CN111141703B - Terahertz metamaterial sensor for cell migration and detection method adopting terahertz metamaterial sensor - Google Patents
Terahertz metamaterial sensor for cell migration and detection method adopting terahertz metamaterial sensor Download PDFInfo
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Abstract
A terahertz metamaterial sensor comprises a substrate, a terahertz metamaterial structure sensing unit and a polydimethylsiloxane enclosure used for limiting a cell growth region, wherein: the terahertz metamaterial structure sensing unit is formed on the substrate, and periodically arranged split rings and two circular ring alignment marks are formed on the terahertz metamaterial structure sensing unit; the polydimethylsiloxane enclosure is located on the substrate and comprises a middle circular hollowed-out polydimethylsiloxane square piece and a solid polydimethylsiloxane cylindrical piece, wherein the inner diameter of the hollow circle of the polydimethylsiloxane square piece is aligned with an outer ring alignment mark on the terahertz metamaterial structure sensing unit, and the outer diameter of the solid polydimethylsiloxane cylindrical piece is aligned with an inner ring alignment mark. The sensor has the advantages of simple preparation process, high detection speed and small required cell amount, and provides a quick and reliable method for detecting cell migration.
Description
Technical Field
The invention relates to the technical field of terahertz metamaterial biosensing, in particular to a terahertz metamaterial sensor for cell migration and a detection method adopting the terahertz metamaterial sensor.
Background
Cell migration assay is one of the commonly used experimental methods in cell biology, and is an important method for evaluating the stimulation effect of stimulating factors on cells, especially in the field of tumor biology research. The traditional cell migration detection experiment is long in time consumption, an experimenter needs to manually set cell wounds, and when the experiment is observed, a distance needs to be measured under a microscope, so that the time consumption is long, and the operation is troublesome; with the development of microfluidic technology, some microfluidic chips for detecting cell migration are gradually developed, but the microfluidic body flow rate and the processing of microstructures are required to be accurately controlled, and the technical requirements are high. Therefore, establishing a cell migration chip and a detection method thereof which are convenient to prepare and detect has important significance for improving the traditional cell migration experiment.
Terahertz (THz) radiation refers to electromagnetic waves with the frequency of 0.1-10 THz and the wavelength of 30-3000 microns, and a Terahertz time-domain spectrometer has the advantages of high signal-to-noise ratio, capability of directly obtaining the refractive index and the like and is widely applied to biomedical research, including cancer imaging and the like. Meanwhile, the photon energy of 1THz is only 4.14meV, and the THz wave can not generate harmful ionization effect when penetrating through cells, so that the THz detection technology belongs to a pure physical, safe and effective non-destructive and label-free detection technology. The literature reports that living cells and dead cells can be successfully distinguished by utilizing different terahertz spectral characteristics, and a relevant theoretical support is provided for the application of terahertz in cell migration detection.
The characteristic structures with different cell numbers on the substrate have different dielectric response characteristics in the THz wave band, so that the cell migration can be detected by utilizing the terahertz spectrum. Terahertz detection has the problem of 'water sensitivity', the existing solution is to use a light transparentizing agent or introduce a microfluidic chip technology, and most of the light transparentizing agents belong to organic solvents and are incompatible with hydrophilic cells; PDMS microfluidic chips can cause large THz light absorption and increase the complexity of the experiment. If the microfluidic chip is not added, the cell growth area can not be limited. In view of the above problems, there is a need to provide a simple THz sensor device that enables rapid detection of cell migration.
Disclosure of Invention
In view of the above, the present invention is directed to a terahertz metamaterial sensor for cell migration and a detection method thereof, so as to partially solve at least one of the above technical problems.
In order to achieve the above object, as an aspect of the present invention, there is provided a terahertz metamaterial sensor comprising a substrate, a terahertz metamaterial structure sensing unit, and a polydimethylsiloxane enclosure for limiting a cell growth region, wherein:
the terahertz metamaterial structure sensing unit is formed on the substrate, and periodically arranged split rings and two circular ring alignment marks are formed on the terahertz metamaterial structure sensing unit;
the polydimethylsiloxane enclosure is located on the substrate and comprises a middle circular hollowed-out polydimethylsiloxane square piece and a solid polydimethylsiloxane cylindrical piece, wherein the inner diameter of the hollow circle of the polydimethylsiloxane square piece is aligned with an outer ring alignment mark on the terahertz metamaterial structure sensing unit, and the outer diameter of the solid polydimethylsiloxane cylindrical piece is aligned with an inner ring alignment mark.
