CN105548114B - A method of based on saccharomycete on-line analysis Atmospheric particulates toxicity - Google Patents

A method of based on saccharomycete on-line analysis Atmospheric particulates toxicity Download PDF

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CN105548114B
CN105548114B CN201511032569.7A CN201511032569A CN105548114B CN 105548114 B CN105548114 B CN 105548114B CN 201511032569 A CN201511032569 A CN 201511032569A CN 105548114 B CN105548114 B CN 105548114B
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saccharomycete
micro
fluidic chip
atmospheric particulates
toxicity
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CN105548114A (en
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要茂盛
魏恺
罗春雄
邱明昊
张荣飞
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Beijing Ding Lan Technology Co ltd
Beijing Zhong Lan Technology Co ltd
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Peking University
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    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

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Abstract

The invention discloses a kind of methods based on saccharomycete on-line analysis Atmospheric particulates toxicity.The saccharomycete different function albumen for having sensitive response to extraneous different stimulated is filtered out first, and obtains the functional protein by the saccharomycete of Green Fluorescent Protein;Its bacterium solution is passed through in micro-fluidic chip and is used as biosensor;Then Atmospheric particulates are acquired, are mixed into the fluid nutrient medium of saccharomycete in real time, are passed through in micro-fluidic chip by peristaltic pump, dynamic observes the Strength Changes of the live body saccharomycete green fluorescence of different zones different function protein labeling in micro-fluidic chip;Numerical value is changed over time by the intensity of quantitative analysis fluorescence, assesses the toxicity of Atmospheric particulates.This method integrates Atmospheric particulates sampling, multichannel micro-fluidic technology and single live body saccharomycete sensor, it realizes the analysis to the real-time of Atmospheric particulates toxicity, original position, full constituent, is provided in the comprehensive analysis method of molecular level for evaluation air pollution health effect Mechanism Study.

