CN103712963B - A kind of fluorescence analysis method and device - Google Patents
A kind of fluorescence analysis method and device Download PDFInfo
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- CN103712963B CN103712963B CN201210378343.2A CN201210378343A CN103712963B CN 103712963 B CN103712963 B CN 103712963B CN 201210378343 A CN201210378343 A CN 201210378343A CN 103712963 B CN103712963 B CN 103712963B
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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 being used to quantitatively detect the determinand in sample, described device includes the absorption pad that the perforated membrane that (1) includes detection zone is located at (2) below perforated membrane.The invention also discloses a kind of detection method of quantitative detection determinand based on the leak detection apparatus and a kind of detection means based on the detection method.
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, and so as to carry out auxiliary diagnosis or monitoring etc. to disease, purposes is very extensive.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, radioactive intensity, solution absorbance or optical signal etc. are finally detected, 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 being established in 1989 by detecting AntiHIV1 RT activity, 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 using film as one kind in the collaurum quick diagnosis technology of carrier, has inspection
Survey it is quick, convenient, do not need special installation, result to judge the advantages that directly perceived.It is non-special because this method need to only visually observe
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：It is applied to a variety of parasitic disease marks, cardiac markers, biography
The detection etc. of infectious diseases mark, poisonous substance, sexually transmitted disease and other biochemical indicators, 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 keeping it quick, easy, cost is cheap, 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 high sensitivity are quantitative accurate.
Present invention also offers a kind of detection means for quantitatively detecting determinand.
In first aspect present invention, there is provided a kind of leak detection apparatus, the leak detection apparatus include：
(a1) perforated membrane of detection zone is included, wherein, described detection zone includes immobilization capturing agent, and
The immobilization capturing agent can be combined with flowable fluorescent material and flowable first bonding agent simultaneously, so as to
Form " fluorescent material-bonding agent of capturing agent-the first " compound；First bonding agent can be combined with determinand, and described
Determinand can be combined with the second bonding agent, so as to form " the first bonding agent-bonding agent of determinand-the second " compound；
So as to after detection zone contacts described fluorescent material, the first bonding agent, determinand and the second bonding agent, detect
Area forms " fluorescent material-bonding agent of capturing agent-the first-bonding agent of determinand-the second " compound, wherein, described the
Two bonding agents are marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(a2) absorption pad below perforated membrane, the absorption pad have absorbability so that add to the sample of detection zone
Product and penetration by liquid perforated membrane are simultaneously absorbed by absorption pad.
In another preference, the immobilization capturing agent is streptavidin, and the fluorescent material is by biotin labeling, institute
The first bonding agent is stated by biotin labeling.
In another preference, the species or quantity of the first described bonding agent and the second bonding agent are one or more；
It is preferred that it can be used to detect one or more determinands simultaneously.
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 the first described bonding agent and the second bonding agent be can
In combination with the antibody of the antigen；Or described determinand is antibody, then the first described bonding agent and second combines
Agent is can be in combination with the antigen of the antibody or the antibody (antiantibody) of the antibody.
In second aspect of the present invention, there is provided a kind of leak detection apparatus, the leak detection apparatus include：
(b1) perforated membrane of detection zone is included, wherein, described detection zone includes immobilization capturing agent, and
The immobilization capturing agent can be combined with flowable fluorescent material and flowable determinand equivalent simultaneously, so as to
Form " fluorescent material-capturing agent-determinand equivalent " compound；The determinand equivalent can be combined with bonding agent, from
And " determinand equivalent-bonding agent " compound is formed, and the bonding agent can be combined with determinand；
So that after the described fluorescent material of detection zone contact, bonding agent, determinand and determinand equivalent, in detection zone
" fluorescent material-capturing agent-determinand equivalent-bonding agent " compound is formed, wherein, described bonding agent is by extinction thing
Matter marks, and the extinction material influences the fluorescence intensity of the fluorescent material；
(b2) absorption pad below perforated membrane, the absorption pad have absorbability so that add to the sample of detection zone
Product and penetration by liquid perforated membrane are simultaneously absorbed by absorption pad.
