CN103293303B - A kind of fluorescence analysis method and device - Google Patents
A kind of fluorescence analysis method and device Download PDFInfo
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- CN103293303B CN103293303B CN201210051830.8A CN201210051830A CN103293303B CN 103293303 B CN103293303 B CN 103293303B CN 201210051830 A CN201210051830 A CN 201210051830A CN 103293303 B CN103293303 B CN 103293303B
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
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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
- G01N21/84—Systems specially adapted for particular applications
- G01N21/8483—Investigating reagent band
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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
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Abstract
The present invention relates to a kind of fluorescence analysis method and device, specifically, the invention discloses the detection method of a kind of detection by quantitative determinand and detection device based on described detection method, described method uses the test sheet of the present invention, and described test sheet includes: (i) can add the sample application zone of sample；(ii) land of sample application zone near-end it is positioned at；(iii) land near-end and the test section of sample application zone far-end it are positioned at；(iv) the sample uptake zone of test section near-end and land far-end it is positioned at.Described method includes step: determinand sample is added to the sample application zone of the test sheet of the present invention by (1)；(2) measure the fluorescence intensity of the test section of described test sheet, thus be scaled the quantity of determinand.Described method is quick, easy, with low cost, and highly sensitive, the most accurately.
Description
Technical field
The invention belongs to vitro detection technical field, more particularly to a kind of based on the analysis measuring fluorescence intensity
Method and apparatus.
Background technology
In field of immunodetection, it is often necessary to all kinds of antigens or antibody are qualitatively or quantitatively detected.Existing
Have in technology, based on " Competitive assays and double antibodies sandwich ", derive panimmunity response analysis method, such as:
Radioimmunology, euzymelinked immunosorbent assay (ELISA), chemoluminescence method, time-resolved fluorescence method and fluorescent immune method etc., can
For determining pathogenic microorganism, the specific proteins detection by quantitative to human body, thus disease is carried out auxiliary and examines
Disconnected or monitoring etc., purposes is widely.Method is analyzed in this kind of immunoreation, is generally fixed by capture antibody
In solid phase carrier, then react with antigen (target protein), react with traget antibody again after washing, washing,
Last detection of radioactive intensity, solution absorbance or optical signal etc., thus report target protein in detection sample
Concentration.The automatization of said method eliminates the loaded down with trivial details of hand washing, but just because of automatization so that instrument
Device is bulky expensive, general only in the use of large-scale experiment room.
Nineteen ninety, the comprehensive gold colloidal such as Beggs and immuno analytical method, establish colloidal gold immunity chromatography
(GICA), for detecting HCG (BEGGS M, the NOVOTNY M, SAMPEDRO in Urina Hominis and serum
S.A selfperforming chromatographic immunoassay for the qualitative
determination of human chorionicgonadotrophin(HCG)in urine and serum[J].Clin
Chem, 1990,36:1084-1085).Hereafter 20 years, people, on the basis of GICA, have developed use
In pathogen (CN 03115143.4), hormone (CN 200610014168.3), cardiac marker (CN
200410011165.5), tumor markers (CN 200510104796.6), autoimmune disease mark (CN
200410027291.X) etc. detectable；Be also applied to simultaneously food, environment (CN 03116692.X) and
Veterinary (CN 02139704.X) field.Only need perusal due to the method, layman also can grasp
Make, make emergency treatment, basic hospital, patient bedside and scene etc. carry out relevant inspection away from the place of large-scale experiment room
Survey is possibly realized, so range of application is the most extensive.
Although this immuno analytical method alreadys more than the development of 20 years, but its basic operation principle does not change
Becoming, which dictates that up to the present, it is served only for qualitative or half-quantitative detection is (fixed according to the detection line gray scale depth
Amount), and sensitivity quantitative immunoassay method the most as the aforementioned, it is further applied and encounters bottleneck.
Therefore, this area is badly in need of changing existing colloidal gold chromatography method, is giving its high sensitivity
With quantitatively while the newest advantage, the feature such as keep it quick, easy, with low cost, thus enter
Step extends its application.
Summary of the invention
The invention provides a kind of test sheet, the determinand in detection by quantitative sample.
The invention provides the detection method of a kind of detection by quantitative determinand, described method is highly sensitive, quantitatively
Accurately.
Present invention also offers the detection device of a kind of detection by quantitative determinand.
First aspect present invention provides a kind of test sheet, and this test sheet includes:
I () can add the sample application zone of sample；
(ii) being positioned at the land of sample application zone near-end, described land comprises:
One or more flowable bonding agent, in described bonding agent, at least one is by extinction material labelling, institute
State bonding agent and can be combined the complex formed containing extinction material with determinand or its equivalent；
(iii) being positioned at land near-end and the test section of sample application zone far-end, described test section comprises immobilized catching
Obtaining agent, described trapping agent moves the bonding agent by extinction material labelling to test section for capturing from land
Or the complex containing extinction material, described test section has fluorescence；With
(iv) being positioned at the sample uptake zone of test section near-end and land far-end, wherein uptake zone has absorption energy
Power, so that the sample adding to sample application zone diffuses to end sample uptake zone from sample application zone；
Wherein, the described bonding agent by extinction material labelling or described containing extinction material is captured when described trapping agent
Complex time, described extinction material affect described test section fluorescence intensity.
In another preference, the fluorescence of described test section is the own fluorescence of described test sheet.
