CN105929155A - Immuno-chromatographic test paper and detection method thereof - Google Patents
Immuno-chromatographic test paper and detection method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N33/532—Production of labelled immunochemicals
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Abstract
The invention discloses immuno-chromatographic test paper which comprises a combination pad, a test line and a quality control line, wherein a marked detection antibody or a long afterglow luminescent nanoparticle for detecting an antigen is arranged on the combination pad; the test line is coated with a capturing antibody or a capturing antigen aiming at a target in a sample to be detected; the quality control line is coated with an antibody for identifying a detection antibody or a detection antigen marked on the long afterglow luminescent nanoparticle. The immuno-chromatographic test paper has the advantages that by the use of the unique long afterglow luminescent performance of a long afterglow luminescent material, an afterglow light signal is used as an accurate quantitative reading detection result; and an interference signal is effectively eliminated, and the detection sensitivity and the quantification accuracy of an object to be detected are greatly improved.
Description
Technical field
The present invention relates to the technical field of immune chromatography test paper, be specifically related to a kind of immune chromatography test paper and detection thereof
Method.
Background technology
It is current in-vitro diagnosis (in the scene of thing to be detected in biological sample, quick, sensitive and detection by quantitative
Vitro diagnosis, IVD) key element pursued.In the last few years, biology based on immuno-chromatographic test paper strip
Medical science detects, and has the advantages such as quick, easy, low cost because of it increasingly by researcher and medical worker
Favor.Immune chromatography test paper technology is based on Ag-Ab specific recognition combination principle, certain having
The nitrocellulose filter in aperture is with under capillarity, and the indicator that marked antibody can specific recognition and combination
Such as the particular test in the biological specimens such as urine sample, blood sample or saliva, and ELISA test strip area stay with
Colour developing, and detection by quantitative can be carried out in conjunction with readout instrument.
In the last few years owing to the constantly innovation of medical industry is with progressive, to involved immuno-chromatographic test paper strip
Detection by quantitative requires also to increased.Apply the earliest early as the immune chromatography test paper of indicator based on gold colloidal
Pregnant detection field, has expanded to multiple field the most, such as biomedicine, food safety, environment, pesticide residues
Deng, achieve huge success.But it is the most helpless in detection by quantitative and super sensitivity detection, and it fills it
Amount can only be defined as sxemiquantitative conventional sense.Therefore, fast development based on society, it is badly in need of one or more energy
The immune chromatography test paper method of rapid sensitive detection by quantitative.
Grew up in recent years based on fluorescent nano particles (as rare earth doped luminophor or organic fluorescence contaminate
The polystyrene nanoparticle of material molecule and fluorescence quantum) although the immune chromatography test paper as indicator is being determined
Improve in amount and Sensitive Detection, but due to during quantitative readout detection, it is necessary to assure exciting light is always
It is radiated on test strips region, due to during excitation light irradiation, the nitrocellulose filter in test strips and life
Thing sample itself also can be excited to send fluorescence, causes quantitative readout to detect the luminous quantity obtained, not only contains work
For the light quantity that is stimulated of the fluorescent nano particles of detection target, further comprises the celluloid as interference signal
Film and the light quantity that is stimulated of biological sample itself, reduce the noise of fluorescent nano particles indicator and background signal
Ratio, thus weaken detection sensitivity and the dosing accuracy treating detectable substance.Especially for detection sensitivity
The detection project having higher requirements with accuracy, such as diseases such as infectious disease, myocardial damage, infection classes, one
As traditional immune chromatography test paper be difficult to meet testing requirement.
Therefore, market is badly in need of a kind of detection sensitivity luminescent indicator immune chromatography test paper the most accurately high, quantitative
And quantitative detecting method.This is the place that the application needs to improve emphatically.
Summary of the invention
The technical problem to be solved is to provide for a kind of effectively elimination and disturbs luminous signal, it is possible to promote
Detection by quantitative sensitivity and the immune chromatography test paper of accuracy.
Another technical problem to be solved by this invention is intended to offer can promote detection by quantitative sensitivity with accurate
The quantitative detecting method of the immune chromatography test paper of degree.
A kind of immunity-chromatography test is the technical scheme is that in order to solve first technical problem of the present invention
Paper, including conjugate pad, p-wire and nature controlling line；Described conjugate pad is provided with marked detection antibody or inspection
Survey the long-persistence luminous nanoparticle of antigen.
In such scheme, described p-wire is coated with for the capture antibody of object in detected sample or capture
Antigen；Described nature controlling line is coated with for identifying marked detection antibody on long-persistence luminous nanoparticle
Antibody or the antibody of detection antigen.
In such scheme, described immune chromatography test paper also includes base plate, sample pad, nitrocellulose filter and water suction
Pad, described base plate overlaps sample pad, conjugate pad, nitrocellulose filter and adsorptive pads, described test successively
Line and nature controlling line are located on described nitrocellulose filter.
In such scheme, described sample pad is glass fibre membrane or polyester fiber film；Described conjugate pad is glass
Fibrous membrane；Described base plate is polyvinyl chloride plastic sheet.
