阅读说明：本技术 一种用于检测dna的免疫层析试纸条及其制备方法 (Immunochromatographic test strip for detecting DNA and preparation method thereof ) 是由 刘国东 余庆才 张静 邱万伟 钱立生 李坤 张学记 于 2020-11-20 设计创作，主要内容包括：本发明提供了一种用于检测DNA的免疫层析试纸条,属于免疫层析检测技术领域；所述免疫层析试纸条的结合垫上喷涂有检测探针与金纳米棒的偶联物；所述检测探针为5’端巯基修饰的单链DNA；所述检测探针与待检测DNA的核苷酸序列互补配对。本发明中,金纳米棒(AuNRs)具有可调谐和更强的可见/近红外吸收、更高的表面增强拉曼散射截面、单向等离子体传播的可能性以及增强的光致发光。本发明基于AuNRs的免疫层析试纸条能够在短时间内对DNA进行灵敏和定量的视觉检测。经过对比试验,基于AuNRs的侧向流动传感器用肉眼能够检测到DNA最小浓度为10pM,比基于金纳米颗粒(AuNPs)的低20倍。(The invention provides an immunochromatographic test strip for detecting DNA, belonging to the technical field of immunochromatographic detection; conjugates of the detection probe and the gold nanorods are sprayed on the bonding pad of the immunochromatographic test strip; the detection probe is single-stranded DNA modified by 5' end sulfydryl; the detection probe is complementary and matched with the nucleotide sequence of the DNA to be detected. In the present invention, gold nanorods (AuNRs) have tunable and stronger visible/near infrared absorption, higher surface-enhanced raman scattering cross-section, the possibility of unidirectional plasma propagation, and enhanced photoluminescence. The immune chromatography test strip based on AuNRs can perform sensitive and quantitative visual detection on DNA in a short time. Through comparative experiments, the AuNRs-based lateral flow sensor can detect DNA with a minimum concentration of 10pM, which is 20 times lower than that of gold nanoparticles (AuNPs) with naked eyes.)

1. An immunochromatographic test strip for detecting DNA, which comprises a base plate, and a sample pad, a combination pad, a test pad and an absorption pad which are connected on the base plate in sequence,

a conjugate of a detection probe and a gold nanorod is sprayed on the bonding pad; the detection probe is single-stranded DNA modified by 5' end sulfydryl; the detection probe is complementary and matched with the nucleotide sequence of the DNA to be detected;

the detection pad is provided with a detection line and a quality control line; a conjugate of a capture probe and streptavidin is sprayed on the detection line; the 3' end of the capture probe is labeled by biotin;

a conjugate of a quality control probe and streptavidin is sprayed on the quality control line; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementary and matched with the nucleotide sequence of the DNA to be detected.

2. The immunochromatographic test strip according to claim 1, wherein the length of the gold nanorods is 20 to 200 nm.

3. The immunochromatographic test strip according to claim 1, wherein the preparation method of the conjugate of the detection probe and the gold nanorod comprises the following steps:

and sequentially adding dATP, a sodium dodecyl sulfate aqueous solution, a sodium chloride aqueous solution and a detection probe into the gold nanorod, performing coupling reaction, centrifuging, collecting precipitate, washing and resuspending to obtain a solution containing the conjugate of the detection probe and the gold nanorod.

4. The immunochromatographic test strip according to claim 3, wherein the volume ratio of the gold nanorods, dATP, aqueous sodium dodecyl sulfate solution, aqueous sodium chloride solution and detection probes is: 500: 5: 7.5: 25: 85 parts by weight; the concentration of dATP is 1 mM; the mass concentration of the sodium dodecyl sulfate aqueous solution is 1 percent; the concentration of the sodium chloride aqueous solution was 2M.

5. The immunochromatographic test strip according to claim 3, wherein the reaction procedure of the coupling reaction comprises: incubate in water bath at 60 ℃ for 3 h.

6. The immunochromatographic test strip according to claim 3, wherein the reagents for resuspension comprise an elution buffer; the elution buffer comprises the following components in parts by mass: na (Na)₃PO₄·12H₂O304 mg, bovine serum albumin 2.0g, sucrose 4.0g, Tween-200.1 g and water 40 g.

