CN101059508A - Homogeneous immune analysis nano device construction method - Google Patents
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- CN101059508A CN101059508A CN 200710040475 CN200710040475A CN101059508A CN 101059508 A CN101059508 A CN 101059508A CN 200710040475 CN200710040475 CN 200710040475 CN 200710040475 A CN200710040475 A CN 200710040475A CN 101059508 A CN101059508 A CN 101059508A
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Abstract
The invention relates to a method for building uniform immunity analyzing nanometer device, composed of a hyperoxide enzyme, an immunity compound, and an energy acceptor nanometer particle cadmium telluride quantum point, which uses the cadmium telluride quantum point as core lighting based on chemical lighting resonance energy transfer theory to be connected with an antigen or an antibody via static or covalence function, and the hyperoxide enzyme is connected with the antibody via covalence, to obtain the biological nanometer device based on antigen-antibody immunity reaction. The invention has better solubility and stability, adjustable emitting wavelength, low background interface, strong light signal via chemical lighting resonance energy transfer, and the application in uniform immunity analysis, without external laser light source.
Description
Technical field
The present invention relates to a kind of construction method of homogeneous immune analysis nano device,, make up the nano-device that is used for homogeneous immunoassay, belong to preparation of biomolecule nano-device and bioassay technique field based on chemiluminescence resonance energy principle of transfer.
Background technology
Immunoassay is based on antibody and antigentic specificity association reaction and the analytical approach of a kind of high selectivity of setting up, and it is widely used in clinical analysis, food analysis, fields such as environmental analysis.Immunoassay mainly adopts detection meanss such as fluorescence, chemiluminescence and radiation at present.Wherein to have a detecting instrument simple for chemiluminescence detection, characteristics such as sensitivity height, but poor selectivity, and the bulk solution autoluminescence is disturbed bigger.In addition, immunoassay mainly adopts heterogeneous pattern at present, needs loaded down with trivial details operation of multistep such as coated plate, separates, and cleans, and analysis time is long.
The immune response of liquid phase method (or being called the homogeneous phase method) and signal measuring step in solution finishes, and do not have the participation of solid phase, so reaction velocity is very fast, operate also corresponding simply, wherein the homogeneous fluorescent immunoassays are most widely used.The homogeneous fluorescent immunoassay is based on fluorescence resonance energy at present changes principle.
Fluorescence quantum (quantum dots, QDs) be a kind of inorganic nanoparticles of forming by II family-VI family or III family-V group element, compare with the conventional fluorescent probe, it has the exciting light spectrum width, emission spectrum is narrow and be symmetrical distribution, can make its characteristics such as light of launching different colours by regulating to form with size, and quantum dot has been used for cell imaging as the research of fluorescence probe mark biomolecule, immunoassay, aspects such as DNA hybridization.Tradition organic fluorescent dye absorption spectrum is narrow, emission spectrum is usually with hangover, can influence the overlapping degree of donor emission spectrum and acceptor absorption spectrum like this, and donor and acceptor emission spectrum generation mutual interference mutually, limited the application of FRET (fluorescence resonance energy transfer) in homogeneous immunoassay.Some up-to-date reports are used for the FRET (fluorescence resonance energy transfer) immunoassay with luminescent quantum dot, have overcome the weak point of organic fluorescent dye.People such as Goldman (Goldman, E.R.; Clapp, A.R.; Anderson, G.P.; Uyeda, H.T.; Mauro, J.M.; Medintz, I.L.; Mattoussi, H.Multiplexed Toxin Analysis Using Four Colors ofQuantum Dot Fluororeagents, Anal.Chem.2004,76,684-688) based on immune response, by FRET (fluorescence resonance energy transfer), the fluorescence quantum of four kinds of different emission of employing detects when having realized four kinds of toxin.
