CN109142291A - A kind of fluorescence sense array and the method that Classification Identification is carried out to microorganism using the array - Google Patents
A kind of fluorescence sense array and the method that Classification Identification is carried out to microorganism using the array Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention belongs to molecular biology Material Fields, disclose a kind of fluorescence sense array and the method using the array to microorganism progress Classification Identification.The fluorescence sense array is made of aggregation-induced emission molecular solution and graphene oxide solution mixing.Fluorescence sense array of the invention directly mixes aggregation-induced emission molecule with graphene oxide, it is able to achieve the Classification Identification to multiple-microorganism, chemical modification is not needed, signal response is completed in 2h, data can be acquired by microplate reader, Fluorescence Spectrometer and digital image-forming, data are handled in conjunction with principal component analysis, with it is safe and simple, efficient, without complicated chemical label, background signal is low and the advantage of high specificity.
Description
Technical field
The invention belongs to molecular biology Material Fields, and in particular to a kind of fluorescence sense array and using the array to micro-
The method that biology carries out Classification Identification.
Background technique
Bacterium and fungi and the mankind have close relationship, on the one hand, the whole world every year because the infection of bacterium or fungi and
Dead number reaches 2,000,000；On the other hand, the bacterium in enteron aisle is in food digestion, the synthesis of nutrient molecule and exempting from for body
Important role is all had in terms of epidemic disease.Based on this, Classification Identification microorganism understands us and has using microorganism important
Meaning.However, current microorganism is based primarily upon microculture, have the shortcomings that time-consuming is waited so long, meanwhile, some microorganisms
Culture need special culture medium.Other technologies, including PCR, gene sequencing, surface-enhanced Raman and mass spectrographic method
It is all more complicated and take time and effort.The microorganism Classification Identification technology for developing novel and high-efficiency has great importance.
Biometrics technology has proven to the strong method for the identification of complex biological molecule parting.?
It is applied to distinguish protein, ion and escaping gas.In general, biological identification technology is based primarily upon molecule sensing battle array
The building of column and carry out.Based on the non-specific interaction between analysans and molecule sensor array to different analytes
Generate specific pattern.Currently, the Classification Identification for realizing bacterium or tumour cell using the technology has been reported.However it is at present
Only, following problem is still remained: 1, commonly use complicated equipment often in signal acquisition, 2, traditional fluorescent molecule is often
With stronger fluorescence signal, 3, the identification differentiation of bacterium often uses viable microbial at present, usually has certain sense
Metachromia.
Summary of the invention
In place of the above shortcoming and defect of the existing technology, the primary purpose of the present invention is that providing a kind of fluorescence
Sensor array.The fluorescence sense array of the present invention couple is by with aggregation-induced emission (aggregation-induced
Emission, AIE) property fluorescent molecule (AIEgens) and graphene oxide (GO) construct fluorescent molecule sensor array.No
It is same as traditional fluorescent molecule, aggregation-induced emission fluorescent molecule shines under dissolved state with very weak, and when molecule is transported
It moves by limited time, fluorescent emission efficiency greatly enhances.Graphene oxide not only can further quench background fluorescence, can be with
Interaction between the biomolecule of introduction of competition, to improve the separating capacity of molecule sensor array.
Classification Identification is carried out to microorganism using above-mentioned fluorescence sense array another object of the present invention is to provide a kind of
Method.
A further object of the present invention is to provide a kind of rapid typification of microorganism prepared using above-mentioned fluorescence sense array
Identification kit.
The object of the invention is achieved through the following technical solutions:
A kind of fluorescence sense array is made of aggregation-induced emission molecular solution and graphene oxide solution mixing；It is described
Aggregation-induced emission molecule has structural formula described in any one of following A1~A13:
(A1,Anal.Chem.2010,82,7035-7043；A2,Chem-Eur.J.2010,16,1232-1245；A3,
Chem.Commun.2017,53,4795-4798；A4,Chem.Commun.2012,48,8637-8639；A5,
J.Med.Chem.2007,50,663-673；A6,Chem.Sci.2017,8,1822-1830；A7,Chem.Sci.2017,8,
1822-1830；A8,Chem-Eur.J.2010,16,1232-1245；A9,Anal.Chem.2015,87,9487-9493；A10,
Chem.Commun.2014,50,8312-8315；A11-A13,CN.Patent.105541660[P].2016-01-15).
