CN103713126B - A kind of fluorescence analysis method and device - Google Patents
A kind of fluorescence analysis method and device Download PDFInfo
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- CN103713126B CN103713126B CN201210378333.9A CN201210378333A CN103713126B CN 103713126 B CN103713126 B CN 103713126B CN 201210378333 A CN201210378333 A CN 201210378333A CN 103713126 B CN103713126 B CN 103713126B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The present invention relates to a kind of fluorescence analysis method and device, specifically, the invention discloses a kind of leak detection apparatus for the determinand being used in quantitatively detection sample, described device includes the absorption pad that the perforated membrane of (1) comprising detection zone and (2) are located at below perforated membrane.The invention also discloses a kind of detection method and a kind of detection means based on the detection method of the quantitative detection determinand based on the leak detection apparatus.
Description
Technical field
The invention belongs to vitro detection technical field, more particularly to a kind of analysis method based on measurement fluorescence intensity and
Device.
Background technology
In field of immunodetection, it is often necessary to which all kinds of antigens or antibody are qualitatively or quantitatively detected.Prior art
In, panimmunity response analysis method is derived based on " Competitive assays and double antibodies sandwich ", such as：It is radioimmunology, enzyme-linked
Immunization, chemoluminescence method, time-resolved fluorescence method and fluorescent immune method etc., available for pathogenic microorganism is determined, to human body
Specific proteins quantitatively detects that, so as to carry out auxiliary diagnosis or monitoring etc. to disease, purposes is widely.This kind of immune response
Analysis method, generally will capture antibody be fixed on solid phase carrier, then with antigen (target protein) react, after washing again with mark
Antibody response, washing finally detects radioactive intensity, solution absorbance or optical signal etc., so as to report target in detection sample
The concentration of albumen.The automation of the above method eliminates the loaded down with trivial details of hand washing, but just because of automation so that equipment instrument is huge
It is big expensive, it is general only to be used in large-scale experiment room.
Dot immuno gold filtration assay technology (dot immunogold filtration assay, DIGFA) is Spielberg
Deng initially in 1989 by detecting AntiHIV1 RT activity foundation, with the high speed development of colloidal gold technique, material technology and computer,
Dot immuno gold filtration assay technology is complete day by day, as one kind in the collaurum quick diagnosis technology using film as carrier, with inspection
Survey is quick, convenient, do not need the advantages of special installation, result judge directly perceived.It is non-special because this method need to only be visually observed
Industry personage is also operable, makes the inspection related to the local development of the remote large-scale experiment room such as scene of emergency treatment, basic hospital, patient bedside
Survey is possibly realized, so application is quite extensive.Such as：A variety of parasitic disease marks, cardiac markers, biography are applied to
Infectious diseases mark, poisonous substance, detection of sexually transmitted disease and other biochemical indicators etc., in other field such as food hygiene, ring
The fields such as border protection all have been reported that.
DIGFA methods already exceed the development of 20 years, but its basic operation principle does not change, which dictates that to being at present
Only, it is served only for qualitative or half-quantitative detection and sensitivity also quantitative immunoassay method not as the aforementioned, further application
Encounter bottleneck.Therefore, this area is badly in need of changing existing DIGFA methods, is assigning its high sensitivity and quantitative standard
While true new advantage, the features such as its is quick, easy, with low cost is kept, so as to further expand its application field.
The content of the invention
The invention provides a kind of leak detection apparatus, for quantitatively detecting the determinand in sample.
The invention provides a kind of detection method for quantitatively detecting determinand, methods described sensitivity is high, quantitative accurate.
Present invention also offers a kind of detection means for quantitatively detecting determinand.
There is provided a kind of leak detection apparatus in first aspect present invention, the leak detection apparatus includes：
(1) perforated membrane of detection zone is included, wherein, described detection zone includes the immobilization marked by fluorescent material and detected
Agent, and
(a1) the immobilization detection agent can be combined with the determinand in sample, so that it is multiple to form " detection agent-determinand "
Compound；Wherein described determinand can also be combined with bonding agent, so that after detection zone contacts sample and bonding agent containing determinand,
In detection zone formation " detection agent-determinand-bonding agent " compound；
Or
(b1) the immobilization detection agent can be combined with bonding agent, so as to form " detection agent-bonding agent " compound；Its
Described in bonding agent can also be combined with determinand so that after detection zone contacts mixed solution containing determinand and bonding agent,
Detection zone formation " detection agent-bonding agent " compound；
Wherein, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence of the fluorescent material strong
Degree；
(2) it is located at the absorption pad below perforated membrane, the absorption pad has absorbability so that add to the sample of detection zone
With penetration by liquid perforated membrane and absorbed by absorption pad.
In another preference, described detection zone includes one or more immobilization detection agents；It is preferred that described
Bonding agent quantity is one or more, so as to simultaneously for detecting one or more determinands.
In another preference, described leak detection apparatus is quantitative testing device.
In another preference, described determinand is antigen or antibody.
In another preference, described determinand is antigen, then described bonding agent and detection agent be can be in combination with
In the antibody of the antigen；Or described determinand is antibody, then described detection agent and bonding agent be can in combination with
The antibody (antiantibody) of the antigen of the antibody or the antibody.
In another preference, described determinand is antigen, then described bonding agent can be combined in the antigen
Antibody, the detection agent is the equivalent of the antigen；Or described determinand is antibody, then described bonding agent is to tie
The antibody (antiantibody) of antigen or the antibody together in the antibody, the detection agent is the equivalent of the antibody.
There is provided a kind of leak detection apparatus in second aspect of the present invention, the leak detection apparatus includes：
(1) perforated membrane of detection zone is included, wherein, described detection zone includes the first fixed detection agent, wherein, it is described
First detection agent is marked by fluorescent material, and compound detection agent can be formed with flowable second detection agent, and
(a2) the compound detection agent can be combined with the determinand in sample, so as to form " compound detection agent-determinand "
Compound；The determinand can be combined with bonding agent, so that after detection zone contacts sample and bonding agent containing determinand, in inspection
Survey area and form " compound detection agent-determinand-bonding agent " compound；
Or
(b2) the compound detection agent can be combined with bonding agent, so as to form " compound detection agent-bonding agent " compound；
Wherein described bonding agent can also be combined with determinand, so that after detection zone contacts the mixed solution containing determinand and bonding agent,
In detection zone formation " compound detection agent-bonding agent " compound；
Wherein, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence of the fluorescent material strong
Degree；
(2) it is located at the absorption pad below perforated membrane, the absorption pad has absorbability so that add to the sample of detection zone
With penetration by liquid perforated membrane and absorbed by absorption pad.
