CN110658154B - Preparation method, detection method and application of reproducible terahertz biological sample detection pool - Google Patents
Preparation method, detection method and application of reproducible terahertz biological sample detection pool 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
- G01N21/25—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
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Abstract
The invention belongs to the technical field of biological sample detection, and particularly discloses an ultrasensitive, label-free and reproducible specific biomacromolecule detection method, which comprises the following steps: (1) The preparation method of the reproducible terahertz biological sample detection pool comprises the steps of substrate cleaning, deep silicon etching, film coating, gluing, exposure, development, post-baking, wet etching and cutting; (2) Modifying the high polymer membrane strip by a polyethyleneimine-glutaraldehyde crosslinking method to prepare an antibody crosslinked detection membrane strip; (3) THz-TDs detection; (4) adding antigen for incubation; (5) THz-TDs detection; calculating the frequency shift change quantity delta f of the frequency shift; (6) Repeating the steps (3) to (5) to detect the biomacromolecule solutions with different concentrations. The method is mainly used for detecting oxidized low-density lipoprotein solutions with different concentrations, solves the problems that the terahertz detection sensitivity is insufficient and the metamaterial is difficult to clean, and realizes specific unmarked antigen detection.
Description
Technical Field
The invention belongs to the technical field of biological sample detection, and particularly discloses a reproducible terahertz biological sample detection pool and an ultrasensitive, specific and quantitative detection method.
Background
The terahertz wave is an electromagnetic wave with the frequency of 0.1-10 THz, and can carry out label-free, non-contact and nondestructive detection on the biological substance based on the THz. The weak interaction (such as hydrogen bond, van der Waals force and the like) among biological macromolecules such as nucleic acid, protein and the like, skeleton vibration, dipole rotation and the like are just positioned in the THZ spectrum range, and each biological macromolecule has a specific THz spectrum fingerprint, so that the THz wave can detect the composition, structure, function and other information of the biological macromolecules, which cannot be obtained by other electromagnetic wave bands. The THz wave can reveal the structure and function information of a plurality of biomacromolecules at the same time node in a pure physical process without a marking mode, thereby possibly providing a revolutionary novel technical means for the unmarked detection of the biomacromolecules. However, some biological macromolecules do not have characteristic absorption peaks in the terahertz range, so that the application of terahertz in biological molecule sensing is limited.
As a periodically arranged sub-wavelength artificial composite electromagnetic material, metamaterials (MMs) have characteristics which are difficult to be compared with natural materials. The metamaterial with a certain structure can excite a high-quality resonance peak in a terahertz frequency domain range, and can effectively sense substances. THz-MMs based sensing still faces the following problems: 1. lack of a specific recognition system and poor specificity; 2. the minority specificity capture is based on the modification of gold on the surface of the metamaterial, and has no reproducibility and higher detection cost; 3. the gold surface area on the surface of the metamaterial is small, the amount of the bound antibody is limited, and the detection sensitivity is limited.
Monoclonal antibodies have high selective specificity, recognize specific antigens, and form stable antigen-antibody complexes. Through the specific capture of the antibody, the specific detection of the antigen can be realized. At the same time, capture by the antibody increases the sensitivity of the detection. At present, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay, chemiluminescence and the like are used as detection methods for antigen-antibody reaction, and all the detection methods need to be marked by enzyme, fluorescein and radioactive elements; this increases the number of steps in the test procedure, making the test more complex.
Disclosure of Invention
The invention aims to provide a reproducible terahertz biological sample detection pool and an ultra-sensitive quantitative detection method, and aims to solve the problems of multiple operation steps, special labeling and complex detection in the antigen specificity detection process.
