CN109342403A - A method for the detection of cell degranulation based on surface-enhanced Raman spectroscopy - Google Patents
A method for the detection of cell degranulation based on surface-enhanced Raman spectroscopy Download PDFInfo
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Abstract
The invention belongs to technical field of biological, are related to a kind of detection method of cell degranulation, and in particular to a method of cell degranulation is detected based on Surface enhanced Raman spectroscopy.The following steps are included: cell degranulation component is generated and is extracted, SERS spectra detects cell degranulation component, the data processing of cell degranulation SERS spectra and characteristic peak and chooses, establishes β-hexosaminidase release rate and cell degranulation degree correlation.The present invention is in the different degrees of degranulation of detection mast cell, compared with traditional detection method i.e. detection β-hexosaminidase, has consistent conclusion, but the method for the present invention has efficient and convenient and fast detection characteristic.
Description
Technical field
The invention belongs to technical field of biological, are related to a kind of detection method of cell degranulation, and in particular to a kind of
The method of Surface enhanced Raman spectroscopy detection cell degranulation.
Background technique
Mast cell (Mast Cells, MCs) is broken up under IL-3 effect by the multi-functional candidate stem cell in marrow
And with blood circulation move to privileged site or tissue is proliferated, is broken up and mature, it eventually enters into connective tissue.Fertilizer
Maxicell degranulation (Degranulation) is mast cell by specific antigen stimulant (such as bacterium, virus and toxin
Deng) or nonspecific stimulation object (such as C48/80, Calcium ionophore and wasp toxin) induction release histamine, serotonin,
The important molecule event of the particles such as heparin, trypsinlike enzyme, cell factor and growth factor, it is thin will to directly affect second order effect
The recruitment and activation of born of the same parents' (such as eosinophil and neutrophil leucocyte), causes the generation of such as inflammation disease, immunological diseases.Closely
Nian Lai, studies have reported that mast cell may promote or inhibit angiogenesis when tumour occurs, this to the research of cancer especially
It is important.Mainly there are the substances such as histamine, trypsinlike enzyme and β-hexosaminidase in mast cell degranulation.Histamine detecting step
It is cumbersome, half-life short, required technical requirements height；Trypsinlike enzyme causes to be not easy to examine because of cell sampling position difference content difference
It surveys；Though β-hexosaminidase half-life period is longer, content is relatively stable, influences when detecting vulnerable to color sample.Due to hypertrophy
Cell degranulation participates in the generation of various biomolecules event, therefore the detection of its degranulation degree and particulate matter is to understanding molecule
Event mechanism and signal path regulation are of great significance.
Differentiate and determination method mainly to have in vivo and vitro detection currently used for mast cell degranulation degree.
It takes determining site tissue that slice dyeing is made mostly in body detecting method, it is imperfect to observe mast cell after birth under the microscope
Number and total cell number ratio calculation degranulation ratio, this detection process is many and diverse, tediously long, and unsuitable clinical diagnosis is promoted.Body
Outer detection method mainly has three classes: 1) detection of degranulation content of material, such as histamine, trypsinlike enzyme and beta-amino hexoside
Enzyme.These three are respectively present half-life short and detection is cumbersome, and content difference is big and activity is relatively low, are influenced by solution colour
The shortcomings that；2) the profile variation detection of mast cell, but it loses activity there are poor reproducibility, cell dyeing and counts by subjectivity
The deficiency that factor influences；3) probe or fluorescent marker, are observed using Laser Scanning Confocal Microscope.Required detection technique requires high, cost
Greatly.So there is an urgent need to a kind of quick, convenient, samples to be not necessarily to pretreatment, and not by the detection technique of analyte Color influences
The method for differentiating for mast cell degranulation degree and analyzing.
