CN117417936A - Fluorescent nano probe, preparation method thereof and method for detecting intestinal probiotics - Google Patents
Fluorescent nano probe, preparation method thereof and method for detecting intestinal probiotics Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention provides a fluorescent nano probe, a preparation method thereof and a method for detecting intestinal probiotics. The fluorescent nano probe comprises a first targeting molecule, a second targeting molecule, a first gold nano particle and a second gold nano particle; the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer; the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2. The fluorescent nano probe is added into a solution to be detected containing target bacteria, the aptamer tends to be combined with the target bacteria, double chains are opened, and the fluorescence intensity is changed, so that the quantitative detection of lactobacillus acidophilus is realized, the specificity is good, the detection sensitivity is high, and the detection limit is as low as 1CFU/mL.
Description
Technical Field
The application relates to the field of nucleic acid detection, in particular to a fluorescent nano probe and a preparation method thereof, and a method for detecting intestinal probiotics.
Background
Lactobacillus acidophilus is one of important probiotics in intestinal tracts, belongs to gram-positive bacillus, is planted in the intestinal tracts, can release lactic acid, acetic acid and some antibiotics, inhibit proliferation of bad microorganisms, regulate flora balance and promote intestinal health. In addition, the additive is also added in some yoghurt products, can help the deregulated intestinal environment to restore to normal, and has good nutrition and health care effects. The research of a novel lactobacillus acidophilus detection method and the research of the distribution situation of the lactobacillus acidophilus detection method at the original position of the intestinal tract are of great significance to the research of the mechanism of the lactobacillus acidophilus to exert health efficacy.
The nucleic acid aptamer is an oligonucleotide sequence obtained from a nucleic acid molecule library by using an in vitro screening technology, i.e. an exponential enrichment ligand system evolution technology. Has the advantages of wide target range, high specificity and affinity, small molecular weight, good environmental tolerance, chemical synthesis, easy chemical modification, small immunogenicity and the like. The optical signal element is modified on the nucleic acid aptamer through chemical synthesis and modification to form an optical nucleic acid aptamer probe, and the corresponding substance to be detected is detected through detecting an optical change signal caused by the recognition of the aptamer and the target substance. The basic composition of a nucleic acid aptamer probe can be divided into a targeting moiety, a signaling molecule, and a linker linking the targeting ligand and the signaling molecule.
The nano gold is a nano material with simple preparation and excellent optical property, and different functions can be endowed to the nano gold by different sizes, shapes, surface modification modes and the like. For example, probes can be constructed based on fluorescence or surface-enhanced Raman principles to detect various target substances, but metal nano materials have certain limitations. For example, the problem of biocompatibility in vivo is controversial, so that the polymer material with good biocompatibility is adopted for surface modification, and the polymer material has potential application prospect in vivo related research. Pectin is rich in plant cell walls, and plant cells such as citrus peel, apple peel, grape peel, beet pulp and the like are the main sources, and the molecular side chains of the pectin contain a large amount of carboxyl groups and are anionic polysaccharides. Chitosan is a polysaccharide produced by deacetylation of chitin, usually obtained from crustacean shells, and is a cationic biopolymer. Since chitosan and pectin are natural polysaccharides, the chitosan and pectin have the advantages of rich yield, low cost, good biocompatibility and the like, and are widely applied to the field of targeted delivery of bioactive substances and some medicaments at present, so that the chitosan and pectin are explored to have certain feasibility for in-vivo targeted delivery of probes.
Disclosure of Invention
The invention aims to provide an effective technical means for detecting intestinal probiotics at an original site in vivo, and in order to achieve the aim, the intestinal probiotics nucleic acid aptamer of the fluorescent dye is modified by adopting nano gold particles, and a biological probe with excellent performance is constructed by the complementary sequence of the intestinal probiotics nucleic acid aptamer to detect the intestinal probiotics.
In one aspect, the present disclosure provides a fluorescent nanoprobe comprising a first targeting molecule, a second targeting molecule, a first gold nanoparticle, and a second gold nanoparticle;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2.