In such scheme, described immune chromatography test paper also includes being arranged on getting stuck of outside, described in get stuck and be provided with
For dripping the sample application zone of detection sample and for observing or read the colour developing district of signal；Described detection sample is blood
Clearly, one or more in whole blood, urine and tissue fluid.
In such scheme, being smaller in size than equal to 300 nanometers of described long-persistence luminous nanoparticle, and be more than
In 5 nanometers.
The persistence of described long-persistence luminous nanoparticle be no less than 30 seconds, preferably greater than 1 minute.
Described long-persistence luminous nanoparticle preferably employs following a few class:
(1) sulfide type long-afterglow material, such as ZnS:Cu2+、CaS:Eu2+、SrGa2S4:Eu2+Or Y2O2S:Eu3+。
(2) aluminate long afterglow materials, especially Eu2+The rare earth aluminate series long-afterglow material activated, its
Middle green light better performances have SrAlO4:Eu2+,Dy3+, blue light-emitting better performances have CaAlO4:Eu2+,Nd3+。
(3) silicate long-afterglow material, such as Ba5Si8O21:Eu2+,Dy3+。
(4) titanate long-afterglow material, such as CaTiO3:Pr3+, and add supplementary doping element or electric charge
The CaTiO of compensation3:Pr3+。
(5) gallate long-afterglow material, such as ZnGa2O4:Cr3+, and add supplementary doping element or electric charge
The ZnGa2O4:Cr of compensation3+。
The present invention utilizes long-persistence luminous nanoparticle as indicator, it is provided that a kind of detection sensitivity and detection standard
The immune chromatography test paper that exactness is high.Concrete scheme is that antibody or the antigen of detection object are passed through chemical coupling
Method is tagged on long-persistence luminous nanoparticle, then by good for labelling antibody or the long-persistence luminous nanometer of antigen
Particle is located on conjugate pad, for detecting the object in testing sample；The survey set up on nitrocellulose filter
Examination line is coated with the capture antibody for object or antigen in advance；The nature controlling line thing set up on nitrocellulose filter
First it is coated with for the antibody being marked on long-persistence luminous nanoparticle, or the antibody of antigen.Above-mentioned conjugate
Pad and be coated with the nitrocellulose filter of p-wire and nature controlling line with other sample pad, adsorptive pads, get stuck and base plate
The immune chromatography test paper forming the present invention is assembled according to tradition reagent paper manufacture method.
Described immune chromatography test paper is the technical scheme is that in order to solve second technical problem of the present invention
Detection method, step is as follows:
(1) in sample pad, detected sample is instilled；
(2) with excitation light irradiation p-wire and nature controlling line；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
In such scheme, the wavelength of exciting light used is 254nm to 488nm, and irradiation power is 50mW/cm2
To 300mW/cm2, irradiation time is 5 seconds to 1 minute.
Superior effect of the present invention is: the present invention uses long-persistence luminous nanoparticle to replace traditional fluorescence to receive
Rice corpuscles is used for traget antibody or antigen, owing to long after glow luminous material has the long-persistence luminous performance of uniqueness,
After it absorbs the energy of excitation light irradiation in advance, close excitation light irradiation and still can launch photon signal for a long time, send out
The time of penetrating is a few hours；But the nitrocellulose filter in test strips and biological sample itself do not have twilight sunset
Performance, will be no longer luminous when exciting light no longer irradiates, and i.e. will not produce the optical signal of interference；So exciting
After light irradiates and closes, recycling photon readout instrument reads the after-glow light signal of long-persistence luminous nanoparticle,
The after-glow light signal arrived is as quantitative readout testing result accurately；Therefore, the present invention effectively eliminates nitric acid fibre
Dimension element film and the interference signal being excited to produce of biological sample itself, the detection that significant increase treats detectable substance is clever
Sensitivity and dosing accuracy.
Accompanying drawing explanation
The Figure of description of the part constituting the application is used for providing a further understanding of the present invention, the present invention
Schematic description and description be used for explaining the present invention, be not intended that inappropriate limitation of the present invention.Attached
In figure:
Fig. 1 is the structural representation of the present invention；
Label declaration in figure
1 PVC base plate；2 sample pad；
3 conjugate pads；4 nitrocellulose filters；
5 adsorptive pads；6 p-wires；
7 nature controlling lines.
Detailed description of the invention
Below in conjunction with accompanying drawing, embodiments of the invention are described in detail, but the present invention can be by claim
The multitude of different ways limited and cover is implemented.
Embodiments of the invention are described in detail below in conjunction with Fig. 1.Fig. 1 shows that the structure of the embodiment of the present invention is shown
It is intended to.
Embodiment 1
The present embodiment is a kind of immune chromatography test paper for detection by quantitative alpha-fetoprotein (AFP), as it is shown in figure 1,
The present invention provides a kind of immune chromatography test paper, including PVC base plate 1, base plate 1 is sequentially provided with sample pad 2,
Conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；On nitrocellulose filter 4 along from sample pad 2 to
The direction of adsorptive pads 5, is also sequentially provided with p-wire 6 and nature controlling line 7.The finger of arrow in chromatography direction such as Fig. 1
Show direction.