In another preference, the immobilization capturing agent is streptavidin, then the fluorescent material is by biotin labeling,
The determinand equivalent is by biotin labeling.
In another preference, the species or quantity of described bonding agent are one or more；It is preferred that it can be used for simultaneously
Detect one or more determinands.
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 can be combined in the antigen
Antibody；Or described determinand is antibody, then described bonding agent is the antigen that can be combined in the antibody or the antibody
Antibody (antiantibody).
In another preference, described leak detection apparatus also includes：
A bottom device below absorption pad be present；
A cap device above perforated membrane also be present, there is the perforate for being available for adding sample on the cap device, and
The perforated membrane is located at 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.
Also include the check plot different from detection zone in another preference, 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.
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, the fluorescent material is selected from the group：Fluorescein, Fluoresceincarboxylic acid, 2- methoxyl groups fluorescein,
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 third aspect present invention, there is provided a kind of fluorescence analysis method for quantitatively detecting determinand, including step：
(a1) the first bonding agent, determinand sample and the second bonding agent are made an addition to the detection of leak detection apparatus respectively
Area, or described detection zone will be made an addition to after the mixing of the first bonding agent, determinand sample and the second bonding agent,
And fluorescent material is made an addition to described detection zone；Wherein, described detection zone includes immobilization capturing agent, and
And the immobilization capturing agent can be combined with flowable fluorescent material and flowable first bonding agent simultaneously, so as to be formed
" fluorescent material-bonding agent of capturing agent-the first " compound；First bonding agent can be combined with determinand, and described to be measured
Thing can be combined with the second bonding agent, so as to form " the first bonding agent-bonding agent of determinand-the second " compound；
So as to after detection zone contacts described fluorescent material, the first bonding agent, determinand and the second bonding agent, detect
Area forms " fluorescent material-bonding agent of capturing agent-the first-bonding agent of determinand-the second " compound, wherein, described the
Two bonding agents are marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) the fluorescence intensity F of the detection zone is detected, so as to measure the presence or absence of determinand or quantity.
In fourth aspect present invention, there is provided a kind of fluorescence analysis method for quantitatively detecting determinand, including step：
(b1) determinand sample, determinand equivalent 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 determinand equivalent, then mixes bonding agent and first
Compound makes an addition to described detection zone successively, or the second mixture that bonding agent and the first mixture are formed makes an addition to institute
The detection zone stated,
Or
3rd mixture of one determinand sample and bonding agent is provided first, then mixed determinand equivalent and the 3rd
Thing makes an addition to described detection zone successively or the 4th mixture for being formed determinand equivalent and the 3rd mixture adds
In described detection zone,
And fluorescent material is made an addition to described detection zone；Wherein, described detection zone includes immobilization capturing agent, and
And the immobilization capturing agent can be combined with flowable fluorescent material and flowable determinand equivalent simultaneously, so as to be formed
" fluorescent material-capturing agent-determinand equivalent " compound；The determinand equivalent can be combined with bonding agent, so as to shape
Into " determinand equivalent-bonding agent " compound, and the bonding agent can be combined with determinand；
So that after detection zone contacts fluorescent material, determinand sample, determinand equivalent and bonding agent, in detection zone shape
Into " fluorescent material-capturing agent-determinand equivalent-bonding agent " compound, wherein, described bonding agent is by extinction material
Mark, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) the fluorescence intensity F of the detection zone is detected, so as to measure the presence or absence of determinand or quantity.
In another preference, described step (2) includes：By the fluorescence intensity F and standard curve of detection zone or with
The initial fluorescent intensity F0 not being loaded 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 as to
The step of making standard curve.
In fifth aspect present invention, there is provided a kind of fluorescence measuring device for quantitatively detecting determinand, described device
Including：
(a) first aspect present invention or the leak detection apparatus described in second aspect of the present invention；With
(b) detector for 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 form new or preferable 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 (F) canonical plotting of embodiment 1.
