CN105891493A - Fluorescence detection device and detection method thereof - Google Patents
Fluorescence detection device and detection method thereof Download PDFInfo
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- CN105891493A CN105891493A CN201610379771.5A CN201610379771A CN105891493A CN 105891493 A CN105891493 A CN 105891493A CN 201610379771 A CN201610379771 A CN 201610379771A CN 105891493 A CN105891493 A CN 105891493A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
The invention provides a fluorescence detection device and a detection method thereof. The device comprises a sample extracting mechanism, a magnetic microsphere gathering mechanism and a fluorescence detection mechanism. The sample extracting mechanism comprises a liquid sucking needle head, a quartz tube and a liquid sucking pump, the liquid sucking needle head is connected with the quartz tube, and the quartz tube is connected with the liquid sucking pump. The magnetic microsphere gathering mechanism is an electromagnet wound with a constant-current-source power supply coil, and one end of the electromagnet faces the quartz tube. The fluorescence detection mechanism comprises an exciting light source, a first photoelectric converter, an exciting light path and a first collecting light path, light of the exciting light source is gathered on the quartz tube through the exciting light path, the gathering point and the position where the electromagnet faces the quartz tube are the same, the first collecting light path is arranged corresponding to the position of the gathering point, and the first photoelectric converter corresponds to the first collecting light path.
Description
Technical field
The invention belongs to technical field of biomedical detection, be specifically related to a kind of fluorescence detection device and detection method thereof.
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 that label is by exempting from by label (spike probe) detection is determined detected material
The coating techniques such as epidemic disease reaction method, chemical reaction method, biochemical reaction method, Streptavidin and biotin, are fabricated to have relatively Gao Te
The probe of the opposite sex, probe is combined with target substance by corresponding reaction;Detecting instrument, by the detection to spike probe, measures
Go out the concentration of target substance.On the other hand coating technique is also in development, changes into using magnetic microsphere conduct from traditional coated elisa plate
Immunoreactive carrier, captures magnetic microsphere by controlling magnetic field, is greatly improved the thing amount that is coated, increase detection sensitivity, reduce
In the response time, facilitating the cleaning in course of reaction, laboratory automation operates, and improves system flexibility and work efficiency, as at present
Popular chemical illumination immunity analysis instrument.
At present, either use magnetic microsphere is as the equipment of immunoreation carrier, such as: immunofluorescence analysis instrument, chemistry are sent out
Light fluorescence analyser;Or with 96 orifice plates as the device of immunoreation carrier, such as: microplate reader or Timed-resolved fluoroimmunoassay
Instrument, needs the sample cell carried out several times to clean in course of reaction, last fluoroscopic examination is all carried out, the most just in reaction tank
It is to detect whole reaction tank fluorescent marker content.The fluorescent labeling detection technique of the most this routine, all having one cannot
The problem overcome is exactly: owing to course of reaction have passed through cleaning step, it is impossible to magnetic microsphere in reaction tank when ensureing every time to test
Quantity does not changes.It is to say, during detection, wash number is different or cleaning error can affect testing result, tool
There is a following defect:
When 1, not cleaning up, the prompt fluorescence that a lot of impurity exist constitutes interference to the specificity of fluorescent marker fluorescence.
2, detection magnetic microsphere is distributed in sample solution with low concentration, is easily affected by the interfering material in sample solution,
Cause detecting error.
Summary of the invention
For solving above-mentioned technical problem, the invention provides a kind of time-resolved fluorescence detection device and detection method thereof.
Magnetic microsphere in sample cell is divided into some parts of equivalent, and completes fluorescent labeling analyte detection again after magnetic microsphere is collected.
