阅读说明：本技术 一种液晶型适配体生物传感器及其制备方法和在检测前列腺特异性抗原中的应用 (Liquid crystal form aptamer biosensor, preparation method thereof and application thereof in detection of prostate specific antigen ) 是由 金丽虹 潘兴隆 吕九宫 牟晓雨 张翔宇 李晶怡 申炳俊 夏冰 高妍 于 2021-05-25 设计创作，主要内容包括：本发明涉及一种液晶型适配体生物传感器及其制备方法和在检测前列腺特异性抗原中的应用,所述生物传感器包括依次排列的经一定步骤处理的上玻片、铜载网和下玻片。本发明为快速检测前列腺特异性抗原(PSA)提供了一种新的方法,PSA液晶型适配体传感器具有特异性好、线性范围宽、灵敏度高、无需标记和检测方便快捷等优点,利于广泛应用。(The invention relates to a liquid crystal form aptamer biosensor, a preparation method thereof and application thereof in detecting prostate specific antigen. The invention provides a new method for quickly detecting Prostate Specific Antigen (PSA), and the PSA liquid crystal type aptamer sensor has the advantages of good specificity, wide linear range, high sensitivity, no need of marking, convenience and quickness in detection and the like, and is beneficial to wide application.)

1. The liquid crystal form aptamer biosensor is characterized by comprising an upper glass slide, a copper carrying net and a lower glass slide which are sequentially arranged; the upper glass slide and the lower glass slide are partially or completely overlapped, and the copper carrying net is arranged between the upper glass slide and the lower glass slide; wherein:

treating the assembled membrane substrate of the lower glass slide by mixed liquid of DMOAP and APTES, performing aldehyde modification on the amino group of the APTES membrane by using one aldehyde group of a dialdehyde compound, and fixing the 5' end of the other aldehyde group to modify an amino PSA adapter;

the copper carrying net is arranged in the center of a detection object surface of the lower glass slide for fixedly carrying the amino PSA aptamer, and 5CB liquid crystal molecules are distributed on the copper carrying net;

the slides were treated with DMOAP to induce vertical alignment of the 5CB liquid crystal molecules.

2. The method for preparing a liquid crystal type aptamer biosensor according to claim 1, comprising the steps of:

(1) pretreatment of glass slide: modifying the surface-cleaned glass slide with DMOAP, cleaning the modified glass slide, and soaking in 0.35% DMOAP water solution at room temperature for 30 min; then, rinsing with ultrapure water, N₂Drying by blowing, and drying for 1h at the constant temperature of 110 ℃ to obtain an upper glass slide on which the DMOAP film is fixed for later use;

(2) pretreatment of a lower glass slide: modifying the lower glass slide subjected to surface cleaning treatment by using a mixed solution of DMOAP and APTES to obtain a lower glass slide self-assembly DMOAP and APTES mixed membrane, cleaning, placing the lower glass slide into an absolute ethyl alcohol mixed solution of 0.3 percent of DMOAP and 0.3 percent of APTES in volume percentage, soaking the lower glass slide for 2 hours at the temperature of 80 ℃, then sequentially washing the lower glass slide by using absolute ethyl alcohol and ultrapure water, and carrying out N-step washing and N-step washing treatment on the lower glass slide by using N-step washing and N-step washing treatment₂Drying, namely drying at the constant temperature of 110 ℃ for 1 h; aldehyde modification is carried out on APTES membrane amino by using one aldehyde group of a dialdehyde compound, and the other aldehyde group is fixed at the 5' end to modify an amino PSA aptamer;

(3) copper net carrying pretreatment: placing a copper carrying net in the center of the detection object surface of the lower glass slide obtained in the step (2) for fixedly carrying the PSA aptamer, and transferring and distributing 5CB liquid crystal molecules to the whole copper carrying net;

(4) and (3) overlapping the upper glass slide and the lower glass slide which are obtained by the pretreatment in the steps (1) and (2), and placing the copper carrying net obtained by the pretreatment in the step (3) between the upper glass slide and the lower glass slide to obtain the biosensor which is formed by sequentially arranging the upper glass slide, the copper carrying net and the lower glass slide after the pretreatment.