The inelastic scattering effect that incident light shines the frequency occurred when sample and direction changes is referred to as Raman and dissipates
Penetrate effect.Raman spectrum, which can provide the large biological molecule such as chemical fingerprint of the ingredients such as protein, nucleotide and lipid and conformation, to be believed
It ceases, then can reflect the molecular chemistry bond structure and vibration information of substance by Spectra peak recognition, can be used for parsing molecular structure letter
Breath.This method have many advantages, such as quickly detect, it is easy to sample nondestructive and sample preparation, can be used as a kind of quick material composition
Analysis method.But the weak output signal that normal Raman spectral collection arrives, the interference of sample itself fluorescence background is big, especially micro dense
When the research of degree sample and precision are researched and analysed, what is generated misses by a mile, and actual requirement is much not achieved in sensitivity.It sends out in recent years
Surface enhanced Raman spectroscopy (Surface-enhanced Raman spectroscopy, SERS) technology that exhibition is got up is not only gram
The disadvantage and deficiency of normal Raman technology are taken, while because enhancing substrate by nano metal material such as Au, Ag, Cu and Pt etc., from
And substantially increase raman spectral signal (104-1014Times).Therefore, SERS method have to detect needed for sample it is few, to sample
The advantages that lossless, high sensitivity, high specific and effective quenching fluorescence is a kind of efficient, quick molecular structure information detection
Analytical technology.
Summary of the invention
It is an object of the invention to propose a kind of spectrum that degranulation molecular events occur using SERS technology detection cell
Detection method.Using C48/80 medicine irritation cell degranulation occurs for this method, detects extracted cell using SERS technology
The Surface enhanced Raman spectroscopy of degranulation component establishes the degranulation material surface enhanced spectrum database of different cell lines, knot
It closes the methods of principal component analysis and linear discriminant analysis (PCA-LDA) and carries out discrimination analysis, obtain under the conditions of different stimulated not
Degranulation substance SERS difference spectrum and two-dimentional scatter diagram with cell line, realize the detection of different cell line degranulations.In addition,
In order to probe into cell and acupuncture, analgesic relationship, also to cell, cell activation is sent out under the laser stimulation of different light dosages for we
Raw degranulation situation and degree carry out SERS detection and analysis, and using the analysis of Multivariate Statistics method after laser irradiation not
With the cell degranulation SERS spectra of time.The present invention has many advantages, such as quick, easy to operate, highly sensitive and inexpensive, can have
Effect ground obtains and detects degranulation SERS spectra and difference of the different cell lines under various concentration C48/80 medicine irritation and swashs
Different cell line degranulation SERS spectras under light irradiation time and the irradiation of different laser powers, differentiate and are detected for cell degranulation
A kind of quickly and efficiently new method is provided.
For achieving the above object, the present invention adopts the following technical scheme:
A method of cell degranulation is detected based on Surface enhanced Raman spectroscopy, is included the following steps:
(1) cell degranulation component is generated and is extracted:
In vitro culture mast cell is centrifuged and is abandoned to it culture solution when being in logarithmic growth division stage, use fresh medium
It is resuspended, adjustment cell concentration is 103-1010A/mL is inoculated in 24 orifice plates or culture dish by the inoculum concentration of 10 ~ 75%v/v, culture
1-72h；500-1500rpm is centrifuged 3-20min after taking-up, abandons supernatant, and addition pH is that the PBS of 6-8,1-20mM are resuspended；To hypertrophy
Cell carries out the stimulation of different pharmaceutical concentration gradient or using 1-24h is cultivated after the irradiation of different optical maser wavelengths, and centrifuging and taking supernatant stores
In centrifuge tube；
(2) SERS spectra detects cell degranulation component:
Using the SERS enhancing substrate concentrating colloidal solution enhancing SERS substrate prepared, take the mast cell extracted in (1) de-
Grain fraction is mixed and is shaken up in equal volume with SERS enhancing substrate concentrating colloidal solution, takes out the substrate for being added dropwise and detecting in SERS
On, cell degranulation component SERS spectra, which is carried out, using Confocal laser-scanning microscopy after natural drying detects；
(3) data processing of cell degranulation SERS spectra and characteristic peak are chosen:
Step (2) mast cell degranulation component SERS spectra data obtained carry out the fitting of five rank multinomials and scratch except fluorescence is carried on the back
Scape noise, then carry out the normalization of spectral intensity peak value or area normalization processing；Using SPSS software combination principal component analysis and
Linear discriminant analysis handles spectroscopic data；To spectroscopic data from 400-3000 cm-1It is the most aobvious that wave-number range chooses 3-4 variation
The spectral peak of work is as mast cell degranulation degree SERS spectra characteristic peak；
(4) β-hexosaminidase release rate and cell degranulation degree correlation are established；
Promoted degranulation drug using microplate reader detection or different optical maser wavelengths act on lower mast cell β-hexosaminidase and release
Rate is put for embodying mast cell degranulation degree；The journey of degranulation occurs as mast cell for β-hexosaminidase release rate
Scale will, for examining SERS spectra technology to detect degranulation component；The different reflection degranulation components of SERS spectra characteristic peak
Middle different biological substance and content；β-hexosaminidase release rate calculates gained according to following formula:
。
Step (1) the different pharmaceutical stimulation includes nonspecific stimulation object and differential stimulus object；The non-specificity
Stimulant includes cell degranulation drug C48/80, calcium ion carrier A 23187, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 or wasp toxin；The specificity thorn
Sharp object includes bacterium, virus and toxin；The concentration range is 0.5-100 μ g/mL or 1-500 μM.
