Spherical nucleic acid-based nanoprobe and preparation method and application of paper-based chip of spherical nucleic acid-based nanoprobe

Abstract

The invention discloses a preparation method and application of a spherical nucleic acid-based nanoprobe and a paper-based chip thereof. The preparation method is characterized by comprising the following steps: preparing the spherical nucleic acid nanoprobe based on a sulfadiazine aptamer by adopting a microwave method; the paper-based chip is prepared by constructing a hydrophobic barrier by adopting a laser printing and high-temperature heating method, a to-be-detected water sample is dropwise added into a sample adding area of the paper-based chip, and the sample firstly reaches a detection area 1 through capillary action and is gathered after acting with a nano probe for detecting aminoglycoside antibiotics, so that the color of the paper-based chip is changed; after the sample continuously flows to a detection area 2 and reacts with the prepared spherical nucleic acid nanoprobe based on the sulfadiazine aptamer, a sodium chloride solution is dropwise added into the area to trigger a particle aggregation reaction, so that the color of the paper-based chip is changed, and simultaneous detection of aminoglycoside and sulfonamide antibiotics is realized. Compared with the prior art, the method has the characteristics of rapidness, convenience, economy and practicability, and is of great significance to environmental risk assessment, pollutant control and public health.

Description

Technical Field

[0001] This invention relates to the field of antibiotic residue detection technology in environmental water samples, specifically to the preparation and application of a spherical nucleic acid (SNA) nanoprobe and its portable paper-based chip. Background Technology

[0002] The overuse and discharge of antibiotics have led to their frequent detection and gradual accumulation in global aquatic environments, posing a potential threat to ecosystems and human health. Among them, sulfonamide antibiotics and aminoglycoside antibiotics are two important antibiotic pollutants in the environment due to their widespread use in livestock and aquaculture.

[0003] Currently, routine detection methods for antibiotics mainly rely on instrumental analysis techniques such as high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). While these methods offer high accuracy, they also have limitations, including complex pretreatment procedures and operations, expensive equipment, long analysis cycles, and the need for specialized personnel, restricting their application in environmental field analysis. Paper-based chips, with their advantages of low cost, portability, ease of operation, and rapid sensing response, show great potential in environmental field detection. However, traditional paper-based sensors often suffer from insufficient sensitivity, poor selectivity, and difficulty in simultaneously detecting multiple targets. Globular nucleic acids (GNIs) are novel nanomaterials composed of an inorganic nanoparticle core (such as gold nanoparticles) and highly oriented, densely packed oligonucleotides. GNIs combine the unique optical and surface chemical properties of the core nanoparticles (such as surface plasmon resonance) with the programmability of the oligonucleotide shell, exhibiting excellent stability and biocompatibility, making them ideal probes for constructing highly sensitive and selective optical sensors.

[0004] In conclusion, developing rapid and sensitive methods for detecting antibiotic residues in water is crucial for environmental risk assessment and pollution control. Therefore, combining spherical nucleic acid nanoprobes with paper-based chips to develop a sensing platform capable of simultaneously, rapidly, and visually detecting multiple antibiotics has significant practical implications. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method and application for the preparation of spherical nucleic acid nanoprobes and their paper-based chips. Spherical nucleic acid nanoprobes based on sulfadiazine aptamers are prepared using a microwave method and loaded onto a paper-based chip with a specific channel design, enabling rapid and visualized detection of antibiotics in environmental water samples. The preparation method is simple and convenient. This invention allows for the screening and application of corresponding aptamers based on the structure of the target analyte, providing a new approach for the analysis of other antibiotics or environmental pollutants. It is of great significance for environmental risk assessment, pollutant control, and public health. It enables rapid on-site screening and semi-quantitative analysis of aminoglycoside antibiotics and sulfonamide antibiotics in environmental water samples, and can be widely applied in environmental monitoring and other fields, becoming an important part of a real-time on-site detection platform. It provides a new platform for the specific visualized detection and pollution assessment of aminoglycoside antibiotics and sulfonamide antibiotics in environmental water samples. This invention has been successfully applied to the spiked detection of actual environmental water samples, with good recovery and low relative standard deviation, demonstrating the accuracy and reliability of the method in complex matrices. It provides an effective technical means for monitoring antibiotic residues in environmental water bodies and has significant application value in environmental monitoring and pollution control.