3. The method of manufacturing according to claim 2, wherein the surface cleaning treatment is: soaking the upper glass slide or the lower glass slide in newly-prepared Piranha solution at 80 ℃ for 2h, cleaning, performing ultrasonic treatment in absolute ethyl alcohol for 30min to remove organic matters on the surfaces of the upper glass slide and the lower glass slide and hydroxylate the surface of the glass slide, then washing with a large amount of ultrapure water to remove residual acid liquor, N₂And (4) drying by blowing, drying at a constant temperature of 110 ℃ for 3-5 h, and taking out for dust prevention for later use.

4. The preparation method according to claim 2, wherein the lower slide pretreatment process is:

modifying the lower glass slide with APTES-GA/DMOAP to obtain the lower glass slide with the substrate of DMOAP and APTES composite membrane, soaking in 0.1 vol% GA water solution at room temperature for 45min to aldehyde-convert APTES on the surface of the lower glass slide, taking out, washing with ultrapure water, and N-washing₂Drying by blowing to obtain the lower glass slide with the modified APTES-GA/DMOAP substrate for later use.

5. The preparation method according to claim 2, wherein the lower slide pretreatment process is:

modifying the lower glass slide with APTES-GA-Apt/DMOAP, dripping 200 μ L of Tris-HCl solution with aptamer Apt concentration of 100pmol/L to the bottom surface of the membrane substrate of the lower glass slide modified with APTES-GA/DMOAP, incubating at 37 ℃ for 12h, washing with sodium dodecyl sulfate cleaning solution and ultrapure water with volume ratio of 0.005% and pH value of 7.0, removing the unfixed excessive aptamer on the lower glass slide, and small flow N₂And drying for later use.

6. The method of any one of claims 2 to 6, further comprising blocking unreacted aldehyde-based active sites on the lower slide: soaking the pretreated lower glass slide in 80mmol/L glycine solution at room temperature for 1h, rinsing with ultrapure water, and N₂The air is dried,removing the interference of non-specific adsorption substances.

7. The method according to any one of claims 2 to 6, wherein the amino PSA aptamer has a base sequence of: 5 '-5 AmMC 6-AATTAAAGCTCGCCATCAAATAGC-3'; wherein, 5AmMC6 refers to the modification of amino group at the 5' end of aptamer.

8. The method according to any one of claims 2 to 6, further comprising the step of incubating the following biosensor and PSA aptamer: 200 mu L of PSA aptamer solution, interferent or serum samples with different concentrations are dripped on the surface of the lower glass slide on which the 5' -end modified amino PSA aptamer is fixed, the reaction is carried out for 1h at room temperature, the lower glass slide is respectively washed by 0.01mol/L PBS buffer solution with pH7.4 and ultrapure water, and the small flow N is carried out₂And (5) drying.

9. Use of the biosensor according to any one of claims 1 to 8 for detecting prostate specific antigen, wherein the biosensor carrying detection samples with different concentrations of PSA is placed on a stage of a polarizing microscope, a digital camera on the polarizing microscope is used to obtain a polarization microscopic image, and PSA rapid label-free detection of the sample is achieved according to the bright area coverage of the polarization microscopic image.

10. The application of claim 9, wherein MATLAB is used to perform horizontal rectification on the polarization microscopic image, segment the liquid crystal film image, and calculate the bright area coverage rate of each detection region, wherein the bright area coverage rate is the bright area/the total interface area, and the bright area and the total interface area are equivalent to the number of pixels of the polarization microscopic image.

Technical Field

The invention relates to the technical field of chemical detection, in particular to a prostate specific antigen liquid crystal form aptamer biosensor with wide linear range and low detection limit, and a preparation method and quantitative detection application thereof.

Background

Prostate Specific Antigen (PSA) is a androgen regulated serine protease with a molecular weight of approximately 33kDa, a member of the kallikrein family, produced in epithelial cells of the Prostate gland and ducts. As the first tumor marker approved by FDA in the united states, PSA is widely used in prostate cancer patient screening and diagnosis, and has a sensitivity of more than 90% to prostate cancer. The concentration of PSA secreted by a healthy prostate into the human circulatory system is generally below 4.0ng/mL, with 4.0ng/mL being the concentration threshold for international general assessment of prostate cancer. For prostatectomy patients, lower serum PSA concentrations are required, and PSA above 0.1ng/mL after surgery indicates a risk of cancer recurrence. Since serum samples are tested before dilution is needed, this directly results in the actual concentration of PSA being measured well below this value. Therefore, the high sensitivity, specificity and low detection limit of the serum PSA detection method are very important for the screening, diagnosis and prognosis evaluation of the prostate cancer.

