CN101482509A - Method for detecting animal active unicellular sample by surface reinforced Raman spectrum - Google Patents
Method for detecting animal active unicellular sample by surface reinforced Raman spectrum Download PDFInfo
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- CN101482509A CN101482509A CNA2009101111299A CN200910111129A CN101482509A CN 101482509 A CN101482509 A CN 101482509A CN A2009101111299 A CNA2009101111299 A CN A2009101111299A CN 200910111129 A CN200910111129 A CN 200910111129A CN 101482509 A CN101482509 A CN 101482509A
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Abstract
The invention relates to an animal active single-cell sample treatment method wherein the animal active single-cell sample is for surface-enhanced raman spectroscopy detection. The treatment method comprises: dissolving solid silver nitrate in deionisation water and heating water to 100 DEG C., dripping sodium citrate solution into water and stirring the water until boiling, centrifuging the boiled water and taking the concentrated silver sol at the lower layer; preparing the single-cell suspension by culturing the animal active single-cell sample to be detected in the cell culture medium; performing the second centrifugation of the prepared silver sol and removing away the supernatant and adding the cell culture medium to prepare silver sol suspension and mixing the silver sol suspension with the single-cell suspension according the volume ratio of 1:4, transfering the mixture into an electric-shock cup sample cell and performing electroporation after ice bathing, and then rinsing the mixture out from the sample cell and culturing the mixture in an incubator after ice bath, finally the sample can be detected using a confocal Raman spectrometer. The treatment method has features of simpleness, quick speed, high reliability, good generality and clear and stable surface-enhanced raman spectroscopy of the inside of active cell.
Description
Technical field
The present invention relates to a kind of unicellular Surface enhanced raman spectroscopy test sample preprocess method, be specifically a kind of will be used for that unicellular Surface enhanced raman spectroscopy detects test sample---the active animal cell carries out pre-service, and utilize electroporation that metal nanoparticle is imported method in the test sample, belong to biomedical sector.
Background technology
Raman spectrum belongs to molecular vibration spectrum, the number of Raman line, the size of shift value and intensity of bands of a spectrum etc. all with the vibration of material molecule and rotate relevant, these message reflections the structure picture and the residing environment thereof of molecule.Raman spectrum can provide the situation about various normal mode of vibration frequencies of intramolecule and relevant vibrational energy level, thereby can identify the functional group that exists in the molecule and the feature structure of molecule.Yet the light intensity of Raman spectrum scattering only is about 10 of incident intensity
-10Since conventional Raman signal too a little less than, detect after need taking technological means that Raman signal is strengthened, Surface enhanced raman spectroscopy (Surface-enhanced Ramanspectroscopy is called for short SERS) is exactly a kind of enhancements with surface selectivity that adopts usually.SERS is meant that their Raman scattering intensity can increase by 10 when some molecules are adsorbed on some coarse metal (as silver, copper, gold etc.) surface
4～10
6Doubly.So-called SERS effect that Here it is.SERS is good with its selectivity, and high sensitivity is high 1,000,000 times to the conventional Raman spectrum of its remolding sensitivity of some molecule, can detect the unimolecular layer and the inferior unimolecular layer that are adsorbed on the metal surface, and abundant molecular structure information is provided, and be widely used.In biomedical research, scientist's employing is at present composed the research competent cell cell interior importing silver or golden nanometer particle to obtain SERS, SERS has the resolution characteristic of non-destructive and finger-print type, aspects such as the selective excitation of the analysis of aqueous sample, sample and signal collection are all demonstrated its unique advantage, therefore aspect biomedical research, shown good prospects for application.Yet it is at present that the means of silver or golden nanometer particle importing competent cell are also few, usually adopt directly and cell is hatched mode, the metallics that utilizes the encytosis of cell will be adsorbed on cell surface is introduced competent cell, the time that this mode needs is long and efficient is not high, cell can be regarded the nano particle that is attached with metal as foreign matter in addition, and very fast these nano particles are removed totally further reduced importing efficient.
Under normal physiological function situation, cell membrane plays the barrier action of internal and external environment, can hinder the transmission of nano particle in the internal and external environment or molecule preferably.But when applying intensity is that micropore can appear in cell membrane when KV/cm, duration being the electric pulse stimulation cell membrane of μ s～ms level, causes cell membrane to hinder the particulate penetrating power and reduces.This cell membrane permeability that is produced by electric pulse is referred to as electroporation usually.If the opening diameter that intensity by the regulating impulse electric field and duration can be controlled cell membrane, under top condition, keep micro-pore diameter in 20～100nm scope, and micropore is healed automatically about 10s, can make cell penetrating effectively, can make the minimum activity that keeps of cellular damage again, reach and to keep cell activity again with in the required metallics transfered cell.