The present invention, in order to realize foregoing invention purpose, adopts the following technical scheme that
A kind of fluorescence detection device, including sampling mechanism, magnetic microsphere collecting mechanism and fluoroscopic examination mechanism;Described sample
Drawing mechanism includes imbibition syringe needle, quartz ampoule and suction pumps, and described imbibition syringe needle connects quartz ampoule, and quartz ampoule connects suction pumps;
Described magnetic microsphere collecting mechanism is the electric magnet that constant current source power supply coil is wound around, and one end of described electric magnet is towards quartz ampoule;
Described fluoroscopic examination mechanism includes that exciting light sources, the first optical-electrical converter, excitation light path and first collect light path;Described sharp
The light of illuminating source is focused on quartz ampoule by excitation light path, and this Rendezvous Point and electric magnet are towards the position phase of quartz ampoule
With, described first collection light path to should the position of Rendezvous Point arrange, described the first optical-electrical converter corresponding first is collected
Light path.
The fluorescence detection device of the present invention passes through the electric magnet magneticaction to magnetic microsphere, and magnetic microsphere is gathered in quartz ampoule
Detection region, make magnetic microsphere improve thousandfold in the concentration of regional area, the fluorescence intensity of fluorescent marker then can improve
Up to ten thousand times, and ambient interferences fluorescence is not correspondingly improved, thus improve the capacity of resisting disturbance of system and highly sensitive.
Fluorescence detection device of the present invention also includes magnetic microsphere Monitoring of Quantity mechanism, including the second collection light path and
Two optical-electrical converters;Described second position collecting the corresponding described convergent point of light path is arranged, the second described optical-electrical converter pair
Answer the second collection light path.
Preferably, the second described collection light path is that scattered light collects light path.Shone by magnetic microsphere at detection Rendezvous Point
Penetrate the scattered light produced when light irradiates, it is achieved magnetic microsphere is collected the real-time monitoring of quantity, it is ensured that magnetic during fluoroscopic examination every time
Microsphere quantity is identical, is effectively increased accuracy and the reliability of detection.
Light source used by magnetic microsphere of the present invention Monitoring of Quantity mechanism can share same light source with time-resolved fluorescence testing agency,
Or scattered light light source or fluorescent magnetic microsphere excitation source are additionally set, can be with the exciting light of time-resolved fluorescence testing agency
Source shares a kind of wavelength, it is also possible to be two kinds of different wave lengths.
Quartz ampoule of the present invention is hollow transparent pipe, and its internal diameter is 0.1-10mm.
Preferably, internal diameter and the external diameter of described quartz ampoule is square.Magnetic microsphere can be limited assemble to extension, make limited
Space in magnetic microsphere be evenly distributed and quantity is stable, and guarantee at magnetic microsphere Rendezvous Point, it is thus achieved that higher magnetic microsphere is total
Number and the accounting of liquid capacity, be so greatly improved system signal noise ratio.
Suction pumps of the present invention is peristaltic pump or air ejector, liquid flowing quartz ampoule is produced pressure reduction, promotes
Liquid flows through quartz ampoule from reaction tank, and flow velocity is controlled.
Excitation light path of the present invention, the first collection light path and second collect light path by convergent lens and optical filter structure
Become.
Present invention also offers the detection method of this fluorescence detection device:
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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, makes
Magnetic microsphere quantity of collecting in quartz ampoule is continuously increased, and forms Rendezvous Point;
3) after magnetic microsphere collects a period of time, starting exciting light sources, light converges in focusing on quartz ampoule by excitation light path
On the magnetic microsphere of collection point, first collects light path collects the fluorescence signal of the fluorescent marker that magnetic microsphere combines, and by the first light
Electric transducer is converted to the signal of telecommunication, carries out fluorescent marker content detection, closes constant-current source;
Repeat the above steps 1)-3), obtain the testing result collecting magnetic microsphere in extraction liquid in batches, and add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
Preferably, after magnetic microsphere collects 0.1 second to 10 seconds, start exciting light sources.Control carrying when collecting of magnetic microsphere
So that every time fluoroscopic examination time, magnetic microsphere to collect quantity the most identical.
It is further preferred that the magnetic force constant of described electric magnet is 1~2000mg, it is ensured that magnetic microsphere is assembled stable.
It is further preferred that the constant flow rate that described liquid is in quartz ampoule is 0.1~100 cels, under this flow velocity
Magnetic microsphere energy rapid aggregation.
