CN109917123A - A kind of residual detection device of agriculture based on DELFIA and detection method - Google Patents
A kind of residual detection device of agriculture based on DELFIA and detection method Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides a kind of residual detection device of the agriculture based on DELFIA and detection method, the analyzer including centrifugal pan and fluorescence detection mechanism, is fixed on centrifugal pan and obtains by centrifugal action the micro-fluidic chip of test liquid；Radial direction inside the micro-fluidic chip along centrifugal pan is disposed with sample well, filter hole, the first reacting hole, the second reacting hole and waste liquid hole from the inside to the outside；Fluid channel connection is passed sequentially through between sample well, filter hole, the first reacting hole, the second reacting hole and waste liquid hole, is respectively arranged with the sugared film of different-thickness at each miniflow road junction, the sugared film of different-thickness is separately turned on fluid channel according to different centrifugal rotational speeds；The sample well is used to accommodate the sample solution of the residual test liquid of agriculture and is stirred evenly by being centrifuged rotation；To detect the oxious component content in agricultural sample, whole process manual operation process is few and easy to operate, realizes the quick detection of the residual sample solution of agriculture.
Description
Technical field
The present invention relates to pesticide residue examine field more particularly to a kind of residual detection device of agriculture based on DELFIA and
Detection method.
Background technique
The development of agriculture industrialization makes the production of agricultural product be increasingly dependent on the allogenes such as pesticide, antibiotic and hormone
Matter.China's pesticide is high in the dosage of agricultural product, and the unreasonable use of these substances will lead to the agriculture in agricultural product
Medicine residual is exceeded, influences consumer's edible safety, will cause consumer when serious and causes a disease, develops abnormal, or even directly results in
It is poisoned to death.Excessive pesticide residues also will affect the trade of agricultural product, and Pesticide Residue is paid much attention in countries in the world, to each
Pesticide residue all defines the limit standard being increasingly stringenter in kind agricultural and sideline product, and China's Agricultural outlet is made to face severe choose
War.
Fast Determination of Pesticide Residue method is many kinds of at present, mainly there is enzyme process, enzyme-linked immunization and a colloidal gold method, and three
Kind of method is by the way that different chemical substances is added step by step, and stepwise reaction finally obtains test liquid, then is tested point
Analysis, comparison obtain result.The residual enzyme-linked immunization inspection principle of agriculture are as follows: under certain condition, organic phosphorus and carbamic acid class agriculture
Medicine has an inhibiting effect to cholinesterase normal function, and the concentration of inhibiting rate and pesticide is positively correlated.Under normal circumstances, enzymatic
Nerve conduction metabolite (acetylcholine) hydrolysis, hydrolysate are reacted with color developing agent, are generated yellow substance, are used spectrophotometric
Absorbance changes with time value under meter measurement 412nm, calculates inhibiting rate, by inhibiting rate may determine that in sample whether
Contain organic phosphorus or carbamate chemicals for agriculture residual.Reagent step during realization are as follows: configuration buffer takes 1 packet
Buffer is added in 500mL distilled water or pure water, and phosphate buffer (pH7.6) is made in stirring and dissolving, is stored at room temperature.Configuration
Color developing agent: taking 1 bottle of color developing agent to add 25mL buffer solution, and when use takes 100 μ L, and 4 DEG C of refrigerators save.Configuration substrate: 1 bottom of bottle is taken
Object adds 12.5mL distilled water or dissolved in purified water, and when use takes 100 μ L, and 4 DEG C of refrigerators save;Or take 1 bottom of bottle object that 2.5mL is added to distill
Water or dissolved in purified water, when use, take 20 μ L, and 4 DEG C of refrigerators save.Configuration cholinesterase: taking 100 μ L when use, 4 DEG C of refrigerators are protected
It deposits.
The above method in operation each step require be added reagent operated again by standard, it is more so as to cause using
Reagent and step it is very much, it is complicated for operation.
Therefore, the existing technology needs to be improved and developed.
Summary of the invention
The technical problem to be solved in the present invention is that in view of the above drawbacks of the prior art, providing a kind of based on DELFIA
The residual detection device of agriculture and detection method, the residual detecting step of agriculture can be simplified, optimizing detection mode realizes the residual quick detection of agriculture.
