CN111394460B - Colloidal gold lateral chromatography test strip for breast cancer detection and colloidal gold lateral chromatography test strip for simultaneous detection of breast cancer and cervical cancer - Google Patents
Colloidal gold lateral chromatography test strip for breast cancer detection and colloidal gold lateral chromatography test strip for simultaneous detection of breast cancer and cervical cancer Download PDFInfo
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Abstract
The invention provides a colloidal gold lateral chromatography test strip for detecting breast cancer, a preparation method thereof and the colloidal gold lateral chromatography test strip for simultaneously detecting the breast cancer and cervical cancer, belonging to the technical field of immunochemistry detection, wherein the colloidal gold lateral chromatography test strip comprises a PVC base plate, a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad; the colloidal gold lateral chromatography test paper can simultaneously detect miRNA markers (miR-16 and miR-191) and protein markers (CA 153 and CA 125) of breast cancer. The invention also provides a colloidal gold lateral chromatography test strip for simultaneously detecting the breast cancer and the cervical cancer, which can overcome the interference of background signals (hybridization reaction among components), realize the specificity detection of two tumor marker groups of different types, namely miRNA group of the cervical cancer and the breast cancer and protein, and the detection specificity reaches 100%.
Description
Technical Field
The invention relates to the technical field of immunochemistry detection, in particular to a colloidal gold lateral chromatography test strip for detecting breast cancer and a colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer.
Background
Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast gland. Currently, breast cancer examination mainly depends on physical examination, imaging examination (B-ultrasound, molybdenum target X-ray, MRI), and histocytology examination. These methods have a high detection rate, but are not only invasive and uncomfortable to the examiner, but also have insufficient specificity and sensitivity of detection and low diagnostic efficacy of detection.
Disclosure of Invention
The invention aims to provide a colloidal gold lateral chromatography test strip for detecting breast cancer and a colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer. The colloidal gold lateral chromatography test strip for simultaneously detecting the breast cancer and the cervical cancer can simultaneously detect miRNA groups and protein tumor marker groups of the breast cancer and the cervical cancer, and can improve the diagnosis efficiency of detection.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a colloidal gold lateral chromatography test strip for breast cancer detection, which comprises a PVC (polyvinyl chloride) base plate, a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad; the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad are sequentially adhered to the PVC base plate and connected in a lap joint mode;
the nitrocellulose membrane is provided with a detection area T area and a quality control area C area; the detection area T area comprises a T1 line, a T2 line, a T3 line and a T4 line; the quality control area C comprises a line C1 and a line C2;
the long side of the nitrocellulose is taken as a row, and the T1 line, the T2 line, the T3 line, the T4 line, the C1 line and the C2 line are distributed in 2 rows and 3 columns; the C1 line and the C2 line are divided into two rows and distributed at one end of each row close to the absorption pad;
a T1 line solution is sprayed on the T1 line; the T1 line solution comprises a miR-16 capture probe; the nucleotide sequence of the miR-16 capture probe is shown in SEQ ID NO:1 is shown in the specification; the 3' end of the miR-16 capture probe is labeled by biotin;
a T2 line solution is sprayed on the T2 line; the T2 line solution comprises a miR-191 capture probe; the nucleotide sequence of the miR-191 capture probe is shown as SEQ ID NO:2 is shown in the specification; the 3' end of the miR-191 capture probe is labeled by biotin;
the T3 line is sprayed with a T3 line solution; the T3 line solution comprises a mouse anti-CA 153 protein monoclonal antibody;
the T4 line is sprayed with a T4 line solution; the T4 line solution comprises a mouse anti-CA 125 protein monoclonal antibody;
the C1 line is sprayed with a C1 line solution; the C1 line solution comprises a miR-16 control probe and a miR-191 control probe; the nucleotide sequence of the miR-16 control probe is shown as SEQ ID NO:3 is shown in the specification; the nucleotide sequence of the miR-191 control probe is shown as SEQ ID NO:4 is shown in the specification; the 3' ends of the miR-16 control probe and the miR-191 control probe are respectively marked by biotin;
the C2 line is sprayed with a C2 line solution; the C2 line solution comprises a secondary antibody;
the binding pad is adsorbed with a colloidal gold-detection probe conjugate; the colloidal gold-detection probe conjugate comprises a colloidal gold-miR-16 detection probe conjugate, a colloidal gold-miR-191 detection probe conjugate, a colloidal gold-CA 153 protein-bovine serum albumin conjugate and a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-16 detection probe is shown as SEQ ID NO:5 is shown in the specification; the nucleotide sequence of the miR-191 detection probe is shown as SEQ ID NO:6 is shown in the specification; the 3' ends of the miR-16 detection probe and the miR-191 detection probe are respectively modified by sulfydryl.
Preferably, the concentration of the mouse anti-CA 153 protein monoclonal antibody in the T3 line solution is 0.8-1.2 mg/mL; the concentration of the mouse anti-CA 125 protein monoclonal antibody in the T4 line solution is 0.8-1.2 mg/mL.
Preferably, the lengths of the overlapped portions of the lap joint are respectively 2 to 3mm.
Preferably, the T1 line, the T3 line and the C1 line are distributed in sequence in a row from the combination pad to the absorption pad, and the T2 line, the T4 line and the C2 line are distributed in sequence in a second row; the distance between every two adjacent sites is 4-6 mm.
Preferably, the preparation method of the colloidal gold-CA 153 protein-bovine serum albumin conjugate or the colloidal gold-CA 125 protein-bovine serum albumin conjugate comprises the following steps:
mixing the nano-gold solution, the HPV protein solution and the bovine serum albumin solution, and coupling to obtain a colloidal gold-CA 153 protein-bovine serum albumin conjugate or a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the HPV protein is CA153 protein or CA125 protein.
