CN103048306B - There is core-shell nano gold bioprobe and the Synthesis and applications of high SERS effect - Google Patents
There is core-shell nano gold bioprobe and the Synthesis and applications of high SERS effect Download PDFInfo
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Abstract
A kind of core-shell nano bioprobe and Synthesis and applications with high SERS effect, by continuing at the certain thickness golden shell of nm of gold core Surface Creation after small particle diameter nm of gold surface-assembled section of DNA sequence and Raman Small molecular, the gap of certain size is there is between nucleocapsid, Raman Small molecular exists in this gap, and obtains efficiently homogeneous SERS signal due to the singularity of this structure.Raman Small molecular exists in the gap of the fixed measure between golden nucleocapsid, thus the region at each molecule place is that " " the SERS signal that region produces is basically identical, and has good repeatability for focus.By this detecting probe surface of preparation assembling biomolecule, target cell surface receptors can be identified specifically, laser Raman spectrometer can be utilized to detect and imaging cell, the surface simultaneously obtained increases Raman scattering value also by the expression of reacting cells surface receptor efficiently, and this probe also can be applicable to the research field such as biology sensor, biomolecule detection in addition.
Description
Technical field
The invention belongs to functionalization and the application of nano material, relate to a kind of preparation method with the core-shell nano gold bioprobe of high SERS effect, can be applicable to the field such as biomolecule detection and cell imaging.
Background technology
Surface enhanced raman spectroscopy (SurfaceEnhancedRamanscattering, SERS) refers to the phenomenon that the Raman signal of the Small molecular being positioned at roughened metal surface itself is enhanced.This phenomenon is widely used in fields such as Surface Science, analysis science and bio-science, the information on molecular level is provided, as differentiated the surface structure of molecule or the bonding of ion on surface, configuration and orientation and material for the structure on the various surface of deep sign (interface) and process.About the enhancing mechanism of SERS, although still there is dispute up till now, but what comparatively approve is Electromagnetic enhancement mechanism, and " focus " (hotspot) related in this mechanism generally refers in the molecular aggregation of some nanoparticles, the region of the space part between adjacent nano particle.The SERS effect in this region is the strongest.How to build the efficiently homogeneous SERS substrate containing more " focus ", become focus and the difficult point of SERS research field.
In present research, the common methods building efficient homogeneous substrate has several as follows:
1, metal electrode active substrate, this uses a kind of substrate comparatively widely at present.Carry out suitable roughening process by electrode surface, roughness can be produced and be substantially in macro-asperity and submicroscopic roughness range.The shortcoming of this mode be most metal after treatment, rough surface dimensional variation scope is larger, cause substrate last some surface enhanced effect to alter a great deal, this structural inhomogeneity directly affects the stability of SERS spectrum and the reappearance of data of binding molecule.
2, the active substrate of chemical etching and chemogenic deposit the atom of metal surface is dissolved by strong corrosive material the object reaching surface roughening by chemical reaction.The shortcoming of this mode is the more difficult control of reaction conditions, the time of deposition, the temperature of reaction, the concentration etc. of reagent all have impact to the roughness of substrate.
Also have lithographic printing, self-service packing technique, ordered fabrication technology etc. to prepare active substrate in addition, but all there is respective shortcoming thus 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 the core-shell nano bioprobe of high SERS effect, except all characteristics itself possessing nano material, the field such as biomolecule detection and cell imaging can also be applied to as the efficient Raman microprobe of one.
A kind of preparation method with the core-shell nano bioprobe of high SERS effect, it is characterized in that, after small particle diameter nm of gold finishing one deck Small molecular, grow again, one deck gold shell is formed again on golden core surface, there is the Small molecular with Raman signal between gap between gold nucleocapsid, step is as follows:
(1) nm of gold core surface-assembled DNA;
(2) assemble potpourri and carry out burin-in process;
(3) solution I is obtained after centrifuge washing;
(4) nm of gold core surface-assembled Small molecular;
(5) solution II is obtained after centrifuge washing;
(6) growth of golden shell is carried out on nm of gold core surface;
(7) golden nuclear structure centrifuge washing;
Described nm of gold core is the nm of gold of particle diameter 5 ~ 15nm.