Present invention also offers the another kind of detection method of this fluorescence detection device, it is characterised in that:
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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, and the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, 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, the second collection
Light path collects the light that at Rendezvous Point, magnetic microsphere produces under excitation light irradiation, and is converted into the signal of telecommunication, thus monitors Rendezvous Point
The gathering quantity of place's magnetic microsphere;
4) when magnetic microsphere gathering quantity reaches certain setting value, collect light path by first and collect the fluorescent labeling that magnetic microsphere combines
The fluorescence signal of thing, 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 add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
Preferably, when magnetic microsphere reaches the setting value between 1~100000 in the quantity that Rendezvous Point is assembled, start and swash
Illuminating source.
Preferably, the inside of described magnetic microsphere adds the fluorescent material different from fluorescent marker wavelength, described
Two collection light paths are the collection light path that magnetic microsphere carries fluorescence.
The particle diameter of magnetic microsphere of the present invention is between 100nm to 50 μm, 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.
The detection object of magnetic microsphere of the present invention includes: immune protein, hormone, enzyme, medicine, trace element,
Tumor marker, gene, DNA and RNA, microorganism and metabolite thereof etc..
The fluoroscopic examination of the present invention, it is also possible to be chemiluminescence fluorescent labeling analyte detection device, chemiluminescence fluorescent labeling
Thing, under the effect of chemical luminous substrate, produces the fluorescence of long-life specific wavelength, and chemiluminescence phosphor collection light path is built 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 exists
After Rendezvous Point gathering quantity reaches setting value, the fluorescence intensity signal of telecommunication of the chemiluminescent labels that record magnetic microsphere is combined;
By extracting liquid in batches, collecting magnetic microsphere, the chemiluminescence fluorescence of detection fluorescent marker, to 2 to 1000 detection knots
Fruit carries out statistics and obtains meansigma methods, it is achieved to fluorescent marker content detection in whole reaction tank.
The beneficial effects of the present invention is:
1, by electric magnet, magnetic microsphere is gathered in detection region, makes the magnetic microsphere concentration in detection region improve thousandfold,
The fluorescence intensity of fluorescent marker then can improve up to ten thousand times, and produced by the impurity in solution, background noise will not be increased
By force, the interference that effectively testing result is the most thoroughly brought by abatement due to cleaning.
2, the purpose that popular response test is cleaned is to reduce impurity, improves fluorescent marker and background signal accounting;This
Bright detection method, by improving magnetic microsphere density, improves fluorescent marker and background signal accounting, improves experimental precision,
It is particularly well-suited to the reaction system detection fluorescent marker content of No clean.
3, the liquid sample of reaction tank is divided into some parts by detection method, carries out fluorescent labeling quality testing the most successively
Survey, with the statistical value of testing result repeatedly, replace the conventional method that fluorescent marker in reaction tank is carried out one-time detection, carry
The high detection accuracy of reaction tank, reduces system detection fluctuation.
4, the quantity that magnetic microsphere is assembled is monitored by arranging magnetic microsphere Monitoring of Quantity mechanism, 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, and experiment can
Control property is higher.
Accompanying drawing explanation
Fig. 1 is the structural representation of embodiment of the present invention 1-4 fluorescence detection device.
Fig. 2 is the structural representation of embodiment of the present invention 1-4 fluorescence detection device detection state.
Fig. 3 is the embodiment of the present invention 1-4 device profile at focal point vertical liquid flow direction.
Fig. 4 is the structural representation of embodiment of the present invention 5-7 fluorescence detection device.
Fig. 5 is the structural representation of embodiment of the present invention 5-7 fluorescence detection device detection state.
Fig. 6 is the embodiment of the present invention 5-7 device profile at focal point vertical liquid flow direction.
Reference: 1, quartz ampoule, 2, magnetic microsphere, 3, exciting light sources, the 4, second optical-electrical converter, the 5, first photoelectricity turns
Parallel operation, 6, electric magnet constant-current source, 7, Rendezvous Point, 8, electric magnet, 9, excitation light path, 10, first collects light path, and 11, second collects
Light path, 12, reaction tank, 13, imbibition syringe needle, 14, suction pumps.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the essentiality content of the present invention is described in further detail.
