CN111060475A - Cancer marker protein biosensors based on Parylene-C and related methods - Google Patents
Cancer marker protein biosensors based on Parylene-C and related methods Download PDFInfo
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- 239000000439 tumor marker Substances 0.000 title claims abstract description 32
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- 238000005406 washing Methods 0.000 claims description 6
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- 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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Abstract
A preparation method of a terahertz metamaterial cancer marker protein sensor comprises the following steps: step 1, growing a parylene C type film on a silicon substrate as a flexible substrate; step 2, cleaning the product obtained in the step 1, spin-coating photoresist on the product and drying the photoresist; step 3, exposing and developing the photoresist by using the designed metamaterial mask plate to obtain a photoresist mask; step 4, growing an adhesion layer and a gold film on the product obtained in the step 3 by using a thermal evaporation method; step 5, putting the product obtained in the step 4 into acetone for soaking and stripping to form a needed metamaterial gold structure; and 6, separating the flexible substrate and the silicon substrate in deionized water. The invention adopts the ultra-thin substrate with low refractive index, thereby improving the sensitivity and reducing the signal loss. And the adsorption capacity of the Parylene-C film is utilized in the modification method, so that the modification test process is simplified, the test data is reduced, and the sensitivity of the sensor is improved.
Description
Technical Field
The invention relates to the technical field of terahertz metamaterial biosensing, in particular to a terahertz metamaterial cancer marker protein biosensor based on a Parylene C (Parylene-C) film and a related method.
Background
The terahertz wave band is located between infrared and microwave, and is the transition field of electronics and photonics, and many biomacromolecule vibration energy levels are located in the terahertz wave band. In recent years, due to the development of nano-fabrication technology and terahertz spectroscopy, research in the terahertz field has also been gradually growing. The terahertz metamaterial is the main research direction, and the resonance peak of the terahertz metamaterial generates frequency shift along with the change of a surface environment, so that the frequency shift can be caused by performing biological modification on the surface of the terahertz metamaterial, and biological sensing is performed. The biosensor based on the terahertz metamaterial gradually becomes an important subject of research in the terahertz field due to the advantages that the biosensor does not damage a sample, does not need to be marked and the like. However, most terahertz metamaterial biosensors are based on a rigid substrate at present, and the application range of the biosensors is greatly limited. And the rigid substrate is accompanied by serious signal loss and echo reflection, so that the sensitivity of the current terahertz metamaterial sensor is low, and the practical application is limited. On the other hand, the surface biological modification method based on covalent bond binding has the disadvantages of complicated steps, long time consumption and low modification efficiency, so that the modification and test time consumption is long.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a terahertz metamaterial cancer marker protein biosensing method based on Parylene-C film, so as to partially solve at least one of the above technical problems.
In order to achieve the above object, as a first aspect of the present invention, there is provided a method for preparing a terahertz metamaterial cancer marker protein biosensor, comprising the steps of:
step 1, growing a parylene C type film on a silicon substrate as a flexible substrate;
step 2, cleaning the product obtained in the step 1, spin-coating photoresist on the product and drying the photoresist;
step 4, growing an adhesion layer and a gold film on the product obtained in the step 3 by using a thermal evaporation method;
step 5, putting the product obtained in the step 4 into acetone for soaking and stripping to form a needed metamaterial gold structure;
and 6, separating the flexible substrate and the silicon substrate in deionized water to prepare the terahertz metamaterial cancer marker protein sensor.
Wherein, in the step 1, parylene is grown by CVD, the growth evaporation temperature is 175 ℃, the cracking temperature is 690 ℃, the vacuum tube temperature is 135 ℃, the air pressure of the chamber is 18 hectopascal, and the thickness of the parylene C type film is 8-15 μm.
And 3, wherein the metamaterial mask pattern selected in the step 3 is a circular split ring.
Wherein, the adhesion layer in the step 4 is chromium with the thickness of 10nm, and the gold film has the thickness of 100 nm.
As a second aspect of the invention, the terahertz metamaterial cancer marker protein sensor prepared by the preparation method is also provided.
