CN110031423B - The method of unmarked assessment aquaporin function based on THz wave technology - Google Patents
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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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3581—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation
- G01N21/3586—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation by Terahertz time domain spectroscopy [THz-TDS]
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- General Health & Medical Sciences (AREA)
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Abstract
The method for the unmarked assessment aquaporin function based on THz wave technology that the present invention relates to a kind of, in the Terahertz Meta Materials chip surface inoculating cell of building, it cultivates to cell adherent growth and is fused into monolayer completely, then using the resonance peak situation of cellular layer in terahertz time-domain spectroscopy instrument measurement isotonic buffer solution；Replace hypotonic buffer liquid, a large number of water molecules enters cell interior by aquaporin, so that initial cell volume increases, cell after forming swelling, resonance peak situation of change of the adherent layer in osmotic pressure variation is obtained using terahertz time-domain spectroscopy instrument in real time, effectively reflection cell degree of swelling, i.e. hydrone passes through the infiltration situation of cellular layer, and the inverse the time required to reaching maximum response is acquired according to real-time response curve, the unmarked assessment for realizing cell aquaporin function situation can be used for the high flux screening of related targeting AQP drug.
Description
Technical field
The invention belongs to Terahertz field of measuring technique, and it is logical to be related to a kind of unmarked assessment water based on THz wave technology
The method of road protein function.
Background technique
Water is Source of life.Aquaporin (aquaporin, AQP) is the transmembrane protein on cell membrane, selectivity
The efficiently channel of transhipment hydrone.Since reporting first aquaporin (AQP1) for the first time since 1991, researcher is mutually secondary
13 kinds of aquaporins of Different Organs are now distributed widely in, for maintaining organ normal physiological function to play vital work
With.AQP expression quantity and a variety of diseases such as functional status and tumour, cranial vascular disease, kidney trouble are closely related.Especially AQP
Up-regulation is expressed in tumor tissues, and tumour cell is entered by rapid transport hydrone and mediates its migration, proliferation and invasion, and is joined
With tumor neovasculature formation.Therefore accurate evaluation cell AQP functional status, the blocking agent for researching and developing targeting AQP, inhibition
Agent or even agonist are the hot spot places of current cancer targeted therapy and imaging.
Cell AQP functional assessment mainly passes through the transhipment that measurement hydrone penetrates after birth under extracellular osmotic pressure situation of change
Situation, macro manifestations are that the volume of cell and water content change.Based on this, assessment cell AQP functional method has light scattering at present
The methods of method, microscopic image measurement method, optical interferometry, ion concentration responsive type fluorescent marker.Light scattering method is limited to artifact letter
Number generate, less application；Microscopic image measurement is only applied to the Xenopus Oocytes of expression AQP in cell swelling process
The analysis of stereomutation, data variation is larger, reliability is lower；Laser interference to the beam alignment of instrument and vibration require compared with
Height, it is not very practical.Research from AQP functional study field leading force Verkman discloses: tradition is based on cubing
AQP function evaluating method reliability is lower, thus is difficult to the drug that Effective selection really inhibits AQP function.Fluorescent marker rule
As reliable appraisal procedure, by carrying out the yellowish green uniformly dyeing color of cytoplasmic calcium to the cellular layer being grown on solid support or turning
Green colouring fluorescin, the variation of fluorescence signal obtains cell when changing using total internal reflection fluorescent platform assay osmotic gradient
Volume kinetics change parameter, have the significant advantage that artifact error is small, photobleaching effect is low and result stability is high.But it is still
It is to there is label to detect, it can damaging cells activity；And since evanescent wave penetration depth limits, it is only capable of obtaining the cell of submicron-scale
Layer partial information.To sum up, still lack a kind of unmarked, reliable cell AQP function evaluating method at present.