In another preference, it is to cause test that described extinction material affects the fluorescence intensity of described test section
The fluorescence intensity in district declines.
In another preference, described determinand is antigen or antibody.
In another preference, the specific binding determinand of described bonding agent or its equivalent；It is preferred that institute
Stating bonding agent is antigen, antibody or oligonucleotide.
In another preference, described land can comprise two kinds of bonding agent, wherein, a kind of by extinction thing
The bonding agent of matter labelling, another kind of by biotin labeled bonding agent, the two bonding agent is simultaneously with to be measured
Thing combines, thus is formed a kind of by the biotin labeled complex containing extinction material.
In another preference, the described complex containing extinction material can contain determinand, it is also possible to contains
Determinand equivalent.
In another preference, the specific binding bonding agent by extinction material labelling of described trapping agent or containing inhale
The complex of stimulative substance.
In another preference, described trapping agent is streptavidin, the antibody of determinand or the equivalent of determinand
Thing.
In another preference, the quantity of described bonding agent is more than the quantity of described determinand；Described trapping agent
Quantity more than moving from land to the bonding agent by extinction material labelling of test section or containing extinction material
The quantity of complex.
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 determinand includes that tumor markers, Applications of Cardiac Markers etc. are specific
Albumen.
In another preference, described test sheet has exciting or emission spectrum and described extinction material of fluorescence by oneself
Absorption spectrum the most overlapping.
In another preference, described extinction material is selected from lower group: gold colloidal, nanometer gold bar, nanometer silver
Rod or a combination thereof.
In another preference, described gold colloidal be mean diameter be the colloid gold particle of 10-70nm.
In another preference, between test section and sample uptake zone, also comprise at least one check plot, institute
Stating check plot and contain immobilized placebo, wherein, described placebo is for specific binding by extinction material
The bonding agent of labelling.
In another preference, described check plot is for disclosing the effectiveness of the testing result of determinand.
Second aspect present invention provides the fluorescence analysis method of a kind of detection by quantitative determinand, including step:
(1) determinand sample is added to the sample application zone testing sheet described in first aspect present invention；
(2) measure the fluorescence intensity of the test section of described test sheet, thus be scaled the quantity of determinand.
Third aspect present invention provides the fluorescence analysis method of a kind of detection by quantitative determinand, including step:
A () provides the test sheet described in a first aspect present invention；
B determinand sample is added to the sample application zone of described test sheet by ()；
C the determinand in () sample moves to land, tie with the bonding agent by extinction material labelling therein
Close, thus form the flowable complex containing extinction material；
What d in () step (c), the remaining bonding agent by extinction material labelling or step (c) obtained is flowable
Complex containing extinction material moves to test section, is combined with trapping agent therein, thus is formed and be fixed on
The complex containing extinction material of test section；
E () measures the fluorescence intensity of described test section, thus be scaled the quantity of determinand.
In another preference, the fluorescence of described test section is the own fluorescence of described test sheet.
In another preference, when the bonding agent by extinction material labelling remaining in trapping agent integrating step (c)
Time, described trapping agent is antibody or the antigen of described bonding agent or is determinand equivalent.
In another preference, when determinand sample is solid phase, step (b) also includes adding solvent (such as water
Or buffer).
In another preference, in step (2) or step (e), by the fluorescence of test section Yu middle, check plot
The ratio of fluorescence intensity F1 of intensity F2 and test section, and standard curve compares, so that it is determined that determinand
Quantity；Or
Fluorescence intensity F1 and the standard curve of test section are compared, so that it is determined that the quantity of determinand.
In another preference, described method also includes surveying with the determinand standard substance of concentration known
Amount, thus make the step of standard curve.
Fourth aspect present invention provides a kind of detection kit, and described test kit includes: a present invention first
Test sheet described in aspect；And operation instructions.
Fifth aspect present invention provides the fluorescence measuring device of a kind of detection by quantitative determinand, described device
Including:
Test sheet described in (a) first aspect present invention；
B () is for the detector of fluorescence intensity；With
C () describes the operation instruction of using method.
In another preference, described device also includes light source and computer.
Described light source is irradiated to detect at line by optical fibers, and the fluorescence inspired is entered by optical fibers
Detector, is carried out data process and analysis by computer.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as embodiment)
Can be combined with each other between each technical characteristic of middle specific descriptions, thus constitute new or preferred technical side
Case.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 is test sheet schematic diagram.
Fig. 2 is detection device schematic diagram.
Fig. 3 is that AFP standard series concentration (C) of embodiment 1 is bent with corresponding fluorescence intensity ratio (F2/F1) standard
Line chart.
Fig. 4 is the logarithm value (lgC) of the CRP standard series concentration of embodiment 2 and corresponding fluorescence intensity ratio
Logarithm value lg (F2/F1) canonical plotting.
Detailed description of the invention
The present inventor is by in-depth study for a long time, it was found that a kind of detection side based on principle of fluorescent quenching
Method, described method judges whether containing determinand not only by the color of range estimation test section；All right
Carrying out detection by quantitative determinand by the way of measuring test section fluorescence intensity, said method can be directly by quenching
Go out the material impact on the own fluorescence intensity of test section, thus measure the quantity of determinand.Described method is not
Only have highly sensitive, the most accurately advantage, and operate the easiest, quick and low cost
Honest and clean.Described method is a kind of test sheet provided based on the present invention, by a kind of containing light source and detector
Device achieves the detection by quantitative to determinand.On this basis, inventor completes the present invention.