Preferably, the preparation method of the colloidal gold-miR-16 complementary sequence probe conjugate or the colloidal gold-miR-191 complementary sequence probe conjugate comprises the following steps:
mixing the nano-gold solution, the dATP solution, the sodium dodecyl sulfate solution, the NaCl aqueous solution and the miRNA complementary sequence probe, and coupling to obtain a colloidal gold-miR-16 complementary sequence probe conjugate or a colloidal gold-miR-191 complementary sequence probe conjugate; the miRNA complementary sequence probe is a miR-16 complementary sequence probe or a miR-191 complementary sequence probe conjugate.
Preferably, the preparation method of the T1 line solution or the T2 line solution comprises the following steps: and mixing the miR-16 partial complementary sequence or miR-191 partial complementary sequence marked by the biotin with a streptavidin solution, and culturing to obtain a T1 line solution or a T2 line solution.
The invention also provides a preparation method of the breast cancer detection colloid Jin Celiu chromatographic test strip, which comprises the following steps:
1) Sticking a nitrocellulose membrane on a PVC (polyvinyl chloride) bottom plate, and fixing a combination pad, a sample pad and an absorption pad in sequence;
2) Adsorbing the colloidal gold-detection probe conjugate on the binding pad, and respectively spraying a T1 line solution, a T2 line solution, a T3 line solution, a T4 line solution, a C1 line solution and a C2 line solution on a T1 line, a T2 line, a T3 line, a T4 line solution, a C1 line solution and a C2 line of the nitrocellulose membrane to obtain the breast cancer detection colloidal Jin Celiu chromatographic test strip.
The invention provides a colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer, which is based on the colloidal gold lateral chromatography test strip for detecting breast cancer in the scheme, wherein a T area of a detection area also comprises a T5 line, a T6 line, a T7 line and a T8 line;
a T5 line solution is sprayed on the T5 line; the T5 line solution comprises a miR-21 capture probe; the nucleotide sequence of the miR-21 capture probe is shown as SEQ ID NO:9 is shown in the figure; the 3' end of the miR-21 capture probe is labeled by biotin;
a T6 line solution is sprayed on the T6 line; the T6 line solution comprises a miR-196a capture probe; the nucleotide sequence of the miR-196a capture probe is shown as SEQ ID NO:10 is shown in the figure; the 3' end of the miR-196a capture probe is labeled by biotin;
the T7 line is sprayed with a T7 line solution; the T7 line solution comprises a mouse anti-HPV E6 protein monoclonal antibody;
the T8 line is sprayed with a T8 line solution; the T8 line solution comprises a mouse anti-HPV E7 protein monoclonal antibody;
the colloidal gold-detection probe conjugate also comprises a colloidal gold-miR-21 detection probe conjugate, a colloidal gold-miR-196 a detection probe conjugate, a colloidal gold-HPV E6 protein-bovine serum albumin conjugate and a colloidal gold-HPV E7 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-21 detection probe is shown as SEQ ID NO:13 is shown in the figure; the nucleotide sequence of the miR-196a detection probe is shown as SEQ ID NO:14 is shown in the figure; the 5' ends of the miR-21 detection probe and the miR-196a detection probe are respectively modified by sulfydryl.
Preferably, the long side of the nitrocellulose is taken as a row, the T1 line, the T2 line, the T3 line, the T4 line, the T5 line, the T6 line, the T7 line, the T8 line, the C1 line and the C2 line are distributed in 2 rows and 3 columns, the T1 line, the T2 line, the T3 line, the T4 line and the C1 line are distributed in sequence in the row from the combining pad to the absorption pad, and the T5 line, the T6 line, the T7 line, the T8 line and the C2 line are distributed in sequence in the second row.
The invention has the beneficial effects that: the invention provides a colloidal gold lateral chromatography test strip for breast cancer detection, which comprises a PVC (polyvinyl chloride) base plate, a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad; the detection probe modified by colloidal gold is adsorbed on the combination pad, and the colloidal gold-detection probe conjugate comprises a colloidal gold-miR-16 complementary sequence probe conjugate, a colloidal gold-miR-191 complementary sequence probe conjugate, a colloidal gold-CA 153 protein-bovine serum albumin conjugate and a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the nitrocellulose membrane is provided with a detection area T area and a quality control area C area; the detection area T area comprises a T1 line, a T2 line, a T3 line and a T4 line; the quality control area C comprises a line C1 and a line C2; a miR-16 capture probe is sprayed on the T1 line; miR-191 capture probes are sprayed on the T2 line; the T3 line is sprayed with a mouse anti-CA 153 protein monoclonal antibody; the T4 line is sprayed with a mouse anti-CA 125 protein monoclonal antibody. The colloidal gold lateral chromatography test paper can simultaneously detect miRNA markers (miR-16 and miR-191) and protein markers (CA 153 and CA 125) of breast cancer, and the specificity reaches 100%.
When a sample solution contains a tumor marker (miRNA or protein), the marker and gold nanoparticles are captured in a test area of the lateral flow sensor by the sandwich type bonding reaction and are red, and an excessive gold nanoparticle complex is captured in a control area and is red. The gray value of the red area of the test area is in direct proportion to the concentration of the tumor marker in the sample solution, and can be used for quantitatively detecting the concentration of the marker in the sample. Compared with the PCR technology and the enzyme-linked immunoassay technology which are generally adopted in the market at present, the colloidal gold lateral chromatography test strip for detecting the breast cancer has better diagnosis efficiency. The colloidal gold lateral chromatography test strip for detecting the breast cancer is a simple, convenient, non-invasive, rapid and simple tool, has high sensitivity and specificity on breast cancer biomarkers, can be used for screening early cancers, monitoring the response to treatment and providing a technology for providing real-time prognosis information of cancer patients.
