CN112359094A - DNA/Fe3O4Nucleic acid detection method combining net structure with magnetic three-phase extraction method - Google Patents
DNA/Fe3O4Nucleic acid detection method combining net structure with magnetic three-phase extraction method Download PDFInfo
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Abstract
The invention discloses a DNA/Fe3O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method comprises the following steps: (1) preparation of Fe3O4Nanosheets; (2) in Fe3O4Modifying single-stranded DNA on a nano-chip to obtain Fe3O4/ssDNA; (3) preparing hyperbranched DNA structural sample, mixing the hyperbranched DNA structural sample with Fe3O4/ssDNA mixing reaction to obtain DNA/Fe3O4A network structure; (4) mixing DNA/Fe3O4The system with the network structure is used as a water phase and mixed with an organic phase, a magnetic rod with extraction liquid drops is immersed into the organic phase to extract DNA/Fe3O4Taking out the reticular structure, monitoring the liquid drop which has the catalytic color reaction by using an ultraviolet-visible spectrometer, and analyzing the ultraviolet-visible spectrum data to obtain the content of the target nucleic acid in the sample to be detected. The method greatly reduces the detection limit of nucleic acid, has high practicability, and has the detection limit of microRNA-122 as low as 0.147aM, detection limit for H-DNA as low as 0.34aM, linear range of 0.5aM to 1pM and 1aM to 1pM (r) respectively2>0.995), the single-drop microextraction technology is utilized, the detection efficiency is high, the operation is simple, and the cost is low.
Description
Technical Field
The invention relates to a nucleic acid detection method, in particular to DNA/Fe3O4A nucleic acid detection method combining a net structure with a magnetic three-phase extraction method.
Background
The components in biological fluid are complex, so that certain nucleic acids (microRNA or DNA) are easily influenced by matrix effect in detection, and the inaccuracy of the existing detection method is not high enough. Most current nucleic acid detection methods rely on hybridization, such as hybridization of a target microRNA or DNA molecule to a complementary labeled oligonucleotide probe. However, nucleic acid hybridization alone does not meet the requirements of sensitivity and accuracy. In recent years, the development of nanotechnology combining DNA and nanomaterials has provided a new signal amplification strategy for biosensors used for early diagnosis. DNA nanotechnology can not only use rigid strands of DNA to build highly ordered structures on programmable nanoscale, but also use controllable and pre-designed methods of DNA in combination with nanomaterials to generate stable amplified signals. However, since the detection relies only on hybridization of DNA, the detection limit is too high, typically in the pM to nM range, thus limiting its practical application.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide DNA/Fe which has low detection limit and high detection efficiency and is suitable for practical application3O4A nucleic acid detection method combining a net structure with a magnetic three-phase extraction method.
The technical scheme is as follows: the invention relates to a hyperbranched DNA/Fe by using peroxidase-like enzyme3O4The method for quantitatively detecting the nucleic acid by combining the net structure with the magnetic three-phase single-drop microextraction technology comprises the following steps:
(1) preparation of Fe3O4Nanosheets;
(2) in Fe3O4Modifying base single-stranded DNA on nano-chip to obtain Fe3O4/ssDNA；
(3) Subjecting a sample to be detected containing target nucleic acid to hyperbranched CHA treatment to obtain a hyperbranched DNA structural sample, and mixing the hyperbranched DNA structural sample with Fe3O4/ssDNA mixing and reacting to obtain DNA/Fe3O4A network structure;
(4) mixing DNA/Fe3O4The system with the network structure is used as a water phase and mixed with an organic phase, a magnetic rod with extraction liquid drops is immersed into the organic phase to extract DNA/Fe3O4And taking out the reticular structure, monitoring the liquid drop subjected to the catalytic color reaction by using an ultraviolet-visible spectrometer, and analyzing ultraviolet-visible spectrum data to obtain the content of the target nucleic acid in the sample to be detected.
Wherein, in the step 2, Fe is mixed by a magnet3O4The supernatant of/ssDNA is separated and discarded, and Fe modified to ssDNA can be used3O4Separation is carried out to avoid influencing subsequent experiments, Fe3O4The volume ratio of the nanosheets to the single-stranded DNA is 1:1 to 10: 1; the single-stranded DNA is a deoxyribonucleic acid sequence containing 10-100 basic groups, the organic phase in the step 4 is alkane, and the extraction liquid drop is TMB-H2O2And (3) solution.