Description

A method of based on saccharomycete on-line analysis Atmospheric particulates toxicity
Technical field
The present invention relates to Atmospheric particulates oxicity analysis technologies, more particularly to a kind of to utilize real-time air sampling, multichannel The Novel Biosensor on-line analysis that microflow control technique is combined with the live body saccharomycete of single Green Fluorescent Protein The method of Atmospheric particulates toxicity can be applied to the real-time online assessment of Atmospheric particulates health effect caused by human body.
Background technology
In recent years, the Chinese severe haze that mostly takes place frequently, many work are dedicated to the mechanism for studying it into haze, wherein coal-fired, Tail gas etc. is classified as important contribution source.Certain damage can be caused for health after Atmospheric particulates sucking, including is drawn Rise respiratory tract infection, allergic reaction, cancer and baby inborn defect etc..Recent years is more and more research shows that air The biological components of particulate matter have angiocardiopathies such as very important influence, including blood pressure increase etc. to its bio-toxicity.It is raw Object aerosol particle has unique health effect as a kind of special particulate matter, and breathing system can be caused by being mainly manifested in it Togetherness dye, three aspects such as lead to allergy and toxic reaction.
The toxicity of Atmospheric particulates is that biology and chemical composition are coefficient as a result, ignoring a part of ingredient of any of which Result of study can all have limitation.Current Atmospheric particulates toxicological study is mainly offline, utilizes mouse or breathing The model of system epithelial cell observes its bio-toxicity and health effect by the Atmospheric particulates of exposure high concentration.It is this General poor in timeliness is studied offline, and collected air sample organizes branch and certain variation occurs at any time, to influence The measurement of its toxicity.In addition, the poor sensitivity of mouse or respiratory system epithelial cell model used, it is often necessary to higher concentration Particulate matter can just observe its biological effect, this has a certain distance with real atmosphere ambient concentration, can not be anti-well Push away Real Atmosphere particulate matter toxic level.The monitoring needs of the toxicity of Atmospheric particulates in real time, original position, full constituent high sensitivity Monitoring system.Still there are no develop this technology at present both at home and abroad.
In recent years, live body saccharomycete increasingly attracts attention as a kind of live body sensor, wherein related saccharomyces cerevisiae The most study of (Saccharomyces cerevisiae).Saccharomycete is completed to pierce external environment by expressing specific protein Sharp response.For example, saccharomycete can significantly increase the specific proteins of extraneous active oxygen (ROS) under the stimulation of extraneous active oxygen It is more, while saccharomycete also has specific protein expression to the stimulation of extraneous metal ion.Research shows that the oxidation of particulate matter is answered Sharp damage is the important way of particulate matter health effect.In addition, multinomial research report, saccharomycete are given birth to as simplest eukaryon Object, a variety of Disease-causing genes and the Disease-causing gene of the mankind are extremely similar, so can using saccharomycete as In vivo detection biology Atmospheric particulates are generalized in the assessment of health effect well.
Invention content
To realize that Atmospheric particulates assess the real-time online of the health effect of human body, the present invention provides a kind of utilizations The method that single live body yeast micro-fluidic biological sensor quickly analyzes Atmospheric particulates toxicity.
Technical scheme is as follows:
A kind of method of on-line analysis Atmospheric particulates toxicity, includes the following steps:
1) the saccharomycete different function albumen for having sensitive response to extraneous different stimulated is filtered out, and obtains the work( The different saccharomycete that energy albumen is marked by green fluorescent protein (GFP);
2) bacterium solution of the saccharomycete for the different function protein labeling that step 1) obtains is each led into micro-fluidic chip not With in channel be used as biosensor;
3) Atmospheric particulates are acquired, and are mixed into the fluid nutrient medium of saccharomycete, are passed through in micro-fluidic chip in real time, Observe the Strength Changes of the live body saccharomycete green fluorescence of different function protein labeling in micro-fluidic chip;
4) numerical value is changed over time to the intensity of fluorescence and carries out record and quantitative analysis, in the horizontal assessment in real time of protein molecular The toxicity of Atmospheric particulates.
Above-mentioned steps 1) in, there is the saccharomycete functional protein of sensitive response can be first from saccharomycete work(environmental stimuli Online open database (the http of energy albumen://www.yeastgenome.org/) in search, what the functional protein was related to Function includes but not limited to oxidative stress response, DNA damage reparation and heavy metal injury repair.In the specific embodiment party of the present invention In formula, 44 kinds of functional proteins are filtered out by online open database, they are:YHR174W、YLR259C、YOR374W、 YCR012W、YLR355C、YLL024C、YAL005C、YAL038W、YLR354C、YOR136W、YMR303C、YNL055C、 YBR072W、YGR254W、YBL015W、YLR034C、YPL060W、YLL015W、YDR135C、YGR281W、YDR270W、 YKL064W、YGL167C、YOL130W、YDR205W、YMR301C、YMR058W、YEL065W、YHL040C、YFL041W、 YMR319C、YMR177W、YOR334W、YBR207W、YMR243C、YOR316C、YLR130C、YPR124W、YER145C、 YGL255W、YGR257C、YBR290W、YJL133W、YKR052C.44 primary yeasts of the functional protein of specific expressed GFP labels Bacteria strain can be bought by commercial sources, and the fusion of GFP and functional protein can also be built by technique for gene engineering, And obtain the yeast strain of conversion.The fusion of GFP and functional protein when saccharomycete is by specific environmental stimuli into Row expression, sends out the green fluorescence of specificity.