But FRET (fluorescence resonance energy transfer) analytic system complex structure, instrument costs an arm and a leg.In addition, autofluorescence can increase the interference of background in the biosome, and its application is subjected to certain restriction.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of construction method of homogeneous immune analysis nano device is provided, the immunoassay nano-device that makes up does not need to add LASER Light Source, emission wavelength tunable, has good water-solubility and stability, shift the strong light signal of generation by the chemiluminescence resonance energy, can be used for homogeneous immunoassay.
For realizing this purpose, the present invention is based on the chemiluminescence resonance energy and shift (chemiluminescenceresonance energy transfer, CRET) principle makes up the immunoassay nano-device, this nano-device is by peroxidase (horseradish peroxidase, HRP), immune complex and energy acceptor nano particle cadmium telluride quantum dot are formed, be with a kind of be core based on the luminous cadmium telluride quantum dot of chemiluminescence resonance energy principle of transfer, be connected with static or covalent effect with antigen or antibody through the cadmium telluride fluorescence quantum, peroxidase and antibody are covalently bound, based on the immunoreactive biological nano device of Ag-Ab.
Method of the present invention specifically comprises the steps:
1. sodium hydrogen telluride preparation:
With mol ratio is that 1: 4 to 4: 1 sodium borohydride and tellurium powder places water, 0-50 ℃ of reaction down, generates sodium hydrogen telluride solution.
2. water soluble cadmium telluride quantum point is synthetic:
With 0.00001-0.1 mol caddy and water-soluble sulfhydryl compound mixing wiring solution-forming; regulate the pH value to 8.0-10.0 with sodium hydroxide solution, logical nitrogen deoxygenation is after 30 minutes, in violent stirring and under nitrogen protection; inject sodium hydrogen telluride solution, form the cadmium telluride mono liquid solution.Divalence cadmium ion in the cadmium telluride mono liquid solution: sodium hydrogen telluride: the mol ratio of water-soluble sulfhydryl compound is 2: 1: 4 to 2: 1: 8, the temperature of reaction of control cadmium telluride mono liquid solution is 70-140 ℃, reaction time 0.5-20 hour, obtain a series of cadmium telluride quantum dot aqueous solution of light emitting region in 500 to 750 nanometers.
3. the preparation of water soluble cadmium telluride quantum point-antigen (or antibody) biomarker solution
With 0.001-5 mg/ml cadmium telluride quantum dot, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride (EDC), antigen (or antibody), placing 0.01 mol, pH value is that 7.0 phosphate buffer mixes, the mol ratio of cadmium telluride quantum dot, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride, antigen (or antibody) is 1: 100: 0.2 to 1: 1000: 6, and mixed solution lucifuge under normal temperature was reacted 2-4 hour.Adopt the ultrafiltration purifying to obtain cadmium telluride quantum dot-antigen (or antibody) biomarker solution.It is standby to place 4 degrees centigrade of refrigerator places to preserve at last.
4. peroxidase and antibody (or antigen) is covalently bound.
It is 7.0 phosphate buffer that 0.001-4 mg/ml peroxidase, EDC and N-maloyl imines are placed 0.01 mol pH value, and about 15 minutes of hybrid reaction adopts this reaction solution of ultrafiltration purifying.In this sublimed solution, add 0.001-3 mg/ml antibody (or antigen) then.The mol ratio of peroxidase, EDC, antibody or antigen is 1: 100: 0.2 to 1: 200: 5, and mixed solution lucifuge under normal temperature was reacted 1-2 hour.Adopt the ultrafiltration purifying to obtain peroxidase-antibody (or antigen) biomarker solution.It is standby to place 4 degrees centigrade of refrigerator places to preserve at last.
5. based on the Ag-Ab immune response, make up peroxidase-immune complex-cadmium telluride quantum dot biological nano device.
Get 5-50 microlitre cadmium telluride quantum dot-antigen (or antibody) biomarker solution respectively and peroxidase-antibody (antigen) biomarker solution places miniature tube, be that 7.4 phosphate buffer is diluted to 500 microlitres with 0.01 mol pH value then, whirlpool mixes, at last to 37 degrees centigrade of constant temperature 2-3 hours, obtain cadmium telluride quantum dot-Ag-Ab-peroxidase immune nano device, or cadmium telluride quantum dot-antibody-antigen-peroxidase immune nano device.
