CN105158477A - Quantum dot fluorescent probe and application thereof - Google Patents
Quantum dot fluorescent probe and application thereof Download PDFInfo
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- CN105158477A CN105158477A CN201510329958.XA CN201510329958A CN105158477A CN 105158477 A CN105158477 A CN 105158477A CN 201510329958 A CN201510329958 A CN 201510329958A CN 105158477 A CN105158477 A CN 105158477A
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to a quantum dot fluorescent probe and application thereof. The invention is characterized in that the quantum dot fluorescent probe utilizes a difunctional cross-linking agent long-chain succinimidyl-4-[N-maieimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate] (SMCC) to undergo a cross-linking reaction with an amino group on the surface of an amino quantum dot; a maleimide group is produced on the surface of the quantum dot; a to-be-labeled antibody is reduced by a reducing agent dithiothreitol (DTT) so as to reduce a disulfide bond into a mercapto group; and the mercapto group and the maleimide group form a covalent bond so as to realize labeling of the quantum dot with the antibody. The quantum dot fluorescent probe is applied to (1) detection of one protein oncofetal antigen or (2) detection of a plurality of protein CEA, euron-specific enolase (NSA) and a cytokerain fragment 19CYFRA21-1, wherein the detection limit of detection (1) is 38 pg/ml, and the detection limit of detection (2) is 0.9 ng/ml.
Description
Technical field
The present invention relates to nano biological detection technique field, particularly a kind of quantum dot fluorescence probe, utilize this probe energy highly sensitive detection various biomolecules simultaneously.
Background technology
Can highly sensitive to biomolecule, height detects specifically, is the direction that people make great efforts all the time, all significant in the field such as early detection, quick diagnosis, health detection, environmental monitoring of disease.
Quantum dot is also called semiconductor nano or semi-conductor nano particles, it is a kind of nano particle be made up of II mono-VI race (as caesium cadmium CdSe, cadmium telluride CdTe) or III mono-V race (as InP, InAs) element, particle diameter is generally 1 ~ 10nm, because electronics and hole are by quantum confinement, continuous print band structure becomes the discrete energy levels structure with molecular characterization, can emitting fluorescence after being excited.As a kind of semiconductor nano material of novelty, compared with traditional fluorescent dye, quantum dot has incomparable advantage: the emission spectrum that can be changed quantum dot by the size changing quantum dot, namely can prepare the quantum dot that multiple difference launches different colours fluorescence; The fluorescence that multiple quantum dot can be excited under same light source to produce different colours realizes multi-channel GPS observations, makes operation become simple, low for equipment requirements; Quantum dot has the narrow and emission spectrum of symmetry, and the mutual interference that its fluorescence signal produced produces is smaller; High and the anti-light quenching ability of fluorescence intensity strong (luminous intensity about 100 times of single particle is better than single organic dye molecule).Above-mentioned superior photoluminescent property makes fluorescence quantum become a kind of desirable fluorescent group of potential replacement conventional organic dyes, and receives researchers' more and more concern in the fields such as analysis detection and biomedical spike imaging.
In addition, significant to biological detecting method as the solid support of biochemical reaction using magnetic microsphere.Because of the superparamagnetism that it possesses, without the need to the equipment (as hydro-extractor) of complexity, only by magnet, just the isolation and purification with other materials can be realized, so be widely used in the fields such as immune detection, cell separation and biological macromolecule purifying in simple and effective ground.The high body surface ratio that magnetic microsphere has, make its surface energy in conjunction with a large amount of antibody, along with slight concussion, magnetic microsphere can be dispersed in system uniformly, substantially increase the joint efficiency of antigen and antibody, have and show as the dynamics that holder is better compared with using flat board.
Reading manner at present for quantum dot signal mainly contains two kinds, spectral analysis and Fluorescence image analysis.Spectral analysis mainly realizes quantitative object by the peak value of record quantum dot emission peak, the method requires large to sample volume amount, the fluorescence signal causing quantum dot to produce can be dispersed in a larger volume interior (normally 0.1 ~ 1mL), so need more quantum dot could produce the signal intensity being different from background signal, detection sensitivity is low; In addition, understand some interference between the fluorescent emission signals of different quantum dot, cause testing result inaccurate.And the present invention intend by fluorescence imaging analysis method to the quantum dot fluorescence signal being enriched in magnetic bead surfaces directly read (comprise color distinguish and fluorescence intensity quantitative), quantum dot signal is enriched in magnetic bead surfaces, and sensitivity is highly improved.So far, the research of this respect rarely has report.