Above-mentioned steps 2) described in micro-fluidic chip be preferably high-throughput micro-fluidic chip, possess 96 it is different logical Road is at best able to be passed through the bacterium solution of 96 kinds of different saccharomycete simultaneously.Common high throughput micro-fluidic chip long 60mm, wide 24mm, The aperture of every microchannel is 150 μm in chip, but size is not limited to these.
After saccharomycete bacterium solution is passed through in micro-fluidic chip by step 2), observation fluorescence intensity is as blank control.
Above-mentioned steps 3) using atmosphere particulate matter sampler the particulate matter in air is collected into saccharomycete from gaseous environment Fluid nutrient medium in, then pass in micro-fluidic chip.Preferably, the liquid containing Atmospheric particulates is trained using peristaltic pump It supports base to be sent into micro-fluidic chip, flow is preferably 100 μ L/min.
Step 3) observes different function egg in micro-fluidic chip simultaneously preferably by inverted fluorescence microscope and CCD camera The Strength Changes for the green fluorescence that the live body saccharomycete of white marker is sent out at any time.Preferably, CCD camera and computer are shown Device is connected, and the image that CCD camera was photographed is watched by computer monitor.CCD camera can observe all choosings simultaneously Determine the fluorescence intensity change situation under the visual field, records a photo within every 5 minutes.
In step 4), the variation numerical value for the fluorescence intensity recorded is read using NIS-Elements AR softwares, for every Kind functional protein carries out quantitative analysis to the change in fluorescence amount of its saccharomycete, and then evaluates the toxicity of Atmospheric particulates.
When data analysis, for each functional protein, photo is sorted sequentially in time, selects to be stabilized multiple Yeast cells monomer reads the variation of its fluorescence intensity, makes the song that the luciferase expression amount of each functional protein changes over time The case where line chart is compared with fluorescence intensity when not being passed through Atmospheric particulates, and analysis saccharomycete is stimulated by Atmospheric particulates.
The present invention sampled by integrated Atmospheric particulates, multichannel micro-fluidic technology and single live body saccharomycete sensor come The detection to the real-time of Atmospheric particulates toxicity, original position, full constituent is realized, by obtaining stabilization to the screening of Saccharomyces protein Particulate matter toxicity detection system (abbreviation SLEPTor systems).The system will provide leather for the research of Atmospheric particulates bio-toxicity New property Principle Method provides for evaluation air pollution health effect Mechanism Study in the comprehensive analysis method of molecular level.This Advantageous effect of the invention is mainly reflected in:
(1) present invention proposes and creates a kind of brand-new real time on-line monitoring based on live body list saccharomycete bio-sensing Atmospheric particulates (mainly PM2.5) bio-toxicity theoretical system;
(2) system of the invention is selective to the ingredient response in Atmospheric particulates, can be specific with real time on-line monitoring Ingredient toxic level, such as heavy metal iron, copper;
(3) it is different from existing Atmospheric particulates toxicity test experiment, the present invention is proposed in single live body saccharomycete level Upper real-time online shows the toxicity of Atmospheric particulates, has the characteristics that highly sensitive, low detection limit;
(4) it is mainly monitored as unit of particulate matter quality concentration to weigh the airborne particulate of air quality different from existing Means, the system can under the specific atmospheric conditions of real time on-line monitoring air bio-toxicity.
Description of the drawings
The operating diagram of Fig. 1 particulate matters toxicity detection (SLEPTor) system, wherein:1- atmosphere particulate matter samplers, 2- peristaltic pumps, 3- micro-fluidic chips, the saccharomycete of 4- functional proteins label, 5- liquid waste collectors, 6- inverted fluorescence microscopes, 7- CCD camera, 8- computer display screens.
In Fig. 2 embodiments 2 with GFP label HSP60 albumen saccharomycete add particulate matter stimulate after 100 minutes (a) and The fluorescence field picture of 250 minutes (b).
Specific implementation mode
The invention will be further described by the following examples, but this is not a limitation of the present invention, this field skill Art personnel basic thought according to the present invention, can make various modifications or improvements, but without departing from the basic of the present invention Thought is all within the scope of the present invention.
Online open database (http of the present invention from saccharomycete functional protein://www.yeastgenome.org/) sieve 44 kinds are selected, but is not limited to these, is related to the saccharomycete of oxidative stress response, DNA damage reparation and heavy metal injury repair Albumen.The title of this 44 kinds of functional proteins is:YHR174W、YLR259C、YOR374W、YCR012W、YLR355C、YLL024C、 YAL005C、YAL038W、YLR354C、YOR136W、YMR303C、YNL055C、YBR072W、YGR254W、YBL015W、 YLR034C、YPL060W、YLL015W、YDR135C、YGR281W、YDR270W、YKL064W、YGL167C、YOL130W、 YDR205W、YMR301C、YMR058W、YEL065W、YHL040C、YFL041W、YMR319C、YMR177W、YOR334W、 YBR207W、YMR243C、YOR316C、YLR130C、YPR124W、YER145C、YGL255W、YGR257C、YBR290W、 YJL133W、YKR052C。
Used GFP marks 44 saccharomycetes of above-mentioned functional protein to derive from from Japanese yeast base in following embodiments Because of fission yeast (Schizosaccharomyces) the probe strains A Y160-14D of source center purchase.Those skilled in the art Technique for gene engineering can be utilized voluntarily to build the saccharomycete of GFP label specific proteins, this method belongs to known technology, not in this It repeats again.
The SLEPTor systems that are used in following experiments as shown in Figure 1, strong oxidizing property gas by atmosphere particulate matter sampler 1 Acquisition enters in liquid phase environment, and collection liquid is transported to by peristaltic pump 2 in the micro-fluidic chip 3 for being loaded with saccharomycete 4, micro-fluidic chip It is placed on observation platform, the waste liquid during which generated flows into liquid waste collector 5.When functional protein label saccharomycete 4 by It sends out fluorescence when the stimulation of extraneous oxidizing substance, is observed by inverted fluorescence microscope 6, microscope imaging figure by CCD camera 7 is taken pictures, and the fluorescent image of its real-time online is external on computer display screen 8 and can be observed in real time.
