CN101551385A - Double labelling Nano-Au probe and preparation method and application thereof - Google Patents
Double labelling Nano-Au probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double labelling Nano-Au probe and a preparation method and application thereof. The probe comprises a Nano-Au particle as a core, the surface of which is connected with antibody and oligonucleotide simultaneously. The preparation method of the double labelling Nano-Au probe includes the following steps: (1) coupled reaction: adding oligonucleotide solution and antibody solution in Nano-Au sol with certain concentration and uniformly mixing the solutions to lead the Nano-Au particle, the antibody and nucleic acid to fully act in the solution; and (2) stabilizing and blocking reaction: preparing PBS buffer solution with the content of SDS being 5-10 percent and then adding the PBS buffer solution in the mixed solution prepared in step (1) and adding sealant to form nano probe sol. The double labelling Nano-Au probe can convert the antigenic detection into the detection of nucleic acid matter and is applied to the detection of tumor marker, hormone and pathogene micro-protein.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of double labelling Nano-Au probe and preparation method thereof and the application of this Nano-Au probe in the ultramicron Detection of antigen of biologically active.
Background technology
Medical diagnostic techniqu develops rapidly in recent years, and immunoassay is also maked rapid progress. and as immunoassay an important branch--the label immunoassay technology is the most active.
Nineteen fifty-nine, American scholar Berson and Yalow set up radiommunoassay (this label immunoassay have been opened up the frontier of label immunoassay for Radio Immunoassay, RIA) method.Because it is highly sensitive that RIA has, high specificity, advantage such as easy, RIA has certain vitality, but because it must use radioactive nuclide, needs protection, prevents to pollute; The label effective storage life is short, is difficult to realize the robotization of operating and measuring etc., and further developing of it is subjected to some limitations, and therefore, many scholars have carried out the exploration of novel marker.
1971, two groups of scholars of Engvall and Perlaman and Weeman and Schuur replaced isotope to prepare enzyme labeling reagent with enzyme, founded the EIA enzyme immunoassay technology (Enzyme Immunosorbent Assay, EIA).So far there are 20 plurality of enzymes to be applied to EIA, wherein based on horseradish peroxidase (HRP) and alkaline phosphatase (AP), EIA is except avoiding radioisotopic harm, prior advantage is that the enzyme labeling thing term of validity is longer, but common EIA sensitivity is not high, and fluoroimmunoassay (the Immunofluorescence Assay that developed afterwards, FIA) and chemiluminescence immune assay (ChemiluminescenceImmunoassay CLIA) improves sensitivity and detect the scope of sending out.
Finding and using new label is one of main direction of studying of immunoassay, and the intervention of nanometer technology also provides infinite imagination space for the development of label immunoassay Study on Technology.Nano particle (English name is nanoparticles) is meant the particle of particle diameter in the 1-100 scope, also claim ultramicron (English name is ultrafine particles), it is in the transitional region of atom and macro object boundary, has special performances.Itself has small-size effect nano particle, surface effect, and the physicochemical characteristic that chemical reactivity etc. are unique can have special interaction with biosome, therefore, is widely used with the method that the thing carrier amplifies reaction that serves as a mark such as nanoparticle, liposome.Wherein, particularly outstanding with the nano gold mark immunoassays.
1971, faulk etc. combine nm of gold with rabbit anti-salmonella serum, detect the surface antigen of salmonella with immunocytochemical technique, start nm of gold mark technology (Faulk, CM.Taylar.An immunocolloidalmethod for the electron microscope.Immunochemistry.1971,8:1080-1084.).1981, Danscher has set up immunogold silver staining (the Immunogold-sliver staining that strengthens the observability of gold grain under the light microscopic with silver-colored developer solution, IGSS), being widely used in medical science and RESEARCH ON CELL-BIOLOGY field at present, is one of responsive method commonly used in the present immunohistochemistry technology.
At present, aspect the tachysynthesis diagnosis, with spot immune gold and silver dyeing (Dot-immunogold silverstaining, Dot-IGSS), tachysynthesis gold percolation (Dot-immunogold filtration assay, DIGFA) and immune golden chromatography (immunochromatography, GICA), three kinds is main.1989, it was the percolation test of label that spielberg etc. have developed at first with the nm of gold, is used to detect antibody of AIDS virus, and establishment spot immune gold percolation (Dot-immunogold filtration assay, DIGFA).Beggs etc. are used for gold size mensuration (the Beggs M of human chorionic gonadotrophin (HCG) at first, Novotny M, Sampedro S, etal.A self-performing chromatog-raphy immunoassay for the quanlitativedetermination ofHCG in urine and serum.Clin.Chem., 1990,36 (6): 1084-1085.)