Summary of the invention
The object of the invention is that just existing method deficiency provides a kind of method of utilizing electroporation metal nanoparticle to be imported fast test sample (active animal cell).Needed compare in 8～24 hours with the pre existing treatment technology, of the present inventionly carry out the needed time of Surface enhanced raman spectroscopy test sample preprocessing process and foreshorten in 30 minutes, have the characteristics simply quick, that reliability is high, versatility is good, it neither influences original cell activity, significant surface-enhanced Raman effect is arranged again, can obtain the inner steady and audible Surface enhanced raman spectroscopy of competent cell.
For realizing that the technical scheme that purpose of the present invention adopts is:
(1) silver sol pre-preparation
The solid nitric acid silver of getting 9～12mg is dissolved in the 50ml deionized water, be heated to 100 ℃, the aqueous solution that then 1ml is contained 1%～3% trisodium citrate dropwise adds, in adition process, stir liquor argenti nitratis ophthalmicus fast, drip the back and continue to keep boiling 2 hours～6 hours, obtain the semifinished product of silver sol, after treating the silver sol natural cooling, with the centrifugal layering of hydro-extractor, supernatant is abandoned, take off the lucifuge sealing at room temperature of the concentrated silver sol of layer to prepare following cell electroporation step.
(2) preparation of single cell suspension
Test sample competent cell to be measured is at first made single cell suspension with commercially available RPMI 1640 cell culture fluids, calculate the content of cell then by blood cell counting plate, the control cell density is 10
5～10
6Individual/ml.
(3) preparation of silver sol-single cell suspension mixed liquor
Get silver sol 1～4ml of preparing in (1) in carrying out with the 8000rpm rotating speed centrifugal 10 minutes of the second time, abandon and in the silver sol that concentrates, add commercially available RPMI 1640 cell culture fluids of 50～200 μ l after the supernatant and make silver sol suspending liquid.The volume ratio of the middle single-cell suspension liquid for preparing in silver sol suspending liquid and (2) by 1: 4 is mixed in the test tube pipe, the mode of the pipettor that utilizes 200 μ l by suction repeatedly be fully with silver sol suspending liquid and single-cell suspension liquid mixing, makes silver sol-single-cell suspension liquid and at room temperature preserve.
(4) Surface enhanced raman spectroscopy test sample preprocessing process
Silver sol-single-cell suspension the liquid that mixes is changed in the electric shock cup sample cell of electric pulse generator, carry out ice bath, the cup that will shock by electricity after 5～10 minutes is put on the electric shock pedestal of electric pulse generator, an electric shock cup electrode gap scope is 1,2,3 or 4mm, voltage continues electric shock time 50ms～100 μ s between 350V～1000V.Behind the electroporation, use the commercially available RPMI1640 cell culture fluid of 300～500 μ l that silver sol-single-cell suspension liquid is flushed out sample cell immediately, put in the ice bath behind 5～10min, transfer in the incubator after the preheating, cultivated 10 minutes for 37 ℃, utilize confocal Raman spectra instrument test sample then to obtain the Surface enhanced raman spectroscopy of sample cell.
Advantage of the present invention is, adopt pulse electric field technology that the metal-sol nano particle is passed through the cell membrane transfered cell smoothly, have transfer efficiency height and pair cell and do not have distinguishing features such as injury, thereby provide effective and easy method for the transfered cell of unicellular Surface enhanced raman spectroscopy silver sol.
Description of drawings
Fig. 1 is that the present invention detects spectrum to the epithelial tumor cell Surface enhanced raman spectroscopy.
Fig. 2 is that the present invention strengthens Raman spectrum detection spectrum to the leukaemia tumor cell surface.
Fig. 3 is that the present invention strengthens Raman spectrum detection spectrum to the nasopharyngeal carcinoma tumor cell surface.
The optical maser wavelength that adopts among Fig. 1, Fig. 2 and Fig. 3 is 785nm, and power is 20mW, and ordinate is the intensity of spectral line, and unit is the peak position of arbitrary unit (a.u) horizontal ordinate for each characteristic spectral line, with wave number (cm
-1) expression.
Embodiment
Measure is further described to the solution of the present invention below.
