CN205691502U - A kind of magnetic microsphere fluorescence detecting system - Google Patents
A kind of magnetic microsphere fluorescence detecting system Download PDFInfo
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- CN205691502U CN205691502U CN201620520566.1U CN201620520566U CN205691502U CN 205691502 U CN205691502 U CN 205691502U CN 201620520566 U CN201620520566 U CN 201620520566U CN 205691502 U CN205691502 U CN 205691502U
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Abstract
This utility model provides a kind of magnetic microsphere fluorescence detecting system, it is characterised in that: include sampling device, electric magnet, magnetic microsphere Monitoring of Quantity device and fluorescent labeling analyte detection device；Described sampling device includes imbibition syringe needle, quartz ampoule and suction pumps, and described imbibition syringe needle connects quartz ampoule, and quartz ampoule connects suction pumps；Described fluorescent labeling analyte detection device includes exciting light sources, optical-electrical converter one, excitation light path and collection light path one；Described magnetic microsphere Monitoring of Quantity device includes collecting light path two and optical-electrical converter two.By electric magnet, magnetic microsphere being gathered in detection region, make the magnetic microsphere concentration in detection region improve thousandfold, the fluorescence intensity of fluorescent marker then can improve up to ten thousand times, the interference that effectively testing result is the most thoroughly brought by abatement due to cleaning.
Description
Technical field
This utility model belongs to technical field of medical detection, is specifically related to a kind of magnetic microsphere fluorescence detecting system.
Background technology
Detection technique of fluorescence is in medical science and the application history of existing nearly 70 years in biology, it and immunoassay technology phase
Engage and form immunofluorence technic, and along with the development of immunological technique and application, become microbiology, immunology, pathology, exempt from
A kind of detection technique the most frequently used, maximally effective in epidemic disease histochemistry and genome chemistry.
Along with clinical medicine and scientific and technological progress, immunofluorence technic develops again multiple practical detection technique, such as electrochemistry
The fluorescence such as fluorescence immunoassay (ECLI), chemiluminescence immune assay (CLIA), time resolved fluoro-immunoassay (TRFIA) are exempted from
Epidemic disease analytical technology.These technology are all by determining detected material to label (spike probe) detection, and label is by immunity
The coating techniques such as reaction method, chemical reaction method, biochemical reaction method, Streptavidin and biotin, are fabricated to have higher special
Property probe, probe is combined with target detection material by corresponding reaction；By luminoscope, spike probe is detected again,
The relevant informations such as the concentration of acquisition target substance.On the other hand coating technique is also in development, in addition to 96 traditional microwell plates are coated,
Also use magnetic microsphere as immunoreactive carrier, capture magnetic microsphere by controlling magnetic field, be greatly improved the amount being coated thing, increase
Adding detection sensitivity, reduce the response time, facilitate the cleaning in course of reaction, laboratory automation operates, and improves system flexibility
And work efficiency, such as the most popular chemical illumination immunity analysis instrument.
For magnetic microsphere as the equipment of immunoreation carrier, such as: immunofluorescence analysis instrument, chemiluminescence fluorescence analysis
Instrument, owing to needing to carry out cleaning several times in course of reaction, the fluorescent marker that reaction tank finally carries out whole cup liquid contains
Amount, to obtain the concentration value of reaction tank detection object., all there is a nothing in the fluorescent labeling detection technique that the above is conventional
The problem that method overcomes is exactly: owing to course of reaction have passed through cleaning step, it is impossible to the magnetic ensured during test every time in reaction tank is micro-
The volume residual of ball keeps consistent.Existence two big defects:
1, detection magnetic microsphere is distributed in sample solution with low concentration, easily because magnetic microsphere skewness or clean not
Thoroughly cause testing result deviation；
Although 2 magnetic microsphere samplings are accurately, but after being cleaned multiple times, in reaction tank, the volume residual of magnetic microsphere is not
Can determine that.
Utility model content
In order to solve above-mentioned technical problem, this utility model provides a kind of time-resolved fluorescence detecting system.By sample
Magnetic microsphere in pond is divided into some parts of equivalent, and completes fluorescent labeling analyte detection again after collecting magnetic microsphere.
