CN103512874A - Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method - Google Patents
Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method Download PDFInfo
- Publication number
- CN103512874A CN103512874A CN201310432508.4A CN201310432508A CN103512874A CN 103512874 A CN103512874 A CN 103512874A CN 201310432508 A CN201310432508 A CN 201310432508A CN 103512874 A CN103512874 A CN 103512874A
- Authority
- CN
- China
- Prior art keywords
- cell
- enhanced raman
- ultrasonic
- cells
- laser optical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004027 cell Anatomy 0.000 title claims abstract description 172
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims description 32
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 claims abstract description 39
- 238000012576 optical tweezer Methods 0.000 claims abstract description 28
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 22
- 230000000694 effects Effects 0.000 claims abstract description 22
- 229910052709 silver Inorganic materials 0.000 claims abstract description 18
- 239000004332 silver Substances 0.000 claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 239000000084 colloidal system Substances 0.000 claims abstract 9
- 238000004458 analytical method Methods 0.000 claims description 29
- 238000004611 spectroscopical analysis Methods 0.000 claims description 22
- 238000010586 diagram Methods 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 13
- 238000001228 spectrum Methods 0.000 claims description 13
- 230000003595 spectral effect Effects 0.000 claims description 11
- 239000006285 cell suspension Substances 0.000 claims description 10
- 238000001237 Raman spectrum Methods 0.000 claims description 9
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 8
- 238000003745 diagnosis Methods 0.000 claims description 8
- 238000002525 ultrasonication Methods 0.000 claims description 7
- 238000012545 processing Methods 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 238000001069 Raman spectroscopy Methods 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000000513 principal component analysis Methods 0.000 claims description 5
- 238000007619 statistical method Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000010606 normalization Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- 230000008676 import Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 5
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 claims 4
- 239000006143 cell culture medium Substances 0.000 claims 3
- 102000004142 Trypsin Human genes 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 claims 1
- 239000012588 trypsin Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 15
- 239000002082 metal nanoparticle Substances 0.000 abstract description 14
- 206010028980 Neoplasm Diseases 0.000 abstract description 11
- 201000011510 cancer Diseases 0.000 abstract description 11
- 210000000170 cell membrane Anatomy 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000002900 effect on cell Effects 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 238000012850 discrimination method Methods 0.000 abstract 1
- 230000001575 pathological effect Effects 0.000 abstract 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 17
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 17
- 239000007788 liquid Substances 0.000 description 17
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 17
- 239000012930 cell culture fluid Substances 0.000 description 11
- 239000008279 sol Substances 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 238000009826 distribution Methods 0.000 description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000002105 nanoparticle Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000004065 semiconductor Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- 238000012935 Averaging Methods 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- XRRQZKOZJFDXON-UHFFFAOYSA-N nitric acid;silver Chemical compound [Ag].O[N+]([O-])=O XRRQZKOZJFDXON-UHFFFAOYSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010073324 Keratinising squamous cell carcinoma of nasopharynx Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 150000001455 metallic ions Chemical class 0.000 description 1
- 208000028133 nasopharyngeal squamous cell carcinoma Diseases 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
一种基于超声波穿孔效应并结合激光光镊表面增强拉曼光谱技术进行细胞判别的方法，是利用超声波对细胞作用产生穿孔效应使细胞膜通透性瞬间增强将金属纳米粒子快速导入细胞作为SERS增强基底，并利用激光光镊技术捕获活细胞同时进行表面增强拉曼光谱检测，以SERS光谱的手段实现对癌细胞的病理检测、筛选功能。本发明包括银胶溶液制备；把高浓度银胶注入活细胞并进行表面增强拉曼光谱测量；建立判别细胞为正常细胞或者癌症细胞表面增强拉曼光谱诊断识别模型。本发明具有简单迅速，可靠性强，灵敏度高的特点，可推广应用于多种癌变细胞的判别。
A cell discrimination method based on the ultrasonic perforation effect combined with laser optical tweezers surface-enhanced Raman spectroscopy technology, which is to use ultrasonic waves to produce a perforation effect on cells to instantly enhance the permeability of cell membranes, and quickly introduce metal nanoparticles into cells as SERS-enhanced substrates , and use laser optical tweezers to capture living cells and perform surface-enhanced Raman spectroscopy detection, and use SERS spectroscopy to realize pathological detection and screening of cancer cells. The invention comprises preparation of silver colloid solution; injecting high-concentration silver colloid into living cells and performing surface-enhanced Raman spectrum measurement; establishing a surface-enhanced Raman spectrum diagnostic identification model for distinguishing cells as normal cells or cancer cells. The invention has the characteristics of simplicity, rapidity, strong reliability and high sensitivity, and can be extended and applied to the discrimination of various cancerous cells.