The reaction principle is as follows: hyperbranched DNA networks are triggered by a series of strand displacement reactions. The target strand first opens the cohesive end of HP1, and then undergoes a strand displacement process to generate a new target/HP 1 hybrid strand. Newly exposing the loop base sequence of HP1 opens HP2 and results in hybridization of HP2 and HP 1. Similarly, unfolding HP2 reopens HP3, thereby initiating a cascade of self-assembly processes until the target/HP 1 hybrid strand is displaced, resulting in the formation of a CHA product branched DNA structure. The released target chain enters the next cycle, so that the cyclic amplification is realized. Meanwhile, newly exposed fragments of HP2 and HP3 opened by CHA are trigger points of HCR, and then, the base sequences exposed by HP2 and HP3 hybridize with HP4 and HP5 of the end-modified tail chain to form hyperbranched having a dendritic structureA DNA network. Reuse of magnetic Fe3O4The ssDNA modified by the nanosheet surface can be hybridized with the tail chains of HP4 and HP5 to enable Fe3O4The nano-sheet is combined with the hyperbranched DNA network structure to obtain the amplified DNA/Fe3O4A net structure. DNA/Fe when the extractant on the bar magnet is immersed in the organic phase in the MTP-SDME process3O4The network structure rapidly passes through the organic phase due to its strong hydrophobic interaction, is extracted from the bottom of the sample phase into the droplets, while unreacted Fe3O4The nano-sheets are blocked by an organic phase due to stronger hydrophilicity, and sink back to the sample solution after the magnetic rods are pumped out. Finally, DNA/Fe3O4The net structure catalyzes TMB in the extractant to generate oxidation color development amplification reaction to generate change of UV-vis signals.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) greatly reduces the detection limit of nucleic acid, has high sensitivity, has the detection limit of microRNA-122 as low as 0.147aM, the detection limit of H-DNA as low as 0.34aM, and has linear ranges of 0.5aM to 1pM and 1aM to 1pM (r2>0.995); (2) the single-drop microextraction technology is utilized, so that the detection efficiency is high; (3) simple operation, low cost and high practicability.
Drawings
FIG. 1 is a reaction scheme of example 1;
FIG. 2 is a TEM image of the network formation of example 1 at miRNA-122 concentrations of 1nM and 10 nM;
FIG. 3 is a calibration curve for miRNA-122 of example 1 and the resulting UV-Vis spectra of miRNA-122 at different concentrations at 650nm after the method;
FIG. 4 is a calibration curve for H-DNA of example 2 and the resulting UV-Vis spectra for different concentrations of DNA at 650nm after the method;
FIG. 5 is the original Fe3O4The nanoplatelets and the TEM image of the network structure formed at a concentration of 100pM H-DNA.
Detailed Description
As a specific embodiment of the invention, the target analytes to be detected are miRNA-122 and H-DNA. The analytes to be detected and the related raw nucleic acids were purchased from Shanghai Biotechnology Ltd and the sequences are as follows.
