CN114410751B - Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle - Google Patents

Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle Download PDF

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CN114410751B
CN114410751B CN202111653244.6A CN202111653244A CN114410751B CN 114410751 B CN114410751 B CN 114410751B CN 202111653244 A CN202111653244 A CN 202111653244A CN 114410751 B CN114410751 B CN 114410751B
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CN114410751A (en
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于庆才
钱立生
汪雁
邱万伟
李田田
张静
刘国东
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Anhui University of Science and Technology
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Abstract

The application discloses a marker nucleic acid probe, a preparation method thereof, a test strip and application of polypyrrole nano particles. The marker nucleic acid probe comprises a marker and a detection probe loaded on the marker, wherein the marker is polypyrrole nano particles. The application creatively adopts the polypyrrole nano particles as the marker, the polypyrrole nano particles have higher storage stability, higher conductivity and high light stability, and based on the lateral chromatography test paper of the polypyrrole nano particles, the sensitive and quantitative visual detection can be carried out on DNA or RNA in a short time, the minimum concentration of the DNA which can be detected by naked eyes is 1pM, and the detection sensitivity of the lateral chromatography test paper is greatly improved.

Description

Marker nucleic acid probe, preparation method thereof, test strip and application of polypyrrole nano particle
Technical Field
The application relates to the field of nucleic acid chromatographic test paper, in particular to a marker nucleic acid probe, a preparation method thereof, a test paper strip and application of polypyrrole nano particles.
Background
Detection of genes (DNA or RNA) is important in gene therapy, clinical diagnosis and various biomedical research. A currently accepted method for gene detection is the Polymerase Chain Reaction (PCR). PCR is highly sensitive and accurate, but it is based on laboratory procedures, requiring relatively trained personnel. In addition, false positive results often occur when small amounts of fragmented DNA are present as contaminants. Therefore, there is an urgent need to develop a rapid on-site detection technique for DNA that is simple, economical, highly sensitive and specific.
Lateral chromatography is a technique for detecting a target substance in a sample based on a hybridization reaction of a nucleic acid probe and a target nucleic acid under capillary chromatography. The lateral chromatography test strip consists of 4 parts, namely a sample pad, a binding pad, a test pad (usually a nitrocellulose membrane) and a water absorption pad, wherein the sample pad is used for rapidly absorbing a sample to be tested; the binding pad is loaded with a marker nucleic acid probe, and the marker nucleic acid probe is bound with a detection target in a sample to be detected to form a detectable complex; a detection line and a quality control line are arranged on the test pad and used for developing the marked compound and observing the detection result; the absorbent pad allows the sample to flow laterally over the test pad.
Among them, the labeled nucleic acid probe is one of the key factors affecting the sensitivity of the chromatographic test paper, and the most widely used colloidal gold labeled probe is the detection probe bound on the surface of the colloidal gold particles. However, the sensitivity of using colloidal gold as a labeling material is low, and it is reported in the literature that the lowest detection concentration of a colloidal gold-based nucleic acid chromatographic test strip is 500pM. How to improve the detection sensitivity of the lateral chromatography test strip is a research hot spot in the current field.
Disclosure of Invention
An object of the present application is to provide a marker nucleic acid probe and a preparation method thereof, which are beneficial to improving the sensitivity of detection of a nucleic acid chromatographic test strip.
Another object of the present application is to provide a nucleic acid chromatographic test strip having high detection sensitivity.
It is a further object of the present application to provide a use of polypyrrole nanoparticles.
To achieve the above object, the present application provides a labeled nucleic acid probe, which includes a label and a detection probe supported by the label, wherein the label is polypyrrole nanoparticles.
Further, the average particle diameter of the polypyrrole nanoparticles is 10nm to 200nm, further the average particle diameter of the polypyrrole nanoparticles is 30nm to 80nm, and further the average particle diameter of the polypyrrole nanoparticles is 50nm to 60nm.
Further, the detection probe is in complementary pairing with the nucleotide sequence at the first end of the detection target, the detection probe is single-stranded DNA, the 5 'end of the detection probe is modified by sulfhydryl, carboxyl or amino, and the detection probe is in complementary pairing with the nucleotide sequence at the 3' end of the detection target. .
The application also provides a preparation method of the marker nucleic acid probe, which comprises the following steps:
s1, providing a dispersion liquid of polypyrrole nano particles;
s2, sequentially adding deoxynucleotide, a first stabilizer and the detection probe into the dispersion liquid to carry out coupling reaction, so that the detection probe is loaded on the polypyrrole nano particle.