Be assembled in nm of gold core described in step (1) and add SH-polyADNA, and final concentration is 0.1 ~ 5 μM, room temperature slight oscillatory spends the night.
The described burin-in process condition of step (2) is: add 0.01 ~ 1M, pH is the phosphate buffer solution (PB) of 7.4, room temperature condition 350rpm/min vibrates 30 ~ 120 minutes, after add 1 ~ 5M sodium chloride, final concentration is 0.1 ~ 0.15M, and point to add for four times, be spaced apart 30 minutes, 350rpm/min shaken overnight under room temperature.
Step (3), (5) described centrifuge washing condition are 4 DEG C, 15000 ~ 12000rpm/min, and 20 minutes, three times, cleansing solution was PB(10mM, pH7.4), rear 0.1M phosphate buffer (PBS, pH7.4) resuspension.
Step (4) described nm of gold core surface-assembled be the Small molecular with obvious characteristic peak, be specially 2,3-benzodiazine (PHTH), 5, one in 5'-bis-sulphur two (2-nitrobenzoic acid) (DTNB), cyanine class dyestuff, fluorescein isothiocyanate (FITC), pyridine or its combination, assembling condition is 1-100 μ L, 0.1 ~ 1M small molecule solution joins in 5 μ L ~ 5mL solution I, and under room temperature, 350rpm/min vibration, adsorbs 2 ~ 3 days.
The growth conditions of the described golden shell of step (6) is: add 0.1 ~ 5% polyvinyl pyrrolidone aqueous solution (PVP) in solution II successively, 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 DEG C, 3000 ~ 10000rpm/min, and 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 core-shell nano Au probe with high SERS effect in the application in biomolecule detection and cell imaging field, can obtain highly sensitive SERS signal.By this detecting probe surface of preparation assembling biomolecule, target cell surface receptors can be identified specifically, utilize laser Raman spectrometer detects cell and imaging obtains Surface enhanced raman spectroscopy signal also by the expression of reacting cells surface receptor efficiently.Its application process is as follows:
(1) alkaline solution adjustment 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 one, solution ph is adjusted to 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 DEG C, 3000-10000rpm/min, 5min, three times, cleansing solution is Milli-Q water;
(4) with the target material Dual culture of biomolecule: target material is the one in cell, protein (antibody), DNA sequence dna.
The present invention passes through at small particle diameter nm of gold surface-assembled section of DNA sequence and Raman Small molecular, by reducing process at the certain thickness golden shell of golden core Surface Creation, the gap of certain size is there is between nucleocapsid, Raman Small molecular just exists in this gap, and obtains efficient SERS signal due to the singularity of this structure.Advantage of the present invention is: Raman Small molecular exists in the gap of the fixed measure between golden nucleocapsid, thus the SERS signal that the region at each molecule place i.e. " focus " region produces is basically identical, and has good repeatability.
Shell nano gold biological probe structure will strengthen micromolecular Raman signal greatly.By this detecting probe surface of preparation assembling biomolecule, target cell surface receptors can be identified specifically, laser Raman spectrometer can be utilized to detect and imaging cell, and the surface simultaneously obtained increases Raman scattering value also by the expression of reacting cells surface receptor efficiently.
Accompanying drawing explanation
Fig. 1 is the Raman spectrogram of the core-shell nano Au probe using different Raman Small molecular to prepare.
Figure a is DTNB, and figure b is Cy3.
Fig. 2 be attachment PHTH micromolecular core-shell nano gold bioprobe when being applied to cell detection cell surface random selecting number point carry out the spectrogram of Raman detection gained.
