CN102621125A - Cell probe complex for detecting human breast cancer cells MCF (Michigan cancer foundation)-7 based on Raman spectrum and method for preparing cell probe complex - Google Patents
Cell probe complex for detecting human breast cancer cells MCF (Michigan cancer foundation)-7 based on Raman spectrum and method for preparing cell probe complex Download PDFInfo
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- CN102621125A CN102621125A CN2012100803769A CN201210080376A CN102621125A CN 102621125 A CN102621125 A CN 102621125A CN 2012100803769 A CN2012100803769 A CN 2012100803769A CN 201210080376 A CN201210080376 A CN 201210080376A CN 102621125 A CN102621125 A CN 102621125A
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Abstract
The invention discloses a cell probe complex for detecting human breast cancer cells MCF (Michigan cancer foundation)-7 based on Raman spectrum and a method for preparing the cell probe complex. The cell probe complex is characterized in that nucleic acid aptamer capable of realizing a targeting effect with the MCF-7 cells is used as a template, bimetal alloy nano particles of gold and silver are deposited on a basic group of a DNA (deoxyribose nucleic acid) chain of the nucleic acid aptamer by a photocatalysis method, and Raman signal molecules rhodamine 6G (Rh6G) is fixedly loaded on the surface of each nano particle. The structure of the probe complex is chain-shaped as shown by a photo of a transmission electron microscope, and the probe complex is capable of realizing the targeting effect with the MCF-7 cells. Characteristic absorption peaks of the rhodamine 6G appear on the Raman spectrum under the effect of the MCF-7 cells and the probe complex, and the human breast cancer MCF-7 cells can be effectively, sensitively and specifically detected.
Description
Technical field
The invention belongs to the cell detection technical field, in particular to a kind of cell probe complex that utilizes surface-enhanced Raman input human breast carcinoma MCF-7 cell and preparation method thereof.
Background technology
Cancer is to cause human dead a kind of major reason, according to World Health Organization's statistics, has every year 7000000 people to die from all kinds of Cancerous diseases approximately.In the middle of the women, breast cancer is the highest Cancerous disease of a kind of incidence of disease, and the incidence of disease of breast cancer is obvious ascendant trend in recent years, and the incidence of disease of global breast cancer in 2008 is up to 41.4%.The early diagnosis and therapy of breast cancer improves disease patient 5 annual survival rates and is extremely important for reducing the breast cancer mortality ratio.
Cancer is a kind of not normal and disease of causing of growth and proliferation of cell mechanism of controlling, and the state of its generation and development and cell is closely related.In all kinds of Cancerous diseases, the growth of cancer cell and split speed be considerably beyond normal cell, normal tissues arround also can locally invading, even transfer to other parts of health via body-internal-circulation system or lymphatic system.Therefore, it is great for early diagnosis, examination, treatment and the prognosis meaning of realizing breast cancer to develop a kind of method of reliable, sensitive detection human breast cancer cell.
In current breast cancer medical diagnostic method, image technology is a kind of the most common effective ways.But image technology also has it significantly not enough, as is difficult to differentiate the identical or close cancer cell of some pathologic characteristics, is difficult to the sensitive cancerous tumor cell of cancer early carcinoma cellular level when low etc. that detect.And the specificity and the sensitivity that improve to detect are great for the early diagnosis therapeutic action of cancer, and therefore increasing work is tended to from unicellular, single molecules level, cancer cell detected.
Technology to unicellular research mainly comprises fluorescence spectrum technology, scanning probe microscopy, microflow control technique, capillary electrophoresis technique etc. at present, but there is invasive in most of technology, can produce destruction by pair cell.Optical technology has and does not have intrusions, no ionising radiation, allow the various modes imaging, can obtain the characteristics of multiple information such as real-time and quantitative, in life science, occupies more and more important position.The spectral technique that in biomedical sector, has been applied at present mainly contains ultraviolet-visible absorption spectroscopy, infrared spectrum, fluorescence spectrum, Raman spectrum etc.Raman spectrum is compared with other spectrum has some outstanding advantages, high like stability, be difficult for quencher, available ruddiness excite, to biological sample damage little, do not receive the interference of biological sample autofluorescence and water etc., thereby be particularly suitable for the research of cell sample.