In another preference, the quantity of the second described detection agent is one or more；It is preferred that described bonding agent
Quantity is one or more, so as to simultaneously for detecting one or more determinands.
In another preference, the compound detection agent is " first the-the second detection agent of detection agent " compound.
In another preference, " compound detection agent-determinand " compound is " the first detection agent-the second is detected
Agent-determinand " compound.
In another preference, " compound detection agent-determinand-bonding agent " compound for " the first detection agent-
Second detection agent-determinand-bonding agent " compound.
In another preference, " compound detection agent-bonding agent " compound is " the first detection agent-the second is detected
Agent-bonding agent " compound.
In another preference, described leak detection apparatus is quantitative testing device.
In another preference, the determinand is antigen or antibody.
In another preference, described determinand is antigen, then described bonding agent and the second detection agent be can be simultaneously
It is incorporated into the antibody of the antigen；Or described determinand is antibody, then the second described detection agent and bonding agent be can be same
When be incorporated into the antigen of the antibody or the antibody (antiantibody) of the antibody.
In another preference, described determinand is antigen, then described bonding agent can be combined in the antigen
Antibody, second detection agent is the equivalent of the antigen；Or described determinand is antibody, then described bonding agent is
The antigen of the antibody or the antibody (antiantibody) of the antibody are can be combined in, second detection agent is being equal for the antibody
Thing.
In another preference, first detection agent is the streptavidin of mark fluorescent material（SA）, then described second
Detection agent is by biotin（biotin）Mark.
In another preference, the leak detection apparatus also includes：
There is a bottom device below absorption pad；
Existing above perforated membrane has the perforate for being available for adding sample, and institute on a cap device, the cap device
Perforated membrane is stated below the perforate.
In another preference, the fluorescent material excite or the absorption spectrum of emission spectrum and the extinction material is complete
Portion partly overlaps.
In another preference, described determinand includes：Albumen, nucleic acid or micromolecular compound.
In another preference, described determinand is liquid phase (solution), suspension or solid phase.
In another preference, described fluorescent material is selected from the group：Fluorescein, Fluoresceincarboxylic acid, 2- methoxyl group fluorescence
Element, 4,5- dimethoxyfluoresceins, rhodamine, phycoerythrin, quantum dot or rare earth element ion or its chelate.
In another preference, described extinction material is selected from the group：Collaurum, nanometer gold bar, Nano Silver rod or its group
Close.
In another preference, described extinction material is nanometer gold bar.
In another preference, the aspect ratio of described nanometer gold bar is 1.5-10, preferably 1.5-5.
In another preference, the length of described nanometer gold bar is 10-200nm, preferably 20-100nm.
In another preference, described collaurum is the colloid gold particle that average grain diameter is 10-70nm.
In another preference, the check plot different from detection zone is also included on the perforated membrane, the check plot is contained
The control agent of immobilization, wherein, the control agent is used to specifically bind the bonding agent marked by extinction material.
In another preference, the check plot is used for the validity for showing the testing result of determinand.
There is provided a kind of fluorescence analysis method for quantitatively detecting determinand, including step in third aspect present invention：
(1) include：
(a1) determinand sample and bonding agent make an addition to the detection zone of leak detection apparatus respectively, or by determinand sample
Described detection zone is made an addition to the mixture of bonding agent,
Wherein, described detection zone includes the immobilization detection agent marked by fluorescent material, and the immobilization is detected
Agent can be combined with the determinand in sample, so as to form " detection agent-determinand " compound；Wherein described determinand can be with knot
Mixture is combined, so that after detection zone contacts sample and bonding agent containing determinand, in detection zone formation " detection agent-to be measured
Thing-bonding agent " compound；
Or
(a2) the second detection agent, determinand sample and bonding agent are made an addition to the detection zone of leak detection apparatus respectively, or
The mixture of second detection agent, determinand sample and bonding agent is made an addition to described detection zone,
Wherein, described detection zone includes the first fixed detection agent, wherein, first detection agent is by fluorescent material mark
Note, can form compound detection agent with flowable second detection agent, and the compound detection agent can be with the determinand in sample
With reference to so as to form " compound detection agent-determinand " compound；The determinand can be combined with bonding agent, so as to work as detection zone
Contact after the second detection agent, the sample containing determinand and bonding agent, in detection zone formation " compound detection agent-determinand-combination
Agent " compound；
Also, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence of the fluorescent material strong
Degree；
(2)：The fluorescence intensity F of the detection zone is detected, so as to measure presence or absence or the quantity of determinand.
There is provided a kind of fluorescence analysis method for quantitatively detecting determinand, including step in fourth aspect present invention：
(1) include：
(b1) determinand sample and bonding agent make an addition to the detection zone of leak detection apparatus successively, or by determinand sample
The mixture of product and bonding agent makes an addition to described detection zone,
Wherein, described detection zone includes the immobilization detection agent marked by fluorescent material, and the immobilization is detected
Agent can be combined with bonding agent, so as to form " detection agent-bonding agent " compound；Wherein described bonding agent can also be with determinand knot
Close, so that after detection zone contact measured thing sample and bonding agent, in detection zone formation " detection agent-bonding agent " compound；
Or
(b2) determinand sample, the second detection agent and bonding agent are made an addition to the detection zone of leak detection apparatus successively,
Or
One is provided first by determinand sample and the first mixture of the second detection agent, then mixes bonding agent and first
Thing makes an addition to described detection zone successively, or the second mixture that bonding agent and the first mixture are formed make an addition to it is described
Detection zone,
Or
The 3rd mixture of a determinand sample and bonding agent is provided first, then by the second detection agent and the 3rd mixture
Described detection zone is made an addition to successively, or the 4th mixture that the second detection agent and the 3rd mixture are formed makes an addition to institute
The detection zone stated,
Wherein, described detection zone includes the first fixed detection agent, wherein, first detection agent is by fluorescent material mark
Note, can form compound detection agent with flowable second detection agent, and the compound detection agent can be combined with bonding agent, so that
Form " compound detection agent-bonding agent " compound；Wherein described bonding agent can be combined with determinand, so that when detection zone contact
After determinand sample, the second detection agent and bonding agent, in detection zone formation " compound detection agent-bonding agent " compound；
Also, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence of the fluorescent material strong
Degree；
(2) the fluorescence intensity F of the detection zone is detected, so as to measure presence or absence or the quantity of determinand.