Optionally, the fluorescent nano probe further comprises a first polysaccharide and a second polysaccharide, which are sequentially coated on the outer layer of the fluorescent nano probe to obtain the polysaccharide coated fluorescent nano probe;
the first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose; in the method, the first polysaccharide and the second polysaccharide are sequentially modified on the outer layer of the fluorescent nano probe, the fluorescent stability of the probe is remarkably improved based on the electrostatic interaction of the first polysaccharide and the second polysaccharide, a novel technical means with prospect is provided for detecting intestinal probiotics at original sites in vivo, and a more visual basis is provided for mechanism research of health efficacy of the intestinal probiotics.
Optionally, the first gold nanoparticle and the second gold nanoparticle are any one selected from the group consisting of gold nanosatellites, gold nanorods, gold nanocages, gold nanoflowers, gold nanobipyramids and gold nanospheres; preferably, the first gold nanoparticles are gold nanospheres, and the second gold nanoparticles are gold nanosatellites;
the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
In another aspect, the present disclosure provides a method of preparing a fluorescent nanoprobe, the method comprising the steps of:
s1, covalently connecting a first targeting molecule with first gold nanoparticles to obtain a first solution;
s2, covalently connecting a second targeting molecule with the second gold nanoparticle to obtain a second solution;
s3, mixing the first solution and the second solution, performing first incubation and centrifugal washing, and then dispersing in a buffer solution again to obtain the fluorescent nano probe;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2.
Optionally, the method further comprises: activating the first targeting molecule by using a thiol reducing agent, and then covalently connecting the activated first targeting molecule with the first gold nanoparticle;
the thiolation reducing agent comprises at least one of TCEP (tri (2-carboxyethyl) phosphine), DTT (dithiothreitol) and DTE (dithioerythritol);
the buffer is preferably Tris-HCl buffer.
Optionally, the first gold nanoparticle and the second gold nanoparticle are any one selected from the group consisting of gold nanosatellites, gold nanorods, gold nanocages, gold nanoflowers, gold nanobipyramids and gold nanospheres; the first gold nanoparticles and the second gold nanoparticles have a size of 20-60nm;
preferably, the first gold nanoparticles are gold nanospheres, and the second gold nanoparticles are gold nanosatellites;
the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
Optionally, the method further comprises: uniformly mixing the fluorescent nano probe with a solution containing first polysaccharide and a solution containing second polysaccharide in sequence to obtain the polysaccharide coated fluorescent nano probe;
the weight ratio of the first targeting molecule, the first gold nanoparticle, the second targeting molecule, the second gold nanoparticle, the first polysaccharide, the second polysaccharide and the buffer solution is 1: (10-1000): (1-10): (10-1000): (10-200): (10-200): (100000-800000).
Optionally, the conditions of the first incubation include: incubating at 35-40 ℃ for 8-12 hours;
the conditions of the centrifugal washing include: centrifugally washing for 3-5 times at 4000-5000rpm for 4-6 min;
the mixing conditions include: magnetically stirring at 25-40deg.C and 400-1000rpm for 10-14 hr.
The first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose.
In another aspect, the present disclosure provides a method of detecting intestinal probiotics, the method comprising:
and (3) carrying out second incubation on the fluorescent nano probe or the fluorescent nano probe prepared by the method and a solution to be detected possibly containing target bacteria, and then measuring the fluorescence intensity.
Optionally, the conditions of the second incubation include: incubating for 1-3h at 35-40 ℃;
the intestinal probiotics comprise at least one of lactobacillus acidophilus, lactobacillus casei and lactobacillus plantarum.
Through the technical scheme, the fluorescent nano probe is provided, the surface of each gold nanoparticle is connected with a nucleic acid aptamer modified with fluorescent dye Cy3, the surface of the other gold nanoparticle is connected with a complementary sequence of the fluorescent aptamer, a probe is constructed based on DNA double-strand hybridization, the fluorescent nano probe is added into a solution to be detected containing target bacteria, the nucleic acid aptamer tends to be combined with the target bacteria, double strands are opened, and the fluorescence intensity is changed, so that the quantitative detection of lactobacillus acidophilus is realized, the fluorescent nano probe has good linearity, good specificity and high detection sensitivity, and the detection limit is as low as 1CFU/mL.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a block diagram of a fluorescent nanoprobe prepared in example 1.
FIG. 2 is a graph of fluorescence enhancement effect of fluorescent nanoprobes T1-T5.