Conjugate pad 3 is provided with the marked long-persistence luminous nanoparticle having detection antibody.This detection antibody is
The antibody (anti-AFP antibody) of energy specific detection alpha-fetoprotein (AFP)；
Another antibody of energy specificity capture alpha-fetoprotein (AFP) it is coated with on p-wire 6；
It is coated with on nature controlling line 7 and can capture the antibody of the antibody of labelling on long-persistence luminous nanoparticle by specificity
(two resist).
The molecular structural formula of long-persistence luminous nanoparticle used is ZnGa2O4:Cr3+, its particle size is 15
Nanometer.The preparation method of this long-persistence luminous nanoparticle is as follows: configuration 1.0M zinc nitrate (Zn (NO3)2) water
Solution, 1.0M Ganite (Fujisawa). (Ga (NO3)3) aqueous solution and 0.001M chromic nitrate (Cr (NO3)3) aqueous solution.Press
Calculating and measure 1mmol zinc nitrate according to above-mentioned concentration, 1mmol nitric acid is transferred, 0.002mmol chromic nitrate,
And with deionized water, system solution volume is increased to 16mL, after stirring and evenly mixing, to above-mentioned in whipping process
Rapidly joining ammonia (28%, wt) in system solution makes system solution pH be 9.5, continues stirring half under room temperature
Proceed to after hour in the polytetrafluoroethyllining lining that capacity is 40mL, then liner put in hydrothermal reaction kettle,
200 DEG C carry out hydro-thermal reaction 12 hours, naturally cool to room temperature, and centrifugal (12000g, 15min) obtains
White depositions, this precipitate 0.02M aqueous hydrochloric acid solution disperses, and obtains optically transparent solution, then to this
Solution adds isopyknic isopropanol, the precipitate vacuum drying that centrifugal (12000g, 20min) obtains
After be long-persistence luminous nanoparticle.Utilize this long-persistence luminous nanoparticle of transmission electron microscope observation
A size of 15nm, the long-persistence luminous nanoparticle obtained is through 405 nanometer LED light source exciting irradiations 30
After Miao, (irradiation power is 100mW/cm2), stop irradiating, through spectrogrph detect its long afterglow launch time up to
15 minutes, a length of 696 nanometers of transmitted wave, fully meet the detection by quantitative of immuno-chromatographic test paper strip readout instrument.
Applying for subsequent bio traget antibody (or antigen), this long-persistence luminous nanoparticle also needs surface to introduce
It is available for the functional group of chemical coupling.It is embodied as follows: long-persistence luminous by size 15 nanometer of above-mentioned acquisition
Nanoparticle (1.0g) dissipates (power 80w, ultrasonic time 5min) by water bath sonicator wavelength-division and is scattered in 20
In mL dehydrated alcohol, add 0.025g 3-aminopropyl triethoxysilane (APTES), be stirred at room temperature anti-
Answer 48 hours.Precipitate is left and taken in centrifugation (10000g, 15min), after utilizing dehydrated alcohol to disperse again
Secondary centrifugal (10000g, 15min), the precipitate obtained is i.e. amidized long-persistence luminous nanoparticle,
Briefly molecular structural formula is ZnGa2O4:Cr3+-NH2,-NH2For the ammonia introduced in long-persistence luminous nanoparticle surface
Base functional group, can be used for antibody labeling.
The antibody labeling of the long-persistence luminous nanoparticle of amination.This antibody labeling method is common method.This
Bright alpha-fetoprotein (AFP) of choosing is illustrated by as a kind of disease model.Labeling method is as follows: weigh above
The long-persistence luminous nanoparticle of amination of 10mg size 15 nanometer prepared, is dispersed in 1.0mL and contains 5%
In the phosphate buffered solution (PBS) of glutaraldehyde (0.01M, pH7.2), room temperature reaction 2 hours；Obtain
The long-persistence luminous nanoparticle of aldehyde radicalization washed by centrifugation after (10000g, 15min), be redispersed in
In 1.0mL PBS (0.01M, pH7.2) solution, add 0.05mg AntiAFP antibody (anti-AFP antibody),
Room temperature reaction 2 hours；By centrifuge washing (10000g, 15min), after removing unreacted free antibodies,
The centrifugal precipitate obtained is i.e. long-persistence luminous nanoparticle antibody complex, the brief molecule knot of this complex
Structure formula is ZnGa2O4:Cr3+-anti-AFP antibody。
Tradition, according to tradition reagent paper manufacture method, is exempted from the present invention by the immune chromatography test paper that the present invention relates to
Colloidal gold antibody complex used in epidemic disease chromatographic test paper, or fluorescence nano latex antibody complex replaces with this
Long-persistence luminous nanoparticle antibody complex prepared by invention, it is therefore an objective to by eliminating background fluorescence signal,
Strengthen signal to noise ratio, be greatly enhanced detection sensitivity and the detection accuracy of thing to be detected.Based on long-persistence luminous
The immune chromatography test paper of nanoparticle antibody complex makes and is briefly described below: by the above-mentioned 5.0mg prepared
Long-persistence luminous nanoparticle antibody complex dissolves and is dispersed in the specific diluent of 10mL (such as borate buffer solution
(50mM, pH8.2)) in, then it is evenly applied on glass fibre element film, i.e. can be used as conjugate pad 3；
Another antibody and two anti-being coated in respectively on nitrocellulose filter 4 of thing to be detected are formed p-wire 6 (T line)
With nature controlling line 7 (C line)；Above-mentioned another involved antibody and the width of two anti-zoned line concentration, T line and C line
And the spacing distance of the two, drying technique for fixing parameter are all with reference to existing mature technology.Finally above-mentioned stroke is had
Another antibody and two anti-nitrocellulose filters, it is coated with the glass of long-persistence luminous nanoparticle antibody complex
Cellulose membrane (conjugate pad), glass fibre or polyester film sample pad 2, adsorptive pads 5 glue according to the schematic diagram of Fig. 1
Patch is assembled on PVC base plate 1, obtains complete immune layer based on long-persistence luminous nanoparticle antibody complex
Analysis reagent paper.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 30 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antibody complex carries out energy supply；
(3) after stopping excitation light irradiation, then believe with luminescence reader read test line 6 and the luminous of nature controlling line 7
Number.Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into the public affairs of " the AFP concentration known-optical signal " demarcated in advance
Formula, draws the concentration of this detected AFP.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence nano material
Unique luminous advantage, it is only necessary to use excitation before reading, and without again in follow-up reading process
Secondary excite；This method eliminates the interference of background fluorescence signal, the highly sensitive detection by quantitative of determinand can be realized.