In another preference, the step (2) includes：By the fluorescence intensity F and standard curve of detection zone or with not
The initial fluorescent intensity F0 of sample-adding is compared, so that it is determined that presence or absence or the quantity of determinand.
In another preference, described determinand is liquid phase (solution), suspension or solid phase.
In another preference, when determinand is solid phase, step (1) also includes adding solvent (such as water or buffer solution)
In another preference, described method also includes being measured with the determinand standard items of concentration known, so that
The step of making standard curve.
There is provided a kind of fluorescence detection device for quantitatively detecting determinand, described device in fifth aspect present invention
Including：
(a) leak detection apparatus described in a first aspect or a second aspect of the present invention；With
(b) one is used for the detector of fluorescence intensity.
In another preference, described device also includes light source and computer.
The light source is irradiated at detection zone, and the fluorescence inspired enters detector, by computer carry out data processing and
Analysis.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Figure 1A is leak detection apparatus schematic diagram.
Figure 1B is leak detection apparatus schematic diagram.
Fig. 2 is detection means schematic diagram.
Fig. 3 is the AFP standard series concentration (lgC) and corresponding fluorescence intensity (lgF) canonical plotting of embodiment 1.
Fig. 4 is the AFP standard series concentration (lgC) and corresponding fluorescence intensity (lgF) canonical plotting of embodiment 2.
Fig. 5 is the CRP standard series concentration (C) and corresponding fluorescence intensity (F) canonical plotting of embodiment 3.
Embodiment
The present inventor's in-depth study by long-term, it was found that a kind of detection method based on principle of fluorescent quenching, institute
The detection agent marked by fluorescent material that method uses immobilization is stated, not only can judge to be by estimating the color of detection zone
It is no containing determinand, can also quantitatively detect determinand by way of fluorescence intensity at detection zone measuring.Methods described is not
Only have sensitivity high, quantitative accurate advantage, and operate still very easy, quick and with low cost.Methods described is
A kind of diafiltration test device provided based on the present invention, is realized to determinand by a kind of device containing light source and detector
Quantitative detection.On this basis, inventor completes the present invention.
Leak detection apparatus
The method and material for manufacturing leak detection apparatus of the present invention are more fully described now.It should be noted that seepage is examined
Surveying the specific configuration of device can change, and this depends on the specific test that intention is carried out with leak detection apparatus.In embodiment
Outside manufacture the variation method of leak detection apparatus, also fall among the scope of the invention.
Leak detection apparatus may include cap device 1, and the cap device has a perforate 11 for being used to be loaded, described
Perforate can be the various shapes such as circular, square.
Perforated membrane 2 (or microporous barrier) is located at below the perforate of cap device 1, and the perforated membrane includes：Detection zone and preferably
Be provided with check plot, and generally have appropriate space between check plot and detection zone.
Absorption pad 3 is located at below perforated membrane.
Also there is a bottom device 4 below absorption pad.
As shown in Figure 1B, perforated membrane 2, absorption pad 3, cap device 1 and bottom device 4 are assembled into diafiltration test dress respectively
Put, wherein, the detection agent marked by fluorescent material has been secured on perforated membrane.
The leak detection apparatus can be that diversified forms are present, such as plate, piece or box etc..By taking box as an example, the cap
Device is lid, and the bottom device is cassette bottom, and perforated membrane can be placed in lid perforate (such as 0.4~0.8cm circular hole)
Underface, absorption pad makes perforated membrane be adjacent to absorption pad at the underface of perforated membrane, tight closure lid, bottom, that is, is prepared into Figure 1A institutes
The leak detection apparatus shown.
The cap device and bottom device can be made of any stabilization, non-porous material.Because many determine is used
Water is as dispersive medium, therefore bottom device is preferably substantially water-impervious, and its intensity should be enough to support and stick at its
Leak detection apparatus.In a preference, backing sheets is made of polymer thing (such as plastics).
The absorption pad can be made with any can absorb as the material of sample and the liquid of buffer solution.Absorbent patch
Absorbability is sufficiently large, to absorb the liquid for being added to leak detection apparatus.Suitable for the example of the material of absorbent patch
Including cellulose and glass fibre.
The perforated membrane can be made of any material, as long as the material has enough porositys to allow on surface and interior
The capillarity of fluid occurs for portion.Perforated membrane should have enough porositys, so as to allow the particle for scribbling antibody or antigen to move
It is dynamic.Perforated membrane can be also soaked (for example, having for waterborne liquid hydrophilic by liquid used in the sample containing analyte to be detected
Property, there is hydrophobicity for organic solvent).For example, by described in United States Patent (USP) No.4,340,482 or No.4,618,533
Method (these methods are described is transformed into water-wetted surface by hydrophobic surface), thus it is possible to vary its hydrophobicity is so as to making it have parent
It is aqueous for use in waterborne liquid.Example of material available for manufacture perforated membrane includes：Polymer PET, cellulose, nitrocellulose,
Cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and polyether sulfone
(polyethersulfone).In a preference, the perforated membrane is nitrocellulose diaphragm
Detection method
Leak detection apparatus of the present invention can be used for a large amount of different seepage analysis methods, and these analysis methods are related to one kind
Or a variety of flowable bonding agents, a kind of immobilization detection agent and optionally it is related to one or more flowable second detection agents.
Described immobilization detection agent is marked by fluorescent material, and at least one bonding agent is marked by extinction material, when fluorescent material mark
Detection agent and extinction material mark bonding agent combine after, the compound containing extinction material and fluorescent material can be formed.Tool
Body, it is of the invention when the extinction material influences and (be partly quenched or be all quenched) fluorescence intensity of the fluorescent material
Detection method can determine the quantity of determinand by detecting the fluorescence intensity of fluorescent material.
As used herein, term " determinand ", " its equivalent " or " analyte " refer to stand-by leak detection apparatus detection with
And be optionally quantitatively determined, any component in sample, determinand or its equivalent or the example of analyte include：Egg
White matter, such as hormone or other secretory proteins, enzyme and cell surface protein；Glycoprotein；Peptide；Small molecule；Polysaccharide；Antibody (including
Monoclonal antibody or polyclonal antibody and its fragment)；Nucleic acid；Medicine (the Cardiac glycosides medicine such as including digoxin)；Toxin；Virus
Or virion；Cell wall composition；Or other have the compound of epitope.