Description
Technical field
The present invention relates to a kind ofly utilize ultrasonic perforating effect and in conjunction with the method for laser optical tweezer Surface enhanced raman spectroscopy (SERS) technical Analysis cell.
Background technology
Multiple biomedical technology has been applied to qualitative, quantitative going and has surveyed, screened normal or sick cell.Such as present radiology, pharmacology, histology, cytology and molecules technology can be applied in the analysis of cell.But these technology are all to have destructively, simultaneously their specificity is not ideal enough, and many times wants multiple technologies coupling just can effectively analyze, but also may be subject to the interference of cell biology means.We are badly in need of a kind of technological means now can overcome above-mentioned several cell analysis technical limitation, can in clinical diagnosis, easily realize accurate cancer cell analysis and screening.
Surface enhanced raman spectroscopy (SERS) technology is compared and can be made Raman signal enhancer reach 10 with conventional Raman spectrum
14, in some condition, even can realize Single Molecule Detection.Meanwhile, SERS technology can also suppress the interference of biological autofluorescence background to a great extent.Therefore, SERS technology has been widely used in the research of biomedical sector.There have been in the world a plurality of research groups to utilize SERS spectrum to carry out cell research, but want to detect cell SERS spectral signal, just must by certain specific process make metal nanoparticle transfered cell inner or make nano particle and cell can adsorbed close together with, the SERS spectral signal of cell so just can be detected.Make nano particle enter cell interior and can carry out SERS detection, more conventional way is metal nanoparticle and cell co-culture, hatches now, through cultivating for a long time (generally wanting about 24 hours), utilize the endocytosis effect of cell, nano particle just can " be gulped down " and be entered cell interior, thereby realize the analysis of cell SERS spectral detection, but this method expends time in very much; Second method is exactly to utilize the autonomous reduction mechanism of biological cell, solution with metallic ion and cell are hatched jointly, at cell interior spontaneous " growing " nano-metal particle, this method need to be carried out cell cultivation equally for a long time, and the metal nanoparticle out-of-shape that is reduced out at cell interior; Third method is to utilize electroporation technology fast metal nanoparticle to be imported to intracellular conveying, but this method needs special electroporation device, operation more complicated, efficiency low, and the distribution of nano particle in cell is very inhomogeneous.
Weak point in view of above the whole bag of tricks, patent of the present invention proposes to utilize the ultrasonic acoustic effect of holes by the quick transfered cell of metal nanoparticle, and laser optical tweezer Raman spectroscopy is combined with this supersonic induced technology can detect and obtain high reproducible cell SERS spectrum, we also further utilize cell SERS spectrum to carry out cancer cell and Normocellular screening study, obtain ideal differentiation effect.