Preferably, the aggregation-induced emission molecule has the described in any item structural formulas of following A1~A7:
Preferably, the concentration of the aggregation-induced emission molecular solution is 1~10mM, and the concentration of graphene oxide solution is
0.2~0.4mg/ml, the volume ratio that aggregation-induced emission molecular solution is mixed with graphene oxide solution are 1:(0.5~5).
A method of Classification Identification is carried out to microorganism using above-mentioned fluorescence sense array, is included the following steps:
Fluorescence sense array is uniformly mixed with microbial lytic liquid and is incubated for, according to the difference of the fluorescence signal of generation
Classification Identification is carried out to microorganism.
Further, the time of the incubation is 2h.
Further, the fluorescence signal of the generation is acquired by microplate reader or Fluorescence Spectrometer, the signal of acquisition
It is realized by the method for principal component analysis (Principal component analysis, PCA) to microbial typing.
Further, the microorganism includes fungi, Gram-negative bacteria and gram-positive bacteria, specifically includes white thought
Pearl bacterium, saccharomyces cerevisiae, pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli and Bacillus subtillis.
A kind of rapid typification of microorganism identification kit prepared using above-mentioned fluorescence sense array.
The principle of the present invention are as follows: by aggregation-induced emission molecule and graphene oxide building fluorescent molecule array with
Fluorescence quenches completely before the effect of microbial lytic liquid, and microbial lytic liquid is used to light fluorescent molecule battle array as analysis substrate
Column.So that fluorescence obtains recovery in various degree after fluorescent molecule array and microbial lytic liquid are incubated for.The fluorescence sense array
It is different to different microbial lytic liquid fluorescence response intensity, to generate specificity fluorescent pattern to specified microorganisms, finally
The differentiation of binding pattern identification technology realization different microorganisms.
Compared with the existing technology, fluorescence sense array of the invention and identification method have the following advantages that and the utility model has the advantages that
(1) fluorescence sense array of the invention directly mixes aggregation-induced emission molecule with graphene oxide, Ji Nengshi
Now to the Classification Identification of multiple-microorganism, chemical modification is not needed, signal response is completed in 2h, can be by microplate reader, glimmering
Photothermal spectroscopic analyzer and digital image-forming are acquired data, handle in conjunction with principal component analysis data, have it is safe and simple,
Efficiently, it is marked without complicated chemical, background signal is low and the advantage of high specificity.
(2) identification method of the invention selects the lysate of microorganism as analysis substrate, compared to the outer membrane of microorganism
For, microbial lytic liquid can be provided about the more molecular informations of microorganism.Meanwhile bacterial lysate can be more easily
It stores for a long time.
Detailed description of the invention
Fig. 1 is that the fluorescence sense array in the embodiment of the present invention 1 is glimmering before and after bacterial lysate (Escherichia coli) are added
Radiograph figure.
Fig. 2 is after the fluorescent molecule sensor array constructed in the embodiment of the present invention 2 is acted on six kinds of microbial lytic liquid
Fluorescence photo figure.X1~X7 respectively represents seven kinds of AIE molecule A1~A7 in abscissa, ordinate Y1~Y6 respectively represent six kinds it is micro-
Biology.
Fig. 3 is to extract after fluorescent molecule sensor array and six kinds of microbial lytic liquid phase interactions in the embodiment of the present invention 2
Fluorescence intensity signals.
Fig. 4 is to carry out principal component analysis by the fluorescence signal that six kinds of microbial lytic liquid generate in the embodiment of the present invention 2
The signal pattern of the six kinds of microorganisms obtained after processing.
Fig. 5 be in the embodiment of the present invention 3 respectively into two sets fluorescent molecule sensor arrays (a, without containing graphene oxide,
Graphene oxide is added in b) the fluorescence photo figure and relatively glimmering after bacterial lysate (pseudomonas aeruginosa, Escherichia coli) is added
Light intensity map.