This utility model, in order to realize foregoing invention purpose, adopts the following technical scheme that
A kind of magnetic microsphere fluorescence detecting system, including sampling device, electric magnet, magnetic microsphere Monitoring of Quantity device and glimmering
Signal analyte detection device；Described sampling device includes imbibition syringe needle, quartz ampoule and suction pumps, and described imbibition syringe needle is even
Connecing quartz ampoule, quartz ampoule connects suction pumps；Described electric magnet is wound around by the coil of constant current source power supply, one end of electric magnet towards
Quartz ampoule；Described fluorescent labeling analyte detection device includes exciting light sources, optical-electrical converter one, excitation light path and collection light path
One, the light of described exciting light sources is focused on quartz ampoule by excitation light path, and this convergent point and electric magnet are towards quartz
The position of pipe is identical, and described collection light path a pair should the position of convergent point be arranged, and described optical-electrical converter a pair is receivable
Collection light path one；Described magnetic microsphere Monitoring of Quantity device includes collecting light path two and optical-electrical converter two, described collection light path
The position of two same corresponding described convergent points is arranged, and described optical-electrical converter two is corresponding collects light path two.
Magnetic microsphere fluorescence detecting system of the present utility model passes through the electric magnet magneticaction to magnetic microsphere, is gathered by magnetic microsphere
Collection, in the detection region of quartz ampoule, makes magnetic microsphere improve thousandfold in the concentration of regional area, and the fluorescence of fluorescent marker is strong
Degree then can improve up to ten thousand times, and ambient interferences fluorescence is not correspondingly improved, thus improves the capacity of resisting disturbance of system and sensitive
Degree height.
Quartz ampoule described in the utility model is hollow transparent pipe, and its internal diameter is 0.1-10mm.Magnetic microsphere can be limited outside
Prolong gathering, make magnetic microsphere in limited space be evenly distributed and quantity is stable, and guarantee at magnetic microsphere Rendezvous Point, it is thus achieved that more
High magnetic microsphere sum and the accounting of liquid capacity, be so greatly improved system signal noise ratio.
Preferably, internal diameter and the external diameter of described quartz ampoule is square.
Suction pumps described in the utility model is peristaltic pump or air ejector, and liquid flowing quartz ampoule is produced pressure reduction,
Promote liquid to flow through quartz ampoule from reaction tank, and flow velocity is controlled.
Excitation light path described in the utility model, collection light path one and collection light path two are by convergent lens and optical filter structure
Become.
The detection method of magnetic microsphere fluorescence detecting system described in the utility model:
Step is as follows:
1) imbibition syringe needle lower end being immersed sample solution to be detected in reaction tank, suction pumps produces pressure reduction to liquid, promotes
Liquid in reaction tank flows in quartz ampoule；Containing combining detected material and fluorescent marker in described sample solution to be detected
Magnetic microsphere；
2) opening constant-current source, electric magnet produces magnetic field, before the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet
End, along with the flowing of liquid, magnetic microsphere quantity of collecting in quartz ampoule is continuously increased, and forms Rendezvous Point；
3) starting exciting light sources, light focuses in quartz ampoule on the magnetic microsphere of Rendezvous Point by excitation light path, and second
Collect light path and collect the light that at Rendezvous Point, magnetic microsphere produces under excitation light irradiation, and be converted into the signal of telecommunication, thus monitor remittance
The gathering quantity of magnetic microsphere at collection point；
4) when magnetic microsphere gathering quantity reaches certain setting value, collect light path by first and collect the fluorescence that magnetic microsphere combines
The fluorescence signal of label, and be converted to the signal of telecommunication by the first optical-electrical converter, carry out fluorescent marker content detection, close
Constant-current source；
Repeat the above steps 1)-4), obtain the testing result collecting magnetic microsphere in extraction liquid in batches, and unite
Meter, completes the content detection of fluorescent marker in whole reaction tank.
The particle diameter of magnetic microsphere described in the utility model is between 0.1um-20um, as the carrier of fluorescent marker.
Fluorescent marker is the probe material of reaction test, is coated with the material that can combine, fluorescence with detection object
Label has the specificity inspiring long-life phosphors under specific wavelength excitation light irradiation.
Preferably, described collection light path two is scattered light light path.
Light source used by magnetic microsphere Monitoring of Quantity device, such as: scattered light light source or fluorescent magnetic microsphere excitation source, can be with glimmering
The excitation source of signal thing fluorescence detecting system shares a kind of wavelength, it is also possible to be two kinds of different wave lengths.
Preferably, described magnetic microsphere adds the fluorescent material different from fluorescent marker wavelength, described collection
Light path two carries the collection light path of fluorescence for magnetic microsphere.
Magnetic microsphere as reaction experiment carrier described in the utility model adds special fluorescent material, such as fluorescence
Dyestuff or Rare Earth Chelate etc., its wavelength of fluorescence is different from fluorescent marker wavelength of fluorescence；Described electric magnet is arranged on quartz ampoule
Middle part, fluorescent magnetic microsphere in working fluid is resided in quartz ampoule by after electricity by electric magnet, forms Rendezvous Point, along with the stream of liquid
Dynamic, the quantity of fluorescent magnetic microsphere is continuously increased；Under described exciting light sources irradiates, fluorescent magnetic microsphere excites carry special
Wavelength fluorescent, magnetic microsphere is carried phosphor collection light path and is built up in quartz ampoule Rendezvous Point side, and is converted into by optical-electrical converter
The signal of telecommunication, signal height reflects the quantity of magnetic microsphere.