Summary of the invention
The object of this invention is to provide a kind of ultrasonic acoustic effect of holes of utilizing and in conjunction with laser optical tweezer Surface enhanced raman spectroscopy technology, carry out the method for cell analysis.It is to utilize high-energy ultrasound effect moment generation acoustic horn effect, make pericellular micro-bubble violent contraction occur, expand and finally collapse and go out, meanwhile the bubble of these generation acute variation and cell generation heat effect and mechanical effect produce perforation effect on cell membrane, at surface of cell membrane, produce reversibility cavernous structure, permeability of cell membrane moment is strengthened greatly; Simultaneously metal nanoparticle forms single dispersion and in high-speed motion state under ul-trasonic irradiation, the aperture that so just can produce by surface of cell membrane brief moment strengthens substrate using the quick transfered cell of metal nanoparticle inside as SERS, and utilize laser optical tweezer technology to catch cell and carry out Surface enhanced raman spectroscopy detection simultaneously, finally the cell SERS spectroscopic data obtaining is carried out to multivariate statistical analysis (PCA-LDA) and process, by the diagnostic equation in scatter diagram, can differentiate cell is cancer or normal cell.We take nasopharyngeal carcinoma cell and normal nasopharyngeal cell as model is to cancer or Normocellularly carry out discriminatory analysis.In measuring process, utilize laser optical tweezer technology can maintain cell and under physiologically active state, carry out SERS spectrum test.The whole processing procedure time of the present invention only needs 10 minutes, simple to operate, quick, reliability is strong; Can make metal nanoparticle very efficiently in transfered cell, and metal nanoparticle distributes more evenly, can obtain the extraordinary cell SERS of reappearance spectrum.To the laser optical tweezer SERS spectrum of normal and cancer cell differentiate, analysis conclusion can be doctor to patient's diagnosis provide fast, objective appraisal standard.The method can be expanded, be applied to other kinds cancer cell and Normocellular discriminatory analysis.
For realizing the technical scheme of object employing of the present invention, be:
(1) get and in the deionized water that silver nitrate powder is dissolved in 200ml, be heated to boiling, then splashing into citric acid three sodium solution, to make the fulmargin that contains Nano silver grain standby;
(2) it is standby that competent cell trypsinization being got off is made single-cell suspension liquid with RPMI1640 cell culture fluid;
(3) by fulmargin with single-cell suspension liquid mixes and utilize ultrasonic perforating effect by the quick transfered cell of Nano silver grain;
(4) to unicellular, carry out the detection of laser optical tweezer Surface enhanced raman spectroscopy maintaining under physiological environment;
(5) set up cell surface and strengthen Raman spectrum data storehouse, utilize multivariate statistical analysis to obtain the scatter diagram corresponding to Surface enhanced raman spectroscopy of variety classes cell;
(6) utilize and to set up cell surface and strengthen Raman spectrum data storehouse, diagnosis cell is not differentiated.
The preparation process of described fulmargin is: get 36mg silver nitrate powder and be dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, splashing into 5ml concentration is 1% citric acid three sodium solution again, continue agitating heating 1 hour, prepare the fulmargin that contains Nano silver grain, and put at room temperature lucifuge and seal up for safekeeping standby;
The preparation process of described single-cell suspension liquid is: the competent cell that trypsinization is got off is made single-cell suspension liquid with RPMI1640 cell culture fluid, then by blood counting chamber, calculates the number of cell, and controlling cell density is 10
5～10
6individual/ml.
The described ultrasonic perforating effect of utilizing by the quick transfered cell internal procedure of Nano silver grain is: get pre-prepared good fulmargin 1 ml and carry out centrifugal, abandon the cell culture fluid that adds 0.5ml after supernatant, in the ratio of 1:1, put into centrifuge tube with the prefabricated active single-cell suspension liquid of getting ready, utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, they to be mixed, make active unicellular elargol mixed liquor; Centrifuge tube is put into ultrasonic pond, and by regulating hyperacoustic power, frequency, the temperature in ultrasonic pond, ultrasonic time parameter, by the ultrasonication of the unicellular elargol mixed liquor of the activity in centrifuge tube; The unicellular elargol mixed liquor of the activity of ultrasonication is mixed with PBS damping fluid and carry out low-speed centrifugal, clean three times, the silver nano-grain that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in RPMI1640 cell culture fluid.
Described hyperacoustic power is: 200W～400W.
Described hyperacoustic frequency is: 25HZ, 40HZ or 59HZ;
The temperature in described ultrasonic pond is: room temperature～60 ℃;
Described ultrasonic time is: 1min～30min.