Step (1) the different optical maser wavelength irradiation are specially the laser irradiation 1-30min using 340-850nm wavelength,
Irradiation power is 1-20mW；The laser is semiconductor laser sending.
Step (2) the SERS enhancing substrate concentrating colloidal solution is fulmargin or gold size solution.
The fulmargin specially weighs 0.01-1g solid nitric acid silver using hydroxylamine hydrochloride restoring method preparation gained
It is completely dissolved in ultrapure water, separately taking sodium hydroxide solution that 1-10mL concentration is 0.01-1M and 1-9mL concentration is 0.01-10M's
Hydroxylamine hydrochloride mixed solution rapidly joins silver nitrate solution, until stirring to solution is uniform newborn grey；Then pass through height
Fast centrifuge centrifugation obtains, and centrifugal rotational speed and time are respectively 6000-15000rpm and 5-20min.
The gold size solution uses 1-20mL concentration for 0.1-10mg/mL gold chloride is soluble in water and the lower heating of stirring strongly
To boiling, the rear 0.1-10mL 0.1-10wt% sodium citrate that is added is reacted, and is kept boiling 5-30min, is become to solution colour
For natural cooling after vinicolor；It is then centrifuged and is obtained by supercentrifuge, centrifugal rotational speed and time are respectively 6000-
15000rpm and 5-20min.
The base material of step (2) SERS detection be 99.99% aluminium flake, quartz, stainless steel, smooth goldleaf, silver foil,
MgF2Or CaF2One of.
The laser power of step (2) SERS spectra detection is 0.2-20mW, laser excitation wavelength 400-850nm,
Chosen spectrum wave-number range is 400-3000 cm-1。
The present invention has the advantages that
1. the present invention, when obtaining the cell secreta of mast cell in vitro culture, step is simple, time-consuming short, and cost is relatively low.
Cell and cell secreta can be used to test simultaneously detection, increase it using degree, and cell secreta SERS spectra is believed
Breath can reflect mast cell degranulation situation.
2. the present invention is detected in the different degrees of degranulation of detection mast cell, with traditional detection methodAmino oneself
Glycosidase is compared, and has consistent conclusion, but the method for the present invention has more efficient and convenient and fast detection characteristic.
3. the present invention detects mast cell degranulation, detection time using the SERS technology that Nano silver grain makees enhancing substrate
It is short, signal is strong, has comparative well, and detect without handling cell itself.Therefore, the present invention have quickly,
Lossless and highly sensitive detection advantage.
4. the present invention detects mast cell degranulation in conjunction with laser irradiation, help further to study the molecular events with
The mechanism of action of analgesic effect, points acupuncture and laser therapy etc. can provide a kind of valuable reference frame for clinical diagnosis and treatment.
5. method of the invention herein in connection with statistics and biochemical method, further studies different mast cell lines
Degranulation situation has certain research significance and application prospect.
Detailed description of the invention
Fig. 1 is that the present invention is thin using the mouse hypertrophy cell oncocyte P815 measured under the effect of different pharmaceutical concentration C 48/80
β-hexosaminidase release rate in born of the same parents' degranulation.
Fig. 2 is that the mouse under C48/80 the and 0.1%Triton X-100 effect for the various concentration that the present invention measures is loose thin
Born of the same parents' oncocyte P815 cell degranulation secretion SERS spectra.Wherein, C48/80 concentration be respectively 0,2,10,20,40 μ g/mL with
And 0.1% Triton X-100.
