CN103048306A - Core-shell nanogold biological probe with high SERS (surface enhanced Raman scattering) effect and preparation and application thereof - Google Patents
Core-shell nanogold biological probe with high SERS (surface enhanced Raman scattering) effect and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a core-shell nanogold biological probe with a high SERS (surface enhanced Raman scattering) effect and a preparation and an application thereof. The preparation comprises the following steps: assembling a section of DNA sequence and Raman micromolecules on the surface of nanogold with a small particle diameter; and further generating a gold shell with a certain thickness on the surface of a core of the nanogold, wherein a gap with a certain size exists between the core and the shell, the Raman micromolecules are in the gap, and due to specificity of the structure, high-efficiency and uniform SERS signals are obtained. The Raman micromolecules are in the gap with the fixed size between the core and the shell of the nanogold, so that the SERS signals generated in areas of the molecules, namely hotspot areas, are basically consistent and have good repeatability; the biological molecules are assembled on the surface of the prepared core-shell nanogold biological probe, so that a surface receptor of a target cell can be recognized specifically, the target cell can be detected and imaged by a laser Raman spectroscopy, and Raman scattering value increase on the obtained surface can show the expression of the surface receptor of the target cell efficiently; and the core-shell nanogold biological probe is also applicable to biological sensor detection, biomolecular detection and other research fields.
Description
Technical field
The invention belongs to functionalization and the application of nano material, relate to a kind of preparation method with core-shell nano gold bioprobe of high SERS effect, can be applicable to the fields such as biomolecule detection and cell imaging.
Background technology
Surface enhanced raman spectroscopy (Surface Enhanced Raman scattering, SERS) refers to be positioned at the phenomenon that the Raman signal of the little molecule of roughened metal surface itself is enhanced.This phenomenon is widely used in fields such as Surface Science, analysis science and bio-science, for the structure on the various surfaces of deep sign (interface) and process provide information on the molecular level, as differentiating that molecule or ion are at bonding, configuration and the orientation on surface and the surface structure of material.Enhancing mechanism about SERS, although still there is up till now dispute, but comparatively approval is Electromagnetic enhancement mechanism, and " focus " that relate in this mechanism (hot spot) generally refers in the molecular aggregation of some nanoparticles, the zone of the space part between the adjacent nano particle.This regional SERS effect is the strongest.How to make up efficient homogeneous and contain the more SERS substrate of " focus ", become focus and the difficult point of SERS research field.
In the present research, the common methods that makes up efficient homogeneous substrate has following several:
1, metal electrode active substrate, this is to use at present comparatively widely a kind of substrate.Carry out suitable roughening by electrode surface and process, can produce roughness and substantially be in macro-asperity and the submicroscopic roughness scope.The shortcoming of this mode be most metals after treatment, rough surface dimensional variation scope is larger, cause last some surface enhanced effect of substrate to alter a great deal, this structural inhomogeneity has directly affected the stability of the SERS spectrum that adsorbs molecule and the reappearance of data.
2, the active substrate of chemical etching and chemogenic deposit is the purpose that by strong corrosive material the atom of metal surface is dissolved to reach surface roughening by chemical reaction.The shortcoming of this mode is that the difficult control of reaction conditions, the time of deposition, the temperature of reaction, the concentration of reagent etc. are all influential to the roughness of substrate.
Also have in addition lithographic printing, self-service packing technique, ordered fabrication technology etc. to prepare active substrate, thereby but all exist separately shortcoming to limit the development and application of SERS technology.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of preparation method with core-shell nano bioprobe of high SERS effect, except all characteristics that itself possess nano material, can also be applied to the fields such as biomolecule detection and cell imaging as a kind of efficient Raman microprobe.
A kind of preparation method with core-shell nano bioprobe of high SERS effect, it is characterized in that, behind the little molecule of small particle diameter nm of gold finishing one deck, grow again, form again the layer of gold shell on gold nuclear surface, have the little molecule with Raman signal between the slit between the gold nucleocapsid, step is as follows:
(1) nm of gold nuclear surface-assembled DNA;
(2) the assembling potpourri carries out burin-in process;
(3) obtain the solution I behind the centrifuge washing;
(4) the little molecule of nm of gold nuclear surface-assembled;
(5) obtain the solution II behind the centrifuge washing;
(6) growth of golden shell is carried out on nm of gold nuclear surface;
(7) golden nuclear structure centrifuge washing;
Described nm of gold nuclear is the nm of gold of particle diameter 5～15nm.