Summary of the invention
The object of the present invention is to provide a kind of quantum dot fluorescence probe, and this quantum dot fluorescence probe is used for highly sensitive detection various biomolecules simultaneously.
Main contents of the present invention comprise: bi-functional cross-linking agent long chain sulfosuccinimidyl 4-[N-citraconic acid]-1-carboxylic cyclohexane (Succinimidyl-4-[N-Maleimidomethyl] cyclohexane-1-carboxy-[6-amidocaproate, SMCC) with the amino group generation cross-linking reaction of amino quantum dot surface, dimaleoyl imino is generated at quantum dot surface.Antibody reducing agent dithiothreitol (Dithiothreitol to be marked, DTT) reduce, disulfide bond reduction is become sulfydryl, sulfydryl forms covalent bond with maleimide base group thus again by antibody labeling on quantum dot, thus obtained quantum dot fluorescence probe both can keep the activity of biomolecule, also prevent the non-specific adsorption that the gathering due to quantum dot and biomolecule brings.When biomolecule detection, using magnetic bead or magnetic microsphere as the solid support of reaction, reaction system is suspended in the liquid phase completely, biomolecule to be measured is caught by solution by immune response or molecular hyridization, go to identify the testing molecule on magnetic bead by quantum dot fluorescence probe again, magnetic bead or magnetic microsphere produce the fluorescence of quantum dot probe owing to combining antigen-antibody reaction thing, the color of this fluorescence and the power of signal have reacted kind and the quantity of biomolecule to be measured, image recognition software can be utilized to carry out identifying and quantitatively.
The step of the preparation and determination methods of quantum dot fluorescence probe of the present invention comprises:
(1) preparation of quantum dot fluorescence probe
First, get the amino quantum dot of 10-50 μ L5-50mM long-chain SMCC to 50-500 μ L and activate, 25 DEG C, 30-60min; The method of desalting column is taked to carry out purifying subsequently; With 10-50mMDTT, 100-500 μ L biomolecule is reduced, 25 DEG C, 30-60min; Remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, be stored in immune response after using column chromatography purifying and strengthen liquid (10mM phosphate buffer PBS, PH7.2,1-10% polyglycol (Polyethyleneglycol, PEG), 1-5% bovine serum albumin(BSA) (Bovineserumalbumin, BSA), 1-10% polyvinylpyrrolidone (Polyvinylpyrrolidone, PVP), in, 4 DEG C of lucifuges are for subsequent use.
(2) detection reaction
5000-10000 magnetic capture probe is added in each reaction, 5-50nM quantum dots characterization probe, by Sample dilution (10mMPBS containing 5-10% calf serum) polishing volume to 10-50ul/ reaction, add in the determinand standard items of the variable concentrations of 10-50ul or blank or sample to be tested subsequently; 30-60min is hatched 37 DEG C of concussions; After biochemical reaction completes, be separated magnetic microsphere with magnetic separator, then use cleaning 1-3 time of cleansing solution (0.05% Tween-20 10mMPBS), carry out observing, taking pictures below microscope.
(3) graphical analysis
Because the quantum dot of different-diameter can present different colors under the exciting of ultraviolet light, as red, green, yellow, under utilizing self-programming software GrayQuantifier first image to be transformed into HSI (HueSaturationIntensity) color space, HIS color model H (tone), S (saturation degree), I (brightness) three parameters represent color, different colors correspond to different H values, by calculating the H value of different colours magnetic bead, realize the differentiation to different colours quantum dot.Second step, on the basis distinguishing different quantum dot colors, adds up its gray-scale value to each magnetic bead, and when quantum dot is brighter time, the gray-scale value corresponding to it is larger.Correlationship between the gray-scale value that ultimate analysis records and target substance concentration, and then realize the quantitative detection to target substance.