As a third aspect of the present invention, there is also provided a modification method comprising the steps of:
step 1, dripping a cancer marker protein antibody on the surface of a sensor for incubation, so that the antibody is physically adsorbed on the parylene C-type membrane, and then washing with phosphate buffered saline;
step 2, dropping bovine serum albumin on the surface of the sensor for incubation, wherein the bovine serum albumin is used for occupying vacant sites on the surface of the membrane, and then washing the membrane with phosphate buffer saline solution;
and 3, dripping the cancer marker protein antigen onto the surface of the sensor for incubation, combining the antigen and the antibody, and then washing with phosphate buffer saline solution.
Wherein, the type of the antibody in the step 1 is selected according to the requirement, the incubation time is 1 hour, and the incubation temperature is 37 ℃.
Wherein, in the step 2, the concentration of the bovine serum albumin is 3%, the incubation time is 1 hour, and the incubation temperature is 37 ℃.
Wherein the antigen selected in the step 3 is consistent with the antibody selected in the step 1, the incubation time is 1 hour, and the incubation temperature is 37 ℃.
As a fourth aspect of the present invention, there is also provided a test method, wherein the terahertz metamaterial cancer marker protein sensor is biologically modified by the modification method described above, the test method comprising the following steps:
step 1, drying a terahertz biosensor incubated with bovine serum albumin by using a nitrogen gun, placing the terahertz biosensor in a terahertz time-domain transmission spectrum testing system, and measuring a resonant frequency f corresponding to a transmission valley of the terahertz biosensor0；
Step 2, drying the terahertz biosensor incubated with the cancer marker protein antigen by using a nitrogen gun, placing the terahertz biosensor in a terahertz time-domain transmission spectrum test system, and measuring the resonant frequency f corresponding to the transmission valley of the terahertz biosensor1；
Based on said resonant frequency f1To the resonance frequency f0The difference is used to detect the change in concentration of the antigen.
Based on the technical scheme, compared with the prior art, the terahertz metamaterial cancer marker protein biosensor based on the Parylene-C film and the method have at least one part of the following beneficial effects:
compared with the existing terahertz sensor, the terahertz sensor has the advantages that the ultra-thin substrate with low refractive index is adopted, so that the sensitivity is improved, and the signal loss is reduced. And the adsorption capacity of the Parylene-C film is utilized in the modification method, so that the modification test process is simplified, the test data is reduced, and the sensitivity of the sensor is further improved.
Drawings
Fig. 1 is a flow chart of a preparation method of a terahertz metamaterial cancer marker protein biosensor based on a circular cleavage ring structure and shown in embodiment 1 of the invention;
FIG. 2 is a schematic structural diagram corresponding to each step of the manufacturing method of the modulation device shown in FIG. 1;
FIG. 3 is a flow chart of modification of a terahertz metamaterial cancer marker protein sensor in embodiment 2 of the present invention;
FIG. 4 is a flow chart of a terahertz metamaterial cancer marker protein sensor test in embodiment 3 of the present invention;
fig. 5 is a schematic three-dimensional structure diagram of the circular split ring structure sensor shown in step S106 in fig. 2;
fig. 6 is an optical microscope physical diagram of the circular split ring structure sensor shown in step S106 in fig. 2.
In the above drawings, the reference numerals have the following meanings:
1-Parylene-C film;
a 2-silicon substrate;
3-AR4340 photoresist;
4-chromium/gold.
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.
The ultra-thin flexible terahertz sensor with the low dielectric constant substrate is obtained by preparing a sub-wavelength terahertz metamaterial structure on a Parylene-C film grown by CVD (Chemical Vapor Deposition). Furthermore, the adsorption capacity and good biocompatibility of the Parylene membrane to the biological sample are utilized, a biological modification method for modifying the cancer marker protein to the sensor is provided, and a sensing test method is provided.
Specifically, the invention provides a preparation method and a biological modification and detection method of a flexible substrate metamaterial cancer marker protein sensor based on a Parylene-C membrane. The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all embodiments, and are not to be taken as limitations on the scope of the present invention.
Example 1
The invention provides a preparation method of a flexible substrate metamaterial cancer marker protein sensor based on a Parylene-C membrane, a preparation flow chart of the preparation method is shown in figure 1, a circular cleavage ring is prepared on the Parylene-C membrane, and the preparation method comprises the following steps:
s101: and (3) putting the clean silicon wafer substrate (2) into a CVD system for growing a Parylene film, setting CVD growth parameters, and growing the Parylene-C film (1). Preferably, the evaporation temperature is 175 ℃, the cracking temperature is 690 ℃, the vacuum tube temperature is 135 ℃, the chamber pressure is 18 hectopascal, and the raw materials are calculated and weighed according to the growth data, so that the thickness of the grown Parylene-C film is 8-15 μm.