The technical proposal for solving the technical problem of the invention is as follows:
A kind of residual detection device of agriculture based on DELFIA includes the analyzer with centrifugal pan and fluorescence detection mechanism, fixed setting
The micro-fluidic chip of test liquid is obtained on centrifugal pan and through centrifugal action；
Radial direction inside the micro-fluidic chip along centrifugal pan is disposed with sample well, filter hole, the first reaction from the inside to the outside
Hole, the second reacting hole and waste liquid hole；
Fluid channel connection is passed sequentially through between sample well, filter hole, the first reacting hole, the second reacting hole and waste liquid hole, each
Miniflow road junction is respectively arranged with the sugared film of different-thickness, and the sugared film of different-thickness is separately turned on micro- according to different centrifugal rotational speeds
Runner；
The sample well is used to accommodate the sample solution of the residual test liquid of agriculture and is stirred evenly by being centrifuged rotation；
The filter hole is used to receive the sample solution in the sample well and filter out macromolecule impurity and absorption is fat-soluble
Impurity；
First reacting hole is for receiving the sample solution in the filter hole and making in sample solution antigen and be located at first
Fluorescence chelating reagent labelled antibody in reacting hole carries out specific immune response；
Second reacting hole is used to receive the sample solution in first reacting hole, by being fixed in the second reacting hole
The antibody of remaining all chelating reagent labels not reacted in antigen capture solution；
The waste liquid hole is used to receive the extra waste liquid in second reacting hole；
Filler is provided in fluid channel between second reacting hole and the waste compartment, the filler after water suction by expanding
And it blocks the second reacting hole and is separated immune multiple with fluorescent marker in the second reacting hole towards the fluid channel between waste liquid hole
Close the waste liquid in object and waste liquid hole.
Further, multilayer is provided in the filter hole for filtering out the filter membrane of macromolecule impurity, two filters that are connected
The filter packing layer for adsorbing oil-soluble impurities is provided between film.
Further, the bottom surface of first reacting hole is uniformly laid with the fluorescence chelating reagent labelled antibody of freeze-drying；It is described glimmering
Light chelating reagent is DTTA-Eu chelating reagent.
Further, the surface cure of second reacting hole is provided with the antigen layer of BSA coupling.
Further, the side of second reacting hole is communicated with redissolution fluid apertures, one end setting of fluid channel by fluid channel
There is the pentasaccharides film for opening and closing fluid channel, the redissolution fluid apertures redissolves liquid and fluorescence enhancement solution for accommodating.
Further, first reacting hole is provided with multiple, and multiple first reacting holes pass through single fluid channel and filter hole
It is connected, second reacting hole is correspondingly arranged on multiple with the first reacting hole.
Further, the micro-fluidic chip appearance profile be sector, fan-shaped micro-fluidic chip be provided with it is multiple and with centrifugation
The rotation center of disk surrounds a circle.
A kind of method of inspection of the residual test liquid of agriculture based on DELFIA is suitable for the residual detection device of agriculture as described above, packet
Include step:
The sample well sample solution of agricultural product being added in micro-fluidic chip；
Sealed sample hole；
Micro-fluidic chip is fixedly mounted on centrifugal pan；
Start centrifugal pan and centrifugally operated is carried out to micro-fluidic chip；
Solution opens fluorescence detection mechanism to the second reacting hole transmitting exciting light after completing centrifugally operated；
Measurement transmitting light simultaneously combines predetermined curve to calculate the antigen concentration in sample.
Further, it is specifically included in the step of starting centrifugal pan carries out centrifugally operated to micro-fluidic chip:
Centrifugal pan is broken through the sugared film at the miniflow road junction between sample well and filter hole by the forward and reverse alternate rotation of First Speed
And sample solution passes through filter hole；
Centrifugal pan rotated backward by second speed make the sugared film at the road junction of the miniflow between filter hole and the first reacting hole broken through and
Sample solution enters the first reacting hole；
Centrifugal pan is rotated forward by third speed makes sample solution hybrid reaction in the first reacting hole；
Centrifugal pan is rotated forward by fourth speed rushes the sugared film at the road junction of the miniflow between the first reacting hole and the second reacting hole
Broken and sample solution enters the first reacting hole；
Centrifugal pan makes sample solution hybrid reaction in the second reacting hole by the 5th velocity reversal rotation；
Centrifugal pan by the 6th velocity reversal rotation make the sugared film at miniflow road junction between the second reacting hole and waste liquid hole broken through and
Redundant sample solution enters waste liquid hole；
Centrifugal pan makes redundant sample solution completely into waste liquid hole by the 7th velocity reversal rotation.
Further, the First Speed is 1500 revs/min, and each runing time of positive and negative rotation is 1 minute；The second speed
It is 3000 revs/min, runing time is 1 minute；The third speed is 3000 revs/min, and runing time is 1 minute；It is described
Fourth speed is 4500 revs/min, and runing time is 1 minute；5th speed is 4500 revs/min, and runing time is 1 point
Clock；6th speed is 6000 revs/min, and runing time is 1 minute；7th speed is 6000 revs/min, when operation
Between be 1 minute.