The invention also provides a colloidal gold lateral chromatography test strip for simultaneously detecting the breast cancer and the cervical cancer, which can simultaneously detect the miRNA groups and the protein tumor marker groups of the breast cancer and the cervical cancer and can improve the diagnosis efficiency of detection. The colloidal gold lateral chromatography test strip for simultaneously detecting the breast cancer and the cervical cancer can overcome the interference of background signals (hybridization reaction among components), realize the specificity detection of the cervical cancer and the breast cancer simultaneously, and the detection specificity reaches 100 percent.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold lateral chromatography test strip for breast cancer detection according to the present invention;
FIG. 2 is a schematic diagram of the colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer in the invention.
Detailed Description
The invention provides a colloidal gold lateral chromatography test strip for breast cancer detection, which comprises a PVC (polyvinyl chloride) base plate, a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad; the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad are sequentially adhered to the PVC base plate and connected in a lap joint mode; the lengths of the overlapped parts are preferably 2-3 mm respectively; the schematic diagram of the colloidal gold lateral chromatography test strip for breast cancer detection is shown in figure 1.
In the present invention, the absorbent pad is preferably lapped on the nitrocellulose membrane; the binding pad is preferably lapped on the nitrocellulose membrane; the sample pad is preferably lapped over the conjugate pad.
The colloidal gold lateral chromatography test strip has the following action principle: the sample flows to the conjugate pad under the action of the buffer solution, is combined with the nano-gold particle DNA probe complex (Au-NP-DNA) or the nano-gold-protein conjugate on the conjugate pad, and moves to the detection area together, namely is hybridized and combined by the probe on the detection area, and the color is developed. The colloidal gold lateral chromatography test strip can simultaneously detect miRNA markers (miR-16 and miR-191) of breast cancer and protein markers (CA 153 and CA 125). When a sample solution contains a tumor marker (miRNA or protein), the marker and gold nanoparticles are captured in a test area of the lateral flow sensor by the sandwich type bonding reaction and are red, and an excessive gold nanoparticle complex is captured in a control area and is red. The gray value of the red area of the test area is in direct proportion to the concentration of the tumor marker in the sample solution, and can be used for quantitatively detecting the concentration of the marker in the sample.
In the invention, the nitrocellulose membrane is provided with a detection zone T zone and a quality control zone C zone; the detection area T area comprises a T1 line, a T2 line, a T3 line and a T4 line; the quality control area C comprises a line C1 and a line C2; the long edge of the nitrocellulose is taken as a row, the T1 line, the T2 line, the T3 line, the T4 line, the C1 line and the C2 line are preferably distributed in 2 rows and 3 columns, the T1 line, the T3 line and the C1 line are sequentially distributed in the row from the combining pad to the absorption pad, and the T2 line, the T4 line and the C2 line are sequentially distributed in the second row; the distance between every two adjacent sites is 4-6 mm, preferably 5mm; the location of the DNA capture probes in front of the protein antibody probes (near the conjugate pad) can reduce non-specific adsorption.
In the invention, a T1 line solution is sprayed on the T1 line; the T1 line solution comprises a miR-16 capture probe; the nucleotide sequence of the miR-16 capture probe is shown as SEQ ID NO:1, specifically: ACGTGCTGCTA; the 3' end of the miR-16 capture probe is labeled by biotin; the nucleotide sequence of the miR-16 target corresponding to the miR-16 capture probe is shown in SEQ ID NO:7, specifically: UAGCAGCACGUAAAUAUUGGCG.
In the invention, a T2 line solution is sprayed on the T2 line; the T2 line solution comprises a miR-191 capture probe; the nucleotide sequence of the miR-191 capture probe is shown as SEQ ID NO:2, specifically: CAGCTGCTTTTG; the 3' end of the miR-191 capture probe is labeled by biotin; the nucleotide sequence of the miR-191 target corresponding to the miR-191 capture probe is shown in SEQ ID NO:8, specifically: CAACGGAAUCCCAAAAGCAGCUG.
The preparation method of the T1 line solution or the T2 line solution comprises the following steps: and mixing the miR-16 partial complementary sequence or miR-191 partial complementary sequence marked by the biotin with a streptavidin solution, and culturing to obtain a T1 line solution or a T2 line solution.
In the specific implementation process of the invention, 50nM biotin-labeled T1/T2 and 200. Mu.L of 2.5mg/ml streptavidin (Shanghai Biotech) are mixed and cultured in 0.01MPBS for 1h, the mixture is transferred to a dialysis tube (molecular weight cut-off 30000), and unbound aptamer probes are removed by centrifugation (6000rpm, 20min,4 ℃) in a refrigerated centrifuge. Adding PBS and repeating the steps twice, and finally collecting the centrifuged solution and fixing the volume to 600 mu L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
In the invention, the T3 line is sprayed with a T3 line solution; the T3 line solution comprises a mouse anti-CA 153 protein monoclonal antibody; the concentration of the mouse anti-CA 153 protein monoclonal antibody in the T3 line solution is preferably 0.8-1.2 mg/mL, and more preferably 1mg/mL.
In the invention, the T4 line is sprayed with a T4 line solution; the T4 line solution comprises a mouse anti-CA 125 protein monoclonal antibody; the concentration of the mouse anti-CA 125 protein monoclonal antibody in the T4 line solution is preferably 0.8-1.2 mg/mL, and more preferably 1mg/mL.
In the invention, the C1 line is sprayed with a C1 line solution; the C1 line solution comprises a miR-16 control probe and a miR-191 control probe; the nucleotide sequence of the miR-16 control probe is shown as SEQ ID NO:3, specifically: AAATATTGGCG; the nucleotide sequence of the miR-191 control probe is shown as SEQ ID NO:4, specifically: CAAAAGCAGCTG; the 3' ends of the miR-16 control probe and the miR-191 control probe are respectively marked by biotin.
In the invention, the C2 line is sprayed with a C2 line solution; the C2 line solution comprises a secondary antibody; the secondary antibody preferably comprises goat anti-mouse IgG.