H-DNA:
5’-CTAGGGGGAGCACCCACGTGTCCTGGCCAA-3’
miRNA-122:
5’-UGGAGUGUGACAAUGGUGUUUG-3’
HP1-miRNA：
5’-GATGCATCCTTTGTACGGAGCAAACACCATTGTCACACTCCACTCCGTA-3’
HP1-DNA:
5’-GATGCATCCTTTGTACGGAGTTGGCCAGGACACGTGGGTGCTCCCCCTAG-CTCCGTA-3’
HP2:
5’-ACTCTCCTCCGTACAAAGGATGCATCTACGGAGGAGAGTGATGCATCCTTTGCAGCTAGTAG-3’
HP3:
5’-ATGCATCACTCTCCTCCGTAGATGCATCCTTTGTACGGAGGAGAGT-3’
HP4:
5’-TTTTTTTTTTTTTTTCAGCTAGTAGTACCGTCTACTAGCTGCAAAGG-3’
HP5:
5’-ACGGTACTACTAGCTGCCTTTGCAGCTAGTAGTTTTTTTTTTTTTTT-3’
ssDNA:
5’-NH2-(CH2)6-CCCCCCCCCCCCCCCAAAAAAAAAAAAA-3’
Example 1
(1) 1.8g of FeCl3·6H2O and 0.9g FeSO4·7H2O was dissolved in a beaker with 50mL of purified water, transferred to a three-necked round bottom flask, magnetically stirred, and added with N2The mixture was purged and 10mL of 25% NH was slowly added dropwise as the reaction temperature rose to 80 deg.C4OH, the solution quickly darkens. The reaction mixture was stirred in a water bath at 80 ℃ for 30min with continuous N2Purging, then separating Fe with magnet3O4Nanosheets and supernatant, and the supernatant was removed and washed 5 times with boiling ultrapure water. Re-dissolving the nanomaterial in 1After 5mL of ultrapure water, 1.25g of citric acid was added, and the mixture was stirred at 90 ℃ for 2 hours, dialyzed against a 12kDa dialysis membrane in ultrapure water for 24 hours, in which water was exchanged every 8 hours, and cooled to room temperature. Then, Fe3O4Washing the nano-sheet at 1000rpm for 10min, and centrifuging to remove Fe3O4Centrifuging the nanosheet solution at 4000rpm for 6min, removing the supernatant, and adding the final Fe3O4Drying the product in a vacuum oven at 60 ℃ for 48h, grinding, collecting, and storing in a sealed bottle;
(2) at room temperature, 0.5mg of Fe was weighed3O4The nanosheets are dissolved in 3mL of deionized water, and the solution is uniformly oscillated. Taking 100 mu L of Fe3O4The nanosheet solution was added 500. mu.L of single stranded DNA, where the single stranded DNA was 1.5. mu.M ssDNA. The mixture was reacted in saturated phosphate buffer for 2h, and ssDNA was modified in Fe by EDC-NHS coupling reaction3O4The nanosheets were surface treated and the supernatant was then separated with a magnet and discarded. Finally, the obtained Fe was washed with saturated phosphate buffer3O4ssDNA, stored at room temperature before use.
(3) Animal serum samples containing miRNA-122 were mixed with 10nM HP1, 100nM HP2, HP3, and 1. mu.M HP4, HP5 (40. mu.L each) and incubated at 37 ℃ for 2 h. And heating all the hairpin chains at 95 ℃ for 5min respectively, and slowly cooling for 1h to 25 ℃ to obtain the hyperbranched DNA structure sample. To 100. mu.L of Fe3O4240 mu L of hyperbranched DNA structure sample is added into the/ssDNA nano-sheet, and the reaction is further carried out for 1h at 25 ℃. The product was then transferred to a new 1.5mL clear chromatography vial. Wherein, TE buffer solution: 50mL of 10mM Tris-HCl, 0.0931g EDTA and 0.6353g MgCl2Mixing and fixing the volume to 250 mL.
(4) To 0.1M NaAc/HAc buffer, 5.0. mu.L of 0.1M TMB and 5. mu.L of 10.0M H were added2O2To obtain TMB-H2O2Solution as extract droplet, take 6.0. mu.L of TMB-H2O2Dropping the solution at the bottom of the magnetic rod, and mixing the hyperbranched DNA structure sample obtained in the step 3 with Fe3O4The reaction product of/ssDNA nano-sheet is used as water phase, 200 mu L alkane is added into 240 mu L water phase, layering occurs, and a magnetic rod with extraction liquid drops is quickly and lightly immersed into the mixtureThe organic phase was kept for 5 seconds, then the extract droplets were taken out and the reaction was monitored with a uv-vis spectrometer and transmission electron microscope and the change in absorbance was recorded at 650 nm.