By the detection of AFP being contrasted with conventional fluorescent immune chromatography test paper, find long afterglow based on the present invention
The immune chromatography test paper of luminescent nanoparticle antibody complex improves 12 times in detection sensitivity；But to detection
Sample treatment requires and does not has significant change on the detection used time.
Embodiment 2
The present embodiment is a kind of immune chromatography test paper for detection by quantitative tuberculosis (TB) antibody, as it is shown in figure 1,
The present invention provides a kind of immune chromatography test paper, including PVC base plate 1, PVC base plate 1 is sequentially provided with sample pad
2, conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；Along from sample pad 2 on nitrocellulose filter 4
To the direction of adsorptive pads 5, also it is sequentially provided with p-wire 6 and nature controlling line 7.
Described conjugate pad 3 is provided with marked has detection antigen and the long-persistence luminous nanoparticle of rabbit igg.
This detection antigen is TB recombinant antigen (CTK company's T B fused antigen, the TB of energy specific detection tuberculosis antibody
antigen)；
The antigen of energy specificity capture tuberculosis antibody it is coated with on described p-wire 6；
It is coated with on described nature controlling line 7 and can capture the rabbit igg of labelling on long-persistence luminous nanoparticle by specificity
Antibody.
Long-persistence luminous nanoparticle used is with embodiment 1.
Applying for subsequent bio labelled antigen, this long-persistence luminous nanoparticle also needs surface to introduce and is available for chemistry occasionally
The functional group of connection.It is embodied as follows: by the long-persistence luminous nanoparticle of size 15 nanometer of above-mentioned acquisition
(1.0g) dissipate (power 80w, ultrasonic time 5min) by water bath sonicator wavelength-division and be scattered in 10mL Sanguis Bovis seu Bubali
In pure albumen (BSA) aqueous solution (2.0%, wt), centrifugation after being stirred at room temperature 6 hours (10000g,
25min), the precipitate obtained is i.e. the long-persistence luminous nanoparticle after BSA surface is modified, and brief molecule is tied
Structure formula is ZnGa2O4:Cr3+-BSA ,-BSA are the bovine serum albumin introduced in long-persistence luminous nanoparticle surface
In vain, with carboxyl functional group, can be used for antigen and rabbit igg labelling.
The antigen of the long-persistence luminous nanoparticle that BSA modifies and rabbit igg labelling.This labeling method is conventional side
Method.This example is chosen tuberculosis antibody and is illustrated by as a kind of disease model.Labeling method is as follows: weigh above
The long-persistence luminous nanoparticle that the BSA of 10mg size 15 nanometer prepared modifies is dispersed in 1.0mL PBS
In (0.01M, pH7.2) solution, add 0.05mg TB recombinant antigen and 0.05mg rabbit igg, mix backward
Above-mentioned system adds 8.0mg/mL carbodiimide hydrochloride (EDC HCl) the aqueous solution 20 μ L of fresh configuration,
After room temperature reaction 2 hours, by centrifuge washing (10000g, 25min), remove unreacted floating preteins
After, the centrifugal precipitate obtained is i.e. long-persistence luminous nanoparticle antigenic compound.
The manufacture method of described immune chromatography test paper is with embodiment 1.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad 3, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 25 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antigenic compound carries out energy supply；
(3) after stopping excitation light irradiation, then believe with luminescence reader read test line 6 and the luminous of nature controlling line 7
Number.Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into prior " the TB antibody concentration known-optical signal " demarcated
Formula, draws the concentration of this detected TB antibody.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence nano material
Unique luminous advantage, it is only necessary to use excitation before reading, and without again in follow-up reading process
Secondary excite；This method eliminates the interference of background fluorescence signal, it is achieved the highly sensitive detection by quantitative of determinand.