Magnetic microsphere Monitoring of Quantity device, it is also possible to utilize and set suitable assemble index, make the microsphere amount field of Rendezvous Point
Territory is saturated, reaches to monitor the purpose of magnetic microsphere quantity.
Fluorescent marker detecting system, it is also possible to be chemiluminescence fluorescent marker detecting system, chemiluminescence fluorescence mark
Note thing, under the effect of chemical luminous substrate, produces the fluorescence of long-life specific wavelength, and chemiluminescence phosphor collection light path is set up
In quartz ampoule magnetic microsphere Rendezvous Point side, the fluorescence signal of collection is converted to the signal of telecommunication through optical-electrical converter；Monitoring magnetic microsphere
After Rendezvous Point gathering quantity reaches setting value, the fluorescence intensity telecommunications of the chemiluminescent labels that record magnetic microsphere is combined
Number；By extracting liquid in batches, collecting magnetic microsphere, the chemiluminescence fluorescence of detection fluorescent marker, to 2 to 1000 detections
Result carries out statistics and obtains meansigma methods, it is achieved to fluorescent marker content detection in whole reaction tank.
The beneficial effects of the utility model are:
1. by electric magnet, magnetic microsphere is gathered in detection region, makes the magnetic microsphere concentration in detection region improve thousands of
Times, the fluorescence intensity of fluorescent marker then can improve up to ten thousand times, and background noise produced by the impurity in solution will not
It is enhanced, the interference that effectively testing result is the most thoroughly brought by abatement due to cleaning.
2. monitor the quantity of magnetic microsphere gathering by arranging magnetic microsphere Monitoring of Quantity device, it is ensured that label fluorescence every time
Detection be all based on identical magnetic microsphere under the conditions of, thus improve the stability of fluorescent marker testing result.
Accompanying drawing explanation
Fig. 1 is the structural representation of this utility model magnetic microsphere fluorescence detecting system.
Fig. 2 is the schematic diagram of this utility model magnetic microsphere fluorescence detecting system detection state.
Fig. 3 is this utility model magnetic microsphere fluorescence detecting system profile of vertical liquid flow direction at Rendezvous Point.
Reference: 1, quartz ampoule, 2: magnetic microsphere, 3, exciting light sources, 4, optical-electrical converter two, 5, optical-electrical converter
One, 6, electric magnet constant-current source, 7, Rendezvous Point, 8, electric magnet, 9, excitation light path, 10, collect light path one, 11, collect light path two,
12, reaction tank, 13, imbibition syringe needle, 14, suction pumps.
Detailed description of the invention
Below in conjunction with detailed description of the invention, essentiality content of the present utility model is described in further detail.
Embodiment 1
As shown in Figure 1, Figure 2 and Figure 3, a kind of magnetic microsphere fluoroscopic examination dress system, including sampling device, electric magnet 8,
Magnetic microsphere Monitoring of Quantity device and fluorescent labeling analyte detection device；Described sampling device includes imbibition syringe needle 13, quartz
Pipe 1 and suction pumps 14, described imbibition syringe needle 13 connects quartz ampoule 1, and quartz ampoule 1 connects suction pumps 14；Described electric magnet 8 is permanent
The coil of stream source 6 power supply is wound around, and one end of electric magnet 8 is towards quartz ampoule 1；Described fluorescent labeling analyte detection device includes exciting
Radiant 3, optical-electrical converter 1, excitation light path 9 and collection light path 1, the light of described exciting light sources 3 passes through exciting light
Road 9 focuses on quartz ampoule 1, and the collection light path one that this convergent point is identical towards the position of quartz ampoule 1 with electric magnet 8, described
10 pairs should the position of convergent point be arranged, and described optical-electrical converter 1 is corresponding collects light path 1；Described magnetic microsphere quantity
Monitoring device includes collecting light path 2 11 and optical-electrical converter 24, the same corresponding described convergent point of described collection light path 2 11
Position arrange, described optical-electrical converter 24 is corresponding collects light path 2 11.
Embodiment 2
The present embodiment is on the basis of embodiment 1:
Described quartz ampoule 1 is hollow transparent pipe, and its internal diameter is 0.1mm.
Embodiment 3
The present embodiment is on the basis of embodiment 1:
Described quartz ampoule 1 is hollow transparent pipe, and its internal diameter is 10mm, and its internal diameter and external diameter are square.
Described suction pumps 14 is peristaltic pump.
