CN105806819B - A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent - Google Patents

A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent Download PDF

Info

Publication number
CN105806819B
CN105806819B CN201610292623.XA CN201610292623A CN105806819B CN 105806819 B CN105806819 B CN 105806819B CN 201610292623 A CN201610292623 A CN 201610292623A CN 105806819 B CN105806819 B CN 105806819B
Authority
CN
China
Prior art keywords
food
borne
spectrum image
micro
fluorescence
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.)
Active
Application number
CN201610292623.XA
Other languages
Chinese (zh)
Other versions
CN105806819A (en
Inventor
陈全胜
李欢欢
欧阳琴
林颢
郭志明
胡薇薇
杨明秀
刘妍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ICTEHI TECHNOLOGY DEVELOPMENT JIANGSU Co.,Ltd.
Original Assignee
Jiangsu University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Jiangsu University filed Critical Jiangsu University
Priority to CN201610292623.XA priority Critical patent/CN105806819B/en
Publication of CN105806819A publication Critical patent/CN105806819A/en
Application granted granted Critical
Publication of CN105806819B publication Critical patent/CN105806819B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
    • C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
    • C09K11/7772—Halogenides
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Composite Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The present invention relates to a kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent.This method are as follows: using food-borne microorganism as research object, construct a set of fluorescence microscopy Hyperspectral imager, by synthesizing polychrome up-conversion nano material, one polychrome namo fluorescence probe with the food-borne microorganism of specific recognition of construction carries out more target labels to multiple-microorganism；Under fluorescent microscopic imaging mode, for the fluorescence spectrum image data for obtaining object to be measured, extract the fluorescence spectrum of area-of-interest (ROI), pass through Data Dimensionality Reduction means, it is preferred that the characteristic spectrum image under particular dimensions, and by image processing means, construct the quantitative detection model of fluorescence intensity change value and food-borne microorganism, detection while realizing a variety of food-borne microorganisms, this method are suitable for the technical fields such as food safety, environmental monitoring.