Test sheet
Now it is more fully described method and the material testing sheet for manufacturing the present invention.It should be noted that test sheet
Specific configuration can change, this depends on the concrete test that intention test sheet carries out.Embodiment it
The outer variation method manufacturing test sheet, also falls among the scope of the invention.
As it is shown in figure 1, test sheet 1 can include backing sheets 2, its length is identical with test sheet.
Sample application zone 3 is positioned at one end of test sheet, and sample pad 31 is positioned at sample application zone, can be pasted by binding agent
In sample application zone.
Uptake zone 7 is positioned at the other end of test sheet, and at the far-end of sample application zone, water suction pad 71 is positioned at uptake zone.
Land 4 between sample application zone and uptake zone, the near-end in sample application zone and the far-end of uptake zone, knot
Close pad 41 and be positioned at land.
Test section 5 (also referred to as detecting line) is between land and uptake zone, and usual test section is arranged on diaphragm 56
On, connect pad and adsorptive pads by described diaphragm.Preferably, described diaphragm is additionally provided with check plot
6 (also referred to as nature controlling lines), check plot is between test section and uptake zone, it is also possible to be positioned at test section and land
Between, and and test section between have suitable space.
Test piece making method, it is preferable that as it is shown in figure 1, respectively by sample pad, bonding pad, film
Sheet, water suction pad are pasted on backer board by binding agent, obtain described test sheet.
Backing sheets can be made with any material stable, atresia, and its intensity should be enough to supporting material and glue
Test sheet in it.Because many mensuration uses water as dispersive medium, therefore backing sheets is the most substantially
Fluid-tight.In a preference, backing sheets polymeric film is made, and is more preferably to use polychlorostyrene second
(such as the PVC offset plate) that alkene film is made.
Sample pad can be made with any absorbent material.Spendable example of material includes: cellulose, nitre
Acid cellulose, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer
/ nylon and polyether sulfone.
Bonding pad or diaphragm can be made with any material, as long as this material has enough porositys thus allows
In surface and the internal capillarity that fluid occurs.Bonding pad or diaphragm should have enough porositys, from
And allow the granule scribbling antibody or antigen to move.Bonding pad or diaphragm also can be by containing analytes to be detected
Liquid moistening used in sample (such as, has hydrophilic for waterborne liquid, has for organic solvent thin
Aqueous).By such as method (these sides described in United States Patent (USP) No.4,340,482 or No.4,618,533
Method describes and hydrophobic surface is transformed into water-wetted surface), thus it is possible to vary its hydrophobicity thus make it have hydrophilic
For use in waterborne liquid.The example of material that can be used for manufacturing bonding pad or diaphragm includes: polymer PET, fibre
Dimension element, celluloid, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, third
Alkene copolymer/nylon and polyether sulfone (polyethersulfone).In a preference, bonding pad is to use
Polymer PET is made, and diaphragm celluloid is made.
Absorbent patch can absorb the material as sample and the liquid of buffer with any energy and make.Absorption pad
The absorbability of sheet is sufficiently large, in order to absorb the liquid added to testing sheet.It is applicable to the material of absorbent patch
The example of material includes cellulose and glass fibre.
Detection method
The present invention tests sheet and can be used for the most different lateral flow assays methods, and these analysis methods are directed to use with
One or more flowable bonding agent and the immobilized trapping agent of one, and utilize own glimmering in test section
The change of light intensity.
Preferably, described method includes step:
(1) determinand sample is added to the sample application zone of test sheet of the present invention；
(2) measure the fluorescence intensity of the test section of described test sheet, thus be scaled the quantity of determinand.
After sample-adding, the determinand in sample moves to land, and bonding agent is contained in described land, described
Determinand is combined with bonding agent therein, thus forms the complex containing extinction material；
Then, the complex containing extinction material formed in remaining bonding agent containing extinction material and land
Move to test section, be combined with trapping agent therein, thus form the test section being enriched with extinction material.
Specifically, when described in the described extinction material impact (part cancellation or all cancellation) being enriched in test section
During the fluorescence intensity of test section (test sheet has fluorescence by oneself), the detection method of the present invention can be tested by detection
District's fluorescence intensity measures the quantity of determinand.
Determinand or its equivalent
As used herein, term " determinand ", " its equivalent " or " analyte " refer to stand-by test sheet
Detection and be optionally quantitatively determined, any component in sample, determinand or its equivalent or point
The example of analysis thing includes: protein, such as hormone or other secretory proteins, enzyme and cell surface protein；Sugar
Albumen；Peptide；Little molecule；Polysaccharide；Antibody (includes monoclonal antibody or polyclonal antibody and fragment thereof)；Core
Acid；Medicine (includes the Cardiac glycosides medicines such as digoxin)；Toxin；Drugs；Virus or virion；Cell
Wall component；Or other have the compound of epi-position.
Preferably, described determinand includes the specific albumen such as tumor markers, Applications of Cardiac Markers, institute
State tumor markers selected from lower group: alpha-fetoprotein (AFP), C reactive protein (CRP), carcinoembryonic antigen (CEA),
Cancer antigen 125 (CA125), CA19-9 (CA19-9), total-PSA (PSA), free before
Row gland specific antigen (f-PSA), NSE (NSE), carbohydrate antigen (CA242),
Cancer antigen (CA15-3) or human chorionic gonadotropin (β-HCG).