7. The immunochromatographic test strip according to any one of claims 1 to 3, wherein the preparation method of the gold nanorods comprises the following steps:

1) adding HAuCl into hexadecyl trimethyl ammonium bromide water solution₄Adding NaBH into the aqueous solution while stirring₄Continuously stirring the solution for 2min to obtain a gold nano seed solution;

2) mixing hexadecyl trimethyl ammonium bromide aqueous solution and HAuCl₄Aqueous solution, AgNO₃And mixing the aqueous solution, the reducing agent and the gold nano-seed solution, and carrying out reduction reaction to obtain the gold nano-rod.

8. The immunochromatographic test strip according to claim 1, wherein the preparation method of the conjugate of the capture probe and streptavidin comprises: mixing a capture probe and a streptavidin aqueous solution, coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS (phosphate buffer solution) with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove the unreacted capture probe, and collecting the supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of the capture probe to the streptavidin aqueous solution is 50 nmol: 80 μ L.

9. The immunochromatographic test strip according to claim 1, wherein the preparation method of the conjugate of the quality control probe and streptavidin comprises: mixing a quality control probe with a streptavidin aqueous solution with the concentration of 2.5mg/mL, coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove unreacted capture probes, and collecting supernatant to obtain a quality control probe solution containing a conjugate of the quality control probe and the streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is 4: 1.

10. the preparation method of the immunochromatographic test strip of any one of claims 1 to 9, comprising the steps of:

s1, 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;

s2, adsorbing a conjugate of a detection probe and a gold nanorod on the bonding pad, spraying a conjugate of a capture probe and streptavidin on a detection line of the nitrocellulose membrane, and spraying a conjugate of a quality control probe and streptavidin on a quality control line to obtain an immunochromatography test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorod is 8-12 cm²。

Technical Field

The invention relates to the technical field of immunochromatography detection, in particular to an immunochromatography test strip for detecting DNA and a preparation method thereof.

Background

The gene is a genetic basic unit, carries DNA or RNA sequence of genetic information, transmits the genetic information to the next generation through replication, and guides the synthesis of protein to express the genetic information carried by the gene, thereby controlling the character expression of organism individuals. Gene detection is a technique for detecting DNA by blood, other body fluids, or cells. DNA detection is very important in gene therapy, clinical diagnosis, and various biomedical research. The most common technique for detecting DNA is the Polymerase Chain Reaction (PCR). PCR has high sensitivity and accuracy, but due to the need for trained personnel, expensive instruments and relatively clean environments, PCR is not effective when the concentration of the sample to be tested is too low.

Disclosure of Invention

The invention aims to provide an immunochromatographic test strip for detecting DNA and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an immunochromatographic test strip for detecting DNA, which comprises a base plate, and a sample pad, a combination pad, a test pad and an absorption pad which are sequentially connected on the base plate;

a conjugate of a detection probe and a gold nanorod is sprayed on the bonding pad; the detection probe is single-stranded DNA modified by 5' end sulfydryl; the detection probe is complementary and matched with the nucleotide sequence of the DNA to be detected;

the detection pad is provided with a detection line and a quality control line; a conjugate of a capture probe and streptavidin is sprayed on the detection line; the 3' end of the capture probe is labeled by biotin;

a conjugate of a quality control probe and streptavidin is sprayed on the quality control line; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementary and matched with the nucleotide sequence of the DNA to be detected.

Preferably, the length of the gold nanorod is 20-200 nm.

Preferably, the preparation method of the conjugate of the detection probe and the gold nanorod comprises the following steps:

and sequentially adding dATP, a sodium dodecyl sulfate aqueous solution, a sodium chloride aqueous solution and a detection probe into the gold nanorod, performing coupling reaction, centrifuging, collecting precipitate, washing and resuspending to obtain a solution containing the conjugate of the detection probe and the gold nanorod.