The sequences used in this example include:
as shown in FIG. 1, hyperbranched DNA networks are formed by a series of strand displacement reaction triggers. The target strand first opens the cohesive end of HP1, and then undergoes a strand displacement process to generate a new target/HP 1 hybrid strand. Newly exposing the loop base sequence of HP1 opens HP2 and results in hybridization of HP2 and HP 1. Similarly, unfolding HP2 reopens HP3, thereby initiating a cascade of self-assembly processes until the target/HP 1 hybrid strand is displaced, resulting in the formation of a CHA product branched DNA structure. The released target chain enters the next cycle, so that the cyclic amplification is realized. Meanwhile, newly exposed fragments of HP2 and HP3 opened by CHA are trigger points of HCR, and then, base sequences exposed by HP2 and HP3 hybridize with HP4 and HP5 of the end-modified tail chain to form a hyperbranched DNA network structure having a dendritic structure. Reuse of magnetic Fe3O4The ssDNA modified by the nanosheet surface can be hybridized with the tail chains of HP4 and HP5 to enable Fe3O4The nano-sheet is combined with the hyperbranched DNA network structure to obtain the amplified DNA/Fe3O4A net structure. DNA/Fe when the extractant on the bar magnet 1 is immersed in the organic phase 2 in the MTP-SDME process3O4The network rapidly passes through the organic phase 2 and is extracted from the bottom of the sample phase into the droplets, while unreacted Fe3O4The nano-sheets are blocked by an organic phase, and the nano-sheets are sunk into the sample solution after the magnetic rods are pumped out. Finally, DNA/Fe3O4The net structure catalyzes TMB in the extractant to generate oxidation reaction and generates change of UV-vis signals.
FIGS. 2(a), (b) are DNA/Fe at concentrations of 1nM and 10nM, respectively, of miRNA-122, the target analyte3O4TEM image of the network structure. It can be seen from FIG. 2 that Fe is present during HCR3O4The nanosheets have been successfully linked to hyperbranched DNA strands to form DNA/Fe3O4A net structure.
Evaluation of DNA/Fe prepared with three-phase SDME Using UV-Vis Nanophotometer3O4Sensitivity of the network structure. From FIGS. 3(a) and (b), it can be seen that there is a linear positive correlation between the concentration of miRNA-122 and the absorbance value. Hyperbranched DNA/Fe based on MTP-SDME binding peroxidase3O4The detection limit of the reticular structure to miRNA-122 is 0.147aM, the linear range is 0.5aM to 1pM, a standard calibration curve shows that in the range of 0.5aM to 1pM, the peak area is related to the logarithm of the concentration of miRNA-122, R20.993. Table 1 shows the detection results of animal serum samples with different miRNA-122 contents, and it can be seen that the relative recovery rates of the samples doped with different miRNA-122 contents are close to 100%, which illustrates that the method can accurately and quantitatively detect the miRNA-122 content in the samples.
TABLE 1 detection results of samples of different miRNA-122 contents
| Amount of miRNA-122 incorporated | Relative | |
| Sample | ||
| 1 | 0 | - |
| |
0.6fM | 102.4 |
| Sample 3 | 6fM | 103.9 |
| Sample No. 4 | 60fM | 103.3 |
| Sample No. 5 | 600fM | 104.4 |
Relative recovery rate (total concentration-blank concentration)/incorporation concentration
Example 2
The difference between this example and example 1 is: in step 3, a sample of animal serum containing DNA is obtained, using the sequences comprising:
table 2 shows the results of the measurement of animal serum samples with different DNA content, from which it can be seen that the relative recovery rates of the samples doped with different amounts of DNA are close to 100%, indicating that the method can accurately quantify the DNA in the samples.