Bonding agent
Bonding agent used by the present invention can be any material that can be incorporated into determinand or its equivalent.Tool
Body ground, for specific binding determinand or the material of its equivalent.In described bonding agent, at least one is inhaled
Stimulative substance labelling, described bonding agent can be combined with determinand or its equivalent and form being combined containing extinction material
Thing；
Various types of molecule is had to can be used as analyte binding agent, including such as: oligonucleotide,
Antibody, engineering protein, peptide, hapten or containing antigen (this antigen has analyte binding site)
The lysate of heterogeneous mixture.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
It is part, then can use the receptor being incorporated into this part, vice versa.
Preferably, described determinand or its equivalent are antigen, and described bonding agent is to can be combined in institute
State the antibody of antigen；Or described determinand or its equivalent are antibody, and described bonding agent is to tie
Antigen or the antibody (anti antibody) of described antibody together in described antibody.
Trapping agent
Trapping agent used by the present invention can be that any energy is in conjunction with the complex containing extinction material and/or bonding agent
Material, including such as: antibody, engineering protein, peptide, hapten or (this antigen has containing antigen
Analyte binding site) the lysate of heterogeneous mixture.
Described trapping agent for capture move from land to test section the complex containing extinction material and/or
The remaining bonding agent by extinction material labelling, so that extinction material is enriched in test section.
Preferably, can be tied with complementary nucleic acid strand specificity by antigen and the combination of antibody or oligonucleotide
The mode closed or by biotin (biotin) and the combination of streptavidin (Streptavidin, SA), will catch
Obtain agent to be combined with the complex containing extinction material and/or the remaining bonding agent by extinction material labelling, thus will
Extinction material enrichment is mixed in test section.
Described trapping agent and containing the complex of extinction material or the remaining bonding agent by extinction material labelling
It is combined into specific binding.Preferably, described trapping agent is streptavidin, the antibody of determinand or to be measured
Thing equivalent.
The selection of extinction material
Extinction material for the present invention should have broad UV, visible light even infrared absorption spectroscopy, and total is former
To be then its absorption spectrum with test sheet have by oneself fluorescence excite or emission spectrum is the most overlapping.
Optimum is completely overlapped, so has higher detection sensitivity.
Representational extinction material includes (but being not limited to): the groups such as gold colloidal, nanometer gold bar, nanometer silver rod
Close, their absorption spectrum ranges is 300～1000nm, as long as the absorption spectrum of selected extinction material with
Test sheet has fluorescence excitation or emission spectrum overlap by oneself.
Colloid gold particle can such as be summarized in G.Frens, 1973Nature with any conventional method manufacture
Method in Physical Science, 241:20 (1973).Additive 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 have certain aspect ratio and be horizontally and vertically in
The gold grain of 5-200 nanometer range.
A kind of particularly preferred extinction material is gold colloidal, and especially particle diameter is the gold colloidal of 20-40nm.
Principle of fluorescent quenching
Containing the material of extinction material labelling, after the capture agent capture in tested district, containing extinction material labelling
Material can be enriched with in test section, when test sheet has exciting or the suction of emission spectrum and this extinction material of fluorescence by oneself
When receiving spectrum completely or partially overlap, because of Resonance energy transfer, test sheet can be had by oneself fluorescence and be produced by extinction material
Raw quenching effect, the i.e. fluorescence intensity of cancellation test section.
Operation principle
In conjunction with Fig. 1 and the operation principle of specific embodiment explanation detection method:
Utilize the own fluorescence of diaphragm 56 itself.At detection line, the fixing material being combined with bonding agent (i.e. captures
Agent).Described bonding agent is positioned at bonding pad, flowable.After described bonding agent is dissolved by sample, chromatographing
In journey, the determinand in sample or its equivalent are combined the complex formed containing extinction material, described complex
Flowing through during detection line captured, determinand is the most, and captured described complex is the most.Specifically,
When described complex (part cancellation or all cancellation) affects the fluorescence intensity at described diaphragm detection line, i.e.
The quantity of determinand can be measured by fluorescence intensity.
The present invention there is provided herein following method for optimizing based on above-mentioned principle:
Method one:
(1.1) sample is added on glass fiber sample pad sheet, and the determinand (such as antigen) in sample is in capillarity
Lower move to water suction pad direction, by way of being loaded with bonding agent (as the golden labeling antibody of antigen, biotin labelling
Antibody) bonding pad after, described bonding agent is redissolved completely, in chromatography process, antigen and bonding agent are formed
3 yuan of complex of " gold labeling antibody-antigen-biotin antibody ", the trapping agent (such as Streptavidin) at tested survey line
Capture；
The own fluorescence at golden cancellation film bar detection line in 3 yuan of complex.
(1.2) unnecessary free gold labeling antibody continues reach, is fixed on the placebo at nature controlling line (such as anti-gold
The antibody of labeling antibody) capture, present redness, illustrate that detection is effectively.
(1.3) the glimmering of fluorescence intensity F1 at film bar detection line and detection line and nature controlling line middle is measured respectively
Light intensity F2, calculates the value of F2/F1, and it is the highest to be worth the biggest explanation testing concentration, otherwise the lowest；Such as sample
Middle time without determinand, it is impossible to form 3 yuan of complex, then F2/F1 ≈ 1.