Further, the deoxynucleotide is selected from a mixture of one or more of the following: dATP, dCTP, dGTP, dTTP, said first stabilizer is selected from the group consisting of a mixture of one or more of: polyvinyl alcohol, sodium lauryl sulfate, sodium docusate, polyethylene glycol, tween40, tween60, and polyvinylpyrrolidone.
Further, in the step S2, the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 to 2.5 wt%, the solution of the deoxynucleotide having a concentration of 0.1 to 10mmol/L is added to the dispersion, the solution of the first stabilizer having a concentration of 0.1 to 10wt% is added, the solution of the detection probe having a concentration of 1 to 10 μg/mL is added, and the volume ratio of the dispersion, the solution of the deoxynucleotide, the solution of the first stabilizer, and the solution of the detection probe is (400 to 600): (1-10): (5-15): (50-100).
The application also provides a test strip, including the bottom plate and set gradually sample pad, binding pad, test pad and the pad that absorbs water on the bottom plate, the load has the aforesaid marker nucleic acid probe of this application on the binding pad.
Further, a detection line and a quality control line are arranged on the test pad, the detection line is close to the binding pad, the quality control line is close to the water absorption pad, a conjugate of a capture probe is arranged on the detection line, the capture probe is complementarily paired with a nucleotide sequence at the second end of the detection target, a conjugate of a quality control probe is arranged on the quality control line, and the quality control probe is complementarily paired with the detection probe. .
Further, the conjugate of the capture probe is a conjugate of the capture probe and streptavidin, the 3 'end of the capture probe is marked by biotin, and the capture probe is complementarily paired with the 5' end nucleotide sequence of the detection target; the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin, and the 3' end of the quality control probe is marked by biotin.
The application also provides application of the polypyrrole nano-particles serving as markers to nucleic acid chromatography test strips.
Compared with the prior art, the beneficial effect of this application lies in: the application creatively applies the polypyrrole nano particles to the marker nucleic acid probe, the polypyrrole nano particles have higher storage stability, higher conductivity and high light stability, the marker nucleic acid probe based on the polypyrrole nano particles is applied to the lateral chromatography test paper, the sensitive and quantitative visual detection can be carried out on DNA or RNA in a short time, the minimum concentration of the DNA which can be detected by naked eyes is 1pM, the minimum detection concentration of the DNA is 20 times smaller than that of the existing gold nano particles (AuNPs), and the detection sensitivity of the detection test paper is greatly improved. The marker nucleic acid probe provides a new thought for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.
Drawings
FIG. 1 is a schematic representation of one embodiment of a test strip of the present application;
FIG. 2 shows the detection of target nucleic acid by the test strip;
FIG. 3 shows the target nucleic acid not detected by the test strip;
fig. 4 is an electron micrograph of polypyrrole nanoparticles of example 1 of this application.
Fig. 5 is an electron micrograph of polypyrrole nanoparticles of example 2 of this application.
FIG. 6 is a photograph showing the test of each test strip of example 6 of the present application.
FIG. 7 is a photograph showing the test of each test strip of example 7 of the present application.
FIG. 8 is a photograph showing the test of each test strip of example 11 of the present application.
FIG. 9 is a photograph showing the test of each test strip of example 12 of the present application.
Detailed Description
The present application will be further described with reference to the specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.
In the description of the present application, it should be noted that, for the azimuth terms such as terms "center", "lateral", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., the azimuth and positional relationships are based on the azimuth or positional relationships shown in the drawings, it is merely for convenience of describing the present application and simplifying the description, and it is not to be construed as limiting the specific protection scope of the present application that the device or element referred to must have a specific azimuth configuration and operation, as indicated or implied.
It should be noted that the terms "first," "second," and the like in the description and in the claims of the present application are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order.
The terms "comprises" and "comprising," along with any variations thereof, in the description and claims of the present application are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements that are expressly listed or inherent to such process, method, article, or apparatus.
The application provides a marker nucleic acid probe, which comprises a marker and a detection probe loaded on the marker, wherein the detection probe is in complementary pairing with a nucleotide sequence at one end of a detection target, and the marker is polypyrrole nano particles.
