CN111337661A - Up-conversion bar code fluorescence sensor for detecting veterinary drug residues in food and preparation method thereof - Google Patents
Up-conversion bar code fluorescence sensor for detecting veterinary drug residues in food and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an up-conversion bar code fluorescence sensor for detecting veterinary drug residues in food and a preparation method thereof. The up-conversion bar code fluorescence sensor comprises a core-shell nanoparticle unit and a competitive ligand unit, wherein the core-shell nanoparticle unit comprises: upconversion core-shell nanoparticles and SiO coated on outer side of upconversion core-shell nanoparticles2Layer, and modification to SiO2NH on the layer2-streptavidin-biotin-detection target aptamer; the competitive ligand unit comprises gold nanoparticles and cDNA (complementary deoxyribonucleic acid) with the 3' end connected with the gold nanoparticles and matched with the detection target aptamer. The detection of the invention has better sensitivity and specificity, and can realize three targets by different colorsDetection of (3).
Description
Technical Field
The invention belongs to the field of food detection, and particularly relates to an up-conversion bar code fluorescent sensor for detecting veterinary drug residues in food and a preparation method thereof.
Background
The pesticide and veterinary drug residues not only seriously affect the health of people, but also hinder the healthy development of food and agricultural product industries. Face the challenges of increasing food contaminants, stricter regulatory requirements, and increasing trade protection and market competition. At present, the methods commonly used for analyzing and detecting the residue of the veterinary drugs comprise: high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, etc. The method has the advantages of high sensitivity, good specificity and the like. However, these methods are expensive in terms of equipment and require specialized operators. Therefore, the method can not meet the requirements of a large number of samples and on-site rapid analysis of pesticide and veterinary drug residues. According to incomplete statistics, the pollutants of food and environment are as high as 20000, and the development of a high-flux rapid screening technology for the pollutants is of great significance, can realize coarse screening of a large number of samples, quickly identify the types of residues, and is of great significance for dealing with food safety emergencies and guaranteeing the environment and food safety.
At present, the most successful fluorescent code high-throughput detection is the Luminex xMAP technology, which is also called flow fluorescence technology, and the maximum of 100 microspheres with different characteristic fluorescence spectra are obtained by adjusting different proportions of two fluorescent dyes, so that the maximum of 100 different detection reactions can be simultaneously completed in one reaction hole. However, in the research process, the organic fluorophore is poor in light stability, not suitable for long-term observation and wide in absorption and emission bands due to the optical barcode mainly using the organic fluorescent dye. In addition, the fluorescent dye usually uses high-energy Ultraviolet (UV) or visible light as excitation light, and when the sample matrix is complex, the interferent has a certain autofluorescence, thereby greatly reducing the detection sensitivity and detection limit. The upconversion nanoparticles have the remarkable advantages of deeper light penetration depth, no background fluorescence interference and the like due to the adoption of near-infrared continuous laser as an excitation source. Therefore, the development of a high-throughput detection technology for converting the fluorescent codes of the nano particles has important significance, the sensitivity and the detection limit of the detection method can be improved, and the trace detection of pollutants is realized.
Disclosure of Invention
Aiming at the problems of complicated sample matrix, larger interference of autofluorescence and the like in the existing fluorescence coding high-flux detection of fluorescent dye, quantum dots and the like. The invention prepares the up-conversion nano bar code with low background and long service life, establishes a high-flux detection technology based on the up-conversion nano bar code, enables the up-conversion nano bar code to realize the multi-element and trace detection of various pollutants when a large amount of samples are detected in a complex matrix, and further improves the specificity of the up-conversion nano bar code through an immunological technology. The technology also has wide application prospect in the fields of environmental monitoring, biomedicine, clinical detection and the like.
In order to achieve the above objects, a first aspect of the present invention provides an upconversion barcode fluorescence sensor for detecting veterinary drug residue in food, the upconversion barcode fluorescence sensor comprising a core-shell nanoparticle unit and a competitor ligand unit, wherein,
the core-shell nanoparticle unit includes: upconversion core-shell nanoparticles and SiO coated on outer side of upconversion core-shell nanoparticles2Layer, and modification to SiO2NH on the layer2-streptavidin-biotin-detection target aptamer;
the competitive ligand unit comprises gold nanoparticles and cDNA (complementary deoxyribonucleic acid) with the 3' end connected with the gold nanoparticles and matched with the detection target aptamer.