Fig. 4 is the CRP standard series concentration (C) and corresponding fluorescence intensity (F) canonical plotting of embodiment 2.
Embodiment
The present inventor is by long-term and in-depth study, it was found that a kind of detection method based on principle of fluorescent quenching, institute
State method to combine flowable fluorescent material and compound containing extinction material by Avidin, can not only pass through and estimate inspection
The color for surveying area judges whether containing determinand, can also quantify detection by way of fluorescence intensity at detection zone measuring
Determinand.Methods described not only has high sensitivity, it is quantitative accurate the advantages of, and operate it is still very easy, quick and into
This is cheap.Methods described be based on it is provided by the invention it is a kind of be percolated test device, by a kind of containing light source and detector
Device realizes the quantitative detection to determinand.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 intention with leak detection apparatus the specific test that carries out.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 easy to sample-adding, 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.
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 make perforated membrane be adjacent to absorption pad, that is, are prepared into Figure 1A institutes at the underface of perforated membrane, tight closure lid, bottom
The leak detection apparatus shown.
The cap device and bottom device can be made of any stabilization, non-porous material.Because many measure are 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 of any material that can be absorbed as 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 so as 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 describe 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 water-based 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 directed to use with
A kind of flowable fluorescent material, one or more bonding agents and a kind of immobilization capturing agent, wherein, at least one bonding agent quilt
Extinction material marks.After the bonding agent of the described fluorescent material of capturing agent capture and extinction material mark, it can be formed containing suction
The compound of stimulative substance and fluorescent material.Specifically, when extinction material influence (be partly quenched or be all quenched) is described glimmering
During the fluorescence intensity of stimulative substance, detection method of the invention can determine determinand by detecting the fluorescence intensity of fluorescent material
Quantity.
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 such as tumor markers, Applications of Cardiac Markers, the tumour mark
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 is incorporated into the part
Acceptor, vice versa.
Mark substance
For the label of bonding agent, the present invention preferentially selects extinction material label (abbreviation extinction material).Work as fluorescence
After material and extinction label are combined by specific mode, when fluorescent material excite or the suction of emission spectrum and extinction material
Receive spectra part or it is completely overlapped when, extinction material can influence the fluorescence of (be partly quenched or all be quenched) fluorescent material, so as to
The quantity of determinand is determined by detecting the fluorescence intensity of fluorescent material.
In a preference, detectable is particle.Workable particle example includes (but being not limited to)：Colloid
Gold grain；Colloid sulfur granules；Colloid granules of selenium；Colloidal barium sulfate particle；Colloid iron sulphate particles；Metal iodate particle；Halogen
Change Argent grain；Silica dioxide granule；Colloid (hydration) metal oxide particle；Colloidal metal sulphides particle；Colloid lead selenide
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 breast
Glue bead.
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. combine, 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, such as 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 is horizontally and vertically in 5-200 nanometer 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 575nm, and the absorption spectrum of 30nm collaurums, in 300 ~ 800nm, broad, maximum absorption band is
At 525 ~ 530nm, overlapped with PE emission spectrum, so selection 30nm collaurums are 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：First, inside compounds extinction material is to fluorescence
Material is quenched；Second, 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,
Also other can be fixed at detection zone can capture the fluorescence labeling material of " sandwich complex ".
(1.1) (it is designated as with an antibody (being designated as Ab1) the mark biotin for determinand (being designated as Ag, such as antigen)
Biotin-Ab1), extinction material G is marked (to be designated as Ab2-G, also referred to as resist with another antibody (being designated as Ab2) for the determinand
Former gold labeling antibody), 2 labeled antibody are mixed with determinand, redissolved, are formed such as biotin-Ab1-Ag-Ab2-G
Compound 1；
(1.2) biotin is marked with a fluorescent material (being designated as F), forms the compound 2 such as biotin-F, wherein G suction
Receive spectrum and F excite and (or) emission spectrum completely and (or) partly overlaps；
(1.3) by well 11, by compound 1 and compound 2 (or various things of the compound 2 with forming compound 1
Matter, compound 1 and compound 2 are still obtained after reaction) it is added on perforated membrane, wait to fully penetrate into, in buffer solution is added dropwise in aperture, treat
Fully penetrate into, fixed capturing agent Avidin (streptavidin, SA) in this captures above-mentioned compound 1 and compound 2, is formed
Deposit containing fluorescent material and extinction material simultaneously, the fluorescence of golden quenching fluorescence material therein.