In the existing marker nucleic acid probe, colloidal gold (or gold nanoparticles) is usually adopted as a marker, polypyrrole nanoparticles are creatively adopted as the marker by the inventor of the application, the polypyrrole nanoparticles have higher storage stability, higher conductivity and high light stability, and based on the lateral chromatography test paper of the polypyrrole nanoparticles, sensitive and quantitative visual detection can be carried out on DNA or RNA in a short time, and the minimum concentration of the DNA which can be detected by naked eyes is 1pM, so that the detection sensitivity of the lateral chromatography test paper is greatly improved. The marker nucleic acid probe based on polypyrrole nanoparticles provides a new thought for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.
The principle that polypyrrole nano particles can be used as markers is that the polypyrrole nano particles have dark colors, a small amount of aggregation in detection lines can form macroscopic black strips, and the more the aggregation is, the darker the color of the detection lines is, so that the polypyrrole nano particles can be used for qualitative or semi-quantitative detection. In addition, the connection of the detection probe and the polypyrrole nanoparticle is mainly adsorption, but the connection of the detection probe and the polypyrrole nanoparticle by chemical bonds is not excluded.
In some embodiments, the polypyrrole nanoparticles have an average particle size of 10nm to 200nm. Preferably, the average particle diameter of the polypyrrole nano particles is 30 nm-80 nm. Further preferably, the average particle diameter of the polypyrrole nanoparticles is 50nm to 60nm.
In some embodiments, the detection target is a single-stranded DNA or RNA with a base number of 15-50, the detection probe is single-stranded DNA, the 5 'end of the detection probe is modified by sulfhydryl, carboxyl or amino, and the detection probe is complementarily paired with the 3' end nucleotide sequence of the detection target.
The marker nucleic acid probes of the present application can be used for various types of gene detection, and thus the present application is not limited to the type of detection probes. For example, the nucleotide sequence of the detection target may be 5'-atgacctatgaattgacagac-3', and the nucleotide sequence of the detection probe may be 5 '-sulfohydroxy-gtctgtcaa-3'; for another example, the nucleotide sequence of the detection target may be 5'-ggcatttgttggggtaaccaactatttgtt-3', and the nucleotide sequence of the detection probe may be SH-C6-5'-aacaaatagttg-3'; for another example, the nucleotide sequence of the detection target may be 5'-UAGCUUAUCAGACUGAUGUUGA-3' (microRNA 21), and the nucleotide sequence of the detection probe may be 5 '-thio-CCCCCTAGACACCGTGTTCAACATCAGT-3'.
The application also provides a preparation method of the marker nucleic acid probe, which comprises the following steps:
s1, providing a dispersion liquid of polypyrrole nano particles;
s2, sequentially adding deoxynucleotide, a first stabilizer and a detection probe into the dispersion liquid to carry out coupling reaction, so that the detection probe is coupled on the polypyrrole nano particle.
In some embodiments, in step S1, the polypyrrole nanoparticles are dispersed in deionized water to obtain the dispersion, where the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 wt%o-2.5 wt%o.
In some embodiments, the polypyrrole nanoparticle is prepared by: and adding pyrrole monomers into a first solution containing a second stabilizer, then adding an oxidant for oxidation polymerization, finally adding a terminator for stopping the reaction, and centrifuging the solution after the reaction to obtain the polypyrrole nano particles. Wherein the solvent of the first solution may be, but is not limited to, water, methanol, diethyl ether; the second stabilizer is selected from the group consisting of a mixture of one or more of the following: polyvinyl alcohol (PVA), sodium Dodecyl Sulfate (SDS), sodium docusate (Sodiumdocusate or AOT), polyethylene glycol (PEG), tween40 (Polyoxyethylenesorbitan monopalmitate or TWEEN 40), tween60 (Poly (ethylene glycol) sorbant monostearate or TWEEN 60)]Polyvinylpyrrolidone (PVP); the oxidant is selected from any one of the following: feCl 3 ，K 2 S 2 O 8 ，H 2 O 2 ，(NH 4 )S 2 O 8 ，AgNO 3 ，CuCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The terminator may be, but is not limited to, methanol. Preferably, the molar ratio of the oxidizing agent to the pyrrole monomer is (1 to 3): 1.
in some embodiments, in step S2, the deoxynucleotide is selected from a mixture of one or more of: dATP, dCTP, dGTP, dTTP; the first stabilizer is selected from the group consisting of a mixture of one or more of the following: polyvinyl alcohol (PVA), sodium Dodecyl Sulfate (SDS), sodium docusate (AOT), polyethylene glycol (PEG), TWEEN40 (polyoxymethylene orbitan monopalmitate or TWEEN 40), TWEEN60 [ poly (ethylene glycol) sorbitan monostearate or TWEEN60], polyvinylpyrrolidone (PVP).