Nano gold mark antibody, and can be by silver-colored development amplifying signal.Duan L etc. utilizes nano gold mark as reporter molecules, it is B-mode to set up a kind of while fast detecting, the protein chip of hepatitis C virus (antibody chip) detection method, compare with conventional ELISA detection method, no significant difference (Duan L, Wang Y, Li SS, et al.Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virusantibodies based on a protein chip assay using nano-gold immunological amplificationand silver staining method.BMC Infect Dis.2005; 5:53.).
From isotope, enzyme, fluorescein, the label that the development and application of collaurum is new, purpose is nothing but for the sensitivity that improves immunoassays, specificity, stability or simplicity.In recent years, also became scientist and studied focus with the nucleic acid thing design method of immunity that serves as a mark.Nm of gold can with the nucleic acid of alkane sulfydryl modification between form very strong Au-S covalent bond and combine, 3-mercaptoethanol or mercaptoethanol can take place to replace again nucleic acid is replaced to get off with the Au-S covalent bond.Based on the amplification of DNA or transcription and translation, by polymerase chain reaction (polymerase chainreaction, PCR) millions of times of a few hours amplifications, the sort signal amplification is far from common labels such as nucleic, enzyme, fluorescein or element and can reach, thereby the dna marker immunoassays have high detection sensitivity.
Chad professor A.Mirkin of nanotechnology research institute of Northwestern Univ USA leader's research group has set up " biological barcode nucleic acid chip detection method " (the Nam JM based on the dna marker immunoassays, Thaxton CS, Mirkin CA.Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection ofProteins.SCIENCE, 2003,301:1884-1886).The detection sensitivity height detects lower limit and is low to moderate 6 orders of magnitude than traditional E LISA method as a result, is not only applicable to DNA and also is applicable to protein.2006, this method of research group's successful Application detects PSA simultaneously, HCG, AFP (Stoeva SI, Lee JS, Smith JE, et al.Multiplexed Detection of Protein Cancer Markers with BiobarcodedNanoparticle Probes.J.AM.CHEM.SOC.2006,128:8378-8379.), in addition, they also detect micro-ADDL albumen in the cerebrospinal fluid with this method, and common detection method is difficult to detect ADDL, illustrate the ADDL protein concentration that is higher than healthy individual in the cerebrospinal fluid that after death is diagnosed as the senile dementia patient, helped early diagnosis (Georganopoulou DG, the Lei Chang of senile dementia, Nam JM, et al.Nanoparticle-based detection in cerebral spinal fluid of a solublepathogenic biomarker for Alzheimer ' s disease.PNAS, 2005,102 (7): 2273-2276).
Application for a patent for invention prospectus (publication number: CN 1339609A; Country origin: China; Open day: on March 13rd, 2002) disclose a kind of nano gold mark gene probe, wherein adopt nm of gold to combine and form probe with the nucleic acid of alkane sulfydryl modification, be used for diagnosis type genetic chip field, but it can not be used for immunoassays, has difficulties on actual applying.
Summary of the invention:
In order to overcome the micro-Detection of antigen technology existing problems of above-mentioned routine, improve the detection sensitivity specificity, the purpose of this invention is to provide a kind of by nm of gold surface covalent bond oligonucleotides single chain molecule simultaneously, Electrostatic Absorption antibody molecule and the Nano-Au probe that constitutes; Another object of the present invention provides a kind of preparation method of nm of gold particulate label probe of the present invention; A further object of the invention is the application of Nano-Au probe in the ultramicron Detection of antigen.
It below is technical scheme of the present invention.
A kind of double labelling Nano-Au probe, this probe comprises: nano Au particle is nuclear, and the surface connects antibody and oligonucleotides simultaneously.
Described Nano-Au probe mean grain size is 20-60nm.
Described Nano-Au probe mean grain size is 30-50nm.Usually, particle size range is between the 10-240nm.
Wherein said antibody is general purpose I gG, and described general purpose I gG is one of polyclonal antibody or monoclonal antibody or its combination.
Wherein said antibody is that goat anti-mouse igg, anti-people PSA polyclonal antibody, anti-people HCG polyclonal antibody, anti-people HBV polyclonal antibody, anti-people HIV polyclonal antibody, anti-people ADDL polyclonal antibody, the anti-popular feeling are received one of element (ANP) polyclonal antibody, anti-people NSE polyclonal antibody, anti-people C-peptide polyclonal antibody or monoclonal antibody or its combination.
