CN112816692A - Liquid crystal biosensor for simultaneously detecting multiple cancer markers and preparation and detection methods thereof - Google Patents
Liquid crystal biosensor for simultaneously detecting multiple cancer markers and preparation and detection methods thereof Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention relates to a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation method and a detection method thereof. The liquid crystal biosensor comprises a multi-channel sample groove and a liquid crystal biosensor interface modified by coding DNA of different cancer markers; the multi-channel sample groove comprises an individual channel and a communicating channel, and the communicating channel is positioned above the individual channel, so that the upper half parts of different individual channels are communicated with each other; the different cancer markers code DNA modified liquid crystal biosensing interfaces constructed in different single channels of the multi-channel sample groove. The invention utilizes different cancer markers to code DNA to modify the liquid crystal biosensor, realizes the simultaneous detection and quantitative detection of various different cancer markers, and also realizes the accurate detection of extreme samples.
Description
Technical Field
The invention relates to a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation method and a detection method thereof, belonging to the technical field of analysis and detection.
Background
With the continuous progress of the technology level, the living standard is continuously improved, the life span of human is continuously prolonged, and cancer gradually becomes one of the diseases which have the greatest threat to the life health of human. The high specificity and high sensitivity cancer marker detection plays an important role in early diagnosis, drug intervention and prognosis treatment of cancer. In practical clinical application, multiple markers are detected to improve the detection accuracy and eliminate false positive results. Therefore, the development of the multiple detection technology can reduce sample consumption, save operation cost, shorten detection time and improve detection flux and reliability. At present, the simultaneous detection of multiple biomarkers is mainly based on means such as fluorescence analysis, surface enhanced Raman, enzyme-linked immunosorbent assay, electrochemical analysis and the like, and the detection methods generally have the problems of high cost, complicated operation steps, need of special instruments and molecular markers and the like. Therefore, development of a high-performance multi-detection biosensor which is low in cost, convenient, rapid, efficient, high in sensitivity, good in selectivity and free of markers is urgently needed to meet the requirement of medical detection diversification of people under the 'big health' background.
The liquid crystal biosensor is a leading-edge biosensing technology and has the advantages of simple structure, low cost, low energy consumption, no need of marking, small sample consumption, high sensitivity and the like. Due to the unique advantages, the liquid crystal biosensor gradually draws the attention of researchers at home and abroad, achieves certain results in the aspect of cancer marker detection, and has wide marketization prospect. However, because the liquid crystal optical signal only has a bright form and a dark form, and is difficult to encode, a liquid crystal sensor capable of simultaneously detecting a plurality of substances is not realized yet, and the practical application of the liquid crystal biosensor in the aspect of clinical cancer diagnosis is severely restricted.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a liquid crystal biosensor for simultaneously detecting multiple cancer markers and a preparation and detection method thereof.
Description of terms:
DMOAP: n, N-dimethyl-N- [3- (trimethylsilyl) propyl ] octadecylammonium chloride, CAS: 27668-52-6;
OTAB: octadecyl trimethyl ammonium chloride;
room temperature: having a meaning well known to those skilled in the art, generally 25. + -. 2 ℃.
The technical scheme of the invention is as follows:
a liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises a multi-channel sample groove and a liquid crystal biosensor interface modified by coding DNA of different cancer markers; the multi-channel sample groove comprises an individual channel and a communicating channel, and the communicating channel is positioned above the individual channel, so that the upper half parts of different individual channels are communicated with each other; the different cancer markers code DNA modified liquid crystal biosensing interfaces constructed in different single channels of the multi-channel sample groove.
Preferably, the cancer markers include, but are not limited to, carcinoembryonic antigen, prostate specific antigen and alpha fetoprotein, wherein the nucleotide sequence of carcinoembryonic antigen encoding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of prostate specific antigen encoding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of alpha fetoprotein encoding DNA is TAG ATA TGG CAG GA.
In the invention, the single channels of the multi-channel sample groove separate the liquid crystal biological sensing interfaces modified by different cancer marker coding DNAs, and the sample to be detected is added into the communication channel to realize the circulation of the sample to be detected among different single channels.
The preparation method of the liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises the following steps:
(1) soaking the glass sheet in a washing solution, soaking for 30-60 min at 70-90 ℃, then respectively washing for 3-6 times by using water, absolute ethyl alcohol and methanol, drying by using nitrogen, drying for 10-20 h at 100-120 ℃, placing in a DMOAP solution with the mass concentration of 0.1-3% for 15-30 min at room temperature, washing for 3-6 times by using distilled water for three times, drying by using nitrogen, solidifying for 3-5 h at 100-110 ℃, and cooling to obtain a glass substrate;
(2)3D printing a multi-channel sample tank, wherein the multi-channel sample tank comprises independent channels and communication channels, and the communication channels are positioned above the independent channels so that the upper half parts of different independent channels are mutually communicated;
(3) fixing the multi-channel sample tank obtained in the step (2) on the glass substrate obtained in the step (1), placing a copper mesh at the bottom of the single channel, and injecting OTAB-doped 4-cyano-4' -pentylbiphenyl into the copper mesh while the sample tank is hot at 35-45 ℃ to construct a liquid crystal biosensing interface, wherein the thickness of the liquid crystal biosensing interface is consistent with that of the copper mesh;
(4) and (3) respectively injecting the coding DNA solutions of various cancer markers into different independent channels of the multi-channel sample tank treated in the step (3) to modify a liquid crystal biosensing interface, standing at room temperature for 10-20 min, and washing free coding DNA with Tris buffer solution to obtain the liquid crystal biosensor for simultaneously detecting various cancer markers.