(1.4) while free gold labeling antibody is captured by control agent (antibody of such as anti-gold labeling antibody), and red is presented, illustrates inspection
Survey effective.
(1.5) determine detection zone at fluorescent material fluorescence intensity, fluorescence it is stronger explanation testing concentration it is lower, it is on the contrary then
The concentration of determinand is higher；As it is nonantigenic in sample when, then the fluorescence intensity at detection zone is raw florescent intensity, most by force.
The present disclosure additionally applies for A competitive inhibition method：
Method two,
(2.1) biotin is marked with the standard antigen (being designated as Ag0) of determinand, forms biotin-Ag0；With for be measured
An antibody (being designated as Ab1) the mark extinction material G of thing (such as antigen, being designated as Ag), forms Ab1-G, by Ab1-G and biotin-Ag0
Mixing, redissolve, form the compound 1 such as biotin-Ag0-Ab1-G；
(2.2) biotin is marked with a fluorescent material (being designated as F), forms the compound 2 such as biotin-F, wherein G suction
Receive spectrum and F excite and (or) emission spectrum completely and (or) partly overlaps；
(2.3) biotin-Ag0, determinand (being designated as Ag), Ab1-G and biotin-F are mixed, will by well 11
The mixture formed is added on perforated membrane, and the capturing agent Avidin (streptavidin, SA) for being fixed on detection zone is caught simultaneously
Above-mentioned compound 1 and compound 2 are obtained, forms the deposit containing fluorescent material and extinction material, golden quenching fluorescence thing therein
The fluorescence of matter；
Preferably, before perforated membrane is added to, after biotin-F, biotin-Ag0 and determinand (being designated as Ag) are mixed,
Mixed again with Ab1-G, or after biotin-Ag0 and determinand (being designated as Ag) are mixed, then mixed with Ab1-G and biotin-F；
(2.4) fluorescence intensity of fluorescent material at detection zone is determined, biotin-Ag0 and Ag competitively ties with Ab1-G
Close, Ag is more, and the compound 1 of generation is fewer, so that fluorescence intensity is stronger.
Therefore, fluorescence is stronger, and testing concentration is higher, conversely, then the concentration of determinand is lower；If without to be measured in sample
Thing, then the compound 1 formed on perforated membrane at detection zone is most, and correspondingly, 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
With any light source that can provide suitable wavelength, include but is not limited to：LED, xenon lamp, halogen tungsten lamp, laser etc..It is a kind of preferable
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..
Optical maser wavelength is relevant with laser medium caused by laser, 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 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 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, the quantity of determinand being determined by measuring the fluorescence intensity of fluorescent material, methods described is quick, easy, cost is cheap,
And high sensitivity, it is quantitative accurate.
(3) present invention also offers a kind of detection means, described device to be 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) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and
Number is calculated by weight.
Reagent and equipment：
For AFP 1 pair of monoclonal antibody（Ab1 and Ab2）, commercially available product；
For C reactive protein（CRP）Monoclonal antibody, commercially available product；
CRP antigens, commercially available product；
AFP antigens are purchased from Biodesign；
30nm collaurum, commercially available product；
PE is purchased from Invitrogen companies；
Streptavidin（SA）, commercially available product；
BSA（BSA）, commercially available product；
Biotin, commercially available product；
Detector：USB4000-FL (Ocean Optics of the U.S.)；
Light source：532nm LASER Light Sources.
