CN108254366A - A kind of method of the Nano silver grain detection microorganism based on phenyl boric acid functionalization - Google Patents
A kind of method of the Nano silver grain detection microorganism based on phenyl boric acid functionalization Download PDFInfo
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- CN108254366A CN108254366A CN201710845810.0A CN201710845810A CN108254366A CN 108254366 A CN108254366 A CN 108254366A CN 201710845810 A CN201710845810 A CN 201710845810A CN 108254366 A CN108254366 A CN 108254366A
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Abstract
The invention belongs to analytical chemistry fields, are related to a kind of method of the Nano silver grain detection microorganism based on phenyl boric acid functionalization.The Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization are added in tested microorganism sample, so that biological surface contains along o-dihydroxy group in detected sample after reaction, inhibit the aggregation of phenyl boric acid functionalization Nano silver grain, make solution colour that different variations occur, the quantitatively or semi-quantitatively detection of microorganism in sample is realized by colorimetric detection or the variation for visually observing solution colour.The present invention has the advantages that simple for process, easy to operate, economical and practical and quick and precisely, and entire detection process can be completed in 20min, the potentiality with great clinical practice.Meanwhile expensive laboratory apparatus is not needed to, and it is of low cost, by the way that the half-quantitative detection of microorganism visually can be realized, there is great popularizing value.
Description
Technical field
The invention belongs to analytical chemistry fields, are related to a kind of Nano silver grain detection microorganism based on phenyl boric acid functionalization
Method.
Background technology
A big threat of the microorganism as fields such as publilc health, environmental monitoring, food security and biochemistry safety is fast
Speed, Sensitive Detection increasingly cause the concern of people.Traditional detection method, such as colony counting method generally require to expend 3-7 days
Time obtain testing result；And although PCR can be quickly obtained testing result, it is complicated for operation,
Expensive instrument is needed simultaneously.Therefore, it is still that we face to develop accurate, quick, sensitive, easy microorganism detection method
A major challenge.
Colorimetric method causes people due to having many advantages, such as simple, stable, economic and potential clinical detection application value
Great interest.Enzyme linked immunoassay as a kind of most widely used colorimetric detection method, is catalyzed using enzyme marker
Substrate develops the color to carry out signal output, realizes the purpose of detection, and the extinction coefficient of chromophore that substrate for enzymatic activity generates can limit
Make its detection sensitivity.In order to improve the detection sensitivity of colorimetric method, in these years, including gold nanoparticle and Nano silver grain
Nano plasma excimer material inside with research of the special optical property to Analytical Chemistry in Life Science field due to bringing
New breakthrough.Colorimetric detection method based on nano plasma excimer material is the gold, silver nanometer by identifying molecular modification
The combination of material and object to change the dispersity of gold, silver nano material, makes it become state of aggregation from dispersed, makes it
Color changes.Traditional identification molecule mainly uses antibody, but there is antibody expensive, different batches quality to deposit
Difference and it is unstable the shortcomings of.
In recent years, phenyl boric acid and its derivative were obtained as application of the identification molecule in the detection of carbohydrate and bacterium
Very big concern.Phenyl boric acid can be artificial synthesized, cheap, and stability is good, can in the lipopolysaccharides and glycoprotein of bacterium surface
The cis- o-dihydroxy structure contained reversibly covalent bond forms cyclic ester through dehydration, therefore, can be used as the knowledge of microorganism
Other molecule.At present, phenyl boric acid and its derivative are chiefly used in as identification molecule in electrochemical sensor, so far still without colorimetric
Method is used for the report of the detection of microorganism.The exciting agent that the present invention creatively assembles phenyl boric acid as nano silver can will divide
The nano silver for dissipating good phenyl boric acid functionalization is converted into state of aggregation；And the nano silver using phenyl boric acid as identification molecule can be coupled to
Bacterium surface, the presence of bacterium can inhibit the aggregation of the nano silver of phenyl boric acid functionalization, and the bacterium of different content is to phenyl boric acid work(
The inhibition level of the nano silver aggregation of energyization is different, makes solution colour that different variations occur.Its color change can pass through naked eyes
It is differentiated, while it is detected using ultraviolet-uisible spectrophotometer.