According to the invention, the washing liquid in the step (1) is preferably prepared from concentrated sulfuric acid with the mass concentration of 98% and hydrogen peroxide with the mass concentration of 30% according to the volume ratio of 7: 3.
According to a preferred embodiment of the invention, the preparation of the OTAB-doped 4-cyano-4' -pentylbiphenyl in step (3) comprises: dissolving OTAB powder in chloroform to prepare 0.1mM OTAB chloroform solution, mixing the OTAB chloroform solution and 4-cyano-4 '-pentylbiphenyl according to the volume ratio of 1:1, swirling at 2500rpm for 1-5 min, and fully mixing, and blowing the chloroform by nitrogen to obtain the OTAB-doped 4-cyano-4' -pentylbiphenyl.
In the invention, in the step (3), in order to enhance the sealing property between the multi-channel sample groove and the glass substrate, a small amount of vaseline is coated between the glass substrate and the multi-channel sample groove.
Preferably, 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 carcinoembryonic antigen encoding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of prostate specific antigen encoding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of alpha fetoprotein encoding DNA is TAG ATA TGG CAG GA.
In the present invention, the liquid crystal biosensor modified with DNA encoded in step (4) has an optical response to the addition of DNA of a specific sequence.
In the invention, the coding DNA solution in the step (4) modifies the liquid crystal biological sensing interface to ensure that the coding DNA of the liquid crystal biological sensing interface reaches a saturated state; preferably, the concentration of the coding DNA solution in the step (4) is more than or equal to 0.1 mu M; further preferably, the concentration of the encoding DNA solution is 0.1 to 100. mu.M.
According to the invention, the pH value of the Tris buffer solution in the step (4) is 7-8.
According to the present invention, the solvent used for the encoding DNA solution in step (4) is 10mM Tris, 20mM NaCl, pH 7.3 buffer.
The detection method of the liquid crystal biosensor for simultaneously detecting a plurality of cancer markers comprises the following steps:
s1, preparation of a capture magnetic bead probe: respectively incubating the magnetic bead dispersion liquid with solutions of aptamers 1 of different cancer markers at 37 ℃ for 0.5-1 h, discarding the solutions, washing magnetic beads for 3-5 times by using a phosphate buffer solution to prepare magnetic bead capturing probes of different cancer markers, and jointly dispersing the magnetic bead capturing probes of different cancer markers in the phosphate buffer solution to obtain a mixed dispersion liquid of the magnetic bead capturing probes;
s2, preparation of a double-stranded signal probe: respectively mixing the aptamer 2 solutions of different cancer markers with the corresponding DNA solutions of the cancer marker signals for 5-10 min, heating to 95 ℃, incubating for 5-10 min, 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, uniformly mixing a sample to be detected and the mixed dispersion liquid of the captured magnetic bead probes in the S1, incubating for 30-60 min at 30-40 ℃, discarding the solution, washing for 3-5 times by using a phosphate buffer solution, dispersing the washed captured magnetic bead probes in the mixed solution of the double-stranded signal probes in the S2, incubating for 30-60 min at 30-40 ℃, taking supernatant, adding the supernatant into a communication channel of a multi-channel sample tank of the liquid crystal biosensor, stabilizing for 10min to observe the optical appearance of liquid crystal, and simultaneously detecting various cancer markers according to the change of the optical appearance of the liquid crystal in different independent channels.
Preferably, 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 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 TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC.
According to a preferred embodiment of the present invention, the magnetic beads in step S1 are modified with streptavidin.
Preferably, according to the present invention, the cancer marker aptamer 1 is biotin-modified in step S1.
In the invention, after the magnetic bead dispersion liquid and the cancer marker aptamer 1 solution are incubated together in the step S1, the magnetic beads reach the saturated adsorption capacity; preferably, in step S1, the ratio of the magnetic beads to the cancer marker aptamer 1 is 400pmol of cancer marker aptamer 1 modified by 1mg of magnetic beads.
According to the present invention, preferably, the concentration of the capture magnetic bead probes of different cancer markers in the mixed dispersion of the capture magnetic bead probes in step S1 is the same, and the total concentration of the capture magnetic bead probes of different cancer markers is greater than or equal to 0.1 mg/mL; further preferably, the total concentration of the capture magnetic bead probes of different cancer markers is 0.1-10 mg/mL.
In the present invention, the solvents of the magnetic bead dispersion and the cancer marker aptamer 1 solution in step S1 are phosphate buffer solutions.