In some embodiments, in step S2, a solution of the deoxynucleotide having a concentration of 0.1mmol/L to 10mmol/L is added to the dispersion, a solution of the first stabilizer having a concentration of 0.1wt% to 10wt% is added to the dispersion, and a solution of the detection probe having a concentration of 1 μg/mL to 10 μg/mL is added to the dispersion.
In some embodiments, the volume ratio of the dispersion, the solution of deoxynucleotides, the solution of the first stabilizer, and the solution of the detection probe is (400-600): (1-10): (5-15): (50-100).
In some embodiments, after each addition of a solution to the dispersion, shaking mixing is performed for a period of 10 to 30 minutes at a temperature of 20 to 30 ℃, preferably for a period of 20 minutes at a temperature of 25 ℃.
In some embodiments, in step S2, the coupling reaction conditions are: the incubation is carried out for 2 to 8 hours by a room temperature shaking table, and the incubation is preferably carried out for 4 hours by a room temperature shaking table.
In some embodiments, in step S2, after the coupling reaction, the labeled nucleic acid probe solution is obtained through further steps of centrifugation, collection of precipitate, washing, and resuspension.
It is noted that the storage temperature of the label nucleic acid probe solution is 2 to 8℃and preferably 4 ℃.
The application also provides a nucleic acid chromatography test strip, as shown in fig. 1, comprising a bottom plate, and a sample pad, a binding pad, a test pad and a water absorption pad which are sequentially arranged on the bottom plate, wherein the binding pad is loaded with the marker nucleic acid probe.
Further, a detection line and a quality control line are arranged on the test pad, the detection line is close to the binding pad, the quality control line is close to the water absorbing pad, a conjugate of a capture probe is arranged on the detection line, the capture probe is complementarily paired with a nucleotide sequence at the second end of the detection target (the nucleotide sequence at the first end of the detection target is complementarily paired with the detection probe, so that the detection target can be combined with the capture probe after being combined with the detection probe), and a conjugate of a quality control probe is arranged on the quality control line, and the quality control probe is complementarily paired with the detection probe.
The application method of the test strip provided by the application is as follows: and immersing the sample pad into a buffer solution containing target DNA or RNA, transferring the liquid to the water absorption pad, visually evaluating a detection area and a quality control area within 10min, wherein the quality control area and the detection area are positive in strip, the quality control area is negative in strip, and the quality control area is ineffective in detection if the quality control area is not provided with strip.
As shown in fig. 2, when the sample pad is immersed in a solution containing a detection target, the detection target first reaches the binding pad to bind to the marker nucleic acid probe, then the marker nucleic acid probe that binds to the detection target and the marker nucleic acid probe that does not bind to the detection target reach the test pad, the detection target binds to the capture probe at the detection line, a band appears at the detection line, and the marker nucleic acid probe that does not bind to the detection target reaches the quality control line to bind to the quality control probe, so that a band appears at the quality control line. The detection line and the quality control line are both developed, which indicates that the sample contains a detection target.
As shown in fig. 3, when the sample pad is immersed in a solution containing no detection target or the concentration of the detection target is low in the solution, the label nucleic acid probe is combined with the quality control probe only when the solution reaches the quality control line based on the principle of lateral chromatography, and a strip appears at the quality control line. Indicating that the sample does not contain the detection target or that the concentration of the detection target in the sample is below the minimum detection limit.
In some embodiments, the conjugate of the capture probe is a conjugate of the capture probe and streptavidin. Further, the 3 'end of the capture probe is labeled by biotin, and the capture probe is complementarily paired with the 5' end nucleotide sequence of the detection target.
In some embodiments, the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin. Further, the 3' end of the quality control probe is labeled with biotin.
In one embodiment, the capture probe or quality control probe is prepared by: 50nmol of capture probe or quality control probe and streptavidin aqueous solution (80 mu L,2.5 mg/mL) are mixed for coupling reaction (preferably incubation for 1h at 25 ℃) to obtain coupling reaction liquid, the coupling reaction liquid is mixed with Phosphate Buffer Solution (PBS) with the concentration of 500mg/L, the mixture is placed in a sample dialysis tube with the molecular weight cut-off of 30000, centrifugation is carried out at 6000rpm for 20min to remove unreacted capture probe or quality control probe, and the cut-off solution is collected to obtain capture probe conjugate solution or quality control probe conjugate solution. Wherein the molar ratio of capture probe or quality control probe to streptavidin is preferably 100:1, a step of; the centrifugation is preferably carried out at a temperature of 4℃and the centrifugation step is preferably repeated three times, each time the centrifuged product is dissolved in 500. Mu.L of PBS and centrifuged in a dialysis tube.