Preferably, the up-conversion nanoparticles are at least one NaYF4: M, wherein M is at least two of Yb, Er and Tm.
Further preferably, the upconversion nanoparticles are multiple and different in color.
Based on the system of the invention, the detection target can be at least one of 17 β -estradiol, bisphenol A and progesterone, and the method of the invention can detect one of the targets independently or simultaneously.
Specifically, the sequence of 17 β -estradiol aptamer is shown as SEQ ID NO. 1, and the sequence of cDNA matched with the aptamer is shown as SEQ ID NO. 2;
5’-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGC GCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3’(SEQ ID NO:1);
5’-CGTGTAATTCAATAAGCTGGAAGCTTTTTTTTTTTT-3’(SEQ ID NO:2)。
specifically, for the detection of bisphenol a: the sequence of the bisphenol A aptamer is shown as SEQ ID NO: 3, the sequence of the cDNA matched with the aptamer is shown as SEQ ID NO: 4 is shown in the specification;
5’-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAG CGCATCACGGGTTCGCACCA-3’(SEQ ID NO:3);
5’-CCCACCTGACCACCCACCGG-3’(SEQ ID NO:4)。
specifically, for the detection of progesterone: the sequence of the progesterone aptamer is shown in SEQ ID NO: 5, the sequence of the cDNA matched with the aptamer is shown as SEQ ID NO: 6 is shown in the specification;
5’-GCATCACACACCGATACTCACCCGCCTGATTAACATTAGCCCAC CGCCCACCCCCGCTGC-3’(SEQ ID NO:5);
5’-TGGGCGGTGG-3’(SEQ ID NO:6)。
the second aspect of the invention provides a preparation method of an up-conversion bar code fluorescence sensor for detecting veterinary drug residues in food, which comprises the following steps:
(1) synthesizing the up-conversion core-shell nano particles by adopting a solvothermal method and a core-shell technology;
(2) by usingThe method is characterized in that a silicon dioxide layer is modified on the surface of the upconversion core-shell nanoparticle;
(3) adding gamma-aminopropyltriethoxysilane, introducing amino on the surface of the nano-particles through chemical reaction, and further coupling streptavidin;
(4) combining the streptavidin-coupled nanoparticles with a biotin-modified detection target aptamer to prepare the core-shell nanoparticle unit;
(5) and modifying complementary strand cDNA of a detection target aptamer on the gold nanoparticles to prepare the competitive ligand unit.
The detection principle schematic diagram of the upconversion bar code fluorescence sensor is shown in fig. 1, an upconversion bar code is formed by utilizing different upconversion luminous colors, and the color corresponds to a target object. The upconversion nanometer particles NaY F4, Yb, Er and Tm consisting of different rare earth elements are synthesized by a solvothermal method, and different luminescent wavelengths are synthesized by selecting the collocation of different elements and adjusting the proportion of the elements. The up-conversion nano particles are synthesized into single up-conversion core-shell nano particles by adopting a core-shell technology, so that the up-conversion nano particles have different fluorescence intensities at different emission wavelengths, and the fluorescence coding of the up-conversion nano particles is realized. Using sol-gel based processesAnd modifying the surface of the upconversion nanometer particle with a silicon dioxide layer. Adding gamma-aminopropyl triethoxy silane (APTES), and introducing amino on the surface of the nano particles through a chemical reaction. The nano-particles are combined with the biotin-modified aptamer after modifying streptavidin, and meanwhile, 2-3nm gold nano-particles are modified on a complementary chain to form a fluorescence internal filtering effect and quench fluorescence. When the target exists, the small molecules compete with the complementary strand, and the complementary strand with the gold particles is replaced from the up-conversion particles, so that the up-conversion particles emit light and develop color.
The up-conversion bar code fluorescence sensor and the preparation method thereof have the following advantages:
1. the up-conversion bar codes with various colors can be formed, so that the detection of different target objects is realized.
2. During detection, the method is simple to operate (no pretreatment is needed), quick in response (stable response signals are obtained in 5 min), and high in sensitivity.