Preferably, the cancer marker aptamer 2 in step S2 includes, but is not limited to, carcinoembryonic antigen aptamer 2, prostate specific antigen aptamer 2, and alpha fetoprotein aptamer 2, wherein the nucleotide sequence of carcinoembryonic antigen aptamer 2 is TAG CTA TAG GGG GTG AAG GGA TAC CC, the sequence of prostate specific antigen aptamer 2 is TAC ATC TGG GAG AGC GGA AGC GUG CUG GGC CGU CAG GUC ACG GCA GCG AAG CUC UAG GCG CGG CCA GUU GCC AUA ACC CAG AGG UCG AUG GAU CCU, and the 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 GGG TCT GCG TGG TCT GTG GTG CTG T.
According to a novel preferred embodiment 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 carcinoembryonic antigen signal DNA is TTC ACC CCC TAT AGC TA, the nucleotide sequence of prostate specific antigen signal DNA is CCG CTC TCC CAG ATG TA, and the nucleotide sequence of alpha fetoprotein signal DNA is GTC TTC CTG CCA TAT CTA.
Preferably, in step S2, the concentrations of the double-stranded signal probes of different cancer markers in the mixed solution of double-stranded signal probes are consistent, and the concentrations of the double-stranded signal probes of different cancer markers are respectively 10pM to 1000 pM.
Preferably, the solvent of the cancer marker aptamer 2 solution and the cancer marker signal DNA solution in step S2 is a buffer solution of 10mM Tris, 130mM NaCl, 10mM KCl, pH 7.3.
In the invention, step S3 is to mix the sample to be detected and the capture magnetic bead probe mixed dispersion liquid uniformly, the cancer markers in the sample to be detected are captured by the capture magnetic bead probes, and the cancer markers captured by the capture magnetic bead probes are combined with the cancer marker aptamer 2 in the double-chain signal probe mixed solution; preferably, in the step S3, the volume ratio of the sample to be detected to the mixed dispersion of the capture magnetic bead probes is 1 (1-10).
According to a preferred embodiment of the invention, the phosphate buffer comprises the following components: 2mM KH2PO4，8mM Na2HPO4，136mM NaCl，2.6mM KCl，pH＝7.3。
According to a preferred embodiment of the present invention, the detection method further comprises: and establishing a working curve according to the standard solution of the cancer markers and the change of the optical appearance of the liquid crystal thereof, thereby realizing the quantitative detection of the multiple cancer markers in the sample to be detected.
The invention has the technical characteristics that:
according to the invention, the multi-channel sample tank is printed in a 3D mode, the multi-channel sample tank comprises the independent channels and the communicating channels, the communicating channels are located above the independent channels, the intervals among the independent channels are eliminated, the upper half parts of different independent channels are communicated with each other, the multi-channel sample tank is fixed on a glass substrate, a liquid crystal biological sensing interface is constructed at the bottom of each independent channel, the liquid crystal biological sensing interface of each independent channel is modified by adopting coding DNA of different cancer markers, and the liquid crystal biosensor capable of detecting multiple cancer markers simultaneously is constructed.
Respectively co-incubating different cancer marker aptamers 1 with magnetic beads to prepare capture magnetic bead probes of different cancer markers, and then jointly dispersing the capture magnetic bead probes in a phosphate buffer solution to obtain a capture magnetic bead probe mixed dispersion solution; respectively annealing the aptamers 2 of different cancer markers with corresponding cancer marker signal DNA to prepare double-stranded signal probe solutions of different cancer markers, and mixing to obtain a double-stranded signal probe mixed solution; mixing a sample to be detected with the mixed dispersion liquid of the capture magnetic bead probe, capturing the cancer marker in the sample to be detected by utilizing the specific interaction of the aptamer 1 and the target cancer marker, and realizing the separation of the cancer marker from other matrixes in the sample to be detected and the enrichment of the cancer marker; dispersing the capture magnetic bead probe capturing the cancer marker into a double-stranded signal probe mixed solution, and separating the aptamer 2 from the signal DNA by utilizing the specific interaction of the aptamer 2 and the target cancer marker, wherein the binding force of the aptamer 2 and the target cancer marker is greater than that of the aptamer 2 and the complementary signal DNA, so that free signal DNA is released by displacement; adding free signal DNA solution into a communication channel of a multi-channel sample groove of a liquid crystal biosensor modified by different cancer marker coding DNAs, hybridizing the signal DNA and the coding DNA with high specificity due to the complementation of the sequences of the signal DNA and the coding DNA, hybridizing the coding DNAs in different independent channels with corresponding signal DNAs by using a DNA in-situ hybridization technology, changing the arrangement mode of liquid crystal, realizing the change of liquid crystal optical signals, and simultaneously detecting various cancer markers in a sample by using the change of the optical signals in different independent channels.
Has the advantages that:
1. the invention utilizes different cancer markers to code DNA to modify the liquid crystal biosensor, realizes the simultaneous detection and quantitative detection of various different cancer markers, and also realizes the accurate detection of extreme samples.