[0006] The specific technical solution for achieving the objective of this invention is: a method for preparing spherical nucleic acid nanoprobes, characterized in that spherical nucleic acid nanoprobes based on sulfadiazine aptamers are prepared using a microwave method, and the specific preparation includes the following steps: 900 μL of 3.5 nM gold nanoparticles (AuNPs) were mixed with 100 μL of 3'-terminal FAM-modified sulfadiazine aptamer at a concentration of 100 nM, and then placed in a microwave chemical reactor and heated for 5 min at an output power of 700 W. After the reaction was completed, the product was brought to the required volume using ultrapure water to obtain spherical nucleic acid nanoprobes based on sulfadiazine aptamers.

[0007] A paper-based chip based on spherical nucleic acid nanoprobes is characterized by integrating a sample application area, two detection areas, and two liquid delivery channels, enabling efficient simultaneous detection of two classes of antibiotics, aminoglycosides and sulfonamides, on a single chip. The overall dimensions of the paper-based chip are 8 mm wide × 40 mm long. Its structural design features a sample application area at one end, with liquid delivery channels sequentially reaching the two detection areas. The sample loading area is located at one end of the paper-based chip. The sample flows in through this area and then flows sequentially to detection area 1 and detection area 2 via the liquid delivery channel, specifically recognizing different antibiotics. The paper base is medium-speed filter paper. Both the sample loading area and the detection area are circular with a diameter of 6 mm, and the liquid delivery channel is a rectangle with a width of 2 mm and a length of 10 mm. The hydrophobic barrier is formed by carbon powder particles deposited on the paper surface melting and penetrating into the pores inside the filter paper fibers after being heated at 200 ℃ for 60 min. The sample loading area, the liquid delivery channel, and the two detection areas together constitute a hydrophilic region, while the remaining part is a hydrophobic region, ensuring that the sample flows directionally within the designated channel.

[0008] The fabrication of a paper-based chip based on spherical nucleic acid nanoprobes specifically includes the following steps: 1) Use AutoCAD software to design the paper-based chip configuration, defining the sample application area, liquid delivery channel, and detection area as hydrophilic regions, and the remaining parts as hydrophobic regions; 2) Cut the medium-speed filter paper to A4 size, and use an HP laser MFP 136w laser printer with an HP110A toner cartridge to print the design pattern on the filter paper at a resolution of 1200 dpi. 3) Place the printed filter paper in an oven at 200 ℃ and heat for 60 min to completely melt the carbon powder on the surface of the filter paper and penetrate into the internal pores of the filter paper to form a stable hydrophobic barrier. 4) The paper-based chip treated in step 3) was immersed in 10 mM, pH 7.0 HEPES buffer and then dried at 60°C for 10 min to obtain a paper-based chip that can detect two classes of antibiotics simultaneously.