Embodiment 1
As shown in Figure 1, Figure 2 and Figure 3, a kind of fluorescence detection device, including sampling mechanism, magnetic microsphere collecting mechanism and fluorescence
Testing agency;Described sampling mechanism includes imbibition syringe needle 13, quartz ampoule 1 and suction pumps 14, and described imbibition syringe needle 13 is even
Connecing quartz ampoule 1, quartz ampoule 1 connects suction pumps 14;Described magnetic microsphere collecting mechanism is the electromagnetism that constant-current source 6 power coil is wound around
Ferrum 8, one end of described electric magnet 8 is towards quartz ampoule 1;Described fluoroscopic examination mechanism includes that exciting light sources the 3, first photoelectricity turns
Parallel operation 5, excitation light path 9 and first collect light path 10, and the light of described exciting light sources 3 focuses on quartz by excitation light path 9
On pipe 1, and this Rendezvous Point 7 is identical towards the position of quartz ampoule with electric magnet, and the first described collection light path 10 is to collecting
The position of point 7 is arranged, and light path 10 collected by described the first optical-electrical converter 5 corresponding first.
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.
The internal diameter of described quartz ampoule 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.
The internal diameter of described quartz ampoule and external diameter are square.
Described suction pumps 14 is air ejector.
Described excitation light path 9 and the first collection light path 10 are made up of convergent lens and optical filter.
Embodiment 5
The present embodiment is on the basis of embodiment 1:
As shown in Figure 4, Figure 5 and Figure 6, described fluorescence detection device also includes magnetic microsphere Monitoring of Quantity mechanism, including the second receipts
Collection light path 11 and the second optical-electrical converter 4;Described second position collecting the corresponding described convergent point 7 of light path 11 is arranged, described
Light path 11 collected by second optical-electrical converter 4 corresponding second.
Embodiment 6
The present embodiment is on the basis of embodiment 5:
The internal diameter of described quartz ampoule 1 is 5mm.
Described suction pumps 14 is air ejector.
Embodiment 7
The present embodiment is on the basis of embodiment 5:
The internal diameter of described quartz ampoule 1 is 5mm.
Described suction pumps 14 is air ejector.
Described excitation light path 9, first is collected light path 10 and second and is collected light path 11 by convergent lens and optical filter structure
Become.
Embodiment 8
The present embodiment is the detection method of fluorescence detection device described in embodiment 1-4, and 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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, makes
Magnetic microsphere quantity of collecting in quartz ampoule is continuously increased, and forms Rendezvous Point;
3) after magnetic microsphere collects a period of time, starting exciting light sources, light converges in focusing on quartz ampoule by excitation light path
On the magnetic microsphere of collection point, first collects light path collects the fluorescence signal of the fluorescent marker that magnetic microsphere combines, and by the first light
Electric transducer is converted to the signal of telecommunication, carries out fluorescent marker content detection, closes constant-current source;
Repeat the above steps 1)-3), obtain the testing result collecting magnetic microsphere in extraction liquid in batches, and add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
Embodiment 9
The present embodiment is on the basis of embodiment 8:
The magnetic force constant of described electric magnet is 1mg.
Described liquid constant flow rate in quartz ampoule is 0.1 cel.
Embodiment 10
The present embodiment is on the basis of embodiment 8:
The magnetic force constant of described electric magnet is 2000mg.
Described liquid constant flow rate in quartz ampoule is 100 cels.
Embodiment 11
The present embodiment is on the basis of embodiment 8:
The magnetic force constant of described electric magnet is 1000mg.
Described liquid constant flow rate in quartz ampoule is 50 cels.
Embodiment 12
The present embodiment is the detection method of fluorescence detection device described in embodiment 5-7, and 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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, and the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, 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, the second collection
Light path collects the light that at Rendezvous Point, magnetic microsphere produces under excitation light irradiation, and is converted into the signal of telecommunication, thus monitors Rendezvous Point
The gathering quantity of place's magnetic microsphere;
4) when magnetic microsphere gathering quantity reaches certain setting value, collect light path by first and collect the fluorescent labeling that magnetic microsphere combines
The fluorescence signal of thing, 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 add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
Embodiment 13
The present embodiment is on the basis of embodiment 12:
The second described collection light path 11 collects light path for scattered light.