According to the difference between the detection targets and the detection probes, proper capture probes and quality control probes are selected.
For example, the nucleotide sequence of the detection target is 5'-atgacctatgaattgacagac-3', the nucleotide sequence of the detection probe is 5 '-sulfohydroxy-gtctgtcaa-3', the nucleotide sequence of the capture probe is 5 '-taggtcat-Biotin-3', and the nucleotide sequence of the quality control probe is 5'-Biotin-MC6-D-ttgacagac-3'.
For another example, the nucleotide sequence of the detection target is 5'-ggc att tgt tgg ggt aac caa cta ttt gtt-3', the nucleotide sequence of the detection probe is SH-C6-5'-aac aaa tag ttg-3', the nucleotide sequence of the capture probe is 5'-cca aca aat gcc-3' -Biotin, and the nucleotide sequence of the quality control probe is Biotin-5'-caa cta ttt gtt-3'.
For another example, the nucleotide sequence of the detection target is 5'-UAG CUUA UCA GAC UGA UGU UGA-3' (microRNA 21), the nucleotide sequence of the detection probe is 5 '-thio-CCCCCT AGA CAC CGT GTT CAA CATC AGT-3', the nucleotide sequence of the capture probe is 5'-CTG ATA AGC TAC CCCC-Biotin-3', and the nucleotide sequence of the quality control probe is 5'-Biotin-ACACGG TGT CTA GGG GG-3'.
The application also provides a preparation method of the test strip, which comprises the following steps:
a1, sticking a nitrocellulose membrane on a bottom plate, and then sequentially fixing a combination pad, a sample pad and a water absorption pad;
a2, loading the marker nucleic acid probe on the binding pad, spraying the capture probe conjugate solution on a detection line of the nitrocellulose membrane, and spraying the quality control probe conjugate solution on a quality control line of the nitrocellulose membrane.
In some embodiments, the label nucleic acid probe solution on the conjugate pad has an adsorption capacity of 8. Mu.L/cm 2 ～12μL/cm 2 The concentration of the marker nucleic acid probe solution is 2×10 -4 mg/uL～8×10 -4 mg/uL, preferably an adsorption amount of 10. Mu.L/cm 2 At a concentration of 6X 10 -4 mg/uL。
The spraying amount of the capture probe conjugate solution and the quality control probe conjugate solution is 0.5 mu L-1 mu L, and the solution concentration is 0.1 nmol/mu L-0.5 nmol/mu L; preferably, the spraying amount is 1. Mu.L, and the solution concentration is 0.2 nmol/. Mu.L.
The application also provides application of the polypyrrole nanoparticle, wherein the polypyrrole nanoparticle serving as a marker is applied to a nucleic acid chromatography test strip.
[ example 1 ]
Preparing polypyrrole nanoparticle dispersion liquid: into a 500mL Erlenmeyer flask was added 100mL of water, PVA (M w =31000) 8g, and stirring at 900rpm for 20min at room temperature; adding 500mg of pyrrole monomer, and stirring at 900rpm at room temperature for 20min; after 50mL (56 mg/mL) of the aqueous solution of ferric chloride was rapidly added, the mixture was stirred at 1500rpm at room temperature for 6 hours, and the reaction was terminated by adding 20mL of methanol, and centrifuged at 15000rpm for 30 minutesThe supernatant was washed three times with hot water and then dispersed in water for use, the mass fraction of polypyrrole nanoparticles in the dispersion was 2.5 wt% and the average particle size of the polypyrrole nanoparticles was 46nm, as shown in fig. 8.
[ example 2 ]
Preparing polypyrrole nanoparticle dispersion liquid: into a 500mL Erlenmeyer flask was added 100mL of water, PVA (M w =9000) 8g, stirring at 900rpm for 20min at room temperature; adding 500mg of pyrrole monomer, and stirring at 900rpm at room temperature for 20min; after 50mL (56 mg/mL) of the aqueous solution of ferric chloride was rapidly added, the mixture was stirred at room temperature for 6 hours at 1500rpm, the reaction was terminated by adding 20mL of methanol, centrifuging at 15000rpm for 30min to remove the supernatant, washing the obtained solid three times with hot water, and then dispersing in water for later use, wherein the mass fraction of polypyrrole nanoparticles in the dispersion was 2.5 wt% and the average particle diameter of the polypyrrole nanoparticles was 89nm, as shown in FIG. 9.