3. The preparation method is simple and convenient: the preparation of the up-conversion nanoparticles is carried out simultaneously with silanization and amination, and then streptavidin is modified.
4. The method can realize the rapid detection of the three hormone pollutants, has good specificity and has good application prospect.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 shows a schematic diagram of the detection principle of the upconversion barcode fluorescence sensor of the present invention.
FIG. 2 shows a characterization of the nanoparticles of the present invention, wherein A is an upconversion core-layer structured nanoparticle; b is up-conversion core-shell structure nano particles; c is an aminated upconversion nanoparticle; d is the upconversion nanoparticles after modification of SA; e is gold nanoparticles; f is a fluorescence spectrogram of the orange UNCPs before and after modification; g is a fluorescence spectrogram before and after cyan UNCPs modification; h is a fluorescence spectrum before and after modification of the green UNCPs.
FIGS. 3A and 3B illustrate aptamer fluorescence sensor detection E2The result is optimized by the condition (2). FIG. 3A shows the optimization results of buffer type, wherein 1 represents 0.01mol/L PBS; 2 represents 0.02mol/L PBS; 3 represents 0.01mol/L PBS +0.1mol/L NaCl; 4 represents 0.01mol/L PBS +0.03mol/L MgCl2(ii) a 5 represents 0.1mol/L Tris-HCl; 6 represents 0.1mol/L Tris-HCl +0.1mol/L NaCl; 7 represents 0.1mol/L Tris-HCl +0.03mol/L MgCl2. FIG. 3B shows the optimized pH of the buffer.
Fig. 4A and 4B show the results of condition optimization for detection of BPA by the aptamer fluorescence sensor. FIG. 4A shows the optimization results of buffer type, wherein 1 represents 0.01mol/L PBS; 2 represents 0.02mol/L PBS; 3 represents 0.01mol/L PBS +0.1mol/L NaCl; 4 represents 0.01mol/L PBS +0.03mol/L MgCl2(ii) a 5 represents 0.1mol/L Tris-HCl; 6 represents 0.1mol/L Tris-HCl +0.1mol/L NaCl; 7 represents 0.1mol/L Tris-HCl +0.03mol/L MgCl2. FIG. 4B shows the optimized pH of the buffer.
Fig. 5A and 5B show the result of condition optimization of aptamer fluorescence sensor detection P4. FIG. 5A shows the optimization results for buffer type, wherein 1 represents 0.01mol/L PBS; 2 represents 0.02mol/L PBS; 3 represents 0.01mol/L PBS +0.1mol/L NaCl; 4 represents 0.01mol/L PBS +0.03mol/L MgCl2(ii) a 5 represents 0.1mol/L Tris-HCl; 6 represents 0.1mol/L Tris-HCl +0.1mol/L NaCl; 7 represents 0.1mol/L Tris-HCl +0.03mol/L MgCl2. FIG. 5B shows the optimized pH of the buffer.
FIGS. 6A-6C show standard curves for aptamer fluorescence sensor detection of three targets, where FIG. 6A represents the standard curve for E2Fig. 6B represents the aptamer fluorescence sensor for BPA, and fig. 6C represents the aptamer fluorescence sensor for P4.
FIGS. 7A-7C show the results of aptamer fluorescence sensor-specific experiments (each at a concentration of 100 ng/mL), wherein FIG. 7A represents the results for E2Fig. 7B represents the aptamer fluorescence sensor for BPA, and fig. 7C represents the aptamer fluorescence sensor for P4.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the invention, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein.
Yttrium chloride (YCl)3·7H2O), ytterbium chloride (YbCl)3·6H2O), erbium chloride (ErCl)3·6H2O), thulium chloride (TmCl)3·6H2O) from Shandong-Xiya chemical industries, Ltd, 17 β -Estradiol (17 β -Estradiol, E)2) Bisphenol a (BPA), Progesterone (progestasterone, P4) were purchased from Sigma company; biphenyldiphenol (BP), 2-bis (4-hydroxyphenyl) Butane (BPC), tetrabromobisphenol A (TB-BPA), 6F-bisphenol A (6F-BPA), bromophenol blue (BPB) available from Shanghai-sourced leaf BiotechLimited company. The experimental water was ultrapure water (18 M.OMEGA.. multidot.cm), and the other reagents were analytical grade. E2Aptamers, BPA aptamers, P4 aptamers, and complementary strand cDNA were synthesized by Shanghai Biotechnology Ltd, and the nucleic acid sequences are shown in Table 1.