Take out nano-device, promptly can be used for chemiluminescence resonance energy transfer analysis.
Biological nano device of the present invention is made up of peroxidase, immune complex, energy acceptor fluorescent nano particles.
Immune complex of the present invention by antigen and antibody generation specificity in conjunction with obtaining (immune complex that obtains as bovine serum albumin(BSA) and bovine serum albumin(BSA) antibodies).Antigen comprises bovine serum albumin(BSA) in the described immune complex, carcinomebryonic antigen, alfafoetoprotein, calcitonin, thyroprotein, cancer antigens c A125, cancer antigens c A 5-3 and cancer antigens c A19-9 etc.
Energy acceptor fluorescent nano particles of the present invention is the cadmium telluride quantum dot that the mercaptopropionic acid of the synthetic different size of water is modified, its emission wavelength is the 500-750 nanometer, quantum yield is 20-60%, its finishing have a plurality of carboxyls (COOH), be used for protein (as antigen) on amino (NH
2) covalently bound.
In the present invention, having set up a kind of is that energy donor and fluorescence quantum are the novel resonance energy transfer of energy acceptor based on chemiluminescent substance, and promptly the chemiluminescence resonance energy shifts, and it shifts comparatively similar to the bioluminescence resonance energy.The chemiluminescence resonance energy shifts the non-radiative resonance energy be meant based between chemical illuminating reagent donor after the reaction substrate oxidation and suitable acceptor and shifts.Difference between it and the FRET (fluorescence resonance energy transfer) mainly is light source that the FRET (fluorescence resonance energy transfer) process need adds and the chemiluminescence resonance energy shifts that (as luminol, luminol) oxidation and producing does not need to add light source based on reaction substrate.Owing to reduced the background fluorescence interference, it has higher sensitivity (being signal to noise ratio (S/N ratio)) for FRET (fluorescence resonance energy transfer).
The immune complex nano-device of preparation can be used for homogeneous immunoassay, comprises two kinds of sandwich method and competition laws.In competition law, fluorescence quantum and peroxidase be a kind of in a pair of antigen of mark, the antibody respectively, form fluorescence quantum-antibody and peroxidase-antigen respectively, they flock together because of immune response, the chemiluminescence resonance energy takes place to be shifted, when having corresponding unlabelled antigen or antibody in the testing sample, just may with the antigen or the antibody competition of mark, take place to replace and generate the immune complex that no chemiluminescence resonance energy shifts, make the detection signal minimizing.
Method cost of the present invention is low, easy and simple to handle, mild condition.Product biological nano device has water-soluble and good stability, the fluorescence quantum yield height, and characteristics such as light emitting region is wide are used for the chemiluminescence resonance energy and shift, and do not need to add LASER Light Source.The immune complex nano-device of preparation can be used for homogeneous immunoassay.
Description of drawings
Fig. 1 is the immunoassay nano-device synoptic diagram based on antigen and antibody mediated immunity reaction.
Fig. 2 is for being respectively the immunoassay nano-device chemiluminescence resonance energy transfer spectrogram of energy acceptor with the cadmium telluride quantum dot of different size.Used quantum dot light emitting wavelength is respectively 557 nanometers, 587 nanometers, 622 nanometers and 657 nanometers.
Embodiment
Below in conjunction with accompanying drawing and by several specific embodiments technical scheme of the present invention is further described.
The biological nano device architecture of the present invention's preparation is made of peroxidase, immune complex and nano particle cadmium telluride quantum dot as shown in Figure 1.In Fig. 1, the chemiluminescent substance luminol is as energy donor, and cadmium telluride quantum dot is as energy acceptor.Cadmium telluride quantum dot is connected with antigen, and peroxidase is connected with antibody.When cadmium telluride quantum dot-antigen and peroxidase-antibody labeling thing generation immune response, the chemiluminescence resonance energy shifts and takes place.
