CN109060729B - Transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance - Google Patents
Transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
Abstract
The present invention relates to a kind of transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance, can be used for carrying out quick, unmarked quantitative detection to the cell of transwell test room lower layer.The detection device includes: terahertz signal generating unit, including femtosecond laser light source, laser beam splitter, hemispherical silicon lens I and photoconductive antenna I；Total reflection prism detection unit from top to bottom includes the cell transwell and total reflection prism, and the bottom of the cell transwell is equipped with permeability porous membrane, and monolayer is adhered in the lower section of porous membrane, and monolayer and total reflection prism are in close contact；Terahertz signal receiving unit, including photoconductive antenna II；Time delay control system, including time delay device；Signal processing unit.
Description
Technical field
The invention belongs to Terahertz detection technique field, it is related to the transwell based on Terahertz Technique of Attenuated Total Reflectance
Detection device and method.
Background technique
Cell and tissue culture technology is the pith in basic life science field.It is supported using filter membrane as cell culture
Medium derives from Grobstein (Natrue, 172:860-872 earliest；1953) correlative study about the induction of rear kidney filter membrane.It is aobvious
So, firm Tissue Culture Dish and culture bottle are compared, permeability support interface is conducive to cell and secretes from its basal surface and top surface
It, can be more natural, metabolic closer to being carried out under concrete conditions in the establishment of a specific crime with absorption molecule.Thus, with permeability film substrate
Cup-formed device, the i.e. cell transwell (transwell chamber), in interaction, migration and the invasion of research cell
There is irreplaceable important function in terms of ability.
Transwell experimental result interpretation depends on pair especially when assessing cell migration, invasive ability
The counting of confluent monolayer cells under the cell transwell, to obtain the data such as cell migration rate.Lower ventricular cell method of counting is main at present
It is divided into direct counting method and indirect counting method.Direct counting method is by removal matrigel and upper chamber cellular layer, using such as crystallizing
Purple, trypan blue, hematoxylin etc. dye lower room attached cell, then randomly select several visuals field using microscope and are counted
Number；According to unit visual field cell distribution situation and overall inoculating cell quantity, the mobility of cell is calculated.This method it is more according to
Rely the determining, analysis of accounts in the selection of researcher's subjectivity, has larger bias, it is difficult to total evaluation cell distribution density.It counts indirectly
Rule reads the correspondence absorbance value of cell pyrolysis liquid, assesses the migration situation of cell by dyeing lower ventricular cell.This method
It is relatively complicated, it is difficult to effectively to meet the needs of unmarked, in situ detection to there is label to detect.
Terahertz (Terahertz, THz) wave is electricity of the frequency in 0.1~10THz (corresponding wavelength range is 30 μm of -3mm)
Magnetic wave, in macroelectronics to the wave band of microcosmic photonics transition, has unmarked inspection between microwave and infrared band
The physical features and unique fingerprint map advantage of survey.Meanwhile time with higher and spatial resolution, photon energy are smaller
(photon energy is about 4meV at 1THz) will not generate destruction to measured matter.However, the Terahertz wave spectrum of living cells is examined
Survey suffers from liquid phase environment and absorbs interference and Terahertz Detection wavelength and the matched restriction of monolayer thickness mistake, Wu Fazhun by force
Really carry out assessment qualitatively and quantitatively.Specific manifestation are as follows: the terahertz time-domain spectroscopy system based on transmission mode is limited to cultivate
The strong absorption of based sols can not accurately obtain in liquid solution monolayer signal in the cell Transwell, and it is even more impossible to realize
Assessment to cell density distribution.Meta Materials based on resonant ring enhance technology, need that cell growth is made to be adhered to resonant ring table
Face, and wipe the extracellular water of cell away and detected, it is unable to satisfy the high-throughput detection demand of living cells.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of transwell based on Terahertz Technique of Attenuated Total Reflectance
Detection device and method can be used for carrying out quick, unmarked quantitative detection to the cell of transwell test room lower layer.