[ example 3 ]
Detecting the nucleotide sequence of the target HPV 16: 5'-ggc att tgt tgg ggt aac caa cta ttt gtt-3'.
HPV16 marker nucleic acid probe preparation: taking 500 mu L of polypyrrole nanoparticle dispersion liquid in example 1, adding 15 mu L of dATP with the concentration of 1mmol/L, vibrating for 30min at 25 ℃, adding 7.5 mu L of sodium dodecyl sulfate aqueous solution with the mass concentration of 1wt% and vibrating for 10min at 25 ℃, then adding 10 mu L of HPV16 detection probe (with the nucleotide sequence of SH-C6-5'-aac aaa tag ttg-3') with the concentration of 1.0OD/mL, and incubating for 4h at a room temperature shaking table; centrifuging at 8000rpm for 8min, collecting precipitate, washing with phosphate buffer solution for 3 times, and re-suspending in elution buffer to obtain HPV16 marker nucleic acid probe solution (concentration about 6X10) -4 mg/. Mu.L) was stored at 4℃for later use.
The preparation method of the elution buffer comprises the following steps: 304mg of Na was added sequentially to 40g of water 3 PO 4 ·12H 2 O, 2.0g of bovine serum albumin, 4.0g of sucrose and 0.1g of Tween-20 are uniformly mixed.
[ example 4 ]
Preparation of HPV16 capture probe conjugate:
(1) 100uL of HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) at a concentration of 0.5nmol/uL was mixed with aqueous streptavidin (80. Mu.L, 2.5 mg/mL) for coupling reaction, the molar ratio of HPV16 capture probe to streptavidin was about 100:1, incubating for 1h at 25 ℃ to obtain a coupling reaction solution;
(2) Mixing the coupling reaction solution with Phosphate Buffer Solution (PBS) with the concentration of 500mg/L, placing the mixture into a sample dialysis tube with the molecular weight cut-off of 30000, centrifuging at 6000rpm for 20min to remove unreacted HPV16, and collecting the cut-off solution;
(3) Step (2) was repeated 3 times and the retentate in the dialysis tube was HPV16 capture probe conjugate solution (concentration about 0.2 nm/. Mu.L).
[ example 5 ]
HPV16 quality control probe conjugate: the difference from example 4 is that the HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced with HPV16 quality control probe (nucleotide sequence: biotin-5'-caa cta ttt gtt-3').
[ example 6 ]
Preparing a test strip: sticking a nitrocellulose membrane on a bottom plate, and then sequentially fixing a combining pad, a sample pad and a water absorption pad; adsorbing the labeled nucleic acid probe solution prepared [ example 3 ] on the conjugate pad in an amount of 10. Mu.L/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The capture probe conjugate solution of example 4 was sprayed on the detection line of the nitrocellulose membrane in an amount of 1 μl, and the quality control probe conjugate solution of example 5 was sprayed on the quality control line of the nitrocellulose membrane in an amount of 1 μl.
Sample solution preparation: solutions of target DNA concentrations of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM and 5000pM were prepared, respectively, and the solvent of the solutions was a buffer (1/4 SSC).
The test strips were immersed in 100. Mu.L of the above sample solution, respectively, and the detection zone and the quality control zone were visually evaluated within 10 minutes. The detection structure is shown in fig. 6. The test strips in FIG. 6 were immersed in solutions having target DNA concentrations of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, 5000pM in this order from left to right. It can be seen that the test strip visually observed that the test line showed that the depth of the test line increased with increasing DNA concentration at 1pM of target DNA concentration.
[ example 7 ]
Preparing a test strip: sticking a nitrocellulose membrane on a bottom plate, and then sequentially fixing a combining pad, a sample pad and a water absorption pad; adsorbing the labeled nucleic acid probe solution prepared [ example 3 ] on the conjugate pad in an amount of 5. Mu.L/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The capture probe conjugate solution of example 4 was sprayed on the detection line of the nitrocellulose membrane in an amount of 1 μl, and the quality control probe conjugate solution of example 5 was sprayed on the quality control line of the nitrocellulose membrane in an amount of 1 μl.