The target property that improves Raman optical spectrum method is very important for improving the specificity and the sensitivity that detect.The surface of cell membrane of cancer cell has albumen or the glycosyl that some and normal cell do not have usually, and mark commonly used comprises EGF-R ELISA, phosphatase and folacin receptor etc.One section oligonucleotide sequence that can combine with target molecule with high affinity and specificity that fit (aptamer) is that repeated screening obtains the random oligonucleotide sequence library that from prosthesis outside, synthesizes with part index concentration method phylogeny (SELEX) technology, can with protein even whole cell generation specificity combination.The aptamer of these cancer cell marks is carried out the functionalization of surface-enhanced Raman and interacts with cancer cell, can realize that the high sensitivity based on SERS, the cancer cell target of high specific detect.
Summary of the invention
The purpose of this invention is to provide a kind of Raman spectrum and detect cell probe complex of human breast cancer cell MCF-7 and preparation method thereof; Breast cancer cell through developing a kind of functionalization is fit-nano composite material (Au-Ag-aptamer); And process cell probe complex (Rh 6G-Au-Ag-aptamer) based on said nano composite material; Because nano composite material has strong surface raman enhancement effect; Strong selectivity effect between fit and cell surface marker; The cell probe complex can be used for high sensitivity, high specific detection based on the breast cancer cell MCF-7 of surface-enhanced Raman signal to the selectivity that human breast cancer cell MCF-7 has height.
The technical scheme of accomplishing the foregoing invention task is:
A kind of cell probe complex (Rh 6G-Au-Ag-aptamer) that detects human breast cancer cell MCF-7 based on Raman spectrum; It is characterized in that; Described probe complex is with aptamer (S2.2; Sequence is 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 ') be template, deposited gold on its base/silver bimetal alloy nano particle, the immobilized Raman signal molecule of nanoparticle surface rhodamine 6G.
Described its particle diameter of cell probe complex Rh 6G-Au-Ag-aptamer is about 20～120nm.
Described cell probe complex Rh 6G-Au-Ag-aptamer is synthetic by following method:
Aptamer S2.2 is dissolved in the PBS (PBS, pH 7.4), adds the NaAuCl of certain volume ratio
4And AgNO
3Solution (mix and stir, and in the dark places more than 12 hours, places illumination 5 min to 60 min under the uviol lamp, forms the Au-Ag-aptamer nano-complex by volume ratio 1 ~ 5:1); Rhodamine 6G (Rh 6G) solution is joined in the Au-Ag-aptamer nano-complex dispersion liquid; Lucifuge stirs the back centrifugal treating; Remove the not Rh 6G molecule of absorption with the PBS washing, obtain described probe complex (being labeled as Rh 6G-Au-Ag-aptamer).
The preparation method of described Rh 6G-Au-Ag-aptamer nano-probe compound substance specifically may further comprise the steps:
1) aptamer (S2.2, sequence is 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 ') is dissolved with 0.1 mol/L phosphate buffer (PBS, pH 7.4), concentration is 300 ng/mL;
2) with a certain amount of NaAuCl
4(1mmol/L) and AgNO
3(1mmol/L) solution mixes in (volume ratio 1 ~ 5:1) joins 1 with mixed liquor) described aptamer solution according to a certain volume, 4 ℃ of following continuous stirring 0.5 – 2 hours, and in the dark keeps in Dark Place more than the 12h, makes Au
3+And Ag
+Be adsorbed onto on the base of aptamer and obtain Au
3+-Ag
+-aptamer compound;
3) with Au
3+-Ag
+-aptamer compound is illumination 5min-60min under the uviol lamp of 254 nm in wave number, and solution colour gradually becomes blue by colourless, obtains the Au-Ag-aptamer nano-complex;
4) Rh 6G solution is joined in the Au-Ag-aptamer nano-complex dispersion liquid; The ultimate density of control Rh 6G is 1 μ mol/L's; Stir 1h 4 ℃ of following lucifuges, after the centrifugal treating (rotating speed is 10000 rev/mins, times 10 min); Remove the not Rh 6G molecule of absorption with the washing of 0.1 mol/L PBS solution (pH 7.4), obtain Rh 6G-Au-Ag-aptamer probe complex.