[0009] An application of the paper-based chip in the simultaneous colorimetric visualization detection and analysis of aminoglycoside antibiotics and sulfonamide antibiotics in water samples includes environmental water samples stored at 4 ℃ in the dark and filtered through a 0.22 μm filter membrane to remove suspended solids and impurities. The application of the paper-based chip in the simultaneous detection of neomycin (an aminoglycoside antibiotic) and sulfadiazine (a sulfonamide antibiotic) in water samples specifically includes the following steps: 1) Drop the nanoprobe for detecting aminoglycoside antibiotics onto detection zone 1 of the paper-based chip and wait for it to dry; 2) The prepared spherical nucleic acid nanoprobes based on sulfadiazine aptamers were dropped onto the detection zone 2 of the paper-based chip and allowed to dry. 3) The water sample to be tested is dropped into the sample application area of ​​the paper-based chip. The sample flows along the liquid transport channel through capillary action and reaches the detection area 1. After interacting with the nanoprobe for detecting aminoglycoside antibiotics, the sample aggregates, causing the color of the paper-based chip to change. 4) After the sample continues to flow to detection zone 2 and reacts with the prepared spherical nucleic acid nanoprobe based on sulfadiazine aptamer, 155 mM sodium chloride solution is added to this zone to trigger the particle aggregation reaction, which causes the paper-based chip to change color. 5) Allow the reaction to stand for a few minutes until the color stabilizes, then use a smartphone to photograph the color development results of the two detection areas of the paper-based chip; 6) Using the color recognition function of a smartphone, obtain the intensity values ​​of the red (R), green (G), and blue (B) color channels in the display area, and calculate the R / B ratio; 7) Compare the R / B ratio with the pre-established standard curve to calculate the concentrations of neomycin (aminoglycoside antibiotic) and sulfadiazine (sulfonamide antibiotic) in the water sample, respectively, to achieve simultaneous detection of aminoglycoside and sulfonamide antibiotics.

[0010] Compared with the prior art, the present invention has the following beneficial technical effects and significant technical progress: 1) The paper-based chip based on spherical nucleic acid nanoprobes designed in this invention employs a one-end sample application area, with the solution flowing to the reaction area via capillary action. The sample first reaches detection area 1, where neomycin (an aminoglycoside antibiotic) reacts with the nanoprobes used to detect aminoglycoside antibiotics, causing aggregation and a color change in the paper-based chip. The remaining sample continues to detection area 2, where a solution containing only sulfadiazine (a sulfonamide antibiotic) reacts with spherical nucleic acid nanoprobes based on sulfadiazine aptamers. A 155 mM sodium chloride solution is then added to this area to trigger particle aggregation, again causing a color change in the paper-based chip. This channel and detection area design effectively avoids interference from neomycin (an aminoglycoside antibiotic) in detection area 2.

[0011] 2) The paper-based chip based on spherical nucleic acid nanoprobes designed in this invention integrates a sample application area, two detection areas and two liquid delivery channels, which can simultaneously detect aminoglycosides and sulfonamide antibiotics on a single paper-based chip, greatly improving detection efficiency.

[0012] 3) The detection process of this invention requires no complex instruments and can complete sample addition, reaction, and result interpretation in a short time. The detection results can be qualitatively or semi-quantitatively analyzed by naked-eye measurement through color changes of the paper-based chip, and can be conveniently quantitatively detected by combining with a smartphone, making it very suitable for rapid on-site screening.

[0013] 4) The raw materials for this invention are readily available, the preparation process is simple and rapid, and it can be mass-produced. The method for synthesizing spherical nucleic acid nanoprobes is simple and rapid. The entire detection system consumes very few reagents, which is in line with the concept of green chemistry.

[0014] 5) This invention has been successfully applied to the spiked detection of actual environmental water samples, with good recovery rate and low relative standard deviation, proving the accuracy and reliability of the method in actual complex matrices, and providing an effective technical means for monitoring antibiotic residues in environmental water bodies.

[0015] 6) This invention uses a microwave method to prepare spherical nucleic acid nanoprobes. The preparation method is simple and rapid, and can be used for the specific visual detection of aminoglycoside antibiotics and sulfonamide antibiotics. It has the characteristics of simple operation, low cost, rapid detection and strong visualization.

[0016] 7) Based on the structure of the target analyte, the present invention can also screen and apply corresponding aptamers to prepare corresponding spherical nucleic acid nanoprobes, providing a new platform for the rapid detection of other environmental pollutants, and has important application value in the fields of environmental monitoring and pollution prevention and control. Attached Figure Description