The clinical detection method for serum PSA is based on the immunological principle, such as radioimmunoassay, enzyme-linked immunoassay and chemiluminescence immunoassay, and the methods have the common problems of complicated operation steps, long reaction time and the like. In recent years, the development of advanced analytical techniques for PSA detection has become one of the important issues worldwide. The high-sensitivity PSA detection method by using antibodies and polypeptides as specific recognition molecules and utilizing the technologies of fluorescence, colorimetry, Dynamic Light Scattering (DLS), electrochemical analysis and the like is reported successively, and the PSA detection limit can reach fg-ng/mL. However, antibodies and polypeptides have the problems of long preparation period, high cost, easy inactivation and the like, which limits the wide clinical application of the antibodies and polypeptides to a certain extent.

An Aptamer (Aptamer) is an artificially selected single-stranded DNA or RNA molecule that can bind to a target with high specificity and high affinity, a process similar to antigen-antibody binding reactions. Compared with antibodies, aptamers have many advantages such as small size, chemical stability, low cost, and non-harsh storage conditions when applied to the field of biosensing as recognition elements. In 2017, Yang et al applied the aptamer to a Surface Enhanced Raman Spectroscopy (SERS) technology, separated free and bound gold nanoparticles by a magnet, and then performed PSA detection according to the SERS signal, wherein the linear range of the PSA detection is 5-500 pg/mL, and the detection limit is as low as 5.0 pg/mL. In 2012, Chen et al used a resonance scattering spectroscopy (RLS) method to achieve quantitative detection of PSA in a linear range of 0.13-110 ng/mL, with a detection limit of 32 pg/mL. However, the SERS and RLS analysis methods have the disadvantages of complicated operation and expensive equipment or consumables, and are difficult to be widely used.

Liquid Crystals (LC) are widely used in sensor research due to their unique properties of Liquid flow and solid anisotropy. The liquid crystal type biosensor utilizes the orientation change condition of liquid crystal molecules on the surface of a sensitive film before and after the sensor detects target molecules and the change of the light refraction capacity of liquid crystal to cause the change of the color and the brightness of the sensor, thereby realizing the detection of the target molecules. In recent years, more and more tumor marker aptamers are screened, and a liquid crystal aptamer biosensor constructed by taking the aptamers as molecular recognition elements and liquid crystal molecules as signal amplification and transduction elements provides a new way for tumor marker detection. Currently, only a few tumor markers, liquid crystal aptamer biosensors, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), mucin (MUC1), and PSA, are studied. In 2020, after magnetic separation of magnetic beads-Apt 1-PSA-Apt2, quantitative detection of PSA is realized according to the difference of the influence of the complementary single strands of the partial double-stranded aptamer 2 and the aptamer 2 on the orientation of liquid crystal molecules by using PSA captured by the coupled magnetic bead aptamer 1(Apt1) by Qi and the like to further bind the aptamer 2(Apt2), and the linear range of the PSA is 0.04-0.5 ng/mL, and the detection limit is 70 pg/mL. The document reports that the PSA liquid crystal type aptamer sensor has the problems of narrow detection linear range, high cost of single-chain and double-chain aptamers, and the like.

The invention provides a preparation and quantitative detection method of a PSA liquid crystal aptamer sensor, which has the advantages of good specificity, high sensitivity, wide detection range, lightness, portability, low cost, simple operation and no need of expensive detection instruments, and aims to solve the problems in the background art.

Disclosure of Invention

The invention aims to provide a liquid crystal form aptamer biosensor, a preparation method thereof and application of the liquid crystal form aptamer biosensor in quantitative detection of PSA (pressure-sensitive adhesive), so as to solve the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

a liquid crystal form aptamer biosensor comprises an upper glass slide, a copper carrying net and a lower glass slide which are sequentially arranged; the upper glass slide and the lower glass slide are partially or completely overlapped, and the copper carrying net is arranged between the upper glass slide and the lower glass slide; wherein:

treating the assembled film substrate of the lower glass slide by using a mixed solution of N, N-dimethyl-N-octadecyl ammonium chloride (DMOAP) and 3-aminopropyl triethoxysilane (APTES), performing aldehyde modification on the amino group of the 3-aminopropyl triethoxysilane film by using one aldehyde group of a dialdehyde compound, and fixing a 5' end modified amino PSA adapter by using the other aldehyde group;

the copper carrying net is arranged in the center of a detection object surface of the lower glass slide for fixedly carrying the PSA aptamer, and 4-cyano-4-pentylbiphenyl (5CB) liquid crystal molecules are transferred to the whole copper carrying net;

the glass slides were treated with N, N-dimethyl-N-octadecylammonium chloride to induce vertical alignment of the liquid crystal molecules of the 4-cyano-4 pentylbiphenyl.