In order to achieve the purpose, the technical scheme of the invention is as follows: a preparation method of a reproducible terahertz biological sample detection cell comprises the following steps:
(1) Cleaning a substrate: cleaning a 4-inch silicon wafer in an ultrasonic cleaning instrument, taking out, cleaning with an organic solvent, rinsing with deionized water, drying the surface of the silicon wafer with air, and drying to obtain a substrate;
(2) Deep silicon etching: a single channel with the length, the width and the depth of 220nm is carved in the center of each 1.5-1.5 cm area on the silicon wafer dried in the step (1);
(3) Film coating: plating a gold film with the thickness of 120nm on the surface of the channel;
(4) Gluing: uniformly spin-coating photoresist on the surface of the gold film, drying, and naturally cooling in a dark room;
(5) Exposure: carrying out periodic structural unit exposure on the uniformly coated surface by using an exposure machine; the unit structure is a single-opening resonance ring, the length and the width are 26 mu m, the line width is 6 mu m, and the opening gap is 2 mu m;
(6) And (3) developing: selecting a developing solution for developing;
(7) Post-baking: baking the developed substrate, removing residual moisture and developing solution, and improving the adhesion between the photoresist and the substrate;
(8) Wet etching: after wet etching is adopted, the gold film which is not protected by the photoresist is corroded, and then is washed by deionized water until the surface etching mixed solution is cleaned;
(9) And cutting, namely cutting the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection pool.
Further, the organic solvent in the step (1) is one of ethanol, acetone and methanol.
Further, the gas used in step (1) is high-pressure nitrogen.
The application of the reproducible terahertz biological sample detection pool can be used for specifically detecting oxidized low-density lipoprotein solutions with different concentrations.
Further, the detecting comprises the steps of:
(1) Photoetching a periodic SRRs metamaterial structure in a detection pool, wherein the unit structure is a single-opening resonance ring, the length and width of the unit structure are 26 micrometers, the line width is 6 micrometers, and the opening gap is 2 micrometers;
(2) Detecting the THz-TDS of the constructed renewable sample pool, and recording the resonant frequency f and the full width at half maximum of a resonant peak;
(3) Adding 1ml of 0.2% polyethyleneimine methanol solution into a reaction tank, placing the reaction tank in a thermostat at 70 ℃ for reacting for 3 hours in a dark place, and cooling to room temperature;
(4) Fully washing with deionized water for 2-3 times; adding 1% glutaraldehyde solution (1 ml), and standing at room temperature in dark for 30min;
(5) Fully washing with deionized water for 2-3 times; adding 1ml of 0.1% DPBS solution, which is 0.1% by weight of 0.001g DDM dissolved in 1ml of 10mmol/L pH7.4 buffer, and soaking for 5 min;
(6) Fully washing with deionized water for 2-3 times; soaking 500 μ l antibody E06 and anti-Ox-LDL solution, and refrigerating in a refrigerator at 4 deg.C overnight for use, wherein the soaking process is wrapped with preservative film to prevent solution volatilization;
(7) Taking out the membrane strip combined with the antibody, drying the membrane strip by using nitrogen, placing the membrane strip on a sample cell, detecting THz-TDS, enabling a resonance peak to generate frequency shift, and recording the resonance frequency f and the full width at half maximum (FWHM) of the resonance peak; calculating the delta f resonance rate variation;
(8) Adding 50 mul of oxidized low-density lipoprotein solution into the reaction tank, and incubating for 4h to ensure that the antigen and the antibody are fully combined;
(9) Taking out the membrane strip to be tested, and drying the membrane strip by nitrogen; placing on a terahertz detection sample pool, and detecting and recording the resonant frequency (f) and the full width at half maximum (FWHM) of a resonant peak by THz-TDS; calculating the delta f resonance rate variation;
(10) The test membrane strip is discarded and the test sample cell can be used for the next sample measurement.
Further, the steps (7) to (9) are sequentially repeated, and oxidized low density lipoprotein solutions with different concentrations are added, so that the oxidized low density lipoprotein solutions with different concentrations can be detected.