Method two:
Trapping agent (as the capture antibody of antigen in determinand) is fixed at detection line.
(1.1) sample is added on glass fiber sample pad sheet, and the determinand (such as antigen) in sample is in capillarity
Lower to water suction pad direction move, after the bonding pad being loaded with bonding agent (such as the golden labeling antibody of antigen),
Being redissolved completely by described bonding agent, in chromatography process, antigen and bonding agent form the 2 of " gold labeling antibody-antigen "
Unit's complex, the capture antibody capture at tested survey line；
The own fluorescence at golden cancellation film bar detection line in 2 yuan of complex.
(1.2) unnecessary free gold labeling antibody continues reach, is fixed on the placebo at nature controlling line (such as anti-gold
The antibody of labeling antibody) capture, present redness, illustrate that detection is effectively.
(1.3) the glimmering of fluorescence intensity F1 at film bar detection line and detection line and nature controlling line middle is measured respectively
Light intensity F2, calculates the value of F2/F1, and it is the highest to be worth the biggest explanation testing concentration, otherwise the lowest；Such as sample
Middle time without determinand, it is impossible to form 2 yuan of complex, then F2/F1 ≈ 1.
Present disclosure additionally applies for A competitive inhibition method:
(1.1) equivalent of determinand is fixed at detection line and fixes as trapping agent, the antibody of bonding agent
At nature controlling line；
(1.2) being dripped at the bonding pad of accompanying drawing 1 by bonding agent (such as gold labeling antibody), sample is added in glass fibers
On dimension sample pad, determinand therein (such as antigen) moves, by way of being loaded with described knot to water suction pad direction
It is also redissolved by the bonding pad of mixture completely, during liquid moves ahead, and the determinand in sample and pad
Bonding agent on sheet forms " the 1st binary complex " of gold labeling antibody-antigen；
(1.3) remaining bonding agent proceeds to, at the detection line of diaphragm 56, form gold with the equivalent of determinand
" the 2nd binary complex " of labeling antibody-determinand equivalent and be captured, testing concentration is the highest, is formed
" the 2nd binary complex " the fewest, correspondingly, captured " the 2nd binary complex " is the fewest；
(1.4) bonding agent at large continues to move ahead with " the 1st binary complex ", is combined by nature controlling line
The antibody capture of agent, nature controlling line display redness, test is effectively；
(1.5) the glimmering of fluorescence intensity F1 at film bar detection line and detection line and nature controlling line middle is measured respectively
Light intensity F2, calculates the value of F2/F1, and it is the least to be worth the biggest explanation testing concentration, otherwise the biggest.
Detection device
The detection device of the present invention is described in conjunction with Fig. 2:
As in figure 2 it is shown, described device may include that test sheet, detector, light source, optical fibers and meter
Calculation machine.The operation instruction of a detection method can also be included.Wherein test the operation principle of sheet as it has been described above,
The detection method of fluorescence intensity can (but being not limited only to this) as described below, any can be used for fluorescence intensity
Method be used equally to the detection device of the present invention.
The detection of fluorescence intensity
Light source is irradiated to detect at line by optical fibers, and the fluorescence inspired enters detection by optical fibers
Device, obtained data are processed by computer and analyze.
Described optical fibers can be Y type, is connected to light source, detection line and detector.
In the present invention, light source is for providing the light of a certain transmitting wavelength, thus excites test sheet to send fluorescence.
Can be selected for any light source that suitable wavelength can be provided, include, but is not limited to: LED, xenon lamp, halogen tungsten lamp,
Laser etc..
A kind of preferably light source is LASER Light Source, and LASER Light Source can be with the method and apparatus of this area routine (as swashed
Light device) produce.Representational laser instrument includes (but being not limited to): semiconductor laser, helium neon laser,
Argon ion laser, also include the optional laser instrument of wavelength, multiple-wavelength laser and dual laser etc..
The optical maser wavelength that laser instrument produces is relevant with laser medium, and common optical maser wavelength see table 1:
Table 1
| 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 (HONGGUANG) | 633 |
| Rhodamine 6G dyestuff (tunable optical) | 570-650 |
| Ruby (CrAlO3) (HONGGUANG) | 694 |
| Neodymium-yttrium-aluminium-garnet (near infrared light) | 1064 |
Detector in the present invention can be (but are not limited to) photomultiplier tube, CCD or light cell etc..
Standard curve
In the present invention, directly the fluorescence intensity at line can be detected by mensuration, so that it is determined that described to be measured
The quantity of thing；Can also be by measuring fluorescence intensity F1 at detection line and detection line and nature controlling line middle
Fluorescence intensity F2, calculates the value of F2/F1, so that it is determined that the quantity of described determinand.
In preference, by comparing with standard curve, thus quantitative result can be obtained.
Standard curve can obtain with following methods:
By the determinand sample of known variable concentrations (C) by above-mentioned detection method after, measure respectively its inspection
Fluorescence intensity (F) at survey line, by each concentration (C) or its logarithm value (log C or lg C) and corresponding fluorescence intensity
(F) or its logarithm value (log F or lg F) mapping, obtain standard curve；Or
By the determinand sample of known variable concentrations (C) by above-mentioned detection method after, measure respectively its inspection
Fluorescence intensity (F1) at survey line, detection line and fluorescence intensity F2 of nature controlling line middle, calculate the value of F2/F1,
By each concentration (C) or its logarithm value (log C or lg C) and corresponding fluorescence intensity ratio (F2/F1) or its logarithm
Value (log F2/F1 or lg F2/F1) mapping, obtains standard curve.
