CN100371713C - Surface functionalization of gold or silver nanoparticle, and colorimetry detection method for molecule by using the same - Google Patents
Surface functionalization of gold or silver nanoparticle, and colorimetry detection method for molecule by using the same Download PDFInfo
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- CN100371713C CN100371713C CNB2006100377639A CN200610037763A CN100371713C CN 100371713 C CN100371713 C CN 100371713C CN B2006100377639 A CNB2006100377639 A CN B2006100377639A CN 200610037763 A CN200610037763 A CN 200610037763A CN 100371713 C CN100371713 C CN 100371713C
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Abstract
The present invention discloses a method for marking, color comparison and detection of biomolecules through gold nanometer particles or silver nanometer particles having functionalized surfaces, which relates to a method using gold nanometer particles or silver nanometer particles having functionalized surfaces as labels for color comparison and detection of biomolecules. A probe molecule for recognising biomolecules and an anchoring molecule for connecting gold nanometer particles or silver nanometer particles are firstly arranged on high molecular hyskon in a covalent modification mode through a bifunctional reagent, and an ornament coated with the gold nanometer particles or silver nanometer particles is formed. The ornament is puritied, and the quantity of the number of the molecules on the ornament is fixed. Gold nanometer particles or silver nanometer particles coated with the hyskon are formed by that the anchoring groups on the ornament carry out self assembly on the surfaces of the gold nanometer particles or silver nanometer particles. The method utilizes metal nanometer particles for color comparison and detection of biomolecules, which avoids complicated detection devices. The present invention has simple method and high sensitivity, and the detection limit reaches an f mol level.
Description
Technical field
The present invention relates to the serve as a mark method of thing colorimetric detection biomolecule of a kind of surface-functionalized gold or Nano silver grain, belong to the nanometer mark and the detection technique field of biomolecule.
Background technology
The labeled analysis method is one of main method of biomolecule (nucleic acid, protein etc.) detection.Many biomolecule since itself can for the character of analyzing a little less than, obtain highly sensitive detection, usually be to obtain measurable signal by external label to analyze.Label commonly used can be divided into luminous marker, enzyme labeling thing, electrochemical label thing or the like.Wherein the luminescent marking method is topmost one big class methods, as various fluorescence labelings, chemiluminescent labeling etc.The luminous intensity of luminous marker is depended in the sensitivity that luminescent marking detects to a great extent.
Nanometer technology will be brought new development to diagnosis, treatment, bioanalysis science with combining of biotechnology.One of application of nano particle is the label as biomolecule.Gold, silver metal nanoparticle chemical property is stable, just is used in immunoassay as the label of antibody, protein in early days.Labelled antibody, protein mainly are to utilize surface electrostatic absorption, adsorb the pH value that firm degree largely depends on solution.Yet the method for this physisorption also is not suitable for the mark of other molecules such as DNA.
The metal nanoparticle mark of dna molecular is to use the golden nanometer particle mark at present, adopt method (the Elghanian R of the oligonucleotides of sulfhydrylation in golden nanometer particle surface self assembly, Storhoff J J, Mucic R C, Letsinger R L, Mirkin C A, Science, 1997,277,1078; Storhoff J J, Elghanian R, Mucic R C, Mirkin C A, J Am Chem Soc, 1998,120,1959).The oligonucleotides of sulfhydrylation is commercial to be obtainable, can not obtain but other molecule of most sulfhydrylations is commercial, and therefore, other molecule is difficult to realize by the golden nanometer particle mark by this method.Method with the metal nanoparticle marker DNA of other kind is not appeared in the newspapers.
Summary of the invention
Technical matters: the purpose of this invention is to provide a kind of new, can make various biomolecules by gold and Nano silver grain mark, and utilize the method for the highly sensitive colorimetric detection biomolecule of color of gold or Nano silver grain.