When magnetic microsphere 2 reaches 1 in the quantity that Rendezvous Point 7 is assembled, start exciting light sources 3.
Embodiment 14
The present embodiment is on the basis of embodiment 12:
The second described collection light path 11 collects light path for scattered light.
When magnetic microsphere 2 reaches 1000 in the quantity that Rendezvous Point 7 is assembled, start exciting light sources 3.
Embodiment 15
The present embodiment is on the basis of embodiment 12:
When magnetic microsphere 2 reaches 100000 in the quantity that Rendezvous Point 7 is assembled, start exciting light sources 3.
Described magnetic microsphere 2 adds the fluorescent material different from fluorescent marker wavelength, the second described collection light
Road 11 carries the collection light path of fluorescence for magnetic microsphere.
Embodiment 16
The present embodiment is on the basis of embodiment 12:
When magnetic microsphere 2 reaches 1000 in the quantity that Rendezvous Point 7 is assembled, start exciting light sources 3.
Described magnetic microsphere 2 adds the fluorescent material different from fluorescent marker wavelength, the second described collection light
Road 11 carries the collection light path of fluorescence for magnetic microsphere.
Embodiment 17
A kind of fluorescence detection device, including sampling mechanism, magnetic microsphere collecting mechanism and fluoroscopic examination mechanism, wherein:
Sampling mechanism includes imbibition syringe needle 13, quartz ampoule 1, suction pumps 14;Reaction tank is immersed in described imbibition syringe needle 13 lower end
In 12 below sample solution liquid level to be detected, upper end connects suction pumps 14 through hose connection quartz ampoule 1, quartz ampoule lower end, inhales
Liquid pump 14 is peristaltic pump or air ejector, liquid flow line is produced pressure reduction, promotes liquid to flow through stone from reaction tank 12
English pipe 1, and flow velocity is controlled, and suction pumps 14 outlet connects waste collection bottle, waste liquid bottle and connection flexible pipe.
Magnetic microsphere collecting mechanism includes electric magnet 8, constant-current source 6;Described electric magnet 8 is arranged in the middle part of control quartz ampoule 1, beats
The magnetic force that after opening constant-current source 6, electric magnet 8 produces, resides in electric magnet 8 front end by the magnetic microsphere 2 flow through in quartz ampoule 1 liquid,
Magnetic force constant and flow rate of liquid constant in the case of, the magnetic microsphere 2 quantity of collecting in quartz ampoule is continuously increased, formed high density
Rendezvous Point 7.
Fluoroscopic examination mechanism includes exciting light sources 3 and fluorescence detection device;Described exciting light sources 3 light is by light path 9
Focusing in quartz ampoule 1 on the magnetic microsphere 2 of Rendezvous Point 7, fluorescent marker produces the specific long-life under exciting light 3 irradiates
Fluorescence;Fluorescence detection device is arranged on quartz ampoule 1 side, and first collects light path 10 is used for collecting fluorescent marker fluorescence, then leads to
Cross the first optical-electrical converter 5 and be converted to the signal of telecommunication.
Described quartz ampoule 1 is square or circular elongated hollow liquid stream pipeline, and internal diameter is between 0.1 to 10mm.
Detection method based on said apparatus is: by the liquid in reaction tank 12 through the continuous suction quartz ampoule 1 of syringe needle 13,
Electric magnet 8 it is provided with, along with the continuous flowing of liquid, before increasing magnetic microsphere 2 is gathered in electric magnet 8 in the middle part of quartz ampoule 1
End;After magnetic microsphere 2 gathers certain time, quantity is basicly stable, closes suction pumps, starts light source 3 and irradiates magnetic microsphere 2 when setting
Close after between, after the delayed setting time, start the first optical-electrical converter 5, it is thus achieved that the fluorescent marker that magnetic microsphere 2 is combined
Time-resolved fluorescence intensity electrical signals.