Embodiment 1：The detection of Serum Alpha Fetoprotein (AFP)（Contain flowable fluorescent material in reaction system）
1st, the preparation of gold labeling antibody
1.1 collaurums-antibody preserves liquid
| Sodium tetraborate | 0.1g |
| BSA (BSA) | 0.25g |
| NaN3 | 0.025g |
PH to 7.4 is adjusted with 6N HCl after being dissolved in water, moisturizing to 250ml, after 0.45 μm of membrane filtration, 4~8 DEG C are protected
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.
1.3 gold labeling antibody（Gold-Ab1）Preparation
1.3.1 20~30nm particle colloid gold liquid 20ml are taken, are slowly added to purified Ab1 antibody under magnetic stirring
1.0ml (1mg/ml), is stirred at room temperature 30min；
1.3.2 plus 10% BSA 0.8ml (final concentration 0.4%), 5min is stirred at room temperature；
1.3.3 plus 10% PEG 0.4ml (final concentration 0.2%), 5min is stirred at room temperature；
1.3.412000~1500r/min centrifuges 60~40min, is carefully sucked away from the heart clearly, precipitation is dissolved in 0.5ml and preserves liquid
In, the optical density of gold labeling antibody（O.D）About 100O.D, the wherein concentration of Ab1 antibody are 1mg/ml, put 4 DEG C and save backup；
2nd, biotin marks Ab2 antibody（biotin-Ab2）
2.1Ab2 pretreatment
2.2 take the μ L of Ab2 10 of above-mentioned pretreatment, add 25 μ L 1mg/ml NHSS-Biotin DMSO solutions, mix,
4 DEG C of refrigerator lucifuges are reacted 2 hours, and dialysed overnight is standby.
3.biotin marks PE（biotin-PE）
Experimental method is with step 2, the difference is that the Ab2 of pretreatment and PE is mixed, redissolves, in gained biotin-PE, PE
Final concentration of 0.5mg/ml.
4th, SA is fixed on microporous barrier
Take SA 2mg/ml phosphate buffer（pH7.2）The center of 0.5 μ l points microporous barrier in fig. ib, lucifuge
Drying at room temperature, it is assembled into the percolating device such as Figure 1A；
5th, prepared by standard curve
5.1 prepare AFP series standard solution with working solution（Concentration is shown in Table 1）；
5.2 each μ l of concentration point standard liquid 50 are separately added into 0.5 μ l Gold-Ab1,1 μ l biotin-Ab2 and 1 μ l
Biotin-PE, mix；
5.3 take 6 percolating devices, horizontal positioned, add 6 kinds of mixed solutions of previous step in aperture respectively, treat to ooze completely
Enter, 2 drop pH7.4 PBS is added dropwise at aperture；
5.4 determine the fluorescence intensity at each percolating device spot after fully penetrating into, and data are shown in Table 1, are made as shown in Figure 3
Standard curve.
Table 1：AFP standard series concentration and corresponding fluorescence intensity
| Standard series concentration (ng/ml) | Fluorescence intensity (F) |
| 0 | 36000 |
| 6.45 | 30500 |
| 32.25 | 23500 |
| 129 | 15000 |
| 430 | 9800 |
| 1290 | 4500 |
6th, pattern detection
With serum sample alternate standard serial solution, 5.2,5.3,5.4 steps are repeated, F is substituted into standard curve, measures 6
The AFP values of individual sample are shown in Table 2.
Table 2：The inventive method testing result and Roche Electrochemiluminescince testing result
| Catalogue number(Cat.No.) | The inventive method（ng/ml） | Roche Electrochemiluminescince（ng/ml） |
| 1 | 12.5 | 10.17 |
| 2 | 34.64 | 43.00 |
| 3 | 122.36 | 98.40 |
| 4 | 148.23 | 175.80 |
| 5 | 520.38 | 496.70 |
| 6 | 698.9 | 796.50 |
The correlation of testing result is good, R2=0.9853。
Embodiment 2：A competitive inhibition method detects C reactive protein（CRP）
1st, CRP monoclonal antibodies standard gold（Gold-Ab）
Process with embodiment 1 step 1, unlike Ab1 replaced using CRP monoclonal antibodies, collaurum O.D values are after mark
75, CRP monoclonal antibody concentration are 1mg/ml.