TABLE 2 detection results of samples of different DNA content
| Amount of incorporated DNA | Relative recovery rate | |
| Sample No. 6 | 0 | - |
| Sample 7 | 0.6fM | 100.7 |
| Sample 8 | 6fM | 104.9 |
| Sample 9 | 60fM | 101.2 |
| Sample 10 | 600fM | 103.8 |
Relative recovery rate (total concentration-blank concentration)/incorporation concentration
From FIGS. 4(a) and (b), it can be seen that there is a linear positive correlation between the concentration of DNA and the absorbance. Hyperbranched DNA/Fe based on MTP-SDME binding peroxidase3O4The detection limit of the reticular structure to DNA is 0.34aM, the linear range is 1aM to 1pM, the standard calibration curve is shown in the range of 1aM to 1pM, R2＝0.996。
Comparative example
(1) 1.8g of FeCl3·6H2O and 0.9g FeSO4·7H2O was dissolved in a beaker with 50mL of purified water, transferred to a three-necked round bottom flask, magnetically stirred, and added with N2The mixture was purged and 10mL of 25% NH was slowly added dropwise as the reaction temperature rose to 80 deg.C4OH, the solution quickly darkens. The reaction mixture was stirred in a water bath at 80 ℃ for 30min with continuous N2Purging, then separating with magnetFree of Fe3O4Nanosheets and supernatant, and the supernatant was removed and washed 5 times with boiling ultrapure water. The nanomaterial was redissolved in 15mL of ultrapure water, 1.25g of citric acid was added, and stirred at 90 ℃ for 2h, dialyzed against 12KDa dialysis membrane in ultrapure water for 24h, where water was exchanged every 8h, cooled to room temperature. Then, Fe3O4Washing the nano-sheet at 1000rpm for 10min, and centrifuging to remove Fe3O4Centrifuging the nanosheet solution at 4000rpm for 6min, removing the supernatant, and adding the final Fe3O4Drying the product in a vacuum oven at 60 ℃ for 48h, grinding, collecting, and storing in a sealed bottle;
(2) mixing an animal serum sample containing miRNA-122 with Fe3O4Mixing the nano sheets;
(3) to 0.1M NaAc/HAc buffer, 5.0. mu.L of 0.1M TMB and 5. mu.L of 10.0M H were added2O2To obtain TMB-H2O2Solution as extract droplet, take 6.0. mu.L of TMB-H2O2Dropping the solution at the bottom of the magnetic bar, and adding Fe in the step 23O4Taking a reaction product of the mixture of the nanosheets and the animal serum sample as a water phase, adding 200 mu L of alkane into 240 mu L of the water phase to generate layering, quickly and gently immersing the magnetic rod with the extraction liquid drops into an organic phase, keeping for 5 seconds, taking out the extraction liquid drops, monitoring the reaction by using an ultraviolet-visible spectrometer and a transmission electron microscope, and recording the change of absorbance at 650 nm.
FIG. 5(a) shows synthesized Fe3O4TEM image of the nanosheets, from which the original Fe can be seen3O4The diameter of the nanosheets was approximately 10nm, and FIG. 5(b) is the hyperbranched DNA/Fe formed3O4TEM image of net structure shows that peroxidase-like hyperbranched DNA/Fe is successfully prepared3O4A net structure.
Claims (6)
1. DNA/Fe3O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized by comprising the following steps of:
(1) preparation of Fe3O4Nanosheets;
(2) in Fe3O4Modifying single-stranded DNA on a nano-chip to obtain Fe3O4/ssDNA；
(3) Subjecting a sample to be detected containing target nucleic acid to hyperbranched CHA treatment to obtain a hyperbranched DNA structural sample, and mixing the hyperbranched DNA structural sample with Fe3O4/ssDNA mixing and reacting to obtain DNA/Fe3O4A network structure;
(4) mixing DNA/Fe3O4The system with the network structure is used as a water phase and mixed with an organic phase, a magnetic rod with extraction liquid drops is immersed into the organic phase to extract DNA/Fe3O4And taking out the reticular structure, monitoring the liquid drop subjected to the catalytic color reaction by using an ultraviolet-visible spectrometer, and analyzing ultraviolet-visible spectrum data to obtain the content of the target nucleic acid in the sample to be detected.
2. DNA/Fe according to claim 13O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized in that in the step 2, Fe is extracted by a magnet3O4The supernatant of/ssDNA was separated and discarded.
3. DNA/Fe according to claim 13O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized in that Fe in the step 23O4The volume ratio of the nanosheet to the base single-stranded DNA is 1:1 to 10: 1.
4. DNA/Fe according to claim 13O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized in that the single-stranded DNA in the step 2 is a deoxyribonucleic acid sequence containing 10-100 basic groups.
5. DNA/Fe according to claim 13O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized in that the organic phase in the step 4 is alkane.
