Liquid crystal biosensor for simultaneously detecting multiple cancer markers and preparation and detection method thereof

Abstract

The present invention relates to a liquid crystal biosensor for detecting multiple cancer markers simultaneously and a preparation and detection method thereof. The liquid crystal biosensor of the present invention comprises a multi-channel sample tank and a liquid crystal biosensor interface modified with DNA encoding different cancer markers; the multi-channel sample tank comprises a single channel and a connecting channel, the connecting channel is located above the single channel, so that the upper parts of different single channels are interconnected; the liquid crystal biosensor interface modified with DNA encoding different cancer markers is constructed in different single channels of the multi-channel sample tank. The present invention utilizes a liquid crystal biosensor modified with DNA encoding different cancer markers to achieve simultaneous detection and quantitative detection of multiple different cancer markers, and also achieves accurate detection of extreme samples. The detection method of the present invention uses a small amount of sample, is convenient to detect, is fast and efficient, has high sensitivity, is low cost, and does not require the use of large instruments.

Description

Technical Field

[0001] The invention relates to a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation and detection method thereof, belonging to the technical field of analysis and detection. Background Art

[0002] With the continuous advancement of science and technology, the continuous improvement of living standards, and the continuous extension of human life span, cancer has gradually become one of the diseases that pose the greatest threat to human life and health. Highly specific and highly sensitive detection of cancer markers plays an important role in the early diagnosis, drug intervention, and prognosis and treatment of cancer. In actual clinical applications, it is often necessary to detect multiple markers to improve the accuracy of detection and exclude false positive results. Therefore, the development of multiple detection technology can reduce sample consumption, save operating costs, shorten detection time, and improve detection throughput and reliability. At present, the simultaneous detection of multiple biomarkers is mainly based on fluorescence analysis, surface enhanced Raman, enzyme-linked immunosorbent, electrochemical analysis and other means. These detection methods usually have high costs, cumbersome operating steps, and require special instruments and molecular markers. Therefore, it is urgent to develop low-cost, convenient, fast, efficient, highly sensitive, selective, and label-free high-performance multiple detection biosensors to meet people's diverse needs for medical testing under the background of "big health".

[0003] Liquid crystal biosensor is a cutting-edge biosensor technology with the advantages of simple structure, low cost, low energy consumption, no labeling, small sample usage, and high sensitivity. Due to its unique advantages, liquid crystal biosensors have gradually attracted widespread attention from researchers at home and abroad, and have achieved certain results in the detection of cancer markers, with broad market prospects. However, since liquid crystal optical signals only have two forms, bright and dark, they are difficult to encode, and liquid crystal sensors that can simultaneously detect multiple substances have not yet been realized, which seriously restricts the practical application of liquid crystal biosensors in clinical cancer diagnosis. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation and detection method thereof. The present invention designs a multi-channel sample slot, including separate channels and connecting channels, and constructs liquid crystal biosensor interfaces modified with DNA encoding different cancer markers in separate channels, constructs liquid crystal biosensors, adds samples to be detected in the connecting channels, and realizes simultaneous detection of multiple cancer markers by analyzing the optical signals in each separate channel.

[0005] Terminology Note:

[0006] DMOAP: N,N-dimethyl-N-[3-(trimethoxysilyl)propyl]octadecyl ammonium chloride, CAS: 27668-52-6;

[0007] OTAB: octadecyltrimethylammonium chloride;

[0008] Room temperature: has a meaning well known to those skilled in the art, generally referring to 25±2°C.

[0009] The technical solution of the present invention is as follows:

[0010] A liquid crystal biosensor for detecting multiple cancer markers simultaneously, the liquid crystal biosensor comprising a multi-channel sample slot and a liquid crystal biosensor interface modified with DNA encoding different cancer markers; the multi-channel sample slot comprises individual channels and connecting channels, the connecting channels are located above the individual channels so that the upper halves of different individual channels are interconnected; the liquid crystal biosensor interface modified with DNA encoding different cancer markers is constructed in different individual channels of the multi-channel sample slot.

[0011] Preferably according to the present invention, the cancer markers include but are not limited to carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, wherein the nucleotide sequence of the DNA encoding the carcinoembryonic antigen is TAG CTA TAG GGG GT, the nucleotide sequence of the DNA encoding the prostate-specific antigen is TAC ATC TGG GAG AG, and the nucleotide sequence of the DNA encoding the alpha-fetoprotein is TAGATA TGG CAG GA.

[0012] In the present invention, the individual channels of the multi-channel sample tank separate the liquid crystal biosensor interfaces modified with DNA encoding different cancer markers, and adding the samples to be detected into the connecting channels can realize the flow of the samples to be detected between different individual channels.