By the detection of TB antibody being contrasted with conventional fluorescent immune chromatography test paper, find more than length based on the present invention
The immune chromatography test paper of brightness luminescent nanoparticle antigenic compound improves 8 times in detection sensitivity；But to inspection
Survey sample treatment require and there is no significant change on the detection used time.
Embodiment 3
The present embodiment is a kind of immune chromatography test paper for detection by quantitative flesh calcium egg I (cTnI), such as Fig. 1 institute
Showing, the present invention provides a kind of immune chromatography test paper, including PVC base plate 1, PVC base plate 1 is sequentially provided with sample
Product pad 2, conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；Along from sample on nitrocellulose filter 4
Product pad 2, to the direction of adsorptive pads 5, is also sequentially provided with p-wire 6 and nature controlling line 7.
Described conjugate pad 3 is provided with the marked long-persistence luminous nanoparticle having detection antibody.This detection resists
Body is the antibody (anti-cTnI antibody) of energy specific detection flesh calcium egg I；
Another antibody of energy specificity capture flesh calcium egg I (cTnI) it is coated with on described p-wire 6；
It is coated with on described nature controlling line 7 and can capture the antibody of labelling on long-persistence luminous nanoparticle by specificity
Antibody (two resist).
The molecular structural formula of long-persistence luminous nanoparticle used is CaTiO3:Pr3+@lpSi02, its particle size
It is 250 nanometers.lpSi02For macroporous silica nanoparticle.The preparation side of this long-persistence luminous nanoparticle
Method is as follows: (1) weighs 0.1 gram of polystyrene-b-polyacrylic acid amphiphilic diblock copolymer
PS (20000)-b-PAA (900), the molecular weight related to is number-average molecular weight, is dissolved in 30 as porogen
In the oxolane of mL, stir and add 60mL deionized water to abundant dissolving, sharp after continuing stirring 2 hours
With adding ammonia by above-mentioned system solution pH regulator to 11.5, add 0.2 gram of TEOS, room temperature reaction 24 hours,
Leaving and taking precipitate after centrifugation (8000g, 15min), disperse with dehydrated alcohol, recentrifuge separates
Leaving and taking precipitate after (8000g, 15min), 80 degrees Celsius are dried, and under 600 degrees Celsius, calcining removes pore
Agent, utilizes high-resolution projection electron microscope to learn the macroporous silica nanometer being obtained a size of 250 nanometers
Particle, utilizes BET surface analysis and N2Absorption-De contamination method learns that the specific surface area of product reaches 450
m2/ g, duct volume is 3.2cm3/ g, macropore pore size about 22.2nm, utilize small angle x ray scattering (SAXS)
Characterize and small-angle x-ray diffractometer is obtained and known that the macropore of obtained macroporous silica nanoparticle has six sides
Pore passage structure, the result observed with projection electron microscope is consistent.Obtained big hole path as micro-reaction
Device is for the synthesis of long-afterglow material.(2) 1.18g calcium nitrate tetrahydrate (Ca (NO is weighed3)2·4H2O),
0.008g praseodymium nitrate (Pr (NO3)3), 1.05g citric acid, (molecular weight is one to 0.5g Polyethylene Glycol PEG
Ten thousand) and 2.2mL butyl titanate, dissolving in 30mL deionized water by the above-mentioned thing that weighs, stirring is to the most molten
Solution state, with the salpeter solution of 0.5M by after the pH regulator of above-mentioned mixed solution to 4.0, adds 2.0g
The above-mentioned macroporous silica nanoparticle prepared, by water bath sonicator wavelength-division dissipate (power 80w, time ultrasonic
Between 5min), be stirred at room temperature 6 hours, so that macroporous silica nanoparticle adsorbing metal ions, then
Centrifugation (8000g, 15min) obtains the large hole nano grade silica particle being filled with metal ion.
The centrifugal precipitate 80 degrees Celsius obtained is dried and within 2 hours, is placed on the argon gas containing 1%～12% reductive hydrogen
In atmosphere, calcine 2 hours in 650 DEG C, i.e. obtain the long afterglow silica dioxide nano particle of about 250nm monodispersity
Son.After 405nm LED light source exciting irradiation 60s, (irradiation power is 100 to obtained nanoparticle
mW/cm2), stop irradiating, detect its long afterglow launch time up to 1.5 hours through spectrogrph, launch wavelength
For 615nm, fully meet readout instrument detection by quantitative.Its brief molecular structural formula is CaTiO3:Pr3+@lpSi02,
Wherein lp is the acronym of macropore (large pore).
Applying for subsequent bio traget antibody, this long-persistence luminous nanoparticle also needs surface to introduce and is available for chemistry occasionally
The functional group of connection.It is embodied as follows: by the long-persistence luminous nanoparticle of size 250 nanometer of above-mentioned acquisition
(1.0g) it is scattered in 20mL by water bath sonicator wavelength-division scattered (power 80w, ultrasonic time 5min) anhydrous
In ethanol, add 0.05g 3-aminopropyl triethoxysilane (APTES), reaction 48 is stirred at room temperature little
Time.Precipitate is left and taken in centrifugation (10000g, 15min), utilizes recentrifuge after dehydrated alcohol dispersion
(10000g, 15min), the precipitate obtained is i.e. amidized long-persistence luminous nanoparticle, briefly divides
Subformula is CaTiO3:Pr3+@lpSi02-NH2,-NH2For the ammonia introduced in long-persistence luminous nanoparticle surface
Base functional group, can be used for antibody labeling.