Description

A kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent Simultaneous Detection
Technical field
The present invention relates to a kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent to examine simultaneously Survey method, and in particular to the unique optical properties of high light spectrum image-forming technology, micro-imaging technique and nanocrystal are combined, are adopted New approaches are detected with the micro- high light spectrum image-forming of nano fluorescent, establish quick, sensitive food-borne microorganism simultaneous quantitative detection side Method.
Background technique
Food-borne pathogens are the first causes for causing food origin disease, cause very big harm to human health, are food The major hidden danger of safety.Common food-borne pathogens analysis method mainly has traditional microbial testing technology, molecule at present Biology techniques, instrument analysis technology and immunological technique.Though these existing analysis methods are each advantageous, all exist certain Limitation perhaps pre-treatment step is complicated, the time is long or instrument and equipment it is huge it is expensive, microorganism cannot be carried out it is visual Change analysis etc..Good working foundation is had accumulated in food field of non destructive testing in view of applicant, especially in high light spectrum image-forming What detection field was done gos deep into the research of technology, the quasi- a set of fluorescence microscopy Hyperspectral imager of building of this project, further investigation Quickly, sensitively food-borne microorganism simultaneous quantitative determination, this method are suitable for the technologies such as food safety, environmental monitoring neck Domain.
Currently, realizing a variety of food-borne microorganism Simultaneous Detections not yet with the micro- high light spectrum image-forming technology of nano fluorescent It appears in the newspapers.The present invention realizes a variety of food-borne microorganisms while examining as a kind of emerging food-borne microorganism quantitative approach Survey analysis.
Summary of the invention
The object of the present invention is to provide a kind of based on a variety of food-borne micro- of the micro- high light spectrum image-forming technology of nano fluorescent Biological Simultaneous Detection, high sensitivity, highly reliable, detection speed is fast, realizes a variety of food-borne microorganisms while examining Analysis is surveyed, the technical fields such as food safety, environmental monitoring are suitable for.
To achieve the goals above, the technical solution adopted by the present invention: using food-borne microorganism as research object, building one Fluorescence microscopy Hyperspectral imager is covered, by synthesizing polychrome up-conversion nano material, construction one there is specific recognition to eat The polychrome namo fluorescence probe of source property microorganism carries out more target labels to multiple-microorganism；Under fluorescent microscopic imaging mode, For the fluorescence spectrum image data for obtaining object to be measured, the fluorescence spectrum of area-of-interest (ROI) is extracted, Data Dimensionality Reduction is passed through Means, the characteristic spectrum image preferably under particular dimensions, and by image processing means, construct fluorescence intensity change value and food source Property microorganism quantitative detection model, realize a variety of food-borne microorganisms while detect.
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, the micro- high light spectrum image-forming technology of the nano fluorescent are by high light spectrum image-forming technology, micro-imaging technique and nanocrystal Unique optical properties combine, meanwhile, fluorescence microscopy Hyperspectral imager be using the 980nm laser of power adjustable as Light irradiation sample to be tested, the sample that the beam expander in system increases laser to the irradiated area of sample, under instantaneous field of view Band finally reaches the slit of imaging spectrometer by microscopical eyepiece and C-Mount interface, using spectrum component Afterwards, the light that sample strip issues occurs scattering in sample strip vertical direction and finally projects EMCCD imaging plane, finally obtains The fluorescence data of one linear array.