Fig. 6 is the schematic illustration of the competitive effect of graphene oxide of the present invention induction.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
A kind of fluorescence sense array of the present embodiment and the method to microorganism progress Classification Identification, the specific steps are as follows:
(1) solution of AIE molecule is configured；Respectively A1 (water dissolution, 1mM), A2 (water dissolution, 10mM), (DMSO is molten by A3
Solution, 1mM), A4 (DMSO dissolution, 1mM), A5 (DMSO dissolution, 10mM), A6 (DMSO dissolution, 1mM), A7 (DMSO dissolution, 1mM)；
The AIE molecule of A1~A7 has the following structure formula:
(2) taking 10 μ L AIE molecular solution that number in 96 orifice plates is added respectively is 1~7, wherein 50 μ are added in 1~No. 6 pipe
5 μ L graphene solutions (concentration 0.2mg/ml) are added in No. 7 pipes in L graphene solution (concentration 0.4mg/ml), are uniformly mixed
After be prepared into fluorescent molecule sensor array.
(3) bacterial lysate (Escherichia coli) of 100 μ L is added into each test tube of step (2), is uniformly mixed, room temperature
It is incubated for 2h, is taken pictures under ultraviolet light irradiation.
Photo such as Fig. 1 institute of the fluorescence sense array before and after bacterial lysate (Escherichia coli) are added in the present embodiment
Show.As can be seen from Figure 1 before bacterial lysate is added, the fluorescence of fluorescence sense array is quenched, only 96 orifice plate materials
Fluorescence, and after bacterial lysate is added, the fluorescence of fluorescence sense array is lit, each hole issues different colours, brightness
Fluorescence.
Embodiment 2
A kind of fluorescence sense array of the present embodiment and the method to microorganism progress Classification Identification, the specific steps are as follows:
(1) solution of AIE molecule is configured；Respectively A1 (water dissolution, 1mM), A2 (water dissolution, 10mM), (DMSO is molten by A3
Solution, 1mM), A4 (DMSO dissolution, 1mM), A5 (DMSO dissolution, 10mM), A6 (DMSO dissolution, 1mM), A7 (DMSO dissolution, 1mM)；
(2) taking 10 μ L AIE molecular solution to be added in 96 orifice plates and number respectively is X1~X7, wherein in X1~X6 pipe
50 μ L graphene solutions (concentration 0.4mg/ml) are added, 5 μ L graphene solutions (concentration 0.2mg/ml) are added in X7 pipe,
It is prepared into fluorescent molecule sensor array after mixing.
(3) parallel six groups of sample-adding, preparation are directed to the fluorescence sense array of six kinds of microorganisms, and number is Y1~Y6.
(4) it is added a kind of 100 μ L of bacterial lysate in each group of hole, totally six kinds.Y1~Y6 respectively correspond Candida albicans,
Saccharomyces cerevisiae, pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli, Bacillus subtillis are uniformly mixed, and room temperature is incubated
2h is educated, is taken pictures under ultraviolet light irradiation.Repetitive operation 5 times.
(5) fluorescence spectrum in each hole is tested by microplate reader, obtains fluorescent molecule sensor array to each microorganism
Lysate response intensity.
(6) analyzing and drawing is carried out to data by principal component analysis (4.5 software of Canoco).
Fluorescence photo of the fluorescence sense array after six kinds of microbial lytic liquid are added in the present embodiment is as shown in Figure 2.
Figure it is seen that response difference of the fluorescence sense array for each microbial lytic liquid, predominantly fluorescent molecule signal
Intensity it is different.
The signal of six kinds of microorganism fluorescence signal response intensities in the present embodiment is as shown in Figure 3.It can from Fig. 3
Out, every kind of microbial lytic liquid can stimulate fluorescent molecule array to generate specific fluorescence intensity array.
Fluorescent signal data in the present embodiment is handled as shown in figure 4, figure 4, it is seen that by principal component analysis
After processing, six kinds of microorganisms are being distinguished well.
Embodiment 3
A kind of fluorescence sense array of the present embodiment and the method to microorganism progress Classification Identification, the specific steps are as follows:
(1) solution of AIE molecule is configured；Respectively A1 (water dissolution, 1mM), A2 (water dissolution, 10mM), (DMSO is molten by A3
Solution, 1mM), A4 (DMSO dissolution, 1mM), A5 (DMSO dissolution, 10mM), A6 (DMSO dissolution, 1mM), A7 (DMSO dissolution, 1mM)；
(2) 1 μ L AIE molecular solution is taken to be added in test tube respectively, test tube number is 1~7, wherein being added 5 in 1~No. 6 pipe
0.5 μ L graphene solution (concentration 0.2mg/ml) is added in No. 7 pipes in μ L graphene solution (concentration 0.4mg/ml), mixes
Fluorescent molecule sensor array is prepared into after uniformly.Two parts of sensor arrays of preparation in parallel.