Invention content
The purpose of the present invention is to provide a kind of methods of the Nano silver grain detection microorganism based on phenyl boric acid functionalization.
To achieve the above object, the present invention is implemented using following technical scheme：
A kind of method of the Nano silver grain detection microorganism based on phenyl boric acid functionalization, adds in tested microorganism sample
Enter the Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization, cause that biological surface contains cis- in detected sample after reaction
O-dihydroxy group inhibits the aggregation of phenyl boric acid functionalization Nano silver grain, makes solution colour that different variations occur, passes through ratio
Color detects or visually observes the quantitatively or semi-quantitatively detection for changing microorganism in realization sample of solution colour.
The Nano silver grain of the phenyl boric acid functionalization and the concentration of mercaptophenyl boronic acid are respectively 5nM and 5-30nM.
The Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization and the condition of microbial reaction are incubated at room temperature 0.5-
2h。
The Nano silver grain preparation process of the phenyl boric acid functionalization is：Into the Nano silver grain of a concentration of 5nM, add in
The mercaptophenyl boronic acid of 10 μ L 5-30nM concentration, reacts 5-30min, you can obtain the nano grain of silver of phenyl boric acid functionalization at room temperature
Son.
The mercaptophenyl boronic acid is 4- mercaptophenyl boronic acids or 3- mercaptophenyl boronic acids.
The microorganism is Gram-negative bacteria.
The beneficial effects of the present invention are：
Colorimetric analysis of the present invention is had using the identification molecule that phenyl boric acid is microorganism, silver nano-grain for signal element
It is simple for process, easy to operate, economical and practical and quick and precisely the advantages of, entire detection process can be completed in 20min, have
The potentiality of great clinical practice.Meanwhile expensive laboratory apparatus is not needed to, and it is of low cost, it is micro- by visually can be realized
The half-quantitative detection of biology has great popularizing value.
Description of the drawings
Fig. 1 is the ultraviolet-visible absorption spectroscopy figure after different form provided in an embodiment of the present invention aggregation, wherein, a is
MPBA-AgNPs, b are combined for MPBA-AgNPs with Escherichia coli, and c adds in excess 4- mercaptophenyl boronic acids for MPBA-AgNPs, and d is
MPBA-AgNPs, which is combined to add with Escherichia coli, adds in excess 4- mercaptophenyl boronic acids.
In the Nano silver grain of Fig. 2 phenyl boric acid functionalization, 1 × 10 is added in7, 5 × 106, 1 × 106, 5 × 105, 1 × 105, 5
×104cfu mL-1Escherichia coli reaction 10min after, add in 10 μ L300 μM mercaptophenyl boronic acid reaction 1min after it is ultraviolet can
See abosrption spectrogram.
Under Fig. 3 detection methods, 1 × 107, 5 × 106, 1 × 106, 5 × 105, 1 × 105, 5 × 104cfu mL-1Large intestine
The absorbance value figure that bacillus measures at 398nm.
Under Fig. 4 detection methods, 1 × 107, 5 × 106, 1 × 106, 5 × 105, 1 × 105, 5 × 104cfu mL-1Large intestine
Color diagram corresponding to bacillus.
Under Fig. 5 detection methods, 1 × 107, 1 × 106, 1 × 105, 1 × 104, 1 × 103, 1 × 102cfu mL-1Sulfuric acid
Color diagram corresponding to salt reducing bacteria.
Specific embodiment
The present invention is further explained in the following with reference to the drawings and specific embodiments.