Step (1) is described to be assembled in the nm of gold nuclear and to add SH-polyA DNA, and final concentration is 0.1～5 μ M, the slight shaken overnight of room temperature.
The described burin-in process condition of step (2) is: add 0.01～1M, pH is 7.4 phosphate buffer solution (PB), room temperature condition 350rpm/min vibration 30～120 minutes, rear adding 1～5M sodium chloride, final concentration is 0.1～0.15M, and minute four addings are spaced apart 30 minutes, 350rpm/min shaken overnight under the room temperature.
Step (3), (5) described centrifuge washing condition are 4 ℃, 15000～12000rpm/min, 20 minutes, three times, cleansing solution was PB(10mM, pH7.4), rear 0.1M phosphate buffer (PBS, pH7.4) resuspension.
What the described nm of gold of step (4) was examined surface-assembled is the little molecule with obvious characteristic peak, be specially 2,3-benzodiazine (PHTH), 5,5'-two sulphur two (2-nitrobenzoic acids) (DTNB), a kind of or its combination in the cyanine class dyestuff, fluorescein isothiocyanate (FITC), pyridine, assembling condition is 1-100 μ L, 0.1 the little molecular solution of～1M joins in 5 μ L～5mL solution I, the 350rpm/min vibration was adsorbed 2～3 days under the room temperature.
The growth conditions of the described golden shell of step (6) is: add successively 0.1～5% polyvinyl pyrrolidone aqueous solution (PVP) in the solution II, oxammonium hydrochloride aqueous solution (NH2OHHCl), 0.01～1% aqueous solution of chloraurate (HAuCl4), mixing vibration 1 minute.
The described golden nuclear structure centrifuge washing condition of step (7) is: 20 ℃, and 3000～10000rpm/min, 6 minutes, three times, cleansing solution was Milli-Q water, rear 10～1000 μ LMilli-Q water resuspensions.
The present invention also provides a kind of core-shell nano Au probe with high SERS effect, and described core-shell nano Au probe particle diameter is 30～50 nanometers, and concentration is 0.1～1nM.
The present invention also provides a kind of and has the core-shell nano Au probe of high SERS effect in the application in biomolecule detection and cell imaging field, can obtain highly sensitive SERS signal.This detecting probe surface assembling biomolecule with preparation, can identify specifically the target cell surface receptor, utilize laser Raman spectrometer that cell is detected and the resulting Surface enhanced raman spectroscopy signal of imaging also with the efficiently expression of reacting cells surface receptor.Its application process is as follows:
(1) alkaline solution is adjusted core-shell nano gold solution pH: alkaline solution is 0.1～2M NaOH, sal tartari (K
2CO
3) or saleratus (KHCO
3) or sodium bicarbonate (NaHCO
3) in a kind of, pH is adjusted into 8～10;
(2) add biomolecule, assemble on its surface: biomolecule is protein (antibody) or polypeptide or DNA(aptamer); Assembling condition is room temperature 30～120 minutes;
(3) centrifuge washing: 4 ℃, 3000-10000rpm/min, 5min, three times, cleansing solution is Milli-Q water;
(4) cultivate altogether with the target material of biomolecule: the target material is a kind of in cell, protein (antibody), the dna sequence dna.
The present invention passes through at small particle diameter nm of gold surface-assembled section of DNA sequence and the little molecule of Raman, examine the certain thickness golden shell of Surface Creation by reducing process at gold, the slit that has certain size between the nucleocapsid, the little molecule of Raman just exists in this slit, and owing to the singularity of this structure obtains efficient SERS signal.Advantage of the present invention is: the little molecule of Raman exists in the slit of the fixed measure between the golden nucleocapsid, thereby the SERS signal of i.e. " focus " zone generation in the zone at each molecule place is basically identical, and good repeatability is arranged.