FIG. 3 is a graph of stability analysis before and after coating polysaccharide with a probe.
FIG. 4 is a graph of stability analysis before and after coating of the polysaccharide with the probe.
FIG. 5 is a fluorescence detection of probe T6 without chitosan and pectin coated for different concentration bacterial suspensions.
FIG. 6 is a graph showing the specificity of probe T6, which is not coated with chitosan and pectin, for intestinal probiotics.
FIG. 7 is a graph of fluorescence detection of chitosan and pectin coated probe T1 for various concentrations of bacterial suspension.
FIG. 8 is a graph showing the specificity of chitosan and pectin coated probe T1 for intestinal probiotics.
FIG. 9 is a graph of fluorescence detection of different concentrations of bacterial suspensions using other aptamer sequences to construct probe T7.
Detailed Description
The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
A first aspect of the present disclosure provides a fluorescent nanoprobe comprising a first targeting molecule, a second targeting molecule, a first gold nanoparticle, and a second gold nanoparticle;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2. The first targeting molecule and the second targeting molecule used in the present disclosure may also include specific nucleic acid aptamers and cdnas of other intestinal probiotics.
In one embodiment, the fluorescent nanoprobe further comprises a first polysaccharide and a second polysaccharide, which are sequentially coated on the outer layer of the fluorescent nanoprobe to obtain the fluorescent nanoprobe coated with the polysaccharide; the first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose; polysaccharides used in the present disclosure may also include other natural polysaccharides and biopolymers.
The polysaccharide is used for coating the fluorescent nano probe, so that the stability of the fluorescent nano probe is remarkably improved, and the application potential in an in-vivo environment is shown.
The first gold nanoparticles and the second gold nanoparticles are any one selected from gold nanostars, gold nanorods, gold nanocages, gold nanoflowers, gold nanobipyramids and gold nanospheres; preferably, the first gold nanoparticles are gold nanospheres, and the second gold nanoparticles are gold nanosatellites;
the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
In another aspect, the present disclosure provides a method of preparing a fluorescent nanoprobe, the method comprising the steps of:
s1, covalently connecting a first targeting molecule with first gold nanoparticles to obtain a first solution;
s2, covalently connecting a second targeting molecule with the second gold nanoparticle to obtain a second solution;
s3, mixing the first solution and the second solution, performing first incubation and centrifugal washing, and then dispersing in a buffer solution again to obtain the fluorescent nano probe;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2.
In one embodiment, the method further comprises: activating the first targeting molecule by using a thiol reducing agent, and then covalently connecting the activated first targeting molecule with the first gold nanoparticle;
the thiolation reducing agent comprises at least one of TCEP (tri (2-carboxyethyl) phosphine), DTT (dithiothreitol) and DTE (dithioerythritol);
the buffer is preferably Tris-HCl buffer.
In one embodiment, the first gold nanoparticle and the second gold nanoparticle are any one selected from the group consisting of gold nanosatellites, gold nanorods, gold nanocages, gold nanoflowers, gold nanobipyramids, and gold nanospheres; the first gold nanoparticles and the second gold nanoparticles have a size of 20-60nm;
in a preferred embodiment, the first gold nanoparticle is a gold nanosphere, and the second gold nanoparticle is a gold nanostar;
the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
In one embodiment, the method further comprises: uniformly mixing the fluorescent nano probe with a solution containing first polysaccharide and a solution containing second polysaccharide in sequence to obtain the polysaccharide coated fluorescent nano probe;
the weight ratio of the first targeting molecule, the first gold nanoparticle, the second targeting molecule, the second gold nanoparticle, the first polysaccharide, the second polysaccharide and the buffer solution is 1: (10-1000): (1-10): (10-1000): (100-200): (10-200): (100000-800000);
preferably 1: (10-100): (1-10): (10-100): (20-50): (50-80): (600000-800000).
In one embodiment, the conditions of the first incubation include: incubating at 35-40 ℃ for 8-12 hours;
the conditions of the centrifugal washing include: centrifugally washing for 3-5 times at 4000-5000rpm for 4-6 min;
the mixing conditions include: magnetically stirring at 25-40deg.C and 400-1000rpm for 10-14 hr.
The first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose.