In order to achieve the above objectives, the invention provides the following technical scheme:
Transwell cell experiment detection device based on Terahertz Technique of Attenuated Total Reflectance, comprising:
Terahertz signal generating unit, including femtosecond laser light source, laser beam splitter, hemispherical silicon lens I and photoconduction
Antenna I, femtosecond laser light source emit laser and are divided into pump beam and detection light beam, pump beam warp under laser beam splitter effect
Cross the focusing of hemispherical silicon lens I, collimation can excite photoconductive antenna I to emit THz wave to total reflection prism detection unit；
Total reflection prism detection unit, from top to bottom includes the cell transwell and total reflection prism, and transwell is small
The bottom of room is equipped with permeability porous membrane, and monolayer is adhered in the lower section of porous membrane, and monolayer is tight with total reflection prism
Contiguity touching, the evanescent wave that the vertical direction distribution that total reflection generates occurs for same total reflection prism surface sufficiently interact,
Modulate reflected terahertz hereby wave；
Terahertz signal receiving unit, including photoconductive antenna II, for receiving the reflection of total reflection prism detection unit
Carry the THz wave of adherent monolayer information；
Time delay control system, including time delay device are set between laser beam splitter and photoconductive antenna I,
THz wave is generated and emitted for controlling terahertz signal generating unit；
Signal processing unit, including computer, for acquiring and analyzing the Terahertz of terahertz signal receiving unit acquisition
Wave completes the detection of transwell cell experiment.
The detection frequency range of above-mentioned detection device is in 0.1~4THz, and circuit ambient humidity is below 3%.
One of as a preferred technical scheme, the femtosecond laser light source is titanium-doped sapphire femto-second laser.
One of as a preferred technical scheme, the terahertz signal receiving unit further includes hemispherical silicon lens II, is used
In collimation and focused probe beam, the received THz wave for carrying monolayer information of excitation photoconductive antenna II.
One of as a preferred technical scheme, the cell transwell selects Terahertz to absorb the poly- carbonic acid of lower 8 μm of micropores
Ester film, polytetrafluoroethylene film or polyester film, further preferably polycarbonate membrane.
One of as a preferred technical scheme, surface emitting Terahertz of the terahertz signal generating unit to total reflection prism
Wave.
One of as a preferred technical scheme, controllable pressure fixture is equipped in the top of the cell transwell.
As further preferred one of technical solution, the controllable pressure fixture is spring controllable pressure fixture.
As further preferred one of technical solution, the controllable pressure fixture is fixed by fixture mount, is totally reflected rib
Mirror is fixed by prism fixed frame, and the material requirement of fixture mount and prism fixed frame: hardness is high, and corrosion-resistant, deformation is small, preferably
Stainless steel material.
As one of technical solution still more preferably, the two sides of prism fixed frame are equipped with circular hole, for THz wave
Normal incidence and outgoing.
As further preferred one of technical solution, the bottom of the controllable pressure fixture is equipped with detachable round pressure
Plate, and contacts at the top of the cell transwell.
One of as a preferred technical scheme, the terahertz signal generating unit is Terahertz generator, Terahertz letter
Number receiving unit is Terahertz receiver.
One of as a preferred technical scheme, the material of the total reflection prism should be chosen in terahertz wave band high saturating
It crosses, high refraction, further preferably silicon or germanium, is still more preferably silicon.
One of as a preferred technical scheme, the signal processing unit uses Picometrix time domain Terahertz system T-
Ray 5000 included signal processing unit.
The transwell cell experiment detection side based on Terahertz Technique of Attenuated Total Reflectance realized using above-mentioned apparatus
Method, the specific steps are as follows:
1) cell transwell of bottom attachment monolayer is placed on total reflection prism, so that monolayer and complete
The intimate surface contact of reflecting prism；
2) terahertz signal generating unit emits THz wave to total reflection prism detection unit；
3) THz wave occurs to be totally reflected and formed to divide vertically upward along total reflection prism surface on total reflection prism surface
The evanescent wave of cloth interacts with the monolayer fitted closely, modulates reflected terahertz hereby wave；
4) hereby wave, time delay control system adjust THz wave to terahertz signal receiving unit reception reflected terahertz
With detection optical path, the terahertz signal of signal processing unit acquisition and analysis monolayer completes the inspection of transwell cell experiment
It surveys.