Sample solution preparation: solutions of target DNA concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM and 5000pM were prepared, respectively, and the solvents of the solutions were buffers prepared from 1/4SSC and serum according to a ratio of 1: 1.
The test strips were immersed in 100. Mu.L of the above sample solution, respectively, and the detection zone and the quality control zone were visually evaluated within 10 minutes. The detection structure is shown in fig. 7. The test strips in FIG. 7 were immersed in solutions of target DNA concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, 5000pM, 10nM in this order from left to right. It can be seen that the test strip visually observed that the test line showed that the depth of the test line increased with increasing DNA concentration at 1pM of target DNA concentration.
[ example 8 ]
Detecting a target microRNA21 nucleotide sequence: 5'-UAG CUUA UCA GAC UGA UGU UGA-3'. Wherein the detection target microRNA21 is synthesized and prepared.
Preparation of microRNA21 marker nucleic acid probe: taking 500 mu L of polypyrrole nanoparticle dispersion liquid in example 2, adding 15 mu L of dATP (data transfer peptide) with the concentration of 1mmol/L, vibrating for 30min at 25 ℃, adding 7.5 mu L of sodium dodecyl sulfate aqueous solution with the mass concentration of 1wt% and vibrating for 10min at 25 ℃, then adding 10 mu L of microRNA21 detection probe (nucleotide sequence: 5 '-thio-CCCCCT AGA CAC CGT GTT CAA CATC AGT-3') with the concentration of 1.0OD/mL, and incubating for 4h at room temperature in a shaking table; centrifuging at 8000rpm for 8min, collecting precipitate, and usingWashing 3 times with phosphate buffer solution, and re-suspending in elution buffer solution to obtain microRNA21 marker nucleic acid probe solution (concentration of about 6×10) -4 mg/. Mu.L) was stored at 4℃for later use.
[ example 9 ]
Preparation of microRNA21 capture probe: the difference from example 4 is that the HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced with the microRNA21 capture probe (nucleotide sequence: 5'-CTG ATA AGC TAC CCCC-Biotin-3').
[ example 10 ]
Preparation of microRNA21 quality control probe: the difference from example 4 is that HPV16 capture probe (nucleotide sequence: 5'-cca aca aat gcc-3' -Biotin) is replaced with microRNA21 quality control probe (nucleotide sequence: 5'-Biotin-ACACGG TGT CTA GGG GG-3').
[ example 11 ]
Preparing a test strip: sticking a nitrocellulose membrane on a bottom plate, and then sequentially fixing a combining pad, a sample pad and a water absorption pad; adsorbing the labeled nucleic acid probe prepared in example 8 onto a conjugate pad in an amount of 5. Mu.L/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The capture probe solution of example 9 was sprayed on the detection line of the nitrocellulose membrane in an amount of 1. Mu.L, and the quality control probe solution of example 10 was sprayed on the quality control line of the nitrocellulose membrane in an amount of 1. Mu.L.
Sample solution preparation: solutions of target microRNA21 nucleic acid concentrations of 0, 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, and 5000pM were prepared, respectively, and the solvent of the solutions was a buffer (1/4 SSC). Wherein microRNA21 is synthesized artificially.
The test strips were immersed in 100. Mu.L of the above sample solution, respectively, and the detection zone and the quality control zone were visually evaluated within 10 minutes. The detection structure is shown in fig. 8. The test strips in FIG. 8 were immersed in solutions having target DNA concentrations of 0, 0.5pM, 1pM, 5pM, 10pM, 100pM, 500pM, 1000pM, 5000pM in this order from left to right. It can be seen that the test strip visually observed that the test line showed that the depth of the test line increased with increasing nucleic acid concentration at 1pM target nucleic acid concentration.
[ example 12 ]
The method for extracting target RNAmicroRNA 21 from cells comprises the following steps: at 37℃with 5% CO 2 MDA-MB-231 cells were cultured in L-15 medium containing 10% fetal bovine serum (Gibco) and 1% antibiotics (100U/ml penicillin and 100mg/ml streptomycin sulfate) in the atmosphere. HeLa cells were inoculated in DMEM medium containing 10% antibiotic under the same conditions. All cells were treated in the logarithmic growth phase and counted by a cell counter prior to treatment. The total RNA (including microRNA 21) was extracted using an RNA extraction kit (chinese tenna) according to the manufacturer's instructions to obtain an extract.
The test strips were prepared in the same manner as in example 11.