In another aspect, the present disclosure provides a method of detecting intestinal probiotics, the method comprising:
and (3) carrying out second incubation on the fluorescent nano probe or the fluorescent nano probe prepared by the method and a solution to be detected possibly containing target bacteria, and then measuring the fluorescence intensity.
In one embodiment, the conditions of the second incubation include: incubating for 1-3h at 35-40 ℃;
the intestinal probiotics comprise at least one of lactobacillus acidophilus, lactobacillus casei and lactobacillus plantarum.
The present disclosure is further illustrated in detail by the following examples.
The starting materials used in the examples are all available commercially.
Example 1
The embodiment provides a preparation method of a polysaccharide-nanogold-based aptamer fluorescent probe, which is used for detecting intestinal probiotics.
(1) First gold nanoparticle AuNPs preparation: 50mL of 0.01% (w/v) HAuCl 4 ·3H 2 The O solution was heated to boil, 0.5mL of 1% (w/v) trisodium citrate solution was added and reacted for 10min, the solution turned from colorless to purplish red, then cooled to room temperature, auNPs preparation completed and stored in the dark at 4 ℃.
(2) Second gold nanoparticle auss preparation: the seed-mediated growth method is adopted, and gold seeds are synthesized first. 100 mL of HAuCl 4 ·3H 2 The O (1 mM) solution was boiled and 15 mL of 1% (w/v) trisodium citrate solution was added. The mixture was reacted for 30 minutes and then cooled to room temperature, and Au seeds were prepared. Subsequently, HCl (0.1 mL, 1M), au seed 2mL was added to HAuCl 4 ·3H 2 O (100 mL, 0.1 mM) solution was magnetically stirred at 600 r/min at room temperature, followed by rapid addition of AgNO 3 (1.5 mL, 2 mM) and L-ascorbic acid (0.5 mL, 100 mM), stirring was stopped after 30s, auNSs preparation completed, and stored in the dark at 4 ℃.
(3) The first targeting molecule thiolated Aptamer-Cy3 (50. Mu.L, 10. Mu.M, available from Shanghai) was reacted with TCEP (100. Mu.L, 2mM, available from SIGMA) for 30min, after which 50mL of AuNPs were added, incubated overnight at 4℃and washed three times with 4500rpm,5min centrifugation, redispersed in 20mL of Tris-HCl and stored in the dark at 4 ℃.
Specific nucleic acid aptamer sequence of lactobacillus acidophilus:
5'-AGCAGCACAGAGGTCAGATGTAGCCCTTCAACATAGTAATATCTCTGCATTCTGTGTGCCTATGCGTGCTACCGTGAA-3' (SEQ ID NO. 1), 5 '-modified-HS-, 3' -modified-Cy 3;
complementary sequence cDNA:
5'-TTCACGGTAGCACGCATAGGCACACAGAATGCAGAGATATTACTATGTTGAAGGGCTACATCTGACCTCTGTGCTGCT-3' (SEQ ID NO. 2), 5' -end modification-HS-.
(4) The second targeting molecule thiolated cDNA (50. Mu.L, 10. Mu.M, available from Shanghai) was reacted with TCEP (100. Mu.L, 2 mM) for 30min, after which 50mL AuNS was added and incubated overnight at 4℃and centrifuged (4500 rpm,5 min) to wash three times, redispersed in 20mL Tris-HCl and stored in the dark at 4 ℃.
(5) Covalently connecting a first targeting molecule with a first gold nanoparticle to obtain a first solution; covalently connecting a second targeting molecule with the second gold nanoparticle to obtain a second solution; the first solution was mixed with the second solution and incubated at 37℃for 12h to allow hybridization. The prepared product was then centrifuged (4500 rpm, 3 min) to remove excess nanoparticles and redispersed in Tris-HCL buffer and stored in the dark at 4 ℃ to give fluorescent nanoprobes.
(6) Mixing the double-stranded hybridized probe with 2mL of chitosan solution with the concentration of 1mg/mL, magnetically stirring for 12h at room temperature, then adding 3.33mL of citrus pectin solution with the concentration of 1mg/mL, magnetically stirring for 12h, and completing the preparation of the polysaccharide-coated fluorescent probe T1, wherein the composition of the probe is shown in figure 1.