2. The invention utilizes the magnetic bead enrichment separation technology to simply and conveniently separate the target cancer marker from other interfering matrixes, greatly improves the specificity of detection and can realize the detection in biological samples such as whole blood, blood plasma, blood serum and the like.
3. The invention adopts a liquid crystal interface and combines a DNA interface hybridization technology, has high sensitivity and can meet the actual detection requirement without amplification.
Drawings
FIG. 1 is a schematic diagram showing the detection principle of carcinoembryonic antigen, prostate specific antigen and alpha fetoprotein;
FIG. 2 is a three-dimensional view and a sectional view of a multi-channel sample cell according to example 1;
FIG. 3 is a graph of the working curves for 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;
FIG. 4 is a bar graph showing the results of detection of carcinoembryonic antigen, prostate specific antigen and alpha fetoprotein of the blood samples of example 3; in the figure, a is the detection result of carcinoembryonic antigen, b is the detection result of prostate specific antigen, and c is the detection result of alpha fetoprotein; the ordinate is the content of cancer markers in blood;
FIG. 5 is a bar graph showing the results of detecting carcinoembryonic antigen in the hemolyzed sample of example 4; a is the comparison of the hemolysis sample and the normal blood sample, and b is the comparison of the hemolysis sample liquid crystal biosensor and the commercial ELISA kit.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the invention is not limited thereto. The drugs mentioned in the examples are all common commercial products unless otherwise specified.
The present invention is further illustrated by examples of three cancer markers, carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA) and Alpha Fetoprotein (AFP).
In the embodiment, the detection principle of carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA), and alpha-fetoprotein (AFP) is shown in fig. 1, and carcinoembryonic antigen aptamer 1, prostate specific antigen aptamer 1, and alpha-fetoprotein aptamer 1 are respectively co-incubated with magnetic beads to prepare capture magnetic bead probes for carcinoembryonic antigen, prostate specific antigen, and alpha-fetoprotein, and the three capture magnetic bead probes are simultaneously dispersed in a phosphate buffer to obtain a capture magnetic bead probe mixed dispersion solution; respectively annealing 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 to prepare a double-chain signal probe of carcinoembryonic antigen, prostate specific antigen and alpha-fetoprotein, and mixing the three double-chain signal probes to obtain a double-chain signal probe mixed solution; mixing the capture magnetic bead probe mixed dispersion liquid with a sample to be detected, capturing a cancer marker in the sample to be detected by utilizing the specific interaction of the aptamer 1 and the target cancer marker, and realizing the separation of the cancer marker from other matrixes in the sample to be detected and the enrichment of the cancer marker; dispersing the capture magnetic bead probe capturing the cancer marker into a double-stranded signal probe mixed solution, and separating the aptamer 2 from the signal DNA by utilizing the specific interaction of the aptamer 2 and the target cancer marker, wherein the binding force of the aptamer 2 and the target cancer marker is greater than that of the aptamer 2 and the complementary signal DNA, so that free signal DNA is released by displacement; adding free signal DNA into a communication channel of a multichannel sample groove of a liquid crystal biosensor modified by coding DNA of carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA) and Alpha Fetoprotein (AFP), hybridizing the signal DNA and the coding DNA with high specificity due to the complementary sequence of the signal DNA and the coding DNA, hybridizing the coding DNA in different independent channels with corresponding signal DNA by using a DNA in-situ hybridization technology, changing the arrangement mode of liquid crystal, realizing the change of liquid crystal optical signals, and simultaneously detecting the carcinoembryonic antigen (CEA), the Prostate Specific Antigen (PSA) and the Alpha Fetoprotein (AFP) in a sample by using the change of the optical signals in different independent channels.