Embodiment 1
(1) sodium hydrogen telluride preparation:
80 milligrams of tellurium powder, 80 milligrams of sodium borohydrides and 2 ml waters are placed in one 10 milliliters the little flask, at room temperature reacted 8 hours, prepare sodium hydrogen telluride solution.
(2) water soluble cadmium telluride quantum point is synthetic:
With 0.00125 mol caddy and 0.003 mol mercaptopropionic acid mixing wiring solution-forming; regulate the pH value to 8.0-9.0 with sodium hydroxide solution, logical nitrogen deoxygenation is after 30 minutes, in violent stirring and under nitrogen protection; inject sodium hydrogen telluride solution, form the cadmium telluride mono liquid solution.The divalence cadmium ion that adds: sodium hydrogen telluride: the mercaptopropionic acid mol ratio is 2: 1: 4.8.The precursor solution that has obtained is put into the micro-wave digestion jar, pass through carry out microwave radiation heating.Control reaction time 0.5-2 hour, obtaining the water soluble cadmium telluride quantum point quantum yield is 20-60%, and emission wavelength is the 500-750 nanometer.
(3) prepare cadmium telluride quantum dot-bovine serum albumin(BSA) biomarker by electrostatic interaction
Contain 5 mg/ml cadmium telluride quantum dots, 6 mg/ml bovine serum albumin(BSA)s mix in the phosphate buffer of 0.01 mol pH7.0, and mixed solution lucifuge under normal temperature was reacted 1-2 hour.Adopt ultrafiltration purifying quanta point biological label.Preserve standby to 4 degrees centigrade of refrigerator places at last.
(4) preparation of the anti-bovine serum albumin antibody of rabbit-peroxidase labelling thing
Contain 4 mg/ml peroxidase, 2 mg/ml EDC and 3 mg/ml N-maloyl imines are about 15 minutes of hybrid reaction in 7.0 the phosphate buffer in 0.01 mol pH value, adopt this reaction solution of ultrafiltration purifying.In the solution of this purifying, add the anti-bovine serum albumin(BSA) antibody-solutions of 10 mg/ml rabbits then.Mixed solution lucifuge under normal temperature was reacted 1-2 hour.Adopt this biomarker of ultrafiltration purifying, preserve standby to 4 degrees centigrade of refrigerator places at last.
(5) prepare the immunoassay nano-device by immune response
Quantum dot-bovine serum albumin(BSA) label solution and the anti-bovine serum albumin(BSA) antibody of the rabbit-peroxidase labelling solution of getting 5 microlitre purifying respectively place miniature tube, be that 7.4 phosphate buffer is diluted to 500 microlitres with 0.01 mol pH value then, whirlpool mixes, to 37 ℃ of constant temperature 2-3 hours, obtain the anti-bovine serum albumin(BSA) antibody of quantum dot-bovine serum albumin(BSA)-rabbit-peroxidase nano device at last.Take out reaction solution and be used for chemiluminescence resonance energy transfer analysis, analysis result as shown in Figure 2.In Fig. 2, adopt four kinds of different size emission wavelengths be 557,587,622 and the cadmium telluride quantum dot of 657 nanometers be used separately as energy acceptor.Experimental result shows when antigen bovine serum albumin(BSA)-cadmium telluride quantum dot label and peroxidase-bovine serum albumin(BSA) antibody labeling thing generation immune response, has produced the transfer of chemiluminescence resonance energy.First peak is luminol glow peak (left side) among Fig. 2, and the peak that the back is four is the cadmium telluride quantum dot glow peak.
Embodiment 2
(1) the sodium hydrogen telluride preparation is with embodiment 1.
(2) water soluble cadmium telluride quantum point is synthetic with embodiment 1.