The feature of fluorescent quantum point probe provided by the invention and detection biomolecule thereof is:
Fluorescent quantum point probe provided by the invention, has that fluorescence signal is decayed by force, not easily, biomolecule activity is good, can realize the highly sensitive detection of various biomolecules.
1) fluorescent quantum point probe quantum dot productive rate provided by the invention is high, fluorescence signal strong, and not easily decays; Quantum dot size used is less, and diameter is 5-8nm, has larger specific surface area, can in conjunction with more biomolecule; Owing to using bi-functional cross-linking agent SMCC by biomolecule leading mark at quantum dot surface, the activity of biomolecule can be kept preferably, avoid the gathering of quantum dot and biomolecule, improve detection sensitivity.Conventional quantum dot marking method adopts 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (1-3-(dimethylamino) propyl-3-ethylcarbodiimidemethiodide, and N-hydroxy thiosuccimide (N-hydroxysulfosuccinimideNHS) activation system EDC), by the carboxyl of quantum dot and the amino-reactive of biomolecule, the carboxyl of antibody also can be activated simultaneously, quantum dot fluorescence productive rate can be caused so low, quantum dot probe is assembled, crosslinked grade for effect is there is between biomolecule, detection sensitivity is caused to reduce.
2) adopt magnetic microsphere to catch biomolecule to be measured and the fluorescence signal of quantum dot fluorescence probe can be enriched in microsphere surface, more weak fluorescence signal just can be detected and obtain, improve the sensitivity of detection.And detection side's rule of routine is dispersed in larger liquid-phase system by limited fluorescence signal molecule, need more fluorescence signal molecule just can detect, detection sensitivity is low.
3) for the quantum dot fluorescence probe of the different-grain diameter be enriched on magnetic microsphere, different colors is presented: green, yellow, red under the exciting of ultraviolet light, employing CCD takes pictures and gray scale scanning software can carry out identification with quantitative to different fluorescence signals very accurately, the interference of signal and background signal between different quantum dot fluorescence probe can be avoided, improve the accuracy and detection sensitivity that detect.And the detection method of the mensuration quantum dot fluorescence spectrum of routine is vulnerable to the fluorescence signal of different quantum dot and measures the interference of solution background signal, cause the accuracy and the sensitivity decline that measure concentration.
Accompanying drawing explanation
Fig. 1 is based on the mensuration schematic flow sheet of fluorescent quantum point probe.A) being wherein a kind of Protein Detection principle schematic, b) is three kinds of Protein Detection principle schematic.
Fig. 2 is based on three kinds of Protein Detection typical curves of fluorescent quantum point probe, wherein Fig. 2 a is the result of the three kind target proteinses of the synchronous detectable concentration scope of the method from 0.9ng/mL to 2000ng/mL, and Fig. 2 b is the linear relationship region (3.9ng/mL to 125ng/mL) in sensing range.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1: the fluorescent quantum point probe that can detect a kind of protein carcinomebryonic antigen (carcinoembryonicantigenCEA)
(1) preparation of quantum dot fluorescence probe
First, get the amino quantum dot 625 (Qdot625) of 10-50 μ L5-50mMSMCC to 50-500 μ L and activate, 25 DEG C, 30-60min; The method of desalting column is taked to carry out purifying subsequently; Detect antibody with 10-50mMDTT to 100-500 μ LCEA to reduce, 25 DEG C, 30-60min; Remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, be stored in 10mMPBS (PH7.2) after separation and purification, 4 DEG C of lucifuges are for subsequent use.
(2) detection reaction
The magnetic capture probe of 5000-10000 in vitro CEA capture antibody mark is added in each reaction, 5-50nMQdot625 detector probe, by Sample dilution (10mMPBS containing 5-10% calf serum) polishing volume to 10-50ul/ reaction, add subsequently the variable concentrations of 10-50ul CEA standard items (2000,1000,500,250,125,62.5,31.2,15.6,7.8,3.9,1.9,0.9,0ng/mL) or sample to be tested in; 30-60min is hatched 37 DEG C of concussions; After biochemical reaction completes, be separated magnetic microsphere with magnetic separator, then use cleaning 1-3 time of cleansing solution (0.05% Tween-20 10mMPBS), carry out observing below microscope, CCD takes pictures.