6. DNA/Fe according to claim 13O4The nucleic acid detection method combining the net structure with the magnetic three-phase extraction method is characterized in that the extraction liquid drops in the step 4 are TMB-H2O2And (3) solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322307A (en) * | 2021-05-24 | 2021-08-31 | 江苏科技大学 | Method for detecting chloramphenicol by inducing formation of three-dimensional nanostructure through nucleic acid cascade amplification |
| CN113640240A (en) * | 2021-08-09 | 2021-11-12 | 江苏科技大学 | Smartphone ultraviolet detection SO based on gold and silver nanostar etching2Method (2) |
| CN113866136A (en) * | 2021-06-16 | 2021-12-31 | 江苏科技大学 | Method for detecting antibiotics through solvent-free single-drop magnetic enrichment-dispersion solid phase extraction method |
| CN114018892A (en) * | 2021-11-19 | 2022-02-08 | 江苏科技大学 | Method for detecting GST by combining magnetic single-drop microextraction fluorescent switch with PDA (personal digital assistant) coating vesicles |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978295A (en) * | 2012-08-30 | 2013-03-20 | 重庆西南医院 | Pathogenic microorganism nucleic acid amplification-free detection and typing method |
| CN106520972A (en) * | 2016-11-25 | 2017-03-22 | 复旦大学 | Method for detecting nucleic acid concentration by using self-assembly system based on nucleic acid-platinum nano-material |
| CN106770143A (en) * | 2017-02-28 | 2017-05-31 | 济南大学 | A kind of biology sensor of detection MiRNA and preparation method thereof |
| CN108940213A (en) * | 2018-08-08 | 2018-12-07 | 武汉谱信环保科技有限公司 | Magnetic Nano material and preparation method thereof for Magnetic solid phases extraction |
| CN110412156A (en) * | 2019-06-28 | 2019-11-05 | 宁波大学 | A method of measurement PCBs in Soil |
-
2020
- 2020-07-27 CN CN202010729287.7A patent/CN112359094B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978295A (en) * | 2012-08-30 | 2013-03-20 | 重庆西南医院 | Pathogenic microorganism nucleic acid amplification-free detection and typing method |
| CN106520972A (en) * | 2016-11-25 | 2017-03-22 | 复旦大学 | Method for detecting nucleic acid concentration by using self-assembly system based on nucleic acid-platinum nano-material |
| CN106770143A (en) * | 2017-02-28 | 2017-05-31 | 济南大学 | A kind of biology sensor of detection MiRNA and preparation method thereof |
| CN108940213A (en) * | 2018-08-08 | 2018-12-07 | 武汉谱信环保科技有限公司 | Magnetic Nano material and preparation method thereof for Magnetic solid phases extraction |
| CN110412156A (en) * | 2019-06-28 | 2019-11-05 | 宁波大学 | A method of measurement PCBs in Soil |
Non-Patent Citations (1)
| Title |
|---|
| HUIMIN WANG等: "Construction of an enzyme-free concatenated DNA circuit for signal amplification and intracellular imaging", CHEMICAL SCIENCE, vol. 9, pages 1 - 8 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322307A (en) * | 2021-05-24 | 2021-08-31 | 江苏科技大学 | Method for detecting chloramphenicol by inducing formation of three-dimensional nanostructure through nucleic acid cascade amplification |
| CN113866136A (en) * | 2021-06-16 | 2021-12-31 | 江苏科技大学 | Method for detecting antibiotics through solvent-free single-drop magnetic enrichment-dispersion solid phase extraction method |
| CN113640240A (en) * | 2021-08-09 | 2021-11-12 | 江苏科技大学 | Smartphone ultraviolet detection SO based on gold and silver nanostar etching2Method (2) |
| CN113640240B (en) * | 2021-08-09 | 2022-10-14 | 江苏科技大学 | Smartphone ultraviolet detection SO based on gold and silver nanostar etching 2 Method (2) |
| CN114018892A (en) * | 2021-11-19 | 2022-02-08 | 江苏科技大学 | Method for detecting GST by combining magnetic single-drop microextraction fluorescent switch with PDA (personal digital assistant) coating vesicles |
| CN114018892B (en) * | 2021-11-19 | 2024-01-30 | 江苏科技大学 | Method for detecting GST by combining magnetic single-drop microextraction fluorescent switch with PDA coating vesicle |
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