CN108254348B - pH sensitive fluorescent sensor for high-throughput detection of active microorganisms and construction method - Google Patents
pH sensitive fluorescent sensor for high-throughput detection of active microorganisms and construction method Download PDFInfo
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- CN108254348B CN108254348B CN201810097374.8A CN201810097374A CN108254348B CN 108254348 B CN108254348 B CN 108254348B CN 201810097374 A CN201810097374 A CN 201810097374A CN 108254348 B CN108254348 B CN 108254348B
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—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
- G01N2021/6417—Spectrofluorimetric devices
Abstract
A pH sensitive fluorescent sensor for detecting active microorganisms in high flux and a construction method thereof comprise the following steps: 1) preparing a fluorescent nano material, dissolving phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescein by using ethanol, fully mixing, and slowly dripping into pure water to prepare a fluorescent nano micelle; 2) preparing a sensing gel, namely adding a fluorescent nano micelle into a 1-3% hot agar solution, fully mixing, and condensing to prepare the fluorescent sensing gel; 3) the sensor is constructed by dividing a container into two parts by using a water-phase filter membrane, filling sensing gel into the lower part of the container, and filling liquid microorganism culture medium into the upper part of the container. The invention has the advantages that the culture unit and the detection unit are separated, the rapid and real-time detection of the active acid-producing and gas-producing microorganism is realized by detecting the change of the fluorescence signal in the sensing gel, compared with the traditional flat plate method, the sensor is based on the traditional culture theory, has convenient operation and short detection time, and can realize the high-flux screening of the microorganism in different materials.
Description
Technical Field
The invention belongs to the field of microbial detection, relates to detection of trace active microbes, and particularly relates to a pH sensitive fluorescent sensor for detecting active microbes in a high-throughput manner and a construction method thereof.
Background
In recent years, food safety incidents at home and abroad are endless, which not only affect the market order, but also bring certain panic to the public, such as the recent microbial contamination incident of the constantly natural milk powder, and although the recent microbial contamination incident is proved not to be botulic bacteria, the social anxiety about the microbial contamination in the food is indirectly shown.
The traditional plate counting method for detecting microorganisms has higher accuracy and sensitivity, but has more involved experiments, complicated operation, longer preparation time and ending time, and a great deal of personnel participation. Therefore, there is an urgent need for an accurate, time, labor and cost-effective rapid assay method, which is currently mainly used in microbiological test paper, biochemical assay, ATP method, flow cytometry, and the like.
Perrifilm of 3M CoTM The Plate series microbial test piece can respectively detect the total number of bacterial colonies, the count of coliform groups, the count of mould and yeast and the like, and the paper sheet method and the national standard method are both based on the traditional culture theory, so that the consistency is better in the detection rate of the microorganisms, the bacterial colonies are typical and easy to judge, but the detection flux is not high, and the sensitivity of the test paper sheet still needs to be further improved.
The PCR technology adopts in vitro enzymatic reaction to synthesize specific DNA fragments, and then identifies bacteria through amplification products. Because PCR sensitivity is high, can detect the copy gene of a bacterium theoretically, therefore only need increase the fungus or even not increase the fungus in the short time in the detection of bacterium, can screen through PCR, saved a large amount of time, but PCR technique needs higher personnel's requirement and laboratory environment, and can't accomplish to living bacterium and dead bacterium and distinguish, and the extraction of target gene in complicated matrix also has certain difficulty simultaneously.
The ATP bioluminescence method utilizes an ATP bioluminescence analysis technology and a somatic cell removal technology to measure bacterial ATP and somatic cell ATP, the quantity of the bacterial ATP is in direct proportion to the number of bacteria, and therefore detection of the microorganisms is achieved.
After the sample is pretreated, the single-cell suspension is prepared, and is analyzed after specific staining, the analysis speed is high, multiple parameters can be analyzed simultaneously, but the instrument is expensive, the analysis concentration needs higher orders of magnitude, and the analysis error of trace microorganisms is larger.
In summary, although many methods and techniques for detecting microorganisms exist at present, most of the methods and techniques have certain problems, and have great difference from the traditional culture theory, so that the results have risks in consistency, and the detection of trace microorganisms still has certain challenges. Therefore, the development of a method which has high sensitivity and is easy to operate and can realize rapid detection of a large number of samples is an urgent problem to be solved.
Disclosure of Invention
The invention aims to overcome the defects of the existing trace microorganism detection technology and develop a pH sensitive fluorescence sensor for detecting active microorganisms at high flux based on real-time fluorescence, simple operation, high sensitivity and high detection flux monitoring analysis technology, and a construction method thereof.