In the invention, the binding pad adsorbs a colloidal gold-detection probe conjugate; the colloidal gold-detection probe conjugate comprises a colloidal gold-miR-16 detection probe conjugate, a colloidal gold-miR-191 detection probe conjugate, a colloidal gold-CA 153 protein-bovine serum albumin conjugate and a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-16 detection probe is shown as SEQ ID NO:5, specifically: CGCCAATATTT; the nucleotide sequence of the miR-191 detection probe is shown as SEQ ID NO:6, specifically: GGATTCCGTTG; the 3' ends of the miR-16 detection probe and the miR-191 detection probe are respectively modified by sulfydryl.
In the present invention, the nitrocellulose membrane includes detection regions (T1 to T4) and control regions (C1, C2). The detection center region of the lateral flow strip was mainly concentrated on the nitrocellulose membrane. The detection result is measured by a T area and a C area, and the color on the T area can be subjected to semi-quantitative or quantitative and qualitative analysis. The C area is used for verifying the effectiveness of the test strip detection. If the C area has no color, the test result of the test strip is not credible.
In the present invention, the material of the bonding pad preferably includes a glass fiber film. The combination pad is used for adsorbing the detection probe (complementary sequences of the antibody and the miRNA part) modified by the colloidal gold and uniformly conveying the sample liquid to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved.
In the present invention, the preparation method of the colloidal gold-CA 153 protein-bovine serum albumin conjugate or the colloidal gold-CA 125 protein-bovine serum albumin conjugate preferably comprises the following steps: and mixing the nano-gold solution, the HPV protein solution and the bovine serum albumin solution, and coupling to obtain a colloidal gold-CA 153 protein-bovine serum albumin conjugate or a colloidal gold-CA 125 protein-bovine serum albumin conjugate.
In the present invention, the HPV protein includes a CA153 protein or a CA125 protein; the invention has no special limit on the nano-gold, and the nano-gold is prepared by adopting a conventional method; the nano gold solution is preferably a five-time concentrated nano gold solution; the particle size of the nano-gold in the nano-gold solution is preferably 15-20 nm; the proportion of the nano-gold solution, the HPV protein solution and the bovine serum albumin solution is 1mL:10 μ L of: 100 mu L of the solution; the concentration of HPV protein in the HPV protein solution is preferably 1mM; the mass fraction percentage of the bovine serum albumin in the bovine serum albumin solution is 10 percent; the temperature of the coupling is preferably 20-30 ℃, and more preferably 25 ℃; the coupling process is preferably accompanied by shaking.
In the specific implementation process of the invention, firstly, mixing the nano-gold solution and the HPV protein solution for first coupling to obtain a colloidal gold-HPV protein conjugate, and mixing the colloidal gold-HPV protein conjugate and a bovine serum protein solution for second coupling to obtain a colloidal gold-HPV protein-bovine serum protein conjugate; the first coupling time was 60min; the time for the second coupling is preferably 30min; after the coupling, the preferable steps comprise centrifuging the conjugate, washing the precipitate by using PBS buffer solution, and dissolving the precipitate in a nanoparticle storage solution for later use; the rotating speed of the centrifugation is preferably 12,000rpm, and the time is preferably 10min; the pH value of the PBS buffer solution is preferably 7.2-7.4; the number of the washing is preferably set to be equal to3 times; the nanoparticle stock solution comprises the following components: 20mmol/LNa 3 PO 4 ·12H 2 O (12 sodium phosphate hydrate), 5% BSA (bovine serum albumin), 0.25% Tween 20 (Tween 20), 10% sucrose.
In the invention, the preparation method of the colloidal gold-miR-16 complementary sequence probe conjugate or the colloidal gold-miR-191 complementary sequence probe conjugate comprises the following steps: mixing the nano-gold solution, the dATP solution, the sodium dodecyl sulfate solution, the NaCl aqueous solution and the miRNA complementary sequence probe, and coupling to obtain a colloidal gold-miR-16 complementary sequence probe conjugate or a colloidal gold-miR-191 complementary sequence probe conjugate; the miRNA complementary sequence probe comprises a miR-16 complementary sequence probe or a miR-191 complementary sequence probe conjugate.
In the specific implementation process of the invention, 1ml of ten-fold concentrated nano gold solution and 10 mu L of dATP with the concentration of 1mM are mixed and mixed for 20min at the temperature of 20-30 ℃ to obtain a first mixture; mixing the first mixture with 10 μ L of SDS at a concentration of 1% for 10min to obtain a second mixture; mixing the second mixture with 50 mu L of NaCl aqueous solution with the concentration of 0.2M, and adding 2 mu L of NaCl aqueous solution every 2-3min during the mixing process to obtain a third mixture; mixing the third mixture with a 1OD miRNA complementary sequence probe, and coupling for 3h at 60 ℃ to obtain a conjugate; centrifuging the conjugate at 12,000rpm for 10min, washing the precipitate with PBS buffer solution, and dissolving the washed precipitate in a nanoparticle storage solution for later use; the pH value of the PBS buffer solution is preferably 7.2-7.4; the number of washing is preferably 3.
In the present invention, the material of the sample pad preferably includes a glass fiber membrane; the sample pad has the function of slowing down the migration speed of a sample to be detected, is beneficial to the uniform distribution of liquid to be detected on the sample pad, and creates a precondition for better flowing to the combination pad.
The invention uses the absorption paper with high absorption efficiency, large capacity and good stability. The absorption pad is used for promoting the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensuring that an object (liquid) to be detected can cross the NC membrane through the last step of siphoning action, improving the output value of a detection signal and reducing the signal-to-noise ratio.