Preferably, described determinand includes the specific albumen, the tumour mark such as tumor markers, Applications of Cardiac Markers
Will thing is selected from the group：Alpha-fetoprotein (AFP), C reactive protein (CRP), carcinomebryonic antigen (CEA), cancer antigen 125 (CA125), sugar
Antigen 1 9-9 (CA19-9), T-PSA (PSA), free prostate gland specificity antigen (f-PSA), neuron are special
Specific enolase (NSE), carbohydrate antigen (CA242), cancer antigen (CA15-3) or human chorionic gonadotrophin (β-HCG).
Bonding agent
Bonding agent used in the present invention can any can be incorporated into determinand or the material of its equivalent.Specifically,
For specific binding determinand or the material of its equivalent.
There is various types of molecule to can be used as analyte binding agent, including for example：Oligonucleotides, antibody, work
Journey albumen, peptide, the lysate of haptens or the heterogeneous mixture containing antigen (antigen has analyte binding site).
P.Holliger et al., Trends in Biotechnology 13:7-9(1995);S.M.Chamow et al., Trends in
Biotechnology 14:52-60(1996).If analyte to be detected is part, then can be used and be incorporated into the part
Acceptor, vice versa.
Detection agent
Detection agent used in the present invention can be it is any can be with the determinand in bonding agent and/or sample or its equivalent knot
The material of conjunction, including for example：Antibody, engineering protein, peptide, haptens or (antigen has analyte bound site containing antigen
Point) heterogeneous mixture lysate.
Preferably, combination that can be by antigen and antibody or oligonucleotides and the side of the single-stranded specific binding of complementary nucleic acid
Formula is combined the material containing fluorescence labeling with the material marked containing extinction material；Can also by biotin (biotin) and
The combination of streptavidin (Streptavidin, SA), so that by the material containing fluorescence labeling with containing extinction material mark
The material of note is combined.
Mark substance
Being connected in the detectable of bonding agent or detection agent includes a large amount of different materials, as long as label can be detected
Survey.The example of detectable includes (but being not limited to)：Particle, luminous marker；Colorimetric marker, fluorescent marker；
Chemical markers；Enzyme；Radioactively labelled substance；Or RF tag thing；Metallic colloid；And chemiluminescent labels.Conventional detection side
The example of method includes (but being not limited to)：Optical means, such as measurement light scattering, single reflection, photometer or photomultiplier；Radiation
Property (being measured with Geiger counter etc.)；Electric conductivity or dielectric (electric capacity)；Electrochemical process detects discharged electric active matter
Matter, such as indium, bismuth, gallium, tellurium ion are [as used Hayes et al. (Analytical Chem.66:Side described in 1860-1865 (1994)
Method], or ferrocyanide is [as used Roberts and Durst (Analytical Chem.67:482-491 (1995) is proposed
Method.Wherein by the way that detergent is added dropwise in detection zone, make to be wrapped in the ferrocyanide in liposome and be released, then electricity consumption
The ferrocyanide of chemical method detection release].As long as other conventional methods are suitable, it is possible to use.
In a preference, detectable is particle.Workable particle example includes (but being not limited to)：Glue
Body gold grain；Colloid sulfur granules；Colloid granules of selenium；Colloidal barium sulfate particle；Colloid iron sulphate particles；Metal iodate particle；
Silver halide particle；Silica dioxide granule；Colloid (hydration) metal oxide particle；Colloidal metal sulphides particle；Colloid selenizing
Lead particle；Colloid cadmium selenide particle；Colloidal metal phosphate particle；Colloidal metal ferrous acid salt particle；Scribble organic or inorganic layer
Any of the above-described kind of colloidal solid；Protein or peptide molecule；Liposome；Or organic polymer latex particle, such as polystyrene
Latex bead.
For the label of detection agent, the present invention is preferential from fluorescent marker (abbreviation fluorescent material), for bonding agent
Label, the present invention is preferential from extinction material label (abbreviation extinction material).When fluorescent marker and extinction label
After being combined by specific mode, when fluorescent material excite or emission spectrum and extinction material absorption spectrum partially or completely
When overlapping, extinction material can influence and (partly be quenched or be all quenched) fluorescence of fluorescent material, so that by detecting fluorescent material
Fluorescence intensity determine the quantity of determinand.
The selection of fluorescent material and extinction material
The fluorescent material and extinction material marked for the present invention should be used cooperatively, and basic principle is exciting for fluorescent material
Or emission spectrum is partially or completely overlapping with the absorption spectrum of extinction material.Optimal is completely overlapped, is so had higher
Detection sensitivity.
Representational fluorescent material includes (but being not limited to)：Fluorescein, Fluoresceincarboxylic acid, 2- methoxyl groups fluorescein, 4,
5- dimethoxyfluoresceins, rhodamine, phycoerythrin (Phycoerythrin, PE), quantum dot, rare earth element ion (such as Eu3+)
And its combination such as chelate, as long as selected fluorescent material excite or the absorption spectrum of emission spectrum and selected extinction material has weight
It is folded.
Representational extinction material includes (but being not limited to)：Collaurum, nanometer gold bar, Nano Silver rod etc. are combined, as long as
The absorption spectrum of selected extinction material and selected fluorescent material excite or emission spectrum is overlapping.
Colloid gold particle can be manufactured with any conventional method, for example, be summarized in G.Frens, 1973 Nature Physical
Science,241:Method in 20 (1973).Other method is described in United States Patent (USP) No.5,578,577,5,141,850,4,
775,636、4,853,335、4,859,612、5,079,172、5,202,267、5,514,602、5,616,467、5,681,
775。
As used herein, term " nanometer gold bar " refers to certain aspect ratio and horizontally and vertically in 5-200 nanometers of models
The gold grain enclosed.
A kind of particularly preferred extinction material is collaurum, and especially particle diameter is 20-40nm collaurum.
Preferably, those skilled in the art often include PE with fluorescence labeling material, PE absorption spectrum 550 ~ 650nm it
Between, maximum emission wavelength is 575nm, and the absorption spectrum of 30nm collaurums is in 300 ~ 800nm, and broad, maximum absorption band is
At 525 ~ 530nm, overlapped with PE emission spectrum, so selection 30nm collaurums are used as corresponding extinction material.
Principle of fluorescent quenching
After material containing fluorescence labeling is combined with the material marked containing extinction material, exciting or sending out when fluorescent material
Penetrate spectrum it is completely or partially overlapping with the absorption spectrum of the extinction material as fluorescence quenching when, because of Resonance energy transfer, inhale
Stimulative substance can produce quenching effect to the fluorescence of the fluorescent material.