Fig. 3 is that five kinds of various concentration drug C48/80 that the present invention measures act on lower cell degranulation secretion SERS spectra
The principal component two dimension scatter plot of PCA-LDA.
Fig. 4 is 633nm laser irradiation 5min, the power 10mW that the present invention measures, small after continuing culture 1h, 2h and 3h
The SERS spectra of mouse tumor mast cell P815 culture solution and respective standard deviation.
Fig. 5 is the SERS difference spectrogram of any two groups of various durations of tri- groups of SERS spectras of Fig. 3.
Fig. 6 is that the present invention uses wavelength for 633nm, laser power 10mW, and irradiation is respectively 0,2,5,10 and 15min's
The SERS spectra figure of mouse tumor mast cell P815 cell degranulation secretion.
Fig. 7 is to be secreted in Fig. 6 using the mouse tumor mast cell P815 cell degranulation of 633nm laser irradiation different time
The two-dimentional scatter plot of the PCA-LDA of object SERS spectra；Wherein figure a, b, c and d be respectively irradiation time be 2,5,10 and 15min with
The cell degranulation secretion SERS two dimension scatter plot of no laser irradiation compares.
Fig. 8 is that the present invention uses power for 10mW, and wavelength is that the laser of 633nm first irradiates application on human skin mast cell HMC-1
5min, then 0h, 12h and cell degranulation secretion SERS spectra measured for 24 hours are persistently cultivated respectively.
Fig. 9 is the cell in Fig. 8 persistently to cultivate different time after 633nm laser irradiation application on human skin mast cell HMC-1
The three-dimensional scatter plot of the PCA-LDA of degranulation secretion SERS spectra.
Figure 10 is the present invention be two kinds of 10mW different optical maser wavelength i.e. 633 and 785nm irradiation 5min using power after
Resulting SERS spectra figure.
Figure 11 is two kinds of different optical maser wavelengths 633 and the 785nm irradiation application on human skin mast cell HMC-1 5min institute of Figure 10
The three-dimensional scatter plot of the PCA-LDA of the SERS spectra of the cell degranulation secretion obtained.
Specific embodiment
Embodiment 1
The degranulation secretion SERS spectra of 48/80 inducing mouse mast cell oncocyte P815 of different pharmaceutical concentration C detects.
(1) degranulation occurs for Cultured Mouse mast cell oncocyte P815 and its induction of different pharmaceutical concentration C 48/80
In vitro culture mast cell P815, every two days one second generations of biography pass on ratio 1:3.When P815 being taken to be in logarithmic growth phase, from
The heart and discarding culture solution, with fresh medium, (final concentration is added in 90%v/v DMEM high glucose medium, 10%v/v fetal calf serum
The penicillin and streptomysin of 100 μ g/mL) it is resuspended, with 106A/mL, by 25%(v/v) inoculum concentration is inoculated in 24 orifice plates, it cultivates
24h.Centrifugation and gurry are taken out, the PBS resuspension of addition pH 7.4,6.7mM, setting blank group (only PBS), control group (are not added
Medicine), dosing group (C48/80 drug concentration is respectively 2 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 40 μ g/mL) and total enzyme
Group (0.1% Triton X-100 of addition, i.e. membranolysis release intracellular all β-hexosaminidases), every group sets
Three multiple holes are set, respectively packet transaction, continues culture and be incubated for 1h.Later, cell secreta is collected respectively in centrifuge tube.
(2) mouse hypertrophy cell oncocyte P815 degranulation degree detecting
β-hexosaminidase substrate is incubated for 60min with the cell secreta being collected into, adds 150 μ L reaction terminating liquids to terminate anti-
It answers, measures each group light absorption value, absorbing wavelength 405nm using microplate reader.β-hexosaminidase release rate formula is as follows:
。
Cellular component after taking mast cell P815 drug-treated is mixed with SERS enhancing matrix fulmargin in equal volume respectively
It closes, is added dropwise and is detected after 99.99% smooth aluminium flake is dry for SERS.Raman spectrometer laser power is 10mW, integral
Time 20s is integrated 2 times, and spectral region is 400-1800 cm-1.The P815 cell degranulation degree and SERS spectra detected point
Not as illustrated in fig. 1 and 2, Fig. 2 ordinate indicates that the intensity at surface increasing Raman spectrum peak, unit are arbitrary unit (a.u.), horizontal seat
Mark indicates the peak position of each feature spectral peak, and unit is with wave number cm-1It indicates.