S102: and putting the grown chip substrate into acetone, alcohol and water in sequence for ultrasonic treatment, then blowing the chip substrate to dry by using a nitrogen gun, and placing the chip substrate on a hot plate for drying. Preferably, the ultrasonic power is 20%, the ultrasonic time is 10 minutes, the hot plate temperature is 110 ℃, and the drying time is 10 minutes.
S103: and putting the substrate into a plasma gluing machine, and bombarding the substrate by using oxygen plasma. Preferably, the power of the degumming machine is 100W, and the bombardment time is 1 minute. The substrate is placed into a spin coater, AR4340 photoresist (3) is spin-coated, and the substrate is placed on a hot plate for prebaking. Preferably, the spin coating conditions are 1000 revolutions per second for 6 seconds at the initial rotation, and 20 seconds at 6000 revolutions per second at the high speed. The prebaking temperature was 110 ℃ and the mixture was heated for 2 minutes.
S104: photoetching a sample by using a mask plate with a circular split ring metamaterial, and performing photoetching by using a KMP PD238 developing solution and water in a ratio of 5: 2, and obtaining an annular photoresist mask. Putting the film on a hot plate for hardening. Preferably, the exposure dose is 123mw and the development time is 90 seconds.
S105: the chrome/gold plating (4) is deposited by a thermal evaporation method. Preferably, chromium is 10nm thick as an adhesion layer and gold is 100nm thick.
S106: and soaking the chip with the metal deposited in acetone to strip the metal on the photoresist. Then, the edge of the silicon substrate is scratched by a knife, the chip is placed in water, and the thin film is slowly separated from the silicon substrate.
Fig. 2 is a schematic structural diagram corresponding to each step of the manufacturing method of the modulation device in the flow of the manufacturing method.
Example 2
The invention provides a flexible substrate metamaterial cancer marker protein sensor biological modification method based on a Parylene-C film, a modification flow chart of the modification method is shown in figure 3, and a cancer marker protein Alpha Fetoprotein (AFP) is modified on a Parylene-C film terahertz metamaterial, and the method comprises the following steps:
and putting the prepared chip into a plasma degumming machine, and bombarding the substrate by using oxygen plasma. Preferably, the power of the degumming machine is 100W, and the bombardment time is 1 minute.
S201: an alpha-fetoprotein (AFP) antibody is added dropwise to the surface of the chip for incubation, preferably in a volume of 200. mu.L, at a temperature of 37 ℃ for 1 hour, and then washed three times with a Phosphate Buffered Saline (PBS) solution.
S202: bovine Serum Albumin (BSA) solution was added dropwise to the chip surface to occupy surface vacancies, preferably in a volume of 200. mu.L, and incubated for 1 hour at 37 ℃, followed by three washes with Phosphate Buffered Saline (PBS) solution.
S203: alpha-fetoprotein (AFP) antigen was added dropwise to the chip surface for incubation, preferably in a volume of 200. mu.L, at a temperature of 37 ℃ for 1 hour, and then washed three times with Phosphate Buffered Saline (PBS) solution.
Example 3
The invention provides a method for testing a flexible substrate terahertz metamaterial cancer marker protein sensor based on a Parylene-C film, a test flow chart of the testing method is shown in figure 4, and the method for testing the peak shift caused by modifying cancer marker protein Alpha Fetoprotein (AFP) on the Parylene-C film terahertz metamaterial sensor comprises the following steps:
s301: and (3) drying the chip modified with Bovine Serum Albumin (BSA) by using a nitrogen gun, and putting the chip into a terahertz time-domain spectroscopy test system to test data. Obtaining a transmission curve by Fourier transformation and comparing with air transmission data, and reading the position frequency f of a transmission valley0。
S302: and (3) drying the chip modified with Alpha Fetoprotein (AFP) by using a nitrogen gun, and putting the chip into a terahertz time-domain spectroscopy test system to test data. Obtaining a transmission curve by Fourier transformation and comparing with air transmission data, and reading the position frequency f of a transmission valley1。
By analysis of f1And f0The difference in (c) can be used to quantify the concentration of the test antigen.