Preferably, the volume ratio of the gold nanorods, the dATP, the sodium dodecyl sulfate aqueous solution, the sodium chloride aqueous solution and the detection probe is as follows: 500: 5: 7.5: 25: 85 parts by weight; the concentration of dATP is 1 mM; the mass concentration of the sodium dodecyl sulfate aqueous solution is 1 percent; the concentration of the sodium chloride aqueous solution was 2M.

Preferably, the reaction sequence of the coupling reaction comprises: incubate in water bath at 60 ℃ for 3 h.

Preferably, the reagents used for resuspension include an elution buffer; the elution buffer comprises the following components in parts by mass: na (Na)₃PO₄·12H₂O304 mg, bovine serum albumin 2.0g, sucrose 4.0g, Tween-200.1 g and water 40 g.

Preferably, the preparation method of the gold nanorods comprises the following steps:

1) adding HAuCl into hexadecyl trimethyl ammonium bromide water solution₄Adding NaBH into the aqueous solution while stirring₄Continuously stirring the solution for 2min to obtain a gold nano seed solution;

2) mixing hexadecyl trimethyl ammonium bromide aqueous solution and HAuCl₄Aqueous solution, AgNO₃And mixing the aqueous solution, the reducing agent and the gold nano-seed solution, and carrying out reduction reaction to obtain the gold nano-rod.

Preferably, the preparation method of the conjugate of the capture probe and the streptavidin comprises the following steps: mixing a capture probe and a streptavidin aqueous solution, coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS (phosphate buffer solution) with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove the unreacted capture probe, and collecting the supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of the capture probe to the streptavidin aqueous solution is 50 nmol: 80 μ L.

Preferably, the quality control probe and a streptavidin water solution with the concentration of 2.5mg/mL are mixed and coupled to obtain a coupling reaction solution, the coupling reaction solution and PBS with the concentration of 500mg/L are mixed and placed in a sample concentration tube with the molecular weight cutoff of 30000, centrifugation is carried out at 6000rpm for 20min to remove unreacted capture probe, and supernate is collected to obtain the quality control probe solution containing the conjugate of the quality control probe and the streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is 4: 1.

the invention also provides a preparation method of the immunochromatographic test strip in the scheme, which comprises the following steps:

s1, 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;

s2, adsorbing a conjugate of a detection probe and a gold nanorod on the bonding pad, spraying a conjugate of a capture probe and streptavidin on a detection line of the nitrocellulose membrane, and spraying a conjugate of a quality control probe and streptavidin on a quality control line to obtain an immunochromatography test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorod is 8-12 cm²。

The invention has the beneficial effects that: the invention provides an immunochromatographic test strip for detecting DNA, wherein a conjugate of a detection probe and a gold nanorod is sprayed on a binding pad of the immunochromatographic test strip; the detection probe is single-stranded DNA modified by 5' end sulfydryl; the detection probe is complementary and matched with the nucleotide sequence of the DNA to be detected. In the present invention, gold nanorods (AuNRs) have tunable and stronger visible/near infrared absorption, higher surface-enhanced raman scattering cross-section, the possibility of unidirectional plasma propagation, and enhanced photoluminescence. The immune chromatography test strip based on AuNRs can perform sensitive and quantitative visual detection on DNA in a short time. Through comparative experiments, the AuNRs-based lateral flow sensor can detect DNA with a minimum concentration of 10pM, which is 20 times lower than that of gold nanoparticles (AuNPs) with naked eyes. The immunochromatographic test strip provides a new idea for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.

Drawings

FIG. 1 is a drawing of gold nanorods with an average length of 25 nm;

FIG. 2 is a gold nanorod with an average length of 60 nm;

FIG. 3 is a gold nanorod with an average length of 80 nm;

FIG. 4 shows the comparison of the results of detecting target DNA (target concentration of 0.2nM) for three gold nanorods with different lengths;

FIG. 5 is a graph showing the results of DNA detection using a test strip based on AuNR (average major diameter of 60 nm);

FIG. 6 shows DNA detection (target concentration 10pM) by gold nanoparticles (A) and gold nanorods (B).