Technical scheme: the surface-functionalized method of gold of the present invention or Nano silver grain with mark, colorimetric detection biomolecule be identification biomolecule probe molecule and be connected gold or the anchoring molecule of Nano silver grain at first by bifunctional reagent by covalent modification on high molecular glucosan, constitute the trim of parcel gold or Nano silver grain, molecular number on the purified and trim of trim quantitatively after, by the self assembly of the anchoring group on the trim, form the gold or the Nano silver grain of dextran molecule parcel again in gold or nano grain of silver sub-surface.With the gold of this functionalization or Nano silver grain colorimetric detection biomolecule is that white polymer microsphere with micron-scale is a carrier, the solution of target molecule solution and excessive gold that is modified with probe molecule or Nano silver grain is joined in the solution of the white polymer microsphere that is mounted with capture molecules and react, gold or Nano silver grain are to be connected the combination that molecule forms sandwich style with white polymer microsphere by target molecule, generation is the microballoon of cash or Nano silver grain color, after centrifugal or isolated by filtration of microballoon and the washing, the depth of microballoon color is directly proportional with the amount of the target molecule that combines, contrast combines the color of the microballoon of known quantity target molecule, and visual or scanning colorimetric is measured the content of target molecule.
Each dextran molecule of having modified probe molecule and anchoring molecule in the such scheme is the surface that is fixed in nano particle by many anchoring groups.Containing the group that can maybe can generate active function groups with bifunctional reagent reactive activity functional group on the dextran molecule, can be glycosaminoglycan or carboxyl glucosan or Sensor Chip CM 5.Probe molecule is a kind of for the biomolecule centering that can discern mutually, the one end contain can with bifunctional reagent reactive activity group, can be oligonucleotides or biotin or antibody or peptide or protein.Anchoring molecule contain the disulfide group that is connected with gold or Nano silver grain (S-S-) or mercapto groups (SH-) and be connected with dextran molecule or with bifunctional reagent reactive activity group.Bifunctional reagent be can with the succinimide ester of amino reaction or can with the carbodiimide of carboxyl reaction or can with the hydrazone compounds of aldehyde radical reaction.Being modified at the UV that probe molecule on the dextran molecule and anchoring molecule number generate chemical bond can be by coupling the time absorbs quantitatively.
When wrapping up the gold or the nano grain of silver period of the day from 11 p.m. to 1 a.m with the dextran molecule of having modified probe molecule and anchoring molecule, the amount of the glucosan that coupling is required records in a certain amount of gold or silver nano-particle solution by the glucosans that contain probe molecule and anchoring molecule that add different amounts.Wrap up when incomplete because the existence of salt in the damping fluid, nano particle is assembled, solution change color, and the UV absorbance changes.Solution filtered and recording solution in the UV absorbance of gold or Nano silver grain can record the minimum glucosan amount of required adding when wrapping up fully.The required minimum glucosan that contains probe molecule and anchoring molecule joins in gold or the silver nano-particle solution when wrapping up fully, and fully mixing promptly generates stable gold or Nano silver grain probe.
During with gold or Nano silver grain probe colorimetric detection biomolecule, use commercially available white polymer microsphere (as Bangs, Dynal, companies such as Molecular Probe) make carrier, be connected the capture molecules binding target molecule specifically on the white polymer microsphere, the one end contain can with the reactive group of microballoon combination, capture molecules can be oligonucleotides or biotin or antibody or peptide or protein.The method that capture molecules is connected with polymer microsphere is provided by microballoon manufacturer.
Beneficial effect: gold or Nano silver grain mark thing have the preparation simple, inexpensive, the advantage that chemical property is very stable, the inventive method is except that having these advantages, also has following distinct advantages: because probe molecule is fixed in nanoparticle surface by a plurality of anchoring molecule that contain sulfydryl, compare with the nano particle that common single sulfydryl probe molecule is modified, the nano particle that many sulfydryls probe molecule is modified has the stability of height, can more use under the exacting terms (as under the high salt concentration, the PCR pyroprocess).Secondly, the molecule that desire connects is modified on the macromolecule, and the preparation process on whole modification top layer is not subjected to the interference of nano particle itself, has high preparation productive rate and is easy to purifying and quantitative test.The 3rd, the method that probe molecule is modified on the macromolecule parcel nano particle is not only applicable to being connected of DNA and nano particle, is applicable to being connected of other biomolecule and nano particle yet.The 4th, detect the color colorimetric estimation that utilizes metal nanoparticle.The color of metallics not time to time change has permanent stability, and complicated checkout equipment has been avoided in colorimetric analysis, and method is simple and have high sensitivity, and detectability reaches the fmol level.