Described is the laser optical tweezer system (model: OTKB) that is accompanied with Raman spectrum module that adopts U.S. Thorlabs company to produce maintaining under physiological environment to the unicellular laser optical tweezer Surface enhanced raman spectroscopy testing process of carrying out, utilize in this laser optical tweezer system acquisition RPMI1640 cell culture fluid active unicellularly, utilize the semiconductor laser that wavelength is 785nm to carry out cell SERS spectral detection as exciting light simultaneously.For obtaining high reappearance SERS spectrum, under the condition of cell, reduce excitation light power as far as possible and carry out cell SERS spectrum test guaranteeing to catch, can obtain high reappearance cell SERS spectrum.
The described cell surface of setting up strengthens Raman spectrum data storehouse model, and its process is: cellularity is differentiated and must be realized by a diagnostic model, and this model is set up by calibration process.For guaranteeing that data have statistical significance, need to gather the cell SERS spectrum of abundant confirmed cases, set up calibration spectra database.On this basis, use main element analysis method and linear discriminant analysis algorithm, set up the diagnostic equation being formed by cell SERS spectrum parameter.First we need cell SERS spectrum to detain fluorescence and intensity normalized, whole piece spectral line data input SPSS software is carried out to major component (PCA) analysis, then utilize independent variable T check to obtain having the PCA score of significant difference, and utilize the PCA score with significant difference to draw the scatter diagram with diagnostic equation, and further utilize linear discriminant analysis (LDA) to draw statistics.
The described built vertical cell surface that utilizes strengthens Raman spectrum data storehouse model, diagnosis cell is not differentiated, its process is: diagnosis cell is not made to the unicellular elargol mixed liquor of required activity according to the method described in technical scheme, and maintaining under physiological environment the unicellular laser optical tweezer Surface enhanced raman spectroscopy detection SERS spectrum that carries out, spectrum is imported to the cell surface having established and strengthen Raman spectrum data storehouse, utilize multivariate statistical analysis to obtain the not scatter diagram corresponding to Surface enhanced raman spectroscopy of diagnosis cell, according to the region that is distributed in diagnostic equation both sides in scatter diagram, differentiate cell.
Advantage of the present invention is to adopt ultrasonic perforation effect to make metal nanoparticle can enter fast cell, have that nano particle imports that efficiency is high, metal nanoparticle is in the cell interior more uniform feature that distributes, combine and can guarantee cytoactive with laser optical tweezer technology, can detect and obtain high reappearance cell SERS spectrum simultaneously; And with this cell SERS spectroscopic data, set up diagnostic model, can be used for the differentiation to nasopharyngeal carcinoma cell.Also the method can be expanded to the discriminatory analysis that is applied to the kinds cancer cells such as cancer of the esophagus, cutaneum carcinoma.
Accompanying drawing explanation
Fig. 1 be the present invention to 63 NP69 normal cells cell SERS spectrum averaging spectrum under ultrasonic perforation effect, shade and shadow partly represents the standard deviation of cell SERS spectrum.
Fig. 2 be the present invention to 67 C666 nasopharyngeal carcinoma cells cell SERS spectrum averaging spectrum under ultrasonic perforation effect, shade and shadow partly represents the standard deviation of cell SERS spectrum.
Fig. 3 is the present invention to 67 C666 cells and 63 NP69 cell SERS spectrum PCA score PC1 and PC2 two dimension scatter diagram.
Fig. 4 is the present invention to 67 C666 cells and 63 NP69 cell SERS spectrum PCA score PC1, PC2 and the three-dimensional scatter diagram of PC3.
Embodiment
Concrete ins and outs of the present invention are implemented to be described below for normal nasopharyngeal cell NP69, two types of cells of differentiated Nasopharyngeal Squamous Cell Carcinoma C666:
Embodiment 1
1, the solid nitric acid silver of getting 36mg is dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, then splashes into 1% trisodium citrate of 5ml, continues agitating heating 1 hour, obtain silver sol solution, and put at room temperature lucifuge sealing preservation in order to follow-up supersonic induced step use; Two kinds of competent cell NP69, C666 with pancreatin, just having digested are made to single-cell suspension liquid with commercially available RPMI1640 cell culture fluid respectively, then by blood counting chamber, calculate the number of cell, controlling cell density is 10
6individual/ml.