Beneficial effect using the above scheme is: a kind of residual detection device of agriculture based on DELFIA proposed by the present invention and inspection
Survey method, by be arranged in a micro-fluidic chip different chambers and by different sugared films successively open different chamber with
Sample solution is reacted, to make the blending process of existing complexity be integrated in different chamber, to only need to add agricultural production
The solution to be measured (sample solution) of product, which can be centrifuged required for obtaining time immunofluorescence chromatography, detects liquid, makes operating procedure
Simply, reagent consumption is few.And pipeline connection is carried out using fluid channel in this method, to realize precise controlling, add compared to artificial
Sample is quantitative more accurate, and amount of solution only needs 50-100 microlitres of sample introduction, about 200 microlitres of reaction reagents is embedded, to reduce reagent
Waste, detection limit is minimum can be to PPt rank, testing result favorable reproducibility, and reaction box can be reserved for, and each chamber is independently arranged, and is kept away
Exempt from cross contamination, it is as a result reproducible.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 2 is a kind of front view of the residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 3 is a kind of micro-fluidic chip cross-sectional view of the residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 4 is a kind of micro-fluidic chip cross-sectional view of the residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 5 is the filter hole cross-sectional view of another residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 6 is the micro-fluidic chip cross-sectional view of another residual detection device embodiment of agriculture based on DELFIA of the invention.
Fig. 7 is a kind of flow chart of the detection method of the residual test liquid of agriculture based on DELFIA of the invention.
In figure: 100, centrifugal pan；200, micro-fluidic chip；210, sugared film；211, the first sugared film；212, the second sugared film；
213, third sugar film；214, tetrose film；215, pentasaccharides film；220, sample well；230, filter hole；231, filter membrane；232, mistake
Filter packing layer；240, the first reacting hole；250, the second reacting hole；260, waste liquid hole；270, filler；280, fluid apertures is redissolved；290,
Fluid channel；300, fixture；400, fluorescence detection mechanism.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer and more explicit, right as follows in conjunction with drawings and embodiments
The present invention is further described.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and do not have to
It is of the invention in limiting.
As shown in Figure 1 and Figure 2, the residual detection device of a kind of agriculture based on DELFIA, including examined with centrifugal pan 100 and fluorescence
The analyzer of mechanism 400 is surveyed, centrifugal pan 100 realizes centrifugation rotation, and fluorescence detection mechanism 400 is used for fluorescence detection.The present embodiment
In centrifugal pan 100 be arranged from the horizontal by certain angle, when work, presents lower heeling condition, passes through on centrifugal pan 100
Fixture 300 is fixedly installed with micro-fluidic chip 200, and the agricultural product solution being placed in micro-fluidic chip 200 passes through centrifugal action point
It is not chemically reacted with the substance in micro-fluidic chip 200, to obtain the test liquid of needs, is directly irradiated and examined by exciting light
Liquid, then comparison is analyzed, to obtain result.
As shown in Figure 3, Figure 4, the radial direction in micro-fluidic chip 200 along centrifugal pan 100 is disposed with sample from the inside to the outside
Hole 220, filter hole 230, the first reacting hole 240, the second reacting hole 250 and waste liquid hole 260；Miniflow is passed sequentially through between each room
Road 290 is connected to, and to flow out convenient for liquid when centrifugation, the middle position of each hole joint face, and fluid channel is arranged in each fluid channel 290
290 diameters are 2 microns, to realize accurate flow control by fine hole.It is set respectively in the runner mouth of each fluid channel 290
It is equipped with the sugared film 210 of different-thickness, the sugared film 210 of different-thickness is separately turned on fluid channel 290 according to different centrifugal rotational speeds；
When centrifugal speed is not up to setting value, sugared film 210 is just fail to open, then fluid channel 290 is blocked state, when speed reaches
The setting value that sugared film 210 is opened, sugared film 210 are washed open by big centrifugal force, open fluid channel 290, and the sugared film of different-thickness
The 210 setting value differences opened, therefore the fluid channel 290 between corresponding cavate can be opened according to different revolving speeds, sugared film
Main ingredient is the polyethylene glycol of paraffin and high polymerization degree, breaks through sugared film by centrifugal impact force, and in the fluid channel of sugared film location
Because narrowing structure, i.e. the fluid channel before sugared film location has one section narrower than normal fluid channel of runner for setting, if solution
Centrifugal force not enough can not just be set so that dual guarantee prevents solution from flowing back by sugared film and narrowed pipeline by narrowed pipeline
Meter, to enable agricultural product solution and each chamber step-reaction, finally obtains the residual detection test liquid of desired agriculture.