Shell nano gold biological probe structure will strengthen micromolecular Raman signal greatly.This detecting probe surface assembling biomolecule with preparation, can identify specifically the target cell surface receptor, can utilize laser Raman spectrometer that cell is detected and imaging, simultaneously resulting surface increases the Raman scattering value also with the efficiently expression of reacting cells surface receptor.
Description of drawings
Fig. 1 is the Raman spectrogram of the core-shell nano Au probe of the little molecule preparation of the different Ramans of use.
Figure a is DTNB, and figure b is Cy3.
Fig. 2 is that cell surface selects access point to carry out the spectrogram of Raman detection gained at random when adhering to the micromolecular core-shell nano of PHTH gold bioprobe and being applied to cell detection.
The a line is for using this invention middle probe gained spectrogram, and the b line is little molecular adsorption gained spectrogram after nm of gold surface and cytosis, and the c line is for to make mutually time spent gained spectrogram without probe and cell.
Fig. 3 is at PHTH characteristic peak place, the SERS effect comparative result of this core-shell nano Au probe and nm of gold-Small-molecule probe.
Embodiment
Embodiment 1:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M PHTH joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is MilliQ water, rear 100 μ LMilliQ water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of phthalazines (PHTH) peak.Get this nucleocapsid probe of 200 μ L, add 20 μ L 0.1M NaOH solution, the pH value is 10 rear adding 20 μ L, 25 μ M polypeptide solutions, and after mixing, room temperature 30min assembling.Rear 4 ℃, 5000rpm/min, 5min washing 3 times, cleansing solution is Milli-Q water, sediment cell culture fluid resuspension.Getting 100 μ L joins in the double dish that contains the 2mL nutrient solution in 37 ℃, the cell culture incubator of 5.0% carbon dioxide and co-culture of cells 1h, rear PBS solution cleans 2 times, adds fixedly 10min of formaldehyde immobile liquid room temperature, MilliQ water flushing 3 times detects under the laser Raman spectrometer.
Embodiment 2:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M phthalazines PHTH joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is MilliQ water, rear 100 μ LMilliQ water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of phthalazines (PHTH) peak.Get this nucleocapsid probe of 200 μ L, add 20 μ L 0.1M NaOH solution, the pH value is 10 rear adding 20 μ L, 2%PEG solution, and after mixing, room temperature 30min assembling.Rear 4 ℃, 5000rpm/min, 5min washing 3 times, cleansing solution is Milli-Q water, sediment cell culture fluid resuspension.Getting 100 μ L joins in the double dish that contains the 2mL nutrient solution in 37 ℃, the cell culture incubator of 5.0% carbon dioxide and co-culture of cells 1h, rear PBS solution cleans 2 times, adds fixedly 10min of formaldehyde immobile liquid room temperature, Milli-Q water flushing 3 times detects under the laser Raman spectrometer.
Embodiment 3:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M DTNB joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of DTNB peak.
Embodiment 4:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M Cy3 joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of Cy3 peak.
Embodiment 5:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M pyridines joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of pyridine peak.
Embodiment 6:
100 μ L, 10nM particle diameter add 4 μ L, 100 μ M SH-polyA DNA, the slight shaken overnight of room temperature in the nm of gold of 15nm; The rear adding liquid 0.1M PB(pH7.4 that wears out) solution 10 μ L, the room temperature condition 350rpm/min 30min that vibrates, rear adding 2M NaCl 20 μ L minutes four times add, are spaced apart 30min, 350rpm/min shaken overnight under the room temperature; 4 ℃, 12000rpm/min carries out centrifuge washing three times under the 20min condition, and cleansing solution is 10mMPB, rear 1mL0.1M PBS resuspension; The little molecular solution of 100 μ L 0.1M FITC joins in the 500 μ L mentioned solutions, and the 350rpm/min vibration was adsorbed 2-3 days under the room temperature.Get 100 μ L mentioned solutions and then add 50 μ L1%PVP, 25 μ L 10mM NH
2OHHCl, 25 μ L 5mM HAuCl
4, behind the mixing vibration 1min 20 ℃, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter that this mode prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at the high Enhanced feature of FITC peak.