2, get the above-mentioned fulmargin 10ml preparing and carry out centrifugally, abandon after supernatant, be divided into two groups, every group adds respectively the cell culture fluid of 2.5ml to mix; Get respectively 0.5ml silver sol and NP69, C666 cell suspending liquid are put into respectively EP pipe separately in the ratio of 1:1 for every group; utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, silver sol suspending liquid and cell suspending liquid to be mixed, make unicellular elargol mixed liquor;
3, the centrifuge tube that silver sol and cell suspending liquid are housed is put into ultrasonic pond, by regulating the ultrasonic power in ultrasonic pond, be that 200W, ultrasonic frequency are 25HZ, 30 ℃ of ultrasonic pond temperature, ultrasonic time 1min, make the mixed liquor ultrasonication under best ultrasound condition in centrifuge tube.
4, the cell of ultrasonication mixed with PBS damping fluid and carry out low-speed centrifugal, cleaning three times, the metal nanoparticle that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in nutrient solution.
5, the sample cell that the cell suspending liquid cleaning is injected to laser optical tweezer Raman detection system carries out light tweezer SERS spectral detection, the semiconductor laser that excitation wavelength is 785nm in the environment of cell culture fluid.Test condition is 100 times of Nikon oil mirrors.
6, by definite normal nasopharyngeal cell and nasopharyngeal carcinoma cell SERS establishment of spectrum calibration spectra database, and all cells SERS spectral line is input to SPSS software carries out PCA analysis, is the concrete computation process that application PCA analyzes below:
(1) first utilize fitting of a polynomial to eliminate fluorescence background 63 normal NP69 nasopharyngeal cells and 67 C666 nasopharyngeal carcinoma cell SERS spectrum;
(2) for reducing experimental error impact, the normal and nasopharyngeal carcinoma cell SERS spectrum of eliminating fluorescence background is carried out to area normalization processing;
(3) utilize SPSS software that the normal and nasopharyngeal carcinoma cell SERS spectrum of processing through (1), (2) is carried out to PCA analysis;
(4) utilize on this basis the T method of inspection in SPSS, select to have most three PCA of significant difference LDA analysis of must assigning to further carry out;
Utilizing T check to obtain three has the PCA score of significant difference most and draws scatter plot distributions.
The present invention confirms that by independent variable T check PCA analyzes rear major component 1, major component 2 and major component 4 these three major components and has significant difference.We further draw major component 1 and the two-dimentional scatter diagram of major component 4 with diagnostic equation, as shown in Figure 3.Diagnostic equation is y=-2.73x-0.43, and this diagnostic equation line can distribute nasopharyngeal carcinoma cell point set to distribute and effectively separate to get up with normal cell point set.If utilize major component 1, major component 2 and the major component 4 in two class cell SERS spectrum PCA analyses with significant difference further to draw three-dimensional scatter diagram, as shown in Figure 4, the data point distribution that can find to represent normal nasopharyngeal cell and represent nasopharyngeal carcinoma cell is in two separate spaces, without any overlapping, distinguish effect very obvious each other.Further utilize follow-up LDA to analyze and draw statistics, the present invention reaches 100% to specificity and the sensitivity of the cancer cell identification in calibration spectra collection.Normal and nasopharyngeal carcinoma cell SERS spectrum PCA score scatter plot distributions is measurement result of the present invention, also can be diagnosis important reference is provided.
Embodiment 2
1, the solid nitric acid silver of getting 36mg is dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, then splashes into 1% trisodium citrate of 5ml, continues agitating heating 1 hour, obtain silver sol solution, and put at room temperature lucifuge sealing preservation in order to follow-up supersonic induced step use; Two kinds of competent cell NP69, C666 with pancreatin, just having digested are made to single-cell suspension liquid with commercially available RPMI1640 cell culture fluid respectively, then by blood counting chamber, calculate the number of cell, controlling cell density is 10
6individual/ml.