Sample well 220 is arranged near the position of 100 rotation center of centrifugal pan, and sample well 220 is for accommodating the residual inspection of agriculture
It tests liquid sample and is stirred evenly by being centrifuged rotation, 220 constant volume of sample well is 200 microlitres, passes through liquid-transfering gun in sample well 220
Agricultural product sample solution is added, and using sealing film sealing (being not drawn into figure)；Abrasive material is added in sample well 220, abrasive material is
Quartz sand material, abrasive material can be fallen during operating by gravity, can be centrifuged by centrifugal force, inclined-plane place from
Cartridge 100 moves up and down abrasive material constantly under the action of two kinds of power, thus the abundant broken wall of more conducively cell.Sample well
Sugared film 210 between 220 and filter hole 230 is the first sugared film 211, and the filter hole 230 passes through fluid channel 290 and sample well
220 connections are connected between filter hole 230 and sample well 220 when centrifugal rotational speed reaches the setting value that the first sugared film 211 is opened,
Filter hole 230 receives the sample solution in the sample well 220 and filters out macromolecule impurity and absorption oil-soluble impurities.
As shown in figure 5, the filter membrane 231 that multilayer is used to filter out macromolecule impurity is provided in filter hole 230, it is described to be connected
The filter packing layer 232 for adsorbing oil-soluble impurities is provided between two filter membranes 231.Filter membrane 231 in the present embodiment is oil
Property filter membrane 231, setting altogether is of five storeys, and filter membrane 231 is for the macromolecule impurity (such as pigment, organic acid) in filtered sample solution, mistake
Filter packing layer 232 be composite PSA (PSA is a kind of filler 270 of SPE solid-phase extraction column, N- propyl ethylenediamine solid-phase adsorbent,
English name: primary secondary amine sorbent), for adsorbing oil-soluble impurities (such as chlorophyll)；By more
Layer filter membrane 231 plus the superposition cooperation of filter packing layer, enable the macromolecule impurity and oil-soluble impurities of interference test in sample solution
It filters out, can remove more impurity with conventional centrifugal partition method；In preferable embodiment, 5 layers of oblique placement of filter membrane 231,
I.e. at an angle with the water (flow) direction of filter hole 230, filter membrane 231 is vertical with water (flow) direction in traditional setting up procedure, still
Impact force is larger under the action of the centrifugal for water flow, and filter membrane 231 is caused to be broken through, and loses filter effect, therefore using inclination filter membrane 231
Setting can be such that water flow flows along filter membrane 231, play the role of buffering water impact.Several filters are provided on every layer of filter membrane 231
Hole, each layer of filter opening aperture successively become smaller from the inside to the outside along the radial direction of centrifugal pan 100, and the filter opening of 5 layers of filter membrane 231 is successively distinguished
It is 100 microns, 50 microns, 10 microns, 5 microns and 0.45 micron, aperture reduces step by step, and filter sample solution gradually, from
And the impurity for filtering out solution is more, and subsequent survey is avoided to interfere, and keeps experimental result more acurrate.
As shown in Figure 3, Figure 4, the first reacting hole 240 is connected to by fluid channel 290 with filter hole 230, the first reacting hole 240
Sugared film 210 between filter hole 230 is the second sugared film 212, and the thickness of the second sugared film 212 is thicker than the first sugared film 211, and connection is worked as
When centrifugal rotational speed reaches the setting value that the second sugared film 212 is opened, it is connected between the first reacting hole 240 and filter hole 230, first is anti-
It answers hole 240 to receive the sample solution in filter hole 230 and makes antigen in sample solution and pass through fluorescence chelating reagent labelled antibody
It is immunoreacted.The fluorescence chelating reagent label that the bottom surface of specially the first reacting hole 240 is uniformly equipped with one layer of freeze-drying is anti-
Body, the fluorescence chelating reagent are DTTA-Eu chelating reagent, and the antibody of antigen and DTTA-Eu label in sample solution carries out
Immune response；DTTA-Eu chelating reagent is the difunctional chelating reagent of isothiocycmatobenzyl diethylenetriamines tetraacethyl europium, one
End chelates Eu3+ and the other end can be connect with-the NH2 of protein.In neutrality or under the conditions of close to neutral pH, DTTA and Eu3+
With enough chelate stabilities, and under enhancement solution (in acidity) effect, DTTA-Eu again can be rapid, thorough by the Eu3+ of chelating
It releases to bottom and enables Eu3+ fluorescence at thousands of times into the hydrophobic inner core of micella with the ligand sequestration in enhancement solution
Ground amplification.
Preferably, it is coated with one layer of tetrafluoroethene film on the first reacting hole 240 and the inner surface of fluid channel 290, is lyophilized
Enhancing fluorescence chelating reagent labelled antibody be laid on tetrafluoroethene film, increasing tetrafluoroethene film can prevent liquid viscous
It is connected on wall and makes solution loss, so as to cause experimental error, and increase tetrafluoroethene film can completely cut off in the first reacting hole 240
Influence of the plasticiser to albumen, the tetrafluoroethene film in certain actual setting could alternatively be other similar material.