[0013] The preparation method of the above-mentioned liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises the following steps:

[0014] (1) Soaking the glass sheet in a washing solution at 70-90° C. for 30-60 min, then washing with water, anhydrous ethanol, and methanol for 3-6 times, drying with nitrogen, and drying at 100-120° C. for 10-20 h, placing the glass sheet in a DMOAP solution with a mass concentration of 0.1%-3% for 15-30 min at room temperature, washing with three distilled water for 3-6 times, drying with nitrogen, and curing at 100-110° C. for 3-5 h, and cooling to obtain a glass substrate;

[0015] (2) 3D printing a multi-channel sample tank, wherein the multi-channel sample tank includes a single channel and a connecting channel, wherein the connecting channel is located above the single channel so that the upper parts of different single channels are connected to each other;

[0016] (3) fixing the multi-channel sample tank of step (2) on the glass substrate of step (1), placing a copper mesh at the bottom of a single channel, injecting OTAB-doped 4-cyano-4'-pentylbiphenyl into the copper mesh while hot at 35-45° C. to construct a liquid crystal biosensor interface, wherein the thickness of the liquid crystal biosensor interface is consistent with the thickness of the copper mesh;

[0017] (4) Injecting the encoding DNA solutions of multiple cancer markers into different individual channels of the multi-channel sample tank treated in step (3) to modify the liquid crystal biosensor interface, leaving the solution at room temperature for 10 to 20 minutes, and washing away the free encoding DNA with Tris buffer to obtain a liquid crystal biosensor capable of simultaneously detecting multiple cancer markers.

[0018] Preferably, according to the present invention, the washing solution in step (1) is prepared by mixing concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30% in a volume ratio of 7:3.

[0019] Preferably, according to the present invention, the preparation method of OTAB-doped 4-cyano-4'-pentylbiphenyl in step (3) is as follows: OTAB powder is dissolved in chloroform to prepare a 0.1 mM OTAB chloroform solution, the OTAB chloroform solution and 4-cyano-4'-pentylbiphenyl are mixed in a volume ratio of 1:1, vortexed at 2500 rpm for 1 to 5 min to fully mix, and the chloroform is dried with nitrogen to obtain OTAB-doped 4-cyano-4'-pentylbiphenyl.

[0020] In the present invention, step (3) is to enhance the sealing performance between the multi-channel sample tank and the glass substrate by applying a small amount of vaseline between the glass substrate and the multi-channel sample tank.

[0021] Preferably according to the present invention, the cancer markers in step (4) include but are not limited to carcinoembryonic antigen, prostate-specific antigen, and alpha-fetoprotein, wherein the nucleotide sequence of the DNA encoding the carcinoembryonic antigen is TAG CTA TAG GGG GT, the nucleotide sequence of the DNA encoding the prostate-specific antigen is TAC ATC TGG GAG AG, and the nucleotide sequence of the DNA encoding the alpha-fetoprotein is TAG ATA TGG CAG GA.

[0022] In the present invention, the liquid crystal biosensor modified with the encoded DNA in step (4) has an optical response to the addition of DNA of a specific sequence.

[0023] In the present invention, in step (4), the coding DNA solution modifies the liquid crystal biosensor interface so that the coding DNA at the liquid crystal biosensor interface reaches a saturated state; preferably, the concentration of the coding DNA solution in step (4) is ≥ 0.1 μM; further preferably, the concentration of the coding DNA solution is 0.1 to 100 μM.

[0024] Preferably according to the present invention, the pH of the Tris buffer in step (4) is 7-8.

[0025] Preferably according to the present invention, the solvent used for the encoding DNA solution in step (4) is a buffer solution of 10 mM Tris, 20 mM NaCl, pH = 7.3.

[0026] The above-mentioned detection method of the liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises the following steps:

[0027] S1. Preparation of capture magnetic bead probes: incubate the magnetic bead dispersion with different cancer marker aptamer 1 solutions at 37° C. for 0.5 to 1 h, discard the solution, wash the magnetic beads with phosphate buffer for 3 to 5 times to obtain capture magnetic bead probes of different cancer markers, and disperse the capture magnetic bead probes of different cancer markers in phosphate buffer to obtain a capture magnetic bead probe mixed dispersion;

[0028] S2. Preparation of double-stranded signal probes: mixing different cancer marker aptamer 2 solutions with corresponding cancer marker signal DNA solutions and other substances respectively, heating to 95° C. and incubating for 5 to 10 minutes, then slowly annealing to room temperature to obtain double-stranded signal probe solutions of different cancer markers, and mixing the double-stranded signal probe solutions of different cancer markers to obtain a double-stranded signal probe mixed solution;

[0029] S3. Evenly mix the sample to be tested with the mixed dispersion of the captured magnetic bead probes in S1, incubate at 30-40°C for 30-60 min, discard the solution, wash with phosphate buffer for 3-5 times, disperse the washed captured magnetic bead probes in the mixed solution of double-stranded signal probes in S2, incubate at 30-40°C for 30-60 min, take the supernatant, add it to the connecting channel of the multi-channel sample slot of the liquid crystal biosensor, stabilize it for 10 min, observe the optical morphology of the liquid crystal, and simultaneously detect multiple cancer markers based on the changes in the optical morphology of the liquid crystal in different individual channels.

[0030] Preferably according to the present invention, the cancer marker aptamer 1 in step S1 includes but is not limited to carcinoembryonic antigen aptamer 1, prostate specific antigen aptamer 1 and alpha-fetoprotein aptamer 1, wherein the nucleotide sequence of carcinoembryonic antigen aptamer 1 is TAT ​​CCA GCT TAT TCA ATT, the nucleotide sequence of prostate specific antigen aptamer 1 is AAT TAA AGCTCG CCA TCA AAT AGC, and the nucleotide sequence of alpha-fetoprotein aptamer 1 is GTG ACG CTC CTA ACG CTG ACTCAG GTG CAG TTC TCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC.

[0031] Preferably according to the present invention, the magnetic beads in step S1 are modified with streptavidin.

[0032] Preferably according to the present invention, the cancer marker aptamer 1 in step S1 is modified with biotin.