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, the polychrome up-conversion nano material (UCNPs), mainly using oleic acid as surfactant, by adding gadolinium (Gd3+) dilute Earth elements adjust the crystal growth of nano-material surface, in suitable reaction condition 30%Gd3+Doping concentration, 300 DEG C of reaction temperature Degree, synthesize under the 1h reaction time six phase of crystal form, size < 100nm, pattern uniformly, good dispersion, fluorescence efficiency it is high UCNPs。
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, the polychrome namo fluorescence probe, the polychrome namo fluorescence probe are by polychrome up-conversion nano material and corresponding The immunoglobulin of specific response occurs for microorganism or oligonucleotides combines.
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, area-of-interest (ROI) extracting method of a kind of similar angle threshold value, by the spectrum of any pixel at different wavelengths Data are combined into a hyperspace vector, and the pixel vector sum and target area pixel of zone of ignorance are calculated using analytic method Angle between vector determines the ownership of zone of ignorance pixel according to the size of angle, in fluorescence spectrum image data Effective segmentation of ROI.
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, Data Dimensionality Reduction means Data Dimensionality Reduction means of the present invention mainly pass through mesh, mark guide selection go out it is several optimal Interval combinations reject variable largely unrelated with detection target in full SPECTRAL REGION；Then, using intelligent search method, from most It is further preferred to variable progress in excellent interval combinations, the redundant variables with height synteny between adjacent wavelength are rejected, thus It is preferred that going out the characteristic spectrum image under particular dimensions.
A kind of above-mentioned a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent side of detection simultaneously Method, this method comprise the following specific steps that:
1) food-borne micro-biological samples prepare: the bacterial strain of microorganism being connected to Luria-Bertani culture medium respectively first In in 37 DEG C culture for 24 hours, then with revolving speed 5000g be centrifuged 5min, abandon supernatant, and with ultrapure water clean three times, distinguish again It is scattered in ultrapure water.Bacterial solution obtained is finally carried out to 10 times of gradient dilutions respectively, obtains the bacterium solution storage of 8 gradients It is spare, while the specific colony counts of bacterium are determined using bacterium colony colony counting method respectively.
2) prepared by polychrome up-conversion nano material: upper conversion nano particle is prepared using high-temperature cracking method, it is prepared to be The nano particle of oleic acid package.Rare earth-iron-boron (0.2M, RE=Y/Gd (78%), the Yb (20%), Er of 2ml methanol dissolution (2%), Tm (2%) or Ho (2%)), with 3ml oleic acid, 7ml1- octadecylene is added to the flask of 50ml.Stir above-mentioned solution And 160 DEG C are heated to, continue 30min, is then cooled to room temperature.The NH of 5ml methanol dissolution is then added4F (1.6mmol) and NaOH (1mmol) stirs 30min.To methanol evaporating completely, above-mentioned solution is heated to 280-300 DEG C under protection of argon gas, and holds Continuous 1.5h.It is cooled to room temperature, is centrifuged to solution, supernatant liquid, the precipitating methanol and ethyl alcohol of acquisition cleans for several times, finally will Precipitating is placed in a vacuum drying oven drying, obtains conversion nano particle white powder on polychrome and stores for future use.
3) prepared by polychrome namo fluorescence probe: upper conversion nano particle and food-borne microorganism specific immunoglobulin or The conjugation connection of oligonucleotides is connected using NHS/EDC Chemical ligation.First with EDC (25 μ l, 2mg/ml) and NHS Upper conversion nano particle (1mg, 5mg/ml) is activated 3h by (12.5 μ l, 2mg/ml) at room temperature.Then with 9350g, 15min from The upper conversion nano particle for being indecisive and changeable, gained precipitating are dissolved in 1ml ultrapure water.Then, the special of 100 μ g is added into above-mentioned solution Property immunoglobulin or oligonucleotides, the overnight incubation on 4 DEG C of shaking tables.With 6000g, 5min centrifugation removes the upper conversion not being coupled Nano particle, and precipitating is cleaned for several times with ultrapure water, it is finally dissolved in 1ml ultrapure water, is placed in 4 DEG C of refrigerators and stores for future use.