Embodiment 1
The silver nitrate of getting 9mg is dissolved in the 50ml deionized water, is heated to 100 ℃ and makes it boiling, is that 3% trisodium citrate dropwise adds then with 1ml concentration, and stirs fast, drips the back and continues to keep boiling 2 hours.After treating this silver sol natural cooling,, supernatant liquor is abandoned, take off the lucifuge sealing at room temperature of the concentrated silver sol of layer and deposit standby with the centrifugal layering of hydro-extractor.
Test sample epithelial tumor cell (A431) to be measured is at first made the single cell suspension of epithelial tumor cell with commercially available RPMI 1640 cell culture fluids, calculate the content of cell then by blood cell counting plate, the control cell density is 10
5Individual/ml.
Get the single cell suspension 80 μ l and the 320 μ l of concentrated silver sol and epithelial tumor cell (A431) respectively, change over to after mixing under the room temperature in the electric shock cup sample cell, electric shock cup electrode gap is 1mm, and wherein cell quantity is about 2 * 10
5Individual.Cup ice bath 5 minutes will shock by electricity, place in the electroporation apparatus then, select manually control, making alive 350V, duration 100ms, behind the electroporation, use 400 μ lRPMI, 1640 cell culture mediums that cell is flushed out sample cell immediately, put in the ice bath behind the 5min, transfer in the incubator after the preheating, cultivated 10 minutes for 37 ℃, utilize the confocal Raman spectra instrument to carry out unicellular Surface enhanced raman spectroscopy then and detect.
Embodiment 2
The silver nitrate of getting 12mg is dissolved in the 50ml deionized water, is heated to 100 ℃ and makes it boiling, is that 1% trisodium citrate dropwise adds then with 1ml concentration, and stirs fast, drips the back and continues to keep boiling 4 hours.After treating this silver sol natural cooling,, supernatant liquor is abandoned, take off the lucifuge sealing at room temperature of the concentrated silver sol of layer and deposit standby with the centrifugal layering of hydro-extractor.
Test sample leukaemia tumour cell (HL60) to be measured is at first made the single cell suspension of epithelial tumor cell with commercially available RPMI 1640 cell culture fluids, calculate the content of cell then by blood cell counting plate, the control cell density is 10
6Individual/ml.
Get respectively and change in the electric shock cup sample cell after mixing under the single cell suspension 120 μ l of concentrated silver sol and leukaemia tumour cell (HL60) and the 480 μ l room temperatures, an electric shock cup electrode gap is 2mm, and wherein cell quantity is about 3 * 10
5Individual.Cup ice bath 8 minutes will shock by electricity, place in the electroporation apparatus then, select manually control, making alive 700V, duration 50ms, behind the electroporation, use 400 μ lRPMI, 1640 cell culture mediums that cell is flushed out sample cell immediately, put in the ice bath behind the 8min, transfer in the incubator after the preheating, cultivated 10 minutes for 37 ℃, utilize the confocal Raman spectra instrument to carry out unicellular Surface enhanced raman spectroscopy then and detect.
Embodiment 3
The silver nitrate of getting 10mg is dissolved in the 50ml deionized water, is heated to 100 ℃ and makes it boiling, is that 2% trisodium citrate dropwise adds then with 1ml concentration, and stirs fast, drips the back and continues to keep boiling 6 hours.After treating this silver sol natural cooling,, supernatant liquor is abandoned, take off the lucifuge sealing at room temperature of the concentrated silver sol of layer and deposit standby with the centrifugal layering of hydro-extractor.
Test sample nasopharyngeal carcinoma tumour cell (C66) to be measured is at first made the single cell suspension of epithelial tumor cell with commercially available RPMI 1640 cell culture fluids, calculate the content of cell then by blood cell counting plate, the control cell density is 10
5Individual/ml.
Change in the electric shock cup sample cell after getting the single cell suspension 160 μ l of concentrated silver sol and nasopharyngeal carcinoma tumour cell (C66) and 640 μ l mixed at room temperature respectively, an electric shock cup electrode gap is 4mm, and wherein cell quantity is about 4 * 10
5Individual.Cup ice bath 10 minutes will shock by electricity, place in the electroporation apparatus then, select manually control, making alive 1000V, duration 1ms, behind the electroporation, use 400 μ lRPMI, 1640 cell culture mediums that cell is flushed out sample cell immediately, put in the ice bath behind the 10min, transfer in the incubator after the preheating, cultivated 10 minutes for 37 ℃, utilize the confocal Raman spectra instrument to carry out unicellular Surface enhanced raman spectroscopy then and detect.