By extracting liquid in batches, collecting magnetic microsphere 2, the fluorescence of detection fluorescent marker, to 2 to 1000 detection knots
Fruit is added up, it is achieved to fluorescent marker content detection in whole reaction tank 12.
By following example, to the method for target detection thing content in fluorescence detection device of the present invention quantitative determination sample solution
It is described in detail.
Embodiment 18
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 microsphere
By 20mg particle diameter 5 μm, magnetic microsphere (purchased from Thermo Scientific company) the Magnet separation supernatant of surface band carboxyl
Liquid, then removes supernatant, the sodium azide in removing buffer 3 times, suspends with 0.1MMES buffer (pH6.8~7.2),
It is diluted to 5ml;Under agitation add 5mg carbodiimide (EDC) and 5mg N-hydroxy-succinamide sulfonate sodium, stir molten
Solve, room temperature reaction 25 minutes;The magnetic microsphere 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
(the BSA(bovine serum albumin containing 5g/L), the Tris(trishydroxymethylaminomethane acetate of 50mM pH8.5) buffering 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 the pH8.0 of 0.2% sodium azide) dissolution precipitation, make mark microsphere examination
Agent.4 DEG C of stored refrigerated.
2, the α-subunit labeling of monoclonal antibody at anti-human-TSH on fluorescent marker
First the 0.05M phosphate buffer solution of α-subunit monoclonal antibody pH7.0 of anti-human for 1mg-TSH, 2~8 DEG C of dialysis
Four times.Taking out dithiothreitol, DTT (DTT) reductase 12 0 minute adding 0.2mg, (time divides to add the cryptate of 0.4mg
Distinguish fluorescent marker), and ethylenediaminetetraacetic acid (EDTA) low-temp reaction of 0.01mg 24 hours.
Marked the monoclonal antibody of cryptate and not at the monoclonal antibody of labelling by Sephadex G-50 column chromatography for separation, use egg
Bai Yi distinguishes the albumen of different molecular weight, and verification mark is to the ratio of the cryptate of monoclonal antibody simultaneously, prepares fluorescence mark
Note reagent, 4 DEG C of stored refrigerated.This reagent resolved fluorometric label can send 610nm glimmering under the exciting of 340nm wavelength light source
Light, and the life-span be more than 2ms.
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 12
In, 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, adds diluent for the last time.
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, fluoroscopic examination
Detecting sample by apparatus of the present invention, in device, exciting light sources 3 is 340nm photodiode, its supply current
For 80mA, irradiating from top, and focal length is directed at reaction tank 12 center, the optical filter of time-resolved fluorescence light path 10 is 610nm
Light arrowband passes through, and the first optical-electrical converter 5 is Bin Song company photomultiplier tube R4220P, and the signal of telecommunication of conversion is through sending out big and filter
After ripple, and to enter analog digital conversion be digital signal.
First take above-mentioned A example reaction pond, after adding fluorescence enhancement solution, imbibition syringe needle 13 lower end is immersed in reaction tank 12
Below liquid level, open suction pumps 14, make liquid flow line produce pressure reduction, promote liquid sample liquids from reaction tank 12 continuous
Flowing through quartz ampoule 1, and flow speed stability is controlled, suction pumps 14 outlet connects waste collection bottle, waste liquid bottle and connection flexible pipe, does not exists
View draws;
Such as Fig. 2, opening constant-current source 6, make electric magnet 8 be produced magnetic force by electricity, in quartz ampoule 1, the magnetic microsphere 2 in the liquid of flowing exists
Under electric magnet 8 magneticaction, gradually collect in quartz ampoule 1 inwall, cumulative and form Rendezvous Point 7, through 35 seconds set
After, the magnetic microsphere 2 that Rendezvous Point 7 is assembled reaches stable quantity, closes suction pumps 14;Meanwhile, exciting light above Rendezvous Point 7 is opened
Source 3, closes after the irradiation of 50 gsec, and the fluorescent marker that the magnetic microsphere 2 of Rendezvous Point 7 is combined, in 390nm illumination
Penetrating down the fluorescence being inspired 610nm wavelength, the time-resolved fluorescence first of aggregated point 7 side collects light path 10 and the first light
Electric transducer 5, fluorescence signal is converted into the signal of telecommunication;After excitation source 3 closes 200 microsecond delay times, when detecting and record
Between resolved fluorometric signal.