Second reacting hole 250 is connected to by fluid channel 290 with the first reacting hole 240, and the second reacting hole 250 is reacted with first
Sugared film 210 between hole 240 is third sugar film 213, and the thickness of third sugar film 213 is thicker than the second sugared film 212, therefore third sugar film
213 unlatching speed setting values are higher than the second sugared film 212, when centrifugal rotational speed reaches the setting value of the unlatching of third sugar film 213, the
It is connected between two reacting holes 250 and the first reacting hole 240, the second reacting hole 250 receives the sample solution in the first reacting hole 240
And pass through the antibody of all chelating reagent labels not reacted remaining in fixed antigen capture solution.Specifically, described
The surface cure of second reacting hole 250 is provided with the antigen layer of BSA coupling, captures sample solution by the antigen layer of BSA coupling
In extra DTTA label antibody.The antibody levels that fluorescence detection mechanism 400 examines the second reaction chamber to capture, practical is total antibody
Number subtracts the second reaction chamber and captures number, obtains the antibody reacted in the first reaction chamber, reacts in the first reaction chamber to extrapolate
Antigen number, i.e., the antigen number in sample solution, to judge the residual situation of agriculture according to antigen number.
Waste liquid hole 260 is connected to by fluid channel 290 with the second reacting hole 250, waste liquid hole 260 and the second reacting hole 250 it
Between sugared film 210 be tetrose film 214, the thickness of tetrose film 214 is thicker than third sugar film 213, thus tetrose film 214 open
Speed setting value is higher than third sugar film 213, when centrifugal rotational speed reaches the setting value of the unlatching of tetrose film 214, waste liquid hole 260
It is connected between the second reacting hole 250, waste liquid hole 260 receives the sample solution in the second reacting hole 250, the second reacting hole 250
Filler 270 is provided in fluid channel 290 between the waste compartment, the filler 270 is the saturation type trehalose that quickly absorbs water
Gel, filler 270 block the second reacting hole 250 and divide towards the fluid channel 290 between waste liquid hole 260 by expanding after water suction
From the waste liquid in test liquid and waste liquid hole 260 in the second reacting hole 250；Therefore when the unlatching of tetrose film 214, the second reacting hole
250 sample solution enters fluid channel 290, so that the moisture content in solution is absorbed by filler 270, filler 270 is absorbing moisture content
Meanwhile a part of moisture content and not with BSA coupling antigen reactive solution can be flowed into waste liquid hole 260 become waste liquid, and
It is the test liquid needed in second reacting hole 250, completes blocking fluid channel 290 when filler 270 expands, thus the second reaction of separation
The waste liquid in test liquid and waste liquid hole 260 in hole 250.
Therefore, agricultural product solution is filtered by filter hole 230 again after carrying out clasmatosis by sample room 220, then is passed through
Antigen when the first reacting hole 240 in sample solution is immunoreacted with the DTTA-Eu antibody marked, then passes through the second reaction
The antibody of extra label, to obtain test solution, then is examined by fluorescence in the antigen layer capture sample solution being coupled in hole 250
The test solution that the exciting light that mechanism 400 emits penetrates the second reacting hole 250 is surveyed, then detects the strong of transmitting light in specific range
Degree verifies the antibody levels that the second reaction chamber captures, finally predetermined curve is combined to calculate the antigen concentration in sample, thus point
Analysis obtains the medicine residual in agricultural product；Launch wavelength in the present embodiment is 340nm exciting light, and 400 microseconds -800 are micro- after transmitting
Measurement emits light at 613nm in second, and detection effect is most stable under the parameter.
It further include passing through the redissolution fluid apertures 280 that fluid channel 290 is connected in the side of the second reacting hole 250 in the present embodiment,
One end of fluid channel 290 is provided with the pentasaccharides film 215 for opening and closing fluid channel 290, and the redissolution fluid apertures 280 is multiple for accommodating
Solution and fluorescence enhancement solution, redissolving liquid can be such that the test liquid in the second reacting hole 250 sufficiently dissolves, and avoid the second reacting hole
Colloidal solid deposition in test liquid in 250 circularizes.The fluorescence of the antibody of the extra DTTA label of enhancing in fluorescence enhancement solution
Effect is easier to be detected when irradiating fluorescence.
As shown in fig. 6, the first reacting hole 240 is provided with multiple, multiple first reactions in preferred embodiment in the present embodiment
Hole 240 is connected by single fluid channel with filter hole 230, and second reacting hole 250 is correspondingly arranged with the first reacting hole 240
Have multiple.In this way, when placing differential responses object in the first reacting hole 240 and the second reacting hole 250, it can be by once adding agricultural production
Product sample solution and multiple groups different experiments can be done, conventional efficient is improved, using being provided with the schemes of multiple first reacting holes 240
When, filter hole 230 is directly connected to by fluid channel 290 with waste liquid hole 260, increases sugared film 210 at miniflow road junction accordingly, micro-
Filler 270 is provided in runner 290, filter hole 230 comes out solution and successively passes through two the first reacting holes 240, then extra
Solution (pore volume of the reaction more than two), the slightly higher waste liquid hole of fluid channel pressure will be entered, make pipeline pressure is slightly higher to lead to
It crosses and narrows fluid channel to realize.