The preparation method of the liquid crystal type aptamer biosensor comprises the following steps:

(1) pretreatment of glass slide: modifying the surface-cleaned glass slide with N, N-dimethyl-N-octadecyl ammonium chloride (DMOAP), cleaning the modified glass slide, and soaking in 0.35 vol% of N, N-dimethyl-N-octadecyl ammonium chloride at room temperatureAlkyl ammonium chloride (DMOAP) aqueous solution for 30 min; then, rinsing with ultrapure water, N₂Drying by blowing, and drying for 1h at the constant temperature of 110 ℃ to obtain an upper glass slide on which an N, N-dimethyl-N-octadecyl ammonium chloride film is fixed for later use;

(2) pretreatment of a lower glass slide: modifying the lower glass slide subjected to surface cleaning treatment by using a mixed solution of N, N-dimethyl-N-octadecyl ammonium chloride (DMOAP) and 3-Aminopropyltriethoxysilane (APTES) to obtain a lower glass slide self-assembly DMOAP and APTES mixed membrane, cleaning, placing the lower glass slide into an absolute ethyl alcohol mixed solution with the volume percentage of 0.3 percent DMOAP and 0.3 percent APTES, soaking at 80 ℃ for 2 hours, sequentially washing with absolute ethyl alcohol and ultrapure water, and performing N-stage washing and N-stage washing to obtain the finished product₂Drying, namely drying at the constant temperature of 110 ℃ for 1 h; then aldehyde group modification is carried out on the amino group of the 3-aminopropyl triethoxysilane (APTES) membrane by using an aldehyde group of a dialdehyde compound, and the 5' end of the other aldehyde group is fixed to modify an amino PSA adapter;

(3) copper net carrying pretreatment: placing a copper carrying net in the center of the detection object surface of the lower glass slide obtained in the step (2) for fixedly carrying the PSA aptamer, and transferring and distributing 4-cyano-4-pentylbiphenyl (5CB) liquid crystal molecules to the whole copper carrying net;

(4) and (3) overlapping the upper glass slide and the lower glass slide which are obtained by the pretreatment in the steps (1) and (2), and placing the copper carrying net obtained by the pretreatment in the step (3) between the upper glass slide and the lower glass slide to obtain the biosensor which is formed by sequentially arranging the upper glass slide, the copper carrying net and the lower glass slide after the pretreatment.

The surface cleaning treatment comprises the following steps: soaking the upper glass slide or the lower glass slide in newly-prepared Piranha solution at 80 ℃ for 2h, cleaning (washing with absolute ethyl alcohol) and performing ultrasonic treatment in absolute ethyl alcohol for 30min to remove organic matters on the surfaces of the upper glass slide and the lower glass slide and hydroxylate the surface of the glass slide, washing with a large amount of ultrapure water to remove residual acid liquor, N₂And (4) drying by blowing, drying at a constant temperature of 110 ℃ for 3-5 h, and taking out for dust prevention for later use.

The copper net is 150 meshes and 20 mu m thick.

The upper and lower slides are cut from the slide, with recommended dimensions of 2cm x 2 cm.

The Piranha solution has a volume ratio of H₂SO₄：H₂O is 7: 3, in water.

The lower slide pretreatment process comprises the following steps:

modifying the lower glass slide with APTES-GA/DMOAP to obtain the lower glass slide with the substrate of DMOAP and APTES composite membrane, soaking in 0.1 vol% GA water solution at room temperature for 45min to aldehyde-convert APTES on the surface of the lower glass slide, taking out, washing with ultrapure water, and N-washing₂Drying by blowing to obtain the lower glass slide with the modified APTES-GA/DMOAP substrate for later use.

The slide pretreatment process may further include the steps of:

the lower slide was modified with APTES-GA-Apt/DMOAP layer, and 200. mu.L of Tris-HCl solution (containing 10mmol/L Tris, 150mmol/L NaCl, 5mmol/L KCl and 5mmol/L MgCl) at a concentration of 100pmol/L aptamer Apt was placed in the slide₂pH7.4) was added dropwise to the surface of the membrane substrate of the lower slide glass in which the APTES-GA/DMOAP layer was modified, incubation was carried out at 37 ℃ for 12 hours, washing was carried out with a Sodium Dodecyl Sulfate (SDS) washing solution and ultra-pure water in a volume ratio of 0.005% and a pH of 7.0, excess aptamer not immobilized on the lower slide glass was removed, and a small flow of N was applied₂And drying for later use.