A line is for using this invention middle probe gained spectrogram, and b line is that Small molecular is adsorbed on gained spectrogram after nm of gold surface and cytosis, gained spectrogram when c line is for acting on mutually with cell without probe.
Fig. 3 is at PHTH characteristic peak place, the SERS Benefit Transfer result of this core-shell nano Au probe and nm of gold-Small-molecule probe.
Embodiment
Embodiment 1:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1MPHTH small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is MilliQ water, rear 100 μ LMilliQ water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at phthalazines (PHTH) high Enhanced feature peak.Get this nucleocapsid probe of 200 μ L, add 20 μ L0.1MNaOH solution, pH value is add 20 μ L, 25 μMs of polypeptide solutions after 10, and after mixing, room temperature 30min assembles.Latter 4 DEG C, 5000rpm/min, 5min wash 3 times, and cleansing solution is Milli-Q water, sediment cell culture fluid resuspension.Get in the double dish that 100 μ L join containing 2mL nutrient solution in 37 DEG C, the cell culture incubator of 5.0% carbon dioxide and co-culture of cells 1h, rear PBS solution cleans 2 times, adds formaldehyde immobile liquid room temperature and fixes 10min, MilliQ water rinses 3 times, detects under laser Raman spectrometer.
Embodiment 2:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1M phthalazines PHTH small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is MilliQ water, rear 100 μ LMilliQ water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at phthalazines (PHTH) high Enhanced feature peak.Get this nucleocapsid probe of 200 μ L, add 20 μ L0.1MNaOH solution, pH value is add 20 μ L, 2%PEG solution after 10, and after mixing, room temperature 30min assembles.Latter 4 DEG C, 5000rpm/min, 5min wash 3 times, and cleansing solution is Milli-Q water, sediment cell culture fluid resuspension.Get in the double dish that 100 μ L join containing 2mL nutrient solution in 37 DEG C, the cell culture incubator of 5.0% carbon dioxide and co-culture of cells 1h, rear PBS solution cleans 2 times, adds formaldehyde immobile liquid room temperature and fixes 10min, Milli-Q water rinses 3 times, detects under laser Raman spectrometer.
Embodiment 3:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1MDTNB small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at DTNB height Enhanced feature peak.
Embodiment 4:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1MCy3 small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at Cy3 height Enhanced feature peak.
Embodiment 5:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1M pyridine small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at pyridine height Enhanced feature peak.
Embodiment 6:
100 μ L, 10nM particle diameter is add 4 μ L in the nm of gold of 15nm, 100 μMs of SH-polyADNA, and room temperature slight oscillatory spends the night; After add Ageing solution 0.1MPB(pH7.4) solution 10 μ L, room temperature condition 350rpm/min vibrate 30min, after add 2MNaCl20 μ L, point to add for four times, be spaced apart 30min, 350rpm/min shaken overnight under room temperature; 4 DEG C, carry out centrifuge washing three times under 12000rpm/min, 20min condition, cleansing solution is 10mMPB, rear 1mL0.1MPBS resuspension; 100 μ L0.1MFITC small molecule solution join in the above-mentioned solution of 500 μ L, 350rpm/min vibration under room temperature, absorption 2-3 days.Get the above-mentioned solution of 100 μ L and then add 50 μ L1%PVP, 25 μ L10mMNH
2oHHCl, 25 μ L5mMHAuCl
4, mixing vibration 1min after 20 DEG C, 4000rpm/min, each 6min centrifuge washing three times, cleansing solution is Milli-Q water, rear 100 μ LMilli-Q water resuspensions; The particle diameter which prepares is at 40-50nm, and concentration is 1nM, possesses a kind of core-shell nano gold Raman microprobe at FITC height Enhanced feature peak.