The working principle and the beneficial effects of the technical scheme are as follows:
(1) The terahertz metamaterial is a sensitive device which can be used in the field of terahertz sensing, but the cleaning and recycling problems of the terahertz metamaterial limit the use of the terahertz metamaterial to a certain extent, so that a renewable terahertz metamaterial sample pool is invented and developed for terahertz sensing.
(2) The terahertz metamaterial biosensor realizes specificity detection of biomolecules, and specificity capture of antibodies needs to be combined, but the modification of the antibodies on the surface of gold/silicon is difficult, the surface area of gold on the surface of the metamaterial is small, the amount of the combined antibodies is limited, the modification efficiency is low, the detection sensitivity is limited, and the detection efficiency is limited; meanwhile, the antibody is modified on the surface of the metamaterial, so that the metamaterial can be used only once, and the cost is high, and the popularization and the application of the metamaterial are not facilitated; PDMS is low in cost and can be modified by various chemical methods, so that the PDMS is convenient for popularization and application.
(3) The specific detection of biomolecules is realized by the specific capture of antibodies, and the current methods for the specific detection of the antibodies mainly comprise enzyme-linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay, chemiluminescence and the like, and all the methods need to be marked by enzyme, fluorescein and radioactive elements; the method is to develop an ultra-sensitive and label-free method for specifically detecting the biomolecules.
Drawings
FIG. 1 is a front view of an embodiment of a reproducible terahertz biological sample detection cell and an ultrasensitive quantitative detection method according to the present invention;
FIG. 2 is a top view of a test sample cell according to the present invention;
FIG. 3 is a top view of the single-split resonating ring of FIG. 2;
FIG. 4 is a schematic side view of the assembly of a universal sample cell and a disposable PDMS membrane strip;
FIG. 5 is a schematic diagram of detection without the test target;
FIG. 6 is a schematic diagram of detection for specifically recognizing a target to be detected;
FIG. 7 is a pictorial view of a test cuvette in accordance with the present invention;
FIG. 8 is a scanning electron micrograph of a single split resonant ring;
FIG. 9 is a pictorial representation of a pdms test strip;
fig. 10 is a graph of the results of terahertz detection thereof.
Detailed Description
The following is further detailed by way of specific embodiments:
referring to fig. 1-9, a method for preparing a reproducible terahertz biological sample detection cell includes the following steps: (1) cleaning a substrate: cutting a silicon wafer, putting the cut silicon wafer into an ultrasonic cleaning instrument for cleaning, taking out, cleaning with an organic solvent, rinsing with deionized water, wherein the organic solvent can be ethanol, acetone or methanol, preferably ethanol, and drying the surface of the silicon wafer with high-purity nitrogen;
(2) Deep silicon etching: etching a single channel with the length and width of 7200 mu m and the depth of 220nm in the center of each 1.5 × 1.5cm region on the silicon wafer dried in the step (1);
(3) Film coating: plating a gold film with the thickness of 120nm on the surface of the channel;
(4) Gluing: uniformly spin-coating photoresist (SU-8) on the surface of the gold film, drying, and naturally cooling in a dark room;
(5) Exposure: carrying out periodic structural unit exposure on the uniformly coated surface by using an exposure machine; as shown in fig. 2 and fig. 3, the cell structure is a single-opening resonant ring, the length and width of the single-opening resonant ring are 26 μm (L =26 μm), the line width of the single-opening resonant ring is 6 μm (W =6 μm), and the opening gap is 2 μm (g =2 μm);
(6) And (3) developing: selecting a developing solution for developing;
(7) Post-baking: baking the developed substrate, removing residual moisture and developing solution, and improving the adhesion between the photoresist and the substrate;
(8) Wet etching: after wet etching is adopted, the gold film which is not protected by the photoresist is corroded, and then is washed by deionized water until the surface etching mixed solution is cleaned;
(9) And cutting, namely cutting the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection pool.
Preparation of detection film strip
And crosslinking the antibody on the surface of the terahertz high polymer membrane strip by a polyethyleneimine-glutaraldehyde crosslinking method.