Example 1: preparation of liquid crystal biosensor
A preparation method of a liquid crystal biosensor for simultaneously detecting multiple cancer markers comprises the following steps:
(1) preparation of glass substrate: soaking the glass slide in 60mL of washing liquid prepared by concentrated sulfuric acid with the mass concentration of 98% and hydrogen peroxide with the mass concentration of 30% according to the volume ratio of 7:3 at 75 ℃ for 30min, then respectively washing with water, absolute ethyl alcohol and methanol for 3 times, drying with nitrogen, drying at 110 ℃ for 12h, placing the glass slide in a DMOAP solution with the mass concentration of 0.5% for 15min at room temperature, washing with distilled water for 5 times for three times, drying with nitrogen, solidifying at 105 ℃ for 3h, and cooling to obtain the glass substrate;
(2) preparation of a multi-channel sample groove: the multi-channel sample groove is printed in a 3D mode, the multi-channel sample groove comprises independent channels and communicating channels, the communicating channels are located above the independent channels, so that the upper half parts of different independent channels are communicated with one another, the view of the multi-channel sample groove is shown in figure 2, the length of the multi-channel sample groove is 2.8cm, the width of the multi-channel sample groove is 1cm, the height of the multi-channel sample groove is 0.6cm, the diameter of each independent channel is 0.6cm, the multi-channel sample groove comprises 3 independent channels which are arranged side by side, the distance between the centers of circles of the two channels is 0.9cm, and the height of;
(3) fixing the multichannel sample groove prepared in the step (2) on the glass substrate prepared in the step (1) by using a dovetail clamp, in order to enhance the airtightness, smearing a little vaseline between the multichannel sample groove and the glass substrate, placing a G75 copper net at the bottom of a single channel, injecting 1.5 mu L of OTAB-doped 4-cyano-4 ' -pentylbiphenyl heated to 35-45 ℃ into the copper net while the OTAB-doped 4-cyano-4 ' -pentylbiphenyl is hot, and removing the redundant OTAB-doped 4-cyano-4 ' -pentylbiphenyl by using a capillary tube with the inner diameter of 0.5mm to construct a liquid crystal biological sensing interface, wherein the thickness of the liquid crystal biological sensing interface is consistent with that of the copper net and is 20 mu m;
the preparation method of the OTAB-doped 4-cyano-4' -pentylbiphenyl comprises the following steps: dissolving OTAB powder in chloroform to prepare 0.1mM OTAB chloroform solution, mixing the OTAB chloroform solution and 4-cyano-4 '-pentylbiphenyl according to the volume ratio of 1:1, swirling at 2500rpm for 3min, fully mixing, and blowing the chloroform with nitrogen to obtain OTAB-doped 4-cyano-4' -pentylbiphenyl;
(4) respectively preparing the coding DNA of the carcinoembryonic antigen, the prostate specific antigen and the alpha fetoprotein into solutions with the concentration of 1 mu M, respectively injecting corresponding coding DNA solutions into separate channels of the carcinoembryonic antigen, the prostate specific antigen and the alpha fetoprotein by using a buffer solution with the solvent of 10mM Tris, 20mM NaCl and pH 7.5, modifying a liquid crystal biosensing interface, standing for 10min at room temperature, then exchanging the coding DNA solutions in the channels by using Tris buffer solution (pH 7-8), exchanging for ten times, and washing away free coding DNA, thus obtaining the liquid crystal biosensor for simultaneously detecting the carcinoembryonic antigen, the prostate specific antigen and the alpha fetoprotein;
wherein, the nucleotide sequence of the carcinoembryonic antigen coding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of the prostate specific antigen coding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of the alpha fetoprotein coding DNA is TAG ATA TGG CAG GA.
Example 2: method for simultaneously detecting multiple cancer markers
A method for simultaneously detecting carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA) and Alpha Fetoprotein (AFP), comprising the steps of:
s1, preparation of a capture magnetic bead probe: respectively co-incubating 10 mu L of a 0.1mg/mL streptomycin avidin modified magnetic bead dispersion liquid with 10 mu L of a 0.4 mu M biotin modified carcinoembryonic antigen aptamer 1 solution, a prostate specific antigen aptamer 1 solution and an alpha fetoprotein aptamer 1 solution at 37 ℃ for 0.5h, then separating magnetic beads from the solutions by using a commercially available magnetic separation rack, discarding the solutions, washing the magnetic beads for 3 times by using a phosphate buffer solution to prepare three captured magnetic bead probes, then simultaneously dispersing the three captured magnetic bead probes (1 mu g each) into 30 mu L of the phosphate buffer solution, shaking up to obtain a captured magnetic bead probe mixed dispersion liquid, wherein the total concentration of the captured magnetic bead probes in the captured magnetic bead probe mixed dispersion liquid is 0.1 mg/mL;
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 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 TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC;
the magnetic bead dispersion liquid and the aptamer 1 solution are both phosphate buffer solutions;
s2, preparation of a double-stranded signal probe: mixing a carcinoembryonic antigen aptamer 2 solution, a prostate specific antigen aptamer 2 solution and an alpha fetoprotein aptamer 2 solution of 60pM with a corresponding carcinoembryonic antigen signal DNA solution, a prostate specific antigen signal DNA solution and an alpha fetoprotein aptamer 2 solution of 60pM respectively in equal volume, heating to 95 ℃, incubating for 5min, slowly annealing to room temperature to prepare three double-chain signal probe solutions, then mixing the three double-chain signal probe solutions (the concentrations of the three double-chain signal probe solutions are all 30pM) in equal volume, shaking up to obtain a double-chain signal probe mixed solution, wherein the concentrations of the three double-chain signal probes in the double-chain signal probe mixed solution are respectively 10 pM;