In the present invention, the reason for causing fluorescent quenching includes two aspects：One is inside compounds extinction material to fluorescence
Material is quenched；Two be being mutually quenched between the compound being packed together, such as：The extinction material of compound first is quenched compound
The fluorescence of compound third is quenched in the fluorescence of thing second, the extinction material of compound second, and compound is quenched in the extinction material of compound third
The fluorescence of first, the rest may be inferred.
Operation principle
Illustrate the operation principle of detection method in conjunction with Figure 1A and Figure 1B and specific embodiment：
Method one,
(1.1) select a detection agent, the detection agent be to determinand specifically bind material (such as determinand is anti-
Original, then detection agent is an antibody of the antigen, is designated as Ab1), and fluorescent material mark, generation warp are carried out to the detection agent in advance
The detection agent (being designated as F-Ab1) of fluorescent material mark, is then fixed on perforated membrane.It is required that the fluorescent material excite with
The absorption spectrum of emission spectrum and extinction material (such as collaurum) is completely or partially overlapping.
(1.2) by well 11, the sample and bonding agent of determinand (such as antigen, be designated as Ag) will be contained (such as antigen
The gold labeling antibody of colloid gold label secondary antibody, referred to as antigen, is designated as Ab2-G) it is added on perforated membrane, the determinand in sample
Combined with the detection agent (F-Ab1) for antigen of bonding agent and fixed fluorescent material mark in this, form 3 yuan of compounds,
That is the fluorescence of the fluorescent material at detection zone is quenched in " F-Ab1-Ag-Ab2-G " sandwich complex, gold therein.
(1.3) gold labeling antibody of dissociating is captured by the control agent (antibody of such as anti-gold labeling antibody) of check plot, is presented red, is said
Bright detection is effective.
(1.4) determine detection zone at fluorescent material fluorescence intensity, fluorescence it is stronger explanation testing concentration it is lower, it is on the contrary then
It is higher；When nonantigenic in such as sample, it is impossible to form " sandwich complex ", then the fluorescence intensity at detection zone is that Raw fluorescence is strong
Degree.
Method two,
Also other can be fixed at detection zone can capture the fluorescence labeling material of " sandwich complex ".
(2.1) sample and two kinds of bonding agent (two kinds of antibody of such as antigen of determinand (such as antigen, be designated as Ag) will be contained：
The gold labeling antibody of the antibody of colloid gold label, also referred to as antigen, is designated as Ab2-G；And the antibody of biotin marks, it is designated as Ab1-
Biotin after) mixing, after the antibody of determinand, gold labeling antibody and biotin marks redissolves completely, such as G-Ab2-Ag-Ab1- is formed
Biotin 3 yuan of compounds.
(2.2) by well 11,3 yuan of compounds are added on perforated membrane, the energy of fixed fluorescence labeling in this
The detection agent (SA of such as fluorescence labeling, be designated as F-SA) combined with biotin captures above-mentioned 3 yuan of compounds, forms such as G-Ab2-
The fluorescence of the fluorescent material at detection zone is quenched in Ag-Ab1-biotin-SA-F 4 yuan of compounds, gold therein.
(2.3) gold labeling antibody of dissociating is captured by control agent (antibody of such as anti-gold labeling antibody), is presented red, is illustrated that detection has
Effect.
(2.4) determine detection zone at fluorescent material fluorescence intensity, fluorescence it is stronger explanation testing concentration it is lower, it is on the contrary then
It is higher；When nonantigenic in such as sample, it is impossible to form " sandwich complex ", then the fluorescence intensity at detection zone is that Raw fluorescence is strong
Degree.
The present disclosure additionally applies for A competitive inhibition method：
Method three,
(3.1) by equivalent (such as determinand is antigen, and the equivalent is its standard antigen, is designated as Ag0) mark of determinand
Remember fluorescence, generate the detection agent (being designated as F-Ag0) marked by fluorescent material, be then fixed on Figure 1B perforated membrane.
(3.2) first will (gold labeling antibody of such as antigen, be designated as containing sample, the bonding agent of determinand (such as antigen, be designated as Ag)
Ab1-G) mix, form the mixture of the compound containing Ag-Ab1-G, when Ab1-G content is more than the content of determinand,
Ab1-G can be remaining.
(3.3) by well 11, the mixture of step (3.2) is added dropwise on perforated membrane and (drips, has treated as being added dropwise 2~3
It is complete to penetrate into, cleaning solution 2~3 can be added dropwise by well 11 again and dripped, wait to fully penetrate into).The detection of fixed fluorescence labeling in this
Agent (such as F-Ag0) captures remaining Ab1-G in above-mentioned mixed liquor, forms the compound such as F-Ag0-Ab1-G, and gold therein is quenched
The fluorescence of fluorescent material at detection zone.
(3.4) fluorescence intensity of fluorescent material at detection zone is determined, fluorescence is stronger, and testing concentration is higher, conversely, then treating
The concentration for surveying thing is lower；If without determinand in sample, on perforated membrane at detection zone formed " F-Ag0-Ab1-G " at most, phase
Ying Di, fluorescence is most weak.
Method four,
(4.1) by equivalent (such as determinand is antigen, and the equivalent is its standard antigen, is designated as Ag0) mark of determinand
Remember biotin (be designated as biotin-Ag0), and the detection agent of fixation mark fluorescent material (is designated as F- on Figure 1B perforated membrane
SA)。
(4.2) will (gold labeling antibody of such as antigen, be designated as containing sample, the bonding agent of determinand (such as antigen, be designated as Ag)
Ab1-G) and be marked with biotin determinand equivalent mix, formed the compound containing biotin-Ag0-Ab1-G mixing
Thing.
In another preferences, first the sample containing determinand, bonding agent are mixed, the compound such as Ag-Ab1-G is formed,
When Ab1-G content is more than Ag content, Ab1-G can be remaining.It is multiple again with the determinand equivalent mixing for being marked with biotin
It is molten, form the mixture of the compound containing biotin-Ag0-Ab1-G.
In another preference, first the sample containing determinand is mixed with the determinand equivalent for being marked with biotin, then
With bonding agent mixing, redissolve, form the mixture of the compound containing biotin-Ag0-Ab1-G.
(4.3) by well 11, the mixture that step (4.2) is obtained be added dropwise on perforated membrane (as be added dropwise 2~3 drip,
Wait to fully penetrate into, cleaning solution 2~3 can be added dropwise by well 11 again and dripped, wait to fully penetrate into).Fixed fluorescence labeling in this
Detection agent (such as F-SA) captures the compound of biotin-Ag0-Ab1-G in above-mentioned mixed liquor, forms such as F-SA-biotin-Ag0-
The fluorescence of the fluorescent material at detection zone is quenched in Ab1-G compound, gold therein.