Described oligonucleotides is one of the single stranded DNA of sulfydryl modification or RNA molecule or its combination.Synthetic and the modification of the design of oligonucleotides single chain molecule and trust, reactive group-sulfydryl that employing is easy to fix with nm of gold microparticle surfaces commissure (SH) is modified the oligonucleotides single chain molecule, sterically hindered problem when connecting for overcoming, oligonucleotide fragment-SH end has increased the alkane hydrogen group, and has connected 10 adenines (A) conducts " arm " so that activity space to be arranged.
The length of the fragment of described oligonucleotides is 10～50bp, and long fragment is wrapped in the nano Au particle surface easily.
The present invention also provides the preparation method of above-mentioned double labelling Nano-Au probe, comprises following step:
1. coupled reaction: in concentration is that adding concentration is 6.6～33ug/ml nucleic acid solution, concentration 4-10ug/ml antibody-solutions in the 2--6nM nano gold sol, mix, in temperature is 4～30 ℃, the control pH value in reaction is 6～9, time is 10～20h, make that nano Au particle and antibody, nucleic acid can fully act in the solution, make mixed liquor;
2. stablize and capping: it is the PBS buffer solution of 5-10% that configuration contains the SDS amount, wherein the amount of PBS is 0.01-0.1M, add then in the 1. made mixed liquor of step, the final concentration of control activating agent SDS is 0.005-0.015%, evenly mixed, the stabilizing agent that adds 0.3-1.2M then gradually, limit edged mixing, the concentration of final control stabilization agent is 0.1M～0.2M, leaves standstill more than the balance 6-12h, at last, add sealer,, the control final concentration is 0.5%～1%, the mixing balance, the colloidal sol of formation nano-probe.
Described nano gold sol concentration is 3.0nM preferably, better then is 3.3nM.
Wherein said stabilizing agent is the NaCl aqueous solution, and described sealer is bovine serum albumin(BSA) (BSA).
Because the stable and sealing of Nano-Au probe self assembly has only strict control reaction conditions, just can make oligonucleotide fragment, antibody molecule is integrated into the nm of gold surface firmly, thereby avoids taking place irreversible coagulation.Use bovine serum albumin (BSA) to be stabilizing agent, seal unnecessary site, reduce the probe non-specific adsorption, and improve the dispersive property of particulate with an amount of surfactant SDS.
Wherein one of the 1. described oligonucleotides of the step single stranded DNA that is sulfydryl modification or RNA molecule or its combination.
Its step 1. before, also comprise step:
(1) trisodium citrate reduction method prepares nm of gold: the HAuCl4 solution of 0.005%--0.04% is heated to boiling, the 0.5%--3% citric acid three sodium solution that adds fast under the high-speed stirred condition, the wherein required HAuCl4 solution and the volume ratio of citric acid three sodium solution are 100: 8--100: 2, continue heated and stirred to mixing, natural cooling forms nano gold sol then.
It also comprises step in step (1) afterwards: step (1) gained nano gold sol 0.18-0.25u m membrane filtration.
Also can adopt the nano gold sol of outsourcing.
Its step 2. after, also comprise step:
Low temperature ultracentrifugation purifying: to the colloidal sol of the Nano-Au probe of step in 2., carry out centrifugal under 12000～16000r/min rotating speed, centrifugation time is 15～45min, temperature is controlled at the 4-8 degree, remove supernatant, with the cleansing solution cyclic washing of 0.01%～0.10% Sodium Azide, 4～8 ℃ of preservations.
Behind the purifying, can carry out phenetic analysis to probe by the method for transmission electron microscope (TEM) and ultraviolet spectrum.
The nano gold biological probe of described preparation, the particulate top layer is coated with the oligonucleotide fragment by Au-S covalent bond strong bonded, and 3-mercaptoethanol (DTT) or mercaptoethanol (MCE) are replaced oligonucleotides as reductive agent.The power of observing the DTT effect with the ultraviolet-visible spectrophotometer length scanning changes.
Concrete steps can be as follows: get the Nano-Au probe 2.5ml for preparing of 2.9nM, adding 0.1M 3-mercaptoethanol (DTT) to final concentration is 0.01M, leave standstill a period of time after UV scanning once, observe absorption peak, absorbance and change in color.The concrete amount of taking can change according to actual conditions.