As shown in Fig. 2, 200 appearance profile of micro-fluidic chip is set as fan-shaped, fan-shaped in preferred embodiment in the present embodiment
Micro-fluidic chip 200 is provided with multiple and surrounds a circle with the rotation center of centrifugal pan 100.In this way, behaviour can be once centrifuged
The experiment for making the different sample solution of realization, improves the scope of application of this extractor, facilitates the experiment of multiple groups difference sample solution
It carries out simultaneously.
Therefore, the residual detection device of a kind of agriculture based on DELFIA proposed by the present invention, by a micro-fluidic chip 200
The different chamber of interior setting is simultaneously successively opened different chamber by different sugared films 210 and is reacted with sample solution, to make
The blending process of existing complexity is integrated in different chamber, to only need to add the solution to be measured (sample solution) of agricultural product just
It can be centrifuged required for obtaining time immunofluorescence chromatography and detect liquid, keep operating procedure simple, reagent consumption is few.And this method
It is middle that pipeline connection is carried out using fluid channel 290, to realize precise controlling, quantitative more accurate, amount of solution is loaded compared to artificial
50-100 microlitres of sample introduction is only needed, about 200 microlitres of reaction reagents are embedded, to reduce the waste of reagent, detection limit is minimum can be to PPt
Rank, testing result favorable reproducibility, and reaction box can be reserved for, and each chamber is independently arranged, and avoids cross contamination, as a result reproducible.
As shown in fig. 7, being suitable for as above the present invention also provides a kind of detection method of residual test liquid of the agriculture based on DELFIA
The residual detection device of the agriculture, includes step:
Step S100, the sample well being added to the sample solution of agricultural product in micro-fluidic chip.
Specific implementation process are as follows: the homogenate solution of 250 microlitres of agricultural product is added to by micro-fluidic chip by liquid-transfering gun
Sample well, sample well constant volume is 200 microlitres, until sample well is filled it up with.
Step S200, sealed sample hole.
Specific implementation process are as follows: after sample well is filled it up with, need to be sealed sample well, using sealing in the present embodiment
Film sealed sample hole, it is easy to operate.
Step S300, micro-fluidic chip is fixedly mounted on centrifugal pan.
Specific implementation process are as follows: micro-fluidic chip is placed on the rotary-tray of centrifugal pan, and is fixed by fixture, is pressed from both sides
Fixing clamp or particular jig can be used in tool, when carrying out multiple groups experiment, can place simultaneously on a rotary-tray
Multiple groups micro-fluidic chip.
Step S400, starting centrifugal pan carries out centrifugally operated to micro-fluidic chip.
Specific implementation process are as follows: open centrifugal pan, make sample solution exist and carrying out centrifugally operated to micro-fluidic chip
Each chamber reacts respectively, obtains test liquid.
Wherein, preferred steps specifically include:
Step S410, centrifugal pan is made the sugar at the miniflow road junction between sample well and filter hole by the forward and reverse alternate rotation of First Speed
Film is broken through and sample solution passes through filter hole.
Specific implementation process are as follows: the First Speed of centrifugal pan is 1500 revs/min, first clockwise 1500 after centrifugal pan unlatching
Rev/min operation 1 minute, 1500 revs/min of another mistake hour hands ran 1 minute, so that the first sugared film is broken through, positive and negative rotation is set
It sets, sample solution is enable to stir evenly.
Step S420, centrifugal pan is rotated backward by second speed makes the road junction of the miniflow between filter hole and the first reacting hole
Sugared film is broken through and sample solution enters the first reacting hole.
Specific implementation process are as follows: the First Speed of centrifugal pan be 3000 revs/min, centrifugal pan is counterclockwise 3000 revs/min
Operation 1 minute, so that the second sugared film is broken through, sample solution enters the first reacting hole.
Step S430, centrifugal pan is rotated forward by third speed makes sample solution hybrid reaction in the first reacting hole.
Specific implementation process are as follows: the third speed of centrifugal pan is 3000 revs/min, 3000 revs/min clockwise of centrifugal pan
The antibody of operation 1 minute, DTTA-Eu label of the antigen in the first reacting hole with the first reacting hole in sample solution is filled
The immune response divided.
Step S440, centrifugal pan is rotated forward the fluid channel made between the first reacting hole and the second reacting hole by fourth speed
The sugared film of mouth is broken through and sample solution enters the first reacting hole；
Specific implementation process are as follows: the fourth speed of centrifugal pan is 4500 revs/min, centrifugal pan 4500 revs/min of operations 1 clockwise
Minute, so that third sugar film is broken through, sample solution enters the second reacting hole.