(3) image and interpretation of result
Qdot625 launches red fluorescence under ultraviolet excitation, the CEA testing result CCD of variable concentrations is taken pictures, self-programming software is utilized to convert HIS pattern to the primaries mode image obtained, it is qualitative that H that is to say that chrominance component can realize multiple color, reaches the object distinguishing target substance.Meanwhile, software also can realize conversion RGB image being changed into gray-scale value, the fluorescence intensity that the quantum dot that each magnetic bead surfaces of statistical study combines produces, and realizes the quantitative detection to target substance CEA.Result shows: CEA concentration carries out linear fit in 0.39 ~ 50ng/mL scope, and equation is: logy=0.5862logx|3.57223 (R
2=0.988), linear relationship is good, higher than the mean value of the signal of blank (not containing the damping fluid of CEA as sample), detection signal is added that its 3 times of standard deviations are defined as detectability by us, calculate the detection of the method is limited to 38pg/mL, far below the clinical threshold value (5ng/mL) of CEA, meet clinical practice demand.
Embodiment 2: the fluorescent quantum point probe that can detect multiple protein CEA, neural enolase (neuron-specificenolaseNSE), cytokeratin fragment 19 (CYFRA21-1)
(1) preparation of quantum dot fluorescence probe
First, get 10-50 μ L5-50mMSMCC amino quantum dot Qdot525, Qdot585, Qdot625 to 50-500 μ L and activate, 25 DEG C, 30-60min; The method of desalting column is taked to carry out purifying subsequently; Detect antibody with 10-50mMDTT to 100-500 μ LNSE, CEA, CYFRA21-1 to reduce, 25 DEG C, 30-60min; Remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, be stored in 10mMPBS (PH7.2) after separation and purification, 4 DEG C of lucifuges are for subsequent use.
(2) detection reaction
The magnetic capture probe of 5000-10000 in vitro NSE, CEA, CYFRA21-1 capture antibody mark is added in each reaction, 5-50nMQdot525, Qdot585, Qdot625 detector probe, by Sample dilution (10mMPBS containing 5-10% calf serum) polishing volume to 10-50ul/ reaction, add in NSE, CEA, CYFRA21-1 standard items of the variable concentrations of 10-50ul or blank or sample to be tested subsequently; 30-60min is hatched 37 DEG C of concussions; After biochemical reaction completes, be separated magnetic microsphere with magnetic separator, then use cleaning 1-3 time of cleansing solution (0.05% Tween-20 10mMPBS), carry out observing, taking pictures below microscope.
Three kinds of quantum dot concentration Qdot-585 to be detected of described variable concentrations, the synchronous concentration range of detection of Qdot-525 and Qdot-625, from 0.9ng/ml to 2000ng/ml, are specially 0.9,1.9,3.9,7.8,15.6,31.2,62.5,125,250,500,1000 and 2000ng/ml;
(3) image and interpretation of result
By detecting the standard items (NSE, CEA, CYFRA21-1) of the variable concentrations containing three kinds of target substances.Result shows, the quantum dot of different size can produce the fluorescence of the different colours clearly can distinguished, the magnetic bead detecting these three kinds of albumen presents respectively green, yellow and red owing to combining quantum dot fluorescence probe (Qdot-525, Qdot-585, Qdot-625), and fluorescence signal intensity weakens gradually along with the reduction of the antigen concentration captured.After utilizing GrayQuantifier to carry out qualitative, quantitative to the fluorescence signal that three kinds are caught on magnetic bead, drawing standard curve.The amount of testing molecule can be determined according to the fluorescence intensity of typical curve and target to be measured.Fig. 2 result shows: NSE, CEA, CYFRA21-1 carry out linear fit in 3.9 ~ 125ng/mL scope, and fit equation is respectively Y=23714.6lgX-7329.6 (coefficient R
2=0.9904), Y=29986.6lgX-7554.8 (coefficient R
2=0.0.9936), Y=10693.2lgX+6746.9 (coefficient R
2=0.9847), linear relationship is good, detects and is limited to 0.9ng/mL, can meet clinical practice and otherwise demand.