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, the mesh in complete paired samples A
The detection of mark thing.
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 1 TSH quantitative microsphere detection fluorescence results
5, the testing result of conventional sense instrument is used
Carry out above-mentioned 1-3 step operation, finally reactant is moved in ELISA Plate, whole reaction tank is detected fluorescence, use Suzhou
The Anytest2000 detector of new ripple, 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, compares the conventional method of TSH reaction tank fluoroscopic examination, by using quantitative microsphere
Detection method not only reduces magnetic microsphere in cleaning process and loses introduced error, and decreases owing to cleaning the most thoroughly introduces
Ambient interferences, be more suitably applied to full-automatic, quick, high sensitivity, high accuracy and high throughput testing.
Embodiment 19
This gives one utilize electric magnet 8 to magnetic microsphere 2 suction constant under conditions of, flow through quartz ampoule by change
The flow rate of liquid of 1, controls the magnetic microsphere 2 method assembling constant number at Rendezvous Point 7.Specifically comprise the following steps that
As shown in Figure 4, sample is detected by device, and in device, exciting light sources 3 is 390nm photodiode, its electricity of powering
Stream is 80mA, irradiates from top, and focal length is directed at reaction tank 12 center, and the optical filter of fluorescence light path 10 is 610nm light arrowband
Passing through, the first optical-electrical converter 5 is Bin Song company photomultiplier tube R4220P, the signal of telecommunication of conversion after sending out big and filtering,
And to enter analog digital conversion be digital signal.
First turning on suction pumps power supply 15, and maintain voltage constant constant, inhalational immunization has reacted and cleaned treating
The liquid of detection sample A, at the uniform velocity flows through quartz ampoule 1.
Opening electromagnetism constant-current source 6, and maintain electric current constant, the magnetic force constant making electric magnet 8 produce is constant;Start simultaneously at note
The change in voltage of the first optical-electrical converter 5 generation that record exciting light sources 3 excitation labeling produce are raw, the detection continuing 1 minute knot
Really, obtain the testing result such as Fig. 3, in figure t be from open electric magnet power 6 time, E is that magnetic microsphere 2 produces at Rendezvous Point 7
Fluorescence intensity level.
Fig. 3 illustrates, when in quartz ampoule 1, flow rate of liquid is constant, powers after 6 from opening electric magnet, the T0 moment, based on
Constant magnetic force and flow rate of liquid, magnetic microsphere 2 quantity on Rendezvous Point 7 reaches balance, collects quantity and can not continue to increase, keeps
Constant.
Experimental result according to this, can be by starting suction pumps 14, and in keeping quartz ampoule, flow rate of liquid is constant, opens electromagnetism ferroelectricity
Source 6, after the delay adjustments time, opens excitation source 3, records the signal of telecommunication of fluorescent marker the first optical-electrical converter 5 output, so
Rear disconnection electric magnet powers 6, and the magnetic microsphere 2 collected flows away with liquid.After completing above-mentioned one-time detection, it is again started up electromagnetism ferroelectricity
Source 6, repeat the above steps carries out fluoroscopic examination, obtains statistical value through being repeated several times, and in complete paired samples, fluorescent marker contains
The detection of amount.
Claims (14)
1. a fluorescence detection device, it is characterised in that: include sampling mechanism, magnetic microsphere collecting mechanism and fluoroscopic examination machine
Structure;Described sampling mechanism includes imbibition syringe needle, quartz ampoule and suction pumps, and described imbibition syringe needle connects quartz ampoule, quartz
Pipe connects suction pumps;Described magnetic microsphere collecting mechanism is the electric magnet that constant current source power supply coil is wound around, the one of described electric magnet
End is towards quartz ampoule;Described fluoroscopic examination mechanism includes that exciting light sources, the first optical-electrical converter, excitation light path and first are received
Collection light path;The light of described exciting light sources is focused on quartz ampoule by excitation light path, and this Rendezvous Point and electric magnet towards
The position of quartz ampoule is identical, described first collection light path to should the position of Rendezvous Point arrange, the first described opto-electronic conversion
Light path collected by device corresponding first.
