CN-122017226-A - Preparation method and application of spherical nucleic acid-based nano probe and paper-based chip thereof

CN122017226ACN 122017226 ACN122017226 ACN 122017226ACN-122017226-A

Abstract

The invention discloses a spherical nucleic acid nano probe based on a sulfadiazine aptamer and a preparation method and application of a paper-based chip thereof, which are characterized in that the spherical nucleic acid nano probe based on the sulfadiazine aptamer is prepared by adopting a microwave method, the paper-based chip is prepared by adopting a laser printing and high-temperature heating method to construct a hydrophobic barrier, a water sample to be detected is dripped into a sample adding area of the paper-based chip, the sample firstly reaches a detection 1 area through capillary action and is aggregated after the action of the nano probe for detecting aminoglycoside antibiotics, so that the color of the paper-based chip is changed, the sample continuously flows to a detection 2 area and is reacted with the prepared spherical nucleic acid nano probe based on the sulfadiazine aptamer, and then sodium chloride solution is dripped into the area to trigger particle aggregation reaction, so that the color of the paper-based chip is changed, and the simultaneous detection of aminoglycoside and sulfadiazine antibiotics is realized. Compared with the prior art, the method has the characteristics of rapidness, convenience, economy and practicability, and has important significance for environmental risk assessment, pollutant control and public health.

Inventors

ZHOU TIANSHU
LI MENGYI
ZHU YINGHUA
ZHANG SHENGDAN
DENG JINGJING

Assignees

华东师范大学

Dates

Publication Date: 20260512
Application Date: 20260305

Claims (4)

1. A preparation method of spherical nucleic acid nano-probe based on sulfadiazine aptamer is characterized by adopting a microwave method to prepare spherical nucleic acid nano-probe based on sulfadiazine aptamer, specifically preparing 900 mu L of gold nano-particle with concentration of 3.5 nM and 100 mu L of sulfadiazine aptamer with 3' -end modified FAM with concentration of 100 nM, then placing the mixture into a microwave chemical reactor, heating the mixture for 5min under the condition of output power of 700W, and after the reaction is completed, using ultrapure water to fix the volume of the product to a required volume to prepare spherical nucleic acid nano-probe based on sulfadiazine aptamer.
2. A paper-based chip prepared by the preparation method based on the spherical nucleic acid nano probe is characterized in that the paper-based chip integrates a sample adding area, two detection areas and two liquid conveying channels, detection of two antibiotics of aminoglycosides and sulfonamides is simultaneously realized on one paper-based chip, the paper-based chip is 8 mm wide by 40 mm long, the structure is designed to adopt the sample adding area at one end of the paper-based chip, the sample adding area, the liquid conveying channels and the detection areas sequentially reach the two detection areas through the two liquid conveying channels, the sample adding area, the liquid conveying channels and the detection areas jointly form a hydrophilic area, the rest part is a hydrophobic area, a sample is ensured to flow directionally in a designated channel, the sample flows in the area through the sample adding area, flows to the detection 1 area and the detection 2 area through the liquid conveying channels, and specifically identifies different antibiotics, the sample adding area and the detection area are circular with diameters of 6. mm, the liquid conveying channel is rectangular with the length of 2mm times of mm, the hydrophobic area and the filter paper is sequentially deposited at 35 ℃ to the inner surface of filter paper, and the filter paper is sequentially subjected to high-temperature deposition to form a porous filter paper at 200 DEG, and the filter paper is subjected to high-temperature deposition.
3. A method for preparing a paper-based chip based on the spherical nucleic acid nanoprobe prepared by the spherical nucleic acid nanoprobe preparation method according to claim 2, wherein the preparation of the paper-based chip specifically comprises the following steps: 1) Using AutoCAD software to carry out configuration design of the paper-based chip, defining a sample adding area, a liquid conveying channel and a detection area as hydrophilic areas, and the rest as hydrophobic areas; 2) Cutting medium-speed filter paper into A4 size, and printing design patterns on the filter paper with a resolution of 1200 dpi by using a HP LASER MFP w laser printer with a toner cartridge model of HP 110A; 3) Heating the printed filter paper in a 200 ℃ oven for 60 min ℃ to enable carbon powder on the surface of the filter paper to be completely melted and permeate into pores inside the filter paper to form a hydrophobic barrier; 4) The paper-based chip treated in step 3) was subjected to an infiltration treatment using 10 mM HEPES buffer at pH 7.0, followed by drying at 60℃for 10 min to prepare a paper-based chip capable of detecting both antibiotics simultaneously.
4. The application of the spherical nucleic acid nano probe prepared by the preparation method based on the spherical nucleic acid nano probe as claimed in claim 3 in preparing a paper-based chip, which is characterized in that the paper-based chip is applied to detection and analysis of aminoglycosides and sulfa antibiotics represented by neomycin and sulfadiazine, an environmental water sample is preserved in a dark place at 4 ℃, the environmental water sample is filtered by a 0.22 mu m filter membrane to be a water sample to be detected, and the paper-based chip is used for simultaneously carrying out colorimetric visual detection and analysis on the aminoglycoside antibiotics and the sulfadiazine antibiotics in the water sample, and the method specifically comprises the following steps: 1) Dripping the nano probe for detecting the aminoglycoside antibiotics into a detection 1 area of the paper-based chip, and waiting for drying; 2) Dripping the spherical nucleic acid nano probe based on the sulfadiazine aptamer prepared in the method of claim 1 to a detection 2 area of a paper-based chip, and waiting for drying; 3) Dripping a water sample to be detected to a sample adding area of the paper-based chip, enabling the sample to flow along a liquid conveying channel through capillary action, and gathering after reaching the detection 1 area and the effect of the nano probe for detecting the aminoglycoside antibiotics, so that the color of the paper-based chip is changed; 4) After the sample continues to flow to the detection 2 area to react with the spherical nucleic acid nano probe based on the sulfadiazine aptamer, dropwise adding 155 mM sodium chloride solution into the area to trigger particle aggregation reaction, so that the color of the paper-based chip is changed; 5) Standing for reacting for a few minutes, and shooting the color development results of the two detection areas on the paper-based chip by using the smart phone when the color is stable; 6) The color channel intensity value of the color development area is obtained through the color recognition function of the smart phone, wherein red is R, green is G, and blue is B, and the R/B ratio is calculated; 7) Comparing the calculated R/B ratio with a pre-established standard curve, and respectively calculating the concentration of neomycin and sulfadiazine in the water sample to realize simultaneous detection of aminoglycosides and sulfadiazine antibiotics, wherein the linear range of neomycin detection is 5.0-25.0 mu M, the detection limit is 1.6 mu M, and the linear range of sulfadiazine detection is 10.0-30.0 mu M, and the detection limit is 3.3 mu M.