The antibody labeling of the long-persistence luminous nanoparticle of amination.This antibody labeling method is common method.This
The bright Troponin I chosen in myocardial injury markers is used as a kind of disease model and is illustrated by.Labeling method
As follows: to weigh the amination long-persistence luminous nanoparticle dispersion of good 20mg size 250 nanometer made above
In 2.0mL contains the phosphate buffered solution (PBS) of 5% glutaraldehyde (0.01M, pH7.2), room temperature is anti-
Answer 2 hours；After the long-persistence luminous nanoparticle of the aldehyde radicalization obtained is washed by centrifugation (8000g, 15
Min), it is redispersed in 1.0mL PBS (0.01M, pH7.2) solution, adds 0.2mg anti-troponin I
Antibody (anti-cTnI antibody), room temperature reaction 2 hours；By centrifuge washing (8000g, 15min),
After removing unreacted free antibodies, the centrifugal precipitate obtained is i.e. that long-persistence luminous nanoparticle antibody is combined
Thing, the brief molecular structural formula of this complex is CaTiO3:Pr3+@lpSi02-anti-cTnI antibody。
The making of described immune chromatography test paper is with embodiment 1.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad 3, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 30 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antibody complex carries out energy supply；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into the public affairs of " the cTnI concentration known-optical signal " demarcated in advance
Formula, draws the concentration of this detected cTnI.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence nano material
Unique luminous advantage, it is only necessary to use excitation before reading, and without again in follow-up reading process
Secondary excite；This method eliminates the interference of background fluorescence signal, it is achieved the highly sensitive detection by quantitative of determinand.
By the detection of AFP being contrasted with conventional fluorescent immuno-chromatographic test paper strip, find more than length based on the present invention
The immune chromatography test paper of brightness luminescent nanoparticle antibody complex improves 16 times in detection sensitivity；But to inspection
Survey sample treatment require and there is no significant change on the detection used time.
Embodiment 4
The present embodiment is a kind of immune chromatography test paper of syphilis (TP) antibody in detection by quantitative blood, such as figure
Shown in 1, the present invention provides a kind of immune chromatography test paper, including PVC base plate 1, PVC base plate 1 sets successively
There are sample pad 2, conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；On nitrocellulose filter 4 along
From sample pad 2 to the direction of adsorptive pads 5, also it is sequentially provided with p-wire 6 and nature controlling line 7.
Described conjugate pad 3 is provided with marked has detection antigen and the long-persistence luminous nanoparticle of rabbit igg.
This detection antibody is the recombinant antigen of syphilis (TP) antibody in energy specific detection blood；
It is coated with on described p-wire 6 and can capture the antigen of syphilis (TP) antibody in blood by specificity；
It is coated with on described nature controlling line 7 and can capture the rabbit igg of labelling on long-persistence luminous nanoparticle by specificity
Antibody.
Long-persistence luminous nanoparticle used and surface functionalization method are with embodiment 3.
The antigenic mark of the long-persistence luminous nanoparticle of amination.This antibody labeling method is common method.This
Bright syphilis antibody of choosing in blood is illustrated by as a kind of disease model.Labeling method is as follows: weigh above system
The long-persistence luminous nanoparticle of amination of 10mg size 250 nanometer got ready is dispersed in 1.5mL and contains 5%
In the phosphate buffered solution (PBS) of glutaraldehyde (0.01M, pH7.2), room temperature reaction 2 hours；Obtain
The long-persistence luminous nanoparticle of aldehyde radicalization washed by centrifugation after (8000g, 15min), be redispersed in
In 1.0mL PBS (0.01M, pH7.2) solution, add 0.15mg TP recombinant antigen and 0.15mg rabbit igg,
Room temperature reaction 2 hours；By centrifuge washing (8000g, 15min), after removing unreacted free antigen,
The centrifugal precipitate obtained is i.e. long-persistence luminous nanoparticle antigenic compound.
The making of described immune chromatography test paper is with embodiment 1.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad 3, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 40 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antigenic compound carries out energy supply；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into prior " the TP antibody concentration known-optical signal " demarcated
Formula, draws the concentration of this detected TP antibody.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence luminous nanometer material
Unique luminous advantage of material, it is only necessary to use excitation before reading, and in follow-up reading process nothing
Need to again excite；This method eliminates the interference of background fluorescence signal, it is achieved the highly sensitive detection by quantitative of determinand.
By the detection of TP being contrasted with conventional fluorescent immune chromatography test paper, find that long afterglow based on the present invention is sent out
The immune chromatography test paper of light nanoparticle antigenic compound improves 12 times in detection sensitivity；But to detection sample
Product process and require and do not have significant change on the detection used time.