[0033] In the present invention, after the magnetic bead dispersion is co-incubated with the cancer marker aptamer 1 solution in step S1, the magnetic beads reach their saturated adsorption capacity; preferably, in step S1, the ratio of the magnetic beads to the cancer marker aptamer 1 is 1 mg of magnetic beads modified with 400 pmol of cancer marker aptamer 1.

[0034] Preferably according to the present invention, the concentrations of capture magnetic bead probes for different cancer markers in the capture magnetic bead probe mixed dispersion in step S1 are consistent, and the total concentration of capture magnetic bead probes for different cancer markers is ≥0.1 mg / mL; further preferably, the total concentration of capture magnetic bead probes for different cancer markers is 0.1-10 mg / mL.

[0035] In the present invention, the solvents of the magnetic bead dispersion and the cancer marker aptamer 1 solution in step S1 are both phosphate buffer.

[0036] Preferably according to the present invention, the cancer marker aptamer 2 in step S2 includes but is not limited to a carcinoembryonic antigen aptamer 2, a prostate-specific antigen aptamer 2, and an alpha-fetoprotein aptamer 2, wherein the nucleotide sequence of the carcinoembryonic antigen aptamer 2 is TAG CTA TAG GGG GTG AAG GGA TAC CC, the sequence of the prostate-specific antigen aptamer 2 is TACATC TGG GAG AGC GGA AGC GUG CUG GGC CGU CAG GUC ACG GCA GCG AAG CUC UAG GCGCGG CCA GUU GCC AUA ACC CAG AGG UCG AUG GAU CCU, and the sequence of the alpha-fetoprotein aptamer 2 is TAGATA TGG CAG GAA GAC AAA CAA GCT TGG CGG CGG GAA GGT GTT TAA ATT CCC GGG TCTGCG TGG TCT GTG GTG CTG T.

[0037] According to a new preference of the present invention, the cancer marker signal DNA in step S2 includes but is not limited to carcinoembryonic antigen signal DNA, prostate specific antigen signal DNA and alpha-fetoprotein signal DNA, wherein the nucleotide sequence of the carcinoembryonic antigen signal DNA is TTC ACC CCC TAT AGC TA, the nucleotide sequence of the prostate specific antigen signal DNA is CCG CTC TCCCAG ATG TA, and the nucleotide sequence of the alpha-fetoprotein signal DNA is GTC TTC CTG CCA TAT CTA.

[0038] Preferably, according to the present invention, the concentrations of the double-stranded signal probes for different cancer markers in the double-stranded signal probe mixed solution in step S2 are consistent, and the concentrations of the double-stranded signal probes for different cancer markers are 10 pM-1000 pM, respectively.

[0039] Preferably according to the present invention, the solvent of the cancer marker aptamer 2 solution and the cancer marker signal DNA solution in step S2 is a buffer solution of 10 mM Tris, 130 mM NaCl, 10 mM KCl, pH=7.3.

[0040] In the present invention, step S3 mixes the sample to be detected and the capture magnetic bead probe mixed dispersion, and the cancer markers in the sample to be detected are all captured by the capture magnetic bead probe, and the cancer markers captured by the capture magnetic bead probe are all bound to the cancer marker aptamer 2 in the double-stranded signal probe mixed solution; preferably, in step S3, the volume ratio of the sample to be detected and the capture magnetic bead probe mixed dispersion is 1:(1-10).

[0041] According to the preferred embodiment of the present invention, the composition of the phosphate buffer is: 2mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 136mM NaCl, 2.6mM KCl, pH=7.3.

[0042] Preferably according to the present invention, the detection method further comprises: establishing a working curve according to changes in the cancer marker standard solution and its liquid crystal optical morphology to achieve quantitative detection of multiple cancer markers in the sample to be detected.

[0043] The technical features of the present invention are as follows:

[0044] In the present invention, a multi-channel sample tank is 3D printed, and the multi-channel sample tank includes a single channel and a connecting channel. The connecting channel is located above the single channel, and the intervals between the single channels are eliminated, so that the upper parts of different single channels are interconnected. Then the multi-channel sample tank is fixed on a glass substrate, and a liquid crystal biosensor interface is constructed at the bottom of each single channel. The liquid crystal biosensor interface of each single channel is modified with coding DNA of different cancer markers to construct a liquid crystal biosensor that can simultaneously detect multiple cancer markers.

[0045] The different cancer marker aptamers 1 are co-incubated with magnetic beads respectively to prepare capture magnetic bead probes of different cancer markers, which are then dispersed together in a phosphate buffer to obtain a capture magnetic bead probe mixed dispersion; the different cancer marker aptamers 2 are annealed with the corresponding cancer marker signal DNA respectively to prepare double-stranded signal probe solutions of different cancer markers, which are mixed to obtain a double-stranded signal probe mixed solution; the sample to be detected is mixed with the capture magnetic bead probe mixed dispersion, and the cancer marker in the sample to be detected is captured by the specific interaction between the aptamer 1 and the target cancer marker, so as to achieve the separation of the cancer marker and other matrices in the sample to be detected and the enrichment of the cancer marker; the capture magnetic bead probe that has captured the cancer marker is dispersed in the double-stranded signal probe mixed solution, and the aptamer 2 is ... the magnetic beads The specific interaction between aptamer 2 and the target cancer marker is used to separate aptamer 2 from the signal DNA. Since the binding force between aptamer 2 and the target cancer marker is greater than the binding force between aptamer 2 and the complementary signal DNA, the free signal DNA is released through displacement. The free signal DNA solution is added to the connecting channels of the multi-channel sample slot of the liquid crystal biosensor modified with different cancer marker encoding DNA. Since the signal DNA and the encoding DNA sequences are complementary, the two undergo highly specific hybridization. Using the DNA in situ hybridization technology, the encoding DNA in different individual channels hybridizes with the corresponding signal DNA, thereby changing the arrangement of the liquid crystal and realizing the change of the liquid crystal optical signal. The change of the optical signal in different individual channels is used to simultaneously detect multiple cancer markers in the sample.