The substrate is a rigid substrate, preferably made of high-resistance silicon or quartz glass, or a flexible substrate, preferably made of polyimide PI or parylene.
Wherein the split rings are E-center symmetric (left-right symmetric) to each other in pairs;
preferably, each of the periodically arranged cleavage rings is composed of two symmetrical square cleavage rings and a metal line, wherein the openings of the two square cleavage rings are centrosymmetric to each other.
Wherein the substrate size is 2 x 2cm2(ii) a The size of the opening of the cleavage ring is 2-4 mu m, and the thickness is 100 nm; the radius of the outer E-ring alignment mark is 0.5cm, the radius of the inner ring alignment mark is 1.5mm, and the width of a ring line is 100 nm.
Wherein the radius of the hollow circle in the polydimethylsiloxane square sheet is 0.5cm, and the size of the square sheet is 2 multiplied by 2cm2The thickness is 3-5 mm;
the radius of the solid polydimethylsiloxane cylindrical sheet is 1.5mm, and the thickness of the solid polydimethylsiloxane cylindrical sheet is 0.5-1 mm;
the polydimethylsiloxane enclosure is prepared by uniformly mixing silicon rubber 184 and a curing agent according to a ratio of 10:1, vacuumizing, pouring into a mold, and curing at 60-80 ℃ for 40 min-3 h by using a hot plate.
The square polydimethylsiloxane sheet and the substrate are irreversibly bonded through oxygen plasma, and the cylindrical polydimethylsiloxane sheet and the substrate are reversibly attached through van der Waals force.
As another aspect of the present invention, there is also provided a detection method of a terahertz metamaterial sensor for cell migration, including the steps of:
carrying out ultraviolet sterilization on the terahertz metamaterial sensor for 20-40 min, adding surface treatment protein, and incubating for 0.5-1.5 h to promote cell adherence;
adding the cell with the concentration of 1 × 106Putting the suspension per mL into an incubator for adherent culture;
when the confluence degree of cell growth reaches 90%, removing the polydimethylsiloxane cylindrical sheet in the middle;
absorbing the culture medium in the sensor, adding a stimulation factor culture medium with gradient concentration, and suspending stimulation at different time nodes;
the medium was aspirated off, cells were washed 3 times with PBS buffer, PBS was aspirated off;
in the terahertz transmission spectrum test system, the size of a light spot is 3mm, and the light spot is matched with a 3mm metal ring on a sensing substrate, so that a test area is accurately calibrated; and testing the transmission spectrum to obtain the transmission spectrum of different time points and different concentrations of stimuli, thereby obtaining the cell mobility in the suspension.
Wherein the surface treatment protein is fibronectin, gelatin or polylysine.
And calculating the cell migration rate through the obtained resonance frequency corresponding to the transmission spectrum based on the proportional relation that the surface dielectric environment change caused by the cell migration distance change is in direct proportion to the resonance frequency on the transmission spectrum.
Based on the technical scheme, compared with the prior art, the terahertz metamaterial sensor for cell migration and the detection method have at least one part of the following beneficial effects:
the invention adopts a symmetrical cleavage ring structure as a metamaterial structure sensing unit, and two pieces of PDMS are adopted to effectively limit a cell growth area and a THz detection area; the sensor has the advantages of simple preparation process, high detection speed and small required cell amount, and provides a quick and reliable method for detecting cell migration.
Drawings
FIG. 1 is a side view of a cell migration detection chip of a terahertz metamaterial according to an embodiment of the invention;
FIG. 2 is a top view of a cell migration detection chip of a terahertz metamaterial according to an embodiment of the invention;
FIG. 3 is a terahertz metamaterial sensing chip unit with a 4-inch substrate in the embodiment of the invention;
FIG. 4 is a 2 × 2cm terahertz metamaterial sensing unit in an embodiment of the present invention;
FIG. 5 is an optical microscope photograph of a cleaved ring structure in an embodiment of the present invention;
FIG. 6 is a flow chart of cell migration of a terahertz metamaterial according to an embodiment of the present invention.