The preparation method also comprises the following steps of blocking unreacted aldehyde active sites on the lower glass slide: soaking the pretreated lower glass slide in 80mmol/L glycine solution at room temperature for 1h, rinsing with ultrapure water, and N₂Drying and removing the interference of non-specific adsorption substances.

The base sequence of the amino PSA aptamer is as follows: 5 '-5 AmMC 6-AATTAAAGCTCGCCATCAAATAGC-3'; wherein, 5AmMC6 refers to the modification of amino group at the 5' end of aptamer.

The preparation method also comprises the following steps of incubating the biosensor and the PSA aptamer: 200 mu L of PSA aptamer solution, interferent or serum samples with different concentrations are dripped on the surface of the lower glass slide on which the 5' -end modified amino PSA aptamer is fixed, the reaction is carried out for 1h at room temperature, the lower glass slide is respectively washed by 0.01mol/L PBS buffer solution with pH7.4 and ultrapure water, and the small flow N is carried out₂And (5) drying.

The 4-cyano-4-pentylbiphenyl (5CB) was treated as follows: heating to > 35 deg.C (e.g., 40 deg.C) yields isotropic liquid 5 CB.

The biosensor is applied to detecting prostate specific antigen, the biosensor which is immobilized with detection samples with PSA of different concentrations (the detection samples can be selected from PBS solution, interferents, serum samples and the like) is placed on a stage of a polarizing microscope (SOPTOP-CX40P, Ningbo Shun, China), a digital camera (SONY-IMX178, Japan) on the polarizing microscope is used for obtaining a polarization microscopic image, and the rapid and label-free detection of the PSA of the sample is realized according to the bright area coverage rate of the polarization microscopic image.

Further, the application comprises the steps of utilizing MATLAB to horizontally correct the polarization microscopic image, segmenting the liquid crystal film area image, and calculating the bright area coverage rate of each detection area, wherein the bright area coverage rate is equal to the bright area/the total interface area, and the bright area and the total interface area are equivalent to the number of pixels of the polarization microscopic image.

When no PSA was present in the sample, the sensor exhibited a completely black polarized microscopic image due to the small volume of PSA aptamer assembled from the lower slide, which was insufficient to disturb the vertical orientation of the liquid crystal 5 CB; when PSA exists in a sample, the PSA can induce the spatial conformation change of the aptamer fixed on the lower glass slide and is specifically combined with the aptamer, and when the concentration of the PSA reaches a certain amount, the vertical alignment arrangement of liquid crystal molecules can be disturbed, so that the color and the brightness of a polarization microscopic image of the liquid crystal type aptamer sensor are changed. The rapid label-free detection of the sample PSA can be realized according to the measured bright area coverage (Br) of the polarized image, and the schematic diagram of the detection PSA of the liquid crystal type aptamer sensor and the assembly process of the liquid crystal cell are shown in FIG. 2.

Compared with the prior art, the invention has the beneficial effects that:

(1) one aptamer single chain is used as a molecular recognition element of the liquid crystal aptamer sensor, and compared with antibodies and polypeptides, the aptamer single chain has the advantages of small volume, chemical stability, low generation cost and non-harsh storage conditions, and the PSA detection operation process is simpler and more convenient than the PSA detection operation process of a liquid crystal sensor assisted by magnetic separation based on one aptamer single chain and one partial double-helix aptamer chain.

(2) The 5CB liquid crystal molecules are used as a signal amplification and transduction element of the liquid crystal type aptamer sensor, the PSA content is obtained according to the bright area coverage rate of the polarized light microscopic image, and expensive instruments are not needed in the detection process.

(3) The linear range and sensitivity of the sensor for detecting PSA are improved and the detection limit of PSA is reduced by increasing the immobilization amount of the lower slide modified aptamer molecular recognition element, automatically identifying MATLAB and calculating the bright area coverage of a polarization microscopic image.

In conclusion, the invention provides a new method for rapidly detecting Prostate Specific Antigen (PSA), and the PSA liquid crystal type aptamer sensor has the advantages of good specificity, wide linear range, high sensitivity, no need of marking, convenience and rapidness in detection and the like.