[0046] Beneficial effects:

[0047] 1. The present invention utilizes liquid crystal biosensors modified with DNA encoding different cancer markers to achieve simultaneous detection and quantitative detection of multiple different cancer markers, and also achieves accurate detection of extreme samples. The detection method of the present invention requires a small amount of sample, is convenient to detect, is fast and efficient, has high sensitivity, and is low in cost. It does not require the use of large instruments and has the potential to be used for the immediate and accurate detection of cancer markers.

[0048] 2. The present invention utilizes magnetic bead enrichment and separation technology to easily separate target cancer markers and other interfering matrices, greatly improving the specificity of detection and enabling detection in biological samples such as whole blood, plasma and serum.

[0049] 3. The present invention adopts a liquid liquid crystal interface and combines DNA interface hybridization technology, which has high sensitivity and can meet actual detection needs without amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a schematic diagram of the detection principle of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein;

[0051] Figure 2 The three views and the cross-sectional view of the multi-channel sample tank of Example 1;

[0052] Figure 3 are working curves of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein; in the figure, a is the working curve of carcinoembryonic antigen, b is the working curve of prostate-specific antigen, and c is the working curve of alpha-fetoprotein;

[0053] Figure 4 The bar graph is a test result bar graph of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein of the blood sample in Example 3; in the figure, a is the test result of carcinoembryonic antigen, b is the test result of prostate-specific antigen, and c is the test result of alpha-fetoprotein; the ordinate is the content of cancer markers in the blood;

[0054] Figure 5 The bar graphs are the results of the carcinoembryonic antigen test of the hemolyzed sample in Example 4; a is the comparison between the hemolyzed sample and the normal blood sample, and b is the comparison between the hemolyzed sample liquid crystal biosensor and the commercial ELISA kit. DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The drugs involved in the examples are all common commercial products unless otherwise specified.

[0056] Examples The present invention is further described by taking three cancer markers, namely carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and alpha-fetoprotein (AFP), as examples.

[0057] The detection principles of carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and alpha-fetoprotein (AFP) in the embodiment are as follows: Figure 1As shown, carcinoembryonic antigen aptamer 1, prostate-specific antigen aptamer 1 and alpha-fetoprotein aptamer 1 are co-incubated with magnetic beads to prepare capture magnetic bead probes of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, and the three capture magnetic bead probes are dispersed in phosphate buffer at the same time to obtain a capture magnetic bead probe mixed dispersion; carcinoembryonic antigen aptamer 2 and carcinoembryonic antigen signal DNA, prostate-specific antigen aptamer 2 and prostate-specific antigen signal DNA, and alpha-fetoprotein aptamer 2 and alpha-fetoprotein signal DNA are annealed to prepare double-stranded signal probes of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, and the three double-stranded signal probes are mixed to obtain a double-stranded signal probe mixed solution; the capture magnetic bead probe mixed dispersion is mixed with a sample to be detected, and the cancer marker in the sample to be detected is captured by the specific interaction between aptamer 1 and the target cancer marker, so as to achieve the separation of the cancer marker from other matrices in the sample to be detected and the enrichment of the cancer marker; the capture of the cancer marker The magnetic bead probe is dispersed into the double-stranded signal probe mixed solution, and the specific interaction between aptamer 2 and the target cancer marker is also used to separate aptamer 2 from the signal DNA. Because the binding force between aptamer 2 and the target cancer marker is greater than the binding force between aptamer 2 and the complementary signal DNA, the free signal DNA is released through displacement. The free signal DNA is added to the connecting channel of the multi-channel sample slot of the liquid crystal biosensor that has been modified with the coding DNA of carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and alpha-fetoprotein (AFP). Since the signal DNA and the coding DNA sequences are complementary, the two undergo highly specific hybridization. Using the DNA in situ hybridization technology, the coding DNA in different individual channels hybridizes with the corresponding signal DNA, changing the arrangement of the liquid crystal to achieve a change in the optical signal of the liquid crystal. The changes in the optical signals in different individual channels are used to simultaneously detect the carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and alpha-fetoprotein (AFP) in the sample.

[0058] Example 1: Preparation of Liquid Crystal Biosensor

[0059] A method for preparing a liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises the following steps:

[0060] (1) Preparation of glass substrate: a glass slide was immersed in 60 mL of a washing solution prepared by mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3 at 75°C for 30 min, then washed with water, anhydrous ethanol, and methanol for 3 times, dried with nitrogen, dried at 110°C for 12 h, placed in a 0.5% DMOAP solution at room temperature for 15 min, washed with three distilled water for 5 times, dried with nitrogen, and cured at 105°C for 3 h. After cooling, a glass substrate was obtained.