2nd, CRP antigenic marks biotin（biotin-CRP）
Process is with the step 2 in embodiment 1, difference, and pretreated Ab2, mark are substituted with purified CRP
CRP antigen concentrations 2mg/ml afterwards.
3rd, biotin marks PE
With the biotin-PE of embodiment 1.
4th, SA is fixed on microporous barrier
Take SA 3mg/ml phosphate buffer（pH7.2）The center of 1 μ l points microporous barrier in accompanying drawing 1B, lucifuge
Drying at room temperature, it is assembled into such as accompanying drawing 1A percolating device；
5th, prepared by standard curve
5.1 prepare 0,1,5,10 μ g/ml CRP series standard solution with working solution；
5.2 each μ l of concentration point standard liquid 10 are separately added into 0.5 μ l Gold-Ab, 0.5 μ l biotin-CRP and 1 μ l
Biotin-PE and 40 μ l pH7.4 PBS, mix；
5.3 take 5 percolating devices, horizontal positioned, add 5 kinds of mixed solutions of previous step in aperture respectively, treat to ooze completely
Enter, 2 drop pH7.4 PBS is added dropwise at aperture；
5.4 determine the fluorescence intensity at each percolating device spot after fully penetrating into, and data are shown in Table 3, are made as shown in Figure 4
Standard curve.
Table 3：CRP standard series concentration and corresponding fluorescence intensity
| Standard series concentration (μ g/ml) | Fluorescence intensity (F) |
| 0 | 4800 |
| 1 | 6000 |
| 5 | 11900 |
| 10 | 21000 |
6th, sample detection
With serum sample alternate standard serial solution, 5.2,5.3,5.4 steps are repeated, F substitution standard curves are measured 6
The CRP values of sample are shown in Table 4.
Table 4：The inventive method testing result and ELISA method testing result
| Catalogue number(Cat.No.) | The inventive method（μg/ml） | ELISA（μg/ml） |
| 1 | 2.5 | 3.17 |
| 2 | 1.64 | 1.80 |
| 3 | 3.36 | 3.64 |
| 4 | 7.23 | 5.80 |
| 5 | 8.64 | 7.33 |
| 6 | 7.86 | 8.70 |
The correlation of testing result is good, R2=0.9001。
All it is incorporated as referring in this application in all documents that the present invention refers to, it is 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 (32)
1. a kind of leak detection apparatus, it is characterised in that the leak detection apparatus includes：
(a1) perforated membrane of detection zone is included, wherein, described detection zone includes immobilization capturing agent, and
The immobilization capturing agent can be combined with flowable fluorescent material and flowable first bonding agent simultaneously, so as to be formed
" fluorescent material-bonding agent of capturing agent-the first " compound；First bonding agent can be combined with determinand, and described to be measured
Thing can be combined with the second bonding agent, so as to form " the first bonding agent-bonding agent of determinand-the second " compound；
So that after the described fluorescent material of detection zone contact, the first bonding agent, determinand and the second bonding agent, in detection zone shape
Into " fluorescent material-bonding agent of capturing agent-the first-bonding agent of determinand-the second " compound, wherein, the second described knot
Mixture is marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(a2) absorption pad below the perforated membrane, the absorption pad have absorbability so that add to detection zone sample and
Penetration by liquid perforated membrane is simultaneously absorbed by absorption pad,
Also, the immobilization capturing agent is streptavidin, and the fluorescent material is by biotin labeling, the first bonding agent quilt
Biotin labeling.
2. leak detection apparatus as claimed in claim 1, it is characterised in that described fluorescent material is phycoerythrin, and is inhaled
Stimulative substance is the collaurum that particle diameter is 20-40nm.
3. leak detection apparatus as claimed in claim 1, it is characterised in that described the first bonding agent and the second bonding agent
Species or quantity are one or more；So as to simultaneously for detecting one or more determinands.