Strong oxidizing property gas is passed through in SLEPTor systems by embodiment 1, and screening has oxidative stress the work(of sensitive response It can albumen
(1) fluid nutrient medium of saccharomycete is delivered into using syringe pump and is loaded with 44 kinds of GFP label different function albumen Yeast strain micro-fluidic chip in, feed flow 0.0001L/min.The micro-fluidic chip is high-throughput chip, tool It is 24 × 60mm to have 96 independent injection orifices, Chip scale, and injection aperture is 1mm, and the aperture in chip interior channel is 150 μ m.Saccharomycete bacterium solution is injected from injection zone, bacterium solution enters the area of observation coverage along the duct of chip.
(2) image that CCD camera was photographed is connected with computer monitor, it is logical chooses 44 miniflows on a display screen The most clearly 44 different visuals field in road, observe the variation of its fluorescence intensity, observation result is as blank control.
(3) it will be mixed with the fluid nutrient medium of strong oxidizing property gas by peristaltic pump while being delivered into and be loaded with 44 kinds of GFP marks In the micro-fluidic chip for remembering yeast strain, saccharomycete is stimulated.Peristaltic pump feed flow is 0.0001L/min.
(4) fluorescence intensity change of 44 groups of saccharomycete was recorded simultaneously every 5 minutes using CCD camera, and led to Cross the variation of external computer monitor real-time online observation saccharomycete fluorescence intensity.
(5) strong oxidizing property micro-tapping is after 3 hours, using NIS-Elements AR softwares for experimental group and blank pair The saccharomycete fluorescence intensity taken in picture captured by group is that sequence is analyzed with the time, makes its fluorescence intensity and changes over time Relational graph.
(6) difference for comparing strong oxidizing property micro-tapping group and blank control group saccharomycete fluorescence intensity change, filters out Saccharomycete has oxidative stress the functional protein of sensitive response.This testing sieve select the albumen that obvious responses to be BDS2, ZRC1、MRS4、MRS3。
Embodiment 2, acquisition Atmospheric particulates, screening have sensitive response to the stimulation of oxidative stress, DNA damage and heavy metal Functional protein
(1) fluid nutrient medium of saccharomycete is delivered into using syringe pump and is loaded with 44 kinds of GFP label different function albumen Saccharomycete micro-fluidic chip in, feed flow 0.0001L/min.
(2) 44 clearly visuals field are chosen on computer display screen, and the variation of its fluorescence intensity is carried out after half an hour Observation, result is as blank control.
(3) it utilizes atmosphere particulate matter sampler to acquire Atmospheric particulates and mixes the fluid nutrient medium of itself and saccharomycete It closes, acquisition flow is 30L/min.
(4) it will be mixed with the fluid nutrient medium of Atmospheric particulates by peristaltic pump while being delivered into and be loaded with 44 kinds of GFP labels In the micro-fluidic chip of saccharomycete, saccharomycete is stimulated.Peristaltic pump feed flow is 0.0001L/min.
(5) fluorescence intensity change of 44 Yeasts was recorded simultaneously every 5 minutes using CCD camera, and led to Cross the variation of external computer monitor real-time online observation saccharomycete fluorescence intensity.The yeast of HSP60 albumen with GFP labels 100 minutes and 250 minutes fluorescence field pictures, can from Fig. 2 respectively as shown in (a) in Fig. 2 and (b) after bacterium adds particulate matter to stimulate Significantly to find out, the time that the expressing quantity of saccharomycete HSP60 is stimulated with particulate matter increases and constantly rises.
(6) after being passed through Atmospheric particulates 4 hours, using NIS-Elements AR softwares for experimental group and blank control Saccharomycete fluorescence intensity in the captured photo of group is analyzed with the time for sequence, makes what its fluorescence intensity changed over time Relational graph.
(7) difference for comparing the fluorescence intensity and blank control group of saccharomycete after being passed through particulate matter, filters out saccharomycete pair Real Atmosphere particulate matter, which stimulates, the functional protein of sensitive response, and filtering out for Atmospheric particulates has the albumen obvious responsed to be HSP60 and SSA1.
Embodiment 3 is detected and comments to the bio-toxicity of national different cities Atmospheric particulates using SLEPTor systems Estimate
(1) fluid nutrient medium of saccharomycete is delivered into using syringe pump and is loaded with 6 kinds of spirits that Examples 1 and 2 filter out In the micro-fluidic chip of the GFP label saccharomycete of quick response, feed flow 0.0001L/min.
(2) 6 clearly visuals field are chosen on computer display screen, and the variation of its fluorescence intensity is seen after half an hour It examines, result is as blank control.
(3) it utilizes on the PM2.5 particles to sampling film in the different cities of the big flow PM2.5 samplers samples whole nation, utilizes Ultrasonic wave cleans sampling film, and the elution of PM2.5 particles is mixed into the fluid nutrient medium of saccharomycete.
(4) it is blended with the fluid nutrient medium of PM2.5 particles by peristaltic pump while being delivered into be loaded with 6 kinds of GFP label ferment In the micro-fluidic chip of female bacterium, saccharomycete is stimulated.Peristaltic pump feed flow is 0.0001L/min.
(5) fluorescence intensity change of 6 Yeasts was recorded simultaneously every 5 minutes using CCD camera, and passed through The variation of external computer monitor real-time online observation saccharomycete fluorescence intensity.
(6) after being passed through PM2.5 particles 4 hours, using NIS-Elements AR softwares for experimental group and blank control group Saccharomycete fluorescence intensity in captured photo is analyzed with the time for sequence, and the pass that its fluorescence intensity changes over time is made System's figure.
(7) difference for comparing the fluorescence intensity and blank control group of saccharomycete after being passed through PM2.5 particles, evaluates each city The difference of the bio-toxicity of particulate matter.