wherein, the nucleotide sequence of the carcinoembryonic antigen aptamer 2 is TAG CTA TAG GGG GTG AAG GGA TAC CC, and the nucleotide sequence of the 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 GCG AAG CUC UAG GCG CGG CCA GUU GCC AUA ACC CAG AGG UCG AUG GAU CCU, the nucleotide sequence of prostate specific antigen signaling DNA is CCG CTC TCC CAG ATG TA; the nucleotide sequence of the alpha fetoprotein aptamer 2 is TAG ATA TGG CAG GAA GAC AAA CAA GCT TGG CGG CGG GAA GGT GTT TAA ATT CCC GGG TCT GCG TGG TCT GTG GTG CTG T, and the nucleotide sequence of the alpha fetoprotein signal DNA is GTC TTC CTG CCA TAT CTA;
the solvents of the aptamer 2 solution and the signal DNA solution are 10mM Tris, 130mM NaCl, 10mM KCl and pH 7.5 buffer solution;
s3, isovolumetrically mixing a carcinoembryonic antigen, a prostate specific antigen and a standard solution of alpha-fetoprotein, wherein the concentration range of the carcinoembryonic antigen in the mixed standard solution is 0.4-1.6 ng/mL, the concentration of the prostate specific antigen is 0.1-0.5 ng/mL, and the concentration of the alpha-fetoprotein is 0.2-0.8 ng/mL, mixing 10 mu L of the mixed standard solution with 0.1mg/mL of a capture magnetic bead probe mixed dispersion prepared by 10 mu L S1, incubating for 30min at 37 ℃, separating magnetic beads by a magnetic frame, discarding the solution, washing for 3 times by phosphate buffer solution, dispersing the washed capture magnetic bead probe into a 10pM double-chain signal probe mixed solution prepared by 100 mu L S2, incubating for 30min at 37 ℃, separating magnetic beads by the magnetic frame, taking supernatant, adding the supernatant into a communication channel of a multi-channel sample tank of the liquid crystal biosensor prepared in example 1, after shaking for 10min in a shaking table, observing the optical appearance of the liquid crystal, and simultaneously detecting various cancer markers according to the change of the optical appearance of the liquid crystal in different independent channels;
the components of the phosphate buffer referred to in the examples were: 2mM KH2PO4，8mM Na2HPO4，136mM NaCl，2.6mM KCl，pH＝7.3。
Data processing: calculating pixels of a bright area by using Photoshop software, obtaining pixels of all liquid crystal areas by using Photoshop, obtaining the ratio of the two pixels as the ratio of the bright area, obtaining the ratio of the bright area of the initial liquid crystal morphology by using the method, obtaining the ratio of the bright area of the liquid crystal after adding a sample by using the method, calculating the difference of the two pixels as the change of the bright area, drawing by using the concentration of a standard substance as an abscissa and the change of the bright area as an ordinate to obtain a working curve, wherein the working curve is shown in FIG. 3, detecting the standard liquid with known concentration, and determining the yield to be 92.1-110.6%;
wherein the carcinoembryonicThe working curve of antigen (CEA) is that y is 44.02x-1.91, R20.985; the detection limit is 0.31 ng/mL;
the working curve for Prostate Specific Antigen (PSA) is y 155.33x-3.90, R20.986; the detection limit is 0.07 ng/mL;
the working curve of alpha-fetoprotein (AFP) is that y is 102.85x-2.23, R20.993; the detection limit is 0.19 ng/mL.
Example 3: detection of unknown concentrations of CEA, PSA and AFP in blood
The capture magnetic bead probes and the double-stranded signal probes were prepared as in example 2.
The test was carried out immediately after the blood sample was taken, and the sample used in this example was a blood sample of a healthy person.
A2 mu L blood sample is taken, diluted by 5 times by using phosphate buffer solution, incubated with 10 mu L of mixed dispersion liquid of 0.1mg/mL capture magnetic bead probes at 37 ℃ for 30min, the magnetic beads are separated by using a magnetic frame, the solution is discarded, washed for 3 times by using the phosphate buffer solution, the washed capture magnetic bead probes are dispersed into 100 mu L of mixed solution of 10pM double-stranded signal probes, incubated at 37 ℃ for 30min, the magnetic beads are separated by using the magnetic frame, supernatant is taken and added into a communication channel of a multi-channel sample tank of the liquid crystal biosensor prepared in example 1, and the optical appearance of liquid crystal is observed after shaking for 10min in a shaking table.
Data processing: the change of the bright area was calculated by using Photoshop software, and the concentration of CEA, PSA and AFP was calculated by substituting the working curve, and the measured concentrations of carcinoembryonic antigen (CEA), Prostate Specific Antigen (PSA) and alpha-fetoprotein (AFP) are shown in FIG. 4, and the relative deviation between them was-11.4% and 13.8% using a commercial ELISA plate as a detection reference (the commercial ELISA was performed according to the instructions).
Example 4: detection of carcinoembryonic antigen (CEA) in a hemolyzed sample
Blood that could not be immediately tested in example 3 was stored in a heparin tube, and the blood sample in the heparin tube was shaken vigorously at the time of testing to obtain a hemolyzed blood sample.
Taking 2 mu L of hemolytic blood sample, diluting the hemolytic blood sample by 5 times by using phosphate buffer solution, incubating the hemolytic blood sample and 10 mu L of mixed dispersion liquid of 0.1mg/mL capture magnetic bead probes at 37 ℃ for 30min, separating magnetic beads by using a magnetic frame, discarding the solution, washing the hemolyzed blood sample for 3 times by using the phosphate buffer solution, dispersing the washed capture magnetic bead probes into 100 mu L of mixed solution of 10pM double-stranded signal probes, incubating the hemolyzed blood sample for 30min at 37 ℃, separating magnetic beads by using the magnetic frame, taking supernatant, adding the supernatant into a communication channel of a multi-channel sample tank of the liquid crystal.