(3) preparation of cadmium telluride quantum dot-cancer antigen 125 (CA125) biomarker
To contain 0.3 mg/ml cadmium telluride quantum dot, 0.6 mg/ml EDC and 0.3 mg/ml CA125 mix in 0.01 mol pH value is 7.0 phosphate buffer, mixed solution lucifuge reaction 2-4 hour under normal temperature.Adopt ultrafiltration purifying quanta point biological label.Preserve standby to 4 degrees centigrade of refrigerator places at last.
(4) preparation of CA125 antibody-peroxidase labelling thing
Get 0.2 mg/ml superoxide enzyme solutions, 0.2 mg/ml EDC solution and 0.3 mg/ml N-maloyl imines are about 15 minutes of 7.0 phosphate buffer hybrid reaction in 0.01 mol pH value, adopt this reaction solution of ultrafiltration purifying.Add the CA125 antibody-solutions then in this sublimed solution, mixed solution lucifuge under normal temperature was reacted 1-2 hour.Label solution places on the magnetic stirring apparatus and at the uniform velocity stirs, and makes its reaction evenly.Adopt this biomarker of ultrafiltration purifying, preserve standby to 4 degrees centigrade of refrigerator places at last.
(5) prepare the immunoassay nano-device by immune response
Quantum dot-CA125 label solution and the CA125 antibody-peroxidase labelling solution of getting 50 microlitre purifying respectively place miniature tube, be that 7.4 phosphate buffer is diluted to 500 microlitres with 0.01 mol pH value then, whirlpool mixes, to 37 degrees centigrade of constant temperature 2-3 hours, obtain quantum dot-CA125-CA125 antibody-peroxidase nano device at last.Take out reaction solution, be used for chemiluminescence resonance energy transfer analysis.
Embodiment 3
(1) the sodium hydrogen telluride preparation is with embodiment 1.
(2) water soluble cadmium telluride quantum point is synthetic with embodiment 1.
(3) preparation of cadmium telluride quantum dot-cancer antigen 1 9-9 (CA19-9) biomarker
To contain 0.05 mg/ml cadmium telluride quantum dot, 0.1 mg/ml EDC and 0.05 mg/ml CA19-9 mix in 0.01 mol pH value is 7.0 phosphate buffer, mixed solution lucifuge reaction 2-4 hour under normal temperature.Adopt ultrafiltration purifying quanta point biological label.Preserve standby to 4 degrees centigrade of refrigerator places at last.
(4) preparation of CA19-9 antibody-peroxidase labelling thing
To contain 0.04 mg/ml superoxide enzyme solutions, 0.04 mg/ml EDC solution and 0.06 mg/ml N-maloyl imines are in 0.01 mol, the pH value is about 15 minutes an of hybrid reaction in 7.0 the phosphate buffer, adopts this reaction solution of ultrafiltration purifying.Add 0.1 mg/ml CA19-9 antibody-solutions then in the solution of this purifying, mixed solution lucifuge under normal temperature was reacted 1-2 hour.Label solution places on the magnetic stirring apparatus and at the uniform velocity stirs, and makes its reaction evenly.Adopt this biomarker of ultrafiltration purifying, preserve standby to 4 degrees centigrade of refrigerator places at last.
(5) prepare the immunoassay nano-device by immune response
Quantum dot-CA19-9 label solution and the CA19-9 antibody-peroxidase labelling solution of getting 50 microlitre purifying respectively place miniature tube, use 0.01 mol phosphate buffer (pH7.4) to be diluted to 500 microlitres then, whirlpool mixes, to 37 degrees centigrade of constant temperature 2-3 hours, obtain quantum dot-CA19-9-CA19-9 antibody-peroxidase nano device at last.Take out reaction solution, be used for chemiluminescence resonance energy transfer analysis.