[0017] Figure 1 This is a TEM characterization image of the spherical nucleic acid nanoprobe based on sulfadiazine aptamers from Example 1. Figure 2 The image shows the TEM characterization of the nanoprobe used to detect aminoglycoside antibiotics in Example 1. Figure 3 This is a schematic diagram of the paper-based chip in Example 2; Figure 4 This is a schematic diagram illustrating the simultaneous detection of neomycin (an aminoglycoside antibiotic) and sulfadiazine (a sulfonamide antibiotic) in Example 2. Figure 5 This is the standard curve for linear fitting in Example 3. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0019] Example 1 1) Preparation, characterization, and interaction mechanism of spherical nucleic acid nanoprobes based on sulfadiazine aptamers with sulfadiazine 900 μL of 3.5 nM gold nanoparticles (AuNPs) were mixed with 100 μL of 100 nM sulfadiazine aptamer with 3'-terminal FAM modification, and then placed in a microwave chemical reactor and heated for 5 min at an output power of 700 W. After the reaction was completed, the product was brought to the desired volume with ultrapure water to prepare spherical nucleic acid nanoprobes based on sulfadiazine aptamers. See Figure 1Figure A shows the dispersed state, and Figure B shows the aggregated state after binding with sulfadiazine (a sulfonamide antibiotic) and inducing salt reaction. Transmission electron microscopy (TEM) was used to investigate the interaction mechanism between the spherical nucleic acid nanoprobe and the target sulfadiazine. The prepared spherical nucleic acid nanoparticles exhibited good monodispersity in the dispersed state. When the spherical nucleic acid nanoprobe specifically bound to sulfadiazine (a sulfonamide antibiotic) and was induced by sodium chloride solution, the system underwent electrostatic imbalance.

[0020] See Figure 1 A. Before the reaction, the spherical nucleic acid nanoprobe particles are well dispersed. When the target analyte sulfadiazine (sulfonamide antibiotic) is present, the sulfadiazine aptamer specifically binds to sulfadiazine (sulfonamide antibiotic) through hydrogen bonding, induction effect and π-π stacking, triggering cross-linking between particles and causing changes in its own configuration and conformation.

[0021] See Figure 1 B. Obvious overlap and aggregation of particles were observed, which was consistent with the color change of the solution, confirming the colorimetric detection mechanism of "sulfadiazine aptamer-sulfadiazine (sulfonamide antibiotic) specific binding → high salt-induced aggregation".

[0022] 2) Characterization of the nanoprobes used to detect aminoglycoside antibiotics before and after interaction with neomycin See Figure 2 Figure A shows the dispersed state, and Figure B shows the aggregated state after reaction with neomycin (an aminoglycoside antibiotic). Transmission electron microscopy (TEM) was used to characterize the morphology of the nanoprobe before and after reaction with the target neomycin (an aminoglycoside antibiotic).

[0023] See Figure 2 A. The probe particles were well dispersed before the reaction, but particle aggregation was triggered after neomycin (an aminoglycoside antibiotic) was applied.

[0024] See Figure 2 B. After the reaction, the nanoprobe showed significant aggregation, which caused a change in the color of the paper-based chip.

[0025] Example 2 A method for fabricating a paper-based chip based on spherical nucleic acid nanoprobes, specifically including: 1) Paper-based chip design and fabrication See Figure 3The paper-based chip pattern was designed using AutoCAD 2021 software, with an overall size of 8 mm × 40 mm. The hydrophilic region includes a 6 mm diameter circular sample loading area, two 2 mm × 10 mm liquid delivery channels, and two 6 mm diameter circular detection areas (Detection Area 1 and Detection Area 2); the remaining area is hydrophobic. The designed pattern was printed on medium-speed filter paper at a resolution of 1200 dpi using an HP Laser MFP 136w laser printer with an HP110A toner cartridge. Subsequently, the printed filter paper was placed in a 200 ℃ oven for 60 min to melt the toner and allow it to fully penetrate into the filter paper fibers, forming a stable hydrophobic barrier. Finally, the chip was wetted with 10 mM HEPES buffer (pH 7.0) and dried at 60 ℃ for 10 min to enhance the liquid transport performance of the hydrophilic region.