Detailed Description

The invention provides an immunochromatographic test strip for detecting DNA, which comprises a base plate, and a sample pad, a combination pad, a test pad and an absorption pad which are sequentially connected on the base plate;

a conjugate of a detection probe and a gold nanorod is sprayed on the bonding pad; the detection probe is single-stranded DNA modified by 5' end sulfydryl; the detection probe is complementary and matched with the nucleotide sequence of the DNA to be detected;

the detection pad is provided with a detection line and a quality control line; a conjugate of a capture probe and streptavidin is sprayed on the detection line; the 3' end of the capture probe is labeled by biotin;

a conjugate of a quality control probe and streptavidin is sprayed on the quality control line; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementary and matched with the nucleotide sequence of the DNA to be detected.

In the present invention, the length of the DNA to be detected is preferably20-30 bp; in the present invention, the 5' end of the quality control probe is preferably further modified with (CH)₂)₆[ 6 methylenes ].

In the invention, the length of the gold nanorod is preferably 20-200 nm, more preferably 25-80 nm, and even more preferably 50-60 nm.

In the present invention, the preparation method of the conjugate of the detection probe and the gold nanorod preferably comprises: and sequentially adding dATP, a sodium dodecyl sulfate aqueous solution, a sodium chloride aqueous solution and a detection probe into the gold nanorod, performing coupling reaction, centrifuging, collecting precipitate, washing and resuspending to obtain a solution containing the conjugate of the detection probe and the gold nanorod.

In the present invention, the volume ratio of the gold nanorods, dATP, an aqueous solution of sodium dodecyl sulfate, an aqueous solution of sodium chloride, and the detection probe is preferably: 500: 5: 7.5: 25: 85 parts by weight; the concentration of dATP is preferably 1 mM; the mass concentration of the sodium dodecyl sulfate aqueous solution is preferably 1%; the concentration of the sodium chloride aqueous solution is preferably 2M; the concentration of the detection probe is preferably 3. mu.g/mL. In the invention, each substance is added into the gold nanorods, and preferably, shaking mixing is carried out; the time for each mixing is preferably 10 to 30min, more preferably 20min, and the temperature is preferably 25 ℃. In the invention, the sodium chloride aqueous solution is preferably added in 5 times, and after each addition, the sodium chloride aqueous solution is added again after being shaken and uniformly mixed. The invention has no special requirements on the adding rates of dATP, the aqueous solution of sodium dodecyl sulfate, the aqueous solution of sodium chloride and the detection probe. In the present invention, the procedure of the coupling reaction is preferably incubation in a water bath at 60 ℃ for 3 h. In the invention, the rotation speed of the centrifugation is preferably 8000rpm, and the time is preferably 8 min; the reagent used for washing the precipitate is preferably Phosphate Buffered Saline (PBS); the number of washing is preferably 3. In the present invention, the reagents used for resuspension preferably include an elution buffer; the elution buffer comprises the following components in parts by mass: na (Na)₃PO₄·12H₂O304 mg, Bovine Serum Albumin (BSA)2.0g, sucrose 4.0g, Tween-200.1 g and water 40 g; the preparation method of the elution buffer is preferableThe method comprises the following steps: sequentially adding Na into water₃PO₄·12H₂O, bovine serum albumin, 4.0g of sucrose and 200.1g of tween-tween are uniformly mixed to obtain an elution buffer solution.

In the present invention, the storage temperature of the conjugate of the detection probe and the gold nanorods is preferably 4 ℃.

In the present invention, the preparation method of the gold nanorods preferably includes the following steps:

1) adding HAuCl into hexadecyl trimethyl ammonium bromide water solution₄Adding NaBH into the aqueous solution while stirring₄Continuously stirring the solution for 2min to obtain a gold nano seed solution;

2) mixing hexadecyl trimethyl ammonium bromide aqueous solution and HAuCl₄Aqueous solution, AgNO₃And mixing the aqueous solution, the reducing agent and the gold nano-seed solution, and carrying out reduction reaction to obtain the gold nano-rod.