The invention also provides a preparation method of the breast cancer detection colloid Jin Celiu chromatographic test strip, which comprises the following steps:
1) Sticking a nitrocellulose membrane on a PVC (polyvinyl chloride) bottom plate, and fixing a combination pad, a sample pad and an absorption pad in sequence;
2) Adsorbing the colloidal gold-detection probe conjugate on the binding pad, and respectively spraying a T1 line solution, a T2 line solution, a T3 line solution, a T4 line solution, a C1 line solution and a C2 line solution on a T1 line, a T2 line, a T3 line, a T4 line solution, a C1 line solution and a C2 line of the nitrocellulose membrane to obtain a breast cancer detection colloidal Jin Celiu chromatographic test strip; the spraying amount of the T1 line solution, the T2 line solution, the T3 line solution, the T4 line solution, the C1 line solution and the C2 line solution is preferably 1 mu L.
The application method of the breast cancer detection colloid Jin Celiu chromatographic test strip preferably comprises the following steps:
after 10 μ L of serum was mixed with 90 μ L of sample buffer (PBS + 1%) and dropped on the sample pad, when the sample solution contained a tumor marker (miRNA or protein), the sandwich-type bonding reaction captured the marker and gold nanoparticles in the test area of the test strip and appeared red, and the excess gold nanoparticle complex was captured in the control area and appeared red. And observing the detection result after 5min, wherein the detection result is invalid after more than 20 min. The gray value of the red area of the test area is in direct proportion to the concentration of the tumor marker in the sample solution, and can be used for quantitatively detecting the concentration of the marker in the sample; the quantitative detection equipment is preferably a test strip gray scale detector.
The invention provides a colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer, which is based on the colloidal gold lateral chromatography test strip for detecting breast cancer, and the T area of the detection area also comprises a T5 line, a T6 line, a T7 line and a T8 line.
In the invention, a T5 line solution is sprayed on the T5 line; the T5 line solution comprises a miR-21 capture probe; the nucleotide sequence of the miR-21 capture probe is shown as SEQ ID NO:9, specifically: CTGATAAGCTA; the 3' end of the miR-21 capture probe is labeled by biotin; the nucleotide sequence of the miR-21 target corresponding to the miR-21 capture probe is shown in SEQ ID NO:15, specifically: UAGCUUAUCAGACCUGAUUGUAUGA.
In the invention, a T6 line solution is sprayed on the T6 line; the T6 line solution comprises a miR-196a capture probe; the nucleotide sequence of the miR-196a capture probe is shown as SEQ ID NO:10, specifically: GAAACTACCTA; the 3' end of the miR-196a capture probe is labeled by biotin; the nucleotide sequence of the miR-196a target corresponding to the miR-196a capture probe is shown in SEQ ID NO:16, specifically: UAGGUAGUUUCAUGUUGUUGGG.
The preparation method of the T5 line solution or the T6 line solution comprises the following steps: and mixing the partial complementary sequence of the miR-21 or the partial complementary sequence of the miR-196a marked by the biotin with a streptavidin solution, and culturing to obtain a T5 line solution or a T6 line solution.
In the specific implementation process of the invention, 50nM biotin-labeled T5/T6 and 200. Mu.L of 2.5mg/ml streptavidin (Shanghai Biotech) are mixed and cultured in 0.01MPBS for 1h, and then the mixture is transferred to a dialysis tube (molecular weight cut-off 30000) and centrifuged (6000rpm, 20min,4 ℃) in a refrigerated centrifuge to remove unbound aptamer probes. Adding PBS and repeating the steps twice, and finally collecting the centrifuged solution and fixing the volume to 600 mu L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
In the invention, the T7 line is sprayed with a T7 line solution; the T7 line solution comprises a mouse anti-HPV E6 protein monoclonal antibody; the concentration of the mouse anti-HPV E6 protein monoclonal antibody in the T7 line solution is preferably 0.8-1.2 mg/mL, and more preferably 1mg/mL.
In the invention, the T8 line is sprayed with a T8 line solution; the T8 line solution comprises a mouse anti-HPV E7 protein monoclonal antibody; the concentration of the mouse anti-HPV E7 protein monoclonal antibody in the T8 line solution is preferably 0.8-1.2 mg/mL, and more preferably 1mg/mL.
In the invention, the colloidal gold-detection probe conjugate also comprises a colloidal gold-miR-21 detection probe conjugate, a colloidal gold-miR-196 a detection probe conjugate, a colloidal gold-HPV E6 protein-bovine serum albumin conjugate and a colloidal gold-HPV E7 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-21 detection probe is shown as SEQ ID NO:13, specifically: TCAACATCAGT; the nucleotide sequence of the miR-196a detection probe is shown as SEQ ID NO:14, specifically: CCCAACAACAT; the 5' ends of the miR-21 detection probe and the miR-196a detection probe are respectively modified by sulfydryl.
The long side of the nitrocellulose is taken as a row, the T1 line, the T2 line, the T3 line, the T4 line, the T5 line, the T6 line, the T7 line, the T8 line, the C1 line and the C2 line are preferably distributed in 2 rows and 3 columns, the T1 line, the T2 line, the T3 line, the T4 line and the C1 line are sequentially distributed in the row from the combining pad to the absorption pad, and the T5 line, the T6 line, the T7 line, the T8 line and the C2 line are sequentially distributed in the second row; the distance between every two adjacent sites is preferably 4-6 mm, and more preferably 5mm; the location of the DNA capture probes in front of the protein antibody probes (near the conjugate pad) can reduce non-specific adsorption.
In the specific implementation process of the invention, 1ml of ten-fold concentrated nano gold solution and 10 mu L of dATP with the concentration of 1mM are mixed and mixed for 20min at the temperature of 20-30 ℃ to obtain a first mixture; mixing the first mixture with 10 μ L of SDS at a concentration of 1% for 10min to obtain a second mixture; mixing the second mixture with 50 mu L of NaCl aqueous solution with the concentration of 0.2M, and adding 2 mu L of NaCl aqueous solution every 2-3min during the mixing process to obtain a third mixture; mixing the third mixture with a 1OD miRNA complementary sequence probe, and coupling for 3h at 60 ℃ to obtain a conjugate; centrifuging the conjugate at 12,000rpm for 10min, washing the precipitate by using a PBS buffer solution, and dissolving the washed precipitate in a nanoparticle storage solution for later use; the pH value of the PBS buffer solution is preferably 7.2-7.4; the number of washing is preferably 3.