Detection of
And adding a sample to be detected into the reaction tank, and placing the sample and the reaction membrane strip for 4 hours at room temperature to ensure that the antigen and the antibody fully react. And taking out the detection membrane strip, placing the detection membrane strip on a renewable detection pool, and detecting the THz-TDS. Obtaining the curve of the concentration changing along with the frequency shift, and quantifying the biological samples with different concentrations.
The detection method of the detection cell comprises the following steps:
referring to fig. 4, 5 and 6, the reproducible sample cell and the specific detection membrane strip are used to detect oxidized low density lipoprotein.
Construction of reproducible detection sample pool
The detection pool is etched by the deep silicon, and the structural parameters are as follows: 7.2mm in length, 7.2mm in width and 220nm in height; 1.2 photoetching a sub-wavelength periodic SRRs metamaterial structure in a detection pool. The unit structure is a single-opening resonance ring, the length and width of the single-opening resonance ring are 26 μm (L =26 μm), the line width is 6 μm (W =6 μm), and the opening gap is 2 μm (g =2 μm); detecting the THz-TDS of the constructed renewable sample pool, and recording the resonant frequency (f) and the full width at half maximum (FWHM) of a resonant peak;
screening for antibodies
E06 was derived from a monoclonal antibody from an immunodeficient ApoE knockout mouse. Ox-PC was recognized, and the epitope was identified as phosphatidylcholine (PhoCho), a hydrophilic portion of lecithin.
E06 antibody was immobilized to PDMS surface;
adding 1ml of 0.2% polyethyleneimine methanol solution into a reaction tank, placing the reaction tank in a thermostat at 70 ℃ for reacting for 3 hours in a dark place, and cooling to room temperature.
Fully washing with deionized water for 2-3 times; adding 1ml of 1% glutaraldehyde solution (l g solute is dissolved in 99ml water, namely the amount concentration is 1%), and placing in the dark at room temperature for 30min;
fully washing with deionized water for 2-3 times; adding 1ml DPBS solution (0.001 g DDM dissolved in 1ml 10mmol/L buffer solution with pH of 7.4) with mass concentration of 0.1%, and soaking for 5min.
Fully washing with deionized water for 2-3 times; soaking 500 μ l antibody (anti-Ox-LDL) solution (wrapped with preservative film to prevent volatilization), and refrigerating in refrigerator at 4 deg.C overnight for use.
Taking out the membrane strip combined with the antibody, drying the membrane strip by using nitrogen, placing the membrane strip on a sample cell, detecting THz-TDS, enabling a resonance peak to generate frequency shift, and recording the resonance frequency (f) and the full width at half maximum (FWHM) of the resonance peak; calculating Δ f (resonance ratio variation); adding 50 mul of oxidized low density lipoprotein solution, and incubating for 4h to fully combine the antigen and the antibody; taking out the membrane strip to be tested, and drying the membrane strip by nitrogen; placing the terahertz sample on a terahertz detection sample pool, and detecting and recording the resonance frequency (f) and the full width at half maximum (FWHM) of a resonance peak of the terahertz sample pool by THz-TDS; Δ f (resonance ratio variation) is calculated. Discarding the detection membrane strip, and continuously using the detection sample pool for measuring the next sample; repeating the steps, adding oxidized low density lipoprotein solutions with different concentrations, wherein the different delta f (resonance rate variation) are different, and detecting the oxidized low density lipoprotein solutions with different concentrations.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent.