Data processing: the CEA concentration was determined by substituting the change in the bright region calculated by Photoshop software into the working curve, and the resulting carcinoembryonic antigen concentration was as shown in fig. 5, and a commercial ELISA plate was used as a detection reference (the commercial ELISA was performed according to the instructions).
The CEA concentration value of the hemolysis sample detected by the liquid crystal biosensor is slightly changed compared with the value measured by the same batch of normal blood samples (figure 5a), while the CEA value of the hemolysis sample detected by the traditional ELISA method is higher, 4 of the 5 samples exceed the cutoff value and show false positive (figure 5b), which shows that the performance of the detection method in the extreme sample detection is better than that of the traditional ELISA method.
The above description is only for the specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (10)
1. The liquid crystal biosensor for simultaneously detecting multiple cancer markers is characterized by comprising a multi-channel sample groove and a liquid crystal biosensor interface modified by coding DNA of different cancer markers; the multi-channel sample groove comprises an individual channel and a communicating channel, and the communicating channel is positioned above the individual channel, so that the upper half parts of different individual channels are communicated with each other; the different cancer markers code DNA modified liquid crystal biosensing interfaces constructed in different single channels of the multi-channel sample groove.
2. The liquid crystal biosensor of claim 1, wherein the cancer marker comprises carcinoembryonic antigen, prostate specific antigen and alpha fetoprotein, wherein the nucleotide sequence of carcinoembryonic antigen encoding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of prostate specific antigen encoding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of alpha fetoprotein encoding DNA is TAG ATA TGG CAG GA.
3. The method for preparing a liquid crystal biosensor for simultaneously detecting multiple cancer markers according to claim 1, comprising the steps of:
(1) soaking the glass sheet in a washing solution, soaking for 30-60 min at 70-90 ℃, then respectively washing for 3-4 times by using water, absolute ethyl alcohol and methanol, drying by using nitrogen, drying for 10-20 h at 100-120 ℃, placing in a DMOAP solution with the mass concentration of 0.1-3% for 15-30 min at room temperature, washing for 5-6 times by using distilled water for three times, drying by using nitrogen, solidifying for 3-5 h at 100-110 ℃, and cooling to obtain a glass substrate;
(2)3D printing a multi-channel sample tank, wherein the multi-channel sample tank comprises independent channels and communication channels, and the communication channels are positioned above the independent channels so that the upper half parts of different independent channels are mutually communicated;
(3) fixing the multi-channel sample tank obtained in the step (2) on the glass substrate obtained in the step (1), placing a copper mesh at the bottom of the single channel, and injecting OTAB-doped 4-cyano-4' -pentylbiphenyl into the copper mesh while the sample tank is hot at 35-45 ℃ to construct a liquid crystal biosensing interface, wherein the thickness of the liquid crystal biosensing interface is consistent with that of the copper mesh;
(4) and (3) respectively injecting the coding DNA solutions of the multiple cancer markers into different independent channels of the multi-channel sample tank treated in the step (3) to modify a liquid crystal biosensing interface, standing at room temperature for 10-20 min, and washing free coding DNA with a Tris buffer solution to obtain the liquid crystal biosensor for simultaneously detecting the multiple cancer markers.
4. The method of claim 3, wherein one or more of the following conditions are satisfied:
i. the washing liquid in the step (1) is prepared from concentrated sulfuric acid with the mass concentration of 98% and hydrogen peroxide with the mass concentration of 30% according to the volume ratio of 7: 3;
the process for preparing the OTAB-doped 4-cyano-4' -pentylbiphenyl of step (3): dissolving OTAB powder in chloroform to prepare 0.1mM OTAB chloroform solution, mixing the OTAB chloroform solution and 4-cyano-4 '-pentylbiphenyl according to the volume ratio of 1:1, swirling at 2500rpm for 1-5 min, and fully mixing, and blowing the chloroform by nitrogen to obtain OTAB-doped 4-cyano-4' -pentylbiphenyl;
the cancer marker in step (4) comprises carcinoembryonic antigen, prostate specific antigen and alpha fetoprotein, wherein the nucleotide sequence of carcinoembryonic antigen encoding DNA is TAG CTA TAG GGG GT, the nucleotide sequence of prostate specific antigen encoding DNA is TAC ATC TGG GAG AG, and the nucleotide sequence of alpha fetoprotein encoding DNA is TAG ATA TGG CAG GA;
the concentration of the coding DNA solution in the step (4) is more than or equal to 0.1 mu M;
v, the pH value of the Tris buffer solution in the step (4) is 7-8;
the encoding DNA solution in step (4) was in a buffer of 10mM Tris, 20mM NaCl, pH 7.5.