4) fluorescence spectrum image acquires: before carrying out image scanning, needing fluorescence microscopy Hyperspectral imager in advance Opening carries out preheating 30 minutes；Meanwhile polychrome namo fluorescence probe being placed in from the microorganism mixing sample of different extension rates On microscopical objective table, under the irradiation of external laser light source 980nm laser, micro- high-spectral data acquisition is carried out.It sweeps Retouching speed is 0.01mm/s.Imaging samples are individually regarded by the slit of imaging spectrometer in an orderly manner with the movement speed of 0.01mm/s Open country, the moving range of X are 8-20mm.The time for exposure of EMCCD camera is set as 3000ms.
5) data processing and inversion: firstly, being carried out to the fluoroscopic image data that system high microspectrum imaging system obtains Area-of-interest (ROI) extracts, and then carries out Data Dimensionality Reduction, obtains characteristic spectrum image, carries out micro- product to characteristic spectrum image Divide fluorescence intensity to obtain, construct the Quantitative Analysis Model of fluorescence intensity change value and food-borne microorganism under different extension rates, Detection while realizing a variety of food-borne microorganisms.
Compared with prior art, the present invention has the advantages that
The micro- high light spectrum image-forming technology of nano fluorescent constructed by the present invention is by high light spectrum image-forming technology, micro-imaging skill Art and the unique optical properties of nanocrystal combine, meanwhile, fluorescence microscopy Hyperspectral imager is with 980nm laser work For light irradiation sample to be tested, the sample strip under instantaneous field of view is finally arrived by microscopical eyepiece and C-Mount interface Up to the slit of imaging spectrometer, after spectrum component, the light that sample strip issues is sent out in sample strip vertical direction Raw scattering finally projects EMCCD imaging plane, finally obtains the fluorescence data of a linear array.
A kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent of the present invention are simultaneously Detection method is easily operated, high sensitivity, and detection speed is fast, is widely applied in technical fields such as food safety, environmental monitorings.
Polychrome up-conversion nano material (UCNPs) of the present invention, mainly using oleic acid as surfactant, by adding Add gadolinium (Gd3+) rare earth element adjust nano-material surface crystal growth, in suitable reaction condition 30%Gd3+Doping concentration, 300 DEG C of reaction temperatures, under the 1h reaction time synthesize six phase of crystal form, size < 100nm, pattern uniformly, good dispersion, fluorescence effect The high UCNPs of rate includes at least upper conversion fluorescent nano particle NaY/GdF4: Yb3+, Er3+(UCNPsEr), NaY/GdF4: Yb3+, Tm3+(UCNPsTm), NaY/GdF4: Yb3+, Ho3+(UCNPsHo)。
Area-of-interest (ROI) extracting method of a kind of similar angle threshold value of the present invention, by any pixel in difference Spectroscopic data under wavelength is combined into a hyperspace vector, using analytic method calculate zone of ignorance pixel vector sum with Angle between the pixel vector of target area, the ownership of zone of ignorance pixel is determined according to the size of angle, to fluorescence light Compose effective segmentation of ROI in image data.
Data Dimensionality Reduction means of the present invention mainly pass through goal orientation and filter out several optimum interval combinations, reject complete Variable largely unrelated with detection target in SPECTRAL REGION；Then, using intelligent search method, to change from optimum interval combination Amount progress further preferably, rejects the redundant variables with height synteny between adjacent wavelength, to it is preferred that go out under particular dimensions Characteristic spectrum image.
Detailed description of the invention
Fig. 1 is that test detects the standard curve of various concentration bacterium solution simultaneously (for detecting Escherichia coli, b is used to detect gold a Staphylococcus aureus)；
Fig. 2 is fluorescence microscopy Hyperspectral imager device figure；
Specific embodiment
Embodiment 1
In order to further verify the method for the invention to a variety of food-borne microorganisms while detect, present example, with For Escherichia coli (E.coli) and staphylococcus aureus (S.aureus), specific steps are as follows:
(1) food-borne micro-biological samples prepare: the bacterial strain of microorganism being connected to Luria-Bertani culture medium respectively first In in 37 DEG C culture for 24 hours, then with revolving speed 5000g be centrifuged 5min, abandon supernatant, and with ultrapure water clean three times, distinguish again It is scattered in ultrapure water.Bacterial solution obtained is finally carried out to 10 times of gradient dilutions respectively, obtains the bacterium solution storage of 8 gradients It is spare, while the specific colony counts of bacterium are determined using bacterium colony colony counting method respectively.
(2) prepared by polychrome up-conversion nano material: upper conversion nano particle is prepared using high-temperature cracking method, it is prepared to be The nano particle of oleic acid package.Rare earth-iron-boron (0.2M, RE=Y/Gd (78%), the Yb (20%), Er of 2ml methanol dissolution (2%), Tm (2%) or Ho (2%)), with 3ml oleic acid, 7ml1- octadecylene is added to the flask of 50ml.Stir above-mentioned solution And 160 DEG C are heated to, continue 30min, is then cooled to room temperature.The NH of 5ml methanol dissolution is then added4F (1.6mmol) and NaOH (1mmol) stirs 30min.To methanol evaporating completely, above-mentioned solution is heated to 300 DEG C under protection of argon gas, and continues 1.5h.It is cooled to room temperature, is centrifuged to solution, supernatant liquid, the precipitating methanol and ethyl alcohol of acquisition is cleaned for several times, will finally be sunk Shallow lake is placed in a vacuum drying oven drying, obtains conversion nano particle white powder on polychrome and stores for future use.
(3) prepared by polychrome namo fluorescence probe: upper conversion nano particle and Escherichia coli and staphylococcus aureus are special Property immunoglobulin conjugation connection using NHS/EDC Chemical ligation connect.First with EDC (25 μ l, 2mg/ml) and NHS Upper conversion nano particle (1mg, 5mg/ml) is activated 3h by (12.5 μ l, 2mg/ml) at room temperature.Then with 9350g, 15min from The upper conversion nano particle for being indecisive and changeable, gained precipitating are dissolved in 1ml ultrapure water.Then, it is added 100 μ g's into above-mentioned solution E.coli antibody, the overnight incubation on 4 DEG C of shaking tables.With 6000g, the upper conversion nano particle that 5min centrifugation removal is not coupled, and Precipitating is cleaned for several times with ultrapure water, is finally dissolved in 1ml ultrapure water, is placed in 4 DEG C of refrigerators and stores for future use.
(4) fluorescence spectrum image acquires: before carrying out image scanning, needing fluorescence microscopy Hyperspectral imager in advance (such as Fig. 1) opening carries out preheating 30 minutes；Meanwhile by the microorganism aggregate sample of polychrome namo fluorescence probe and different extension rates Originally it is placed on microscopical objective table, under the irradiation of external laser light source 980nm laser, carries out micro- high-spectral data and obtain It takes.Scanning speed is 0.01mm/s.Imaging samples individually pass through imaging spectrometer with the movement speed of 0.01mm/s in an orderly manner The slit visual field, the moving range of X are 8-20mm.The time for exposure of EMCCD camera is set as 3000ms.
(5) data processing and inversion: firstly, being carried out to the fluoroscopic image data that system high microspectrum imaging system obtains Area-of-interest (ROI) extracts, and then carries out Data Dimensionality Reduction, obtains characteristic spectrum image, carries out micro- product to characteristic spectrum image Divide fluorescence intensity to obtain, constructs the Quantitative Analysis Model of fluorescence intensity change value and food-borne microorganism under different extension rates (such as Fig. 2) is detected while realizing a variety of food-borne microorganisms.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " illustrative examples ", The description of " example ", " specific example " or " some examples " etc. means specific features described in conjunction with this embodiment or example, knot Structure, material or feature are included at least one embodiment or example of the invention.In the present specification, to above-mentioned term Schematic representation may not refer to the same embodiment or example.Moreover, specific features, structure, material or the spy of description Point can be combined in any suitable manner in any one or more of the embodiments or examples.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this The range of invention is defined by the claims and their equivalents.