One of as a preferred technical scheme, in step 1), the cell transwell that monolayer is adhered in bottom is to pass through
What following methods obtained: the bottom of the cell transwell is equipped with permeability porous membrane, using porous membrane as boundary, transwell
Cell is divided into the upper and lower, and matrigel is added in upper layer and cell suspension, lower layer are filled with cell culture medium, and porous membrane soaks completely
Enter in cell culture medium, after completing cell culture, take out the cell transwell, wipes the matrigel on upper layer and cell and clean,
Draw the moisture of lower layer.
As one of technical solution still more preferably, the specific method is as follows: matrigel, aseptic condition is added dropwise to upper layer
It is lower to stay overnight, it is ensured that matrigel polymerize completely, and cell suspension is added, is filled with cell culture medium to lower layer, takes out after cell culture
The cell transwell, after wiping matrigel and the cell on upper layer with cotton swab head, PBS buffer solution is cleaned 3 times, uses aseptic filter paper
The moisture of gentle aspiration lower layer.
One of as a preferred technical scheme, in step 1), applies pressure using controllable pressure fixture and make monolayer
With the intimate surface contact of total reflection prism.
One of as a preferred technical scheme, in step 4), it can be obtained in prism surface by formula (1) by incident Terahertz
Wave generates the skin depth of evanescent wave when being totally reflected and cellular layer thickness matches, therefore being emitted THz wave can completely take
Response signal with the adherent dielectric material of prism surface；
Wherein, dpFor the skin depth of evanescent wave, λ is the wavelength of incident THz wave, nSiFor the refraction of total reflection prism
Rate, θ are incidence angle of the THz wave in prism surface, nemFor the refraction of prism surface sample to be examined (i.e. adherent monolayer)
Rate；
Simplify dielectric model according to the binary of EFFECTIVE MEDIUM THEORY, such as formula (2), as acquired in outgoing terahertz signal
The equivalent complex dielectric permittivity ε of substance at prism boundaryeffIt is related with the spatial volume score of culture medium and cell, that is, determining εmedium
And εcellIn the case of, it can be by measuring εeffSignal, obtain the cell transwell lower layer cell density distribution situation, into
And realize the markless detection to cell quantity；
Wherein, εeffFor the equivalent complex dielectric permittivity of substance at prism boundary acquired in outgoing terahertz signal, εcellIt is thin
The complex dielectric permittivity of born of the same parents, VcellFor the cellular layer volume of detection zone, εmediumFor culture medium complex dielectric permittivity, VmediumFor detection
The volume of region culture medium.
The beneficial effects of the present invention are:
THz wave Technique of Attenuated Total Reflectance provides secure support for living cells markless detection, is existed using THz wave
The evanescent wave generated when being totally reflected inside prism obtains the Terahertz wave spectrum letter of substance within the scope of prism surface some tens of pm
Breath；Evanescent wave penetration depth matches with cellular layer thickness, effectively excludes the signal interference of liquid phase environment, obtains monolayer
THz wave spectrum signature, to be applied to the distinctive subpicosecond scale aquation kinetic parameter of research cell, after birth infiltration sexually revises
Response and the apoptosis-induced process of oxidative stress.Correlated results shows that Terahertz decaying total reflection platform can effectively distinguish culture medium
With the complex dielectric permittivity feature of monolayer, the especially imaginary part of complex dielectric permittivity；It can be to monolayer water content and existence shape
State generates sensitive response.Thus, the cell transwell film lower part cell distribution density can be reduced to have different multiple dielectrics normal
The cell and culture medium binary mixture of number feature, and then the total reflection platform that can be decayed by Terahertz realizes accurate evaluation point
Analysis.