Wherein the detection method of the overlay capacity of oligonucleotide fragment can be the fluorescence labeling detection method.
The oligonucleotides of FAM mark is incorporated into gold particle, adopts fluorospectrophotometer to detect the FAM fluorescence volume, and calculate the connection overlay capacity of DNA.Concrete steps can be as follows: get 3.0nM, the Nano-Au probe that 4ml prepares, adding 0.1M 3-mercaptoethanol (DTT) to final concentration is 0.01M, leave standstill abundant effect, centrifuging and taking gets supernatant, the preparation of FAM typical curve: in identical pH, ionic strength, under the condition of DTT concentration, detect the oligonucleotides (FAM) of concentration known and the supernatant of unknown concentration.The concrete amount of taking can change according to actual conditions.
The articles of reference of related detection method, be Kim EY, Jennifer Stanton, Vega RA, et al.Areal-time PCR-based method for determining the surface coverage ofthiol-capped oligonucleotides bound onto gold nanoparticles.NucleicAc ids Research, 2006,34 (7): 47-54.
The nano gold biological probe of described preparation, the nano Au particle top layer is connected with polyclonal antibody, adopts the diafiltration of spot immune gold to measure antibody activity.Concrete steps can be as follows: 5 μ l antigens are incorporated on the nitrocellulose filter of treated mistake, with the 5 negative contrasts of μ l goat-anti rabbit and not point sample be blank, drying at room temperature, place the self-control percolation box.Add 0.01M PBS (containing 1%BSA) 100 μ l sealing, add 50 μ l nano gold biological probes, hatch 30min, PBST cleansing solution washing back observations through 37 ℃.The concrete amount of taking can change according to actual conditions.
In general, antibody connection amount and input amount are proportionate within the specific limits, therefore, detect antibody absorption situation on the novel immunoglobulin g-gold with the Bradford method.Pass through formula: probe antibody connection amount=antibody input amount-antibody residual volume, the rate that is connected of rough calculation nano Au particle and antibody.The nano gold biological probe of described preparation, the minimum steady that nm of gold combines with polyclonal antibody quantitatively are 5 μ g/ml, and the suitableeest stable quantity is 6-7 μ g/ml.
By the method for PCR detection oligonucleotide fragment, detect the Nano-Au probe surface oligonucleotide fragment labelled antigen antibody response of described preparation.Concrete steps can be as follows: get 35 μ l, and 100 μ g/ml immune magnetic microspheres, the PBS repeated washing adds a certain amount of Au probe (2.5nM), PBS is settled to 2ml, 37 ℃ of water-bath vibration 1h, and separate in magnetic field, cleansing solution washing (0.01M, pH7.4PBS, 0.15M NaCl), repeat for several times.Collect magnetic field separator (immunomagnetic beads and Au probe connector).Add the DTT effect, ultracentrifugation is obtained supernatant, as the pcr amplification sample.After the amplification product is handled with saturated urea, 55 ℃ of water-bath 10min of high temperature, 18% the polyacrylamide gel electrophoresis analysis that contains 7M urea is done in quick-frozen immediately.
The application of described double labelling Nano-Au probe, it is that this probe can be converted into detection of antigens the detection to nucleic acid substances, is applied to the detection of tumor marker will thing, hormone, pathogen trace protein.
Unspecified percentage composition is weight percentage among the present invention, and the solution of Te Bieshuominging all is not aqueous solution.
By above we as can be seen, the present invention combines the synthetic package technique of Nanosurface, immunological technique, Measurement for Biochemistry, the gordian technique that solves the nm of gold finishing and respectively be connected simultaneously with polyclonal antibody and oligonucleotide fragment, require neither to influence the specificity combination of antigen-antibody in the immune response, do not influence oligonucleotide fragment connection and detection again.The present invention is by open Nano-Au probe, the preparation method of this kind Nano-Au probe, the application of this kind Nano-Au probe, just can come labelled antigen and antibody with oligonucleotide fragment, and then oligonucleotides is judged by round pcr, the oligonucleotides amount that detection signal amplifies, can know the ultramicron protein of combination, thereby improve detection sensitivity greatly.The present invention is applicable to all kinds of detection of antigens, comprises denier detection of antigens such as early carcinoma antigen and some neuropeptide.But the characteristics of maximum of the present invention are oligonucleotides labelled antigen antibody responses, the detection of materials such as antigen is converted into mensuration to nucleic acid substances, thereby improves detection sensitivity, and characteristics such as the tool method of operating is easy, quick.Can be used for ultramicron Detection of antigen such as early carcinoma antigen and some neuropeptide, the good clinical using value is arranged.
