaling molecule in pseudomonas aeruginosa increases, the absorption intensity under 277nm wavelength strengthens gradually.As shown in Figure 3, in the absorption intensity at 277nm place and pseudomonas aeruginosa, the concentration of signaling molecule has good linear relationship, proves Cu
2+dMSO/H
2o solution, in 2.0 μMs of-100 μMs of concentration ranges, can quantitatively detect signaling molecule in pseudomonas aeruginosa.
Embodiment 3
A kind ofly detect the method for signaling molecule in pseudomonas aeruginosa to comprise step as follows:
At CH
3oH and H
2o volume ratio is (pH=6.5) in the mixed solvent of 4:1, adds the CuCl that initial concentration is 1.0mM
22H
2o, then, the initial concentration adding different amount is successively signaling molecule in the pseudomonas aeruginosa of 1.0mM, makes Cu in solution
2+concentration be 5.0x10
-4m, in pseudomonas aeruginosa, the concentration of signaling molecule is respectively 1.0 μMs, 2.0 μMs, 5.0 μMs, 10.0 μMs, 20.0 μMs, 50.0 μMs, 100 μMs, 200 μMs, 300 μMs, 400 μMs, 500 μMs, do not add signaling molecule in pseudomonas aeruginosa in contrast, to leave standstill 30min system and reach balance.
Test the uv-visible absorption spectra in different pseudomonas aeruginosa under signaling molecule condition with ultraviolet-ray visible absorbing instrument, result as shown in Figure 4.As shown in Figure 4, along with the concentration of signaling molecule in pseudomonas aeruginosa increases, the absorption intensity under 277nm wavelength strengthens gradually.As shown in Figure 5, in the absorption intensity at 277nm place and pseudomonas aeruginosa, the concentration of signaling molecule has good linear relationship, proves Cu
2+cH
3oH/H
2o solution, in the concentration range of 100 μMs-500 μMs, can quantitatively detect signaling molecule in pseudomonas aeruginosa.
Embodiment 4
A kind ofly detect the method for signaling molecule in pseudomonas aeruginosa to comprise step as follows:
At DMF and H
2o volume ratio is (pH=6.5) in the mixed solvent of 4:1, adds the CuCl that initial concentration is 1.0mM
22H
2o, then, the initial concentration adding different amount is successively signaling molecule in the pseudomonas aeruginosa of 1.0mM, makes Cu in solution
2+concentration be 5.0x10
-4m, in pseudomonas aeruginosa, the concentration of signaling molecule is respectively 1.0 μMs, 2.0 μMs, 5.0 μMs, 10.0 μMs, 20.0 μMs, 50.0 μMs, 100 μMs, 200 μMs, 500 μMs, do not add signaling molecule in pseudomonas aeruginosa in contrast, to leave standstill 30min system and reach balance.
Test the uv-visible absorption spectra in different pseudomonas aeruginosa under signaling molecule condition with ultraviolet-ray visible absorbing instrument, result as shown in Figure 6.As shown in Figure 6, along with the concentration of signaling molecule in pseudomonas aeruginosa increases, absorption intensity at a wavelength of 280 nm strengthens gradually.As shown in Figure 7, in the absorption intensity at 280nm place and pseudomonas aeruginosa, the concentration of signaling molecule has good linear relationship, proves Cu
2+dMF/H
2o solution, in the concentration range of 1.0 μMs-50 μMs, can quantitatively detect signaling molecule in pseudomonas aeruginosa.
Claims (4)
1. one kind is detected signaling molecule N-(3-oxododecanoyl) homoserinelactone (3OC in pseudomonas aeruginosa
12-HSL) method, it is characterized in that, signaling molecule and Cu in pseudomonas aeruginosa
2+after coordination combines, there is following structural formula:
2. detect a method for signaling molecule in pseudomonas aeruginosa, it is characterized in that, comprise the following steps:
At DMSO and H
2o volume ratio is in the mixed solvent of 4:1, adds the CuCl that initial concentration is 1.0mM
22H
2o, then, the initial concentration adding different amount is successively signaling molecule in the pseudomonas aeruginosa of 1.0mM, makes Cu in solution
2+concentration be 5.0x10
-4m, in pseudomonas aeruginosa, the concentration of signaling molecule is respectively 1.0 μMs, 2.0 μM, 5.0 μM, 10.0 μMs, 20.0 μMs, 50.0 μMs, 100 μMs, 200 μMs, 300 μMs, 400 μMs, 500 μMs, not add in pseudomonas aeruginosa signaling molecule in contrast, leave standstill 30min system and reach balance, with the uv-visible absorption spectra under signaling molecule condition in uv-visible absorption spectra instrument test variable concentrations pseudomonas aeruginosa, signaling molecule in pseudomonas aeruginosa is detected in the mode that 277nm and 280nm place absorption peak strength in uv-visible absorption spectra obviously changes, described DMSO and H
2the pH value of O mixed solvent is by dripping the pH to 6.5 of hydrochloric acid and NaOH adjustment detection system.
3. a kind of method detecting signaling molecule in pseudomonas aeruginosa as claimed in claim 2, is characterized in that, be applied to Cu
2+cH
3oH/H
2in O solution, the detection of signaling molecule in pseudomonas aeruginosa, detects signaling molecule in pseudomonas aeruginosa in the mode that 277nm place absorption peak strength in uv-visible absorption spectra obviously changes.
4. a kind of method detecting signaling molecule in pseudomonas aeruginosa as claimed in claim 2, is characterized in that, be applied to Cu
2+dMF/H
2in O solution, the detection of signaling molecule in pseudomonas aeruginosa, detects signaling molecule in pseudomonas aeruginosa in the mode that 280nm place absorption peak strength in uv-visible absorption spectra obviously changes.
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