Main advantages of the present invention have:
(1) the invention provides a kind of test sheet.
(2) the invention provides the detection method of a kind of above-mentioned test sheet, described method is former based on fluorescent quenching
Reason, has the quantity of fluorescent strength determining determinand by oneself by measuring test sheet, described method is quick, easy,
With low cost, and highly sensitive, the most accurately.
(3) present invention also offers a kind of detection device, described device, can be extensive based on above-mentioned detection method
For quantitative detection field.
Below in conjunction with being embodied as, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate
The present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, 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 built according to manufacturer
The condition of view.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Embodiment
Reagent and equipment:
For 1 pair of monoclonal antibody (Ab1 and Ab2) of AFP, commercially available product；
For the monoclonal antibody of C reactive protein (CRP), commercially available product；
CRP antigen, commercially available product；
AFP antigen is purchased from Biodesign；
The gold colloidal of 30nm, commercially available product；
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 Source.
Embodiment 1: the detection (utilizing the own fluorescence of diaphragm) of Serum Alpha Fetoprotein (AFP)
1, the preparation of gold labeling antibody
1.1 gold colloidals-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 membrane filtration,
4～8 DEG C of preservations.
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 |
After being dissolved in water, use 6N HCL tune pH to 7.0～7.5 moisturizings are to 1000ml, with 0.45 μm membrane filtration
After, 4～8 DEG C of preservations.
The preparation of 1.3 gold medal labeling antibodies (Gold-Ab1)
1.3.1 take 20～30nm particle colloid gold liquid 20ml, be slowly added to purification under magnetic stirring
Ab1 antibody 1.0ml (0.6mg/ml), is stirred at room temperature 30min；
1.3.2 the BSA 0.8ml (final concentration 0.4%) adding 10%, is stirred at room temperature 5min；
1.3.3 the PEG 0.4ml adding 10% (final concentration 0.2%), 5min is stirred at room temperature；
～1500r/min is centrifuged 60～40min 1.3.412000, being carefully sucked away from clear in the heart, precipitation is dissolved in 0.5ml
Preserving in liquid, the optical density (O.D) of gold labeling antibody is about 100O.D, and wherein the concentration of Ab1 antibody is
1mg/ml, puts 4 DEG C and saves backup；
2.biotin labelling Ab2
2.1Ab2 pretreatment
The 2.2 Ab210 μ L taking above-mentioned pretreatment, add the 1mg/ml NHSS-Biotin DMSO of 25 μ L
Solution, mixes, and 4 DEG C of refrigerator lucifuges are reacted 2 hours, and dialysed overnight is standby.
3.SA point film with upload Gold-Ab1, biotin-Ab2
Phosphate buffer (pH7.2) the 0.5 μ l point taking SA 1mg/ml detects line position, lucifuge room at accompanying drawing 1
Temperature is dried；
Take Gold-Ab1, biotin-Ab20.5 μ l to drip on the bonding pad of accompanying drawing 1.
4. prepared by standard curve
4.1 prepare AFP series standard solution (concentration is shown in Table 2) with working solution；
4.2 take 5 test sheets, and horizontal positioned, the 50 μ l standards adding 5 kinds of concentration respectively on each sample pad are molten
Liquid；
After 4.310min, measure and detect fluorescence intensity F1 of 605nm at line on each test sheet and detect line and matter
Fluorescence intensity F2 of control line middle 605nm, the Value Data calculating F2/F1 is shown in Table 2, and standard curve is shown in Fig. 3.
Table 2AFP standard series concentration and corresponding fluorescence intensity
5, pattern detection
With serum sample alternate standard serial solution, repeat 4.2,4.3 steps, F2/F1 substituted into standard curve,
The AFP value recording 6 samples is shown in Table 3.
Table 3 the inventive method testing result and Roche Electrochemiluminescince testing result
The inventive method testing result is good with the dependency of Roche Electrochemiluminescince testing result,
R2=0.9637.
Embodiment 2: based on A competitive inhibition method detection C reactive protein (CRP)
1.CRP monoclonal antibody standard gold (Gold-CRP)
Process is with embodiment 1 step 1, and difference is, substitutes Ab1 antibody with the CRP monoclonal antibody of purification,
After labelling, gold colloidal O.D value is 75, and CRP monoclonal antibody concentration is 1mg/ml.
2., with the pure antigen of CRP of PBS-TBN preparation 1mg/ml, take 0.2 μ l point at detection line；With
The antibody of the CRP antibody of PBS-TBN preparation 1mg/ml, takes 0.2 μ l point at nature controlling line；Take 0.5 μ l
Gold-CRP is added on pad, and drying at room temperature is standby.
3, prepared by standard curve
3.1 prepare CRP series standard solution (concentration is shown in Table 4) with working solution；
3.2 take 5 test sheets, and horizontal positioned, the 50 μ l standards adding 5 kinds of concentration respectively on each sample pad are molten
Liquid；
After 3.310min, measure and detect fluorescence intensity F1 of 605nm at line on each test sheet and detect line and matter
Fluorescence intensity F2 of control line middle 605nm, the Value Data calculating F2/F1 is shown in Table 4, and standard curve is shown in Fig. 4.