TABLE 1
HT7700 TEM transmission electron microscope (japan electronics corporation); TU-1901 double-beam UV-visible spectrophotometer (Beijing Pujingyo general instruments, Inc.); an F97 Pro fluorescence spectrophotometer (Shanghai prism technology Co., Ltd.), a 980nm excitation light source (Haite photoelectricity Co., Ltd.); TGL-16C centrifuge (Shanghai' an pavilion scientific instruments factory); ZWY-240 constant temperature culture shaker (Shanghai Zhicheng analytical instruments manufacturing, Inc.); 98-II-B magnetic stirring electric heating jacket (Tester instruments, Inc., Tianjin); model KQ-500E ultrasonic cleaner (ultrasonic instruments ltd, kunshan).
Example 1
1. Synthetic core-shell structure upconversion nanoparticles
(1) Respectively preparing 1mol/L YCl3·7H2O、0.25mol/L YbCl3·6H2O、0.1mol/L TmCl3·6H2O、0.1mol/L ErCl3·6H2O, 1mol/L NaCl in ethylene glycol.
(2) 0.5g of PVP was weighed, and 8mL of ethylene glycol was added thereto and stirred until dissolved. Then taking the prepared YCl according to the core layer proportion in the table 23Solutions, ErCl3Solution, YbCl3Solution, TmCl3The solution, NaCl solution, was added to the PVP solution. Magnetically stir at room temperature for 2 hours. 8mL of a solution containing 0.470g of NH were added4And F, stirring for 30 minutes.
(3) Transferring the mixed solution into a reaction kettle for reaction, wherein the temperature is set to be 180 DEG CThe reaction time was 2 hours. After the reaction is finished, cooling to room temperature to obtain the NaYF-containing material4Glycol solution of nanoparticles.
(4) Dissolving 0.25g PVP in 3mL of ethylene glycol, adding YCl according to the core layer ratio shown in Table 23Solution, YbCl3Solutions, ErCl3The solution, NaCl solution, was magnetically stirred at ambient temperature for 2 h.
(5) Adding 9mL of NaYF synthesized in the step (3) into the solution obtained in the step (4)4The ethylene glycol stock solution of the crystal nucleus was vigorously stirred for 2 hours.
(6) 0.47g of KF was dissolved in 4mL of ethylene glycol, and the above reaction solution was added and stirred for 1 hour.
(7) And (4) transferring the mixed solution obtained in the step (6) into a reaction kettle, reacting for 24 hours at 220 ℃, precipitating with acetone, adding deionized water, centrifuging at 11000rpm and 25 ℃ for 15min, and repeating for 3 times. And drying at 80 ℃ for 24 hours to obtain white powder, namely the UCNPs nano particles.
TABLE 2 upconversion luminescence color of different doped upconversion nanoparticles
2. Functionalization of upconverted nanoparticle barcodes
2.1 Ammonia-in-silicon
(1) Weighing 10mg of UCNPs nano particles into a round-bottom flask, adding 20mL of ethanol and 1.5mL of water, and carrying out ultrasonic treatment for 30 minutes to uniformly disperse the nano particles.
(2) 0.3mL of ammonia water was added, and the mixture was magnetically stirred at 40 ℃ for 10 minutes. 10mL of absolute ethanol in which 0.025mL of TEOS was dissolved was slowly dropped into the system, and the stirring was continued overnight.
(3) Add 50. mu.L ATPES, sonicate for 5 minutes, charge nitrogen to remove oxygen for 30 minutes, and mechanically stir at 70 ℃ for 6 hours. Washing with ethanol for 3 times, and drying at 60 deg.C for 24 hr to obtain UCNPs @ SiO2@NH2And (4) granulating for later use.
2.2 packet SA
(1) Weighing 5mg of UCNPs @ SiO2@NH2The particles were dispersed in 2.5mL of 0.01mol/L PBS bufferAnd (5) performing ultrasonic dispersion for 15 min.