Embodiment 5
The present embodiment is a kind of immune chromatography test paper of c reactive protein (CRP) in detection by quantitative blood, as
Shown in Fig. 1, the present invention provides a kind of immune chromatography test paper, including PVC base plate 1, on PVC base plate 1 successively
It is provided with sample pad 2, conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；Edge on nitrocellulose filter 4
From sample pad 2 to the direction of adsorptive pads 5, be also sequentially provided with p-wire 6 and nature controlling line 7.
Described conjugate pad 3 is provided with the marked long-persistence luminous nanoparticle having detection antibody.This detection resists
Body is the antibody (anti-CRP antibody) of c reactive protein (CRP) in energy specific detection blood；
Another antibody of energy specificity capture c reactive protein (CRP) it is coated with on described p-wire 6；
It is coated with on described nature controlling line 7 and can capture the antibody of labelling on long-persistence luminous nanoparticle by specificity
Antibody (two resist).
The molecular structural formula of long-persistence luminous nanoparticle used is ZnGaGe2O4:Cr3+, its particle size is 25
Nanometer.The preparation method of this long-persistence luminous nanoparticle is as follows: configuration 1.0M zinc nitrate (Zn (NO3)2) water
Solution, 1.0M Ganite (Fujisawa). (Ga (NO3)3) aqueous solution, 1.0M nitric acid germanium (Ge (NO3)2) aqueous solution, 0.001
M chromic nitrate (Cr (NO3)3) aqueous solution.Calculate and measure 2mmol zinc nitrate, 3mmol according to above-mentioned concentration
Nitric acid is transferred, 0.8mmol nitric acid germanium, 0.002mmol chromic nitrate, and with deionized water by system solution volume
Increase to 20mL, after stirring and evenly mixing, rapidly join in above-mentioned system solution in whipping process ammonia (28%,
Wt) making system solution pH is 9.0, proceeds to, after continuing to stir half an hour, the polytetrafluoroethyl-ne that capacity is 40mL under room temperature
In alkene liner, then being put into by liner in hydrothermal reaction kettle, 220 DEG C carry out hydro-thermal reaction 10 hours, the coldest
But to room temperature, centrifugal (10000g, 15min) obtains white depositions, this precipitate 0.01M hydrochloric acid
Aqueous dispersion, obtains optically transparent solution, then adds isopyknic isopropanol in this solution, centrifugal
It is long-persistence luminous nanoparticle after the precipitate vacuum drying that (10000g, 20min) obtains.Utilize
The size of this long-persistence luminous nanoparticle of transmission electron microscope observation is 25 nanometers, and the long afterglow obtained is sent out
After 405nm LED light source exciting irradiation 40s, (irradiation power is 100mW/cm to light nanoparticle2), stop
Only irradiate, detect its long afterglow launch time up to 1 hour through spectrogrph, a length of 697nm of transmitted wave, completely
Meet immune chromatography test paper readout instrument detection by quantitative.Its brief molecular structural formula is ZnGaGe2O4:Cr3+。
The surface functionalization method of this long-persistence luminous nanoparticle is with embodiment 2.
The antibody labeling of the long-persistence luminous nanoparticle that BSA modifies.This labeling method is common method.This reality
Example is chosen c reactive protein in blood and is illustrated by as a kind of disease model.Labeling method is as follows: weigh above
The long-persistence luminous nanoparticle that the BSA of 15mg size 25 nanometer prepared modifies is dispersed in 1.0mL PBS
In (0.01M, pH7.2) solution, add 0.08mg anti crp antibody, mix backward above-mentioned system and add new
8.0mg/mL carbodiimide hydrochloride (EDC HCl) the aqueous solution 20 μ L of fresh configuration, room temperature reaction 2 is little
Shi Hou, by centrifuge washing (10000g, 25min), after removing unreacted free antibodies, is centrifuged and obtains
Precipitate be i.e. long-persistence luminous nanoparticle antibody complex.
The making of described immune chromatography test paper is with embodiment 1.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad 3, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 30 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antibody complex carries out energy supply；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into the public affairs of " the CRP concentration known-optical signal " demarcated in advance
Formula, draws the concentration of this detected CRP.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence luminous nanometer material
Unique luminous advantage of material, it is only necessary to use excitation before reading, and in follow-up reading process nothing
Need to again excite；This method eliminates the interference of background fluorescence signal, it is achieved the highly sensitive detection by quantitative of determinand.
By the detection of CRP being contrasted with conventional fluorescent immune chromatography test paper, find long afterglow based on the present invention
The immune chromatography test paper of luminescent nanoparticle antibody complex improves 15 times in detection sensitivity；But to detection
Sample treatment requires and does not has significant change on the detection used time.
Embodiment 6
The present embodiment is a kind of immune chromatography test paper of HSP70 antibody in detection by quantitative human blood, such as Fig. 1
Shown in, the present invention provides a kind of immune chromatography test paper, including PVC base plate 1, PVC base plate 1 is sequentially provided with
Sample pad 2, conjugate pad 3, nitrocellulose filter 4 and adsorptive pads 5；On nitrocellulose filter 4 along from
Sample pad 2, to the direction of adsorptive pads 5, is also sequentially provided with p-wire 6 and nature controlling line 7.
Described conjugate pad 3 is provided with marked has detection antigen and the long-persistence luminous nanoparticle of rabbit igg.