The present invention establishes the Terahertz decaying total reflection platform of integrated spring controllable pressure fixture, is applied to for the first time
The quantitative detecting analysis of transwell experimental thin films lower layer cell distribution density, it is innovative that cellular layer is grown on Terahertz suction
On the lower permeability film of yield, to solve existing Terahertz cell detection needs, in sensing interface, (Meta Materials surface is declined
Subtract total reflection prism surface) growth monolayer face cultivate take long time, detect the single problem of sample, high pass can be met
Measure the demand of Terahertz cell analysis.The apparatus structure is simple, is easy to integrate, and it is convenient to operate, and detection is quick.
Transwell cellular invasiveness or transfer ability assessment are basilar memebrane (permeability of the observation cell by simulation
Support film) distribution of movement situation, be different from regular growth suspension growth and adherent cell growth, and compare it is existing count point
Analysis method has following advantage:
1, it is not necessarily to any dye marker, decaying total reflection platform evanescent wave skin depth matches with cellular layer thickness, and
Cellular layer and culture medium have apparent Terahertz absorption spectrum difference；
2, quickly detection, single sample, which obtains, stablized tera-hertz spectra time-consuming within 1 minute, much smaller than direct or indirect
Dyeing time needed for sizing technique；
3, living cells in-situ investigation, cell need not move through dehydration and fix, detected under existing state, after can facilitating
Continuous collect carries out other experiments.
4, the terahertz time-domain spectroscopy system under transmission mode, it is difficult in the case that culture medium solution there are realize to slender
The signal of born of the same parents' layer.Terahertz Technique of Attenuated Total Reflectance is the reliable method of current cellular layer detection, using THz wave in rib
Generated when mirror interface is totally reflected evanescent wave that skin depth exponentially decays within the scope of some tens of pm and vertically upward into
The detection of row adherent layer can exclude the interference of cellular layer top culture medium absorption, obtain monolayer complex dielectric permittivity
Information.
5, the present invention for existing Terahertz cell detection monolayer need to be grown on sensing interface (Meta Materials surface or
Decaying total reflection prism surface) when face culture and take long time, detect the single problem of sample, it is innovative to grow cellular layer
In on the lower permeability film of Terahertz absorptivity, by high-volume cell culture, high-throughput Terahertz cell point can be met
The demand of analysis.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 is the Terahertz decaying total reflection platform schematic diagram of integrated spring controllable pressure fixture；
Fig. 2 is the detection principle diagram that transwell cell count is carried out based on Terahertz Technique of Attenuated Total Reflectance；
Fig. 3 is the terahertz signal value of confluent monolayer cells under the cell transwell after different pharmaceutical concentration is handled；
Fig. 4 is the normalization terahertz signal value of confluent monolayer cells under cell after different pharmaceutical concentration is handled 1.0THz at；
Fig. 5 is Terahertz decaying total reflection signal intensity value and MTT experiment absorbance value variation tendency fitted figure；
Wherein, 1 is controllable pressure fixture, and 2 be fixture mount, and 3 be detachable circular pressing plate, and 4 be total reflection prism, 5
It is the cell transwell for prism fixed frame, 6,7 be monolayer, and 8 be porous membrane.
Dotted line frame on the left of Fig. 2 indicates that cell culture medium, solid arrow indicate that incident THz wave, dotted arrow represent
THz wave is penetrated, right side waveform indicates evanescent wave.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Embodiment:
As depicted in figs. 1 and 2, the transwell cell experiment detection device based on Terahertz Technique of Attenuated Total Reflectance, packet
It includes:
Terahertz signal generating unit, including femtosecond laser light source, laser beam splitter, hemispherical silicon lens I and photoconduction
Antenna I, femtosecond laser light source emit laser and are divided into pump beam and detection light beam, pump beam warp under laser beam splitter effect
Cross the focusing of hemispherical silicon lens I, collimation can excite photoconductive antenna I to emit THz wave to total reflection prism detection unit；
Total reflection prism detection unit from top to bottom includes the cell transwell 6 and total reflection prism 4, transwell
The bottom of cell 6 is equipped with permeability porous membrane 8, and monolayer 7, monolayer 7 and total reflection are adhered in the lower section of porous membrane 8
Prism 4 is in close contact, and the abundant phase of evanescent wave for the vertical direction distribution that total reflection generates occurs for same 4 surface of total reflection prism
Reflected terahertz hereby wave is modulated in interaction；
Terahertz signal receiving unit, including photoconductive antenna II, for receiving the reflection of total reflection prism detection unit
Carry the THz wave of adherent monolayer information；
Time delay control system, including time delay device are set between laser beam splitter and photoconductive antenna I,
THz wave is generated and emitted for controlling terahertz signal generating unit；
Signal processing unit, including computer, for acquiring and analyzing the Terahertz of terahertz signal receiving unit acquisition
Wave completes the detection of transwell cell experiment.