(2) 625 mu L of 25% glutaraldehyde solution is added, the reaction is slowly shaken at room temperature for 2h, after the reaction is finished, the precipitate is centrifugally separated, washed three times by PBS buffer, and the precipitate is resuspended in 0.01mol/L PBS.
(3) Adding 0.033mg/mL streptavidin solution, slowly shaking at room temperature for 12h, centrifuging after the reaction is finished, collecting supernatant, washing the material, and drying the precipitate at 37 ℃ for 12h to obtain the streptavidin-coupled up-conversion fluorescent nanoparticles UCNPs @ SiO2@NH2@SA。
Preparation of 2.33' -AuNP-cDNA
(1) 3mL of AuNPs were centrifuged at 4200rpm for 40min, the supernatant was removed, and the pellet was redispersed in 3mL of deionized water.
(2) To 3. mu.L of SH-cDNA (100. mu.M, whose sequence is shown in Table 1), TCEP (5mM) and 50. mu.L of Tris-HCl buffer (pH 7.00) were added in equal volumes, mixed, and allowed to stand at room temperature for 1 hour.
(3) Coupling, adding activated SH-cDNA into 3mL of AuNPs after centrifugal purification treatment, and standing and reacting for 24h at 4 ℃.
(4) Resuspending, in order to remove unreacted SH-cDNA, the conjugate system after the previous reaction was centrifuged at 4200rpm for 40min, the supernatant was discarded, and the bottom precipitate was redispersed in 3mL of deionized water to obtain Au-cDNA conjugate, which was stored at 4 ℃ for further use.
2.43 'end of complementary strand (3' -AuNP-cDNA) modified with colloidal gold is connected with aptamer
Modification of E on orange UNCPs2The aptamer of (1), wherein the aptamer of BPA is modified on cyan UNCPs, and the aptamer of P4 is modified on green UNCPs.
(1) Weighing 0.2mg of UCNPs @ SiO2@NH2@ SA particles were dissolved in 500. mu.L of 0.01mol/L PBS with shaking and mixed well to a final concentration of 0.4 mg/mL.
(2) Then 5 mu L of biotin-modified aptamer (bio-X, the specific sequence is shown in Table 1) solution is added, the mixture is slowly shaken at 37 ℃ for reaction for 1h, centrifuged and washed three times by PBS, the unattached aptamer is removed, and the mixture is resuspended in 500 mu L of hybridization buffer solution, so that the aptamer-linked UCNPs (aptamer-UCNPs) are obtained.
(3) Adding 5 mu L of 3' -AuNP-cDNA, slowly shaking for reaction at 37 ℃ for 1h, centrifuging, washing with PBS for three times, removing unconnected complementary strands, and suspending into 500 mu L of hybridization buffer solution to obtain the cDNA-aptamer-UCNPs.
The UCNPs synthesized by the hydrothermal method have uniform particle size and good particle dispersibility, the particle size is about 50nm, and the UCNPs are hexagonal, and are shown in a figure 2A; as can be seen from fig. 2B-2C, the modified UCNPs have a uniform surface coating of silicon shell with a thickness of about 10 nm; as can be seen in fig. 2D, the surface of the SA-modified uncaps became rough, indicating that SA was coupled to the particles. The AuNPs transmission electron microscope picture (figure 2E) shows that the synthesized AuNPs have uniform grain diameter and good grain dispersibility, and the grain diameter is about 2-3 nm. FIGS. 2F-2H show that the synthesized UNCPs with three colors have better fluorescence signals and better quenching effect.