Drawings

FIG. 1 is a schematic structural view of a liquid crystal type aptamer biosensor in example 4.

FIG. 2 is a schematic diagram of the detection of prostate specific antigen by the liquid crystal type aptamer sensor and the assembly process of the liquid crystal cell in example 7.

FIG. 3 is a polarizing microscope image of the slide glass after DMOAP modification in example 7.

FIG. 4 is a polarization micrograph of APTES/DMOAP modified slides at different concentration ratios from example 1.

FIG. 5 is a (APTES-GA)/DMOAP modified slide glass polarization microscope image of different concentrations of GA in example 2.

FIG. 6 is a (APTES-GA-Apt)/DMOAP modified lower slide polarization microscope image of aptamers of different concentrations in example 3.

Fig. 7 is a polarization microscopic image of the liquid crystal type aptamer biosensor in example 4 with different concentrations of PSA.

FIG. 8 is a graph of the linear relationship between the PSA concentration and the bright area coverage (Br) of the polarization microscopic image of the liquid crystal type aptamer biosensor in example 4.

FIG. 9 shows the level correction of the polarization microscopic image by the MATLAB software in example 5.

FIG. 10 is an image segmentation of the liquid crystal film region by the MATLAB software of example 5.

FIG. 11 is the MATLAB software UI interface for polarized light microscopy image processing in example 5.

FIG. 12 is a graph showing the calculation of the bright area coverage of each cell detection area by the MATLAB software in example 5.

FIG. 13 is a polarization microscopic image of the LC type aptamer biosensor under the interferent in example 6.

FIG. 14 is a polarization micrograph of an aptamer biosensor in liquid form under a sample of PSA serum in example 8.

Detailed Description

The following detailed description of the embodiments of the present invention will be made with reference to the accompanying drawings, which are not intended to limit the scope of the invention. All equivalent changes, modifications and the like made within the scope of the present invention shall fall within the protection and coverage of the present invention. In addition, it should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

Example 1: proportion optimization of APTES/DMOAP mixed membrane on surface of sensor substrate in self-assembly mode

In order to ensure uniform vertical orientation of liquid crystal molecules of the liquid crystal sensor 5CB, more aptamer molecules are fixedly carried on the lower glass slide substrate of the sensor to improve the PSA detection range, and the proportion optimization of APTES and DMOAP of the APTES/DMOAP mixed film self-assembled on the surface of the lower glass slide substrate of the sensor is carried out.

(1) The concentration of DMOAP modified by the lower slide was fixed at 0.3% (v/v), and the APTES concentrations were varied at 0.3%, 1.5% and 3.0%, respectively. Soaking the cleaned lower glass slide in the absolute ethyl alcohol mixed solution of APTES and DMOAP, soaking at 80 deg.C for 2h, washing with a large amount of absolute ethyl alcohol and ultrapure water, and sequentially washing with N₂And (4) drying by blowing, drying at the constant temperature of 110 ℃ for 1h, and finishing the self-assembly of the APTES/DMOAP mixed membrane on the surface of the lower slide substrate of the sensor.

(2) The APTES/DMOAP concentration ratio is 1: 1, the lower slide polarization microscope image appeared uniformly full black (see fig. 4 a); the APTES/DMOAP concentration ratio is 5: 1, the lower slide polarization microscope image still appeared uniformly full black (see fig. 4 b); the APTES/DMOAP concentration ratio is 10: 1, the lower slide polarization microscope image appeared visibly bright texture (see fig. 4 c). It can be seen that when the ratio of the APTES/DMOAP concentration is 1: 1 and 5: 1, the polarizing microscopic image is uniformly black, 1: 1 and 5: 1 is the optimal concentration ratio of the APTES/DMOAP mixed membrane self-assembled on the surface of the sensor substrate.

Example 2: APTES/DMOAP ═ 1: 1-hour sensor substrate surface GA and aptamer concentration optimization

In order to immobilize the amino group-modified aptamer to the surface of the lower slide, aldehyde treatment of APTES modified on the surface of the lower slide is required. Slides of self-assembled APTES/DMOAP mixed membranes were incubated with 0.1% and 0.5% (v/v) GA, respectively.

(1) The lower slide of the obtained (APTES-GA)/DMOAP mixed film was treated with 0.1% GA to further couple with aminated aptamers, at an aptamer concentration of 100nmol/L, significant colored and bright areas appeared in the polarization microscopy images (see FIG. 5a), and the 5CB molecular orientation within the copper carrier network was affected by excess aptamer; when the aptamer concentration is 1nmol/L, part of light spots still exist in the image (see figure 5 b); when the aptamer concentration was reduced to 100pmol/L, the polarization microscopy image appeared uniformly black (see FIG. 5 c).