Description of drawings
Fig. 1 is embodiment 1 nm of gold and 1.2M NaCl mixed transmission Electronic Speculum figure.
Fig. 2 is embodiment 5 double labelling Nano-Au probe particle diameter and distribution plans.
Fig. 3 is the double labelling Nano-Au probe transmission electron microscope picture of embodiment 5.
Fig. 4 is the ultraviolet-visible spectrophotometer scanning result figure of embodiment 5.
Fig. 5 is the spot immune gold diafiltration of embodiment 5.
Fig. 6 is the time changing curve figure that the DTT of embodiment 6 replaces sulfydryl DNA on the probe.
Fig. 7 is oligonucleotides PAGE result on the probe of embodiment 6.
Fig. 8 is the ssDNA addition and the amount of connection situation synoptic diagram of embodiment 7.
Fig. 9 is the immunogold silver staining observations figure of embodiment 7.
Embodiment
One, the preparation of nm of gold
Embodiment 1:
Trisodium citrate (Shanghai traditional Chinese medicines) reduction HAuCl
4Legal system is equipped with collaurum.HAuCl with 100ml 0.01%
4Solution is heated to boiling; (1200rpm) adds 5ml 1% trisodium citrate (Na fast under the high-speed stirred condition
3C
6H
5O
72H
2O), continue heated and stirred 30min, natural cooling returns to original volume with ultrapure water.0.22 μ m membrane filtration, 4 ℃ keep in Dark Place standby.
As shown in Figure 1, the gained nm of gold is observed the pattern of particle with transmission electron microscope (TEM), measure its size and size distribution with laser light scattering instrument, with ultraviolet-visible spectrophotometer scanning absorption peak and according to the concentration of Lambert-Beer's law estimation nm of gold.
Experimental result gained nm of gold has maximum absorption peak at 518nm, epigranular, and favorable dispersibility, main distribution range is at 10 ± 3nm, and the general concentration of gold size is 3.7nM.
Notice that the not mentioned part of following examples is identical with embodiment 1.
Embodiment 2:
The HAuCl4 solution of getting 100ml 0.01% is heated to boiling; Add 2.5-4.5ml 1% trisodium citrate (Na under the high-speed stirred condition fast
3C
6H
5O
72H
2O), continue heated and stirred 30min, natural cooling returns to original volume with ultrapure water.0.22 μ m membrane filtration, 4 ℃ keep in Dark Place standby.
Experimental result gained nano Au particle particle diameter has maximum absorption peak about 520-534nm, and particle diameter is about 15-30nm.
Two, the best pH of antibodies nm of gold and determining of the suitableeest stable quantity
Embodiment 3:
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced is got several 1.5ml small test tubes, adds the 1ml nano-Au solution respectively; Use Na
2CO
3PH is adjusted to 3,4,5,6,7,8,9,10 respectively successively; Get one 96 orifice plates, respectively above-mentioned collaurum is got 100 μ l from low to high respectively by pH and added in the hand-hole, repeat; . it is antigen or the antibody of 1mg/ml that every hole adds 2 μ l concentration respectively, mixes, and places 10-15min under the room temperature; Then every hole adds the NaCl solution of a certain amount of 1.2M respectively, mixes, and places 10min under the room temperature; . observing colloid gold change color, record keeps red minimum pH (X); And the above step of repetition, pH gradient are X-0.6; X-0.3; X; X+0.3; X+0.6; X+1.Observing colloid gold change color was placed under room temperature 2 hours, and record still keeps the red minimum pH that is.
Experimental result records nano-Au solution and the stable best pH of antibodies is 8-9.
Embodiment 4:
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced is got 5-10 and is propped up test tube, adds collaurum 1ml respectively, 0.1M Na
2Co
3Adjust pH to 9; Each pipe adds the IgG (1,2,4,8,10,16 multiple proportions) of different amounts successively, and mixing is placed 15min under the room temperature; Add a certain amount of NaCl (final concentration is 2.5M), place 20min under the room temperature; At last, still to keep red minimum albumen consumption be minimum protein concentration to color.For guaranteeing accuracy as a result, repeated experiments.
The minimum protein concentration of experimental result gained is 5ug, and the best combination amount often is 130% of a Cmin, i.e. 6.5 μ g/ml.