(3) a set of fluorescent molecule sensor array without containing graphene oxide is separately done, the specific steps are as follows:
(a) solution of AIE molecule is configured；Respectively A1 (water dissolution, 1mM), A2 (water dissolution, 10mM), (DMSO is molten by A3
Solution, 1mM), A4 (DMSO dissolution, 1mM), A5 (DMSO dissolution, 10mM), A6 (DMSO dissolution, 1mM), A7 (DMSO dissolution, 1mM)；
(b) 1 μ L AIE molecule (A1~A7) solution is taken to be added in seven test tubes respectively, test tube number is 1~7, wherein 1~
5 μ L aqueous solutions are added in No. 6 pipes, 0.5 μ L aqueous solution is added in No. 7 pipes, is prepared into the fluorescence of not containing graphene after mixing
Molecule sensor array.Two parts of sensor arrays of preparation in parallel.
(c) bacterial lysate (pseudomonas aeruginosa, Escherichia coli) are added into two sets of fluorescent molecule sensor arrays respectively,
It is incubated for 2 hours, takes pictures under ultraviolet light irradiation.
Fluorescence photo data in the present embodiment as shown in figure 5, a, pseudomonas aeruginosa and Escherichia coli bacteria lysis
Liquid and the AIE interaction of molecules for not containing graphene oxide, are above fluorescence photo figure, and lower is what two kinds of bacterial lysates generated
Relative intensity of fluorescence figure.B, two kinds of bacterial lysates and the fluorescent molecule sensor array that graphene oxide is added interact, on
For fluorescence photo figure, the lower relative intensity of fluorescence figure generated for two kinds of bacterial lysates.From figure 5 it can be seen that without aerobic
The fluorescent molecule sensor array of graphite alkene has response to two kinds of bacterial lysates, and the degree of response is identical, cannot distinguish between two
Kind bacterium.And the fluorescent molecule array that graphene oxide is added can be very good to distinguish two kinds of bacterial lysates.The reason for this is that oxygen
Influence of the competitive effect of graphite alkene induction for promotion sensor array selectivity.The competitive of graphene oxide induction is made
Schematic illustration is as shown in Figure 6.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. a kind of fluorescence sense array, it is characterised in that: the fluorescence sense array is by aggregation-induced emission molecular solution and oxygen
Graphite alkene solution mixing composition；The aggregation-induced emission molecule has structural formula described in any one of following A1~A13:
2. a kind of fluorescence sense array according to claim 1, it is characterised in that: the aggregation-induced emission molecule has
The described in any item structural formulas of following A1~A7:
3. a kind of fluorescence sense array according to claim 1, it is characterised in that: the aggregation-induced emission molecular solution
Concentration be 1~10mM, the concentration of graphene oxide solution is 0.2~0.4mg/ml, aggregation-induced emission molecular solution and oxygen
The volume ratio of graphite alkene solution mixing is 1:(0.5~5).
4. a kind of side for carrying out Classification Identification to microorganism using fluorescence sense array according to any one of claims 1 to 3
Method, it is characterised in that include the following steps:
Fluorescence sense array is uniformly mixed with microbial lytic liquid and is incubated for, according to the difference of the fluorescence signal of generation to micro-
Biology carries out Classification Identification.
5. the method that fluorescence sense array according to claim 4 carries out Classification Identification to microorganism, it is characterised in that: institute
The time for stating incubation is 2h.
6. the method that fluorescence sense array according to claim 4 carries out Classification Identification to microorganism, it is characterised in that: institute
The fluorescence signal for stating generation is acquired by microplate reader or Fluorescence Spectrometer, the method that the signal of acquisition passes through principal component analysis
It realizes to microbial typing.
7. the method that fluorescence sense array according to claim 4 carries out Classification Identification to microorganism, it is characterised in that: institute
Stating microorganism includes fungi, Gram-negative bacteria and gram-positive bacteria.
8. the method that fluorescence sense array according to claim 4 carries out Classification Identification to microorganism, it is characterised in that: institute
Stating microorganism includes Candida albicans, saccharomyces cerevisiae, pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli and withered grass
Bacillus.
9. a kind of rapid typification of microorganism using fluorescence sense array according to any one of claims 1 to 3 preparation is identified
Kit.
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