The present invention adds in the Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization, sulfydryl benzene in containing microbiological specimens
Boric acid can effectively promote phenyl boric acid functionalization Nano silver grain that aggtegation occurs, and the antimicrobial surface in sample contains
Cis- o-dihydroxy group can be combined with phenyl boric acid group, inhibit the aggregation of phenyl boric acid functionalization Nano silver grain, and different
The microbes of content combine different amounts of mercaptophenyl boronic acid, make solution colour that different variations occur, by detecting or seeing
The quantitatively or semi-quantitatively detection of microorganism in sample is realized in the variation for examining solution colour.The present invention has simple for process, operation letter
Just it is, economical and practical and quick and precisely the advantages of, entire detection process can be completed in 20min, have great clinical practice
Potentiality.Meanwhile expensive laboratory apparatus is not needed to, and it is of low cost, it is examined by the sxemiquantitative that microorganism visually can be realized
It surveys, there is great popularizing value.
Embodiment 1
The preparation of the Nano silver grain of phenyl boric acid functionalization
By 1mL 100mM silver nitrate solutions and 1mL 100mM citric acid three sodium solutions under intense agitation, it is added to
36.8mL in ultra-pure water.Then, the 100mM sodium borohydrides that 1.2mL is newly prepared are added in.Solution colour can gradually become yellow, instead
Should be after about 10min, aging 2 days under the conditions of moving it into 4 DEG C.Nano silver grain prepared by 1mL is taken to dilute 16 times, then adds in 10
μ L 4- mercaptophenyl boronic acids react 30min to get the Nano silver grain of phenyl boric acid functionalization has been arrived (referring to Fig. 1).
As shown in Figure 1, a is the ultraviolet-visible absorption spectroscopy figure of the Nano silver grain of phenyl boric acid functionalization, at 398nm
There is a sharp absorption peak；B is to add in a concentration of 10 to the Nano silver grain of phenyl boric acid functionalization7cfu mL-1Escherichia coli
Ultraviolet-visible absorption spectroscopy figure afterwards, the absorbance at 398nm are declined slightly, but are occurred without other peaks；C is to benzene boron
The ultraviolet-visible absorption spectroscopy figure after mercaptophenyl boronic acid is added in the silver nano-particle solution of acid functionalization, compared with a, c exists
Absorbance at 398nm is substantially reduced, and has certain red shift, meanwhile, there is a wider absorption in 550nm or so
Peak, this is because after mercaptophenyl boronic acid adds in so that the Nano silver grain of phenyl boric acid functionalization has occurred what is largely assembled rapidly
Phenomenon；D is after adding in mercaptophenyl boronic acid in the silver nano-particle solution of the phenyl boric acid functionalization after Escherichia coli are added
Ultraviolet-visible absorption spectroscopy figure, compared with b, absorbances of the d at 398nm is declined slightly, but the reduction amplitude much smaller than c, together
When there is an absorption peak in 500nm or so in it, this all shows the poly- of the Nano silver grain of the phenyl boric acid functionalization in its solution
Collection degree is less than the solution for being added without Escherichia coli, caused by this further proves that the addition of bacterium can inhibit mercaptophenyl boronic acid
Phenyl boric acid functionalization Nano silver grain aggregation.
Embodiment 2
Detection based on UV, visible light extinction spectrum to Escherichia coli
Into the Nano silver grain of 1mL phenyl boric acid functionalization, 100 μ L Escherichia coli are added in, after reacting 10min, add in 10 μ
The mercaptophenyl boronic acid that 300 μM of L, after reacting 1min, using ultraviolet-uisible spectrophotometer to its range in 300nm-700nm
Interior progress spectral scan (referring to Fig. 2), as seen from Figure 2 with the increase of e. coli concentration, the maximum extinction at 398nm
Angle value gradually increases.Its absorbance value and the logarithm of e. coli concentration are 5 × 104cfu mL-1To 1 × 107cfu mL-1's
(referring to Fig. 3) in a linear relationship in concentration range.
