阅读说明：本技术 一种可再生的太赫兹生物样品检测池的制备和应用 (Preparation and application of reproducible terahertz biological sample detection cell ) 是由 张阳 谢明真 府伟灵 于 2019-09-29 设计创作，主要内容包括：本发明申请属于生物样品检测技术领域,具体公开了一种超灵敏、无标记、可再生的特异性生物大分子检测方法,包括以下步骤：(1)可再生的太赫兹生物样品检测池的制备,包括衬底基片清洗、深硅刻蚀、镀膜、涂胶、曝光、显影、后烘、湿法刻蚀、切割；(2)通过聚乙烯亚胺－戊二醛交联法修饰高聚物膜条,制备抗体交联的检测膜条；(3)THz-TDs检测；(4)加入抗原孵育；(5)THz-TDs检测；计算其频移改变量Δf；(6)重复上述(3)-(5)步骤,可检测不同浓度的生物大分子溶液。本发明主要用于检测不同浓度的氧化低密度脂蛋白溶液,解决了太赫兹检测灵敏度不够、超材料的较难清洗的问题、实现了特异性、无标记的抗原检测。(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.)

1. The preparation method of the reproducible terahertz biological sample detection cell is characterized by comprising the following steps of:

(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 1.5-1.5 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 the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection cells.

2. The preparation method of the reproducible terahertz biological sample detection cell according to claim 1, wherein the organic solvent in step (1) is one of ethanol, acetone and methanol.

3. The preparation method of the reproducible terahertz biological sample detection cell according to claim 1, wherein the gas used in step (1) is high-pressure nitrogen.

4. The use of the renewable terahertz biological sample detection cell according to any one of claims 1 to 3, wherein the detection of oxidized low density lipoprotein through specific capture of an antibody can be used for specific sensing of biomolecules.

5. The application of the renewable terahertz biological sample detection cell as claimed in claim 4, wherein the detection comprises the following steps:

(1) and photoetching a periodic SRRs metamaterial structure in the detection cell. The unit structure is a single-opening resonance ring, the length and width of the single-opening resonance ring are 26 mu m, the line width is 6 mu m, and the opening gap is 2 mu m;

(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 reacting for 3 hours in a dark place, and cooling to room temperature;

(4) fully washing with deionized water for 2-3 times; adding 1ml of 1% glutaraldehyde solution, and standing at room temperature in the dark for 30 min;

(5) fully washing with deionized water for 2-3 times; adding 1ml of 0.1% DPBS solution 0.001g DDM, dissolving in 1ml of 10mmol/L PH7.4 buffer solution, 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 (wrapped with preservative film to prevent volatilization, and refrigerating in refrigerator at 4 deg.C overnight for use;

(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 the 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 sample pool by THz-TDS; calculating the delta f resonance rate variation;

(10) the test membrane strip is discarded and the test sample cell can continue to be used for the next sample measurement.

6. The use of the renewable terahertz biological sample detection cell as claimed in claim 5, wherein the 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.

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 be used for label-free, non-contact and nondestructive detection of biological substances based on THz. The weak interaction (such as hydrogen bond, Van der Waals force and the like), skeleton vibration, dipole rotation and the like among biological macromolecules such as nucleic acid, protein and the like are exactly 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.

The metamaterial (MMs) is a periodically arranged sub-wavelength artificial composite electromagnetic material and has the characteristics which are not comparable to 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. But 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 selection 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: the preparation method of the reproducible terahertz biological sample detection cell comprises the following steps:

the method 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 1.5-1.5 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 the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection cells.

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) and photoetching a periodic SRRs metamaterial structure in the detection cell. The unit structure is a single-opening resonance ring, the length and width of the single-opening resonance ring are 26 mu m, the line width is 6 mu m, and the opening gap is 2 mu m;

(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 reacting for 3 hours in a dark place, and cooling to room temperature;

(4) fully washing with deionized water for 2-3 times; adding 1ml of 1% glutaraldehyde solution, and standing at room temperature in the dark for 30 min;

(5) fully washing with deionized water for 2-3 times; adding 1ml of 0.1% DPBS solution 0.001g DDM, dissolving in 1ml of 10mmol/L PH7.4 buffer solution, 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 (wrapped with preservative film to prevent volatilization, and refrigerating in refrigerator at 4 deg.C overnight for use;

(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 the 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 sample pool by THz-TDS; calculating the delta f resonance rate variation;

(10) the test membrane strip is discarded and the test sample cell can continue to 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 use of the terahertz metamaterial is limited to a certain extent due to the problems of cleaning and recycling, so that the invention aims to develop a renewable terahertz metamaterial sample cell 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, and the metamaterial can be used only once, so that the cost is high, and the popularization and 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 biomolecules mainly comprise enzyme-linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay, chemiluminescence and the like, and all the methods need to be labeled by enzyme, fluorescein and radioactive elements; it is intended to develop an ultrasensitive, label-free method for specifically detecting a biomolecule.

(2)

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 resonant 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 illustration 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 to 9, a preparation method of 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 x 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 (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 unit structure is a single-aperture resonance ring, and has a length and a width of 26 μm (L ═ 26 μm), a line width of 6 μm (W ═ 6 μm), and an aperture gap of 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 the 4-inch silicon wafer into chips with the size of 1.5 x 1.5cm to obtain the detection cells.

Preparation of detection membrane 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 reproducible 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.

Application of the detection cell:

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 cell

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 micrometers (L is 26 micrometers), the line width is 6 micrometers (W is 6 micrometers), and the opening gap is 2 micrometers (g is 2 micrometers); 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 is recognized, and the epitope is phosphatidylcholine (PhoCho), a hydrophilic part in 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 (the solute of l g is dissolved in 99ml of water, namely the concentration is 1%), and standing at room temperature in a dark place for 30 min;

fully washing with deionized water for 2-3 times; 1ml of a 0.1% DPBS solution (0.001g of DDM in 1ml of 10mmol/L, PH 7.4.4 buffer) was added and soaked for 5 min.

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.