Embodiment 4
The present embodiment is on the basis of embodiment 1:
Described quartz ampoule 1 is hollow transparent pipe, and its internal diameter is 1mm, and its internal diameter and external diameter are square.
Described suction pumps 14 is air ejector.
Described excitation light path, collection light path one and collection light path two are constituted by convergent lens and optical filter.
Embodiment 5
The present embodiment is on the basis of embodiment 1:
Described quartz ampoule 1 is hollow transparent pipe, and its internal diameter is 5mm, and its internal diameter and external diameter are square.
Described suction pumps is peristaltic pump or air ejector.
Described excitation light path, collection light path one and collection light path two are constituted by convergent lens and optical filter.
Described collection light path 2 11 is scattered light light path.
The detection method of this utility model magnetic microsphere fluorescence detecting system, as in figure 2 it is shown, step is as follows:
1) imbibition syringe needle lower end being immersed sample solution to be detected in reaction tank, suction pumps produces pressure reduction to liquid, promotes
Liquid in reaction tank flows in quartz ampoule；Containing combining detected material and fluorescent marker in described sample solution to be detected
Magnetic microsphere；
2) opening constant-current source, electric magnet produces magnetic field, before the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet
End, along with the flowing of liquid, magnetic microsphere quantity of collecting in quartz ampoule is continuously increased, and forms Rendezvous Point；
3) starting exciting light sources, light focuses in quartz ampoule on the magnetic microsphere of Rendezvous Point by excitation light path, and second
Collect light path and collect the light that at Rendezvous Point, magnetic microsphere produces under excitation light irradiation, and be converted into the signal of telecommunication, thus monitor remittance
The gathering quantity of magnetic microsphere at collection point；
4) when magnetic microsphere gathering quantity reaches certain setting value, collect light path by first and collect the fluorescence that magnetic microsphere combines
The fluorescence signal of label, and be converted to the signal of telecommunication by the first optical-electrical converter, carry out fluorescent marker content detection, close
Constant-current source；
Repeat the above steps 1)-4), obtain the testing result collecting magnetic microsphere in extraction liquid in batches, and unite
Meter, completes the content detection of fluorescent marker in whole reaction tank.
Embodiment 6
As it is shown in figure 1, a kind of magnetic microsphere fluorescence detecting system, including sampling system, magnetic microsphere Monitoring of Quantity device
With label fluorescence detecting system, wherein:
Sampling system includes imbibition syringe needle 13, quartz ampoule 1, suction pumps 14；Described imbibition syringe needle 13 lower end is immersed anti-
Answering in pond 12 below sample solution liquid level to be detected, upper end, through flexible pipe, connects quartz ampoule 1, and quartz ampoule lower end connects suction pumps
14, suction pumps 14 is peristaltic pump or air ejector, liquid flow line is produced pressure reduction, promotes liquid to flow from reaction tank 12
Crossing quartz ampoule 1, and flow velocity is controlled, suction pumps 14 outlet connects waste collection bottle, waste liquid bottle and connection flexible pipe, the most in the view
Draw；
Magnetic microsphere quantitative control system includes that electric magnet 8, scattered light collect light path 11 and optical-electrical converter 4；Described quartz
It is provided with electric magnet 8 in the middle part of pipe 1, electric magnet 8 is wound around coil, open constant-current source 6 and make electric magnet 8 be produced magnetic force by electricity；Quartz ampoule 1
Magnetic microsphere 2 in the liquid of interior flowing, under electric magnet 8 magneticaction, collects in quartz ampoule 1 inwall, cumulative and shape gradually
Become Rendezvous Point 7.As in figure 2 it is shown, Rendezvous Point 7 is arranged over excitation source 3, by excitation light path 9, the light focusing of light source is existed
On the microsphere 2 of Rendezvous Point 7, Rendezvous Point 7 side is provided with scattered light and collects light path, i.e. collects light path 2 11 and optical-electrical converter 24,
It is used for detecting the light intensity of the magnetic microsphere 2 scattered light wavelength when light source 3 irradiates；Magnetic microsphere at Rendezvous Point 7 collects quantity
The most, the scattered light detected is the strongest, is converted into the signal of telecommunication by optical-electrical converter 4, when the signal of telecommunication reaches setting value, opens
Dynamic first order fluorescence detection.