The purpose of the invention is realized by the following technical scheme: a pH sensitive fluorescence sensor for detecting active microorganism in high flux comprises sensing gel, a water phase filter membrane and a liquid microorganism culture medium which are assembled in a special container, wherein the sensing gel and the liquid microorganism culture medium are separated by the water phase filter membrane, the sensing gel is positioned at the bottom of the container, and the sensing gel is composed of fluorescent nano-micelles fixed in agar gel.
The special container is a PE plastic pipe with a sealing cover, the specific structure is shown in figure 2, the special container comprises 3 parts, the bottom part is a sensor, the middle part is a microorganism culture part, the upper part is a sealing cover, the bottom part can permeate fluorescence and is isolated from the middle part by a water-based filter membrane.
The construction method comprises the following steps:
1) dissolving phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescent yellow in ethanol, fully mixing, and gradually dropping into pure water to prepare the fluorescent nano micelle;
2) preparing agar hot solution, adding the fluorescent nano-micelle prepared in the step 1), fully mixing, and fixing the fluorescent nano-micelle in agar gel after cooling;
3) and separating the sensing gel from the specific liquid culture medium by adopting a water-phase filter membrane to prepare the fluorescent sensor suitable for detecting different kinds of microorganisms.
The construction method comprises the following specific steps:
1) preparing fluorescent nano-micelle: phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescein were used as 1: 10-1: dissolving the mixture in chromatographic grade absolute ethyl alcohol according to a molar ratio of 50, fully mixing, gradually dropping the mixture into pure water to prepare fluorescent nano-micelle, dialyzing and purifying the prepared nano-micelle by using 20 mM PBS, and storing the purified nano-micelle at 4 ℃;
2) preparation of sensing gel: preparing 1-3% hot agar solution at 50-70 ℃ by using agar, wherein the volume ratio (hot agar solution/fluorescent nano micelle mother solution) of the hot agar solution is 10: 1-30: 1, cooling and fixing the fluorescent nano-micelle to prepare the sensing gel with high transparency, wherein the particle size of the nano-micelle is 20 nm-200 nm, and excitation and emission are respectively 480 nm and 520 nm. The concentration range of the prepared agar gel is 1% -3%, and the agar gel is used for fixing the fluorescent nano micelle and preparing the sensing gel with high transparency.
3) Preparing a liquid culture medium: referring to the preparation method of LB (Luria-Bertani) medium, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl were added to 950 ml of deionized water, and the vessel was shaken until the solute was dissolved; adjusting the pH value to 7.0 by using 5 mol/L NaOH (about 0.2 ml); using deionized water to fix the volume to 1L; at 15 psi (1.05 kg/cm)2) Steam sterilizing under high pressure for 20 min;
4) assembling the sensor: and (3) filling the sensing gel into the bottom of a special container, and separating the sensing gel from the liquid culture medium by using a 0.22-0.45 mu m water-phase filter membrane.
In the invention, the fluorescent nano-micelle is a self-assembled pH-sensitive fluorescent substance, wherein phospholipid polyethylene glycol is used as a micelle carrier, 5- (N-hexadecanoyl) amino fluorescein is self-assembled on the surface of the nano-microsphere due to the hydrophobic fatty chain, and the 5- (N-hexadecanoyl) amino fluorescein is sensitive to pH and can reflect the growth speed of microorganisms in real time.
Compared with the prior art, the invention has the beneficial effects that:
1) novel pH-sensitive fluorescent nanomicelle: the pH sensitive fluorescent dye is fixed by the fluorescent nano-micelle in a self-assembly mode, the particle size of the fluorescent nano-micelle is controllable, a large amount of fluorescent substances can be wrapped, and the fluorescent substances can be effectively fixed by combining the fluorescent nano-micelle with the agar gel.
2) The consistency with the traditional method is higher: based on the traditional culture theory, the liquid culture medium can be prepared by referring to a national standard method, the rapid detection of most microorganisms can be realized, viable bacteria and dead bacteria can be effectively distinguished, and the consistency of the traditional flat plate counting method is high.
3) Real-time monitoring: the sensor comprises a liquid culture medium and a sensing gel, wherein the liquid culture medium is used for culturing microorganisms, and the sensing gel can be used for observing the growth dynamics of the microorganisms in real time.
4) The sensitivity is high: the sensing gel contains a high-sensitivity fluorescent dye substance, the fluorescent signal of the sensing gel is changed due to the growth of microorganisms, the rapid detection of the microorganisms is realized, theoretically, one viable bacterium exists, the detection time is shortened as the bacterium content is higher, and compared with a flat plate counting method, the analysis time is greatly shortened.