In the above drawings, the reference numerals have the following meanings:
100. a terahertz metamaterial structure sensing region;
200. PDMS square pieces;
300. a PDMS wafer;
400. a cell;
101. bonding the PDMS square piece to an alignment ring;
102. attaching the PDMS wafer to the alignment ring;
103. cleaving the ring structure.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments. It should be noted, however, that the following detailed description is only a partial example of the present invention and should not be taken as limiting the scope of the present invention.
The invention discloses a terahertz metamaterial sensor which comprises a substrate, a terahertz metamaterial structure sensing unit and a polydimethylsiloxane fence used for limiting a cell growth area, wherein the terahertz metamaterial structure sensing unit comprises a plurality of terahertz metamaterial units, the polydimethylsiloxane fence comprises a plurality of terahertz metamaterial units, and the polydimethylsiloxane fence comprises the following components in percentage by weight:
the terahertz metamaterial structure sensing unit is formed on the substrate, and periodically arranged split rings and two circular ring alignment marks are formed on the terahertz metamaterial structure sensing unit;
the polydimethylsiloxane enclosure is located on the substrate and comprises a middle circular hollowed-out polydimethylsiloxane square piece and a solid polydimethylsiloxane cylindrical piece, wherein the inner diameter of the hollow circle of the polydimethylsiloxane square piece is aligned with an outer ring alignment mark on the terahertz metamaterial structure sensing unit, and the outer diameter of the solid polydimethylsiloxane cylindrical piece is aligned with an inner ring alignment mark.
The substrate is a rigid substrate, preferably made of high-resistance silicon or quartz glass, or a flexible substrate, preferably made of polyimide PI or parylene.
Wherein the split rings are E-center symmetric (left-right symmetric) to each other in pairs;
preferably, each of the periodically arranged cleavage rings is composed of two symmetrical square cleavage rings and a metal line, wherein the openings of the two square cleavage rings are centrosymmetric to each other.
Wherein the substrate size is 2 x 2cm2(ii) a The size of the opening of the cleavage ring is 2-4 mu m, and the thickness is 100 nm; the radius of the outer E-ring alignment mark is 0.5cm, the radius of the inner ring alignment mark is 1.5mm, and the width of a ring line is 100 nm.
Wherein the radius of the hollow circle in the polydimethylsiloxane square sheet is 0.5cm, and the size of the square sheet is 2 multiplied by 2cm2The thickness is 3-5 mm;
the radius of the solid polydimethylsiloxane cylindrical sheet is 1.5mm, and the thickness of the solid polydimethylsiloxane cylindrical sheet is 0.5-1 mm;
the polydimethylsiloxane enclosure is prepared by uniformly mixing silicon rubber 184 and a curing agent according to a ratio of 10:1, vacuumizing, pouring into a mold, and curing at 60-80 ℃ for 40 min-3 h by using a hot plate.
The square polydimethylsiloxane sheet and the substrate are irreversibly bonded through oxygen plasma, and the cylindrical polydimethylsiloxane sheet and the substrate are reversibly attached through van der Waals force.
The invention also discloses a detection method of the terahertz metamaterial sensor for cell migration, which comprises the following steps:
carrying out ultraviolet sterilization on the terahertz metamaterial sensor for 20-40 min, adding surface treatment protein, and incubating for 0.5-1.5 h to promote cell adherence;
adding the cell with the concentration of 1 × 106Putting the suspension per mL into an incubator for adherent culture;
when the confluence degree of cell growth reaches 90%, removing the polydimethylsiloxane cylindrical sheet in the middle;
absorbing the culture medium in the sensor, adding a stimulation factor culture medium with gradient concentration, and suspending stimulation at different time nodes;
the medium was aspirated off, cells were washed 3 times with PBS buffer, PBS was aspirated off;
in the terahertz transmission spectrum test system, the size of a light spot is 3mm, and the light spot is matched with a 3mm metal ring on a sensing substrate, so that a test area is accurately calibrated; and testing the transmission spectrum to obtain the transmission spectrum of different time points and different concentrations of stimuli, thereby obtaining the cell mobility in the suspension.
Wherein the surface treatment protein is fibronectin, gelatin or polylysine.
And calculating the cell migration rate through the obtained resonance frequency corresponding to the transmission spectrum based on the proportional relation that the surface dielectric environment change caused by the cell migration distance change is in direct proportion to the resonance frequency on the transmission spectrum.