(2) The lower slide of the obtained (APTES-GA)/DMOAP mixed film was treated with 0.5% GA to further couple aminated aptamers, and at an aptamer concentration of 100nmol/L, the polarization microscopic image showed colored and bright areas (see FIG. 5d), and the excess aptamers affected the orientation of the 5CB molecules in the copper carrier network; when the aptamer concentration is 1nmol/L, part of light spots still exist in the image (see figure 5 e); when the aptamer concentration was reduced to 100pmol/L, the polarization microscopy image appeared uniformly black (see FIG. 5 c).

Thus, APTES/DMOAP ═ 1: the concentration of aldehyde group-modified GA in the sensor substrate modified by 1 is 0.1%. Example 3: APTES/DMOAP ═ 5: 1-hour sensor substrate surface GA and aptamer concentration optimization

In order to increase the number of aptamer molecules immobilized on the surface of the glass slide substrate under the sensor, the number of aldehyde-formed molecules of APTES needs to be increased. The surface of the lower slide substrate was incubated with 0.1% and 0.5% (v/v) GA for self-assembled APTES/DMOAP mixed membranes, respectively. 0.1% (v/v) GA aldehydized (APTES-GA)/DMOAP composite layer lower glass slide, the polarization microscopic image presents a uniform black color (see figure 6a), further fixing aptamer; when the aptamer concentration was 100pmol/L, the polarization microscope image appeared as a large area of color and brightness (see FIG. 6 b). In order to fix more aptamer molecules on the surface of a lower glass slide of a sensor substrate to improve the sensitivity of the liquid crystal aptamer biosensor in detecting PSA, the optimized conditions of embodiment 2 are adopted in the patent, namely the conditions of a sensor substrate self-assembly (APTES-GA)/DMOAP film are as follows: 0.3% APTES, 0.3% DMOAP and 0.1% GA, at an aptamer concentration of 100 pmol/L.

Example 4: sensor sensitivity investigation

(1) Dripping PSA solutions with different concentrations onto an aptamer-immobilized lower glass slide, soaking the lower glass slide with 80mmol/L glycine solution, and sealing aldehyde group non-specific active sites which are not coupled with the aptamer on the surface of the lower glass slide; the glass plate is assembled with a DMOAP decorated upper glass slide face to form a liquid crystal box, a 5CB liquid crystal molecule copper carrying net is clamped at the central part, and the assembly structure is shown in an instruction book figure 1.

(2) The higher the solution PSA concentration, the more aptamer molecules bound to it, the greater the disruption of the vertical alignment of the 5CB liquid crystal molecules, and the more distinct the colored bright areas and bright textures in the polarized microscopic image. At a PSA concentration of 1. mu.g/mL, the polarization micrograph exhibits a color texture morphology (see FIG. 7 a); when the PSA concentration is 100ng/mL, 1ng/mL and 1pg/mL, the polarization microscopic image still has a color texture form (see FIGS. 7b, c and d), which is because the PSA molecules bound on the surface of the sensor substrate are larger, the damage to the vertical orientation of the 5CB liquid crystal molecules is still larger, and the birefringence phenomenon is obvious; when the PSA concentration is as low as 1fg/mL, there is still a clear optical signal response (see FIG. 7 e); at PSA concentrations as low as 0ng/mL, the polarization microscopy images appear black with no optical signal response (see fig. 7 f).

(3) The MATLAB automatically identifies and calculates the bright area coverage of the polarized microscopic image with PSA concentration in the range of 0-1 mug/mL, and the result is shown in figure 8. The logarithm of PSA concentration has a linear relation with the bright area coverage (Br), and the linear equation is Br 7.869998logCPSA +6.50541, R2 is 0.99413; the lower detection limit of the liquid crystal aptamer biosensor is 1fg/L, and the detection limit is 81 fg/L.

Example 5: automatic identification liquid crystal type aptamer biosensor polarization microscopic image and calculation of bright area coverage

Using MATLAB to perform horizontal correction of polarization microscopic images, segmenting images of liquid crystal film regions, and calculating the bright area coverage rate of each detection region, wherein the bright area coverage rate (Br) is the area of a bright area/the total area of an interface, and the results are shown in FIGS. 9 to 12.