The detection frequency range of above-mentioned detection device is in 0.1~4THz, and circuit ambient humidity is below 3%.
Femtosecond laser light source in terahertz signal generating unit is titanium-doped sapphire femto-second laser.
Terahertz signal receiving unit further includes hemispherical silicon lens II, for collimation and focused probe beam, exciting light
The received THz wave for carrying monolayer information of conductance antenna II.
The cell transwell 6 selects Terahertz to absorb lower 8 μm of microporous polycarbonate films.
Surface emitting THz wave of the terahertz signal generating unit to total reflection prism 4.
Controllable pressure fixture is equipped in the top of the cell transwell 6.
Controllable pressure fixture 1 is spring controllable pressure fixture.
Controllable pressure fixture 1 is fixed by fixture mount 2, and total reflection prism 4 is fixed by prism fixed frame 5,2 He of fixture mount
Prism fixed frame 5 is stainless steel material.
The two sides of prism fixed frame 5 are equipped with circular hole, for THz wave normal incidence and outgoing.
The bottom of controllable pressure fixture 1 is equipped with detachable circular pressing plate 3, connects with the top of the cell transwell 6
Touching.
Terahertz signal generating unit is Terahertz generator, and terahertz signal receiving unit is Terahertz receiver.
The material of total reflection prism 4 is silicon.
The transwell cell experiment detection side based on Terahertz Technique of Attenuated Total Reflectance realized using above-mentioned apparatus
Method, the specific steps are as follows:
1) cell transwell 6 of bottom attachment monolayer 7 is placed on total reflection prism 4, so that monolayer 7
With the intimate surface contact of total reflection prism 4；
2) terahertz signal generating unit emits THz wave to total reflection prism detection unit；
3) THz wave occurs to be totally reflected and formed along 4 surface of total reflection prism vertically upward on 4 surface of total reflection prism
The evanescent wave of distribution interacts with the monolayer 7 fitted closely, modulates reflected terahertz hereby wave；
4) hereby wave, time delay control system adjust THz wave to terahertz signal receiving unit reception reflected terahertz
With detection optical path, the terahertz signal of signal processing unit acquisition and analysis monolayer 7 completes transwell cell experiment
Detection.
In step 1), the cell transwell 6 of bottom attachment monolayer 7 obtains by the following method:
The bottom of the cell transwell 6 is equipped with permeability porous membrane 8, is boundary with porous membrane 8, it is upper that the cell transwell 6, which divides,
Matrigel and cell suspension is added in layer and lower layer, upper layer, and lower layer is filled with cell culture medium, and porous membrane 8 is completely immersed in cell training
It supports in base, after completing cell culture, takes out the cell transwell 6, wipe the matrigel on upper layer and cell and clean, under absorption
The moisture of layer.
The specific method is as follows: matrigel is added dropwise to upper layer, stays overnight under aseptic condition, it is ensured that matrigel polymerize completely, is added
Cell suspension is filled with cell culture medium to lower layer, and the cell transwell is taken out after cell culture, wipes matrix with cotton swab head
After glue and the cell on upper layer, PBS buffer solution is cleaned 3 times, with the moisture of aseptic filter paper gentle aspiration lower layer.