[0061] (2) Preparation of a multi-channel sample tank: 3D printing a multi-channel sample tank, the multi-channel sample tank includes a single channel and a connecting channel, the connecting channel is located above the single channel, so that the upper parts of different single channels are connected to each other. The multi-channel sample tank of this embodiment is shown in FIG. Figure 2 As shown, the multi-channel sample tank is 2.8 cm long, 1 cm wide, 0.6 cm high, and has a single channel diameter of 0.6 cm. It includes 3 parallel single channels, with a center distance of 0.9 cm between two channels, and a connecting channel height of 0.2 cm.

[0062] (3) The multi-channel sample trough prepared in step (2) is fixed to the glass substrate prepared in step (1) with a dovetail clamp. To enhance the airtightness, a small amount of vaseline is applied between the multi-channel sample trough and the glass substrate. A G75 copper mesh is placed at the bottom of a single channel. 1.5 μL of OTAB-doped 4-cyano-4'-pentylbiphenyl heated to 35-45° C. is injected into the copper mesh while hot. Then, a capillary with an inner diameter of 0.5 mm is used to remove excess OTAB-doped 4-cyano-4'-pentylbiphenyl to construct a liquid crystal biosensor interface. The thickness of the liquid crystal biosensor interface is consistent with the thickness of the copper mesh, which is 20 μm.

[0063] The preparation method of the OTAB-doped 4-cyano-4'-pentylbiphenyl is as follows: OTAB powder is dissolved in chloroform to prepare a 0.1 mM OTAB chloroform solution, the OTAB chloroform solution and 4-cyano-4'-pentylbiphenyl are mixed in a volume ratio of 1:1, vortexed at 2500 rpm for 3 min to fully mix, and the chloroform is dried with nitrogen to obtain OTAB-doped 4-cyano-4'-pentylbiphenyl;

[0064] (4) The coding DNA of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein were prepared into solutions with a concentration of 1 μM, respectively, and the solvent used was 10 mM Tris, 20 mM NaCl, pH = 7.5 buffer solution. The corresponding coding DNA solutions were injected into the separate channels of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, respectively, to modify the liquid crystal biosensor interface, and the solution was allowed to stand at room temperature for 10 minutes. Subsequently, the coding DNA solution in the channel was exchanged with Tris buffer (pH 7-8), and the free coding DNA was washed away after ten exchanges, thereby obtaining a liquid crystal biosensor for simultaneous detection of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein;

[0065] Among them, the nucleotide sequence of the carcinoembryonic antigen encoding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of the prostate specific antigen encoding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of the alpha-fetoprotein encoding DNA is TAG ATATGG CAG GA.

[0066] Example 2: Method for simultaneous detection of multiple cancer markers

[0067] A method for simultaneously detecting carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and alpha-fetoprotein (AFP), comprising the following steps:

[0068] S1. Preparation of capture magnetic bead probes: 10 μL of 0.1 mg / mL streptavidin-modified magnetic bead dispersion was incubated with 10 μL of 0.4 μM biotin-modified carcinoembryonic antigen aptamer 1 solution, prostate-specific antigen aptamer 1 solution and alpha-fetoprotein aptamer 1 solution at 37°C for 0.5 h, and then the magnetic beads were separated from the solution using a commercially available magnetic separation stand, the solution was discarded, and the magnetic beads were washed 3 times with phosphate buffer to obtain three capture magnetic bead probes. Then, the three capture magnetic bead probes (1 μg each) were simultaneously dispersed in 30 μL phosphate buffer and shaken to obtain a capture magnetic bead probe mixed dispersion. The total concentration of the capture magnetic bead probes in the capture magnetic bead probe mixed dispersion was 0.1 mg / mL.

[0069] Among them, the nucleotide sequence of carcinoembryonic antigen aptamer 1 is TAT ​​CCA GCT TAT TCA ATT, the nucleotide sequence of prostate-specific antigen aptamer 1 is AAT TAA AGC TCG CCA TCA AAT AGC, and the nucleotide sequence of alpha-fetoprotein aptamer 1 is GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGATGT GGG TCC TGT CCG TCC GAA CCA ATC;

[0070] The solvents of the magnetic bead dispersion and aptamer 1 solution were both phosphate buffer;

[0071] S2. Preparation of double-stranded signal probes: 60 pM carcinoembryonic antigen aptamer 2 solution, prostate-specific antigen aptamer 2 solution, and alpha-fetoprotein aptamer 2 solution were mixed with equal volumes of corresponding 60 pM carcinoembryonic antigen signal DNA solution, prostate-specific antigen signal DNA solution, and alpha-fetoprotein signal DNA solution, respectively, heated to 95° C. and incubated for 5 min, and then slowly annealed to room temperature to obtain three double-stranded signal probe solutions, and then the three double-stranded signal probe solutions (each with a concentration of 30 pM) were mixed with equal volumes and shaken to obtain a double-stranded signal probe mixed solution, wherein the concentrations of the three double-stranded signal probes in the double-stranded signal probe mixed solution were 10 pM respectively;

[0072] Among them, the nucleotide sequence of carcinoembryonic antigen aptamer 2 is TAG CTA TAG GGG GTG AAG GGA TAC CC, and the nucleotide sequence of carcinoembryonic antigen signal DNA is TTC ACC CCC TAT AGC TA; the nucleotide sequence of prostate-specific antigen aptamer 2 is TAC ATC TGG GAG AGC GGA AGC GUG CUG GGC CGU CAG GUC ACG GCA GCGAAG CUC UAG GCG CGG CCA GUU GCC AUA ACC CAG AGG UCG AUG GAU CCU, and the nucleotide sequence of prostate-specific antigen signal DNA is CCG CTC TCC CAG ATG TA; the nucleotide sequence of alpha-fetoprotein aptamer 2 is TAG ATA TGG CAG GAA GAC AAA CAA GCT TGG CGG CGG GAA GGT GTT TAA ATT CCC GGGTCT GCG TGG TCT GTG GTG CTG T, and the nucleotide sequence of alpha-fetoprotein signal DNA is GTC TTC CTG CCATAT CTA;