Fig. 5 is a schematic three-dimensional structure diagram of the circular split ring structure sensor shown in step S106 in fig. 2, and fig. 6 is an optical microscope object diagram of the circular split ring structure sensor shown in step S106 in fig. 2.
According to the flexible substrate metamaterial cancer marker protein biosensor based on the Parylene-C film and the method, the terahertz metamaterial device is prepared on the Parylene-C film grown by CVD by using a traditional photoetching method, and the sensing device with the advantages of low-refractive-index flexible substrate, low loss, high sensitivity and the like can be obtained. The physical adsorption capacity and biocompatibility of the Parylene-C film are utilized, the modification method is simplified, and the modification and test time is reduced.
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. A preparation method of a terahertz metamaterial cancer marker protein sensor is characterized by comprising the following steps:
step 1, growing a parylene C type film on a silicon substrate as a flexible substrate;
step 2, cleaning the product obtained in the step 1, spin-coating photoresist on the product and drying the photoresist;
step 3, exposing and developing the photoresist by using the designed metamaterial mask plate to obtain a photoresist mask;
step 4, growing an adhesion layer and a gold film on the product obtained in the step 3 by using a thermal evaporation method;
step 5, putting the product obtained in the step 4 into acetone for soaking and stripping to form a needed metamaterial gold structure;
and 6, separating the flexible substrate and the silicon substrate in deionized water to prepare the terahertz metamaterial cancer marker protein sensor.
2. The method according to claim 1, wherein parylene is grown by CVD in step 1 at an evaporation temperature of 175 deg.C, a cracking temperature of 690 deg.C, a vacuum tube temperature of 135 deg.C, a chamber pressure of 18 kPa, and a thickness of 8-15 μm for parylene C.
3. The method according to claim 1, wherein the metamaterial mask pattern selected in step 3 is a circular split ring.
4. The method according to claim 1, wherein the adhesion layer in step 4 is chromium having a thickness of 10nm and the gold film has a thickness of 100 nm.
5. The terahertz metamaterial cancer marker protein sensor prepared by the preparation method of any one of claims 1 to 4.
6. The modification method of the terahertz metamaterial cancer marker protein sensor as claimed in claim 5, comprising the following steps:
step 1, dripping a cancer marker protein antibody on the surface of a sensor for incubation, so that the antibody is physically adsorbed on the parylene C-type membrane, and then washing with phosphate buffered saline;
step 2, dropping bovine serum albumin on the surface of the sensor for incubation, wherein the bovine serum albumin is used for occupying vacant sites on the surface of the membrane, and then washing the membrane with phosphate buffer saline solution;
and 3, dripping the cancer marker protein antigen onto the surface of the sensor for incubation, combining the antigen and the antibody, and then washing with phosphate buffer saline solution.
7. The modification method according to claim 6, wherein the type of antibody in step 1 is selected as required, and the incubation time is 1 hour and the incubation temperature is 37 ℃.
8. The modification method according to claim 6, wherein the bovine serum albumin concentration in step 2 is 3%, the incubation time is 1 hour, and the incubation temperature is 37 ℃.
9. The modification method of claim 6, wherein the antigen selected in step 3 is identical to the antibody selected in step 1, and the incubation time is 1 hour and the incubation temperature is 37 ℃.
10. The testing method of the terahertz metamaterial cancer protein sensor as claimed in claim 5, wherein the terahertz metamaterial cancer marker protein sensor is biologically modified by the modification method of any one of claims 6 to 9, and the testing method comprises the following steps:
step 1, drying a terahertz biosensor incubated with bovine serum albumin by using a nitrogen gun, placing the terahertz biosensor in a terahertz time-domain transmission spectrum testing system, and measuring a resonant frequency f corresponding to a transmission valley of the terahertz biosensor0；
Step 2, drying the terahertz biosensor incubated with the cancer marker protein antigen by using a nitrogen gun, placing the terahertz biosensor in a terahertz time-domain transmission spectrum test system, and measuring the resonant frequency f corresponding to the transmission valley of the terahertz biosensor1；
Based on said resonant frequency f1To the resonance frequency f0The difference is used to detect the change in concentration of the antigen.
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