In the present invention, the material of the sample pad preferably includes a glass fiber membrane; the sample pad has the function of slowing down the migration speed of a sample to be detected, is beneficial to the uniform distribution of liquid to be detected on the sample pad, and creates a precondition for better flowing to the combination pad.
The invention uses the absorption paper with high absorption efficiency, large capacity and good stability. The absorption pad is used for promoting the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensuring that an object (liquid) to be detected can cross the NC membrane through the last step of siphoning action, improving the output value of a detection signal and reducing the signal-to-noise ratio.
The structural schematic diagram of the colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer is shown in figure 2.
The preparation method and the use method of the colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer refer to the colloidal gold lateral chromatography test strip for detecting breast cancer in the scheme, and the detailed description is omitted here.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The sources of reagents used in the examples of the invention are as follows:
chloroauric acid, sodium phosphate dodecahydrate (Na) 3 PO 4 ·12H 2 O), bovine Serum Albumin (BSA), sucrose (sucrose), tween-20 (Tween 20), sodium chloride (NaCl), tris-HCl, phosphate buffered saline (PBS, pH 7.4,0.01M), all available from Sigma-Aldrich.
DNA probes were purchased from Shanghai Producers.
Cellulose fiber membranes (CFSP 001700), glass fiber membranes (GFCP 000800), nitrocellulose membranes (HFB 18004) and laminates (HF 000MCl 00) were all available from Millipore.
Other reagents were analytically pure. All water used in the experiment was ultrapure water (> 18M. Omega. Cm).
TABLE 1 nucleotide sequences involved in the detection of breast cancer according to the invention
TABLE 2 nucleotide sequences involved in cervical cancer detection of the invention
The test paper preparation process comprises the following steps: spraying the colloidal gold coupled antibody (or detection probe) on the bonding pad; and spraying the antibody (or streptavidin coupled probe) on the corresponding position of the NC membrane. Then assembling: the combination pad is lapped on the nitrocellulose membrane; the sample pad is lapped on the combination pad; the absorption pad is lapped on the nitrocellulose membrane.
Embodiment 1 preparation method and application method of colloidal gold lateral chromatography test strip for breast cancer detection
1. Antibody-labeled colloidal gold: adding 10 μ L of 1mM antibody (CA 125 antibody, CA153 antibody, secondary antibody) into 1mL of five-fold concentrated nanogold (15-20 nm) solution, shaking with a shaker at room temperature for 60min, adding 100 μ L of 10% BSA, shaking for 30min, centrifuging at 12,000rpm,10min, removing supernatant, washing with PBS buffer (pH7.2-7.4) for 3 times, and dissolving the precipitate in 1mL of nanoparticle stock solution (20 mmol/LNa) 3 PO 4 ·12H 2 O,5% BSA,0.25% Tween 20, 10% sucralose), the conjugate solution was stored in a refrigerator at 4 ℃ until use.
2. Labeling colloidal gold with a DNA sequence: taking 1mL ten-fold concentrated nanogold (15-20 nm) solution, adding 10 mu L1mM dATP, oscillating for 20min by an oscillator at room temperature, then adding 15 mu L1% SDS, after oscillating and culturing for ten min, adding 50 mu L0.2M NaCl (the speed is controlled to be 2 mu L every 2-3 min), then adding 1OD miRNA complementary sequence probe, reacting for 3h at 60 ℃, centrifuging by a centrifuge (the rotating speed is 12,000rpm, 10min), removing supernatant, washing for 3 times by PBS buffer solution (pH7.2-7.4), finally dissolving the precipitate in 1mL nano particle storage solution (20 mmol/L Na) 3 PO 4 ·12H 2 O,5% BSA,0.25% Tween 20, 10% sucralose), the conjugate solution was stored in a refrigerator at 4 ℃ until use.
3. The T3/T4 protein monoclonal antibody (1 mg/mL) can be directly sprayed on an NC membrane;
4. T1/T2 DNA sequence processing steps: after 50nM biotin-labeled T1/T2 was incubated with 200. Mu. L2.5mg/ml streptavidin (Shanghai Producer) in 0.01MPBS for 1h, the mixture was transferred to a dialysis tube (molecular weight cut-off 30000) and centrifuged (6000rpm, 20min,4 ℃) in a refrigerated centrifuge to remove unbound aptamer probe. Adding PBS and repeating the steps twice, and finally collecting the centrifuged solution and fixing the volume to 600 mu L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
5. Spraying colloidal gold labeled antibody (detection probe) on the bonding pad; and a streptavidin coupled T1/T2 capture probe, a T3/T4 CA125/CA153 antibody, a C1 control probe and a C2 secondary antibody are sprayed at corresponding positions of the NC membrane.
6. A nitrocellulose membrane (NC membrane) was adhered to the PVC base plate, then the conjugate pad was fixed in position and ensured to have 2mm overlap with the NC membrane, then the sample pad was adhered in position and ensured to have 2mm overlap with the conjugate pad, and finally the absorbent pad was adhered and ensured to have 2mm overlap with the NC membrane to ensure smooth liquid flow.
A detection step: after 10 μ L of serum was mixed with 90 μ L of sample buffer (PBS + 1%) and dropped on the sample pad, when the sample solution contained a tumor marker (miRNA or protein), the sandwich-type bonding reaction captured the marker and gold nanoparticles in the test area of the test strip and appeared red, and the excess gold nanoparticle complex was captured in the control area and appeared red. And observing the detection result after 5min, wherein the detection result is invalid after more than 20 min.