2, get the above-mentioned fulmargin 10ml preparing and carry out centrifugally, abandon after supernatant, be divided into two groups, every group adds respectively the cell culture fluid of 2.5ml to mix; Get respectively 0.5ml silver sol and NP69, C666 cell suspending liquid are put into respectively EP pipe separately in the ratio of 1:1 for every group; utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, silver sol suspending liquid and cell suspending liquid to be mixed, make unicellular elargol mixed liquor;
3, the centrifuge tube that silver sol and cell suspending liquid are housed is put into ultrasonic pond, by regulating the ultrasonic power in ultrasonic pond, be that 250W, ultrasonic frequency are 40HZ, 24 ℃ of ultrasonic pond temperature, ultrasonic time 20min, make the mixed liquor in centrifuge tube under best ultrasound condition, carry out ultrasonication.
4, the cell of ultrasonication mixed with PBS damping fluid and carry out low-speed centrifugal, cleaning three times, the metal nanoparticle that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in nutrient solution.
5, the sample cell that the cell suspending liquid cleaning is injected to laser optical tweezer Raman detection system carries out light tweezer SERS spectral detection, the semiconductor laser that excitation wavelength is 785nm in the environment of cell culture fluid.The object lens of test use are 100 times of Nikon oil mirrors.
6, by definite normal nasopharyngeal cell and nasopharyngeal carcinoma cell SERS establishment of spectrum calibration spectra database, and all cells SERS spectral line is input to SPSS software carries out PCA analysis, is the concrete computation process that application PCA analyzes below:
(1) 1) 1 first utilize fitting of a polynomial to eliminate fluorescence background 120 normal NP69 nasopharyngeal cells and 130 C666 nasopharyngeal carcinoma cell SERS spectrum;
(2) for reducing experimental error impact, the normal and nasopharyngeal carcinoma cell SERS spectrum of eliminating fluorescence background is carried out to area normalization processing;
(3) utilize SPSS software that the normal and nasopharyngeal carcinoma cell SERS spectrum of processing through (1), (2) is carried out to PCA analysis;
(4) utilize on this basis the T method of inspection in SPSS, select to have most three PCA of significant difference LDA analysis of must assigning to further carry out;
(5) utilizing T check to obtain three has the PCA score of significant difference most and draws scatter plot distributions.
The present invention confirms that by independent variable T check PCA analyzes rear major component 1, major component 2 and major component 4 these three major components and has significant difference.We further draw major component 1 and the two-dimentional scatter diagram of major component 4 with diagnostic equation.This diagnostic equation line can distribute nasopharyngeal carcinoma cell point set to distribute and effectively separate to get up with normal cell point set.If utilize major component 1, major component 2 and the major component 4 in two class cell SERS spectrum PCA analyses with significant difference further to draw three-dimensional scatter diagram, the data point distribution that can find to represent normal nasopharyngeal cell and represent nasopharyngeal carcinoma cell is in two separate spaces, without any overlapping, distinguish effect very obvious each other.Further utilize follow-up LDA to analyze and draw statistics, the present invention nearly all reaches 100% to specificity and the sensitivity of the cancer cell identification in calibration spectra collection.Normal and nasopharyngeal carcinoma cell SERS spectrum PCA score scatter plot distributions is measurement result of the present invention, also can be diagnosis important reference is provided.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310432508.4A CN103512874A (en) | 2013-09-22 | 2013-09-22 | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310432508.4A CN103512874A (en) | 2013-09-22 | 2013-09-22 | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103512874A true CN103512874A (en) | 2014-01-15 |