[0073] The solvents of the aptamer 2 solution and the signal DNA solution were both 10 mM Tris, 130 mM NaCl, 10 mM KCl, pH = 7.5 buffer;

[0074] S3, mix equal volumes of standard solutions of carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, the concentration of carcinoembryonic antigen in the mixed standard solution ranges from 0.4 to 1.6 ng / mL, the concentration of prostate-specific antigen is 0.1 to 0.5 ng / mL, and the concentration of alpha-fetoprotein is 0.2 to 0.8 ng / mL, mix 10 μL of the mixed standard solution with 10 μL of the 0.1 mg / mL capture magnetic bead probe mixed dispersion prepared in S1, incubate at 37°C for 30 min, separate the magnetic beads with a magnetic stand, discard the solution, wash three times with phosphate buffer, and disperse the washed capture magnetic bead probe into 100 μL The mixed solution of 10 pM double-stranded signal probe prepared in S2 was incubated at 37° C. for 30 min, the magnetic beads were separated by a magnetic rack, the supernatant was taken, and added to the connecting channel of the multi-channel sample tank of the liquid crystal biosensor prepared in Example 1, and the optical morphology of the liquid crystal was observed after shaking on a shaker for 10 min, and multiple cancer markers were detected simultaneously according to the changes in the optical morphology of the liquid crystal in different individual channels;

[0075] The components of the phosphate buffer involved in the embodiment are: 2mM KH 2 PO 4 , 8 mM Na 2HPO 4 , 136mM NaCl, 2.6mM KCl, pH=7.3.

[0076] Data processing: Photoshop software was used to calculate the bright area pixels, and Photoshop was also used to obtain the pixels of all liquid crystal areas. The ratio of the two was the bright area ratio. This method was used to obtain the bright area ratio of the initial liquid crystal morphology. This method was then used to obtain the bright area ratio of the liquid crystal after the sample was added. The difference between the two was calculated as the bright area change. A graph was drawn with the standard concentration as the horizontal axis and the bright area change as the vertical axis to obtain a working curve. The working curve is shown in Figure 1. Figure 3 As shown, the standard solutions with known concentrations were subsequently tested, and the yields were determined to be between 92.1% and 110.6%;

[0077] Among them, the working curve of carcinoembryonic antigen (CEA) is y=44.02x-1.91, R 2 =0.985; detection limit is 0.31ng / mL;

[0078] The working curve of prostate specific antigen (PSA) is y=155.33x-3.90, R 2 =0.986; detection limit is 0.07ng / mL;

[0079] The working curve of alpha-fetoprotein (AFP) is y=102.85x-2.23, R 2 =0.993; detection limit is 0.19 ng / mL.

[0080] Example 3: Detection of unknown concentrations of CEA, PSA and AFP in blood

[0081] The preparation of capture magnetic bead probe and double-stranded signal probe is the same as in Example 2.

[0082] The blood samples were tested immediately after being drawn. The samples used in this embodiment were blood samples from healthy people.

[0083] Take 2 μL of blood sample, dilute it 5 times with phosphate buffer, incubate it with 10 μL of 0.1 mg / mL capture magnetic bead probe mixed dispersion at 37°C for 30 min, separate the magnetic beads with a magnetic rack, discard the solution, wash it three times with phosphate buffer, disperse the washed capture magnetic bead probe into 100 μL of 10 pM double-stranded signal probe mixed solution, incubate it at 37°C for 30 min, separate the magnetic beads with a magnetic rack, take the supernatant, add it to the connecting channel of the multi-channel sample tank of the liquid crystal biosensor prepared in Example 1, and observe the liquid crystal optical morphology after shaking on a shaker for 10 min.

[0084] Data processing: Photoshop software was used to calculate the changes in the bright area and bring it into the working curve to obtain the concentrations of CEA, PSA and AFP. The measured concentrations of carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and alpha-fetoprotein (AFP) were as follows: Figure 4 As shown, a commercial ELISA plate was used as a detection reference (the operation of the commercial ELISA was carried out according to the instructions), and the relative deviation between the two was between -11.4% and 13.8%.

[0085] Example 4: Detection of carcinoembryonic antigen (CEA) in hemolyzed samples

[0086] The blood that could not be tested immediately in Example 3 was stored in a heparin tube. During the test, the blood sample in the heparin tube was vigorously shaken to obtain a hemolyzed blood sample.

[0087] Take 2 μL of hemolyzed blood sample, dilute it 5 times with phosphate buffer, incubate it with 10 μL of 0.1 mg / mL capture magnetic bead probe mixed dispersion at 37°C for 30 min, separate the magnetic beads with a magnetic rack, discard the solution, wash it three times with phosphate buffer, disperse the washed capture magnetic bead probe into 100 μL of 10 pM double-stranded signal probe mixed solution, incubate it at 37°C for 30 min, separate the magnetic beads with a magnetic rack, take the supernatant, add it to the connecting channel of the multi-channel sample tank of the liquid crystal biosensor prepared in Example 1, and observe the liquid crystal optical morphology after shaking on a shaker for 10 min.