Claims (5)

1. a kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent, feature It is, using food-borne microorganism as research object, constructs a set of nano fluorescent high microspectrum imaging system, by synthesizes polychrome Up-conversion nano material, one polychrome namo fluorescence probe with the food-borne microorganism of specific recognition of construction, to a variety of micro- Biology carries out more target labels；Under fluorescent microscopic imaging mode, for the fluorescence spectrum image data for obtaining object to be measured, mention The fluorescence spectrum for taking area-of-interest selects the characteristic spectrum image under particular dimensions by Data Dimensionality Reduction means, and by figure As processing means, the quantitative detection model of fluorescence intensity change value and food-borne microorganism is constructed, realizes a variety of food-borne micro- lifes It is detected while object, this method is suitable for food safety, environmental monitoring technology field；The micro- EO-1 hyperion of constructed nano fluorescent Imaging system be the unique optical properties of high light spectrum image-forming technology, micro-imaging technique and nanocrystal are combined, meanwhile, it is glimmering Light high microspectrum imaging system is using the 980nm laser of power adjustable as light irradiation sample to be tested, in system Beam expander increases laser to the irradiated area of sample, and the sample strip under instantaneous field of view passes through microscopical eyepiece and C- Mount interface finally reaches the slit of imaging spectrometer, and after spectrum component, the light that sample strip issues is in sample Band vertical direction occurs scattering and finally projects EMCCD imaging plane, finally obtains the fluorescence data of a linear array；
Simultaneous Detection comprises the following specific steps that:
The food-borne micro-biological samples of step 1) prepare: the bacterial strain of microorganism being connected to Luria-Bertani culture medium respectively first In in 37 DEG C culture for 24 hours, then with revolving speed 5000g be centrifuged 5min, abandon supernatant, and with ultrapure water clean three times, distinguish again It is scattered in ultrapure water, bacterial solution obtained is finally carried out to 10 times of gradient dilutions respectively, obtains the bacterium solution storage of 8 gradients It is spare, while the specific colony counts of bacterium are determined using bacterium colony colony counting method respectively；
The preparation of step 2) polychrome up-conversion nano material: preparing upper conversion nano particle using high-temperature cracking method, prepared to be The nano particle of oleic acid package, 2ml methanol dissolve the rare earth-iron-boron of 0.2M, wherein the Rare Earth Y Cl for being 78% containing mass ratio3 And GdCl3, mass ratio be 20% Rare Earth Y bCl3, mass ratio be 2% ErCl3 、TmCl3Or HoCl3, with 3ml oil Acid, 7ml 1- octadecylene, is added to the flask of 50ml, stirs above-mentioned solution and is heated to 160 DEG C, continues 30min, then cold But room temperature is arrived, the NH of the 1.6mmol of 5ml methanol dissolution is then added4The NaOH of F and 1mmol stirs 30min, complete to methanol Evaporation, above-mentioned solution is heated to 280-300 DEG C under protection of argon gas, and continues 1.5h, is cooled to room temperature to solution, is centrifuged, and abandons Supernatant liquid, the precipitating methanol and ethyl alcohol of acquisition clean for several times, and precipitating is finally placed in a vacuum drying oven drying, is obtained more Conversion nano particle white powder stores for future use on color；
The preparation of step 3) polychrome namo fluorescence probe: upper conversion nano particle and food-borne microorganism specific immunoglobulin or Oligonucleotides conjugation connection using NHS/EDC Chemical ligation connect, first with 25 μ l concentration be 2mg/ml EDC and The NHS that 12.5 μ l concentration are 2mg/ml converts upper 1mg concentration and activates 3h at room temperature into the nano particle of 5mg/ml, then with The upper conversion nano particle of 9350g, 15min centrifugation activation, gained precipitating are dissolved in 1ml ultrapure water, then, add into above-mentioned solution The specific immunoglobulin or oligonucleotides for entering 100 μ g, the overnight incubation on 4 DEG C of shaking tables, with 6000g, 5min centrifugation removal The upper conversion nano particle not being coupled, and precipitating is cleaned for several times with ultrapure water, it is finally dissolved in 1ml ultrapure water, is placed in 4 DEG C of refrigerators In store for future use；
The acquisition of step 4) fluorescence spectrum image: it before carrying out image scanning, needs fluorescence microscopy Hyperspectral imager in advance Opening carries out preheating 30 minutes；Meanwhile polychrome namo fluorescence probe being placed in from the microorganism mixing sample of different extension rates On microscopical objective table, under the irradiation of external laser light source 980nm laser, micro- high-spectral data acquisition is carried out, is swept Retouching speed is 0.01mm/s, and imaging samples are individually regarded by the slit of imaging spectrometer in an orderly manner with the movement speed of 0.01mm/s The moving range of open country, X is 8-20mm, and the time for exposure of EMCCD camera is set as 3000ms；
Step 5) data processing and inversion: firstly, being carried out to the fluoroscopic image data that system high microspectrum imaging system obtains Then region of interesting extraction carries out Data Dimensionality Reduction, obtain characteristic spectrum image, carries out calculus fluorescence to characteristic spectrum image Intensity obtains, and constructs the Quantitative Analysis Model of fluorescence intensity change value and food-borne microorganism under different extension rates, realizes more It is detected while kind food-borne microorganism.
2. a kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent according to claim 1 Simultaneous Detection, which is characterized in that the polychrome up-conversion nano material leads to mainly using oleic acid as surfactant Cross addition gadolinium Gd3+Rare earth element adjusts the crystal growth of nano-material surface, in suitable reaction condition 30%Gd3+It adulterates dense Degree, 300 DEG C of reaction temperatures synthesize six phase of crystal form, size < 100nm polychrome up-conversion nano material under the 1h reaction time, until It less include upper conversion fluorescent nano particle UCNPsEr, UCNPsTm, UCNPsHo。
3. a kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent according to claim 1 Simultaneous Detection, which is characterized in that the polychrome namo fluorescence probe, be by polychrome up-conversion nano material with it is corresponding micro- The immunoglobulin of specific response occurs for biology or oligonucleotides combines, and includes at least UCNPsErEscherichia coli immune globulin White, UCNPsTmStaphylococcus aureus immunity globulin, UCNPsHoImmunized With Salmonella. Typhimurium globulin, UCNPsErIt is big Enterobacteria oligonucleotides, UCNPsTmStaphylococcus aureus oligonucleotides, UCNPsHoSalmonella typhimurium oligonucleotides.
4. a kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent according to claim 1 Simultaneous Detection, which is characterized in that the fluorescence spectrum that area-of-interest is extracted using a kind of similar angle threshold method, it will be any The spectroscopic data of pixel at different wavelengths is combined into a hyperspace vector, and the picture of zone of ignorance is calculated using analytic method Angle between first vector sum and target area pixel vector, the ownership of zone of ignorance pixel is determined according to the size of angle, With effective segmentation to area-of-interest in fluorescence spectrum image data.
5. a kind of a variety of food-borne microorganisms based on the micro- high light spectrum image-forming technology of nano fluorescent according to claim 1 Simultaneous Detection, which is characterized in that the Data Dimensionality Reduction means mainly pass through goal orientation and filter out several optimum intervals Variable largely unrelated with detection target in full SPECTRAL REGION is rejected in combination；Then, using intelligent search method, from optimal area Between further preferably reject the redundant variables between adjacent wavelength with height synteny to variable in combination, thus it is preferred that Characteristic spectrum image under particular dimensions out.
CN201610292623.XA 2016-05-04 2016-05-04 A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent Active CN105806819B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610292623.XA CN105806819B (en) 2016-05-04 2016-05-04 A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610292623.XA CN105806819B (en) 2016-05-04 2016-05-04 A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent

Publications (2)

Publication Number Publication Date
CN105806819A CN105806819A (en) 2016-07-27
CN105806819B true CN105806819B (en) 2019-01-08

Family

ID=56456404

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610292623.XA Active CN105806819B (en) 2016-05-04 2016-05-04 A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent

Country Status (1)

Country Link
CN (1) CN105806819B (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106706595B (en) * 2017-01-05 2020-06-09 江苏大学 Food-borne pathogenic microorganism detection method based on fusion spectrum technology
CN109211803B (en) * 2018-09-17 2020-10-09 中国科学院生态环境研究中心 Device for rapidly identifying micro plastic based on microscopic multispectral technology
CN109856099A (en) * 2019-03-15 2019-06-07 浙江工业大学 A method of based on alcoholic strength in upconverting fluorescent material detection wine
CN112903658B (en) * 2021-01-29 2022-11-18 江苏大学 Pathogenic bacteria rapid detection method based on gold-silver magnetic-quaternary ammonium salt optical sensing
CN114689523A (en) * 2022-03-25 2022-07-01 江苏省农业科学院 System and method for optically detecting food-borne pathogenic bacteria

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102788756A (en) * 2012-07-13 2012-11-21 上海凯度机电科技有限公司 Multi-modal biological microscope analyzer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI391664B (en) * 2009-02-05 2013-04-01 Univ Nat Taiwan Ocean Micro - point image velocimetry and micro - point image capture method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102788756A (en) * 2012-07-13 2012-11-21 上海凯度机电科技有限公司 Multi-modal biological microscope analyzer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CytoViva纳米荧光高光谱显微成像系统;新发现科技（中国）有限公司;《http://www.ybzhan.cn/st51602/product_8488897.html》;20150430;第1页倒数第1-2行，第2页第2-3、12-13、18-23行 *
稀土上转换荧光纳米材料的制备与生物应用;单爽 等;《生物化学与生物物理进展》;20131231;第40卷(第10期);第925-934页 *