Fluorescence detecting system includes fluorescent marker fluorescence light path, i.e. collects light path 1 and optical-electrical converter 1, described
Collect light path 1 and include focused light passages and the optical filter that only specificity fluorescent wavelength can pass through, be built up in the remittance of magnetic microsphere 2
Collection point 7 sides are used for collecting fluorescent marker fluorescence；When fluorescent marker is by the irradiation of exciting light sources 3, it is inspired
The fluorescence of specificity wavelength, and focus on by collecting light path 1, optical-electrical converter 1 that the fluorescent light of specific wavelength is made a gift to someone
Be converted to the signal of telecommunication；Scattered light light intensity signal in optical-electrical converter 24 output arrives the setting value moment, and fluorescence detecting system is remembered
The fluorescent intensity signal of record optical-electrical converter 1 output；And by changing and again collecting magnetic microsphere 2, it is achieved fluorescence repeatedly
Label fluorescent intensity detects, it is thus achieved that average intensity values, completes the detection to the object in reaction tank 12.
By following example, corresponding with target detection thing content in above-mentioned fluorescence detecting system quantitative determination sample solution
Method be described in detail.
Embodiment 7
The detection of double antibody sandwich method detection by quantitative TSH (thyrotropin)
1, the β of anti-human TSH-subunit monoclonal antibody is coated on magnetic bead
The magnetic particle of 20mg particle diameter 5 μm surface band carboxyl (purchased from Thermo Scientific company) is separated with Magnet
Supernatant, then removes supernatant, the sodium azide in removing buffer 3 times, hangs with 0.1MMES buffer (pH6.8～7.2)
Floating, it is diluted to 5ml；Under agitation add 5mg carbodiimide (EDC) and 5mg N-hydroxy-succinamide sulfonate sodium, stirring
Dissolve, room temperature reaction 25 minutes；The magnetic bead Magnet separation supernatant after activation, then supernatant being removed, it is unnecessary to remove
EDC and N-hydroxy-succinamide sulfonate sodium.Clean with 50mM borate buffer (pH8.2～9), use borate buffer afterwards
Liquid is suspended to 3 milliliters.
Add the β-subunit monoclonal antibody of the anti-human TSH that 1mg 50mM borate buffer (pH8.2～9) was dialysed,
Stir, room temperature reaction 16 to 24 hours；React after terminating with Magnet separation supernatant, supernatant is removed, adds confining liquid
(BSA (bovine serum albumin), the Tris (trishydroxymethylaminomethane acetate) of 50mM pH8.5 containing 5g/L buffer fluid-tight
Close about 8 hours, with Magnet separation supernatant, supernatant removed, then with diluent (BSA, the 5g/L's containing 5g/L
PVP (polyvinylpyrrolidone), the Tris buffer 50mM of pH8.0 of 0.2% sodium azide) dissolution precipitation, make mark microsphere
Reagent.4 DEG C of stored refrigerated.
2, the α-subunit labeling of monoclonal antibody at anti-human-TSH on fluorescent marker
The sodium azide being centrifuged off in buffer by the fluorescent nano particles of 20mg particle diameter 100nm surface band carboxyl, with front
State step 1 preparation and be coated method (remarks: separate fluorescent particle mode and change high speed centrifugation into, remaining same step 1), the general of magnetic microsphere
The α of the anti-human-TSH of another strain-subunit labeling of monoclonal antibody is on nano fluorescent microgranule, with diluent (containing 5g/L's
The PVP of BSA, 5g/L, the Tris buffer 50mM of pH8.0 of 0.2% sodium azide) dissolution precipitation, prepare fluorescent labeling examination
Agent, 4 DEG C of stored refrigerated.This fluorescent marker can send 610nm fluorescence under the exciting of 390nm wavelength light source.
3, prepared by immunoreation sample
Take each 50 μ l of standard sample 3 parts that TSH concentration is 0.09IU/ml, be separately added into 3 type plastic tube example reaction ponds
In 12, and label A, B and C.
Be separately added in above-mentioned 3 sample cells take step 1 preparation magnetic microsphere reagent 20 μ l (solid content is ten thousand/
Two), utilizing electromagnetic field to vibrate, the β the being coated anti-human TSH-subunit monoclonal antibody of the TSH antigen in sample and magnetic microsphere is exempted from
Epidemic disease is reacted, and is attached to magnetic microsphere surface, and the antigen in sample solution is the most, then assemble the most at mark microsphere surface.Instead
After answering 30 minutes, utilizing magnetic field to separate, remove the sample that supernatant is unnecessary, add 200 μ l cleanout fluid, magnetic field disperses,
Recycling magnetic field separates, and repeatedly cleans 5 times, is eventually adding diluent.
Sample cell B and C is carried out repeated washing 6 times, 7 times respectively by above-mentioned steps.
Being separately added into the label reagent 50 μ l of step 2 preparation in above-mentioned 3 sample cells, fluorescent marker can be by mark
α-subunit the monoclonal antibody of the anti-human-TSH of note is combined with the TSH antigen being attached on magnetic microsphere, forms magnetic microsphere-target
The complex of thing-label, after about 30 minutes, utilizes magnetic field to separate, removes the fluorescent marker that supernatant is unnecessary, then add
Entering 200 μ l cleanout fluid, magnetic field disperses, and recycling magnetic field separates, and repeatedly cleans 5 times, is eventually adding diluent.