5. The method for detecting a liquid crystal biosensor which simultaneously detects a plurality of cancer markers according to claim 1, comprising the steps of:
s1, preparation of a capture magnetic bead probe: respectively incubating the magnetic bead dispersion liquid with solutions of aptamers 1 of different cancer markers at 37 ℃ for 0.5-1 h, discarding the solutions, washing magnetic beads for 3-4 times by using a phosphate buffer solution to prepare magnetic bead capturing probes of different cancer markers, and jointly dispersing the magnetic bead capturing probes of different cancer markers in the phosphate buffer solution to obtain a mixed dispersion liquid of the magnetic bead capturing probes;
s2, preparation of a double-stranded signal probe: respectively mixing the aptamer 2 solutions of different cancer markers with the corresponding DNA solutions of the cancer marker signals for 5-10 min, heating to 95 ℃, incubating for 5-10 min, 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, uniformly mixing a sample to be detected and the mixed dispersion liquid of the captured magnetic bead probes in the S1, incubating for 30-50 min at 35-40 ℃, discarding the solution, washing for 3-5 times by using a phosphate buffer solution, dispersing the washed captured magnetic bead probes in the mixed solution of the double-stranded signal probes in the S2, incubating for 30-50 min at 34-40 ℃, taking supernatant, adding the supernatant into a communication channel of a multi-channel sample tank of the liquid crystal biosensor, stabilizing for 10min to observe the optical appearance of liquid crystal, and simultaneously detecting various cancer markers according to the change of the optical appearance of the liquid crystal in different independent channels.
6. The detection method according to claim 5, wherein the cancer marker aptamer 1 in step S1 comprises 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 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 TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC.
7. The detection method according to claim 5, wherein one or more of the following conditions are satisfied:
(i) in the step S1, the magnetic beads are modified by streptomycin avidin;
(ii) the cancer marker aptamer 1 is biotin-modified in step S1;
(iii) in step S1, the ratio of the magnetic beads to the cancer marker aptamer 1 is 1mg magnetic beads modified 400pmol cancer marker aptamer 1;
(iv) in the step S1, the concentrations of the capture magnetic bead probes of different cancer markers in the mixed dispersion liquid of the capture magnetic bead probes are consistent, and the total concentration of the capture magnetic bead probes of different cancer markers is more than or equal to 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 the step S2 are consistent, and the concentrations of the double-stranded signal probes of different cancer markers are respectively 10pM-1000 pM;
(vi) the solvent of the cancer marker aptamer 2 solution and the cancer marker signal DNA solution in step S2 is 10mM Tris, 130mM NaCl, 10mM KCl, pH 7.5 buffer;
(vii) in the step S3, the volume ratio of the sample to be detected to the mixed dispersion liquid of the capture magnetic bead probe is 1 (1-10);
(viii) the phosphate buffer solution comprises the following components: 2mM KH2PO4，8mM Na2HPO4，136mM NaCl，2.6mM KCl，pH＝7.3。
8. The detection method according to claim 5, wherein the cancer marker aptamer 2 in step S2 comprises carcinoembryonic antigen aptamer 2, prostate specific antigen aptamer 2, and alpha fetoprotein aptamer 2, wherein the nucleotide sequence of carcinoembryonic antigen aptamer 2 is TAG CTA TAG GGG GTG AAG GGA TAC CC, the sequence of prostate specific antigen aptamer 2 is TAC ATC TGG GAG AGC GGA AGC GUG CUG GGC CGU CAG GUC ACG GCA GCG AAG CUC UAG GCG CGG CCA GUU GCC AUA ACC CAG AGG UCG AUG GAU CCU, and the 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 GGG TCT GCG TGG TCT GTG GTG CTG T.
9. The detection method according to claim 5, wherein the cancer marker signal DNA in the step S2 includes carcinoembryonic antigen signal DNA, prostate specific antigen signal DNA and alpha fetoprotein signal DNA, wherein the nucleotide sequence of carcinoembryonic antigen signal DNA is TTC ACC CCC TAT AGC TA, the nucleotide sequence of prostate specific antigen signal DNA is CCG CTC TCC CAG ATG TA, and the nucleotide sequence of alpha fetoprotein signal DNA is GTC TTC CTG CCA TAT CTA.
10. The detection method of claim 5, further comprising: and establishing a working curve according to the standard solution of the cancer markers and the change of the optical appearance of the liquid crystal thereof, thereby realizing the quantitative detection of the multiple cancer markers in the sample to be detected.
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| CN113406011A (en) * | 2021-05-25 | 2021-09-17 | 长春理工大学 | Liquid crystal form aptamer biosensor, preparation method thereof and application thereof in detection of prostate specific antigen |
| CN114235714A (en) * | 2022-02-28 | 2022-03-25 | 之江实验室 | Method for realizing one-step nucleic acid detection by using liquid crystal sensor |
| CN114235714B (en) * | 2022-02-28 | 2022-05-27 | 之江实验室 | Method for realizing one-step nucleic acid detection by liquid crystal sensor |
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