In step 1), applies pressure using controllable pressure fixture 1 and make monolayer 7 and the surface of total reflection prism 4 tight
Contiguity touching.
In step 4), it can be obtained by formula (1) and be generated suddenly by incident THz wave when being totally reflected in prism surface
The skin depth of wave that dies and cellular layer thickness match, therefore the adherent dielectric material of prism surface can completely be carried by being emitted THz wave
Response signal；
Wherein, dpFor the skin depth of evanescent wave, λ is the wavelength of incident THz wave, nSiFor the refraction of total reflection prism
Rate, θ are incidence angle of the THz wave in prism surface, nemFor the refraction of prism surface sample to be examined (i.e. adherent monolayer)
Rate；
Simplify dielectric model according to the binary of EFFECTIVE MEDIUM THEORY, such as formula (2), as acquired in outgoing terahertz signal
The equivalent complex dielectric permittivity ε of substance at prism boundaryeffIt is related with the spatial volume score of culture medium and cell, that is, determining εmedium
And εcellIn the case of, it can be by measuring εeffSignal, obtain the cell transwell lower layer cell density distribution situation, into
And realize the markless detection to cell quantity；
Wherein, εeffFor the equivalent complex dielectric permittivity of substance at prism boundary acquired in outgoing terahertz signal, εcellIt is thin
The complex dielectric permittivity of born of the same parents, VcellFor the cellular layer volume of detection zone, εmediumFor culture medium complex dielectric permittivity, VmediumFor detection
The volume of region culture medium.
Test example
With COX-2 inhibitors celecoxib to triple negative breast cancer MDA-MB-231 cell (HTB-
26TM) for invasive ability impact evaluation, it is close using Terahertz confluent monolayer cells under the total reflection cell detection of platform transwell of decaying
Degree distribution, application also extend to Cell migration assay or other results statistics dependent on the detection cell transwell lower layer
The experiment of cell quantity, specific step is as follows for detection:
1,10% fetal calf serum of the percentage of accounting containing volume, streptomysin (100 μ g/mL) and penicillin cell culture: are used
The DMEM in high glucose complete medium of (100U/mL), by MDA-MB-231 cell be placed in 37 DEG C, carbon dioxide volume fraction be
In 5% cell incubator, to 25cm2When cell fusion degree reaches 85% or so in culture bottle, with 1mL0.25% tryptose
Enzyme-EDTA had digestive transfer culture, next day change liquid, primary every passage in 3-4 days.
2, inoculating cell: diluting celecoxib using DMSO, be diluted to 0,50,100 μm of ol/L with DMEM complete medium,
Ensure DMSO final concentration lower than 0.1%.DMEM culture medium without fetal calf serum is pressed into 3:1 ratio with Matrigel (50mg/L) glue
Example is diluted；The Matrigel glue that 300 μ L have diluted is added dropwise on the cell Transwell upper layer, is stayed overnight under aseptic condition, it is ensured that
Glue polymerize completely.The MDA-MB-231 after 0,50,100,150 μm of ol/L celecoxib handles 24 hours respectively is collected by centrifugation
Cell is 2 × 10 with the DMEM culture medium adjustment concentration of cell suspension without fetal calf serum5400 μ are added in a cell/mL, every hole
L cell suspension is supplemented to 1mL in cell upper layer, and using the DMEM culture medium without fetal calf serum；It is complete that lower room is filled with DMEM
Culture medium.After cultivating about 24 hours in cell incubator, decays for Terahertz and be totally reflected the detection of platform.
3, Terahertz detects: successively cleaning total reflection prism surface with acetone, dehydrated alcohol and ultrapure water, is dried with nitrogen
Afterwards；Terahertz detection system shifts to an earlier date 1 hour and is switched on, and is filled with dry air, keeps humidity 3% hereinafter, average 2048 times are adopted
Collect and save the spectrum of background signal.The cell transwell is carefully taken out from culture plate, wipes Matrigel glue with cotton swab head
And after the cell on upper layer, PBS buffer solution is cleaned 3 times.With the moisture of aseptic filter paper gentle aspiration cell lower layer.Cell is placed
In total reflection prism surface intermediate region, make it that THz wave incidence focal zone be completely covered；Adjust the spring of detection module
Formula pressure controllable fixture is to suitable position, and making the plastic frame of cell lower layer abutting prism surface and cell, deformation occurs.