Three: the preparation of double labelling Nano-Au probe
Embodiment 5
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced.According to the pH value that embodiment 4 regulates nano-Au solution, embodiment 5 determines the suitableeest stable quantity scope of antibodies.Promptly get the 2.5ml colloidal gold solution, use 0.1M, 75 μ l Na
2Co
3Adjust pH to 9 adds a certain amount of 3 '-oligonucleotides (0.40D/ml) (it is synthetic that the worker is given birth in Shanghai) that end alkane hydrogen mercaptan is modified in proportion, at once vortex mixing half an hour, add mouse IgG (7 μ g/ml) (Beijing ancient cooking vessel state company) again, the vortex mixing, jog 1-3h, 4 ℃ of lucifuge standing over night.
Add 0.01M, it is 0.01% that the PBS of pH7.4,10% SDS make its final concentration, the vortex mixing places shaking table jog 30min, and it is 0.15M that the NaCl solution that adds 1.2M gradually makes final concentration, limit edged vortex mixing leaves standstill more than the balance 12h, and stable oligonucleotides connects.At last, it is 1% that the bovine serum albumin(BSA) (BSA) of adding 5% makes ultimate density, the mixing balance.
Get the above-mentioned probe for preparing, 12000r/min, 40min, 4 ℃, gently go supernatant to remove unconjugated antibody and oligonucleotides, 0.01M, the red oily precipitation of the PBS of pH7.4 and 0.15M NaCl solution (containing 0.1%BSA) repeated washing, it is standby to add 4 ℃ of preservations of storage liquid at last.
With TEM, ultraviolet spectrum and laser light scattering instrument it is carried out morphology observation, phenetic analysis and granularity Detection.As shown in Figure 2, be particle diameter and the distribution plan that detects the double labelling Nano probe that obtains with laser light scattering instrument.Fig. 3 is the double labelling Nano-Au probe transmission electron microscope picture that TEM does.Fig. 4 is the ultraviolet spectrogram of making of ultraviolet-visible spectrophotometer.
Spot immune gold percolation can be used to observe antibody activity on the probe, and Fig. 5 is spot immune gold diafiltration figure.Available 3-mercaptoethanol (DTT) (Sigma company) effect probe carries out dynamic analysis, and the coverage rate of fluorescent marker method detector probe surface oligonucleotides adopts the existence that has detected the gold surface oligonucleotides with the method for gel electrophoresis simultaneously.
As the appearance of testing gained nano Au particle as a result is for spherical, and mean grain size is 10 ± 3nm, and is dispersed and have good stability.After the nano Au particle surface connects antibody and oligonucleotides, the visible absorption peak by the 518nm red shift to 524nm, probe has antibody biologically active preferably, and detecting probe surface oligonucleotides coverage rate is about (92 ± 20) bar, and transmission electron microscope and ultraviolet spectrophotometer are observed its phenogram 2.
Notice that the not mentioned part of following examples is identical with embodiment 5.
Embodiment 6
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced.According to the pH value that embodiment 4 regulates nano-Au solution, embodiment 5 determines the suitableeest stable quantity scope of antibodies.Get the 2.5ml colloidal gold solution, use 0.1M, 75 μ l Na
2Co
3Adjust pH to 9 adds mouse IgG (7 μ g/ml) (Beijing ancient cooking vessel state company) in proportion, and the vortex mixing adds the oligonucleotides (0.40D/ml) (it is synthetic that the worker is given birth in Shanghai) that a certain amount of 3 '-end alkane hydrogen mercaptan is modified, vortex mixing, lucifuge standing over night behind the 30min.
Add 0.01M, it is 0.01% that the PBS of pH7.4,10% SDS make its final concentration, the vortex mixing places shaking table jog 30min, and it is 0.1M that the NaCl solution that adds 1.2M gradually makes final concentration, limit edged vortex mixing leaves standstill more than the balance 12h, and stable oligonucleotides connects.At last, it is 1% that the bovine serum albumin(BSA) (BSA) of adding 5% makes ultimate density, the mixing balance.
The appearance of experimental result gained nano Au particle is spherical, and mean grain size is 10 ± 3nm, and is dispersed and stable good.Probe has antibody biologically active preferably, and detecting probe surface oligonucleotides coverage rate is about (70 ± 20) bar.
Available 3-mercaptoethanol (DTT) (Sigma company) effect probe carries out dynamic analysis, and Fig. 6 is the time changing curve figure that the DTT of embodiment 6 replaces sulfydryl DNA on the probe.