Table 4CRP standard series concentration and corresponding fluorescence intensity
5, pattern detection
With serum sample alternate standard serial solution, repeat 3.2,3.3 steps, F2/F1 substituted into standard curve,
The CRP value recording 6 samples is shown in Table 5.
Table 5 inventive method testing result and euzymelinked immunosorbent assay (ELISA) method testing result
The inventive method testing result is good with the dependency of euzymelinked immunosorbent assay (ELISA) method testing result, R2=0.9586.
Conclusion:
The method of the invention is only had the impact of fluorescence intensity by oneself and is surveyed by measuring quencher confrontation test section
Determine the quantity of determinand, both overcome the interference of the fluorescence signal that own fluorescence brings, and need not add
The reagent of any fluorescent material labelling, significantly reduces the use of raw material, has saved Financial cost, and experiment
Method is easier.
Visible, the method for the invention not only has highly sensitive, the most accurately advantage, and fast,
Easy and simple to handle, with low cost.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention,
The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally
Please appended claims limited range.
Claims (30)
1. a test sheet, it is characterised in that this test sheet includes:
I () can add the sample application zone of sample；
(ii) being positioned at the land of sample application zone near-end, described land comprises:
One or more flowable bonding agent, in described bonding agent, at least one is by extinction material labelling, institute
State bonding agent and can be combined the complex formed containing extinction material with determinand or its equivalent；
(iii) being positioned at land near-end and the test section of sample application zone far-end, described test section comprises immobilized catching
Obtaining agent, described trapping agent moves the bonding agent by extinction material labelling to test section for capturing from land
Or the complex containing extinction material, described test section has fluorescence；With
(iv) being positioned at the sample uptake zone of test section near-end and land far-end, wherein uptake zone has absorption energy
Power, so that the sample adding to sample application zone diffuses to end sample uptake zone from sample application zone；
Wherein, the described bonding agent by extinction material labelling or described containing extinction material is captured when described trapping agent
Complex time, described extinction material affect described test section fluorescence intensity；
Further, the fluorescence of described test section is the own fluorescence of described test sheet.
Test sheet the most as claimed in claim 1, it is characterised in that described determinand is antigen.
Test sheet the most as claimed in claim 1, it is characterised in that described extinction material affects described survey
The fluorescence intensity in examination district is that the fluorescence intensity causing test section declines.
Test sheet the most as claimed in claim 1, it is characterised in that described determinand is antibody.
Test sheet the most as claimed in claim 1, it is characterised in that described bonding agent is specific binding to be measured
Thing or its equivalent.
Test sheet the most as claimed in claim 1, it is characterised in that described bonding agent be antigen, antibody or
Oligonucleotide.
Test sheet the most as claimed in claim 1, it is characterised in that described land comprises two kinds of bonding agent,
Wherein, a kind of bonding agent by extinction material labelling, another kind of by biotin labeled bonding agent, described two
Plant bonding agent to be combined with determinand simultaneously, thus formed a kind of by biotin labeled being combined containing extinction material
Thing.
Test sheet the most as claimed in claim 1, it is characterised in that the described complex containing extinction material contains
There are determinand or determinand equivalent.
Test sheet the most as claimed in claim 1, it is characterised in that described trapping agent is specific binding to be inhaled
The bonding agent of stimulative substance labelling or the complex containing extinction material.
Test sheet the most as claimed in claim 1, it is characterised in that described trapping agent is streptavidin, treats
Survey antibody or the equivalent of determinand of thing.
11. test sheet as claimed in claim 1, it is characterised in that the quantity of described bonding agent is more than described
The quantity of determinand；The quantity of described trapping agent more than move from land to test section by extinction material mark
The bonding agent of note or the quantity of the complex containing extinction material.
12. test sheet as claimed in claim 1, it is characterised in that described determinand includes: albumen,
Nucleic acid or micromolecular compound.
13. test sheet as claimed in claim 1, it is characterised in that described determinand is liquid phase, suspension
Liquid or solid phase.
14. test sheet as claimed in claim 1, it is characterised in that described determinand includes selected from lower group
Specific albumen: tumor markers, Applications of Cardiac Markers.
15. test sheet as claimed in claim 1, it is characterised in that described test sheet has exciting of fluorescence by oneself
Or emission spectrum is the most overlapping with the absorption spectrum of described extinction material.
16. test sheet as claimed in claim 1, it is characterised in that described extinction material is selected from lower group:
Gold colloidal, nanometer silver rod or a combination thereof.
17. test sheet as claimed in claim 16, it is characterised in that described gold colloidal is mean diameter
Colloid gold particle for 10-70nm.
18. test sheet as claimed in claim 1, it is characterised in that between test section and sample uptake zone
Also comprising at least one check plot, immobilized placebo, wherein, described placebo are contained in described check plot
For the specific binding bonding agent by extinction material labelling.
19. test sheet as claimed in claim 18, it is characterised in that described check plot is used for disclosing to be measured
The effectiveness of the testing result of thing.
The fluorescence analysis method of 20. 1 kinds of detection by quantitative determinands, it is characterised in that include step:
(1) determinand sample adds to test as claimed in claim 1 the sample application zone of sheet；
(2) measure the fluorescence intensity of the test section of described test sheet, thus be scaled the quantity of determinand.