Step S450, centrifugal pan makes sample solution hybrid reaction in the second reacting hole by the 5th velocity reversal rotation.
Specific implementation process are as follows: the 5th speed of centrifugal pan be 4500 revs/min, centrifugal pan is counterclockwise 4500 revs/min
Operation 1 minute, the antibody of the extra DTTA label in the sample solution antigen capture fixed by the second reacting hole, reacts fully
It carries out.
Step S460, centrifugal pan makes the miniflow road junction between the second reacting hole and waste liquid hole by the 6th velocity reversal rotation
Sugared film is broken through and redundant sample solution enters waste liquid hole.
Specific implementation process are as follows: the 6th speed of centrifugal pan be 6000 revs/min, centrifugal pan is counterclockwise 6000 revs/min
Operation 1 minute, so that tetrose film is broken through, extra sample solution enters waste liquid hole.
Step S470, centrifugal pan makes redundant sample solution completely into waste liquid hole by the 7th velocity reversal rotation.
Process was embodied are as follows: the 7th speed of centrifugal pan is 6000 revs/min, 6000 revs/min clockwise of centrifugal pan
Clock is run 1 minute, and the redundant sample solution in sample solution realizes the separation of test liquid and waste liquid completely into waste liquid hole.
It further include having step S480, step S490 after step S470；
Step S480, centrifugal pan is rotated forward the sugar for making to redissolve the miniflow road junction between fluid apertures and the second reacting hole by the 8th speed
Film enters the second reacting hole by the solution for making to redissolve fluid apertures is broken through.
Step S490, centrifugal pan makes the solution entered in the second reacting hole from redissolution fluid apertures exist by the 9th velocity reversal rotation
Hybrid reaction in second reacting hole.
Specific implementation process are as follows: the 8th speed of centrifugal pan is 7500 revs/min, 7500 revs/min clockwise of centrifugal pan
Operation 1 minute makes to redissolve the solution in fluid apertures and enters the second reacting hole, redissolve molten in fluid apertures so that pentasaccharides film is broken through
Liquid is 100 microlitres of acid fluorescence enhancement solutions, and later, the 9th speed of centrifugal pan is 7500 revs/min, and centrifugal pan is with counterclockwise
7500 revs/min run 1 minute, and redissolution liquid and fluorescence-enhancing agent in redissolution fluid apertures and the sample solution in the second reacting hole are anti-
It answers, to obtain the display better test liquid of fluorescence.
S500, solution open fluorescence detection mechanism to the second reacting hole transmitting exciting light after completing centrifugally operated.
S600, measurement transmitting light simultaneously combine predetermined curve to calculate the antigen concentration in sample.
Specific implementation process are as follows: opening fluorescence detector is 340nm exciting light to the second reacting hole launch wavelength, is
Measurement transmitting light at 613nm, combines presetting curve to calculate antigen concentration in sample by emitted luminescence intensity.To detect farming
Oxious component content in object sample.
In conclusion this method is by being arranged different chambers and passing through the difference on centrifugal pan in a micro-fluidic chip
Speed opens different sugared films successively, so that the substance in different chamber be made to be reacted with sample solution, setting is primary
Centrifugally operated program can obtain test liquid, open fluorescence detector and irradiate to the test liquid for being located at the second reacting hole, by emitting
Luminous intensity combines presetting curve to calculate antigen concentration in sample.To detect the oxious component content in agricultural sample,
Whole process manual operation process is few and easy to operate, realizes the quick detection of the residual sample solution of agriculture.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention
Protect range.
Claims (10)
1. a kind of residual detection device of agriculture based on DELFIA, the analyzer including having centrifugal pan and fluorescence detection mechanism is special
Sign is: further including having the micro-fluidic chip for being fixed on centrifugal pan and obtaining test liquid by centrifugal action；
Radial direction inside the micro-fluidic chip along centrifugal pan is disposed with sample well, filter hole, the first reaction from the inside to the outside
Hole, the second reacting hole and waste liquid hole；
Fluid channel connection is passed sequentially through between sample well, filter hole, the first reacting hole, the second reacting hole and waste liquid hole, each
Miniflow road junction is respectively arranged with the sugared film of different-thickness, and the sugared film of different-thickness is separately turned on micro- according to different centrifugal rotational speeds
Runner；
The sample well is used to accommodate the sample solution of the residual test liquid of agriculture and is stirred evenly by being centrifuged rotation；
The filter hole is used to receive the sample solution in the sample well and filter out macromolecule impurity and absorption is fat-soluble
Impurity；
First reacting hole is for receiving the sample solution in the filter hole and making in sample solution antigen and be located at first
Fluorescence chelating reagent labelled antibody in reacting hole carries out specific immune response；
Second reacting hole is used to receive the sample solution in first reacting hole, by being fixed in the second reacting hole
The antibody of remaining all chelating reagent labels not reacted in antigen capture solution；
The waste liquid hole is used to receive the extra waste liquid in second reacting hole；
Filler is provided in fluid channel between second reacting hole and the waste compartment, the filler after water suction by expanding
And it blocks the second reacting hole and is separated immune multiple with fluorescent marker in the second reacting hole towards the fluid channel between waste liquid hole
Close the waste liquid in object and waste liquid hole.