Description

Preparation method and application of spherical nucleic acid-based nano probe and paper-based chip thereof Technical Field The invention relates to the technical field of detection of antibiotic residues in environmental water samples, in particular to a spherical nucleic acid (SPHERICAL NUCLEIC ACIDS, SNA) based nano probe and preparation and application of a portable paper-based chip thereof. Background The abuse and emission of antibiotics has led to their frequent detection and gradual accumulation in global aqueous environments, which pose a potential threat to the ecosystem and human health. Among them, sulfonamide antibiotics and aminoglycoside antibiotics are two important antibiotic pollutants in the environment due to their wide application in livestock breeding and aquaculture. At present, the conventional detection method of antibiotics mainly depends on the instrument analysis technology such as high performance liquid chromatography-tandem mass spectrometry. The methods have high accuracy, but also have the limitations of complex pretreatment program and operation steps, long equipment expensive analysis period, need of professional operation and the like, and limit the application of the methods in environmental field analysis. The paper-based chip has the advantages of low cost, portability, simple operation, rapid sensing response and the like, and has great potential in the field of environmental field detection. However, conventional paper-based sensors often suffer from insufficient sensitivity, poor selectivity, difficulty in achieving simultaneous detection of multiple targets, and the like. Spherical nucleic acid is a novel nanomaterial consisting of an inorganic nanoparticle core (e.g., gold nanoparticle) and highly oriented, densely arranged oligonucleotides. The spherical nucleic acid has unique optical and surface chemical properties (such as surface plasmon resonance effect) of the core nano particles and programmability of the oligonucleotide shell, shows excellent stability and biocompatibility, and is an ideal probe for constructing a high-sensitivity and high-selectivity optical sensor. In summary, developing a method capable of rapidly and sensitively detecting antibiotic residues in a water body is important for environmental risk assessment and pollution control. Therefore, the spherical nucleic acid nano probe is combined with the paper-based chip, and a sensing platform capable of simultaneously, rapidly and visually detecting various antibiotics is developed, so that the method has important practical significance. Disclosure of Invention The invention aims to provide a spherical nucleic acid nano probe based on sulfadiazine aptamer and a preparation method and application of a paper-based chip thereof, aiming at the defects of the prior art, and the spherical nucleic acid nano probe based on sulfadiazine aptamer is prepared by a microwave method and is loaded on the paper-based chip with a specific channel design, so that the visualized rapid detection of antibiotics in an environmental water sample can be realized, and the preparation method is simple and convenient. The invention can screen and apply corresponding aptamer according to the structure of target analyte, provides a new thought for analysis of other antibiotics or environmental pollutants, has important significance for environmental risk assessment, pollution control and public health, can realize rapid on-site screening and semi-quantitative analysis of aminoglycoside antibiotics and sulfanilamide antibiotics in environmental water sample, can be widely applied to the fields of environmental monitoring and the like, becomes an important part of an instant on-site detection platform, and provides a new platform for specific visual detection and pollution assessment of aminoglycoside antibiotics and sulfanilamide antibiotics in environmental water sample. The method is successfully applied to the labeling detection of the actual environmental water sample, has good recovery rate and low relative standard deviation, proves the accuracy and reliability of the detection of the method in the actual complex matrix, provides an effective technical means for the monitoring of the antibiotic residues in the environmental water body, and has important application value in the fields of environmental monitoring and pollution control. The specific technical scheme for realizing the aim of the invention is that the preparation method of the spherical nucleic acid nano probe is characterized in that the spherical nucleic acid nano probe based on sulfadiazine aptamer is prepared by adopting a microwave method, and the specific preparation method comprises the following steps: Mixing 900 mu L of gold nanoparticles (AuNPs) with the concentration of 3.5 nM with 100 mu L of sulfadiazine aptamer with the concentration of 100 nM and 3' -end modified FAM, placing the mixture in a microwave chemical reactor, heating the mixtur