Claims (4)
1. The detection method of the reproducible terahertz biological sample detection cell is characterized by comprising the following steps of:
(1) Photoetching a periodic SRRs metamaterial structure in a detection pool, wherein the unit structure is a single-opening resonance ring, the length and width of the unit structure are 26 micrometers, the line width is 6 micrometers, and the opening gap is 2 micrometers;
(2) Detecting the THz-TDS of the constructed renewable sample pool, and recording the resonant frequency f and the half-height width of the resonant peak;
(3) Adding 1ml of 0.2% polyethyleneimine methanol solution into a reaction tank, placing the reaction tank in a thermostat at 70 ℃ for dark reaction for 3 hours, and cooling to room temperature;
(4) Fully washing with deionized water for 2-3 times; adding 1% glutaraldehyde solution (1 ml), and standing at room temperature in dark for 30min;
(5) Fully washing with deionized water for 2-3 times; adding 1ml of 0.1% DPBS solution, wherein 1ml of 0.1% DPBS solution is 0.001g DDM dissolved in 1ml of 10mmol/L pH7.4 buffer, and soaking for 5 min;
(6) Fully washing with deionized water for 2-3 times; soaking 500 μ l antibody E06 and anti-Ox-LDL solution, and refrigerating in a refrigerator at 4 deg.C overnight for use, wherein the soaking process is wrapped with preservative film to prevent solution volatilization;
(7) Taking out the membrane strip combined with the antibody, drying the membrane strip by using nitrogen, placing the membrane strip on a sample cell, detecting THz-TDS, enabling a resonance peak to generate frequency shift, and recording the resonance frequency f and the full width at half maximum (FWHM) of the resonance peak; calculating the delta f resonance rate variation;
(8) Adding 50 mul of oxidized low density lipoprotein solution into a reaction tank, and incubating for 4h to ensure that the antigen and the antibody are fully combined;
(9) Taking out the membrane strip to be tested, and drying the membrane strip by nitrogen; placing the terahertz sample on a terahertz detection sample pool, and detecting and recording the resonant frequency f and the full width at half maximum (FWHM) of a resonant peak of the terahertz sample pool by THz-TDS; calculating the delta f resonance rate variation;
(10) Discarding the detection membrane strip, and continuously using the detection sample pool for measuring the next sample;
the preparation method of the detection cell in the step (1) comprises the following steps:
(1) cleaning a substrate: cleaning a 4-inch silicon wafer in an ultrasonic cleaning instrument, taking out, cleaning with an organic solvent, rinsing with deionized water, drying the surface of the silicon wafer with air, and drying to obtain a substrate;
(2) deep silicon etching: etching a single channel with the length and width of 7.2mm and the depth of 220nm in the center of each 1.5 × 1.5cm region on the silicon wafer dried in the step (1);
(3) film coating: plating a gold film with the thickness of 120nm on the surface of the channel;
(4) gluing: uniformly spin-coating the photoresist on the surface of the gold film, drying, and naturally cooling in a dark room;
(5) exposure: carrying out periodic structural unit exposure on the uniformly coated surface by using an exposure machine; the unit structure is a single-opening resonance ring, the length and the width are 26 mu m, the line width is 6 mu m, and the opening gap is 2 mu m;
(6) and (3) developing: selecting a developing solution for developing;
(7) post-baking: baking the developed substrate, removing residual moisture and developing solution, and improving the adhesion between the photoresist and the substrate;
(8) wet etching: after wet etching is adopted, the gold film which is not protected by the photoresist is corroded, and then is washed by deionized water until the surface etching mixed solution is cleaned;
(9) and cutting, namely cutting the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection pool.
2. The detection method for the reproducible terahertz biological sample detection cell as claimed in claim 1, wherein steps (7) - (9) are repeated in sequence, and oxidized low density lipoprotein solutions with different concentrations are added, so that oxidized low density lipoprotein solutions with different concentrations can be detected.
3. The detection method of the reproducible terahertz biological sample detection cell as claimed in claim 1, wherein the organic solvent in step (1) of the preparation method of the detection cell is one of ethanol, acetone and methanol.
4. The detection method for the reproducible terahertz biological sample detection cell as claimed in claim 1, wherein the gas used in step (1) of the preparation method of the detection cell is high-pressure nitrogen.
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