[0088] Data processing: Photoshop software was used to calculate the change in the bright area, and the CEA concentration was obtained by inserting it into the working curve. Figure 5 As shown, a commercial ELISA plate was used as a detection reference (the operation of the commercial ELISA was carried out according to the instruction manual).

[0089] The CEA concentration values ​​of hemolyzed samples detected by liquid crystal biosensor are compared with the values ​​of normal blood samples from the same batch, and the values ​​do not change much ( Figure 5 a), while the CEA values ​​in hemolytic samples measured by the traditional ELISA method were all too high, and 4 out of 5 samples exceeded the cutoff value, showing false positives ( Figure 5 b), indicating that the detection method of the present invention performs better than the traditional ELISA method in detecting extreme samples.

[0090] The above is only a specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined by the claims. SEQUENCE LISTING <110> Shandong Analysis and Testing Center Shandong University <120> Liquid crystal biosensor for simultaneously detecting multiple cancer markers and preparation and detection method thereof <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> DNA <213> Homo sapiens <400> 1 tagctatagg gggt 14 <210> 2 <211> 14 <212> DNA <213> Homo sapiens <400> 2 tacatctggg agag 14 <210> 3 <211> 14 <212> DNA <213> Homo sapiens <400> 3 tagatatggc agga 14 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <400> 4 tatccagctt attcaatt 18 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <400> 5 aattaaagct cgccatcaaa tagc 24 <210> 6 <211> 75 <212> DNA <213> Artificial sequence <400> 6 gtgacgctcc taacgctgac tcaggtgcag ttctcgactc ggtcttgatg tgggtcctgt 60 ccgtccgaac caatc 75 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 tagctatagg gggtgaaggg ataccc 26 <210> 8 <211> 96 <212> DNA <213> Artificial Sequence <400> 8 tacatctggg agagcggaag cgugcugggc cgucagguca cggcagcgaa gcucuaggcg 60 cggccaguug ccauaaccca gaggucgaug gauccu 96 <210> 9 <211> 79 <212> DNA <213> Artificial Sequence <400> 9 tagatatggc aggaagacaa acaagcttgg cggcgggaag gtgtttaaat tcccgggtct 60 gcgtggtctg tggtgctgt 79 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <400> 10 ttcaccccct atagcta 17 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <400> 11 ccgctctccc agatgta 17 <210> 12 <211> 18 <212> DNA <213> Artificial sequence <400> 12 gtcttcctgc catatcta 18

Claims

1. A liquid crystal biosensor for simultaneously detecting multiple cancer markers, characterized in that: The liquid crystal biosensor comprises a multi-channel sample tank and a liquid crystal biosensor interface modified with DNA encoding different cancer markers; the multi-channel sample tank comprises a single channel and a connecting channel, the connecting channel is located above the single channel, so that the upper parts of different single channels are interconnected; the liquid crystal biosensor interface modified with DNA encoding different cancer markers is constructed in different single channels of the multi-channel sample tank; The liquid crystal biosensor also includes a capture magnetic bead probe and a double-stranded signal probe; during detection, the sample to be detected is added to the connecting channel of the multi-channel sample tank after being processed; The processing of the sample to be detected comprises the following steps: S1. Preparation of capture magnetic bead probes: incubate the magnetic bead dispersion with different cancer marker aptamer 1 solutions at 37° C. for 0.5 to 1 h, discard the solution, wash the magnetic beads with phosphate buffer for 3 to 4 times to obtain capture magnetic bead probes of different cancer markers, and disperse the capture magnetic bead probes of different cancer markers in phosphate buffer to obtain a capture magnetic bead probe mixed dispersion; S2. Preparation of double-stranded signal probes: mixing different cancer marker aptamer 2 solutions with corresponding cancer marker signal DNA solutions and other substances respectively, heating to 95° C. and incubating for 5 to 10 minutes, then slowly annealing to room temperature to obtain double-stranded signal probe solutions of different cancer markers, and mixing the double-stranded signal probe solutions of different cancer markers to obtain a double-stranded signal probe mixed solution; S3, mix the sample to be tested with the capture magnetic bead probe mixed dispersion in S1, incubate at 35-40°C for 30-50 minutes, discard the solution, wash with phosphate buffer 3-5 times, disperse the washed capture magnetic bead probe in the double-stranded signal probe mixed solution in S2, incubate at 34-40°C for 30-50 minutes, take the supernatant, and obtain the treated sample to be tested; The cancer markers include carcinoembryonic antigen, prostate-specific antigen and alpha-fetoprotein, wherein the nucleotide sequence of the DNA encoding carcinoembryonic antigen is TAG CTA TAG GGG GT, the nucleotide sequence of the DNA encoding prostate-specific antigen is TACATC TGG GAG AG, and the nucleotide sequence of the DNA encoding alpha-fetoprotein is TAG ATA TGG CAG GA; The cancer marker aptamer 1 includes a carcinoembryonic antigen aptamer 1, a prostate-specific antigen aptamer 1 and an alpha-fetoprotein aptamer 1, wherein the nucleotide sequence of the carcinoembryonic antigen aptamer 1 is TAT ​​CCA GCT TAT TCA ATT, the nucleotide sequence of the prostate-specific antigen aptamer 1 is AAT TAA AGC TCG CCA TCA AAT AGC, and the nucleotide sequence of the alpha-fetoprotein aptamer 1 is GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGATGT GGG TCC TGT CCG TCC GAA CCAATC; The cancer marker aptamer 2 includes a carcinoembryonic antigen aptamer 2, a prostate-specific antigen aptamer 2, and an alpha-fetoprotein aptamer 2, wherein the nucleotide sequence of the carcinoembryonic antigen aptamer 2 is TAG CTATAG GGG GTG AAG GGA TACCC, the sequence of the prostate-specific antigen aptamer 2 is TAC ATC TGG GAG AGC GGAAGC GUG CUG GGC CGUCAG GUC ACG GCA GCG AAG CUC UAG GCG CGG CCA GUU GCC AUA ACC CAG AGG UCG AUGGAU CCU, and the sequence of the alpha-fetoprotein aptamer 2 is TAG ATA TGG CAG GAA GAC AAACAAGCT TGG CGG CGGGAAGGT GTT TAAATT CCC GGG TCT GCG TGG TCT GTG GTG CTG T; The cancer marker signal DNA includes carcinoembryonic antigen signal DNA, prostate specific antigen signal DNA and alpha-fetoprotein signal DNA, wherein the nucleotide sequence of the carcinoembryonic antigen signal DNA is TTC ACC CCC TAT AGC TA, the nucleotide sequence of the prostate specific antigen signal DNA is CCG CTC TCC CAG ATG TA, and the nucleotide sequence of the alpha-fetoprotein signal DNA is GTC TTC CTG CCATAT CTA.