500 μ L DMEM complete mediums are added to cell upper layer at this time, then acquires and the tera-hertz spectra for saving the sample is (average
2048 times)；Sample and acquisition spectrum time-consuming are placed less than 1 minute.It repeats above method acquisition each of the above sample and DMEM is complete
The Terahertz decaying total reflection signal value of full culture medium.
4, mtt assay detects: being herein the reliability of verification result, while counting method detection Terahertz is surveyed indirectly using tradition
Sample after amount.1mL MTT containing 5g/L complete medium is added in every hole in 12 orifice plates, and cell is placed therein, lower layer is made
Film is completely immersed in wherein, is taken out after placing 4h in cell incubator.1mL DMSO is added in every hole, and cell is impregnated wherein shake
Swinging 10min dissolves MTT sufficiently.Cell is taken out, using DMEM complete medium as blank control, elution is detected in microplate reader
The OD value of liquid.
5, interpretation of result: the Terahertz for extracting various concentration drug effect group decays total reflection signal (ATR log [dB]),
As shown in figure 3, that is, culture medium, which absorbs, is greater than cellular layer absorption since the complex dielectric permittivity imaginary part of culture medium is higher than cellular layer, therefore
Its total reflection signal intensity value of decaying is minimum.Celecoxib can generate MDA-MB-231 vitro invasion ability to be made compared with high inhibition
With, and dose dependent is presented.Increase so that with drug concentration, the decline of cell invasion ability, the cell density of lower layer in cell
It can significantly reduce, medium body fraction rises.Decaying total reflection signal intensity value rises with drug concentration, and cell lower layer is thin
Born of the same parents' density, which reduces to present, is gradually reduced trend.Each group sample decaying total reflection signal value (ATR log [dB]) is complete with DMEM
Culture medium signal value subtracts each other, and can get this group of sample signal intensity situation after the processing of corresponding concentration medicine group, such as Fig. 4 institute
Show, signal strength indication, which changes trend, can be used to assessment of the acute drug to cell invasion capacity.The same manner processing
The obtained absorbance value signal of MTT experiment, the signal value variation tendency of two kinds of detection methods is fitted, as shown in figure 5, base
It is in the transwell cell quantification detection method of Terahertz decaying total reflection platform and the linearly dependent coefficient of traditional mtt assay
0.996, good relationship, and this platform has the advantage of quick, unmarked living cells in situ detection.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (6)
1. the transwell cell experiment detection device based on Terahertz Technique of Attenuated Total Reflectance characterized by comprising
Terahertz signal generating unit, including femtosecond laser light source, laser beam splitter, hemispherical silicon lens I and photoconductive antenna
I, femtosecond laser light source emits laser and is divided into pump beam and detection light beam under laser beam splitter effect, and pump beam is by super
Hemisphere silicon lens I focuses, collimation can excite photoconductive antenna I to emit THz wave to total reflection prism detection unit；
Total reflection prism detection unit, from top to bottom include the cell transwell and total reflection prism, the cell transwell
Bottom is equipped with permeability porous membrane, and monolayer is adhered in the lower section of porous membrane, and monolayer closely connects with total reflection prism
Touching, the evanescent wave that the vertical direction distribution that total reflection generates occurs for same total reflection prism surface sufficiently interact, modulate
Reflected terahertz hereby wave；
Terahertz signal receiving unit, including photoconductive antenna II, for receiving the carrying of total reflection prism detection unit reflection
The THz wave of adherent monolayer information；
Time delay control system, including time delay device are set between laser beam splitter and photoconductive antenna I, are used for
It controls terahertz signal generating unit and generates and emit THz wave；
Signal processing unit, including computer, it is complete for acquiring and analyzing the THz wave of terahertz signal receiving unit acquisition
It is detected at transwell cell experiment；
Controllable pressure fixture is equipped in the top of the cell transwell；The bottom of the controllable pressure fixture is equipped with detachable
Circular pressing plate, and contacts at the top of the cell transwell.