It is sufficiently molten specially to weigh 1g solid nitric acid silver using hydroxylamine hydrochloride restoring method preparation gained for the fulmargin
Solution separately takes the sodium hydroxide solution that 10mL concentration is 1M and the hydroxylamine hydrochloride that 9mL concentration is 10M to be uniformly mixed molten in ultrapure water
Liquid rapidly joins silver nitrate solution, until stirring to solution is uniform newborn grey；It is then centrifuged and is obtained by supercentrifuge,
Centrifugal rotational speed and time are respectively 15000rpm and 20min.
(3) it statisticallys analyze
β-hexosaminidase release is calculated as mast cell P815 degranulation feelings after embodiment different pharmaceutical processing by formula
Condition, as shown in Figure 1.The a plurality of mouse hypertrophy cell oncocyte P815 degranulation component SERS spectra data of acquisition are deducted
Fluorescence background and normalization, it is as shown in Figure 2 to obtain averaged spectrum.PCA-LDA analysis is carried out using SPSS software, obtains each master
The score and contribution rate of ingredient more can intuitively find out two principal component two dimension scatterplots of significant difference after different pharmaceutical processing
Figure is as shown in figure 3, Χ axis and Y-axis are respectively score value corresponding to each autonomic elements, to realize fertilizer after different pharmaceutical processing
The degranulation degree difference of maxicell P815 differentiates.
Embodiment 2
The detection of degranulation SERS spectra occurs for P815 cell under various laser irradiation time effect
(1) extraction of laser irradiation mast cell P815 component
When the mouse tumor mast cell P815 of in vitro culture is in logarithmic growth phase, 800rpm is centrifuged 5min, abandons supernatant, and use is fresh
(penicillin and strepto- of 100 μ g/mL of final concentration is added in 90%v/v DMEM high glucose medium, 10%v/v fetal calf serum to culture solution
Element) it is resuspended, with 106A/mL is inoculated in 24 orifice plates, every hole 1mL, continues culture for 24 hours.Supernatant is abandoned in centrifugation, adds fresh PBS
(pH 7.4,6.7mM), then be centrifuged once, it abandons supernatant addition PBS and is resuspended, progress 633nm laser irradiation laser power is 10mW,
Irradiation time is 5min, then continues to cultivate 1h, 2h and 3h respectively.800rpm is centrifuged 5min, extracts supernatant, deposits in centrifuge tube
In.In addition, using power be 10mW, wavelength be 633nm laser, irradiate mast cell time be respectively set as 2min,
5min, 10min, 12min and 15min, and without laser irradiation control group, there are also total enzyme groups of 0.1% Triton X-100.No
It puts back to incubator with the cell after laser irradiation to continue to cultivate 3h, supernatant is extracted in centrifugation later, deposits in centrifuge tube.
(2) mouse hypertrophy cell oncocyte P815 degranulation degree detecting and SERS spectra detection
Same irradiation time (5min) different time gathered is persistently cultivated and the groups of cells of different irradiation time culture 3h
Divide and mixed respectively with isometric fulmargin, is added dropwise and is detected after 99.99% smooth aluminium flake is dry for SERS, Raman
Spectrometer laser power is 5mW, time of integration 10s, is integrated 2 times, spectral region 400-1800cm-1.Respectively such as Fig. 4 and Fig. 6
Shown, Fig. 5 is the spectrogram poor two-by-two of same irradiation time difference incubation time, and wherein ordinate indicates surface increasing Raman spectrum peak
Intensity, unit is arbitrary unit (a.u.), and abscissa indicates the peak position of each feature spectral peak, and unit is with wave number cm-1It indicates.No
β-hexosaminidase is measured with the cellular component of irradiation time culture 3h and 0.1% Triton the X-100 total enzyme group handled
Release rate, characterizes mast cell degranulation situation, and method is same as Example 1.