(4.4) fluorescence intensity of fluorescent material at detection zone is determined, fluorescence is stronger, and testing concentration is higher, conversely, then treating
The concentration for surveying thing is lower；If without determinand in sample, on perforated membrane at detection zone formed " F-Ag0-Ab1-G " at most, phase
Ying Di, fluorescence is most weak.
Detection means
Illustrate the detection means of the present invention in conjunction with Fig. 2：
As shown in Fig. 2 described device can include：Leak detection apparatus, detector, light source, optical fiber and computer.
The operation instruction of a detection method can also be included.Wherein the operation principle of leak detection apparatus is as described above, fluorescence intensity
Detection method can as described below (but being not limited only to this), any method available for fluorescence intensity is used equally for this hair
Bright detection means.
Exciting light is irradiated at the detection zone of perforated membrane by 1 branch of Y type optical fibers, and the fluorescence inspired passes through
Another the 1 of Y type optical fibers branches into detector, and data processing and analysis are carried out by computer.
In the present invention, light source is used for the light for providing a certain launch wavelength, so as to excite fluorescent material to send fluorescence.It is optional
The light source of suitable wavelength can be provided with any, including but not limited to：LED, xenon lamp, halogen tungsten lamp, laser etc..It is a kind of preferred
Light source is LASER Light Source, and LASER Light Source can be produced with the conventional method and apparatus (such as laser) in this area.Representational laser
Device includes (but being not limited to)：Semiconductor laser, He-Ne laser, argon ion laser in addition to the optional laser of wavelength
Device, multiple-wavelength laser and dual laser etc..
The optical maser wavelength that laser is produced is relevant with laser medium, and common optical maser wavelength see the table below：
| Laser species | Wavelength (nanometer) |
| Argon fluorine laser (ultraviolet light) | 193 |
| Krypton fluorine laser (ultraviolet light) | 248 |
| Xenon chlorine laser (ultraviolet light) | 308 |
| N_2 laser (ultraviolet light) | 337 |
| Argon laser (blue light) | 488 |
| Argon laser (green glow) | 514 |
| He-Ne Lasers (green glow) | 543 |
| He-Ne Lasers (feux rouges) | 633 |
| Rhodamine 6G dyestuff (tunable optical) | 570-650 |
| Ruby (CrAlO3) (feux rouges) | 694 |
| Neodymium-yttrium-aluminium-garnet (near infrared light) | 1064 |
Detector in the present invention can be (but are not limited to) photomultiplier, CCD, avalanche diode or photocell etc..
Standard curve
In the present invention, the quantity of the determinand can be determined directly by determining the fluorescence intensity at detection zone.
, can be by being compared with standard curve, so as to obtain quantitative result in preference.
Standard curve can be obtained with following methods：The determinand sample of known various concentrations (C) is passed through into above-mentioned detection
After method, its fluorescence intensity (F) at detection zone is measured respectively, by each concentration or its logarithm value (log C) and corresponding glimmering
Luminous intensity or the mapping of its logarithm value (log F), obtain standard curve.
Main advantages of the present invention have：
(1) the invention provides a kind of leak detection apparatus.
(2) the invention provides a kind of detection method of above-mentioned leak detection apparatus, it is former that methods described is based on fluorescent quenching
Reason, by measuring the fluorescence intensity of fluorescent material so as to determine the quantity of determinand, methods described is quick, easy, with low cost,
It is quantitative accurate and sensitivity is high.
(3) present invention also offers a kind of detection means, described device is based on above-mentioned detection method, can be widely used for quantitative
Detection field.
With reference to specific implementation, the present invention is expanded on further.It should be understood that these embodiments be merely to illustrate the present invention and
It is not used in limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition,
Such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and
Number is calculated by weight.
Reagent and equipment：
For AFP paired monoclonal antibody G4/C2, commercially available product；
For C reactive protein (CRP) monoclonal antibody, commercially available product；
CRP antigens, commercially available product；
AFP antigens are purchased from Biodesign；
Biotin is purchased from Roche Holding Ag；
30nm collaurum, commercially available product；
SA-PE is purchased from Invitrogen companies；
Detector：USB4000-FL (Ocean Optics of the U.S.)
Light source：532nm LASER Light Sources.
Embodiment 1：The detection (being fixed on perforated membrane after PE labelled antibodies) of Serum Alpha Fetoprotein (AFP)
1st, the preparation of gold labeling antibody
1.1 collaurums-antibody preserves liquid
| Sodium tetraborate | 0.1g |
| BSA (BSA) | 0.25g |
| NaN3 | 0.025g |
With 6N HCl tune pH to 7.4 after being dissolved in water, moisturizing to 250ml, after 0.45 μm of membrane filtration, 4~8 DEG C of guarantors
Deposit.
1.2 working solution
| Na2HPO4·12H2O | 6.1g |
| NaCl | 8.5g |
| PVP40 | 5.0g |
| Boric acid | 2.1g |
| PEG | 1.0g |
| 10%BSA | 50ml |
| NaN3 | 0.2g |
With 6N HCl tune pH to 7.0~7.5 moisturizings to 1000ml, after 0.45 μm of membrane filtration, 4~8 after being dissolved in water
DEG C preserve.
The preparation of 1.3 gold labeling antibodies (Gold-C2)
1.3.1 the golden liquid 20ml of 20~30nm particle colloids is taken, purified C2 antibody is slowly added under magnetic stirring
1.0ml (0.6mg/ml), is stirred at room temperature 30min；
1.3.2 the BSA 0.8ml (final concentration 0.4%) for plus 10%, are stirred at room temperature 5min；
1.3.3 the PEG 0.4ml (final concentration 0.2%) for plus 10%, are stirred at room temperature 5min；
1.3.4 12000~1500r/min centrifuges 60~40min, sucts clear liquid, and precipitation is dissolved in 0.5ml and preserved in liquid, gold
The optical density (O.D) of labeling antibody is about 60O.D, and wherein the concentration of C2 antibody is 1mg/ml, puts 4 DEG C and saves backup；
2nd, PE marks G4 antibody
2.1 G4 are pre-processed
2.2 biotin mark G4
The G4 of above-mentioned pretreatment is taken, 25 μ L 1mg/ml NHSS-Biotin DMSO solutions are added, mixed, 4 DEG C of refrigerators are kept away
Light reaction 2 hours, dialysed overnight is standby.