Terahertz (Terahertz, THz) wave technology is a kind of novel markless detection technology.It is frequency 0.1~
The electromagnetic wave of 10THz (corresponding wavelength range is 30 μm of -3mm) has markless detection in microwave to the wave band of infrared transition
Frequency spectrum advantage: magnetic resonance, the scattering of quasi- elastic neutron and incoherent neutron are compared in the corresponding 0.16 picosecond of rotation of 1THz frequency of oscillation
The conventional methods such as scattering, THz technology are not necessarily to hydrogen deuterium exchange or sub-cooled, can will test moisture movement scale intracellular from femtosecond
Level is promoted to picosecond and subpicosecond is horizontal, thus realize it is accurate, analyze intracellular Free water in real time and combine the aquation of water dynamic
Mechanics has the potential assessed in real time applied to cell AQP function.But THz wave still faces THz applied to living cells detection
Existing scale loses in matching problem and liquid phase environment between wavelength (1THz~300 μm) and cellular layer thickness (about 5~10 μm)
Absorbing interference strongly to water, (water absorption coefficient is about 230cm at 1THz-1) the problems such as.It there is no at present by THz wave technology application
In the relevant report of the unmarked evaluation areas of cell AQP function.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of unmarked assessment aquaporin egg based on THz wave technology
The method of contour painting energy.
In order to achieve the above objectives, the invention provides the following technical scheme:
The method of unmarked assessment aquaporin function based on THz wave technology, the specific steps are as follows:
(1) Terahertz Meta Materials chip is constructed；
(2) the Meta Materials chip surface inoculating cell constructed by step (1), culture is to cell adherent growth and melts completely
Monolayer is synthesized, then using the resonance peak situation of cellular layer in terahertz time-domain spectroscopy instrument measurement isotonic buffer solution；
(3) hypotonic buffer liquid is replaced, a large number of water molecules enters cell interior by aquaporin, so that initial cell body
Product increases, the cell after forming swelling, and it is humorous when osmotic pressure changes to obtain adherent layer using terahertz time-domain spectroscopy instrument
The infiltration situation that vibration peak situation of change, effectively reflection cell degree of swelling, i.e. hydrone pass through cellular layer, and according to real-time response
Curve acquires the inverse the time required to reaching maximum response, realizes the unmarked assessment of cell aquaporin function situation.
One of as a preferred technical scheme, the specific method of step (1) is: using the High Resistivity Si of twin polishing as substrate,
Then the double layer of metal film on its surface successively sputter etches insensitive to THz wave polarization direction narrow on metal film
Crack structure, so that the bottom of slit is High Resistivity Si.
As further preferred one of technical solution, the material of the metal film is in gold, titanium, silver, copper, aluminium or nickel
It is any.
As further preferred one of technical solution, the narrow slit structure is selected from circular ring shape, rectangular or cruciform shape
Any one of.
As further preferred one of technical solution, using the High Resistivity Si of twin polishing as substrate, successively splashed on its surface
20nm titanium film and 200nm golden film are plated, then etches circular ring shape slit periodic structure in golden film using ultraviolet photolithographic technology,
So that the bottom of slit is High Resistivity Si, wherein High Resistivity Si with a thickness of 500 μm, resistivity is greater than 10000 Ω cm, annulus
Overall diameter is 80 μm, and interior diameter is 72 μm, and ring width (gap) is 4 μm, and the period is 120 μm.Slit sizes and cell size herein
Match, can also be adjusted.
One of as a preferred technical scheme, in step (2), first Meta Materials chip is carried out at cleaning before inoculating cell
Reason, specific method is: first using washes of absolute alcohol Meta Materials chip, reuses U.S.'s Harrick plasma cleaner processing 3
Minute, it is then impregnated in ultrapure water after ten minutes, finally using being dried with nitrogen Meta Materials chip；Wherein, the plasma is clear
The power of washing machine is 100W, vacuum degree 0.3mBar.