Firstly, HAuCl is added into hexadecyl trimethyl ammonium bromide aqueous solution₄Adding NaBH into the aqueous solution while stirring₄And continuously stirring the solution for 2min to obtain the gold nano-seed solution.

In the present invention, the aqueous cetyltrimethylammonium bromide solution, HAuCl₄Aqueous solution and NaBH₄The proportion of the solution is preferably (3-8) mL: (3-8) mL (500-600) of [ mu ] L, more preferably 5 mL: 5mL of: 600 mu L; the concentration of the hexadecyl trimethyl ammonium bromide aqueous solution is preferably 180-220 nM, and more preferably 200 nM; the HAuCl₄The concentration of the aqueous solution is preferably 0.3-0.8 nM, and more preferably 0.5 nM; NaBH thus prepared₄The concentration of the solution is preferably 8 to 12nM, and more preferably 10 nM. In the present invention, the rotation speed of the stirring is preferably 1000 rpm.

After obtaining the gold nano-seed solution, the invention uses Cetyl Trimethyl Ammonium Bromide (CTAB) aqueous solution and HAuCl₄Aqueous solution, AgNO₃And mixing the aqueous solution, the reducing agent and the gold nano-seed solution, and carrying out reduction reaction to obtain the gold nano-rod. In the present invention, the order of mixing is preferably such that cetyltrimethylammonium bromide in water, HAuCl₄Aqueous solution, AgNO₃And sequentially mixing the aqueous solution, the reducing agent and the gold nano-seed solution. In the present invention, the reducing agent is preferably selected from vitamin C or hydroquinone; the reducing agent functions to reduce HAuCl₄. In the present invention, the aqueous CTAB solution and HAuCl are used₄Aqueous solution, AgNO₃The ratio of the aqueous solution, the reducing agent and the gold nanoparticle seed solution is preferably: 5mL of: 5mL of: (30-80) μ L: (55-250) μ L: (8.2-80) mu L; the concentration of the CTAB aqueous solution is preferably 0.1-0.2M; the HAuCl₄Au in aqueous solution³⁺Is preferably 10 mM; the AgNO₃Ag in aqueous solution⁺Is preferably 10 mM; when the reducing agent is vitamin C, the concentration of the vitamin C is preferably 78.8 mM; when the reducing agent is hydroquinone, the concentration of hydroquinone is preferably 0.1M. In the present invention, the procedure of the reduction reaction preferably includes: stirring and mixing for 0-2 min; the rotation speed of the stirring is preferably 200-500 rpm, and more preferably 300 rpm; stirring, mixing, standing and storing; the temperature for the preservation is preferably 28 ℃.

In the present invention, the preparation method of the capture probe and streptavidin conjugate preferably comprises: the preparation method of the conjugate of the capture probe and the streptavidin comprises the following steps: mixing a capture probe and a streptavidin aqueous solution, coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS (phosphate buffer solution) with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove the unreacted capture probe, and collecting the supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of the capture probe to the streptavidin aqueous solution is preferably 50 nmol: 80 μ L, i.e. the molar ratio of capture probe to streptavidin is preferably 4: 1. in the present invention, the temperature of the coupling is preferably 1h, and the temperature is preferably 25 ℃. In the invention, the temperature of the centrifugation is preferably 4 ℃, the rotating speed is preferably 6000rpm, and the time is preferably 20 min; the centrifugation serves to remove unbound capture probes. In the specific implementation process of the invention, the step of mixing the coupling reaction solution and PBS with the concentration of 500mg/L and collecting the supernatant is repeated three times, namely, the supernatant is collected and then PBS is added for repeated centrifugation, so as to remove the probe without coupling.