Description of drawings
Fig. 1. the nano particle that probe molecule is modified and target molecule and the microballoon sandwich style that is mounted with capture molecules combine synoptic diagram.1 is capture molecules, and 2 is white microballoon, and 3 is target molecule, and 4 is probe molecule, and 5 is nano particle.
Fig. 2. (A) golden nanometer particle of diameter 10nm detects the result of DNA, and visual detection is limited to 500fmol.Be respectively 200pmol from top to bottom, 50pmol, 10pmol, 5pmol, 500fmol, the color of 0mol target molecule correspondence.(B) Nano silver grain of diameter 20nm detects DNA result, and visual detection is limited to 500fmol.Be respectively 200pmol from top to bottom, 50pmol, 10pmol, 5pmol, 500fmol, the color of 0mol target molecule correspondence.
Fig. 3. the surface has connected the TEM figure of the white polymer microsphere of golden nanometer particle.(a) (b) for adding the sample of target molecule, (c) be the contrast of no target molecule.
Embodiment
Embodiment 1: the oligonucleotide probe of golden nanometer particle mark is measured DNA
The coupling of oligonucleotides and glucosan: 0.16ml, 25mg/ml 4-Yan acid succinimide ester hydrazine acetone hydrazone (C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone) and 25mg/ml N-succinimide-3-(2-pyridine two sulfo-s)-propionic ester (SPDP, N-succinimidyl3-(2-pyridyldithio) propionate) anhydrous DMSO solution is added to 4ml, reacts 2 hours under the room temperature in 1 * PBS solution of 2mg/ml glycosaminoglycan (MW70KDa).The glucan conjugate that generates is an eluent Sephadex G25 post purifying with 0.1M NaAc (pH=4.5).Two sulphur that generate and the amount of hydrazone functional group can be respectively with 1, and 4-dithionthreitol (Dithiothreitol) and 4-nitrobenzaldehyde (4-nitrobenzaldehyde) are quantitative.Excessive aldehyde group modified oligonucleotides joins in 0.1M NaAc (pH 4.5) solution of glucan conjugate behind the reaction 12h, removes unreacted oligonucleotides and H with centrifugal filter (Millipore) separation of 30KDa weight shutoff
2O washes twice.The covalent bond that hydrazone functional group forms on oligonucleotides and the glucosan is at the absorption peak (ε of 360nm
360=1.8 * 10
4) can be quantitative to the oligonucleotide molecules number on modifying.
The preparation of the oligonucleotide probe of golden nanometer particle mark: the disulfide bond in the PDP molecule of modification can be on the golden nanometer particle surface self assembly and form the nano particle of glucosan parcel.In a certain amount of solution of gold nanoparticles, add the oligonucleotides of different amounts and the glucosan that PDP modifies, solution writes down golden nanometer particle 520nm behind the membrane filtration of 0.20 μ m UV absorbs, and mixes the oligonucleotide probe that generates the golden nanometer particle mark with solution of gold nanoparticles with the required glucosan amount that records.The golden nanometer particle probe that generates does not have repurity.Concrete steps are: the glucosan that 16 μ l probe molecules are modified is added to 0.75ml, 10nm, 5.7 * 10
12In Au nano particle/ml solution, fully mix the back and add 0.375ml 1.5M NaCl 30mM phosphate (pH 7.0) damping fluid, and add 1.2mg BSA, it is standby that 1.2mg glucose and 5.6 μ lTween-20 mix the back.
White microballoon (the 560nm of the probe assay DNA:200 μ g streptavidin parcel of golden nanometer particle mark, Bangs Laboratories, Fishers IN) capture oligo with excessive biotin modification reacts 0.5h under the room temperature in 1 * PBS solution, after the centrifuging with 0.5%Tween-201 * PBS solution washing three times.The oligonucleotide probe and the target DNA that add excessive golden nanometer particle mark are hybridized 1h under hybridization solution (1M NaCl20mM sodium citrate) room temperature, reaction is finished back usefulness 0.5%Tween-201 * PBS solution washing three times.Precipitation is suspended in 20 μ lH again
2Among the O and suspending liquid is injected the microwell plate colorimetric record target DNA concentration.