Part of the procedure is as follows:

flag1＝0；flag2＝0；flag3＝0；flag4＝0；

% upper part

% lower part

% left part

% right part

Example 6: sensor specificity survey

Five serum tumor markers, namely Human Serum Albumin (HSA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 19-9(CA19-9), carbohydrate antigen 12-5(CA12-5) and carbohydrate antigen 72-4(CA72-4), are selected to be used as specific control experiments. The results of the experimental control group showing that HSA concentration of 42g/L, CEA concentration of 5. mu.g/L, AFP concentration of 20. mu.g/L, CA19-9 concentration of 37kU/L, CA12-5 concentration of 35kU/L and CA72-4 concentration of 7U/L were obtained in the same experimental method as PSA are shown in FIG. 13. It can be seen that the polarization microscope images of the six control experiments all exhibited a completely black background, and that there were small scattered bright spots in the polarization microscope images of the individual interferents, probably due to some unreacted material remaining on the sensor substrate slide. Therefore, the liquid crystal aptamer biosensor has good specificity for PSA detection.

Example 7: construction of the biosensor

(1) Cutting the glass slide into 2cm × 2cm to obtain upper and lower glass slides, soaking in newly-prepared Piranha solution at 80 deg.C for 2h, washing with anhydrous ethanol, ultrasonic treating in anhydrous ethanol for 30min to remove organic substances on the surfaces of the upper and lower glass slides to hydroxylate the surfaces of the glass slides, washing with large amount of ultrapure water to remove residual acid solution, and N₂And (4) drying by blowing, drying at a constant temperature of 110 ℃ for 3-5 h, and taking out for dust prevention for later use.

(2) Soaking the surface hydroxylated glass slide in 0.35 volume percent of DMOAP aqueous solution at room temperature for 30 min; then, rinsing with ultrapure water, N₂Blow-drying, drying at 110 deg.C for 1h to obtain DMOAP film-fixed glass slide, and making the polarized light microscopic image appear black (see FIG. 3).

(3) Soaking the lower glass slide with hydroxylated surface in the mixed solution of 0.3% (v/v) APTES and 0.3% DMOAP in absolute ethyl alcohol, soaking at 80 deg.C for 2h, washing with a large amount of absolute ethyl alcohol and ultrapure water, sequentially washing, and N₂And (4) drying by blowing, drying at the constant temperature of 110 ℃ for 1h, and finishing the self-assembly of the APTES/DMOAP mixed membrane on the surface of the lower slide substrate of the sensor.

(4) And treating the lower glass slide of the APTES-GA/DMOAP mixed membrane by using 0.1% GA, and further coupling an aminated aptamer to obtain the APTES-GA-Apt/DMOAP modified lower glass slide when the aptamer concentration is 100 pmol/L.

(5) Dripping 1ng/mLPSA solution onto an aptamer-immobilized lower glass slide, then soaking the lower glass slide by using 80mmol/L glycine solution, and sealing aldehyde group non-specific active sites which are not coupled with the aptamer on the surface of the lower glass slide; and assembling the glass plate and the upper glass plate decorated with DMOAP face to form a liquid crystal box, and clamping a 5CB liquid crystal molecule copper carrying net at the central part. Schematic diagram of liquid crystal type aptamer sensor for detecting prostate specific antigen and assembly process of liquid crystal cell are shown in fig. 2.

(6) And automatically identifying and calculating the bright area coverage of the PSA concentration polarization microscopic image by MATLAB, and obtaining the concentration of the PSA solution of 0.95 +/-0.06 according to the linear equation of the bright area coverage and the PSA concentration of Br-7.869998 log CPSA +6.50541 and R2-0.99413.

Example 8: serum sample testing

Serum samples with PSA concentrations of 50pg/mL, 100pg/mL, 4ng/mL, 10ng/mL and 100ng/mL are obtained by adding a certain amount of PSA into serum samples of healthy women, the method for detecting the serum samples by the liquid crystal type aptamer biosensor is the same as that in example 4, and polarization microscopic images are shown in FIGS. 14 a-c. The PSA concentrations of the serum samples obtained from the linear relationship of the log PSA concentration of example 3 to the bright field coverage (Br) are shown in the following table:

CPSA(ng/mL)	CPSAdetection value (mean value + -RSD ng/mL)
		0.05	0.045±0.02
0.10	0.097±0.04
		4.00	3.97±0.57
10.00	96.80±1.10
		100.00	102.21±1.51