Claims (6)
1. one kind has the preparation method of the core-shell nano bioprobe of high SERS effect, it is characterized in that, after small particle diameter nm of gold finishing one deck Small molecular, grow again, one deck gold shell is formed again on golden core surface, there is the Small molecular with Raman signal between gap between gold nucleocapsid, step is as follows:
(1) nm of gold core surface-assembled DNA;
(2) assemble potpourri and carry out burin-in process;
(3) solution I is obtained after centrifuge washing;
(4) nm of gold core surface-assembled Small molecular;
(5) solution II is obtained after centrifuge washing;
(6) growth of golden shell is carried out on nm of gold core surface;
(7) golden nuclear structure centrifuge washing;
Be assembled in nm of gold core described in step (1) and add SH-polyADNA, and final concentration is 0.1 ~ 5 micromoles per liter, room temperature slight oscillatory spends the night;
The described burin-in process condition of step (2) is: add 0.01 ~ 1M, pH is the phosphate buffer solution (PB) of 7.4, room temperature condition 350rpm/min vibrates 30 ~ 120 minutes, after add 1 ~ 5M sodium chloride, final concentration is 0.1 ~ 0.15M, and point to add for four times, be spaced apart 30 minutes, 350rpm/min shaken overnight under room temperature;
Step (4) described nm of gold core surface-assembled be the Small molecular with obvious characteristic peak, be specially 2,3-benzodiazine (PHTH), 5, one in 5'-bis-sulphur two (2-nitrobenzoic acid) (DTNB), cyanine class dyestuff, fluorescein isothiocyanate (FITC), pyridine or its combination, assembling condition is 1-100 microlitre, 0.1 ~ 1M small molecule solution joins in 5 microlitres ~ 5mL solution I, and under room temperature, 350rpm/min vibration, adsorbs 2 ~ 3 days;
The growth conditions of the described golden shell of step (6) is: add 0.1 ~ 5% polyvinyl pyrrolidone aqueous solution (PVP) in solution II successively, oxammonium hydrochloride aqueous solution (NH2OHHCl), 0.01 ~ 1% aqueous solution of chloraurate (HAuCl4), mixing vibration 1 minute.
2. have the preparation method of the core-shell nano bioprobe of high SERS effect according to claim 1, it is characterized in that, described nm of gold core is the nm of gold of particle diameter 5 ~ 15nm.
3. there is the preparation method of the 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 DEG C, 15000 ~ 12000rpm/min, 20 minutes, three times, cleansing solution is 10 mM/ls, and pH is the phosphate buffer of 7.4; After centrifuge washing process terminates, working concentration is 0.1 mol/L, and pH is the phosphate buffer resuspension of 7.4.
4. there is the preparation method of the 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 DEG C, 3000 ~ 10000rpm/min, 6 minutes, three times, cleansing solution is Milli-Q water, and centrifuge washing process terminates rear use 10 ~ 1000 microlitre Milli-Q water resuspension.
5. the core-shell nano Au probe with high SERS effect prepared by the method described in Claims 1 to 4 any one, described core-shell nano Au probe particle diameter is 30 ~ 50 nanometers, and concentration is 0.1 ~ 1nM.
6., by the application of core-shell nano Au probe described in claim 5 with high SERS effect, be applied in biomolecule detection and cell imaging field, highly sensitive SERS signal can be obtained; By this detecting probe surface of preparation assembling biomolecule, target cell surface receptors can be identified specifically, utilize laser Raman spectrometer detects cell and imaging obtains Surface enhanced raman spectroscopy signal also by the expression of reacting cells surface receptor efficiently; Its application process is as follows:
(1) alkaline solution adjustment 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 one, solution ph is adjusted to 8 ~ 10;
(2) add biomolecule, assemble on its surface: biomolecule is protein antibody or polypeptide or DNA; Assembling condition is room temperature 30 ~ 120 minutes;
(3) centrifuge washing: 4 DEG C, 3000-10000rpm/min, 5min, three times, cleansing solution is Milli-Q water;
(4) with the target material Dual culture of biomolecule: target material is the one in cell, protein antibody, DNA sequence dna.
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