In the present invention, the method for preparing the conjugate of the quality control probe and streptavidin preferably comprises: mixing a quality control probe and a streptavidin aqueous solution with the concentration of 2.5mg/mL, coupling to obtain a coupling reaction solution, mixing the coupling reaction solution and PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cutoff of 30000, centrifuging at 6000rpm for 20min to remove unreacted capture probes, and collecting supernatant to obtain a quality control probe solution containing a conjugate of the quality control probe and the streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is preferably 4: 1. in the present invention, the temperature of the coupling is preferably 1h, and the temperature is preferably 25 ℃. In the invention, the temperature of the centrifugation is preferably 4 ℃, the rotating speed is preferably 6000rpm, and the time is preferably 20 min; the centrifugation serves to remove unbound capture probes. In the specific implementation process of the invention, the step of mixing the coupling reaction solution and PBS with the concentration of 500mg/L and collecting the supernatant is repeated three times, namely, the supernatant is collected and then PBS is added for repeated centrifugation, so as to remove the probe without coupling.

The invention also provides a preparation method of the immunochromatographic test strip in the scheme, which comprises the following steps:

s1, 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;

s2, adsorbing a conjugate of a detection probe and a gold nanorod on the bonding pad, spraying a conjugate of a capture probe and streptavidin on a detection line of the nitrocellulose membrane, and spraying a conjugate of a quality control probe and streptavidin on a quality control line to obtain an immunochromatography test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorod is 8-12 cm²Preferably 10cm²。

In the present invention, the method for using the immunochromatographic test strip preferably includes: the sample pad is immersed in a buffer containing the DNA to be detected, the liquid migrates towards the absorbent pad, and the test and control zones are evaluated visually within 10 min. In the present invention, SSC (sodium citrate buffer) at a concentration of 1/4 is preferable in the buffer; the SSC is derived from conventional commercial sources. The present invention is not particularly limited with respect to the time of immersion. In the invention, when strips appear in both the quality control area and the detection area, the strips are positive; when the quality control area has a strip and the detection area has no strip, the detection area is negative; if the quality control area has no strip, the test strip detection is invalid.

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.

Example 1 gold nanorod preparation

1. Preparation of gold nano-seeds

Under vigorous stirring (1000rpm), freshly prepared NaBH is added₄The solution (10ml, 0.5mM) was added to HAuCl containing 0.1M cetyltrimethylammonium bromide (CTAB)₄In solution (10ml, 0.5 mM). (CTAB, NaBH4 and HAuCl4 are in a molar ratio of 200:1:1) stirring for 2min, and storing at 28 ℃ for 2h to obtain a gold nano seed solution.

2. Growth of gold nanorods

Mixing appropriate amount of CTAB aqueous solution and HAuCl₄Aqueous solution, AgNO₃The aqueous solution, the reducing agent and the gold nanoparticle seed solution were added to the round-bottom flask in sequence, and the amounts used are shown in table 1. The specific method comprises the following steps: aqueous CTAB solution, HAuCl₄Aqueous solution and AgNO₃The aqueous solutions were added sequentially to a round bottom flask. Adding a reducing agent into the solution, and slightly stirring at 200-500 rpm to change the solution from bright yellow to colorless. And finally, adding the gold nanorod solution, slightly stirring for 2min under the condition of 200-500 rpm, and standing for 12h at 28 ℃ to obtain a solution containing the gold nanorods.

TABLE 1 reaction System for reduction reaction

The transmission electron microscope images of the gold nanorods with three different lengths are respectively shown in figures 1-3. FIG. 1 is a drawing of gold nanorods with an average length of 25 nm; FIG. 2 is a gold nanorod with an average length of 60 nm; FIG. 3 is a gold nanorod with an average length of 80 nm.

Example 2 detection of cervical cancer marker HPV16

Target HPV16 sequence: 5-ggc att tgt tgg ggt aac caa ctattt gtt-3, the nucleotide sequence is shown as SEQ ID No. 1;

HPV16 detection probe sequence: SH-C6-5-aac aaa tag ttg-3, the nucleotide sequence is shown as SEQ ID No. 2;

HPV16 capture probe sequence: 5-cca aca aat gcc-3-Biotin, the nucleotide sequence is shown in SEQ ID No. 3;

HPV16 quality control probe sequence: biotin-5-caa cta ttt gtt-3, the nucleotide sequence is shown in SEQ ID No. 4.