Family
ID=49895937
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310432508.4A Pending CN103512874A (en) | 2013-09-22 | 2013-09-22 | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103512874A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ305600B6 (en) * | 2014-11-10 | 2015-12-30 | Univerzita Palackého v Olomouci | Process for preparing layers of silver colloidal particles on glass substrate for the purpose of surface enhanced Raman spectroscopy, a layer of silver colloidal particles on a glass substrate and the use of such a layer |
| CN106645079A (en) * | 2016-09-30 | 2017-05-10 | 福建师范大学 | Human blood type identifying method based on erythrocyte laser tweezers Raman spectroscopy |
| CN114235773A (en) * | 2020-09-09 | 2022-03-25 | 南京大学 | A Raman imaging method for dynamic monitoring of cell membrane repair process |
| CN118690990A (en) * | 2023-10-08 | 2024-09-24 | 深圳市黑金工业制造有限公司 | A production data analysis management system and method based on cloud computing |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240401A1 (en) * | 2005-01-27 | 2006-10-26 | Clarke Richard H | Handheld raman body fluid analyzer |
| CN101339132A (en) * | 2008-08-27 | 2009-01-07 | 福建师范大学 | A method for detecting the components of traditional Chinese medicine decoction using surface-enhanced Raman spectroscopy |
| CN101482509A (en) * | 2009-03-03 | 2009-07-15 | 福建师范大学 | Method for detecting animal active unicellular sample by surface reinforced Raman spectrum |
| CN101799421A (en) * | 2010-04-19 | 2010-08-11 | 福建师范大学 | A detection method of body fluid surface-enhanced Raman spectroscopy |
| CN101806740A (en) * | 2010-04-19 | 2010-08-18 | 福建师范大学 | Detection method of human plasma surface enhanced raman spectroscopy by integrating main component analysis |
| US20110230760A1 (en) * | 2007-05-04 | 2011-09-22 | The Board Of Trustees Of The Leland Stanford Junior University | Raman imaging devices and methods of molecular imaging |
| US20120302940A1 (en) * | 2011-05-26 | 2012-11-29 | Jackson State University | Popcorn Shape Gold Nanoparticle For Targeted Diagnosis, Photothermal Treatment and In-Situ Monitoring Therapy Response for Cancer and Multiple Drug Resistance Bacteria |
-
2013
- 2013-09-22 CN CN201310432508.4A patent/CN103512874A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240401A1 (en) * | 2005-01-27 | 2006-10-26 | Clarke Richard H | Handheld raman body fluid analyzer |
| US20110230760A1 (en) * | 2007-05-04 | 2011-09-22 | The Board Of Trustees Of The Leland Stanford Junior University | Raman imaging devices and methods of molecular imaging |
| CN101339132A (en) * | 2008-08-27 | 2009-01-07 | 福建师范大学 | A method for detecting the components of traditional Chinese medicine decoction using surface-enhanced Raman spectroscopy |
| CN101482509A (en) * | 2009-03-03 | 2009-07-15 | 福建师范大学 | Method for detecting animal active unicellular sample by surface reinforced Raman spectrum |
| CN101799421A (en) * | 2010-04-19 | 2010-08-11 | 福建师范大学 | A detection method of body fluid surface-enhanced Raman spectroscopy |
| CN101806740A (en) * | 2010-04-19 | 2010-08-18 | 福建师范大学 | Detection method of human plasma surface enhanced raman spectroscopy by integrating main component analysis |
| US20120302940A1 (en) * | 2011-05-26 | 2012-11-29 | Jackson State University | Popcorn Shape Gold Nanoparticle For Targeted Diagnosis, Photothermal Treatment and In-Situ Monitoring Therapy Response for Cancer and Multiple Drug Resistance Bacteria |
Non-Patent Citations (5)
| Title |
|---|
| JUQIANG LIN,ET AL.: "Rapid delivery of silver nanoparticles into living cells by electroporation for surface-enhanced Raman spectroscopy", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 冉海涛 等: "超声波空化效应对体外培养细胞细胞膜作用的实验研究", 《中华超声影像学杂志》 * |
| 冯尚源 等: "基于SERS技术结合多变量统计分析胃癌患者血浆拉曼光谱", 《中国科学：生命科学》 * |
| 王雁军 等: "人肝癌组织细胞的激光光镊拉曼光谱研究", 《光谱学与光谱分析》 * |