The following sample solutions were respectively prepared: a sample solution with a target concentration of 0, a sample solution with a target concentration of 50pM (wherein RNAmicroRNA 21 is synthesized artificially), a sample solution containing HeLa cell total RNA extract, a sample solution containing MDA-MB-231 cell extract.
In FIG. 9, the test strips from left to right were immersed in a sample solution having a target concentration of 0, a sample solution having a target concentration of 50pM (wherein microRNA21 is synthesized artificially), a sample solution containing HeLa cell total RNA extract, and a sample solution containing MDA-MB-231 cell extract, respectively. The test result of fig. 9 can demonstrate that the test strip of the present application has good applicability and can detect microRNA21 in cancerous cells.
The foregoing has outlined the basic principles, main features and advantages of the present application. It will be appreciated by persons skilled in the art that the present application is not limited to the embodiments described above, and that the embodiments and descriptions described herein are merely illustrative of the principles of the present application, and that various changes and modifications may be made therein without departing from the spirit and scope of the application, which is defined by the appended claims. The scope of protection of the present application is defined by the appended claims and equivalents thereof.

Claims (6)

1. The utility model provides a test paper strip, includes the bottom plate and sets gradually sample pad, combination pad, test pad and the pad that absorbs water on the bottom plate, its characterized in that:
the binding pad is loaded with a marker nucleic acid probe, the marker nucleic acid probe comprises a marker and a detection probe loaded on the marker, the marker is polypyrrole nano particles, the average particle size of the polypyrrole nano particles is 30-80 nm, the detection probe is single-stranded DNA, the 5 'end of the detection probe is modified by sulfhydryl, carboxyl or amino, and the detection probe is complementarily paired with a 3' end nucleotide sequence of a detection target;
the detection line is close to the binding pad, the quality control line is close to the water absorbing pad, a conjugate of a capture probe is arranged on the detection line, the capture probe is complementarily paired with a 5' -end nucleotide sequence of a detection target, a conjugate of a quality control probe is arranged on the quality control line, and the quality control probe is complementarily paired with the detection probe;
the test strip can visually observe a detection line when the detection target concentration is 1 pM.
2. The test strip of claim 1, wherein the conjugate of the capture probe is a conjugate of the capture probe and streptavidin, and the 3' end of the capture probe is labeled with biotin; the conjugate of the quality control probe is a conjugate of the quality control probe and streptavidin, and the 3' end of the quality control probe is marked by biotin.
3. The test strip of claim 1, wherein the method for preparing the marker nucleic acid probe comprises the steps of:
s1, providing a dispersion liquid of polypyrrole nano particles;
s2, sequentially adding deoxynucleotide, a first stabilizer and the detection probe into the dispersion liquid to carry out coupling reaction, so that the detection probe is loaded on the polypyrrole nano particle, wherein the first stabilizer is selected from the following mixture of one or more of the following components: polyvinyl alcohol, sodium lauryl sulfate, sodium docusate, polyethylene glycol, tween40, tween60, and polyvinylpyrrolidone.
4. A test strip according to claim 3, wherein in step S2 the deoxynucleotide is selected from the group consisting of a mixture of one or more of: dATP, dCTP, dGTP, dTTP.
5. The test strip according to claim 3, wherein in the step S2, the mass fraction of the polypyrrole nanoparticles in the dispersion is 1.5 to 2.5 wt%, the solution of the deoxynucleotide having a concentration of 0.1 to 10mmol/L is added to the dispersion, the solution of the first stabilizer having a concentration of 0.1 to 10wt% is added, the solution of the detection probe having a concentration of 1 to 10 μg/mL is added, and the volume ratio of the dispersion, the solution of the deoxynucleotide, the solution of the first stabilizer, and the solution of the detection probe is (400 to 600): (1-10): (5-15): (50-100).
6. The test strip of any one of claims 1-5, wherein the polypyrrole nanoparticle is prepared by the following steps: adding pyrrole monomer into a first solution containing a second stabilizer, then adding an oxidant for oxidation polymerization, finally adding methanol for terminating reaction, and centrifuging the solution after the purification reaction to obtain polypyrrole nano particles, wherein the second stabilizer is selected from one or more of the following mixtures: polyvinyl alcohol, sodium dodecyl sulfate, sodium docusate, polyethylene glycol, tween40, tween60, polyvinylpyrrolidone, the oxidant is selected from any one of the following: feCl 3 ，K 2 S 2 O 8 ，H 2 O 2 ，(NH 4 )S 2 O 8 ，AgNO 3 ，CuCl 2 。
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