Claims (9)

1. a kind of method of on-line analysis Atmospheric particulates toxicity, includes the following steps:
1) the saccharomycete different function albumen for having sensitive response to extraneous different stimulated is filtered out, and obtains the function egg In vain by the different saccharomycete of Green Fluorescent Protein;
2) difference that the bacterium solution of the saccharomycete for the different function protein labeling that step 1) obtains is each led into micro-fluidic chip is logical Biosensor is used as in road;
3) Atmospheric particulates are acquired, and are mixed into the fluid nutrient medium of saccharomycete, are passed through in micro-fluidic chip in real time, are observed The Strength Changes of the live body saccharomycete green fluorescence of different function protein labeling in micro-fluidic chip;
4) numerical value is changed over time to the intensity of fluorescence and carries out record and quantitative analysis, in the horizontal assessment air in real time of protein molecular The toxicity of particulate matter.
2. the method as described in claim 1, which is characterized in that step 1) is screened from the open database of saccharomycete functional protein Go out following 44 kinds and oxidative stress response, DNA damage reparation and/or the relevant functional protein of heavy metal injury repair: YHR174W、YLR259C、YOR374W、YCR012W、YLR355C、YLL024C、YAL005C、YAL038W、YLR354C、 YOR136W、YMR303C、YNL055C、YBR072W、YGR254W、YBL015W、YLR034C、YPL060W、YLL015W、 YDR135C、YGR281W、YDR270W、YKL064W、YGL167C、YOL130W、YDR205W、YMR301C、YMR058W、 YEL065W、YHL040C、YFL041W、YMR319C、YMR177W、YOR334W、YBR207W、YMR243C、YOR316C、 YLR130C、YPR124W、YER145C、YGL255W、YGR257C、YBR290W、YJL133W、YKR052C。
3. the method as described in claim 1, which is characterized in that micro-fluidic chip described in step 2) is high-throughput micro-fluidic core Piece possesses 96 different channels, is at best able to be passed through the bacterium solution of 96 kinds of different saccharomycete simultaneously.
4. the method as described in claim 1, which is characterized in that after saccharomycete bacterium solution is passed through in micro-fluidic chip by step 2), Fluorescence intensity is observed as blank control.
5. the method as described in claim 1, which is characterized in that step 3) utilizes atmosphere particulate matter sampler by air Grain object is then passed to from the fluid nutrient medium that gaseous environment collects saccharomycete in micro-fluidic chip.
6. method as claimed in claim 5, which is characterized in that step 3) utilizes peristaltic pump by the liquid containing Atmospheric particulates Culture medium is sent into micro-fluidic chip.
7. the method as described in claim 1, which is characterized in that step 3) is same using inverted fluorescence microscope and CCD camera When observation micro-fluidic chip in the Strength Changes of green fluorescence that send out at any time of different function protein labeling saccharomycete.
8. the method for claim 7, which is characterized in that CCD camera is connected by step 3) with computer monitor, passes through The image that computer monitor viewing CCD camera was photographed.
9. the method as described in claim 1, which is characterized in that step 4) reads record using NIS-Elements AR softwares Under florescent intensity value quantitative analysis is carried out to the change in fluorescence amount of its saccharomycete, and then to big for each functional protein The toxicity of aerated particle object is evaluated.
CN201511032569.7A 2015-12-31 2015-12-31 A method of based on saccharomycete on-line analysis Atmospheric particulates toxicity Expired - Fee Related CN105548114B (en)

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