2. detection device according to claim 1, which is characterized in that it is lower that the cell transwell selects Terahertz to absorb
8 μm of microporous polycarbonate films, polytetrafluoroethylene film or polyester film.
3. detection device according to claim 1, which is characterized in that the controllable pressure fixture is fixed by fixture mount,
Total reflection prism is fixed by prism fixed frame.
4. using any one of claims 1 to 33 described device realize based on Terahertz Technique of Attenuated Total Reflectance
Transwell cell experiment detection method, which is characterized in that specific step is as follows:
1) cell transwell of bottom attachment monolayer is placed on total reflection prism, so that monolayer and total reflection
Culture medium is added to cell upper layer in the intimate surface contact of prism；
2) terahertz signal generating unit emits THz wave to total reflection prism detection unit；
3) THz wave occurs to be totally reflected and formed to be distributed vertically upward along total reflection prism surface on total reflection prism surface
Evanescent wave interacts with the monolayer fitted closely, modulates reflected terahertz hereby wave；
4) terahertz signal receiving unit receives reflected terahertz hereby wave, and time delay control system adjusts THz wave and occurs and visit
Optical path is surveyed, the terahertz signal of signal processing unit acquisition and analysis monolayer completes the detection of transwell cell experiment；
In step 1), the cell transwell of bottom attachment monolayer obtains by the following method: transwell is small
The bottom of room is equipped with permeability porous membrane, and using porous membrane as boundary, the cell transwell is divided into the upper and lower, and upper layer adds
Enter matrigel and cell suspension, lower layer is filled with cell culture medium, and porous membrane is completely immersed in cell culture medium, completes cell training
After supporting, the cell transwell is taken out, wipe the matrigel on upper layer and cell and is cleaned, the moisture of lower layer is drawn；
In step 1), apply the intimate surface contact that pressure makes monolayer and total reflection prism using controllable pressure fixture.
5. detection method according to claim 4, which is characterized in that the specific method is as follows: matrigel, nothing is added dropwise to upper layer
Under the conditions of bacterium overnight, it is ensured that matrigel polymerize completely, and cell suspension is added, cell culture medium is filled with to lower layer, through cell culture
The cell transwell is taken out afterwards, and after wiping matrigel and the cell on upper layer with cotton swab head, PBS buffer solution is cleaned 3 times, with nothing
The moisture of bacterium filter paper gentle aspiration lower layer.
6. detection method according to claim 4, which is characterized in that in step 4), can be obtained by formula (1) in prism surface
The skin depth of evanescent wave is generated when being totally reflected by incident THz wave for place and cellular layer thickness matches, therefore is emitted too
Hertz wave can completely carry the response signal of the adherent dielectric material of prism surface；
Wherein, dpFor the skin depth of evanescent wave, λ is the wavelength of incident THz wave, nSiFor the refractive index of total reflection prism, θ
Incidence angle for THz wave in prism surface, nemFor the refractive index of prism surface sample to be examined；
Simplify dielectric model, such as formula (2), the prism as acquired in outgoing terahertz signal according to the binary of EFFECTIVE MEDIUM THEORY
The equivalent complex dielectric permittivity ε of interface substanceeffIt is related with the spatial volume score of culture medium and cell, that is, determining εmediumWith
εcellIn the case of, it can be by measuring εeffSignal, obtain the cell transwell lower layer cell density distribution situation, in turn
Realize the markless detection to cell quantity；
Wherein, εeffFor the equivalent complex dielectric permittivity of substance at prism boundary acquired in outgoing terahertz signal, εcellFor cell
Complex dielectric permittivity, VcellFor the cellular layer volume of detection zone, εmediumFor culture medium complex dielectric permittivity, VmediumFor detection zone
The volume of culture medium.
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