Claims (10)
1. a quantum dot fluorescence probe, it is characterized in that utilizing bi-functional cross-linking agent long chain sulfosuccinimidyl 4-[N-citraconic acid]-1-carboxylic cyclohexane Succinimidyl-4-[N-Maleimidomethyl] cyclohexane-1-carboxy-[6-amidocaproate, the amino group generation cross-linking reaction of SMCC and amino quantum dot surface, dimaleoyl imino is generated at quantum dot surface, antibody reducing agent dithiothreitol Dithiothreitol to be marked, DTT reduces, disulfide bond reduction is become sulfydryl, sulfydryl forms covalent bond with maleimide base group thus again by antibody labeling on quantum dot, obtain quantum dot fluorescence probe thus.
2. by probe according to claim 1, it is characterized in that when biomolecule detection, using magnetic bead or magnetic microsphere as the solid support of reaction, reaction system is suspended in the liquid phase completely, biomolecule to be measured is caught by solution by immune response or molecular hyridization, go to identify the testing molecule on magnetic bead by quantum dot fluorescence probe again, magnetic bead or magnetic microsphere produce the fluorescence of quantum dot probe owing to combining antigen-antibody reaction thing, the color of fluorescence and the power of signal have reacted kind and the quantity of biomolecule to be measured, image recognition software is utilized to carry out identifying and quantitatively.
3., by probe according to claim 1, it is characterized in that described lateral size of dots is 5-8nm.
4., by probe according to claim 2, it is characterized in that the color of described quantum dot probe is for green, yellow or red; Fluorescence signal intensity reduces gradually with the reduction of the antigen concentration captured.
5. prepare the method for probe as claimed in claim 1, it is characterized in that:
1. first, get the amino quantum dot of 10-50 μ L5-50mMSMCC to 50-500 μ L and activate, 25 DEG C, 30-60min;
2. the method for desalting column is taked to carry out purifying subsequently; With 10-50mMDTT, 100-500 μ L biomolecule is reduced, 25 DEG C, 30-60min;
3. remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, be stored in after separation and purification in 10mMPH7.2PBS, 4 DEG C of lucifuges are for subsequent use.
6. the probe according to any one of claim 1-4 is used for detecting, and it is characterized in that:
1. detect a kind of protein carcinomebryonic antigen or
2. multiple protein CEA, neural enolase NSE and cytokeratin fragment 19CYFRA21-1 is detected.
7., by detection according to claim 6, it is characterized in that detecting a kind of protein carcinomebryonic antigen, the testing process of its method is:
(1) preparation of quantum dot fluorescence probe
First, get the amino quantum dot 625 of 10-50 μ L5-50mMSMCC to 50-500 μ L and activate, 25 DEG C, 30-60min; The method of desalting column is taked to carry out purifying subsequently; Detect antibody with 10-50mMDTT to 100-500 μ LCEA to reduce, 25 DEG C, 30-60min; Remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, be stored in after separation and purification in the PBS of 10mMPH7.2,4 DEG C of lucifuges are for subsequent use;
(2) detection reaction
The magnetic capture probe of 5000-10000 in vitro CEA capture antibody mark is added in each reaction, 5-50nMQdot625 detector probe, the 10mMPBS dilution polishing volume containing 5-10% calf serum by sample to 10-50ul/ reaction, in the CEA standard items adding the variable concentrations of 10-50ul subsequently or sample to be tested; 30-60min is hatched 37 DEG C of concussions; After biochemical reaction completes, be separated magnetic microsphere with magnetic separator, then use cleaning 1-3 time of cleansing solution, carry out observing below microscope, CCD takes pictures;
(3) image and interpretation of result
Qdot625 launches red fluorescence under ultraviolet excitation, is taken pictures by the CEA testing result CCD of variable concentrations, converts HIS pattern to the primaries mode image obtained, and what realize multiple color is qualitative, reaches the object distinguishing target substance.Meanwhile, software also can realize conversion RGB image being changed into gray-scale value, the fluorescence intensity that the quantum dot that each magnetic bead surfaces of statistical study combines produces, and realizes the quantitative detection to target substance CEA.