One of as a preferred technical scheme, in step (2), the specific method of inoculating cell is: by configured cell
Suspension is added dropwise in Meta Materials chip surface, and after corresponding culture medium is added, is placed in cell incubator and grows, and pastes to cell
After wall grows and is fused into monolayer completely, Meta Materials chip is taken out from incubator.
One of as a preferred technical scheme, in step (2), the Meta Materials chip after inoculating cell utilizes AQP blocking agent
Processing further preferably uses parachloromercuribenzene sulfonate salt (p-chloromercuribenzene sulphonate, pCMBS)
Solution is handled 20 minutes.PCMBS is reported reliable AQP function blocking agent, can inhibit the function of cell membrane surface AQP.
Corresponding THz Meta Materials chip measurement result: MCF-7 cell is under Hyposmolality environment after pCMBS is handled, resonance peak
Live signal variation degree is far below the control group signal without any processing, and the 1/t value of pCMBS processing group cell is significant
Lower than control group.Through comparing with Meta Materials chip resonance response trend when FDTD sunykatuib analysis cellular water changes of contents, sufficiently
Illustrate that the degree of swelling of the MCF-7 cell after pCMBS is handled weakens, swelling rate reduces, and required time extends, via AQP
Hydrone transport function be suppressed.
One of as a preferred technical scheme, PBS solution is added dropwise in Meta Materials chip surface in step (2), acquires and saves
The terahertz light spectrum signal of initial cell；Step (3) is added dropwise ultrapure water and mixes to Meta Materials chip surface forms hypotonic environment,
Carry out terahertz light spectrometry.
As further preferred one of technical solution, after the terahertz light spectrometry under hypotonic environment, digestion solution
From Meta Materials chip surface cell, Meta Materials chip surface is cleaned, drying is placed in Terahertz light path and PBS is added dropwise
Solution obtains the resonance peak response of PBS solution.
As further preferred one of technical solution, the time-domain spectroscopy signal of phase point when extracting cell differential responses
The second peak information reflected from Meta Materials chip surface, carries out fast Fourier transform analysis, calculates the super of different time phases
The reflectivity of material chip simultaneously acquires Log (1/R) value, for reflecting the real-time change of intracellular water content, by different time phases
Reflection Log (1/R) value subtract each other with initial value, can get cell swelling process chip real-time response variation diagram, calculate and reach
The time required to when to maximum changing value (1/t) reciprocal, for indicating the aquaporin function inhibitio situation of cell, i.e. cell membrane
Opposite water transparency.
One of as a preferred technical scheme, in step (2), the detection method of terahertz time-domain spectroscopy instrument are as follows: select THz
The reflecting module of time-domain spectroscopy instrument is detected, the 1 hour in advance dry air that is switched on and is filled with, and humidity is 3% in maintenance optical path
Hereinafter, measuring the reflectance spectrum signal of the gold-plated film silicon wafer without any narrow slit structure first, spectrum is 128 times average, as reference
Signal；Meta Materials chip level is then placed in THz light path, so that THz hot spot is directed at central detection area domain, in chip list
PBS solution is added dropwise in face, is averaged 128 times and acquires and save the THz spectral signal of initial cell.
One of as a preferred technical scheme, the specific method of step (3) is: peer is added dropwise to Meta Materials chip surface
Product ultrapure water is simultaneously mixed to form osmotic pressure as the hypotonic environment of 150mOsm/L, immediately begins to THz spectral measurement, and average 128
It is secondary, time interval 1 second, 50 secondary reflection time-domain spectroscopy signal of continuous acquisition；After measurement, is digested and dissociated using pancreas enzyme -EDTA
Chip surface cell successively cleans Meta Materials chip surface with dehydrated alcohol, ultrapure water, after being dried with nitrogen, is placed in THz detection
In optical path and PBS solution, the resonance peak response of average 128 acquisitions PBS solution is added dropwise.