Detected the existence of gold surface oligonucleotides with the method for gel electrophoresis, Fig. 7 is oligonucleotides PAGE result on the probe of embodiment 6.
Embodiment 7
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced.According to the pH value that embodiment 4 regulates nano-Au solution, embodiment 5 determines the suitableeest stable quantity scope of antibodies.Get the 2.5ml colloidal gold solution, use 0.1M, 75 μ l Na
2Co
3Adjust pH to 9 adds mouse IgG (7 μ g/ml) (Beijing ancient cooking vessel state company) in proportion, and the vortex mixing adds the oligonucleotides (0.40D/ml) (it is synthetic that the worker is given birth in Shanghai) that a certain amount of 3 '-end alkane hydrogen mercaptan is modified, vortex mixing, lucifuge standing over night behind the 30min.
Add 0.1M, it is 0.01% that the PBS of pH7.4,10% SDS make its final concentration, the vortex mixing places shaking table jog 30min, and it is 0.15M that the NaCl solution that adds 1.2M gradually makes final concentration, limit edged vortex mixing leaves standstill more than the balance 12h, and stable oligonucleotides connects.At last, it is 1% that the bovine serum albumin(BSA) (BSA) of adding 5% makes ultimate density, the mixing balance.
The appearance of experimental result gained nano Au particle is spherical, and mean grain size is 10 ± 3nm, and is dispersed and stable good.Serious adherent phenomenon, dispersiveness and bad stability appear in the probe purge process.
The coverage rate of fluorescent marker method detector probe surface oligonucleotides, Fig. 8 is the ssDNA addition and the amount of connection situation synoptic diagram of embodiment 7.
Fig. 9 is the immunogold silver staining observations figure of embodiment 7.
Embodiment 8
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced.According to the pH value that embodiment 4 regulates nano-Au solution, embodiment 5 determines the suitableeest stable quantity scope of antibodies.Get the 2.5ml colloidal gold solution, use 0.1M, 75 μ l Na
2Co
3Adjust pH to 9, add mouse IgG (7 μ g/ml) (Beijing ancient cooking vessel state company) in proportion, the vortex mixing, room temperature leaves standstill 30min-1h, and adding 5%BSA solution, to make its final concentration be 1%, 12000r/min, 40min, gently goes supernatant to remove unconjugated antibody by 4 ℃, the repetitive operation secondary, 0.01M the PBS of pH7.4 (containing 1%BSA) redissolves, and adds the oligonucleotides (0.40D/ml) that a certain amount of 3 '-end alkane hydrogen mercaptan is modified, add 0.1M, it is 0.01% that the PBS of pH7.4,10% SDS make its final concentration, the vortex mixing, place shaking table jog 30min, it is 0.15M that the NaCl solution that adds 1.2M gradually makes final concentration, 12000r/min, 40min, 4 ℃, gently go supernatant to remove unconjugated oligonucleotides.
Experimental result gained Nano-Au probe is dispersed and have good stability.The probe antibody biologically active is good, and detecting probe surface oligonucleotides coverage rate is about (50 ± 18) bar.Adherent phenomenon appears occurring once in a while in the probe purge process.
Embodiment 9
The nm of gold that adopts optimized preparation condition embodiment 1 to be produced.According to the pH value that embodiment 4 regulates nano-Au solution, embodiment 5 determines the suitableeest stable quantity scope of antibodies.Get the 2.5ml colloidal gold solution, add the oligonucleotides (0.40D/ml) that a certain amount of 3 '-end alkane hydrogen mercaptan is modified, add 0.1M, the PBS of pH7.4, it is 0.01% that 10% SDS makes its final concentration, and the vortex mixing places shaking table jog 30min, it is 0.2M that the NaCl solution that adds 1.2M gradually makes final concentration, 12000r/min, 40min, 4 ℃, use 0.1M, 75 μ l Na
2Co
3Adjust pH to 9 adds mouse IgG (7 μ g/ml) (Beijing ancient cooking vessel state company), the vortex mixing in proportion, room temperature leaves standstill 30min-1h, and adding 5%BSA solution, to make its final concentration be 1%, 12000r/min, 40min, 4 ℃, gently go supernatant to remove unconjugated antibody, repetitive operation secondary, 0.01M, the PBS of pH7.4 (containing 1%BSA) redissolves, and stores.
Experimental result gained Nano-Au probe is dispersed and have good stability.Detecting probe surface oligonucleotides coverage rate is about (110 ± 12) bar, and antibody connection rate reduces.