2.3 biotin-G4 mark PE
Take and marked biotin-G4, add in pH7.4 phosphate buffers, the final concentration of 5 μ g/ml of G4, add simultaneously
Final concentration of 60 μ g/ml, G4-PE (i.e. G4-biotin-SA-PE) cumulative volume of SA-PE, PE be 5ml, 4 DEG C be kept in dark place it is standby.
3rd, PE-G4 points film
The μ l points of PE-G4 1 are taken to assemble percolating device behind microporous barrier center, lucifuge drying at room temperature.
4th, prepared by standard curve
4.1 prepare AFP series standard solution with working solution, are shown in Table 1；
4.2 take 5 percolating devices, horizontal positioned, respectively at aperture plus 50 μ l standard liquids of 5 kinds of concentration, after being percolated,
At aperture be added dropwise 2 drip pH7.4 PBSs, treat diafiltration at aperture plus 1O.D the μ l of gold labeling antibody solution 50, diafiltration
Add 50 μ l PBS again afterwards；
After 4.3 diafiltrations, the fluorescence intensity at each percolating device spot is determined, data are shown in Table 1, and the mark shown in Fig. 3 is made
Directrix curve.
Table 1
| Standard series (ng/ml) | Fluorescence intensity F |
| 0 | 27000 |
| 9.87 | 22100 |
| 31.25 | 18100 |
| 96.54 | 13420 |
| 308.62 | 11200 |
5th, pattern detection
With serum sample alternate standard serial solutions, 4.2,4.3 steps are repeated, fluorescence intensity level is substituted into standard curve,
The AFP values for measuring 6 samples are shown in Table 2.
The inventive method testing result of table 2 and Roche Electrochemiluminescince testing result
| Catalogue number(Cat.No.) | The inventive method (ng/ml) | Roche Electrochemiluminescince (ng/ml) |
| 1 | 7.2 | 10.17 |
| 2 | 9.9 | 13.00 |
| 3 | 20.6 | 18.40 |
| 4 | 30.8 | 45.80 |
| 5 | 152.3 | 253.34 |
| 6 | 285.6 | 346.70 |
The correlation of the inventive method testing result and Roche Electrochemiluminescince testing result is good, R2=0.9666。
Embodiment 2：The detection (being fixed on microporous barrier after PE marks SA) of Serum Alpha Fetoprotein (AFP)
1st, Gold-C2 preparation
With the Gold-C2 of embodiment 1.
2nd, biotin marks G4
2.1 G4 antibody and processing
2.2 biotin mark G4
The μ L of G4 antibody 10 of above-mentioned pretreatment are taken, 25 μ L 1mg/ml NHSS-Biotin DMSO solutions are added, mixed, 4
DEG C refrigerator lucifuge is reacted 2 hours, and dialysed overnight is standby, final concentration of 550 μ g/ml.
3rd, Gold-C2 and biotin-G4 mixing
5 μ l Gold-C2, biotin-G4 are respectively taken, antibody mixed liquor is mixed to obtain；
4th, SA-PE is fixed on microporous barrier
SA-PE (PE concentration is 55 μ g/ml) 1 μ l points are taken to be assembled behind Figure 1B microporous barrier center, lucifuge drying at room temperature
Percolating device.
5th, prepared by standard curve
5.1 prepare AFP series standard solution with working solution, are shown in Table 3；
Add 2 μ l antibody mixed liquors in 50 μ l standard liquids of 5.2 each concentration points, 5 diafiltration dresses are separately added into after mixing
In the aperture put, after being percolated, 2 PBSs for dripping pH7.4 are added dropwise at aperture；
5.3 after being percolated, and determines the fluorescence intensity at each percolating device spot, data are shown in Table 3, and are made shown in Fig. 4
Standard curve.
Table 3
| AFP standard series (ng/ml) | Fluorescence intensity F |
| 0 | 19600 |
| 9.87 | 17100 |
| 31.25 | 12800 |
| 96.54 | 9800 |
| 308.62 | 7200 |
6th, sample detection
With serum sample alternate standard serial solutions, 5.2,5.3 steps are repeated, fluorescence intensity level is substituted into standard curve,
The AFP values for measuring 6 samples are shown in Table 4.
The inventive method testing result of table 4 and Roche Electrochemiluminescince testing result
| Catalogue number(Cat.No.) | The inventive method (ng/ml) | Roche Electrochemiluminescince (ng/ml) |
| 1 | 10.2 | 10.17 |
| 2 | 16.2 | 13.00 |
| 3 | 18.6 | 18.40 |
| 4 | 40.8 | 45.80 |
| 5 | 112.3 | 253.34 |
| 6 | 235.6 | 346.70 |
The correlation of the inventive method testing result and Roche Electrochemiluminescince testing result is good, R2=0.935。
Embodiment 3：A competitive inhibition method detection C reactive protein (CRP)
1st, CRP monoclonal antibodies standard gold (Gold-CRP)
The step 1 of process be the same as Example 1, the difference is that antibody uses collaurum O.D values after CRP monoclonal antibodies, mark to be 75,
CRP monoclonal antibodies concentration is 1mg/ml.
2nd, CRP antigenic marks biotin (biotin-CRP)
The step 2 of process be the same as Example 2, the difference is that replacing CRP antigen concentrations after G4 antibody, mark with CRP antigens
2mg/ml。
3rd, SA-PE is fixed on microporous barrier
SA-PE (PE concentration is 55 μ g/ml) 1 μ l points are taken to be assembled behind Figure 1B microporous barrier center, lucifuge drying at room temperature
Percolating device.
4th, prepared by standard curve
4.1 prepare 0,1,5,10 μ g/ml CRP series standard solution with working solution；
4.2 take the μ l of standard liquid 50 of 4 concentration points respectively, and the Gold-CRP with 1 μ l is mixed respectively, adds 2 μ l's
In the aperture that 5 percolating devices are separately added into after biotin-CRP, mixing, after being percolated, it is added dropwise 2 drop pH7.4's at aperture
PBS；
4.3 after being percolated, and determines the fluorescence intensity at each percolating device spot, data are shown in Table 5, and are made shown in Fig. 5
Standard curve.
Table 5
| Standard series (μ g/ml) | Fluorescence intensity F |
| 0 | 4650 |
| 1 | 6200 |
| 5 | 12000 |
| 10 | 18000 |
5th, sample detection
With serum sample alternate standard serial solution, 4.2,4.3 steps are repeated, fluorescence intensity level is substituted into standard curve surveys
The CRP values for obtaining 6 samples are shown in Table 6.