It is sufficiently molten specially to weigh 1g solid nitric acid silver using hydroxylamine hydrochloride restoring method preparation gained for the fulmargin
Solution separately takes the sodium hydroxide solution that 10mL concentration is 1M and the hydroxylamine hydrochloride that 9mL concentration is 10M to be uniformly mixed molten in ultrapure water
Liquid rapidly joins silver nitrate solution, until stirring to solution is uniform newborn grey；It is then centrifuged and is obtained by supercentrifuge,
Centrifugal rotational speed and time are respectively 15000rpm and 20min.(3) it statisticallys analyze
It carries out scratching fluorescence to the mast cell degranulation component SERS spectra data that 3h is obtained are cultivated under the various laser irradiation time
Background and normalized carry out PCA-LDA analysis using SPSS software, obtain the score and contribution rate of each principal component, can
With more intuitively find out significant difference two principal component two dimension scatter plots as shown in fig. 7, Χ axis and Y-axis be respectively it is each independently at
Score value corresponding to point, to realize that the degranulation degree difference of mast cell P815 under various laser irradiation differentiates.
Embodiment 3
Laser irradiation acts on the detection of human skin's mast cell HMC-1 degranulation SERS spectra
(1) extraction of application on human skin mast cell HMC-1 degranulation component
Vitro culture of human Cutaneous mast cell HMC-1, the RPMI culture of cell culture fluid Hyclone containing 89%v/v company production
Dual anti-and 10%v/v the fetal calf serum of base, 1%v/v, passage in every two days is primary, and cell concentration is controlled 105-106A/mL.It takes
Number growth period cell 800rpm is centrifuged 5min, is resuspended with fresh medium, with every hole 106A/mL is inoculated in 24 orifice plates, and every hole is total
Volume is 1mL, 3 multiple holes of every group of setting.It the use of optical maser wavelength is 633nm, power is that 10mW carries out irradiation 5min, is then proceeded to
Cultivate 0h, 12h and for 24 hours.It takes out and 800rpm is centrifuged 5min, collect supernatant in centrifuge tube.
(2) application on human skin mast cell HMC-1 degranulation SERS spectra detects
It takes the supernatant for continuing different time culture after 633nm laser irradiation to mix in equal volume with fulmargin, is coated in
On 99.99% aluminium flake, detected after natural drying for SERS spectra.Being copolymerized burnt micro Raman spectra excitation wavelength is that 785nm is partly led
Body laser uses silicon wafer 520cm-1The calibration of eigen vibration peak.Wave-length coverage is 400-1800cm-1, object lens be 20 ×, integral
Time 30s, integral number of times 2 times, 10 mW of laser power.The SERS spectra detected is sat as shown in figure 8, wherein indulging after processing
Mark indicates that the intensity at surface increasing Raman spectrum peak, unit are arbitrary unit (a.u.), and abscissa indicates the peak position of each feature spectral peak
It sets, unit is with wave number cm-1It indicates.
The fulmargin specially weighs 0.5g solid nitric acid silver sufficiently using hydroxylamine hydrochloride restoring method preparation gained
It is dissolved in ultrapure water, separately takes the sodium hydroxide solution that 5mL concentration is 0.5M and the hydroxylamine hydrochloride that 5mL concentration is 5M to be uniformly mixed molten
Liquid rapidly joins silver nitrate solution, until stirring to solution is uniform newborn grey；It is then centrifuged and is obtained by supercentrifuge,
Centrifugal rotational speed and time are respectively 12000rpm and 15min.
(3) it statisticallys analyze
Five rank multinomials fitting background correction fluorescence signal is carried out to original SERS spectra background to the SERS spectra data of acquisition
Correction, while in order to reduce system bring error, using Labview software will deduct the SERS spectra of fluorescence background into
Row spectrum area normalization, as shown in Figure 8.Meanwhile dimensionality reduction is carried out to SERS spectra data, extract principal component combination linear discriminant
Analysis, obtains the score and contribution rate of each principal component using SPSS software, and three principal component three-dimensionals for drawing significant difference dissipate
Point diagram, as shown in figure 9, to realize that laser irradiation application on human skin mast cell HMC-1 difference continues degranulation journey under incubation time
Difference is spent to differentiate.Wherein X-axis, Y-axis and Z axis are respectively score value corresponding to each autonomic elements, and percentage value is corresponding PC value pair
The contribution rate of corresponding cell degranulation secretion SERS spectra.