2. The liquid crystal biosensor according to claim 1, characterized in that: Meet one or more of the following conditions: (i) the magnetic beads described in step S1 are modified with streptavidin; (ii) the cancer marker aptamer 1 in step S1 is modified with biotin; (iii) the ratio of the magnetic beads to the cancer marker aptamer 1 in step S1 is 1 mg of magnetic beads modified with 400 pmol of cancer marker aptamer 1; (iv) the concentrations of the capture magnetic bead probes of different cancer markers in the capture magnetic bead probe mixed dispersion in step S1 are consistent, and the total concentration of the capture magnetic bead probes of different cancer markers is ≥ 0.1 mg / mL; (v) the concentrations of the double-stranded signal probes of different cancer markers in the double-stranded signal probe mixed solution in step S2 are consistent, and the concentrations of the double-stranded signal probes of different cancer markers are 10 pM-1000 pM respectively; (vi) The solvent of the cancer marker aptamer 2 solution and the cancer marker signal DNA solution in step S2 is a buffer solution of 10 mM Tris, 130 mM NaCl, 10 mM KCl, pH=7.5; (vii) the volume ratio of the sample to be detected and the capture magnetic bead probe mixed dispersion in step S3 is 1:(1-10); (viii) The composition of the phosphate buffer is: 2 mM KH2PO4, 8 mM Na2HPO4, 136 mM NaCl, 2.6 mM KCl, pH = 7.

3.

3. The method for preparing the liquid crystal biosensor for simultaneously detecting multiple cancer markers according to claim 1, characterized in that: Here are the steps: (1) Soaking the glass sheet in a washing solution at 70-90° C. for 30-60 min, then washing with water, anhydrous ethanol, and methanol for 3-4 times, drying with nitrogen, and drying at 100-120° C. for 10-20 h, placing it in a DMOAP solution with a mass concentration of 0.1%-3% for 15-30 min at room temperature, washing it with three distilled water for 5-6 times, drying with nitrogen, and curing it at 100-110° C. for 3-5 h, and cooling it to obtain a glass substrate; (2) 3D printing a multi-channel sample tank, wherein the multi-channel sample tank includes a single channel and a connecting channel, wherein the connecting channel is located above the single channel so that the upper parts of different single channels are connected to each other; (3) fixing the multi-channel sample tank of step (2) on the glass substrate of step (1), placing a copper mesh at the bottom of a single channel, injecting OTAB-doped 4-cyano-4'-pentylbiphenyl into the copper mesh while hot at 35-45° C. to construct a liquid crystal biosensor interface, wherein the thickness of the liquid crystal biosensor interface is consistent with the thickness of the copper mesh; (4) Injecting the encoding DNA solutions of multiple cancer markers into different individual channels of the multi-channel sample tank treated in step (3) to modify the liquid crystal biosensor interface, leaving the solution at room temperature for 10 to 20 minutes, and washing away the free encoding DNA with Tris buffer to obtain a liquid crystal biosensor capable of simultaneously detecting multiple cancer markers.

4. The preparation method according to claim 3, characterized in that: Meet one or more of the following conditions: i. The washing solution in step (1) is prepared by a volume ratio of 7:3 of 98% concentrated sulfuric acid and 30% hydrogen peroxide; ii. Preparation method of OTAB-doped 4-cyano-4'-pentylbiphenyl in step (3): dissolving OTAB powder in chloroform to prepare a 0.1 mM OTAB chloroform solution, mixing the OTAB chloroform solution and 4-cyano-4'-pentylbiphenyl in a volume ratio of 1:1, vortexing at 2500 rpm for 1 to 5 min to fully mix, and drying the chloroform with nitrogen to obtain OTAB-doped 4-cyano-4'-pentylbiphenyl; iii. The concentration of the encoding DNA solution in step (4) is ≥ 0.1 μM; iv. The pH of the Tris buffer in step (4) is 7 to 8; v. The solvent used for the encoding DNA solution in step (4) is a buffer solution of 10 mM Tris, 20 mM NaCl, pH = 7.5.

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