Embodiment 2: the oligonucleotide probe of Nano silver grain mark is measured DNA
The coupling of oligonucleotides and glucosan: 0.16ml, 25mg/ml 4-Yan acid succinimide ester hydrazine acetone hydrazone (C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone) and 25mg/ml N-succinimide-3-(2-pyridine two sulfo-s)-propionic ester (SPDP, N-succinimidyl3-(2-pyridyldithio) propionate) anhydrous DMSO solution is added to 4ml, reacts 2 hours under the room temperature in 1 * PBS solution of 2mg/ml glycosaminoglycan (MW 70KDa).The glucan conjugate that generates is an eluent Sephadex G25 post purifying with 0.1MNaAc (pH=4.5).Two sulphur that generate and the amount of hydrazone functional group can be used l respectively, and 4-dithionthreitol (Dithiothreitol) and 4-nitrobenzaldehyde (4-nitrobenzaldehyde) are quantitative.The oligonucleotide probe that excessive CHO-modifies joins in 0.1M NaAc (pH 4.5) solution of glucan conjugate behind the reaction 12h, removes unreacted oligonucleotides and H with centrifugal filter (Millipore) separation of 30KDa weight shutoff
2O washes twice.
The oligonucleotide probe preparation of Nano silver grain mark: in a certain amount of silver nano-particle solution, add the oligonucleotides of different amounts and the glucosan that PDP modifies, solution writes down Nano silver grain 408nm behind the membrane filtration of 0.20 μ m UV absorbs, and mixes the oligonucleotide probe that generates the Nano silver grain mark with silver nano-particle solution with the required glucosan amount that records.The Nano silver grain probe that generates does not have repurity.Concrete steps are: the glucosan that 12 μ l probe molecules are modified is added to 0.75ml, 20n m, 7 * 10
10In Ag nano particle/ml solution, fully mix the back and add 0.375ml 1.5M NaCl 30mM phosphate (pH 7.0) damping fluid, and add 1.2mg BSA, it is standby that 1.2mg glucose and 5.6 μ l Tween-20 mix the back.
White microballoon (the 560nm of the probe assay DNA:200 μ g streptavidin parcel of Nano silver grain mark, Bangs Laboratories, Fishers IN) capture oligo with excessive biotin modification reacts 0.5h under the room temperature in 1 * PBS solution, after the centrifuging with 0.5%Tween-201 * PBS solution washing three times, the oligonucleotide probe and the target DNA that add excessive Nano silver grain mark are hybridized 1h under hybridization solution (1M NaCl 20mM sodium citrate) room temperature, reaction is finished the back with 0.5%Tween-201 * PBS solution washing three times, precipitates to be suspended in 20 μ l H again
2Among the O and suspending liquid is injected the microwell plate colorimetric record target DNA concentration.
Embodiment 3: golden nanometer particle mark biotin molecule
The coupling of biotin and glucosan: 5mg N-succinimide-3-(2-pyridine two sulfo-s)-propionic ester (SPDP) is dissolved in 100 μ l, the anhydrous DMSO solution of 50mg/ml biotin lpsilon-N-hydroxy-succinamide ester (Biotinamidohexanic acid N-hydroxysuccinimide ester), this solution under agitation dropwise is added to 2.5ml, the NaHCO of 2mg/ml glycosaminoglycan (MW 70KDa)
3(50mM) reacted 2 hours under the room temperature in the solution.The glucan conjugate that generates is standby behind the dialysis purification in aqueous solution.(4 '-hydroxyazobenzene-2-carboxylicacid) quantitatively for the available HABA of the amount of biotin on the glucan conjugate.
The biotin of golden nanometer particle mark-glucan conjugate probe preparation: the biotin-glucan conjugate that in a certain amount of solution of gold nanoparticles, adds different amounts, solution writes down golden nanometer particle 520nm behind the membrane filtration of 0.20 μ m UV absorbs, and mixes the biotinylated probes that generates the golden nanometer particle mark with solution of gold nanoparticles with the required glucosan amount that records.Concrete steps are that the glucosan that 16 μ l probe molecules are modified is added to 0.75ml, 10nm, 5.7 * 10
12In Au nano particle/ml solution, fully mix the back and add 0.375ml 1.5M NaCl 30mM phosphate (pH 7.0) damping fluid, and add 1.2mg BSA, it is standby that 1.2mg glucose and 5.6 μ lTween-20 mix the back.