3. Study of fluorescence quenching efficiency by buffer
3.1 specific procedures for optimizing the type of hybridization buffer are as follows:
(1) weighing UCNPs @ SiO2@NH2@ SA particles 3.4mg dissolved in 10.5mL of 0.01mol/L PBS, vortexed until completely dissolved, and dispensed into 21 2mL centrifuge tubes containing 500. mu.L of UCNPs @ SiO2@NH2@ SA particles (7 in each group, three groups, each group using the same hybridization buffer, 0.01mol/L PBS, 0.02mol/L PBS, 0.01mol/L PBS +0.1mol/L NaCl, 0.01mol/L PBS +0.003mol/L MgCl2,0.1mol/L Tris-HCl,0.1mol/L Tris-HCl+0.003mol/L MgCl2Three parallel groups); then 5 mu L of 5 mu mol/L bio-X is added into each of 21 centrifuge tubes, and the mixture is slowly shaken at 37 ℃ for reaction for 2 hours;
(2) centrifuging, discarding the supernatant, and washing with 0.01mol/L PBS for multiple times to ensure that bio-X which is not connected with the nano material is removed;
(3) finally, respectively resuspending the obtained up-conversion nanoparticles connected with bio-X in 500 mu L of different types of hybridization buffer solutions;
(4) adding 5 mu L of 5 mu mol/L3 '-AuNPs-cDNA into each of 15 tubes, slowly oscillating at 37 ℃ for reaction for 1h, centrifugally separating, discarding supernatant, and washing with 0.01mol/L PBS for multiple times to ensure removal of 3' -AuNPs-cDNA which is not connected with particles;
(5) the resuspended particles were pelleted in 500. mu.L of buffer and the fluorescence intensity was measured using a fluorescence spectrophotometer.
3.2 specific procedures for optimizing the pH of the hybridization buffer are as follows:
(1) weighing UCNPs @ SiO2@NH2@ SA particles 4.8mg dissolved in 12mL of 0.01mol/L PBS, vortexed until completely dissolved, and dispensed into 24 2mL centrifuge tubes, 500. mu.L of UCNPs @ SiO were added to each tube2@NH2@ SA particles containing 0.2mg of UCNPs @ SiO2@NH2@ SA particles (one group of 8 tubes each, three groups of 8 pH values, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, three parallel groups, respectively);
(2) 5 mu L of 5 mu mol/L bio-X is added into each of 24 centrifuge tubes, and the mixture is slowly vibrated at 37 ℃ for reaction for 2 hours;
(3) centrifuging, discarding the supernatant, and washing with 0.01mol/L PBS for multiple times to ensure that bio-X which is not connected with the nano material is removed;
(4) finally, respectively resuspending the obtained up-conversion nanoparticles connected with bio-X in 500 mu L of hybridization washes with different pH values;
(5) adding 5 mu L of 5 mu mol/L3 '-AuNPS-cDNA into each of 24 tubes, slowly oscillating at 37 ℃ for reaction for 1h, centrifugally separating, discarding supernatant, and washing with 0.01mol/L PBS for multiple times to ensure that 3' -AuNPS-cDNA which is not connected with the nano material is removed;
(6) the resuspended particles were pelleted in 500. mu.L of buffer and the fluorescence intensity was measured using a fluorescence spectrophotometer.
3.3 study of aptamer content on fluorescence quenching efficiency
(1) Weighing 1.8mg of UCNPs @ SiO2@NH2@ SA particles, 4mL of 0.01mol/L PBS was added, dissolved by vortexing, and mixed well to a final concentration of 0.4 mg/mL.
(2) And (3) taking 9 centrifuge tubes, and adding 500 mu L of the particle solution with the concentration of 0.4mg/mL mixed in the step (1) into each centrifuge tube.
(3) Subsequently, 5. mu.L of 1. mu. mol/L bio-X was added to the first tube, 5. mu.L of 2. mu. mol/L bio-X was added to the second tube, 5. mu.L of 3. mu. mol/L bio-X was added to the third tube, 5. mu.L of 4. mu. mol/L bio-X was added to the fourth tube, 5. mu.L of 5. mu. mol/L bio-X was added to the fifth tube, and the reaction was carried out at 37 ℃ for 1 hour with slow shaking.
(4) Centrifuging, discarding the supernatant, washing with 0.01mol/L PBS for several times to ensure that bio-X not connected with the nano material is removed, and finally resuspending the obtained particles connected with bio-X in 500. mu.L hybridization buffer respectively.
(5) Then, 5. mu.L of 1. mu. mol/L3 '-AuNPs-cDNA was added to the first tube, 5. mu.L of 2. mu. mol/L3' -AuNPs-cDNA was added to the second tube, 5. mu.L of 3. mu. mol/L3 '-AUNPS-cDNA was added to the third tube, 5. mu.L of 4. mu. mol/L3' -AuNPs-cDNA was added to the fourth tube, 5. mu.L of 5. mu. mol/L3 '-AuNPs-cDNA was added to the fifth tube, the reaction was slowly shaken at 37 ℃ for 1h, centrifuged, the supernatant was discarded, and washed with 0.01mol/L PBS several times to ensure removal of 3' -AuNPs-cDNA not bound to the nanomaterial.