A kind of fluorescence detection device the most according to claim 1, it is characterised in that: described fluorescence detection device also includes
Magnetic microsphere Monitoring of Quantity mechanism, including the second collection light path and the second optical-electrical converter;Described second collects described in light path correspondence
The position of convergent point is arranged, and light path collected by described the second optical-electrical converter corresponding second.
A kind of fluorescence detection device the most according to claim 2, it is characterised in that: the second described collection light path is scattering
Light collects light path.
A kind of fluorescence detection device the most according to claim 1, it is characterised in that: described quartz ampoule is hollow transparent pipe,
Its internal diameter is 0.1-10mm.
A kind of fluorescence detection device the most according to claim 4, it is characterised in that: internal diameter and the external diameter of described quartz ampoule are equal
For square.
A kind of fluorescence detection device the most according to claim 1, it is characterised in that: described suction pumps is peristaltic pump or sky
Gas jet pump.
7. according to a kind of fluorescence detection device described in claims 1 or 2, it is characterised in that: described excitation light path, first
Collect light path and second and collect light path by convergent lens and optical filter composition.
8. the detection method of fluorescence detection device described in claim 1, it is characterised in that:
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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, makes
Magnetic microsphere quantity of collecting in quartz ampoule is continuously increased, and forms Rendezvous Point;
3) after magnetic microsphere collects a period of time, starting exciting light sources, light converges in focusing on quartz ampoule by excitation light path
On the magnetic microsphere of collection point, first collects light path collects the fluorescence signal of the fluorescent marker that magnetic microsphere combines, and by the first light
Electric transducer is converted to the signal of telecommunication, carries out fluorescent marker content detection, closes constant-current source;
Repeat the above steps 1)-3), obtain the testing result collecting magnetic microsphere in extraction liquid in batches, and add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
The detection method of fluorescence detection device the most according to claim 8, it is characterised in that: treat that magnetic microsphere collects 0.1 second extremely
After 10 seconds, start exciting light sources.
The detection method of fluorescence detection device the most according to claim 9, it is characterised in that: the magnetic force of described electric magnet is permanent
It is set to 1~2000mg.
The detection method of 11. fluorescence detection devices according to claim 9, it is characterised in that: described liquid is in quartz ampoule
Constant flow rate be 0.1~100 cels.
The detection method of fluorescence detection device described in 12. claim 2, it is characterised in that:
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 reaction
Liquid in pond flows in quartz ampoule;Containing the magnetic combining detected material and fluorescent marker in described sample solution to be detected
Microsphere;
2) opening constant-current source, electric magnet produces magnetic field, and the magnetic microsphere flow through in quartz ampoule liquid is resided in electric magnet front end, 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, the second collection
Light path collects the light that at Rendezvous Point, magnetic microsphere produces under excitation light irradiation, and is converted into the signal of telecommunication, thus monitors Rendezvous Point
The gathering quantity of place's magnetic microsphere;
4) when magnetic microsphere gathering quantity reaches certain setting value, collect light path by first and collect the fluorescent labeling that magnetic microsphere combines
The fluorescence signal of thing, 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 add up, complete
The content detection of fluorescent marker in paired whole reaction tank.
13. according to the detection method of fluorescence detection device described in claim 12, it is characterised in that: when magnetic microsphere gathers at Rendezvous Point
When the quantity of collection reaches the setting value between 1~100000, start exciting light sources.
14. according to the detection method of fluorescence detecting system described in claim 12, it is characterised in that: the inside of described magnetic microsphere adds
Having entered the fluorescent material different from fluorescent marker wavelength, the second described collection light path is the collection light that magnetic microsphere carries fluorescence
Road.
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