Claims (10)
1. the surface-functionalized method of gold or Nano silver grain, the anchoring molecule that it is characterized in that discerning the probe molecule of biomolecule and connect gold or Nano silver grain at first by bifunctional reagent by covalent modification on high molecular glucosan, constitute the trim of parcel gold or Nano silver grain, molecular number on the purified and trim of trim quantitatively after, by the self assembly of the anchoring group on the trim, form the gold or the Nano silver grain of dextran molecule parcel again in gold or nano grain of silver sub-surface.
2. the surface-functionalized method of gold according to claim 1 or Nano silver grain, each dextran molecule that it is characterized in that modifying probe molecule are the surfaces that is fixed in nano particle by many anchoring groups.
3. the surface-functionalized method of gold according to claim 1 or Nano silver grain is characterized in that probe molecule is a kind of of the biomolecule centering that can discern mutually, the one end contain can with bifunctional reagent reactive activity group.
4. the surface-functionalized method of gold according to claim 3 or Nano silver grain is characterized in that probe molecule is oligonucleotides or biotin or antibody or peptide or protein.
5. the surface-functionalized method of gold according to claim 1 or Nano silver grain, it is characterized in that anchoring molecule contains the disulfide group that is connected with gold or Nano silver grain or mercapto groups and be connected with dextran molecule or with bifunctional reagent reactive activity group.
6. the surface-functionalized method of gold according to claim 1 or Nano silver grain is characterized in that the glucosan macromolecule that wraps up gold or Nano silver grain contains the group that can maybe can generate active function groups with bifunctional reagent reactive activity functional group.
7. the surface-functionalized method of gold according to claim 1 or Nano silver grain, it is characterized in that bifunctional reagent be can with the succinimide ester of amino reaction or can with the carbodiimide of carboxyl reaction or can with the hydrazone compounds of aldehyde radical reaction.
8. the surface-functionalized method of gold according to claim 1 or Nano silver grain is characterized in that glucosan is a kind of in glycosaminoglycan, carboxyl glucosan or the Sensor Chip CM 5.
9. the method for the colorimetric detection biomolecule of gold as claimed in claim 1 or Nano silver grain, it is characterized in that the white polymer microsphere with micron-scale is a carrier, the solution of target molecule solution and excessive gold that is modified with probe molecule or Nano silver grain is joined in the solution of the white polymer microsphere that is mounted with capture molecules and react, gold or Nano silver grain are to be connected the combination that molecule forms sandwich style with white polymer microsphere by target molecule, generation is the microballoon of cash or Nano silver grain color, after centrifugal or isolated by filtration of microballoon and the washing, the depth of microballoon color is directly proportional with the amount of the target molecule that combines, contrast combines the color of the microballoon of known quantity target molecule, and visual or scanning colorimetric is measured the content of target molecule.
10. the method for the colorimetric detection biomolecule of gold according to claim 9 or Nano silver grain is characterized in that capture molecules binding target molecule specifically, the one end contain can with the reactive group of microballoon combination.
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| CN100510704C (en) * | 2007-08-30 | 2009-07-08 | 桂林工学院 | Method for measuring vitamin C by gold nano particulate spectrophotometry |
| AU2010289483A1 (en) * | 2009-09-01 | 2012-03-29 | Northwestern University | Delivery of therapeutic agents using oligonucleotide-modified nanoparticles as carriers |
| CN102141567B (en) * | 2010-12-19 | 2014-03-12 | 浙江大学 | Method for preparing enzyme labelling antibody |
| CN104020084A (en) * | 2014-06-17 | 2014-09-03 | 大连理工大学 | Method for recognizing precious metal nano particles from dielectric medium scattering background |
| CN104391019B (en) * | 2014-10-29 | 2017-08-18 | 安徽师范大学 | A kind of aptamers electrochemica biological sensor, its preparation method and purposes |
| CN106257271B (en) * | 2015-06-19 | 2019-08-06 | 中国科学院宁波材料技术与工程研究所 | A kind of composite material and preparation method based on Surface enhanced Raman scattering technology |
| CN108254366B (en) * | 2017-09-19 | 2020-08-25 | 中国科学院海洋研究所 | Method for detecting microorganisms based on phenylboronic acid functionalized silver nanoparticles |
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