Drawings
FIG. 1 is a graph showing the change of fluorescence signals in a buffer solution of pH-sensitive fluorescent micelles at different pH values;
FIG. 2 is a schematic diagram of a pH-sensitive fluorescence sensor, in which: 1. a water phase filter membrane; 2.pH sensitive fluorescent nano-micelle; 3. a sensing gel; 4. a liquid culture medium; 5. sealing the upper cover;
FIG. 3 is a graph showing the response of fluorescence signals during the growth of E.coli, wherein the upper solid black line shows a high concentration microbial sample, and the lower solid gray line shows a low concentration microbial sample.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1 preparation of pH-sensitive fluorescent nanomicelle
The preparation method of the pH-sensitive fluorescent nano-micelle provided by the embodiment of the invention comprises the following steps: phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescein were used as 1: 10-1: dissolving 50 mol ratio in ethanol, fully mixing, dropwise adding into pure water, fully stirring to enable phospholipid polyethylene glycol to self-assemble into nano-micelle, and fixing a fluorescence signal group on the surface of the nano-micelle by the action of a hydrophobic chain of 5- (N-hexadecanoyl) amino fluorescein.
The final concentration of the fluorescent nano-micelle in the aqueous solution is 1-10 mg/mL, the particle size of the nano-micelle is increased by excessively high concentration, and the particle size of the nano-micelle is 20-200 nm.
The particle size of the nano micelle is increased along with the increase of the stirring time, and the stirring time used in the invention is 1-3 hours.
The fluorescent nano micelle is sensitive to pH, the pH response range is between pH 5 and pH 9, and the change of the fluorescence response is shown in figure 1. The typical fluorescent dye is 5- (N-hexadecanoyl) amino fluorescein, the tail end of the fluorescein yellow contains a long-chain alkyl chain, the fluorescein yellow has a strong hydrophobic effect, and the fluorescence excitation and the fluorescence emission of the nano-micelle are 480 nm and 520 nm respectively.
Example 2 construction of pH sensitive fluorescent sensor
The construction of the pH sensitive fluorescent sensor provided by the embodiment of the invention comprises the following steps: 1.pH sensitive fluorescent nano-micelle; 2. a sensing gel; 3, liquid culture medium; 4. a water phase filter membrane; 5. and sealing the upper cover. Firstly, preparing sensing gel by utilizing pH sensitive fluorescent nano micelle and agar, placing the sensing gel at the bottom of a special container shown in figure 2, separating the sensing gel from a liquid culture medium by using a 0.22-0.45 mu m water-phase filter membrane, and separating the sensing gel from the outside by using a sealing upper cover to complete the construction of the pH sensitive fluorescent sensor.
The pH-sensitive fluorescent nanomicelle is prepared according to the steps of example 1, and the fluorescent nanomicelle can wrap and fix a plurality of fluorescent dye molecules, provide a high-intensity fluorescent signal, and fix the fluorescent dye molecules so that the fluorescent dye molecules do not diffuse into a liquid medium.
The sensing gel is prepared by mixing 1-3% agar hot solution with the pH sensitive fluorescent nano micelle, and is mixed, stirred and cooled when the temperature of the agar solution is lower than 70 ℃, so that the sensing gel is high in transparency and can be matched with a fluorescence spectrum instrument to read a fluorescent signal.
The aperture size of the water-phase filter membrane is 0.22-0.45 mu m, so that common microorganisms can be effectively isolated, and the microorganisms are prevented from diffusing into the sensing gel.
The liquid culture medium is prepared according to the traditional culture theory, and has certain selectivity aiming at different microorganisms.
Example 3 real-time detection of E.coli
The embodiment of the invention provides a method for detecting escherichia coli by using a pH sensitive fluorescent sensor, which comprises the following steps: 1. pretreating a sample; 2. placing the processed sample into a liquid culture medium; 3. putting the sensor into a special fluorescence detector; 4. reading the fluorescence signal value to obtain a real-time growth curve of the microorganism, wherein the result of the growth curve is shown in FIG. 3，The black solid line is a high concentration microorganism sample, the gray solid line is a low concentration microorganism sample, and the result shows that the higher the microorganism content is, the faster the fluorescence signal increases.
The pre-sample pretreatment requires a treatment for the form (solid, liquid, etc.) of the sample, and if necessary, a simple enrichment treatment.