Claims (5)
1, a kind of Surface enhanced raman spectroscopy detects the unicellular sample treatment of active animal, it is characterized in that:
(1) silver sol pre-preparation
The solid nitric acid silver of getting 9～12mg is dissolved in the 50ml deionized water, be heated to 100 ℃, the aqueous solution with the 1ml trisodium citrate dropwise adds then, stirs the back fast and continues boiling 2 hours～6 hours, obtain the semifinished product of silver sol, the silver sol that centrifuging and taking lower floor concentrates is standby;
(2) preparation of single cell suspension
Test sample competent cell to be measured is at first made single cell suspension with commercially available RPMI 1640 cell culture fluids;
(3) preparation of silver sol-single cell suspension mixed liquor
Get the silver sol 1～4ml for preparing in (1) and carry out centrifugal 10 minutes of the second time, abandon and add commercially available RPMI 1640 cell culture fluids of 50～200 μ l after the supernatant and make silver sol suspending liquid, with the single-cell suspension liquid of preparation in silver sol suspending liquid and (2) volume ratio mixings mixing, make the also preservation at room temperature of silver sol-single-cell suspension liquid by 1: 4;
(4) Surface enhanced raman spectroscopy test sample preprocessing process
Silver sol-single-cell suspension the liquid that mixes is changed in the electric shock cup sample cell, carry out ice bath, carry out electroporation after 5～10 minutes, then flush out sample cell immediately, put in the ice bath behind 5～10min, transfer in the incubator 37 ℃ and cultivated 10 minutes, can utilize confocal Raman spectra instrument test sample.
2, Surface enhanced raman spectroscopy according to claim 1 detects the unicellular sample treatment of active animal, it is characterized in that: contain 1%～3% lemon olive acid trisodium in the aqueous solution of described lemon olive acid trisodium.
3, Surface enhanced raman spectroscopy according to claim 1 detects the unicellular sample treatment of active animal, and it is characterized in that: cell density is 10 in the described single cell suspension
5～10
6Between individual/ml.
4, Surface enhanced raman spectroscopy according to claim 1 detects the unicellular sample treatment of active animal, it is characterized in that: a described electric shock glass electrode gap scope is 1,2,3 or 4mm.
5, Surface enhanced raman spectroscopy according to claim 1 detects the unicellular sample treatment of active animal, it is characterized in that: described electroporation electric shock voltage continues electric shock time 50ms～100 μ s between 350V～1000V.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101799421A (en) * | 2010-04-19 | 2010-08-11 | 福建师范大学 | A kind of body fluid surface strengthens the detection method of Raman spectrum |
| CN102175664A (en) * | 2011-02-17 | 2011-09-07 | 福建师范大学 | Method for detecting surface enhanced Raman spectra of blood RNA |
| WO2011130938A1 (en) * | 2010-04-19 | 2011-10-27 | 福建师范大学 | Detection method for human plasma by surface enhanced raman spectroscopy combined with principal component analysis |
| CN103357885A (en) * | 2012-04-01 | 2013-10-23 | 深圳市宇驰检测技术有限公司 | Preparation method and application for Raman-enhanced silver colloid |
| CN103512874A (en) * | 2013-09-22 | 2014-01-15 | 福建师范大学 | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method |
| US11358984B2 (en) | 2018-08-27 | 2022-06-14 | Regeneran Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
-
2009
- 2009-03-03 CN CNA2009101111299A patent/CN101482509A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101799421A (en) * | 2010-04-19 | 2010-08-11 | 福建师范大学 | A kind of body fluid surface strengthens the detection method of Raman spectrum |
| WO2011130937A1 (en) * | 2010-04-19 | 2011-10-27 | 福建师范大学 | Detection method for body fluid by surface enhanced raman spectroscopy |
| WO2011130938A1 (en) * | 2010-04-19 | 2011-10-27 | 福建师范大学 | Detection method for human plasma by surface enhanced raman spectroscopy combined with principal component analysis |
| CN102175664A (en) * | 2011-02-17 | 2011-09-07 | 福建师范大学 | Method for detecting surface enhanced Raman spectra of blood RNA |
| CN103357885A (en) * | 2012-04-01 | 2013-10-23 | 深圳市宇驰检测技术有限公司 | Preparation method and application for Raman-enhanced silver colloid |
| CN103357885B (en) * | 2012-04-01 | 2016-06-22 | 深圳市宇驰检测技术有限公司 | A kind of Raman strengthens preparation method and the application thereof of elargol |
| CN103512874A (en) * | 2013-09-22 | 2014-01-15 | 福建师范大学 | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method |
| US11358984B2 (en) | 2018-08-27 | 2022-06-14 | Regeneran Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
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Open date: 20090715 |