Described step 2) in, mixes Ag in the aptamer solution of back
+Be preferably 1:5 with the amount ratio of aptamer base.
The present invention utilizes light-catalysed method to the fit functionalization of carrying out of human breast carcinoma MCF-7 cell; The bimetallic alloy nano particle (Au-Ag NPs) of synthetic gold and silver on fit nucleic acid chains; And modify and go up the molecular probe that Raman signal molecule Rh 6G prepares the detection of MCF-7 cell Raman spectrum; Gold/silver bimetal the alloy nano particle on selectively targeted binding ability and fit surface that utilizes fit and MCF-7 cell can be used for the effective detection to the MCF-7 cell to the humidification of Rh 6G molecule Raman signal.
Aptamer S2.2 and MCF-7 cell have selectively targeted binding ability, and the fit S2.2 of related nucleic acid can be referring to .C. Yu, Y.Hu, J. H. Duan, W. Yuan, C. Tang, H. Y. Xu, X. D. Yang, Plos One, 2011,6, e24077.
Described probe complex have a height with the target binding ability of human breast cancer cell MCF-7, and high surface reinforced Raman active.The characteristic absorption peak that has the rhodamine molecule on the SERS of this probe complex.
Can be by the method for the following stated, with Rh 6G-Au-Ag-aptamer probe complex and MCF-7 cytosis and strengthen Raman spectrum and detect:
1) cultivation of human breast cancer cell MCF-7: the MCF-7 cell attachment is grown in DMEM (Dulbecco ' the s modified Eagle medium) nutrient solution; Wherein contain ox embryo serum 10% (v/v), penicillin 100 U/mL, streptomysin 100 μ g/mL, in 37oC, 5% incubator, cultivate.The observation of cell growth makes when about cell length to 90% and goes down to posterity.When going down to posterity, remove nutrient solution,, add the trypsase 37 oC constant temperature digestion 3min of 1 mL 0.25%, get an amount of cell suspension and add and continue in the new nutrient culture media to cultivate, change liquid every other day, control number of cells 1 * 10 with cell counter with PBS solution washing 3 times
5Individual/mL, remove nutrient solution, behind PBS solution washing 3 times, cell is for use.
2) said Rh 6G-Au-Ag-aptamer probe complex being disperseed concentration in (pH 7.4) in PBS solution is 0.1mg/mL; Get 100 μ L and join 1) described in cell in; At room temperature hatch 30 min; Use 3 mL PBS solution washing cells 3-5 time successively, remove not probe complex with cytosis.
3) in said step 2) in cell in, add the trypsase of 1 mL 0.25%, digestion 3 min under the room temperature; Centrifugal 10 min under 1000 rev/mins remove supernatant, add 1 mL PBS; Make it form uniform cell suspension, get 10 these cell suspensions of μ L, keep in Dark Place its mica sheet surface that is coated onto prepared fresh; Adopt SERS technology for detection MCF-7 cell, detecting wavelength is 785 nm, and detecting wave-number range is 400-4000 cm
-1, acquisition time is 10 s.
The Rh 6G-Au-Ag-aptamer compound that makes according to above method is the distribution of DNA chain in typical case on transmission electron microscope picture; After cytosis; The surface of pair cell is carried out SERS and is detected the characteristic Raman signal peak of the tangible signaling molecule Rh 6G of appearance on the collection of illustrative plates.
The present invention has the following advantages: the probe complex Rh 6G-Au-Ag-aptamer nano-complex of the human breast carcinoma MCF-7 cell based on the surface-enhanced Raman signal of the present invention has high specific recognition capability and Raman signal to strengthen ability to the MCF-7 cell, can realize the detection to the MCF-7 cancer cell through the monitoring to Raman signal.This cell probe and detection method thereof have characteristics such as high specificity, sensitivity height, and be significant in the research of physiology, pathology, clinical medicine.