[0026] 2) Loading of spherical nucleic acid nanoprobes on paper substrates The nanoprobes for detecting aminoglycoside antibiotics and the spherical nucleic acid nanoprobes based on sulfadiazine aptamers prepared in Example 1 were respectively added to the corresponding detection zone 1 and detection zone 2, and dried and fixed at room temperature.

[0027] Example 3 A paper-based chip based on spherical nucleic acid nanoprobes for the detection of antibiotics and the establishment of a standard curve: 1) Testing process See Figure 4 A mixed standard solution containing different concentrations of neomycin (an aminoglycoside antibiotic) and sulfadiazine (a sulfonamide antibiotic) was added dropwise to a circular sample application area at one end of a paper-based chip. The sample solution flowed first to detection area 1 via capillary action, reacting with the nanoprobes used to detect aminoglycoside antibiotics. If neomycin was present, the probe aggregated directly, causing a color change in the paper-based chip. The sample continued to detection area 2, reacting with a prepared spherical nucleic acid nanoprobe based on a sulfadiazine aptamer. Then, 155 mM sodium chloride solution was added to this area. If sulfadiazine was present, it aggregated under salt induction, causing a color change in the paper-based chip. After reacting for a few minutes until the color stabilized, a photograph was taken with a smartphone to record the results, enabling simultaneous detection of aminoglycosides and sulfonamide antibiotics. This channel and detection area design effectively avoided interference from neomycin in detection area 2.

[0028] 2) Signal analysis and establishment of standard curve: Use software to analyze the RGB values ​​of the detection area in the photo and calculate the R / B ratio.

[0029] See Figure 5A standard linear fitting curve for the detection of (A) neomycin and (B) sulfadiazine using the paper-based chip of this invention was plotted. The R / B ratio was plotted on the ordinate (Y) and the antibiotic concentration on the abscissa (X). The results showed that the linear range for neomycin detection was 5.0-25.0 μM, the detection limit was 1.6 μM, and the linear equation was Y = 0.00656x + 1.02467, R... 2 = 0.995; the linear range for the detection of sulfadiazine was 10.0–30.0 μM, the limit of detection was 3.3 μM, and the linear equation was Y = 0.0089x + 0.9281, R0 = 0.995. 2 = 0.994.

[0030] Example 4 This embodiment demonstrates the application of the paper-based chip of the present invention in a spiked actual water sample.

[0031] The water sample was filtered through a 0.22 μm aqueous filter membrane and then subjected to a spiked recovery experiment. Two concentration levels of neomycin and sulfadiazine standards were added, one low and one high, respectively, and the analysis was performed according to the procedure in Example 2. Each concentration level was measured in triplicate, and the results are detailed in Table 1 below.

[0032] Table 1. Determination results of paper-based chips in water samples

[0033] As shown in Table 1 above, the average recovery rate of neomycin was 100.9%–105.6%, with a relative standard deviation (RSD) of less than 9.8%; the average recovery rate of sulfadiazine was 98.6%–99.9%, with an RSD of less than 11.8%. These results indicate that the paper-based chip has good accuracy and reliability and can be used for the rapid detection of antibiotics in water samples from complex environments.

[0034] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing spherical nucleic acid nanoprobes, characterized in that, Spherical nucleic acid nanoprobes based on sulfadiazine aptamers were prepared using a microwave method. Specifically, 900 μL of 3.5 nM gold nanoparticles were mixed with 100 μL of 3'-terminal FAM-modified sulfadiazine aptamers (100 nM). The mixture was then placed in a microwave chemical reactor and heated for 5 min at an output power of 700 W. After the reaction was completed, the product was diluted to the desired volume using ultrapure water to obtain spherical nucleic acid nanoprobes based on sulfadiazine aptamers.