In a preferred embodiment, the invention discloses a terahertz metamaterial sensor for cell migration, as shown in fig. 1, which comprises a terahertz metamaterial structure sensing unit substrate 100 and two pieces of Polydimethylsiloxane (PDMS) for limiting a cell growth area. The terahertz metamaterial structure sensing unit, as shown in fig. 4, includes a symmetric square split ring 101 and two ring alignment marks 102 and 103, which are periodically arranged. The PDMS, as shown in fig. 2, includes a PDMS square 200 with a central circular blank area and a solid PDMS disc 300.
The preparation method of the terahertz metamaterial sensor for cell migration comprises the following steps:
1) and preparing a regionalized split-ring metamaterial structural unit on the substrate by utilizing a photoetching process, as shown in FIG. 3.
The split ring metamaterial is composed of a substrate and metal open resonant rings periodically arranged on the substrate, each period is composed of two symmetrical square split rings and a metal line, and the openings of the split rings are centrosymmetric as shown in FIG. 5;
the substrate can be a rigid substrate (high-resistance silicon, quartz glass and the like) or a flexible substrate (polyimide (PI), Parylene (Parylene) and the like);
the size of the substrate is a square piece of 2 x 2 cm;
the size of the opening of the cleavage ring is 2-4 mu m, and the thickness is 100 nm;
the 2 x 2cm sensing unit is provided with two circular rings as alignment marks of the PDMS sheet, the radius of the outer ring is 0.5cm, the radius of the inner ring is 1.5mm, and the line of the circular ring is 100 nm.
2) A PDMS slab matched to the patterned sensing unit was prepared to confine the cell growth area.
The PDMS sheet for limiting the cell growth area consists of a square sheet and a solid circular sheet, wherein the square sheet and the solid circular sheet are circular and hollow inside;
the radius of the inner hollow circle of the square sheet with the circular inner part is 0.5cm, the size of the square sheet is 2 multiplied by 2cm, and the thickness of the square sheet is 3-5 mm;
the solid wafer has the radius of 1.5mm and the thickness of 0.5-1 mm;
the PDMS is prepared by uniformly mixing silicon rubber 184 and a curing agent according to a ratio of 10:1, vacuumizing, pouring into a mold, heating to 80 ℃ for 40min to realize curing, and carefully taking off the mold.
3) And combining the sensing unit substrate with the PDMS sheet to form a sensing chip.
The PDMS square piece and the substrate realize irreversible bonding through oxygen plasma;
and the PDMS wafer and the substrate are reversibly attached by Van der Waals force.
The detection method of the terahertz metamaterial sensor for cell migration in the embodiment comprises the following steps:
1) performing ultraviolet sterilization on the sensor for 30 min;
2) adding surface treatment protein, and incubating for 1h to promote cell adherence;
3) removing adhesion promoting protein by suction, and washing the sensor by PBS;
4) adding the cell with the concentration of 1 × 106Putting the suspension per mL into an incubator for adherent culture;
5) when the confluence degree of cell growth reaches 90%, removing the PDMS wafer in the middle;
6) absorbing the culture medium in the sensor, adding a culture medium containing a concentration gradient stimulating factor, and suspending stimulation at different time nodes;
7) the medium was aspirated off, cells were washed 3 times with PBS buffer, PBS was aspirated off;
8) in the terahertz transmission spectrum test system, the size of a light spot is 3mm, and the light spot is matched with a 3mm metal ring on a sensing substrate, so that a test area is accurately calibrated. Testing the transmission spectrum to obtain the transmission spectrum stimulated by different time points and different concentrations;
wherein, in fig. 1 and 2, 400 represents a cell;
FIG. 6 is a schematic diagram showing the gradual migration and growth of cells towards the center after removing the PDMS disc.
Two metal rings are arranged on the sensing substrate, so that the subsequent alignment with the PDMS sheet can be realized conveniently; the regional growth of cells can be realized by two pieces of PDMS; in addition, the mark of the inner 3mm circular ring is set to be matched with the size of the THz light spot, the testing area is in the 3mm circular ring every time, in-situ detection is realized, and the stability and the accuracy of the testing result are improved.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The terahertz metamaterial sensor is characterized by comprising a substrate, a terahertz metamaterial structure sensing unit and a polydimethylsiloxane enclosure for limiting a cell growth area, wherein:
the terahertz metamaterial structure sensing unit is formed on the substrate, and periodically arranged split rings and two circular ring alignment marks are formed on the terahertz metamaterial structure sensing unit;
the polydimethylsiloxane enclosure is located on the substrate and comprises a middle circular hollowed-out polydimethylsiloxane square piece and a solid polydimethylsiloxane cylindrical piece, wherein the inner diameter of the hollow circle of the polydimethylsiloxane square piece is aligned with an outer ring alignment mark on the terahertz metamaterial structure sensing unit, and the outer diameter of the solid polydimethylsiloxane cylindrical piece is aligned with an inner ring alignment mark.