The present invention can summarize with other the concrete form without prejudice to spirit of the present invention or principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, therefore, in implication suitable and any change in the scope, all should think to comprise within the scope of the present invention with claims of the present invention.
Claims (15)
1, a kind of double labelling Nano-Au probe, this probe comprises: nano Au particle is nuclear, and the surface connects antibody and oligonucleotides simultaneously.
2, Nano-Au probe according to claim 1, described Nano-Au probe mean grain size is 20-60nm.
3, Nano-Au probe according to claim 1, described Nano-Au probe mean grain size is 30-50nm.
4, according to the arbitrary described Nano-Au probe of right 1--3, wherein said antibody is general purpose I gG, and described general purpose I gG is one of polyclonal antibody or monoclonal antibody or its combination.
5, according to the arbitrary described Nano-Au probe of right 1--3, wherein said antibody is that goat anti-mouse igg, anti-people PSA polyclonal antibody, anti-people HCG polyclonal antibody, anti-people HBV polyclonal antibody, anti-people HIV polyclonal antibody, anti-people ADDL polyclonal antibody, the anti-popular feeling are received one of plain ANP polyclonal antibody, anti-people NSE polyclonal antibody, anti-people C-peptide polyclonal antibody or monoclonal antibody or its combination.
6, according to the arbitrary described Nano-Au probe of right 1-3, described oligonucleotides is one of the single stranded DNA of sulfydryl modification or RNA molecule or its combination.
7, according to the arbitrary described Nano-Au probe of right 1---3, the length of the fragment of described oligonucleotides is 10～50bp.
8, the preparation method of the arbitrary described double labelling Nano-Au probe of a kind of claim 1--7 comprises following step:
1. coupled reaction: in concentration is that adding concentration is 6.6～33ug/ml oligonucleotides solution, concentration 4-10ug/ml antibody-solutions in the 2--6nM nano gold sol, mix, in temperature is 4～30 ℃, the control pH value in reaction is 6～9, time is 10～20h, make that nano Au particle and antibody, nucleic acid can fully act in the solution, make mixed liquor;
2. stablize and capping: the amount that preparation contains SDS is the PBS buffer solution of 5-10%, wherein the amount of PBS is 0.01-0.1M, add then in the 1. made mixed liquor of step, the final concentration of control activating agent SDS is 0.005-0.015%, evenly mixed, the stabilizing agent that adds 0.3-1.2M then gradually, limit edged mixing, the concentration of final control stabilization agent is 0.1M～0.2M, leaves standstill more than the balance 6-12h, and is last, add sealer, the final concentration of control sealer is 0.5%～1%, and mixing is even, forms the colloidal sol of nano-probe.
9, preparation method according to claim 8, its step 1. before, also comprise step:
(1) trisodium citrate reduction method prepares nano gold sol: with the HAuCl of 0.005%--0.04%
4Solution is heated to boiling, the 0.5%--3% citric acid three sodium solution that adds fast under the high-speed stirred condition, wherein required HAuCl
4The volume ratio of solution and citric acid three sodium solution is 100: 8--100: 2, continue heated and stirred to mixing, and natural cooling forms nano gold sol then.
10, preparation method according to claim 9, it also comprises step in step (1) afterwards:
Step (1) gained nano gold sol 0.1-0.25 μ m membrane filtration.
11, preparation method according to claim 8, its step 2. after, also comprise step: low temperature ultracentrifugation purifying: to the colloidal sol of the nano-probe of step in 2., carry out centrifugal under 12000～16000r/min rotating speed, centrifugation time is 15～45min, and temperature is controlled at the 4-8 degree, removes supernatant, with the cleansing solution cyclic washing of 0.01%～0.10% Sodium Azide, 4～8 ℃ of preservations.
12, preparation method according to claim 8, wherein said stabilizing agent is the NaCl aqueous solution, described sealer is bovine serum albumin(BSA) (BSA).
13, preparation method according to claim 8, wherein step 1. described mix into: earlier use the vortice mixing, adopt shaking table 50--900r/min jog then.
14, preparation method according to claim 8, wherein one of the 1. described oligonucleotides of the step single stranded DNA that is sulfydryl modification or RNA molecule or its combination.
15, the application of the arbitrary described double labelling Nano-Au probe of a kind of claim 1---7, it is that this probe can be converted into detection of antigens the detection to nucleic acid substances, is applied to the detection of tumor marker will thing, hormone, pathogen trace protein.
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