The fluorescence analysis method of 21. 1 kinds of detection by quantitative determinands, it is characterised in that include step:
A () provides the test sheet described in a claim 1；
B determinand sample is added to the sample application zone of described test sheet by ()；
C the determinand in () sample moves to land, tie with the bonding agent by extinction material labelling therein
Close, thus form the flowable complex containing extinction material；
What d in () step (c), the remaining bonding agent by extinction material labelling or step (c) obtained is flowable
Complex containing extinction material moves to test section, is combined with trapping agent therein, thus is formed and be fixed on
The complex containing extinction material of test section；
E () measures the fluorescence intensity of described test section, thus be scaled the quantity of determinand.
22. methods as described in claim 20 or 21, it is characterised in that in test section and sample uptake zone
Between also comprise at least one check plot, immobilized placebo is contained in described check plot, wherein, described right
According to agent for the specific binding bonding agent by extinction material labelling.
23. methods as claimed in claim 21, it is characterised in that when residue in trapping agent integrating step (c)
By the bonding agent of extinction material labelling time, described trapping agent is antibody or the antigen of described bonding agent or is
Determinand equivalent.
24. methods as claimed in claim 21, it is characterised in that when determinand sample is solid phase, step
Suddenly (b) also includes adding solvent.
25. methods as claimed in claim 22, it is characterised in that in step (2) or step (e), will survey
The ratio of fluorescence intensity F1 of examination district and fluorescence intensity F2 of middle, check plot and test section, and standard curve
Compare, so that it is determined that the quantity of determinand；Or
Fluorescence intensity F1 and the standard curve of test section are compared, so that it is determined that the quantity of determinand.
26. methods as claimed in claim 25, it is characterised in that described method also includes with known dense
The determinand standard substance of degree measure, thus make the step of standard curve.
27. 1 kinds of detection kit, it is characterised in that described test kit includes: claim 1-19 is appointed
One described test sheet；And operation instructions.
The fluorescence measuring device of 28. 1 kinds of detection by quantitative determinands, it is characterised in that described device includes:
Test sheet described in (a) any one of claim 1-19；
B () is for the detector of fluorescence intensity；With
C () describes the operation instruction of using method.
29. devices as claimed in claim 28, it is characterised in that described device also includes light source and calculating
Machine.
30. devices as claimed in claim 29, it is characterised in that described light source is irradiated by optical fibers
At detection line, the fluorescence inspired enters detector by optical fibers, computer carry out data process
And analysis.
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| PCT/CN2012/078714 WO2013127144A1 (en) | 2012-03-01 | 2012-07-16 | Fluorescence analysis method and device |
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| CN106841623A (en) * | 2015-12-03 | 2017-06-13 | 上海鑫谱生物科技有限公司 | A kind of fluorescence analysis method and device |
| JP6709745B2 (en) * | 2017-03-14 | 2020-06-17 | デンカ生研株式会社 | Immunochromatographic test strip for extracting and measuring sugar chain antigens that can control the development of specimens |
| CN110146693B (en) * | 2019-05-09 | 2022-07-29 | 润和生物医药科技(汕头)有限公司 | Nitrocellulose membrane with fluorescent dye and production method thereof |
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| EP0811626B1 (en) * | 1996-06-04 | 2002-08-14 | Roche Diagnostics GmbH | Use of metallo-porphyrin conjugates for the detection of biological substances |
| KR20040064054A (en) * | 2003-01-09 | 2004-07-16 | 바디텍메드 주식회사 | Strips and fluoresence scanner for the measurement of high sensitivity c-reactive protein in serum and whole blood |
| US20050112703A1 (en) * | 2003-11-21 | 2005-05-26 | Kimberly-Clark Worldwide, Inc. | Membrane-based lateral flow assay devices that utilize phosphorescent detection |
| CN1563951A (en) * | 2004-03-19 | 2005-01-12 | 中国科学院长春应用化学研究所 | Double marking fast immunodetection test paper for collidal gold and semiconductor fluorescent nano particle |
| CN1645146A (en) * | 2005-02-03 | 2005-07-27 | 厦门大学 | Immune chromatography with fluorescent rare earth nanometer particle as marker and detecting testing paper strip |
| CN100367034C (en) * | 2005-10-20 | 2008-02-06 | 上海交通大学 | Method for measuring immunologic colloidal gold particle fluorescence quenching |
| US7732153B2 (en) * | 2006-05-09 | 2010-06-08 | Beckman Coulter, Inc. | Nonseparation assay methods |
| US20090104635A1 (en) * | 2007-10-19 | 2009-04-23 | Tom Cheng Xu | Fluorescent Dry Test Strip Biosensor |
| WO2009152209A2 (en) * | 2008-06-10 | 2009-12-17 | Rapid Pathogen Screening, Inc. | Combined visual/fluorescence analyte detection test |
| JP5100541B2 (en) * | 2008-07-04 | 2012-12-19 | 古河電気工業株式会社 | Immunochromatographic conjugate pad containing fluorescent particles and colored particles as labeled particles, immunochromatographic test strip using the same, and inspection method |
| CN102033124A (en) * | 2009-12-25 | 2011-04-27 | 北京博晖创新光电技术股份有限公司 | Immunofluorescence detection device and detection method |
| CN102103145B (en) * | 2011-02-24 | 2013-01-16 | 南京基蛋生物科技有限公司 | Colloidal gold test strip for double-amplification system and preparation method thereof |
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