1. Preparation of conjugate of HPV16 detection probe and gold nanorod

Respectively taking 500 mu L of gold nanorods with the lengths of 25nm, 60nm and 80nm prepared by the method, adding 15 mu L of 1mM dATP, oscillating for 30min at the temperature of 25 ℃, adding 7.5 mu L of Sodium Dodecyl Sulfate (SDS) aqueous solution with the mass concentration of 1% and oscillating for 10min at the temperature of 25 ℃, adding 25 mu L of 2M sodium chloride aqueous solution in 5 batches, and then adding 85 mu L of HPV16 detection probe. Finally, incubation was carried out in a water bath at 60 ℃ for 3 h. Centrifuging at 8000rpm for 8min, washing with Phosphate Buffered Saline (PBS) for 3 times, and suspending in elution buffer (specifically prepared by adding Na into 40mL water sequentially₃PO₄·12H₂O304 mg, Bovine Serum Albumin (BSA)2.0g, sucrose 4.0g and Tween-200.1 g. After uniform mixing), 3 kinds of conjugates of HPV16 detection probes and gold nanorods are obtained, namely a first conjugate (prepared by gold nanorods with the size of 25 (+ -10) multiplied by 8 (+ -3) and a second conjugate (prepared by gold nanorods with the size of 60 (+ -15) multiplied by 28 (+ -5)Obtained) and a third conjugate (prepared from gold nanorods of dimensions 80(± 20) × 10(± 1). The conjugate solution of HPV16 detection probe and gold nanorods was stored at 4 ℃ for subsequent use.

2. Preparation of Capture Probe solution

50nmol of HPV16 capture probe was mixed with 80uL of 2.5mg/mL streptavidin at a molar ratio of about 4: incubating for 1h at the temperature of 1 and 25 ℃; adding 500mg/L PBS500, μ L, 4 deg.C into the mixed solution, centrifuging at 6000rpm in sample concentration tube with molecular weight cutoff of 30000 for 20min, removing unbound capture probe, and repeating the above steps for 3 times; the upper solution of the concentration pipe is the capture probe solution, and the rest solution is the capture probe solution.

3. Preparation of quality control Probe solution

Mixing 50nmol of HPV16 capture probe with 80uL of 2.5mg/mL streptavidin at a molar ratio of about 100:1, and incubating for 1h at 25 ℃; adding 500mg/LPBS into the mixed solution, centrifuging at 4 deg.C for 20min at 6000rpm with a sample concentration tube with molecular weight cutoff of 30000, removing unbound capture probe, and repeating the above steps for 3 times; the upper solution of the concentration pipe is a quality control probe solution.

4. Preparation method of test strip

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) adsorption of HPV16 detection Probe-gold nanorod conjugates (10. mu.L/cm) on a conjugate pad²) And respectively spraying 1 mu L of capture probe solution and 1 mu L of quality control probe solution on the detection line and the quality control line of the nitrocellulose membrane to obtain three gold nanorod lateral flow chromatography test strips for detecting nucleic acid.

Example 3

To the sample pad of the three gold nanorod lateral flow assay strips prepared in example 2, 3 μ L of HPV16 detection probe conjugate to gold nanorods was added dropwise, and the sample pad was immersed in 1 100 μ L of buffer (1/4 concentration SSC, from commercially available 1/4 diluted with deionized water to original concentration) containing the target HPV16 DNA (0.2nM) and migrated toward the absorbent pad. The test and control zones can be visually evaluated within 10 min. Referring to FIG. 4, three different lengths of gold nanorods (25nM, 60nM, 80nM) were used as labels to detect DNA, and the 60nM gold nanorod test line was the most concentrated when the concentration of DNA was 0.2 nM. Therefore, 60nm gold nanorods can be used as an optimal label.