2. The terahertz metamaterial sensor according to claim 1, wherein the substrate is a rigid substrate made of high-resistance silicon or quartz glass, or a flexible substrate made of polyimide PI or parylene.
3. The terahertz metamaterial sensor of claim 1, wherein the split rings are bilaterally symmetric to each other in pairs.
4. The terahertz metamaterial sensor according to claim 1, wherein each periodically arranged split ring is composed of two symmetrical square split rings and one metal line, wherein the openings of the two square split rings are centrosymmetric to each other.
5. The terahertz metamaterial sensor of claim 1, wherein the substrate size is 2 x 2cm2(ii) a The size of the opening of the cleavage ring is 2-4 mu m, and the thickness is 100 nm; the radius of the outer ring alignment mark is 0.5cm, the radius of the inner ring alignment mark is 1.5mm, and the width of the ring line is 100 nm.
6. The terahertz metamaterial sensor according to claim 1, wherein the radius of the hollow circle inside the polydimethylsiloxane square piece is 0.5cm, and the size of the square piece is 2 x 2cm2The thickness is 3-5 mm;
the radius of the solid polydimethylsiloxane cylindrical sheet is 1.5mm, and the thickness of the solid polydimethylsiloxane cylindrical sheet is 0.5-1 mm;
the polydimethylsiloxane enclosure is prepared by uniformly mixing silicon rubber 184 and a curing agent according to a ratio of 10:1, vacuumizing, pouring into a mold, and curing at 60-80 ℃ for 40 min-3 h by using a hot plate.
7. The terahertz metamaterial sensor as claimed in claim 1, wherein the square polydimethylsiloxane sheet and the substrate are irreversibly bonded through oxygen plasma, and the cylindrical polydimethylsiloxane sheet and the substrate are reversibly attached through van der Waals force.
8. A cell migration detection method using the terahertz metamaterial sensor as claimed in any one of claims 1 to 7, comprising the following steps:
carrying out ultraviolet sterilization on the terahertz metamaterial sensor for 20-40 min, adding surface treatment protein, and incubating for 0.5-1.5 h to promote cell adherence;
adding the cell with the concentration of 1 × 106Putting the suspension per mL into an incubator for adherent culture;
when the confluence degree of cell growth reaches 90%, removing the polydimethylsiloxane cylindrical sheet in the middle;
absorbing the culture medium in the sensor, adding a stimulation factor culture medium with gradient concentration, and suspending stimulation at different time nodes;
the medium was aspirated off, cells were washed 3 times with PBS buffer, PBS was aspirated off;
in the terahertz transmission spectrum test system, the size of a light spot is 3mm, and the light spot is matched with a 3mm metal ring on a sensing substrate, so that a test area is accurately calibrated; and testing the transmission spectrum to obtain the transmission spectrum of different time points and different concentrations of stimuli, thereby obtaining the cell mobility in the suspension.
9. The detection method according to claim 8, wherein the surface-treated protein is fibronectin, gelatin, or polylysine.
10. The detection method according to claim 8, wherein the cell migration rate is obtained from the resonance frequency corresponding to the obtained transmission spectrum based on a proportional relationship that the change in the surface dielectric environment caused by the change in the distance of cell migration is proportional to the resonance frequency on the transmission spectrum.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010014994.8A CN111141703B (en) | 2020-01-07 | 2020-01-07 | Terahertz metamaterial sensor for cell migration and detection method adopting terahertz metamaterial sensor |
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| CN202010014994.8A CN111141703B (en) | 2020-01-07 | 2020-01-07 | Terahertz metamaterial sensor for cell migration and detection method adopting terahertz metamaterial sensor |
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| CN111141703A CN111141703A (en) | 2020-05-12 |
| CN111141703B true CN111141703B (en) | 2020-12-15 |
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