Claims (10)
1. preparation method with core-shell nano bioprobe of high SERS effect, it is characterized in that, behind the little molecule of small particle diameter nm of gold finishing one deck, grow again, form again the layer of gold shell on gold nuclear surface, have the little molecule with Raman signal between the slit between the gold nucleocapsid, step is as follows:
Nm of gold nuclear surface-assembled DNA;
The assembling potpourri carries out burin-in process;
Obtain the solution I behind the centrifuge washing;
The little molecule of nm of gold nuclear surface-assembled;
Obtain the solution II behind the centrifuge washing;
The growth of golden shell is carried out on nm of gold nuclear surface;
Gold nuclear structure centrifuge washing.
2. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1 is characterized in that, described nm of gold nuclear is the nm of gold of particle diameter 5～15nm.
3. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1 is characterized in that, described being assembled in the nm of gold nuclear of step (1) adds SH-polyA DNA, and final concentration is 0.1～5 μ M, the slight shaken overnight of room temperature.
4. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, the described burin-in process condition of step (2) is: add 0.01～1M, pH is 7.4 phosphate buffer solution (PB), room temperature condition 350rpm/min vibration 30～120 minutes, rear adding 1～5M sodium chloride, final concentration is 0.1～0.15M, and minute four addings are spaced apart 30 minutes, 350rpm/min shaken overnight under the room temperature.
5. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, step (3), (5) described centrifuge washing condition are 4 ℃, 15000～12000rpm/min, 20 minutes, three times, cleansing solution is PB(10mM, pH7.4), rear 0.1M phosphate buffer (PBS, pH7.4) resuspension.
6. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, what the described nm of gold of step (4) was examined surface-assembled is the little molecule with obvious characteristic peak, be specially 2,3-benzodiazine (PHTH), 5,5'-two sulphur two (2-nitrobenzoic acid) (DTNB), cyanine class dyestuff, fluorescein isothiocyanate (FITC), a kind of or its combination in the pyridine, assembling condition is 1-100 μ L, 0.1 the little molecular solution of～1M joins in 5 μ L～5mL solution I, the 350rpm/min vibration was adsorbed 2～3 days under the room temperature.
7. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, the growth conditions of the described golden shell of step (6) is: add successively 0.1～5% polyvinyl pyrrolidone aqueous solution (PVP) in the solution II, oxammonium hydrochloride aqueous solution (NH2OHHCl), 0.01～1% aqueous solution of chloraurate (HAuCl4), mixing vibration 1 minute.
8. described preparation method with core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, the described golden nuclear structure centrifuge washing condition of step (7) is: 20 ℃, 3000～10000rpm/min, 6 minutes, three times, cleansing solution is Milli-Q water, rear 10～1000 μ LMilli-Q water resuspensions.
9. core-shell nano Au probe with high SERS effect that is prepared by the described method of claim 1～8 any one, described core-shell nano Au probe particle diameter is 30～50 nanometers, concentration is 0.1～1nM.
10. have the core-shell nano Au probe of high SERS effect in the application in biomolecule detection and cell imaging field by claim 9 is described, can obtain highly sensitive SERS signal; This detecting probe surface assembling biomolecule with preparation, can identify specifically the target cell surface receptor, utilize laser Raman spectrometer that cell is detected and the resulting Surface enhanced raman spectroscopy signal of imaging also with the efficiently expression of reacting cells surface receptor; Its application process is as follows:
(1) alkaline solution is adjusted core-shell nano gold solution pH: alkaline solution is 0.1～2M NaOH, sal tartari (K
2CO
3) or saleratus (KHCO
3) or sodium bicarbonate (NaHCO
3) in a kind of, pH is adjusted into 8～10;
(2) add biomolecule, assemble on its surface: biomolecule is protein (antibody) or polypeptide or DNA(aptamer); Assembling condition is room temperature 30～120 minutes;
(3) centrifuge washing: 4 ℃, 3000-10000rpm/min, 5min, three times, cleansing solution is Milli-Q water;
(4) cultivate altogether with the target material of biomolecule: the target material is a kind of in cell, protein (antibody), the dna sequence dna.
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