Embodiment 2 preparation method and application method of colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer
1. Antibody-labeled colloidal gold: adding 10 μ L of 1mM antibody (CA 125 antibody, CA153 antibody, E6 antibody, E7 antibody, secondary antibody) into 1mL of five-fold concentrated nanogold (15-20 nm) solution, shaking with a shaker at room temperature for 60min, adding 100 μ L of 10% BSA, shaking for 30min, centrifuging at 12,000rpm,10min, removing supernatant, washing with PBS buffer (pH 7.2-7.4) for 3 times, and dissolving the precipitate in 1mL of nanoparticle stock solution (20 mmol/L Na) 3 PO 4 ·12H 2 O,5% BSA,0.25% Tween 20, 10% sucralose), the conjugate solution was stored in a refrigerator at 4 ℃ until use.
2. Labeling colloidal gold with a DNA detection probe: taking 1mL ten-fold concentrated nanogold (15-20 nm) solution, adding 10 mu L1mM dATP, shaking for 20min by a shaker at room temperature, then adding 15 mu L1% SDS, shaking for ten min, adding 50 mu L0.2M NaCl (the speed is controlled to be 2 mu L added every 2-3 min), then adding 1OD miRNA complementary sequence probe, reacting for 3h at 60 ℃, centrifuging by a centrifuge (the rotating speed is 12,000rpm, 10min), removing supernatant, washing for 3 times by PBS buffer solution (pH7.2-7.4), and finally dissolving the precipitate in 1mL nanoparticle stock solution (20 mmol/L Na/20 mmol Na) 3 PO 4 ·12H 2 O,5% BSA,0.25% Tween 20, 10% sucralose), the conjugate solution was stored in a refrigerator at 4 ℃ until use.
3. The T3/T4/T7/T8/C2 antibody (CA 125 antibody, CA153 antibody, E6 antibody, E7 antibody, second antibody) (1 mg/mL) can be directly sprayed on the NC membrane;
4. T1/T2/T5/T6/C1 DNA capture probe treatment step: after 50nM biotin-labeled T1/T2/T5/T6/C1 was mixed with 200. Mu.L of 2.5mg/ml streptavidin (Shanghai Biotech) in 0.01MPBS for 1h, the mixture was transferred to a dialysis tube (molecular weight cut-off 30000) and centrifuged (6000rpm, 20min,4 ℃) in a refrigerated centrifuge to remove unbound aptamer probe. Adding PBS and repeating the steps twice, and finally collecting the centrifuged solution and fixing the volume to 600 mu L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
5. Spraying colloidal gold labeled antibody (detection probe) on the bonding pad; streptavidin-coupled T1/T2/T5/T6 capture probes, T3/T4/T7/T8 antibodies, C1 control probes and C2 secondary antibodies are sprayed on the corresponding positions of the NC membrane.
6. A nitrocellulose membrane (NC membrane) was adhered to the PVC base plate, then the conjugate pad was fixed in position and ensured to have 2mm overlap with the NC membrane, then the sample pad was adhered in position and ensured to have 2mm overlap with the conjugate pad, and finally the absorbent pad was adhered and ensured to have 2mm overlap with the NC membrane to ensure smooth liquid flow.
A detection step: after 10 μ L of serum was mixed with 90 μ L of sample buffer (PBS + 1%) and dropped on the sample pad, when the sample solution contained a tumor marker (miRNA or protein), the sandwich-type bonding reaction captured the marker and gold nanoparticles in the test area of the test strip and appeared red, and the excess gold nanoparticle complex was captured in the control area and appeared red. And observing the detection result after 5min, wherein the detection result is invalid after more than 20 min.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (9)
1. A colloidal gold lateral chromatography test strip for breast cancer detection is characterized by comprising a PVC bottom plate, a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad; the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad are sequentially adhered to the PVC base plate and connected in a lap joint mode;
the nitrocellulose membrane is provided with a detection area T area and a quality control area C area; the detection area T area comprises a T1 line, a T2 line, a T3 line and a T4 line; the quality control area C comprises a line C1 and a line C2;
taking the long side of the nitrocellulose as a row, wherein the T1 line, the T2 line, the T3 line, the T4 line, the C1 line and the C2 line are distributed in 2 rows and 3 columns; the T1 line, the T3 line and the C1 line are sequentially distributed in the first row according to the direction from the combination pad to the absorption pad; the T2 lines, the T4 lines and the C2 lines are sequentially distributed in the second row according to the direction from the combination pad to the absorption pad;
a T1 line solution is sprayed on the T1 line; the T1 line solution comprises a miR-16 capture probe; the nucleotide sequence of the miR-16 capture probe is shown as SEQ ID NO:1 is shown in the specification; the 3' end of the miR-16 capture probe is labeled by biotin;
a T2 line solution is sprayed on the T2 line; the T2 line solution comprises a miR-191 capture probe; the nucleotide sequence of the miR-191 capture probe is shown as SEQ ID NO:2 is shown in the specification; the 3' end of the miR-191 capture probe is labeled by biotin;
the T3 line is sprayed with a T3 line solution; the T3 line solution comprises a mouse anti-CA 153 protein monoclonal antibody;
the T4 line is sprayed with a T4 line solution; the T4 line solution comprises a mouse anti-CA 125 protein monoclonal antibody;
the C1 line is sprayed with a C1 line solution; the C1 line solution comprises a miR-16 control probe and a miR-191 control probe; the nucleotide sequence of the miR-16 control probe is shown as SEQ ID NO:3 is shown in the specification; the nucleotide sequence of the miR-191 control probe is shown as SEQ ID NO:4 is shown in the specification; the 3' ends of the miR-16 control probe and the miR-191 control probe are respectively marked by biotin;
the C2 line is sprayed with a C2 line solution; the C2 line solution comprises a secondary antibody;
the binding pad is adsorbed with a colloidal gold-detection probe conjugate; the colloidal gold-detection probe conjugate comprises a colloidal gold-miR-16 detection probe conjugate, a colloidal gold-miR-191 detection probe conjugate, a colloidal gold-CA 153 protein-bovine serum albumin conjugate and a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-16 detection probe is shown as SEQ ID NO:5 is shown in the specification; the nucleotide sequence of the miR-191 detection probe is shown as SEQ ID NO:6 is shown in the specification; the 3' ends of the miR-16 detection probe and the miR-191 detection probe are respectively modified by sulfydryl.