Comparative example 1
Fluorescent nanoprobes were prepared in the same manner as in example 1 except that steps (2) and (4) were not included in the preparation method, and AuNS-cDNA was not included in the prepared fluorescent nanoprobe T2.
Comparative example 2
Fluorescent nanoprobes were prepared in the same manner as in example 1 except that steps (1) and (3) were not included in the preparation method, and AuNP-Apt-Cy3 was not included in the prepared fluorescent nanoprobe T3.
Comparative example 3
A fluorescent nanoprobe was prepared in the same manner as in example 1 except that in the preparation method, in the step (5), auNP-Apt-Cy3 and AuNS-cDNA were simply mixed without double-strand hybridization, and the fluorescent nanoprobe T4 was prepared.
Comparative example 4
A fluorescent nanoprobe was prepared in the same manner as in example 1 except that the preparation method does not include the step (2), and the cDNA and AuNP-Apt-Cy3 were mixed in the step (5) to perform double-strand hybridization, thereby preparing a fluorescent probe T5.
Comparative example 5
Fluorescent nanoprobes were prepared in the same manner as in example 1 except that step (6) was not included in the preparation method, and fluorescent nanoprobe T6 of uncoated polysaccharide was obtained.
Comparative example 6
Polysaccharide coated fluorescent nanoprobe T7 was prepared in the same manner as in example 1 except that the nucleotide sequence of the aptamer of this comparative example (purchased from the division of bioengineering (Shanghai)) was: GCAGTTGATCCTTTGGATACCCTGG (SEQ ID NO. 3); the nucleotide sequence of the aptamer cDNA (available from Biotechnology (Shanghai) Co., ltd.) was CCAGGGTATCCAAAGGATCAACTGC (SEQ ID NO. 4).
Test case
Fluorescent nanoprobes prepared by using example 1 and comparative examples 1 to 5 respectively were used with bacterial suspensions (10) 1 -10 7 CFU/mL) was incubated at 37 ℃ for 1h, and the fluorescence intensity was measured, and the results are shown in fig. 2.
The results show that the fluorescence intensity of T1 is strongest, T4 times, T2 and T5 are similar, and T3 is lowest. The comparison of the results of T1 and T3 shows that the gold nano-star has a fluorescence enhancement effect, the comparison of the results of T1 and T4 shows that the double-chain hybridization has a better fluorescence enhancement effect than the simple mixing, the similar results of T2 and T5 show that cDNA has no obvious effect on fluorescence, and the fluorescence intensity of T3 is the lowest due to unconnected fluorescent dye.
The result shows that the fluorescence enhancement effect of the gold nanostar on the fluorescent dye after double-chain hybridization in the fluorescent nanoprobe prepared in the embodiment 1 can realize higher initial fluorescence, and is favorable for realizing the detection of target bacteria based on different degree weakening of fluorescence.
Figures 3-4 are stability assays before and after probe coating of polysaccharide: the fluorescent nano probe comprises metal salt ions, temperature, simulated gastric juice and simulated intestinal juice environment (the left column is formed by coating chitosan and pectin, the right column is formed by coating chitosan and pectin), and the stability of the fluorescent nano probe is obviously improved after the coated polysaccharide is seen.
FIG. 5 shows fluorescence detection of bacterial suspensions of different concentrations by probe T6 without chitosan and pectin coating.
Fig. 6 is a diagram showing the specificity detection of probe T6, which is not coated with chitosan and pectin, on lactobacillus acidophilus, in order from left to right: lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus acidophilus+lactobacillus casei+lactobacillus plantarum, lactobacillus acidophilus+lactobacillus plantarum, and the three bacteria are mixed. Wherein the fluorescence intensity of the target bacteria, namely lactobacillus acidophilus group, is the lowest, which indicates that the aptamer is more prone to be combined with the lactobacillus acidophilus when the lactobacillus acidophilus exists, the double chain is opened more, the fluorescence intensity is reduced to a greater extent, and further indicates that the prepared aptamer fluorescent probe T1 can specifically identify the lactobacillus acidophilus.
Fig. 7 is a fluorescence detection diagram of the probe T1 coated with chitosan and pectin on bacterial suspensions with different concentrations, and detection of target bacteria can still be achieved after coating chitosan and pectin, but the detection effect is poorer than that of fig. 5 because chitosan and pectin occupy some sites on the surface of nanogold and influence the combination of aptamer and lactobacillus acidophilus to a certain extent, but polysaccharide is further degraded in an in-vivo environment, so that the detection effect equivalent to that of fig. 5 can be expected.