The inventive method testing result of table 6 and ELISA method testing result
| Catalogue number(Cat.No.) | The inventive method (μ g/ml) | ELISA (μ g/ml) |
| 1 | 0.63 | 1.17 |
| 2 | 2.2 | 2.9 |
| 3 | 1.88 | 2.40 |
| 4 | 4.8 | 3.80 |
| 5 | 7.3 | 6.34 |
| 6 | 8.6 | 8.70 |
The correlation of the inventive method testing result and ELISA method testing result is good, R2=0.953。
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (20)
1. a kind of leak detection apparatus, it is characterised in that the leak detection apparatus includes：
(1) perforated membrane of detection zone is included, wherein, described detection zone includes the first fixed detection agent, wherein, described first
Detection agent is marked by fluorescent material, can form compound detection agent with flowable second detection agent,
And the compound detection agent can be combined with the determinand in sample, it is combined so as to form " compound detection agent-determinand "
Thing；The determinand can be combined with bonding agent, so that after detection zone contacts sample and bonding agent containing determinand, in detection zone
Form " compound detection agent-determinand-bonding agent " compound；
Wherein, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) it is located at the absorption pad below perforated membrane, the absorption pad has absorbability so that add to the sample and liquid of detection zone
Body is absorbed through perforated membrane and by absorption pad；
Also, described leak detection apparatus is quantitative testing device,
And first detection agent is the streptavidin of mark fluorescent material, then second detection agent is by biotin labeling；
Also, described fluorescent material is phycoerythrin, and extinction material is the collaurum that particle diameter is 20-40nm.
2. leak detection apparatus as claimed in claim 1, it is characterised in that the quantity of the second described detection agent to be a kind of or
It is a variety of；Described bonding agent quantity is one or more, so as to simultaneously for detecting one or more determinands.
3. leak detection apparatus as claimed in claim 1, it is characterised in that described determinand is antigen or antibody.
4. leak detection apparatus as claimed in claim 1, it is characterised in that the compound detection agent for " the first detection agent-
Second detection agent " compound.
5. leak detection apparatus as claimed in claim 1, it is characterised in that " compound detection agent-determinand " compound
For " first the-the second detection agent of detection agent-determinand " compound.
6. leak detection apparatus as claimed in claim 1, it is characterised in that " the compound detection agent-determinand-combination
Agent " compound is " first the-the second detection agent of detection agent-determinand-bonding agent " compound.
7. a kind of leak detection apparatus, it is characterised in that the leak detection apparatus includes：
(1) perforated membrane of detection zone is included, wherein, described detection zone includes the first fixed detection agent, wherein, described first
Detection agent is marked by fluorescent material, can form compound detection agent with flowable second detection agent,
And the compound detection agent can be combined with bonding agent, so as to form " compound detection agent-bonding agent " compound；Wherein
The bonding agent can also be combined with determinand, so that after detection zone contacts the mixed solution containing determinand and bonding agent, in inspection
Survey area and form " compound detection agent-bonding agent " compound；
Wherein, the bonding agent is marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) it is located at the absorption pad below perforated membrane, the absorption pad has absorbability so that add to the sample and liquid of detection zone
Body is absorbed through perforated membrane and by absorption pad；
Also, described leak detection apparatus is quantitative testing device,
And first detection agent be mark fluorescent material streptavidin, and second detection agent is by biotin labeling, and institute
The fluorescent material stated is phycoerythrin, and extinction material is the collaurum that particle diameter is 20-40nm.
8. leak detection apparatus as claimed in claim 7, it is characterised in that the quantity of the second described detection agent to be a kind of or
It is a variety of；Described bonding agent quantity is one or more, so as to simultaneously for detecting one or more determinands.
9. leak detection apparatus as claimed in claim 7, it is characterised in that the compound detection agent for " the first detection agent-
Second detection agent " compound.
10. leak detection apparatus as claimed in claim 7, it is characterised in that " the compound detection agent-bonding agent " is combined
Thing is " first the-the second detection agent of detection agent-bonding agent " compound.
11. leak detection apparatus as claimed in claim 7, it is characterised in that the determinand is antigen or antibody.
12. the leak detection apparatus as described in claim 3 or 11, it is characterised in that described determinand is antigen, then described
Bonding agent and the second detection agent be can be in combination with the antibody of the antigen；Or described determinand is antibody, then institute
The second detection agent and bonding agent stated are can be in combination with the antigen in the antibody or the antibody of the antibody.
13. the leak detection apparatus as described in claim 3 or 11, it is characterised in that described determinand is antigen, then described
Bonding agent be the antibody that can be combined in the antigen, second detection agent is the equivalent of the antigen；Or it is described
Determinand is antibody, then described bonding agent is the antibody of the antigen or the antibody that can be combined in the antibody, described second
Detection agent is the equivalent of the antibody.
14. the leak detection apparatus as described in claim 1 or 7, it is characterised in that the leak detection apparatus also includes：
There is a bottom device below absorption pad；
Existing above perforated membrane has the perforate for being available for adding sample on a cap device, the cap device, and described many
Pore membrane is located at below the perforate.
15. the leak detection apparatus as described in claim 1 or 7, it is characterised in that the fluorescent material excites or launches light
Spectrum is completely or partially overlapping with the absorption spectrum of the extinction material.
16. the leak detection apparatus as described in claim 1 or 7, it is characterised in that described determinand includes：Albumen, nucleic acid
Or micromolecular compound.
17. the leak detection apparatus as described in claim 1 or 7, it is characterised in that described determinand is liquid phase, suspension
Or solid phase.
18. the leak detection apparatus as described in claim 1 or 7, it is characterised in that also include being different from inspection on the perforated membrane
The check plot in area is surveyed, the control agent of immobilization is contained in the check plot, wherein, the control agent is used to specifically bind by extinction
The bonding agent of mass signatures.
19. leak detection apparatus as claimed in claim 18, it is characterised in that the check plot is used for the inspection for showing determinand
Survey the validity of result.
20. a kind of fluorescence detection device for quantitatively detecting determinand, it is characterised in that described device includes：
(a) leak detection apparatus described in a claim 1 or 7；With
(b) one is used for the detector of fluorescence intensity；
Also, described device also includes：Light source, optical fiber and computer, and described optical fiber is fine for Y types light guide
Dimension, the exciting light that the light source is sent is irradiated at the detection zone of perforated membrane by 1 branch of Y type optical fibers, inspired
Fluorescence branch into detector by another the 1 of Y type optical fibers, data processing and analysis are carried out by computer.
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