Embodiment 4
Different optical maser wavelength effect human skin's mast cell HMC-1 degranulation SERS spectra detections
(1) extraction of application on human skin mast cell HMC-1 degranulation component
When vitro culture of human Cutaneous mast cell HMC-1 is in logarithm division stage, 800rpm is centrifuged 5min, uses fresh medium
(the RPMI culture medium of 89%v/v, the fetal calf serum of 10%v/v and 1%v/v's is dual anti-) is resuspended, with every hole 106A/mL is inoculated in
24 orifice plates, every hole total volume are 1mL, 3 multiple holes of every group of setting.It the use of optical maser wavelength is respectively 633 and 785nm, power is
10mW carries out irradiation 5min, then proceedes to culture for 24 hours.It takes out and 800rpm is centrifuged 5min, collect supernatant in centrifuge tube.
(2) application on human skin mast cell HMC-1 degranulation SERS spectra detects
The supernatant of collection is mixed in equal volume with fulmargin, is coated on 99.99% aluminium flake, carries out SERS after natural drying
Spectral detection, being copolymerized burnt micro Raman spectra excitation wavelength is 785nm semiconductor laser, uses silicon wafer 520cm-1Eigen vibration
Peak calibration.Wave-length coverage is 400-1800cm-1, object lens be 20 ×, time of integration 30s, integral number of times 2 times, laser power 10
mW.The SERS spectra and its difference spectrum for obtaining two kinds of different optical maser wavelength effect human skin's mast cell HMC-1 degranulations are as schemed
Shown in 10, wherein ordinate indicates that the intensity at surface increasing Raman spectrum peak, unit are arbitrary unit (a.u.), and abscissa indicates each
The peak position of feature spectral peak, unit is with wave number cm-1It indicates.
The fulmargin specially weighs 0.5g solid nitric acid silver sufficiently using hydroxylamine hydrochloride restoring method preparation gained
It is dissolved in ultrapure water, separately takes the sodium hydroxide solution that 5mL concentration is 0.5M and the hydroxylamine hydrochloride that 5mL concentration is 5M to be uniformly mixed molten
Liquid rapidly joins silver nitrate solution, until stirring to solution is uniform newborn grey；It is then centrifuged and is obtained by supercentrifuge,
Centrifugal rotational speed and time are respectively 12000rpm and 15min.
(3) it statisticallys analyze
Five rank multinomials are carried out to the multiple groups SERS spectra data of acquisition and are fitted background correction fluorescence, then carry out area normalization
It obtains as shown in Figure 10.Meanwhile taking accounting weight using SPSS software is more than three principal components drafting three of 80% and significant difference
Scatter plot is tieed up, as shown in figure 11, X-axis, Y-axis and Z axis are respectively score value corresponding to each autonomic elements, so as to realize two
Degranulation degree difference differentiates under the different optical maser wavelengths irradiation application on human skin mast cell HMC-1 of kind.
The process that the present invention implements example is only limitted to illustrate technical solution of the present invention, and enlightenment according to the present invention can also
Degranulation event is occurred to other mast cell lines and other promote the degranulation inspection under degranulation drug and optical maser wavelength effect
It surveys, processing and differentiation differentiate.So all those skilled in the art are because of technical inspiration involved in the present invention, and use and equally replace
It changes or technical solution that equivalent deformation mode is formed is fallen within the scope of protection of the present invention.
Claims (8)
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| CN112362637A (en) * | 2020-12-07 | 2021-02-12 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface enhanced Raman technology |
| US11358984B2 (en) | 2018-08-27 | 2022-06-14 | Regeneran Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11358984B2 (en) | 2018-08-27 | 2022-06-14 | Regeneran Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
| US12398176B2 (en) | 2018-08-27 | 2025-08-26 | Regeneron Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
| CN112362637A (en) * | 2020-12-07 | 2021-02-12 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface enhanced Raman technology |
| CN112362637B (en) * | 2020-12-07 | 2023-05-23 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface-enhanced Raman technology |
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