Sample cell B and C is carried out repeated washing 6 times, 7 times respectively by above-mentioned steps.
Impurity in the most solution of wash number is the fewest in theory, and ambient interferences is the lowest, but causes magnetic microsphere quantity to be lost
Probability the biggest.
4, label fluoroscopic examination
Such as Fig. 2, in device, exciting light sources 3 is 390nm photodiode, and its supply current is 80mA, irradiates from top,
And focal length is directed at reaction tank 12 center, the optical filter of fluorescence light path 10 is that 610nm light arrowband passes through, and optical-electrical converter 5 is shore
Song company photomultiplier tube R4220P, the signal of telecommunication of conversion is through amplifying and after filtering, and to enter analog digital conversion be digital signal.
First take into the example reaction pond A of above-mentioned 4 steps, liquid level in reaction tank 12 is immersed in imbibition syringe needle 13 lower end
Hereinafter, open suction pumps 14, make liquid flow line produce pressure reduction, promote liquid sample liquids from reaction tank 12 constantly to flow through
Quartz ampoule 1, and flow speed stability is controlled, and suction pumps 14 outlet connects waste collection bottle, waste liquid bottle and connection flexible pipe, not at view
In draw；
Open constant-current source 6, make electric magnet 8 in the middle part of quartz ampoule 1 be produced magnetic force by electricity, in quartz ampoule 1 in the liquid of flowing
Magnetic microsphere 2, under electric magnet 8 magneticaction, collects in quartz ampoule 1 inwall gradually, cumulative and form Rendezvous Point 7；
Such as Fig. 3, opening excitation source 3 above Rendezvous Point 7, exciting light sources is the light emitting diode of 390nm wavelength, and one
Straight with 80mA constant current source power supply；By light path 9 by the light focusing of light source on the microsphere 2 of Rendezvous Point 7, side on the right side of Rendezvous Point 7
Face is provided with scattered light and collects light path 11 and optical-electrical converter 4, and the optical filter of light path 11 is that 390nm light arrowband passes through, opto-electronic conversion
Device 4 is light cell, and the light intensity of the scattered light wavelength of magnetic microsphere 2 is converted into the signal of telecommunication；Magnetic microsphere at Rendezvous Point 7 collects
Quantity is the most, and it will be the highest that optical-electrical converter 4 is converted into the signal of telecommunication.
Fluorescence detection device is arranged on the left side of Rendezvous Point 7, when the fluorescent marker of magnetic microsphere 2 combination is at 390nm wavelength
The irradiation of exciting light sources 3 time, the fluorescence of specificity 610nm wavelength will be inspired, fluorescence light path 10 is by after fluorescent foci
After the optical filter that 610nm wavelength can pass through, enter optical-electrical converter 5 and be converted to the signal of telecommunication；Monitoring optical-electrical converter 4 exports
Scattered light light intensity signal arrive setting value time, record fluorescent marker the fluorescent intensity signal of telecommunication.
After completing one-time detection, closing electric magnet 8, magnetic microsphere flows away with liquid, is then again turned on, and enters by above-mentioned steps
Row fluorescent labeling analyte detection, and testing result is added up after repeating 30 times, finally obtain mean fluorecence label content knot
Really, reach to detect the purpose of object.
So far, after completing the detection to sample A, imbibition syringe needle 13 is carried out, prevents cross-contamination.By above-mentioned same
Sample B and C is detected by quadrat method successively, obtains testing result as follows.
Table 1 TSH quantitative microsphere detection fluorescence results
5, conventional sense instrument is used to carry out fluoroscopic examination
Carry out above-mentioned 1-3 step operation, finally reactant is moved in ELISA Plate, whole reaction tank is detected fluorescence, use
PE Victor3 V1420-040 detector, the fluorescence signal of detection A, B and C reaction tank, result is as follows:
Table 2 TSH reaction tank detection fluorescence results
The experimental data explanation of the present embodiment, by monitoring magnetic microsphere in the quantity of Rendezvous Point, makes fluorescent labeling analyte detection
Result decreases magnetic microsphere in cleaning process and loses the error caused；And make magnetic microsphere converge in residing in quartz ampoule by magnetic force
The density of collection point, higher than the density in reaction tank 10 to 10000 times, decrease owing to cleaning the background the most thoroughly introduced dry
Disturb, be more suitably applied to full-automatic, quick, high sensitivity, high accuracy and high throughput testing.