The working principle of the invention:
For through annulus slit shape Meta Materials under FDTD (Finite-Difference Time-Domain Method) sunykatuib analysis above structure parameter on the right side of Fig. 1
The distribution map of the electric field of chip；Incident THz wave can excite the resonance electric field at annulus slit, and the resonance electric field of enhancing is from Slot bottom
Along Z-direction rapid decay, skin depth is 1.5 μm, and vertical distribution height is close to 10 μm.Meanwhile applicant uses FDTD points
Analysis modeling, the Meta Materials chip surface under THz reflective-mode add the liquid level conduct of the cellular layer and 500 μ m-thicks of 10 μ m-thicks
Liquid phase cell detection model, and use the liquid level of 510 μ m-thicks as background signal detection model merely.Cellular layer and liquid level
Complex refractivity index parameter is according to having delivered (Shiraga K, Ogawa Y, Suzuki T, the et al.Applied of data in document
Physics Letters,2013,102(5):053702.Zhao X,Zhang M,Wei D,et al.[J].Biomedical
Optics Express,2017,8(10):4427-4437.).The complex refractivity index difference of cell and PBS solution focuses primarily upon multiple
Imaginary index, that is, extinction coefficient, it is main to influence Meta Materials chip resonance peak strength difference, as shown in Fig. 2 analog result, liquid phase
The resonance peak intensity of cellular layer is apparently higher than the resonance peak intensity of PBS solution layer, and is displaced and changes without obvious resonant frequency.
It is Free water that applicant, which assumes that cell swelling process enters hydrone, and enters free water volume (Δ intracellular
VFlow into water) it is the increased volume of cell swelling process, this swelling process can be obtained using the binary model based on EFFECTIVE MEDIUM THEORY
In cellular layer complex refractivity index imaginary values, i.e. formula 1.Using water content intracellular during FDTD analysis cell AQP functional assessment
The response variation of Meta Materials chip when variation.As a result as shown in figure 4, cellular layer changes percentage in water contentWhen increase, the reflection resonance peak intensity of Meta Materials chip is gradually decreased, and moves closer to PBS solution
The signal of background.Linear fit further is carried out to resonance peak Strength Changes situation and water content variation percentage, as a result such as Fig. 5
Shown, linear equation is linear dependence preferably (R2For 0.99874).Thus, the used THz Meta Materials reflectance spectrum of the present invention
Resonance peak situation of change of the adherent layer when osmotic pressure changes is obtained in real time, can effectively reflect cell degree of swelling, i.e. water
Molecule passes through the infiltration situation of cellular layer, and the inverse the time required to reaching maximum response can be acquired according to real-time response curve
(1/t) realizes the unmarked assessment of cell AQP function situation.
Wherein, κIt is swollen cellFor the complex refractivity index imaginary part of cell in swelling process, κCellFor the complex refractivity index imaginary part of initial cell,
κFree waterFor the complex refractivity index imaginary part of Free water, Δ VFlow into waterTo enter Free water total volume intracellular, V in real timeInitial cellFor the first of cell
Initial body product.
The beneficial effects of the present invention are:
Terahertz Meta Materials chip constructed by the present invention, sensing principle are based on superpower light transmission phenomenon: i.e. Terahertz
Resonance peak frequency and the neighbouring equivalent refractive index variation of slit when wave and narrow slit structure reach wave vector exact matching is related.THz wave
It interacts with Meta Materials chip, especially interacts with having the Meta Materials chip of periodical second wavelength metallic structure, it will
The local enhancing electric field for generating a few micrometers of scales can effectively solve THz wave cell detection and face scale mistake matching problem.Meanwhile
The liquid such as cell culture medium can be overcome to absorb interference by force in conjunction with the Meta Materials chip of THz reflectance spectrum, effectively acquisition liquid phase environment
The dielectric response information of middle cellular layer.