Also Published As

Publication number Publication date
CN105806819A (en) 2016-07-27

Similar Documents

Publication Publication Date Title
CN105806819B (en) A kind of a variety of food-borne microorganism Simultaneous Detections based on the micro- high light spectrum image-forming technology of nano fluorescent
CN106018357B (en) A kind of food-borne microorganism visual analysis method based on the micro- high light spectrum image-forming technology of nano fluorescent
Wang et al. Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy
Baride et al. Near infrared-to-near infrared upconversion nanocrystals for latent fingerprint development
CN105758838B (en) The detection of induced with laser enhancing Raman spectrum and method for separating and its device in liquid
Nevin et al. Time-resolved photoluminescence spectroscopy and imaging: new approaches to the analysis of cultural heritage and its degradation
CN104977272B (en) Terahertz Meta Materials and biological sample method for amplifying signal associated with nanogold particle
US20220357217A1 (en) Odmr temperature measurement method
CN108956556A (en) A kind of luminescence probe and time-resolved fluorescence detection system
US10031133B2 (en) Biological sample signal amplification method using both terahertz metamaterials and gold nanoparticles
Monteleone et al. Label-free identification and differentiation of different microplastics using phasor analysis of fluorescence lifetime imaging microscopy (FLIM)-generated data
CN111307782A (en) Rapid bacterium detection method based on laser-induced breakdown spectroscopy
Sumriddetchkajorn et al. Two-dimensional fruit ripeness estimation using thermal imaging
Ansari et al. Assessment of biological leaf tissue using biospeckle laser imaging technique
Maffren et al. Crack detection in high-pressure turbine blades with flying spot active thermography in the SWIR range
Gao et al. The video-rate imaging of sub-10 nm plasmonic nanoparticles in a cellular medium free of background scattering
Qu et al. Imaging of cancer cells by multiphoton microscopy using gold nanoparticles and fluorescent dyes
Loumaigne et al. Influence of polarization and wavelength on two-photon excited luminescence of single gold nanospheres
Wood et al. Molecular imaging of red blood cells by raman spectroscopy
JP2005065570A (en) Method for detecting viable cell and apparatus for counting viable cell
Sulowska et al. Real-time fluorescence sensing of single photoactive proteins using silver nanowires
Alzahrani et al. Efficient lithium-based upconversion nanoparticles for single-particle imaging and temperature sensing
CN209961688U (en) Rapid detection instrument for pathogenic microorganism drug sensitivity
Jianmin et al. Design of on-line detection system for apple early bruise based on thermal properties analysis
Robison et al. Imaging white blood cells using a snapshot hyperspectral imaging system

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Chen Quansheng

Inventor after: Li Huanhuan

Inventor after: Ou Yangqin

Inventor after: Lin Hao

Inventor after: Guo Zhiming

Inventor after: Hu Weiwei

Inventor after: Yang Mingxiu

Inventor after: Liu Yan

Inventor before: Li Huanhuan

Inventor before: Chen Quansheng

Inventor before: Ou Yangqin

Inventor before: Lin Hao

Inventor before: Guo Zhiming

Inventor before: Hu Weiwei

Inventor before: Yang Mingxiu

Inventor before: Liu Yan

CB03 Change of inventor or designer information
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20211009

Address after: 214024 floor 28, national sensing information center, No. 855, Nanhu Avenue, Liangxi District, Wuxi City, Jiangsu Province

Patentee after: ICTEHI TECHNOLOGY DEVELOPMENT JIANGSU Co.,Ltd.

Address before: Zhenjiang City, Jiangsu Province, 212013 Jingkou District Road No. 301

Patentee before: JIANGSU University

TR01 Transfer of patent right