In popular response is tested, the purpose of cleaning is to reduce impurity, improves fluorescent marker and background signal accounting, this
The method that utility model is given, can improve fluorescent marker and background signal accounting, this enforcement by improving magnetic microsphere density
Example illustrates that this utility model is applicable to the reaction system detection fluorescent marker content of No clean.
Embodiment 8
Fluorescent magnetic microsphere carries fluoroscopic examination
Use the magnetic microsphere containing fluorescent material can be inspired under excitation light irradiation as reaction carriers, magnetic microsphere
Specificity fluorescent.
This fluorescent magnetic microsphere in the present embodiment adds Sm by dyeing3+Chelate, preparation technology is summarized as follows: with third
Ketone dissolves β-naphthoyltrifluoroacetone and three the most pungent phosphine oxides, then Sm3+Add and form chelate, then Sm3+Chelate puts into appearance
Device soaks magnetic microsphere 10 hours, then separates with magnetic field, and clear liquid does not enters into the Sm within magnetic microsphere repeatedly3+Chela and thing,
This fluorescent magnetic microsphere can launch, under the exciting of 340nm light, the fluorescence that wavelength is 644nm.
And the material as fluorescent marker is Eu3+Chelate, fluorescent marker can be sent out under the irradiation of 340nm light
Injection wavelength is 610nm fluorescence.
Fluorescent microsphere and fluorescent marker can share an excitation source, excite wavelength of fluorescence be respectively 644nm and
610nm。
Specifically comprise the following steps that
Immunoreation test procedure is with embodiment 10, and light source 3 is xenon lamp, and uses the exciting light of 340nm bandpass filter
Road 9, focuses on the Rendezvous Point 7 of the magnetic microsphere 2 of quartz ampoule 1 by hot spot；It is glimmering that quartz ampoule 1 Rendezvous Point 7 side is provided with fluorescent marker
Light collecting circuit 10, the narrow band pass filter light path 10 passed through by 610nm wavelength is focused on photoelectric sensor 5 and is converted to telecommunications
Number；Quartz ampoule 1 Rendezvous Point 7 side is additionally provided with fluorescent magnetic microsphere fluorescence and collects light path 18, by 644nm wavelength band pass filter light
Road 18 focuses on electric transducer 19 and is converted to the signal of telecommunication.
With reference to Fig. 2, first turn on suction pumps power supply 15, and maintain voltage constant constant, suck and complete as described in Example 1
Immunoreation the liquid of cleaned detected sample A, at the uniform velocity flow through quartz ampoule 1.Open the constant-current source 6 of electric magnet 8, and tie up
Hold electric current constant, magnetic microsphere 2 under electric magnet 8 produces magneticaction on Rendezvous Point 7 quantity be gradually increased；Open light source 3, prison
Surveying the output signal of fluorescent magnetic microsphere fluorescence transducer 19, intensity is the highest, and to have reacted the quantity that magnetic microsphere 2 collects the most, work as conversion
When device 19 output valve reaches set point, record under light source 3 irradiates, the fluorescent marker fluorescence intensity inspired, i.e. change
The magnitude of voltage of device 5 output, obtains first order fluorescence markers tests result.
By the method for above-mentioned detection fluorescent marker fluorescence, draw the magnetic microsphere in reaction tank in batches, carry out repeatedly
Detection, obtains meansigma methods through statistics, reaches to detect the purpose of the fluorescent marker content in sample cell.
Embodiment 9
Time-resolved fluorescence markers tests
Detecting sample with this utility model device, such as Fig. 2, in device, exciting light sources 3 is 390nm photoelectricity two pole
Pipe, its supply current is 80mA, irradiates from top, and focal length is directed at reaction tank 12 center, time-resolved fluorescence light path 10
Optical filter is that 610nm light arrowband passes through, and optical-electrical converter 5 is Bin Song company photomultiplier tube R4220P, the signal of telecommunication warp of conversion
Cross after sending out big and filtering, and to enter analog digital conversion be digital signal.
Fluorescent marker uses Eu3+Chelate, has time resolution specificity, under the irradiation of 390nm light after, inspire
Wavelength is 610nm fluorescence, and fluorescence lifetime is more than 5mS.