Claims (4)
1, a kind of construction method of homogeneous immune analysis nano device is characterized in that comprising the steps:
(1) sodium hydrogen telluride preparation
With mol ratio is that 1: 4 to 4: 1 sodium borohydride and tellurium powder places water, 0-50 ℃ of reaction down, generates sodium hydrogen telluride solution;
(2) water soluble cadmium telluride quantum point is synthetic
With 0.00001-0.1 mol caddy and water-soluble sulfhydryl compound mixing wiring solution-forming, regulate the pH value to 8.0-10.0 with sodium hydroxide solution, logical nitrogen deoxygenation is after 30 minutes, stirs and under nitrogen protection, inject sodium hydrogen telluride solution, form the cadmium telluride mono liquid solution; Divalence cadmium ion in the cadmium telluride mono liquid solution: sodium hydrogen telluride: the mol ratio of water-soluble sulfhydryl compound is 2: 1: 4 to 2: 1: 8; The temperature of reaction of control cadmium telluride mono liquid solution is 70-140 ℃, reaction time 0.5-20 hour, obtains the cadmium telluride quantum dot aqueous solution of light emitting region in 500 to 750 nanometers;
(3) preparation of water soluble cadmium telluride quantum point-antigen or antibody biomarker solution
With 0.001-5 mg/ml cadmium telluride quantum dot, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride, antigen or antibody, placing 0.01 mol, pH value is that 7.0 phosphate buffer mixes, the mol ratio of cadmium telluride quantum dot, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride, antigen or antibody is 1: 100: 0.2 to 1: 1000: 6, mixed solution lucifuge under normal temperature was reacted 2-4 hour, adopt the ultrafiltration purifying to obtain cadmium telluride quantum dot-antigen or antibody biomarker solution, it is standby to place 4 ℃ of refrigerators to preserve;
(4) peroxidase, antibody or antigen is covalently bound
With 0.001-4 mg/ml peroxidase, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride, it is 7.0 phosphate buffer that N-maloyl imines places 0.01 mol pH value, hybrid reaction 15 minutes, adopt this reaction solution of ultrafiltration purifying, in this sublimed solution, add 0.001-3 mg/ml antibody or antigen then, peroxidase, ethyl [3-(dimethylamino) propyl group] carbodiimide hydrochloride, the mol ratio of antibody or antigen is 1: 100: 0.2 to 1: 200: 5, mixed solution lucifuge under normal temperature was reacted 1-2 hour, adopt the ultrafiltration purifying to obtain peroxidase-antibody or antigen biomarker solution, it is standby to place 4 degrees centigrade of refrigerators to preserve;
(5), make up peroxidase-immune complex-cadmium telluride quantum dot biological nano device based on the Ag-Ab immune response:
Get 5-50 microlitre cadmium telluride quantum dot-antigen or antibody biomarker solution and peroxidase-antibody respectively or antigen biomarker solution places miniature tube, be that 7.4 phosphate buffer is diluted to 500 microlitres with 0.01 mol, pH value then, whirlpool mixes, at last to 37 degrees centigrade of constant temperature 2-3 hours, obtain cadmium telluride quantum dot-Ag-Ab-peroxidase immune nano device, or cadmium telluride quantum dot-antibody-antigen-peroxidase immune nano device.
2, according to the construction method of the homogeneous immune analysis nano device of claim 1, it is characterized in that described antigen is bovine serum albumin(BSA), carcinomebryonic antigen, alfafoetoprotein, calcitonin, thyroprotein, cancer antigens c A 125, cancer antigens c A 15-3 or cancer antigens c A19-9.
3, the homogeneous immune analysis nano device that makes up according to the method for claim 1 or 2 is characterized in that this biological nano device is made up of peroxidase, antibody or antigen, energy acceptor fluorescent nano particles.
4, according to the homogeneous immune analysis nano device of claim 3, it is characterized in that described energy acceptor fluorescent nano particles is the cadmium telluride quantum dot of the mercaptopropionic acid modification of the synthetic different size of water, its emission wavelength is the 500-750 nanometer, quantum yield is 20-60%, its finishing have a plurality of carboxyls (COOH), be used for protein on amino (NH
2) covalently bound.
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