4. leak detection apparatus as claimed in claim 1, it is characterised in that described leak detection apparatus is to determine amount detection device
Put.
5. leak detection apparatus as claimed in claim 1, it is characterised in that the determinand is antigen or antibody.
6. leak detection apparatus as claimed in claim 5, it is characterised in that described determinand is antigen, then described
One bonding agent and the second bonding agent are can be in combination with the antibody of the antigen；Or described determinand is antibody, then institute
The first bonding agent and the second bonding agent stated are can be in combination with the antigen of the antibody or the antibody of the antibody.
7. a kind of leak detection apparatus, it is characterised in that the leak detection apparatus includes：
(b1) perforated membrane of detection zone is included, wherein, described detection zone includes immobilization capturing agent, and the immobilization is caught
Obtain agent can while be combined with flowable fluorescent material and flowable determinand equivalent, so as to form " fluorescent material-capture
Agent-determinand equivalent " compound；The determinand equivalent can be combined with bonding agent, and so as to be formed, " determinand is equivalent
Thing-bonding agent " compound, and the bonding agent can be combined with determinand；
So as to after detection zone contacts described fluorescent material, bonding agent, determinand and determinand equivalent, be formed in detection zone
" fluorescent material-capturing agent-determinand equivalent-bonding agent " compound, wherein, described bonding agent is by extinction material mark
Note, and the extinction material influences the fluorescence intensity of the fluorescent material；
(b2) absorption pad below the perforated membrane, the absorption pad have absorbability so that add to detection zone sample and
Penetration by liquid perforated membrane is simultaneously absorbed by absorption pad,
Also, the immobilization capturing agent is streptavidin, then the fluorescent material is equal by biotin labeling, the determinand
Thing is by biotin labeling.
8. leak detection apparatus as claimed in claim 7, it is characterised in that described fluorescent material is phycoerythrin, and is inhaled
Stimulative substance is the collaurum that particle diameter is 20-40nm.
9. leak detection apparatus as claimed in claim 7, it is characterised in that the species or quantity of described bonding agent are one kind
It is or a variety of；So as to simultaneously for detecting one or more determinands.
10. leak detection apparatus as claimed in claim 7, it is characterised in that described leak detection apparatus is quantitative detection
Device.
11. leak detection apparatus as claimed in claim 7, it is characterised in that the determinand is antigen or antibody.
12. leak detection apparatus as claimed in claim 11, it is characterised in that described determinand is antigen, then described
Bonding agent is the antibody that can be combined in the antigen；Or described determinand is antibody, then described bonding agent is to combine
In the antigen of the antibody or the antibody of the antibody.
13. the leak detection apparatus as described in claim 1 or 7, it is characterised in that described leak detection apparatus also includes：
A bottom device below absorption pad be present；
A cap device above perforated membrane also be present, there is the perforate for being available for adding sample on the cap device, and it is described
Perforated membrane is located at below the perforate.
14. 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.
15. 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.
16. leak detection apparatus as claimed in claim 15, it is characterised in that the check plot is used for the inspection for showing determinand
Survey the validity of result.
17. 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.
18. 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.
19. the leak detection apparatus as described in claim 1 or 7, it is characterised in that the fluorescent material is selected from the group：Fluorescence
Element, rhodamine, phycoerythrin, quantum dot or rare earth element ion or its chelate.
20. leak detection apparatus as claimed in claim 19, it is characterised in that the fluorescein is selected from the group：Carboxyl fluorescence
Element, 2- methoxyl groups fluorescein and 4,5- dimethoxyfluorescein.
21. the leak detection apparatus as described in claim 1 or 7, it is characterised in that described extinction material is selected from the group：Glue
Body gold, nanometer gold bar, Nano Silver rod or its combination.
22. leak detection apparatus as claimed in claim 21, it is characterised in that described collaurum is that average grain diameter is 10-
70nm colloid gold particle.