Such as Fig. 3, first take above-mentioned A example reaction pond, after adding fluorescence enhancement solution, reaction is immersed in imbibition syringe needle 13 lower end
In pond 12 below liquid level, open suction pumps 14, make liquid flow line produce pressure reduction, promote liquid sample liquid from reaction tank 12
Body constantly flows through quartz ampoule 1, and flow speed stability is controlled, and it is soft that suction pumps 14 outlet connects waste collection bottle, waste liquid bottle and connection
Pipe, draws the most in the view.Open constant-current source 6, make electric magnet 8 be produced magnetic force by electricity, the magnetic in the liquid of flowing in quartz ampoule 1
Microsphere 2, under electric magnet 8 magneticaction, collects in quartz ampoule 1 inwall gradually, cumulative and form Rendezvous Point 7, through setting
15 seconds after, the magnetic microsphere 2 that Rendezvous Point 7 is assembled reaches stable quantity, closes suction pumps 14, opens and excites above Rendezvous Point 7
Light source 3, closes after the irradiation of 50 gsec, after 200 microsecond delay times, opens and recording light electric transducer 5
Continuous detecting 600 gsec resolved fluorometric signal, it is thus achieved that time-resolved fluorescence testing result.
After completing one-time detection, close electric magnet 8, magnetic microsphere flows away with liquid, is then again turned on, repeat 30 times above-mentioned
Collect magnetic microsphere 2, detection record fluorescence intensity process, finally carry out statistics and obtain average intensity values, in complete paired samples A
The detection of object.
Clean imbibition syringe needle 13 and fluid path inner-walls of duct, prevent cross-contamination.The most successively to sample B and
C detects, and obtains testing result as follows.
Table 3 TSH quantitative microsphere detection fluorescence results
Time-resolved fluorescence labelling compares prompt fluorescence label, has higher specificity, has more anti-background interference
Feature.
Claims (6)
1. a magnetic microsphere fluorescence detecting system, it is characterised in that: include that sampling device, electric magnet, magnetic microsphere quantity are supervised
Survey device and fluorescent labeling analyte detection device；Described sampling device includes imbibition syringe needle, quartz ampoule and suction pumps, described
Imbibition syringe needle connects quartz ampoule, and quartz ampoule connects suction pumps；Described electric magnet is wound around by the coil of constant current source power supply, electric magnet
One end towards quartz ampoule；Described fluorescent labeling analyte detection device includes exciting light sources, optical-electrical converter one, excitation light path
With collection light path one, the light of described exciting light sources focuses on quartz ampoule by excitation light path, focuses on the convergent point formed
Identical towards the position of quartz ampoule with electric magnet, described collection light path a pair should the position of convergent point be arranged, described light
Electric transducer should collect light path 1 a pair；Described magnetic microsphere Monitoring of Quantity device includes collecting light path two and optical-electrical converter
Two, the position of the same corresponding described convergent point of described collection light path two is arranged, and described optical-electrical converter two is corresponding collects light
Road two.
A kind of magnetic microsphere fluorescence detecting system the most according to claim 1, it is characterised in that: described quartz ampoule is that hollow is saturating
Bright pipe, its internal diameter is 0.1-10mm.
A kind of magnetic microsphere fluorescence detecting system the most according to claim 2, it is characterised in that: the internal diameter of described quartz ampoule and
External diameter is square.
A kind of magnetic microsphere fluorescence detecting system the most according to claim 1, it is characterised in that: described suction pumps is for wriggling
Pump or air ejector.
A kind of magnetic microsphere fluorescence detecting system the most according to claim 1, it is characterised in that: described excitation light path, receipts
Collection light path one and collection light path two are constituted by convergent lens and optical filter.
A kind of magnetic microsphere fluorescence detecting system the most according to claim 1, it is characterised in that: described collection light path two is
Scattered light light path.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891493A (en) * | 2016-06-01 | 2016-08-24 | 章健 | Fluorescence detection device and detection method thereof |
| CN113029919A (en) * | 2021-03-13 | 2021-06-25 | 长春长光辰英生物科学仪器有限公司 | Cell enrichment and fluorescence counting detection device and detection and counting method |
| CN113125694A (en) * | 2019-12-31 | 2021-07-16 | 深圳市帝迈生物技术有限公司 | Detection system for realizing classification and quantitative analysis and detection method for immune multi-joint detection |
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2016
- 2016-06-01 CN CN201620520566.1U patent/CN205691502U/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891493A (en) * | 2016-06-01 | 2016-08-24 | 章健 | Fluorescence detection device and detection method thereof |
| CN105891493B (en) * | 2016-06-01 | 2017-10-24 | 章健 | A kind of fluorescence detection device and its detection method |
| CN113125694A (en) * | 2019-12-31 | 2021-07-16 | 深圳市帝迈生物技术有限公司 | Detection system for realizing classification and quantitative analysis and detection method for immune multi-joint detection |
| CN113029919A (en) * | 2021-03-13 | 2021-06-25 | 长春长光辰英生物科学仪器有限公司 | Cell enrichment and fluorescence counting detection device and detection and counting method |
| CN113029919B (en) * | 2021-03-13 | 2024-04-02 | 长春长光辰英生物科学仪器有限公司 | Cell enrichment and fluorescence counting detection device and detection and counting method |
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