2. the residual detection device of the agriculture according to claim 1 based on DELFIA, it is characterised in that: setting in the filter hole
There is multilayer for filtering out the filter membrane of macromolecule impurity, is provided between two filter membranes that are connected for adsorbing oil-soluble impurities
Filter packing layer.
3. the residual detection device of the agriculture according to claim 2 based on DELFIA, it is characterised in that: first reacting hole
Bottom surface is uniformly laid with the fluorescence chelating reagent labelled antibody of freeze-drying；The fluorescence chelating reagent is DTTA-Eu chelating reagent.
4. the residual detection device of the agriculture according to claim 3 based on DELFIA, it is characterised in that: second reacting hole
Surface cure is provided with the antigen layer of BSA coupling.
5. the residual detection device of the agriculture according to claim 1 based on DELFIA, it is characterised in that: second reacting hole
Side is communicated with redissolution fluid apertures by fluid channel, and one end of fluid channel is provided with the pentasaccharides film for opening and closing fluid channel, described
It redissolves fluid apertures and redissolves liquid and fluorescence enhancement solution for accommodating.
6. the residual detection device of the agriculture according to claim 5 based on DELFIA, it is characterised in that: first reacting hole is set
It is equipped with multiple, multiple first reacting holes are connected by single fluid channel with filter hole, and second reacting hole is reacted with first
Hole is correspondingly arranged on multiple.
7. the residual detection device of the agriculture according to claim 1 based on DELFIA, it is characterised in that: outside the micro-fluidic chip
Shape profile is sector, and fan-shaped micro-fluidic chip is provided with multiple and with centrifugal pan rotation center and surrounds a circle.
8. a kind of method of inspection of the residual test liquid of agriculture based on DELFIA, it is characterised in that: be suitable for as claim 1-7 is any
The residual detection device of the agriculture, includes step:
The sample well sample solution of agricultural product being added in micro-fluidic chip；
Sealed sample hole；
Micro-fluidic chip is fixedly mounted on centrifugal pan；
Start centrifugal pan and centrifugally operated is carried out to micro-fluidic chip；
Solution opens fluorescence detection mechanism to the second reacting hole transmitting exciting light after completing centrifugally operated；
Measurement transmitting light simultaneously combines predetermined curve to calculate the antigen concentration in sample.
9. the detection method of the residual test liquid of the agriculture according to claim 8 based on DELFIA, which is characterized in that opened described
It is specifically included in the step of dynamic centrifugal pan carries out centrifugally operated to micro-fluidic chip:
Centrifugal pan is broken through the sugared film at the miniflow road junction between sample well and filter hole by the forward and reverse alternate rotation of First Speed
And sample solution passes through filter hole；
Centrifugal pan rotated backward by second speed make the sugared film at the road junction of the miniflow between filter hole and the first reacting hole broken through and
Sample solution enters the first reacting hole；
Centrifugal pan is rotated forward by third speed makes sample solution hybrid reaction in the first reacting hole；
Centrifugal pan is rotated forward by fourth speed rushes the sugared film at the road junction of the miniflow between the first reacting hole and the second reacting hole
Broken and sample solution enters the first reacting hole；
Centrifugal pan makes sample solution hybrid reaction in the second reacting hole by the 5th velocity reversal rotation；
Centrifugal pan by the 6th velocity reversal rotation make the sugared film at miniflow road junction between the second reacting hole and waste liquid hole broken through and
Redundant sample solution enters waste liquid hole；
Centrifugal pan makes redundant sample solution completely into waste liquid hole by the 7th velocity reversal rotation.
10. the method for the residual detection of the agriculture according to claim 9 based on DELFIA, which is characterized in that the First Speed
It is 1500 revs/min, each runing time of positive and negative rotation is 1 minute；The second speed is 3000 revs/min, and runing time is 1 point
Clock；The third speed is 3000 revs/min, and runing time is 1 minute；The fourth speed is 4500 revs/min, when operation
Between be 1 minute；5th speed is 4500 revs/min, and runing time is 1 minute；6th speed is 6000 revs/min
Clock, runing time are 1 minute；7th speed is 6000 revs/min, and runing time is 1 minute.
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