The application announces one kind based on cellular layer water content under THz Meta Materials reflectance spectrum technology measurement liquid phase environment for the first time
The method of variation, the ingenious sensitivity using THz wave to the strong absorption characteristic of water, for changing to micron level cellular layer water content
Response, and Meta Materials resonance peak magnitude parameters are selected in the difference feature of complex refractivity index imaginary part according to THz wave band inner cell and water,
As the index for reflecting the variation degree of intracellular water content in extracellular osmotic pressure change procedure, can be used for cell AQP function
Unmarked, real-time assessment.Have advantage following prominent:
1. without label, the method for being not required to transfect by any fluorescent staining or fluorescin is simple and efficient and to cell
Any damage is not generated, can effectively assess the functional status of cell AQP；
2. full cell dimensions detection, the local enhancing electric field of Meta Materials can obtain entire in nearly 10 μm of vertical direction distribution
The dielectric response of cellular layer changes；
3. high sensitivity, THz wave are very sensitive to water content variation, the faint water content of cellular layer can be obtained in real time and is become
Change.
To sum up, what this patent proposed can unmarked, real-time assessment cell AQP based on Terahertz Meta Materials reflectance spectrum technology
Functional status can be used for the high flux screening of related targeting AQP drug.
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 schematic diagram and distribution map of the electric field of annulus slit shape Meta Materials chip；
Fig. 2 is the response of FDTD sunykatuib analysis cellular layer and PBS solution on Meta Materials chip；
Fig. 3 is Meta Materials chip markless detection cell AQP function (swelling process under hypotonic environment) schematic diagram；
Fig. 4 is cellular layer water content percentage change procedure response diagram on FDTD sunykatuib analysis Meta Materials chip；
Fig. 5 changes for the response variation of FDTD sunykatuib analysis Meta Materials chip with corresponding cellular layer water content percentage linear
Fitted figure；
Fig. 6 is that the MCF-7 cell of pCMBS processing group and control group real-time response on Meta Materials chip becomes under hypotonic environment
Change；
The time required to Fig. 7 reaches maximum response for the MCF-7 cell of pCMBS processing group and control group under hypotonic environment
(1/t) comparison diagram reciprocal；
Wherein, 1 is golden film, and 2 be silicon base, and 3 be cell (inoculation original state), and 4 be isotonic buffer solution, after 5 is swellings
Cell, 6 be hypotonic buffer liquid.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Illustrate appraisal procedure of the invention for the AQP Function detection of following human breast cancer cell (MCF-7), needs to illustrate
, the invention is not limited to this kind of specific cells.
1, cell culture: percentage by volume is added in high glycoform DMEM culture medium and is 10% fetal calf serum and body is added
The dual anti-working solution of penicillin streptomycin (Gibco, article No. 15140163) that product percentage is 1% forms complete medium, cell
Incubator temperature be set as 37 DEG C, carbon dioxide volume fraction be 5%, to (the raw work biology work in Shanghai of MCF-7 cell in culture bottle
Journey limited liability company, article No. D611026-0001) degrees of fusion is when reaching 85% or so, with trypsase-EDTA digestive juice (its
Middle pancreatin mass concentration is that 0.25%, EDTA mass concentration is 0.02%) to digest 2 minutes in the incubator, is gradually taken off to cell
When falling (culture bottle is in ground-glass-like), complete medium used above is added and terminates digestion, and piping and druming obtains cell repeatedly
Suspension, centrifugation is configured to concentration after being resuspended be 1 × 106A cell/mL suspension is spare.