(6) The resuspended particles were pelleted in 500. mu.L of buffer and the fluorescence intensity was measured using a fluorescence spectrophotometer.
Test example 1
Adding target substances with different concentrations into a mixed system of the up-conversion aptamer nanoparticles and the gold nanoparticles to enable the final system to be 400 mu L, incubating for 1h at room temperature, centrifuging, exciting a detection system by using 980nm light, and detecting the fluorescence intensity of the detection system. The UNCPs connected with the aptamer is quenched after being combined with AuNP-cDNA, so that the condition with the lowest fluorescence value is selected when the detection condition is optimized.
(1) The detection target is estradiol
Optimizing the detection system through the step 3, selecting 0.01mol/L PBS (phosphate buffer solution) +0.1mol/L NaCl buffer solution with pH7.8, selecting 5 mu mol/L aptamer concentration (as shown in figure 3A and figure 3B), and sequentially adding estradiol standard solution according to the concentration gradient (0.001ng/L-10 mu g/L) to obtain a standard curve with the concentration corresponding to the peak intensity, as shown in figure 6A. The standard curve range is 120ng/mL-200ng/mL (y is 703.74-2065.2ln (x-116.76), R20.997), detection limit 118.79 ng/mL. For an actual sample, any pretreatment is not needed, the detection system is directly added, a stable signal can be obtained within 5min, the operation is simple, and the response is rapid.
(2) The detection target is bisphenol A
Optimizing the detection system through the step 3, selecting 0.02mol/L PBS buffer solution with pH7.3, selecting 4 mu mol/L aptamer concentration (as shown in figure 4A and figure 4B), and sequentially adding bisphenol A standard solution according to the concentration gradient (0.001ng/L-10 mu g/L) to obtain a standard curve of which the concentration corresponds to the peak intensity, as shown in figure 6B. The nominal range is 5ng/mL-120ng/mL (y ═ 2604.15+1138.94ln (x +11.22), R20.985), detection limit 4.78 ng/mL. For an actual sample, any pretreatment is not needed, the detection system is directly added, a stable signal can be obtained within 5min, the operation is simple, and the response is rapid.
(3) The detection target is progesterone
Optimizing the detection system through the step 3, selecting 0.01mol/L PBS buffer solution with pH7.6, selecting 5 mu mol/L aptamer concentration (as shown in figure 5A and figure 5B), and sequentially adding progesterone standard solution according to the concentration gradient (0.001ng/L-10 mu g/L) to obtain a standard curve corresponding to the concentration and the peak intensity, as shown in figure 6C. The nominal linear range is 0.0001ng/mL-1000ng/mL (y is 91.89+124.68ln (x +1.92), R20.984), detection limit 0.0001 ng/mL. For an actual sample, any pretreatment is not needed, the detection system is directly added, and after the fluorescence intensity is obtained, the content of a corresponding target object can be converted through a standard curve. The response time is short, and only 5min is needed.
Test example 2
The influence of structural analogs or interfering substances on the detection system was examined. As shown in fig. 7A, at E2In the interference experiment, 6F-BPA, TB-BPA, BP, BPB, BPC and BPA are selected as structural analogues, and the result shows that only E is added2When the fluorescence response is maximum, other structural analogs with the same concentration also have certain influence; as shown in FIG. 7B, in the BPA interference experiment, 6F-BPA, TB-BPA, BP, BPB, BPC, E were selected2As structural analogs, the fluorescence response is maximal only when BPA is added, othersThe structural analogs with the same concentration have no obvious influence on the result; as shown in FIG. 7C, in the P4 interference experiment, 6F-BPA, TB-BPA, BP, BPB, BPC, BPA, E were selected2As a structural analogue, the fluorescence response was maximal only when P4 was added, and no other structural analogue at the same concentration had a significant effect on the results. The above results show that the method of the present invention has good specificity, and particularly has excellent selectivity for detecting P4.