Example 4

3 μ L of the conjugate of the HPV16 detection probe and the gold nanorods are dropped on the binding pad of the prepared test strip, the sample pad is immersed in 1 100 μ L of buffer solution (1/4SSC) containing different concentrations of target HPV16 DNA, and the liquid migrates to the absorption pad. The test and control zones can be visually evaluated within 10 min. Positive stripes appear in the quality control area and the test area; the quality control area has strips, and the test area has no strips, which is negative; if the quality control area has no strip, the test strip detection is invalid. The results are shown in FIG. 5. In FIG. 5, 1 to 8 represent the target DNA concentrations of 0, 10pM, 20pM, 50pM, 100pM, 200pM, 500pM and 1nM, respectively. When the concentration of the target substance is zero, no obvious strip exists in the test area, the higher the concentration is, the darker the strip color is, and strips exist in the quality control area of all the test strips, so that the prepared test strips can normally and well work, and the visual detection limit is 10 pM. Therefore, the prepared test strip can rapidly, accurately and sensitively detect DNA.

Comparative example 1

1. Preparation of gold nanoparticles

100mL of 0.01% chloroauric acid (HAuCl)₄) Heating the solution to boiling, adding 2mL of 1% sodium citrate solution while stirring at high speed (1000rpm), continuing stirring and boiling for 15min, and cooling to 25 deg.C.

2. Preparation of conjugate of detection probe and gold nanoparticles

Taking 500 mu L of gold nanoparticles, adding 15 mu L of 1mM dATP, then shaking for 30min at 25 ℃, adding 7.5 mu L of Sodium Dodecyl Sulfate (SDS) aqueous solution with the mass concentration of 1% and shaking for 10min at 25 ℃, and then slowly and sequentially adding 25 mu L of 2M sodium chloride solution and 85 mu L of HPV16 detection probe. Finally, incubation was carried out in a water bath at 60 ℃ for 3 h. Centrifuging at 8000rpm for 8min, washing with Phosphate Buffered Saline (PBS) for 3 times, and suspending in elution buffer (specifically prepared by adding Na into 40mL water sequentially₃PO₄·12H₂O304 mg, Bovine Serum Albumin (BSA)2.0g, sucrose 4.0g and Tween-200.1 g. Mixing ofAfter being mixed evenly). The conjugate solution of the detection probe and the gold nanoparticles was stored at 4 ℃ for subsequent use.

3. Capture probe solution preparation

Mixing 50nM HPV16 capture probe with 2.5mg/mL streptavidin and incubating for 1h at 25 ℃; adding 500mg/LPBS into the mixed solution, centrifuging at 4 deg.C and 6000rpm for 20min, removing unbound capture probe, and repeating the above steps for 3 times; the upper solution of the concentration tube is the quality control probe solution.

4. Quality control probe solution preparation

Mixing 50nM HPV16 quality control probe with 2.5mg/mL streptavidin, and incubating for 1h at 25 ℃; adding 500mg/LPBS into the mixed solution, centrifuging at 4 deg.C and 6000rpm for 20min, removing unbound capture probe, and repeating the above steps for 3 times; the upper solution of the concentration tube is the quality control probe solution.

5. Preparation method of test strip

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 a conjugate solution (10. mu.L/cm) of detection probe and gold nanoparticles on a conjugate pad²) And respectively spraying 1 mu L of capture probe solution and 1 mu L of quality control probe solution on the detection line and the quality control line of the nitrocellulose membrane to obtain the gold nanoparticle lateral flow chromatography test strip for detecting nucleic acid.

Example 5

The sample pads of the gold nanorod lateral flow chromatography test strip prepared in example 2 and the gold nanoparticle lateral flow chromatography test strip prepared in comparative example 1 were immersed in 1 buffer (1/4SSC) containing 100 μ L of target HPV16 DNA (10pM), respectively, (purchased SSC buffer, 1/4 diluted with deionized water to the original concentration) and the liquid migrated toward the absorbent pad. The test and control zones can be visually evaluated within 10 min. (the test strip is positive when the strip appears in both the quality control area and the test area, the strip appears in the quality control area and the strip does not appear in the test area, and the test strip is ineffective when the strip does not appear in the quality control area) the test result is shown in FIG. 6. As can be seen from FIG. 6, in the same condition, when the gold nanoparticles and the gold nanorod test strip are compared to detect DNA, 10pM can be detected by observing the gold nanorods with naked eyes.

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.

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