The liquid culture medium needs to be designed with certain selectivity according to a culture theory aiming at the types of detected microorganisms, so that the growth of mixed bacteria is avoided.
The fluorescence detector has an excitation wavelength of 480 nm, can detect emission waves at 520 nm, can observe the change of fluorescence signals in the sensing gel in real time, and draws a curve.
Claims (7)
1. A pH sensitive fluorescence sensor for high-flux detection of active microorganisms is characterized in that: the sensor gel is separated from the liquid microorganism culture medium through the water phase filter membrane and is positioned at the bottom of the container, and the sensor gel is composed of fluorescent nano-micelles fixed in agar gel; the fluorescent nano micelle is prepared by dissolving phospholipid polyethylene glycol covalently labeled with 5- (N-hexadecanoyl) amino fluorescent yellow in ethanol, fully mixing, and gradually dropping into pure water; the fluorescent nano-micelle is a self-assembled pH sensitive fluorescent substance, wherein phospholipid polyethylene glycol is used as a micelle carrier, 5- (N-hexadecanoyl) amino fluorescent yellow is self-assembled on the surface of the nano-microsphere due to the hydrophobic fatty chain, and the 5- (N-hexadecanoyl) amino fluorescent yellow is sensitive to pH and can reflect the growth speed of microorganisms in real time.
2. The sensor of claim 1, wherein: the special container is a plastic pipe made of PE material and provided with a sealing cover.
3. The method for constructing a pH-sensitive fluorescence sensor for high-throughput detection of active microorganisms according to claim 1, wherein: the construction method comprises the following steps:
1) dissolving phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescent yellow in ethanol, fully mixing, and gradually dropping into pure water to prepare the fluorescent nano micelle;
2) preparing agar hot solution, adding the fluorescent nano-micelle prepared in the step 1), fully mixing, and fixing the fluorescent nano-micelle in agar gel after cooling;
3) and separating the sensing gel from the specific liquid culture medium by adopting a water-phase filter membrane to prepare the fluorescent sensor suitable for detecting different kinds of microorganisms.
4. The construction method according to claim 3, wherein: the method comprises the following specific steps:
1) preparing fluorescent nano-micelle: phospholipid polyethylene glycol and 5- (N-hexadecanoyl) amino fluorescein were used as 1: 10-1: dissolving the mixture in chromatographic grade absolute ethyl alcohol according to a molar ratio of 50, fully mixing, gradually dropping the mixture into pure water to prepare fluorescent nano-micelle, dialyzing and purifying the prepared nano-micelle by using 20 mM PBS, and storing the purified nano-micelle at 4 ℃;
2) preparation of sensing gel: preparing 1-3% hot agar solution at 50-70 ℃ by using agar, wherein the volume ratio of the hot agar solution is 10: 1-30: 1, adding the fluorescent nano-micelle, cooling, fixing the fluorescent nano-micelle, and preparing the sensing gel with high transparency;
3) preparing a liquid culture medium: referring to the preparation method of LB (Luria-Bertani) medium, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl were added to 950 ml of deionized water, and the vessel was shaken until the solute was dissolved; adjusting the pH value to 7.0 by NaOH; using deionized water to fix the volume to 1L; sterilizing with high pressure steam;
4) assembling the sensor: and (3) filling the sensing gel into the bottom of a special container, and separating the sensing gel from the liquid culture medium by using a 0.22-0.45 mu m water-phase filter membrane.
5. The construction method according to claim 3 or 4, characterized in that: the sensing gel prepared in the step 2 has high transparency, wherein the particle size of the nano-micelle is 20 nm-200 nm, and excitation and emission are 480 nm and 520 nm respectively.
6. The construction method according to claim 3 or 4, characterized in that: the fluorescent nano-micelle is a self-assembled pH sensitive fluorescent substance, wherein phospholipid polyethylene glycol is used as a micelle carrier, 5- (N-hexadecanoyl) amino fluorescent yellow is self-assembled on the surface of the nano-microsphere due to the hydrophobic fatty chain, and the 5- (N-hexadecanoyl) amino fluorescent yellow is sensitive to pH and can reflect the growth speed of microorganisms in real time.
7. The construction method according to claim 3 or 4, characterized in that: the concentration range of the prepared agar gel is 1% -3%, and the agar gel is used for fixing the fluorescent nano micelle and preparing the sensing gel with high transparency.
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| CN109030439A (en) * | 2018-07-09 | 2018-12-18 | 广州华澳生物科技有限公司 | A kind of preparation method and purposes of magnetism rare-earth fluorescent microballoon |
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