| 陈谦 等: "超声波声孔效应中气泡动力学的研究", 《物理学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ305600B6 (en) * | 2014-11-10 | 2015-12-30 | Univerzita Palackého v Olomouci | Process for preparing layers of silver colloidal particles on glass substrate for the purpose of surface enhanced Raman spectroscopy, a layer of silver colloidal particles on a glass substrate and the use of such a layer |
| CN106645079A (en) * | 2016-09-30 | 2017-05-10 | 福建师范大学 | Human blood type identifying method based on erythrocyte laser tweezers Raman spectroscopy |
| CN106645079B (en) * | 2016-09-30 | 2019-07-23 | 福建师范大学 | A kind of human body blood group discrimination method based on red blood cell optical tweezer Raman spectroscopy |
| CN114235773A (en) * | 2020-09-09 | 2022-03-25 | 南京大学 | A Raman imaging method for dynamic monitoring of cell membrane repair process |
| CN114235773B (en) * | 2020-09-09 | 2024-02-09 | 南京大学 | Raman imaging method for dynamic monitoring of cell membrane repair process |
| CN118690990A (en) * | 2023-10-08 | 2024-09-24 | 深圳市黑金工业制造有限公司 | A production data analysis management system and method based on cloud computing |
| CN118690990B (en) * | 2023-10-08 | 2025-04-18 | 深圳市黑金工业制造有限公司 | A production data analysis management system and method based on cloud computing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107446807B (en) | Integrated form Terahertz superstructure nano biological chip and its application and method | |
| CN103512874A (en) | Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method | |
| Yu et al. | Leukemia cells detection based on electroporation assisted surface-enhanced Raman scattering | |
| Bhattacharyya et al. | Gold nanoparticle–mediated detection of circulating cancer cells | |
| CN103472029B (en) | A kind of pick-up unit for food additive component | |
| Harvey et al. | Classification of fixed urological cells using Raman tweezers | |
| WO2008115521A1 (en) | Photoacoustic indicators | |
| CN104502303B (en) | For the Asia-Pacific hertz nano biological sensor and its detection method of fast frame inspection bacterium | |
| US10935495B2 (en) | Detection and analysis method for urine-modified nucleoside based on surface-enhanced resonance Raman spectroscopy | |
| CN103822868A (en) | A device for detecting particle size of phytoplankton in seawater and its detection method | |
| CN104535479B (en) | For the Asia-Pacific hertz nano biological sensor of single or a small amount of cell detection | |
| Chen et al. | Diagnosis and staging of diffuse large B-cell lymphoma using label-free surface-enhanced Raman spectroscopy | |
| Li et al. | Vertical gold nanowires-based surface-enhanced Raman scattering for direct detection of ocular bacteria | |
| JP2012068108A (en) | Cell analyzer | |
| CN109738417B (en) | A method for detecting tumor cells using porous gold nanospheres | |
| Ma et al. | Label-free breast cancer detection and classification by convolutional neural network-based on exosomes surface-enhanced raman scattering | |
| CN103487425A (en) | Method for distinguishing cancer cells through surface enhanced Raman spectroscopy | |
| Gutierrez‐Juarez et al. | Detection of melanoma cells in vitro using an optical detector of photoacoustic waves | |
| Jiang et al. | High precision qualitative identification of yeast growth phases using molecular fusion spectra | |
| CN104027132A (en) | Device and method based on multispectral photoacoustic tomography | |
| JP7457710B2 (en) | Systems and methods for measuring vibrational spectra of biological cells and tissues over time | |
| Li et al. | Non-invasive detection of haemoglobin, platelets, and total bilirubin using hyperspectral cameras | |
| CN106645079B (en) | A kind of human body blood group discrimination method based on red blood cell optical tweezer Raman spectroscopy | |
| CN101482509A (en) | Method for detecting animal active unicellular sample by surface reinforced Raman spectrum | |
| CN205538588U (en) | Unmarked tumor cells detection diagnosis device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140115 |