2. The colloidal gold lateral chromatography test strip of claim 1, wherein the concentration of the murine anti-CA 153 protein monoclonal antibody in the T3 line solution is 0.8-1.2 mg/mL; the concentration of the mouse anti-CA 125 protein monoclonal antibody in the T4 line solution is 0.8-1.2 mg/mL.
3. The colloidal gold lateral chromatography test strip of claim 1, wherein the overlapping portions are each 2-3 mm in length.
4. The colloidal gold lateral chromatography test strip of claim 1, wherein the T1 line, the T3 line and the C1 line are sequentially distributed from the binding pad to the absorption pad, and the T2 line, the T4 line and the C2 line are sequentially distributed from the second row, and the distance between every two adjacent sites is 4-6 mm.
5. The colloidal gold lateral chromatography test strip of claim 1, wherein the preparation method of the colloidal gold-CA 153 protein-bovine serum albumin conjugate or the colloidal gold-CA 125 protein-bovine serum albumin conjugate comprises the following steps:
mixing the nano-gold solution, the HPV protein solution and the bovine serum albumin solution, and coupling to obtain a colloidal gold-CA 153 protein-bovine serum albumin conjugate or a colloidal gold-CA 125 protein-bovine serum albumin conjugate; the HPV protein is CA153 protein or CA125 protein.
6. The colloidal gold lateral chromatography test strip of claim 1, wherein the preparation method of the colloidal gold-miR-16 complementary sequence probe conjugate or the colloidal gold-miR-191 complementary sequence probe conjugate comprises the following steps:
mixing the nano-gold solution, the dATP solution, the sodium dodecyl sulfate solution, the NaCl aqueous solution and the miRNA complementary sequence probe, and coupling to obtain a colloidal gold-miR-16 complementary sequence probe conjugate or a colloidal gold-miR-191 complementary sequence probe conjugate; the miRNA complementary sequence probe is a miR-16 complementary sequence probe or a miR-191 complementary sequence probe conjugate.
7. The colloidal gold lateral chromatography test strip of claim 1, wherein the preparation method of the T1 line solution or the T2 line solution comprises the following steps: and mixing the miR-16 partial complementary sequence or miR-191 partial complementary sequence marked by the biotin with a streptavidin solution, and culturing to obtain a T1 line solution or a T2 line solution.
8. The preparation method of the breast cancer detection colloid Jin Celiu chromatographic test strip of any one of claims 1 to 7, comprising the steps of:
1) Sticking a nitrocellulose membrane on a PVC (polyvinyl chloride) bottom plate, and fixing a combination pad, a sample pad and an absorption pad in sequence;
2) Adsorbing the colloidal gold-detection probe conjugate on the binding pad, and respectively spraying a T1 line solution, a T2 line solution, a T3 line solution, a T4 line solution, a C1 line solution and a C2 line solution on a T1 line, a T2 line, a T3 line, a T4 line solution, a C1 line solution and a C2 line of the nitrocellulose membrane to obtain the breast cancer detection colloidal Jin Celiu chromatographic test strip.
9. A colloidal gold lateral chromatography test strip for simultaneously detecting breast cancer and cervical cancer is characterized in that based on the colloidal gold lateral chromatography test strip for detecting breast cancer of claim 1, a T area of a detection area further comprises a T5 line, a T6 line, a T7 line and a T8 line;
a T5 line solution is sprayed on the T5 line; the T5 line solution comprises a miR-21 capture probe; the nucleotide sequence of the miR-21 capture probe is shown as SEQ ID NO:9 is shown in the figure; the 3' end of the miR-21 capture probe is labeled by biotin;
a T6 line solution is sprayed on the T6 line; the T6 line solution comprises a miR-196a capture probe; the nucleotide sequence of the miR-196a capture probe is shown as SEQ ID NO:10 is shown in the figure; the 3' end of the miR-196a capture probe is labeled by biotin;
the T7 line is sprayed with a T7 line solution; the T7 line solution comprises a murine anti-HPVE 6 protein monoclonal antibody;
the T8 line is sprayed with a T8 line solution; the T8 line solution comprises a murine anti-HPVE 7 protein monoclonal antibody;
the colloidal gold-detection probe conjugate also comprises a colloidal gold-miR-21 detection probe conjugate, a colloidal gold-miR-196 a detection probe conjugate, a colloidal gold-HPVE 6 protein-bovine serum albumin conjugate and a colloidal gold-HPVE 7 protein-bovine serum albumin conjugate; the nucleotide sequence of the miR-21 detection probe is shown as SEQ ID NO:13 is shown in the figure; the nucleotide sequence of the miR-196a detection probe is shown as SEQ ID NO:14 is shown in the figure; the 5' ends of the miR-21 detection probe and the miR-196a detection probe are respectively modified by sulfydryl.
Priority Applications (1)
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|---|---|---|---|
| CN202010255516.6A CN111394460B (en) | 2020-04-02 | 2020-04-02 | Colloidal gold lateral chromatography test strip for breast cancer detection and colloidal gold lateral chromatography test strip for simultaneous detection of breast cancer and cervical cancer |
Applications Claiming Priority (1)
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| CN109270269A (en) * | 2018-09-05 | 2019-01-25 | 河南省生物工程技术研究中心 | A kind of fluorescence immune chromatography detection card, kit for the multi-joint detection of early-stage breast cancer |
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