Description of drawings
The transmission electron microscope picture of Fig. 1 Au-Ag-aptamer nano-complex.
After Fig. 2 Rh 6G-Au-Ag-aptamer compound probe and the MCF-7 cytosis, the transmission electron microscope picture of cell surface.
Fig. 3 is based on the Raman spectrogram of the human breast carcinoma MCF-7 cell of probe complex surface-enhanced Raman signal.
Embodiment
Embodiment 1
With 0.1 mol/L phosphate buffer (PBS, pH 7.4) dissolving, concentration is 300 ng/mL with aptamer (S2.2, sequence is 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 '); NaAuCl with 1mol/L
4With 1mol/L AgNO
3Solution for after 1:1 mixes, joins in the aptamer solution Au in this mixed liquor by volume
3+, Ag
+And the amount of base is than being 1:1:5; 4 ℃ of following continuous stirring 0.5 – 2 hours, and the 12h that in the dark keeps in Dark Place was transferred to it in quartz colorimetric utensil of 1cm * 1cm with this mixed liquor; Be illumination 30min under the uviol lamp of 254 nm in wave number, obtain the Au-Ag-aptamer nano-complex; Dispose the Rh 6G methanol solution of 0.1 mol/L, and it is joined in the Au-Ag-aptamer nano-complex dispersion liquid, the ultimate density of control Rh 6G is 1 μ mol/L's; Stir 1h 4 ℃ of following lucifuges; With centrifugal 10 min of 10000 rev/mins rotating speed,, remove the not Rh 6G molecule of absorption with 0.1 mol/L PBS solution (pH 7.4) washing precipitation totally 3 times; Obtain Rh 6G-Au-Ag-aptamer probe complex; The TEM photo shows that the compound of gained is chain and distributes, and uniform particle diameter is about 20 nm.
Embodiment 2
With 0.1 mol/L phosphate buffer (PBS, pH 7.4) dissolving, concentration is 300 ng/mL with aptamer (S2.2, sequence is 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 '); NaAuCl with 1mol/L
4With 1mol/L AgNO
3Solution for after 2:1 mixes, joins in the aptamer solution Au in this mixed liquor by volume
3+, Ag
+And the amount of base is than being 2:1:5; 4 ℃ of following continuous stirring 0.5 – 2 hours, and the 12h that in the dark keeps in Dark Place was transferred to it in quartz colorimetric utensil of 1cm * 1cm with this mixed liquor; Be illumination 30min under the uviol lamp of 254 nm in wave number, obtain the Au-Ag-aptamer nano-complex; Dispose the Rh 6G methanol solution of 0.1 mol/L, and it is joined in the Au-Ag-aptamer nano-complex dispersion liquid, the ultimate density of control Rh 6G is 1 μ mol/L's; Stir 1h 4 ℃ of following lucifuges; With centrifugal 10 min of 10000 rev/mins rotating speed,, remove the not Rh 6G molecule of absorption with 0.1 mol/L PBS solution (pH 7.4) washing precipitation totally 3 times; Obtain Rh 6G-Au-Ag-aptamer probe complex; The TEM photo shows that the compound of gained is chain and distributes, and uniform particle diameter is about 40 nm.
Embodiment 3
With 0.1 mol/L phosphate buffer (PBS, pH 7.4) dissolving, concentration is 300 ng/mL with aptamer (S2.2, sequence is 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 '); NaAuCl with 1mol/L
4With 1mol/L AgNO
3Solution for after 4:1 mixes, joins in the aptamer solution Au in this mixed liquor by volume
3+, Ag
+And the amount of base is than being 4:1:5; 4 ℃ of following continuous stirring 0.5 – 2 hours, and the 12h that in the dark keeps in Dark Place was transferred to it in quartz colorimetric utensil of 1cm * 1cm with this mixed liquor; Be illumination 30min under the uviol lamp of 254 nm in wave number, obtain the Au-Ag-aptamer nano-complex; Dispose the Rh 6G methanol solution of 0.1 mol/L, and it is joined in the Au-Ag-aptamer nano-complex dispersion liquid, the ultimate density of control Rh 6G is 1 μ mol/L's; Stir 1h 4 ℃ of following lucifuges; With centrifugal 10 min of 10000 rev/mins rotating speed,, remove the not Rh 6G molecule of absorption with 0.1 mol/L PBS solution (pH 7.4) washing precipitation totally 3 times; Obtain Rh 6G-Au-Ag-aptamer probe complex; The TEM photo shows that the compound of gained is chain and distributes, and uniform particle diameter is about 100 nm.