Test example 3
Spiked recovery assay
The accuracy and precision of the detection method were evaluated by a spiking recovery experiment. As can be seen from table 3, the average recovery of 3 spiked levels is: e2100.21-101.79%, BPA 92.65-105.65% and P4 96.31-109.40%, which shows that the precision and accuracy of the method meet the requirement of rapid quantitative detection of three targets.
TABLE 3 three target spiking recovery experiments
The invention establishes a detection method for detecting three small molecular hormone pollutants, namely 17 β -estradiol, bisphenol A and progesterone, by using an aptamer fluorescence sensor based on a multicolor upconversion nano material.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
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Claims (6)
1. An up-conversion barcode fluorescence sensor for detecting veterinary drug residues in food, which is characterized by comprising a core-shell nanoparticle unit and a competitive ligand unit, wherein,
the core-shell nanoparticle unit includes: upconversion core-shell nanoparticles and SiO coated on outer side of upconversion core-shell nanoparticles2Layer, and modification to SiO2NH on the layer2-streptavidin-biotin-detection target aptamer;
the competitive ligand unit comprises gold nanoparticles and cDNA (complementary deoxyribonucleic acid) with the 3' end connected with the gold nanoparticles and matched with the detection target aptamer.
2. The upconversion barcode fluorescence sensor for detecting veterinary residue in food according to claim 1, wherein the upconversion nanoparticle is at least one NaYF4: M, wherein M is at least two of Yb, Er and Tm.
3. The upconversion barcode fluorescence sensor for detecting veterinary drug residue in food according to claim 2, wherein the upconversion nanoparticle is a plurality of types and has different colors.
4. The up-conversion barcode fluorescence sensor for detecting veterinary drug residues in food according to claim 1, wherein the detection target is at least one of 17 β -estradiol, bisphenol a and progesterone.
5. The up-conversion barcode fluorescence sensor for detecting veterinary drug residues in food according to claim 4, wherein the sequence of the 17 β -estradiol aptamer is shown as SEQ ID NO. 1, and the sequence of cDNA paired with the aptamer is shown as SEQ ID NO. 2;
5’-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3’(SEQ ID NO:1);
5’-CGTGTAATTCAATAAGCTGGAAGCTTTTTTTTTTTT-3’(SEQ ID NO:2);
the sequence of the bisphenol A aptamer is shown as SEQ ID NO: 3, the sequence of the cDNA matched with the aptamer is shown as SEQ ID NO: 4 is shown in the specification;
5’-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-3’(SEQID NO:3);
5’-CCCACCTGACCACCCACCGG-3’(SEQ ID NO:4);
the sequence of the progesterone aptamer is shown in SEQ ID NO: 5, the sequence of the cDNA matched with the aptamer is shown as SEQ ID NO: 6 is shown in the specification;
5’-GCATCACACACCGATACTCACCCGCCTGATTAACATTAGCCCACCGCCCACCCCCGCTGC-3’(SEQ IDNO:5);
5’-TGGGCGGTGG-3’(SEQ ID NO:6)。
6. the method for preparing an up-conversion barcode fluorescence sensor for detecting veterinary drug residue in food according to any one of claims 1 to 5, comprising the following steps:
(1) synthesizing the up-conversion core-shell nano particles by adopting a solvothermal method and a core-shell technology;
(2) by usingThe method is characterized in that a silicon dioxide layer is modified on the surface of the upconversion core-shell nanoparticle;
(3) adding gamma-aminopropyltriethoxysilane, introducing amino on the surface of the nano-particles through chemical reaction, and further coupling streptavidin;
(4) combining the streptavidin-coupled nanoparticles with a biotin-modified detection target aptamer to prepare the core-shell nanoparticle unit;
(5) and modifying complementary strand cDNA of a detection target aptamer on the gold nanoparticles to prepare the competitive ligand unit.
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CN112362875A (en) * | 2020-11-10 | 2021-02-12 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Avidin-biotin amplification up-conversion fluorescence detection method and kit for simultaneously detecting multiple small molecules |
CN112630439A (en) * | 2020-12-11 | 2021-04-09 | 郑州轻工业大学 | Splitting aptamer sensor based on nanogold and preparation method and application thereof |
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