23. leak detection apparatus as claimed in claim 21, it is characterised in that the aspect ratio of described nanometer gold bar is 1.5-
10。
24. leak detection apparatus as claimed in claim 21, it is characterised in that the length of described nanometer gold bar is 10-
200nm。
25. a kind of fluorescence analysis method for quantitatively detecting determinand, it is characterised in that including step：
(a1) the first bonding agent, determinand sample and the second bonding agent are made an addition to the detection zone of leak detection apparatus respectively, or
Described detection zone will be made an addition to after the mixing of first bonding agent, determinand sample and the second bonding agent,
And fluorescent material is made an addition to described detection zone；Wherein, described detection zone includes immobilization capturing agent, and institute
State immobilization capturing agent can while be combined with flowable fluorescent material and flowable first bonding agent, so as to form " fluorescence
Material-the bonding agent of capturing agent-the first " compound；First bonding agent can be combined with determinand, and the determinand can be with
Second bonding agent combines, so as to form " the first bonding agent-bonding agent of determinand-the second " compound；
So that after the described fluorescent material of detection zone contact, the first bonding agent, determinand and the second bonding agent, in detection zone shape
Into " fluorescent material-bonding agent of capturing agent-the first-bonding agent of determinand-the second " compound, wherein, the second described knot
Mixture is marked by extinction material, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) the fluorescence intensity F of the detection zone is detected, so as to measure the presence or absence of determinand or quantity,
Also, the immobilization capturing agent is streptavidin, and the fluorescent material is by biotin labeling, the first bonding agent quilt
Biotin labeling.
26. a kind of fluorescence analysis method for quantitatively detecting determinand, it is characterised in that including step：
(b1) determinand sample, determinand equivalent 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 determinand equivalent, then by bonding agent and the first mixture
Make 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
3rd mixture of one determinand sample and bonding agent is provided first, then by determinand equivalent and the 3rd mixture according to
Secondary the 4th mixture for making an addition to described detection zone or being formed determinand equivalent and the 3rd mixture makes an addition to institute
The detection zone stated,
And fluorescent material is made an addition to described detection zone；Wherein, described detection zone includes immobilization capturing agent, and institute
State immobilization capturing agent can while be combined with flowable fluorescent material and flowable determinand equivalent, so as to form " fluorescence
Material-capturing agent-determinand equivalent " compound；The determinand equivalent can be combined with bonding agent, " be treated so as to be formed
Survey thing equivalent-bonding agent " compound, and the bonding agent can be combined with determinand；
So as to after detection zone contacts fluorescent material, determinand sample, determinand equivalent and bonding agent, be formed in detection zone
" fluorescent material-capturing agent-determinand equivalent-bonding agent " compound, wherein, described bonding agent is by extinction material mark
Note, and the extinction material influences the fluorescence intensity of the fluorescent material；
(2) the fluorescence intensity F of the detection zone is detected, so as to measure the presence or absence of determinand or quantity,
Also, the immobilization capturing agent is streptavidin, then the fluorescent material is equal by biotin labeling, the determinand
Thing is by biotin labeling.
27. the method as described in claim 25 or 26, it is characterised in that step (2) includes：By the fluorescence intensity F of detection zone
With standard curve or compared with the initial fluorescent intensity F0 not being loaded, so that it is determined that presence or absence or the number of determinand
Amount.
28. the method as described in claim 25 or 26, it is characterised in that described determinand is liquid phase, suspension or solid phase.
29. method as claimed in claim 28, it is characterised in that when determinand is solid phase, it is molten that step (1) also includes addition
Agent.
30. the method as described in claim 25 or 26, it is characterised in that described method also includes using the to be measured of concentration known
Thing standard items measure, the step of so as to make standard curve.
31. a kind of fluorescence measuring 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) detector for fluorescence intensity.
32. fluorescence measuring device as claimed in claim 31, it is characterised in that described device also includes light source and computer,
The light source is irradiated at detection zone, and the fluorescence inspired enters detector, and data processing and analysis are carried out by computer.
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