2, inoculating cell: using six pieces of the washes of absolute alcohol Meta Materials chips with batch production, and the U.S. is used
Harrick plasma cleaner handles 3 minutes (power 100W, vacuum degree 0.3mBar), impregnates 10 minutes in ultrapure water
Afterwards, using being dried with nitrogen Meta Materials chip.Configured MCF-7 cell suspension is added dropwise on Meta Materials surface, and is trained to cell
It supports after 3mL complete medium is added in ware, is placed in cell incubator and grows.It is fused into list to cell adherent growth and completely
After cellular layer, Meta Materials chip is taken out from incubator, is divided into processing group and control group.Processing group uses 1mM AQP
Blocking agent-parachloromercuribenzene sulfonate salt (p-chloromercuribenzene sulphonate, pCMBS) solution is handled 20 minutes
After take out, and control group does not take any processing, and whole chips use PBS buffer solution (wherein NaCl 136.89mM, KCl
2.67mM,Na2HPO4 8.1mM,KH2PO4 1.76mM；PH is that 7.4) cleaning is rear twice spare.As shown in figure 3, completing the process
Meta Materials chip (including: golden film 1, silicon base 2) surface seeding cell 3, then according to subsequent step measure isotonic buffer solution 4
The resonance peak situation of middle cellular layer.
3, THz is detected: being selected the reflecting module of THz time domain spectrum instrument to be detected, is switched on and is filled with drying within 1 hour in advance
Air, maintain optical path in humidity 3% hereinafter, first measure the gold-plated film silicon wafer without any narrow slit structure reflectance spectrum letter
Number, spectrum is 128 times average, as reference signal.Meta Materials chip level is then placed in THz light path, makes THz hot spot
It is directed at central detection area domain, PBS solution is added dropwise in chip surface, average 128 times the THz spectrum for acquiring and saving initial cell is believed
Number.As shown in figure 3, replacement hypotonic buffer liquid 6, a large number of water molecules enters cell interior by AQP, increases initial cell volume
Greatly, the cell 5 after swelling is formed, specific method is: equal volume ultrapure water is added dropwise to Meta Materials chip surface and mixes with shape
The hypotonic environment for being 150mOsm/L at osmotic pressure, immediately begins to THz spectral measurement, 128 times average, and time interval 1 second, continuously
Acquire 50 secondary reflection time-domain spectroscopy signals.After measurement, chip list is dissociated using pancreas enzyme -EDTA digestive juice used above
Face cell successively cleans Meta Materials chip surface with dehydrated alcohol, ultrapure water, after being dried with nitrogen, is placed in THz light path
And PBS solution is added dropwise, the resonance peak response of average 128 acquisitions PBS solution.The measurement of whole chips is obtained using same method
As a result.
4, Meta Materials chip list interpretation of result: is come from when extracting two groups of cell differential responses in the time-domain spectroscopy signal of phase point
Second peak information of face reflection, carries out fast Fourier transform analysis, is reference with gold-plated film silicon wafer signal, when calculating different
The reflectivity R of the Meta Materials chip of phase point simultaneously acquires Log (1/R) value.By reflection Log (1/R) value of different time phases and initially
Value is subtracted each other, and can get the chip real-time response variation diagram of cell swelling process, as shown in Figure 6.The reflection Log of Meta Materials chip
(1/R) value can reflect the real-time change of the intracellular water content of MCF-7, reciprocal the time required to calculating when reaching maximum changing value
(1/t) is used to indicate the AQP function inhibitio situation of MCF-7, i.e., cell membrane is with respect to water transparency, as shown in Figure 7.
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 (9)
1. the method for the unmarked assessment aquaporin function based on THz wave technology, which is characterized in that specific steps are such as
Under:
(1) Terahertz Meta Materials chip is constructed；
(2) the Meta Materials chip surface inoculating cell constructed by step (1), culture is to cell adherent growth and is fused into completely
Monolayer, then using the resonance peak situation of cellular layer in terahertz time-domain spectroscopy instrument measurement isotonic buffer solution；
(3) hypotonic buffer liquid is replaced, a large number of water molecules enters cell interior by aquaporin, so that initial cell volume increases
Greatly, the cell after swelling is formed, it is humorous when osmotic pressure changes to obtain adherent layer in real time using terahertz time-domain spectroscopy instrument
The infiltration situation that vibration peak situation of change, effectively reflection cell degree of swelling, i.e. hydrone pass through cellular layer, and according to real-time response
Curve acquires the inverse the time required to reaching maximum response, realizes the unmarked assessment of cell aquaporin function situation；
The specific method of step (1) is: using the High Resistivity Si of twin polishing as substrate, the double layer of metal on its surface successively sputter
Then film etches the narrow slit structure insensitive to THz wave polarization direction on metal film, so that the bottom of slit is height
Hinder silicon.