Embodiment 3
The naked eyes detection of Escherichia coli
Into the Nano silver grain of 1mL phenyl boric acid functionalization, 100 μ L Escherichia coli are added in, after reacting 10min, add in 10 μ
The mercaptophenyl boronic acid of L 3mM after reacting 1min, observes solution colour (referring to Fig. 4).As shown in Figure 4, with e. coli concentration
Reduction, color showed from the faint yellow variation tendency to light red.It can be limited to 5 by the lowest detection of discernable by eye ×
105cfu mL-1。
Embodiment 4
The naked eyes detection of sulfate reducing bacteria
Into the Nano silver grain of 1mL phenyl boric acid functionalization, 100 μ L sulfate reducing bacterias are added in, after reacting 30min, are added
Enter the mercaptophenyl boronic acid of 300 μM of 20 μ L, after reacting 1min, observe solution colour (referring to Fig. 5).As shown in Figure 5, with large intestine
The reduction of bacillus concentration, color are showed from the faint yellow variation tendency to brown color.It can be by the minimum inspection of discernable by eye
Survey is limited to 1 × 104cfu mL-1。
Claims (6)
- A kind of 1. method of the Nano silver grain detection microorganism based on phenyl boric acid functionalization, it is characterised in that：Treating micrometer life The Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization are added in object sample, biological table in detected sample is caused after reaction Cis- o-dihydroxy group is contained in face, inhibits the aggregation of phenyl boric acid functionalization Nano silver grain, makes solution colour that different changes occur Change, the quantitatively or semi-quantitatively detection of microorganism in sample is realized by colorimetric detection or the variation for visually observing solution colour.
- 2. the method for the Nano silver grain detection microorganism according to claim 1 based on phenyl boric acid functionalization, feature It is：The Nano silver grain of the phenyl boric acid functionalization and the concentration of mercaptophenyl boronic acid are respectively 5nM and 5-30nM.
- 3. the method for the Nano silver grain detection microorganism according to claim 1 based on phenyl boric acid functionalization, feature It is：The Nano silver grain and mercaptophenyl boronic acid of phenyl boric acid functionalization and the condition of microbial reaction are incubated at room temperature 0.5-2h.
- 4. the method for the Nano silver grain detection microorganism according to claim 1 or 2 based on phenyl boric acid functionalization, special Sign is：The Nano silver grain preparation process of the phenyl boric acid functionalization is：Into the Nano silver grain of a concentration of 5nM, add in The mercaptophenyl boronic acid of 10 μ L 5-30nM concentration, reacts 5-30min, you can obtain the nano grain of silver of phenyl boric acid functionalization at room temperature Son.
- 5. the method for the Nano silver grain detection microorganism according to claim 1 based on phenyl boric acid functionalization, feature It is：The mercaptophenyl boronic acid is 4- mercaptophenyl boronic acids or 3- mercaptophenyl boronic acids.
- 6. the method for the Nano silver grain detection microorganism according to claim 1 based on phenyl boric acid functionalization, feature It is：The microorganism is Gram-negative bacteria.
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| CN201710845810.0A CN108254366B (en) | 2017-09-19 | 2017-09-19 | Method for detecting microorganisms based on phenylboronic acid functionalized silver nanoparticles |
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Cited By (2)
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| CN111157521A (en) * | 2020-02-15 | 2020-05-15 | 信阳学院 | Nano-gold colorimetric method based on agglomeration resistance and determination of silver ions |
| CN113109265A (en) * | 2021-03-18 | 2021-07-13 | 温州医科大学 | Bacterial photo-thermal detection reagent, kit and detection method |
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| JP2024511054A (en) * | 2021-03-24 | 2024-03-12 | トラスティーズ オブ タフツ カレッジ | Boronic acid compounds, compositions and methods |
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| CN113109265A (en) * | 2021-03-18 | 2021-07-13 | 温州医科大学 | Bacterial photo-thermal detection reagent, kit and detection method |
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