This detection antigen is the antigen of HSP70 antibody in energy specific detection blood；
The antigen of energy specificity capture HSP70 antibody it is coated with on described p-wire 6；
It is coated with on described nature controlling line 7 and can capture the rabbit igg of labelling on long-persistence luminous nanoparticle by specificity
Antibody.
Long-persistence luminous nanoparticle used and surface functionalization method thereof are with embodiment 5.
The antigenic mark of the long-persistence luminous nanoparticle that BSA modifies.This labeling method is common method.This reality
Example is chosen HSP70 antibody in blood and is illustrated by as a kind of disease model.Labeling method is as follows: weigh above
The long-persistence luminous nanoparticle that the BSA of 25mg size 25 nanometer prepared modifies is dispersed in 3.0mL PBS
In (0.01M, pH7.2) solution, add 0.05mg HSP70 antigen and 0.04mg rabbit igg, mix backward
Above-mentioned system adds 8.0mg/mL carbodiimide hydrochloride (EDC HCl) the aqueous solution 25 μ L of fresh configuration,
After room temperature reaction 2 hours, by centrifuge washing (10000g, 25min), remove unreacted free antigen
After, the centrifugal precipitate obtained is i.e. long-persistence luminous nanoparticle antigenic compound.
The making of described immune chromatography test paper is with embodiment 1.
The quantitative detecting method of the present embodiment is as follows:
(1) on conjugate pad 3, detected sample is instilled；
(2) with excitation light irradiation p-wire 6 and nature controlling line 7；Specifically irradiate C with the LED light source of 405 nanometers
Line and T line region 45 seconds, irradiation power is 100mW/cm2, C line is received with the long-persistence luminous of combination on T line
Rice corpuscles antigenic compound carries out energy supply；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
Specifically utilize luminescence reader, detect the after-glow light signal of long-persistence luminous nanoparticle antibody complex.
Finally the optical signal data recorded is substituted into " the HSP70 antibody concentration known-light letter demarcated in advance
Number " formula, draw the concentration of this detected HSP70 antibody.
The present embodiment utilizes Ag-Ab specific recognition principle, in conjunction with prepared by long-persistence luminous nanometer material
Unique luminous advantage of material, it is only necessary to use excitation before reading, and in follow-up reading process nothing
Need to again excite；This method eliminates the interference of background fluorescence signal, it is achieved the highly sensitive detection by quantitative of determinand.
By the detection of HSP70 antibody being contrasted with conventional fluorescent immune chromatography test paper, find based on the present invention
The immune chromatography test paper of long-persistence luminous nanoparticle antigenic compound improves 9 times in detection sensitivity；But
Detection sample treatment is required and there is no significant change on the detection used time.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for this area
Technical staff for, the present invention can have various modifications and variations.All within the spirit and principles in the present invention,
Any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (9)
1. an immune chromatography test paper, including conjugate pad, p-wire and nature controlling line；It is characterized in that: institute
State conjugate pad and be provided with marked detection antibody or the long-persistence luminous nanoparticle of detection antigen.
Immune chromatography test paper the most according to claim 1, it is characterised in that: described p-wire is coated with pin
To in detected sample object capture antibody or capture antigen；Described nature controlling line is coated with to be marked for identification
The antibody of note detection antibody on long-persistence luminous nanoparticle or the antibody of detection antigen.
Immune chromatography test paper the most according to claim 1, it is characterised in that: described immune chromatography test paper is also
Including base plate, sample pad, nitrocellulose filter and adsorptive pads, described base plate overlaps sample pad, combination successively
Thing pad, nitrocellulose filter and adsorptive pads, described p-wire and nature controlling line are located on described nitrocellulose filter.
Immune chromatography test paper the most according to claim 3, it is characterised in that: described sample pad is glass fibers
Dimension film or polyester fiber film；Described conjugate pad is glass fibre membrane；Described base plate is polyvinyl chloride plastic sheet.
Immune chromatography test paper the most according to claim 3, it is characterised in that: described immune chromatography test paper is also
Including be arranged on outside get stuck, described in get stuck be provided with for drip detection sample sample application zone and for observing
Or read the colour developing district of signal；Described detection sample is the one in serum, whole blood, urine and tissue fluid or many
Kind.
Immune chromatography test paper the most according to claim 1, it is characterised in that: described long-persistence luminous nanometer
Being smaller in size than equal to 300 nanometers of particle, and more than or equal to 5 nanometers.
Immune chromatography test paper the most according to claim 1, it is characterised in that: described long-persistence luminous nanometer
The persistence of particle is no less than 30 seconds.
8. the detection method of immune chromatography test paper as claimed in claim 1, step is as follows:
(1) in sample pad, detected sample is instilled；
(2) with excitation light irradiation p-wire and nature controlling line；
(3) stop after excitation light irradiation, then with luminescence reader read test line and the luminous signal of nature controlling line.
The detection method of immune chromatography test paper the most according to claim 8, it is characterised in that: used sharp
Luminous wavelength is 254nm to 488nm, and irradiation power is 50mW/cm2To 300mW/cm2, irradiation time
It it is 5 seconds to 1 minute.
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Application publication date: 20160907 |