Embodiment 4
Among the embodiment 4, except that light application time under uviol lamp is 5 min, other operations are all identical with instance 3, demonstrated the minute quantity particulate material on the TEM photo and existed.
Embodiment 5
Among the embodiment 5, except that light application time under uviol lamp is 15 min, other operations are all identical with instance 3, demonstrated small quantities of particles shape material on the TEM photo and existed, and are not chain and distribute.
Embodiment 6
Among the embodiment 6, light application time is 60 min under uviol lamp, and other operations are all identical with instance 3, and the coagulation phenomenon appears in compound, and the TEM photo shows that the compound of gained is dendroid.
Embodiment 7
With the Rh 6G-Au-Ag-aptamer probe complex and the MCF-7 cytosis of embodiment 3 preparations, utilize SERS technology for detection MCF-7 cell.
At first cultivate the MCF-7 cell: the MCF-7 cell attachment is grown in DMEM (Dulbecco ' the s modified Eagle medium) nutrient solution; Wherein contain ox embryo serum 10% (v/v), penicillin 100 U/mL, streptomysin 100 μ g/mL, in 37 ℃, 5% incubator, cultivate.The observation of cell growth makes when about cell length to 90% and goes down to posterity.When going down to posterity, remove nutrient solution,, add 37 ℃ of constant temperature digestion 3min of trypsase of 1 mL 0.25%, get an amount of cell suspension and add and continue in the new nutrient culture media to cultivate, change liquid every other day, control number of cells 1 * 10 with cell counter with PBS solution washing 3 times
5Individual/mL, remove nutrient solution, behind PBS solution washing 3 times, cell is for use.
Prepared Rh 6G-Au-Ag-aptamer probe complex among the embodiment 3 is disperseed in (pH 7.4) in PBS solution, and concentration is 0.1mg/mL, gets 100 μ L dispersion liquids and joins 1 * 10
5In the individual MCF-7 cell, at room temperature hatch 30 min, inhale the probe complex go not with cytosis with suction pipe, and with 3 mL PBS solution washing cells 3-5 time.
To at room temperature digest 3 min with the trypsase of 1 mL 0.25% with cell after the probe complex effect, behind 1000 rev/mins of centrifugal 10 min, remove supernatant; Add 1 mL PBS solution again; Make it form uniform cell suspension, get 10 these cell suspensions of μ L, adopt SERS technology for detection MCF-7 cell its mica sheet surface that is coated onto prepared fresh; Detecting wavelength is 785 nm, wave-number range 400-4000 cm
-1, acquisition time is 10 s.Experimental result shows, occurs the Raman peaks of tangible Raman signal molecule Rh 6G on the Raman spectrogram of MCF-7 cell.
Embodiment 8
The Rh 6G-Au-Ag-aptamer probe complex that embodiment 1,2 is prepared under the condition identical with embodiment 7 with the MCF-7 cytosis, utilize SERS technology for detection MCF-7 cell.Experimental result shows also can access the comparatively Raman signal of weak Rh 6G of the similar but intensity of characteristic peak positions on their Raman spectrograms.
SEQUENCE?LISTING
< 110>Nanjing Normal University
< 120>a kind of cell probe complex that detects human breast cancer cell MCF-7 based on Raman spectrum and preparation method thereof
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial
<220>
< 223>synthetic outside prosthesis with part index concentration method phylogeny (SELEX) technology, with MCF-7 cell tool
Specific binding capacity is arranged
<400> 1
gcagttgatc?ctttggatac?cctgg 25
Claims (6)
1. cell probe complex that detects human breast cancer cell MCF-7 based on Raman spectrum; It is characterized in that; Described probe complex is that the aptamer S2.2 of 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 ' is a template with sequence; Deposited gold on its base/silver bimetal alloy nano particle, the immobilized Raman signal molecule of nanoparticle surface Rh 6G.