2. A paper-based chip fabricated from spherical nucleic acid nanoprobes prepared by the method described in claim 1, characterized in that, The paper-based chip integrates a sample loading area, two detection areas, and two liquid delivery channels, enabling simultaneous detection of two classes of antibiotics, aminoglycosides and sulfonamides, on a single chip. The chip measures 8 mm wide × 40 mm long. Its design utilizes a sample loading area at one end, with samples flowing sequentially to the two detection areas via the two liquid delivery channels. The sample loading area, liquid delivery channels, and detection areas together form a hydrophilic region, while the remaining portion is a hydrophobic region, ensuring directional sample flow within designated channels. The sample loading area, located at one end of the chip, allows the sample to flow in and then sequentially to detection areas 1 and 2 via the liquid delivery channels, specifically identifying different antibiotics. Both the sample loading area and detection areas are circular with a diameter of 6 mm, and the liquid delivery channels are rectangular, 2 mm wide × 10 mm long. The paper substrate is medium-speed filter paper. The hydrophobic region is formed by carbon powder particles deposited on the paper substrate surface melting and penetrating into the pores of the filter paper fibers, creating a hydrophobic barrier.

3. A method for preparing paper-based chips using spherical nucleic acid nanoprobes prepared according to claim 2, characterized in that, The fabrication of this paper-based chip specifically includes the following steps: 1) Use AutoCAD software to design the paper-based chip configuration, defining the sample application area, liquid delivery channel, and detection area as hydrophilic regions, and the remaining parts as hydrophobic regions; 2) Cut the medium-speed filter paper to A4 size, and use an HP laser MFP 136w laser printer with an HP110A toner cartridge to print the design pattern on the filter paper at a resolution of 1200 dpi. 3) Place the printed filter paper in an oven at 200 ℃ and heat for 60 min to completely melt the carbon powder on the surface of the filter paper and penetrate into the internal pores of the filter paper to form a hydrophobic barrier. 4) The paper-based chip treated in step 3) was immersed in 10 mM, pH 7.0 HEPES buffer and then dried at 60 °C for 10 min to obtain a paper-based chip that can detect two classes of antibiotics simultaneously.

4. The application of the spherical nucleic acid nanoprobe prepared by the method of claim 3 for preparing spherical nucleic acid nanoprobes in the fabrication of paper-based chips, characterized in that, The application of paper-based microarrays in the detection and analysis of aminoglycoside and sulfonamide antibiotics, represented by neomycin and sulfadiazine, involves storing environmental water samples at 4°C in the dark and filtering them through a 0.22 μm filter membrane to obtain the test water samples. The paper-based microarrays simultaneously perform colorimetric and visual detection and analysis of aminoglycoside and sulfonamide antibiotics in the water samples, specifically including the following steps: 1) Drop the nanoprobe for detecting aminoglycoside antibiotics onto detection zone 1 of the paper-based chip and wait for it to dry; 2) The spherical nucleic acid nanoprobe based on sulfadiazine aptamer prepared according to claim 1 is dropped onto the detection region 2 of the paper-based chip and left to dry; 3) The water sample to be tested is dropped into the sample application area of ​​the paper-based chip. The sample flows along the liquid transport channel through capillary action and reaches the detection area 1. After interacting with the nanoprobe for detecting aminoglycoside antibiotics, the sample aggregates, causing the color of the paper-based chip to change. 4) After the sample continues to flow to detection zone 2 and reacts with the spherical nucleic acid nanoprobe based on sulfadiazine aptamer, 155 mM sodium chloride solution is added to this zone to trigger the particle aggregation reaction, which causes the paper-based chip to change color; 5) Let the reaction stand for a few minutes until the color stabilizes, then use a smartphone to photograph the color development results of the two detection areas on the paper-based chip; 6) Using the color recognition function of a smartphone, obtain the color channel intensity values ​​of the display area, where red is R, green is G, and blue is B, and calculate the R / B ratio; 7) Compare the calculated R / B ratio with the pre-established standard curve to calculate the concentrations of neomycin and sulfadiazine in the water sample, respectively, to achieve simultaneous detection of aminoglycosides and sulfonamide antibiotics; the linear range for neomycin detection is 5.0-25.0 μM, and the limit of detection is 1.6 μM; the linear range for sulfadiazine detection is 10.0-30.0 μM, and the limit of detection is 3.3 μM.

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