8., by detection according to claim 7, it is characterized in that:
1. the CEA standard concentration of variable concentrations is respectively 2000,1000,500,250,125,62.5,31.2,15.6,7.8,3.9,1.9,0.9 and 0ng/mL;
2. described cleansing solution is 0.05% Tween-20 10mMPBS;
3. CEA concentration carries out matching within the scope of 0.39-50ng/ml, and its equation is logy=0.5869logx+3.57223, demonstrates good linear relationship; Coefficient R
2=0.988;
4. described method detection limit is 38pg/ml, the clinical threshold values of this 5ng/ml lower than CEA.
9., by detection according to claim 6, it is characterized in that testing process when detecting multiple protein CEA, NSE and CYFRA21-1 is:
(1) preparation of quantum dot fluorescence probe
First, get 10-50 μ L5-50mMSMCC amino quantum dot Qdot525, Qdot585, Qdot625 to 50-500 μ L and activate, 25 DEG C, 30-60min; The method of desalting column is taked to carry out purifying subsequently; Detect antibody with 10-50mMDTT to 100-500 μ LNSE, CEA, CYFRA21-1 to reduce, 25 DEG C, 30-60min; Remove after unreacted antibody and other impurity until desalting column, the activation quantum dot after itself and purifying is hatched in hybridization instrument, 25 DEG C, 30-60min; Add 5-50 μ L beta-mercaptoethanol cessation reaction, being stored in 10mMPH after separation and purification is in the PBS of 7.2, and 4 DEG C of lucifuges are for subsequent use;
(2) detection reaction
The magnetic capture probe of 5000-10000 in vitro NSE, CEA, CYFRA21-1 capture antibody mark is added in each reaction, 5-50nMQdot525, Qdot585, Qdot625 detector probe, the dilution polishing volume containing the 10mMPBS of 5-10% calf serum by sample, to 10-50ul/ reaction, adds in NSE, CEA, CYFRA21-1 standard items of the variable concentrations of 10-50ul or blank or sample to be tested subsequently; 30-60min is hatched 37 DEG C of concussions; After biochemical reaction completes, be separated magnetic microsphere with magnetic separator, then use cleaning 1-3 time of cleansing solution (0.05% Tween-20 10mMPBS), carry out observing, taking pictures below microscope;
(3) image and interpretation of result
By detecting the standard items of the variable concentrations containing three kinds of target substances, obtain a series of fluoroscopic image, the quantum dot of different size produces the fluorescence of the different colours clearly can distinguished, owing to combining quantum dot fluorescence probe Qdot-525 on the magnetic bead detecting these three kinds of albumen, Qdot-585, Qdot-625 and present green respectively, yellow or red, and fluorescence signal intensity weakens gradually along with the reduction of the antigen concentration captured, and then after utilizing GrayQuantifier to carry out qualitative, quantitative to the fluorescence signal that three kinds are caught on magnetic bead, drawing standard curve.The amount of testing molecule can be determined according to the fluorescence intensity of typical curve and target to be measured.
10., by detection according to claim 9, it is characterized in that:
1. the synchronous concentration range of detection of three kinds of variable concentrations quantum dot concentration Qdot-585 to be detected, Qdot-525 and Qdot-625 is from 0.9ng/ml to 2000ng/ml, is specially 0.9,1.9,3.9,7.8,15.6,31.2,62.5,125,250,500,1000 and 2000ng/ml;
2. described cleansing solution concentration is 0.05% Tween-20 10mMPBS;
3. NSE, CEA, CYFRA21-1 carry out linear fit in 3.9 ~ 125ng/mL scope, and fit equation is respectively Y=23714.6lgX-7329.6, coefficient R
2=0.9904, Y=29986.6lgX-7554.8, coefficient R
2=0.0.9936, Y=10693.2lgX+6746.9, coefficient R
2=0.9847, linear relationship is good;
4. detect and be limited to 0.9ng/mL.
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