2. cell probe complex according to claim 1 is characterized in that, described its particle diameter of cell probe complex is 20～120nm.
3. cell probe complex according to claim 1 is characterized in that, described cell probe complex is synthetic by following method: aptamer S2.2 is dissolved in the PBS of pH 7.4, adds the NaAuCl of volume ratio 1 ~ 5:1
4And AgNO
3Solution mixes and stirs, and in the dark places more than 12 hours, places illumination 5 min to 60 min under the uviol lamp, forms the Au-Ag-aptamer nano-complex; Rh 6G solution is joined in the Au-Ag-aptamer nano-complex dispersion liquid, and lucifuge stirs the back centrifugal treating, removes the not Rh 6G molecule of absorption with the PBS washing, obtains described probe complex.
4. the preparation method of the cell probe complex of the described human breast cancer cell MCF-7 of claim 1; It is characterized in that; With sequence is that the aptamer S2.2 of 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 ' is dissolved in the PBS of pH 7.4, adds the NaAuCl of volume ratio 1 ~ 5:1
4And AgNO
3Solution mixes and stirs, and in the dark places more than 12 hours, places illumination 5 min to 60 min under the uviol lamp, forms the Au-Ag-aptamer nano-complex; Rh 6G solution is joined in the Au-Ag-aptamer nano-complex dispersion liquid, and lucifuge stirs the back centrifugal treating, removes the not Rh 6G molecule of absorption with the PBS washing, obtains described probe complex.
5. the preparation method of the cell probe complex of human breast cancer cell MCF-7 according to claim 4 is characterized in that, may further comprise the steps:
1) with sequence be the aptamer S2.2 of 5 ’ – GCA GTT GAT CCT TTG GAT ACC CTG G – 3 ' with 0.1 mol/L phosphate buffer (PBS, pH 7.4) dissolving, concentration is 300 ng/mL;
2) with 1mol/L NaAuCl
4With 1mol/L AgNO
3Solution 1 ~ 5:1 by volume mixes, and joins in the aptamer solution in the step 1), 4 ℃ of following continuous stirring 0.5 – 2 hours, and in the dark keeps in Dark Place more than the 12h, makes Au
3+And Ag
+Be adsorbed onto on the base of aptamer, obtain Au
3+-Ag
+-aptamer compound;
3) with Au
3+-Ag
+-aptamer compound is illumination 5min-60min under the uviol lamp of 254 nm in wave number, and solution colour gradually becomes blue by colourless, obtains the Au-Ag-aptamer nano-complex;
4) Rh 6G solution is joined in the Au-Ag-aptamer nano-complex dispersion liquid; The ultimate density of control Rh 6G is 1 μ mol/L; Stir 1h 4 ℃ of following lucifuges; Remove the not Rh 6G molecule of absorption with the washing of 0.1 mol/L PBS solution (pH 7.4) after the centrifugal treating, obtain described probe complex Rh 6G-Au-Ag-aptamer.
6. the preparation method of the cell probe complex of human breast cancer cell MCF-7 according to claim 5 is characterized in that, described step 2) in, Ag in the mixed liquor
+With the amount of the base of fit DNA than being 1:5.
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| CN106282194A (en) * | 2016-08-19 | 2017-01-04 | 东南大学 | Breast cancer lines specific nucleic acid aptamers and preparation detection, diagnose and treat the application in human breast carcinoma preparation |
| CN106282194B (en) * | 2016-08-19 | 2019-03-12 | 东南大学 | Breast cancer lines specific nucleic acid aptamers and its application in preparation detection, diagnosing and treating human breast carcinoma preparation |
| CN108265055A (en) * | 2018-03-21 | 2018-07-10 | 东南大学 | Specifically bind DNA aptamers and its application of human breast cancer cell line Bcap-37 |
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