2. the method according to claim 1, wherein in step (2), first to Meta Materials chip before inoculating cell
It starts the cleaning processing, specific method is: first using washes of absolute alcohol Meta Materials chip, reusing U.S.'s Harrick plasma
Cleaning machine is handled 3 minutes, is then impregnated in ultrapure water after ten minutes, finally using being dried with nitrogen Meta Materials chip；Wherein, institute
The power for stating plasma cleaner is 100W, vacuum degree 0.3mBar.
3. the method according to claim 1, wherein the specific method of inoculating cell is in step (2): will match
The cell suspension set is added dropwise in Meta Materials chip surface, and after corresponding culture medium is added, is placed in cell incubator raw
It is long, after being fused into monolayer after cell adherent growth and completely, the taking-up Meta Materials chip from incubator.
4. the method according to claim 1, wherein the Meta Materials chip after inoculating cell utilizes in step (2)
The processing of AQP blocking agent.
5. the method according to claim 1, wherein step (2) Meta Materials chip surface be added dropwise PBS solution,
Acquire and save the terahertz light spectrum signal of initial cell；Step (3) is added dropwise ultrapure water to Meta Materials chip surface and mixes shape
At hypotonic environment, tera-hertz spectra real-time measurement is carried out.
6. according to the method described in claim 5, it is characterized in that, the tera-hertz spectra real-time measurement under hypotonic environment terminates
Afterwards, digestion dissociation Meta Materials chip surface cell, cleans Meta Materials chip surface, and drying is placed in Terahertz light path
And PBS solution is added dropwise, obtain the resonance peak response of PBS solution.
7. according to the method described in claim 6, it is characterized in that, extract cell differential responses when phase point time-domain spectroscopy signal
In from Meta Materials chip surface reflection second peak information, carry out fast Fourier transform analysis, calculate different time phases
Meta Materials chip reflectivity and acquire Log (1/R) value, for reflecting the real-time change of intracellular water content, when will be different
Reflection Log (1/R) value of phase point is subtracted each other with initial value, can get the chip real-time response variation diagram of cell swelling process, is calculated
Required time inverse 1/t when reaching maximum changing value out, for indicating the aquaporin function inhibitio situation of cell, i.e. cell
Film is with respect to water transparency.
8. the method according to claim 1, wherein in step (2), the detection method of terahertz time-domain spectroscopy instrument
Are as follows: select the reflecting module of THz time domain spectrum instrument to be detected, the 1 hour in advance dry air that is switched on and is filled with maintains in optical path
Humidity is 3% hereinafter, the reflectance spectrum signal of pure gold-plated film silicon wafer of the measurement without any narrow slit structure first, spectrum average 128
It is secondary, as reference signal；Meta Materials chip level is then placed in THz light path, THz hot spot is made to be directed at central detection area
Domain is added dropwise PBS solution in chip surface, is averaged 128 times and acquires and save the THz spectral signal of initial cell.
9. the method according to claim 1, wherein the specific method of step (3) is: to Meta Materials chip surface
Equal volume ultrapure water is added dropwise and mixes to form osmotic pressure as the hypotonic environment of 150mOsm/L, immediately begins to the survey of THz spectrum
Amount, it is 128 times average, time interval 1 second, 50 secondary reflection time-domain spectroscopy signal of continuous acquisition；After measurement, pancreatin-is used
EDTA digestion dissociation chip surface cell, successively cleans Meta Materials chip surface with dehydrated alcohol, ultrapure water, after being dried with nitrogen,
It is placed in THz light path and is added dropwise PBS solution, the resonance peak response of average 128 acquisitions PBS solution.
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