FIG. 8 is a diagram showing the specificity of the probe T1 coated with chitosan and pectin to Lactobacillus acidophilus. Similarly, the detection effect is inferior to that of fig. 6, but it is expected that the detection effect equivalent to that of fig. 6 can be obtained in vivo.
FIG. 9 is a graph of fluorescence detection of bacterial suspensions of different concentrations using other aptamer sequences to construct probe T7 of comparative example 6, where no obvious linearity is shown, indicating that non-specific aptamer using intestinal probiotics cannot achieve effective detection, and the necessity of using target specific aptamer is laterally verified.
According to the fluorescent nanoprobe provided by the invention, the nucleic acid aptamer of the fluorescent dye Cy3 is modified on the surface of the gold nanoparticle, the complementary sequence of the fluorescent aptamer is connected on the surface of the other gold nanoparticle, the fluorescent nanoprobe is added into a solution to be detected containing target bacteria based on DNA double-strand hybridization, the nucleic acid aptamer tends to be combined with the target bacteria, double strands are opened, and the fluorescence intensity is changed, so that the quantitative detection of lactobacillus acidophilus is realized, the fluorescent nanoprobe has good linearity, good specificity and high detection sensitivity, and the detection limit is as low as 1CFU/mL.
Further, the outer surface of the fluorescent nano probe provided by the disclosure can be coated with polysaccharide capable of generating electrostatic interaction, so that the structure of the fluorescent nano probe is more stable, the fluorescent nano probe is suitable for in vivo environment, and the probe can achieve good detection effect after the polysaccharide is gradually degraded in vivo environment.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. A fluorescent nanoprobe, characterized in that the fluorescent nanoprobe contains a first targeting molecule, a second targeting molecule, a first gold nanoparticle and a second gold nanoparticle;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2.
2. The fluorescent nanoprobe according to claim 1, wherein the fluorescent nanoprobe further comprises a first polysaccharide and a second polysaccharide, which are sequentially coated on the outer layer of the fluorescent nanoprobe to obtain the fluorescent nanoprobe coated with the polysaccharide;
the first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose;
the first gold nanoparticles and the second gold nanoparticles are any one selected from gold nanostars, gold nanorods, gold nanocages, gold nanoflowers, gold nanobipyramids and gold nanospheres; the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
3. A method of preparing a fluorescent nanoprobe, said method comprising the steps of:
s1, covalently connecting a first targeting molecule with first gold nanoparticles to obtain a first solution;
s2, covalently connecting a second targeting molecule with the second gold nanoparticle to obtain a second solution;
s3, mixing the first solution and the second solution, performing first incubation and centrifugal washing, and then dispersing in a buffer solution again to obtain the fluorescent nano probe;
the first targeting molecule is a fluorescent dye modified thiolated aptamer, and the second targeting molecule is cDNA of the thiolated aptamer;
the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the cDNA of the aptamer is shown as SEQ ID NO. 2.
4. A method according to claim 3, wherein the method further comprises: activating the first targeting molecule by using a thiol reducing agent, and then covalently connecting the activated first targeting molecule with the first gold nanoparticle;
the thiol reducing agent comprises at least one of TCEP, DTT, DTE;
the buffer solution is Tris-HCl buffer solution.
5. The method of claim 3, wherein the first and second gold nanoparticles are any one selected from the group consisting of gold nanostars, gold nanorods, gold nanocages, gold nanoflowers, gold bipyramids, and gold nanospheres; the first gold nanoparticles and the second gold nanoparticles have a size of 20-60nm;
the fluorescent dye comprises any one of Cy3, cy5, cy7 and indocyanine green.
6. A method according to claim 3, wherein the method further comprises: uniformly mixing the fluorescent nano probe with a solution containing first polysaccharide and a solution containing second polysaccharide in sequence to obtain the polysaccharide coated fluorescent nano probe;
the weight ratio of the first targeting molecule, the first gold nanoparticle, the second targeting molecule, the second gold nanoparticle, the first polysaccharide, the second polysaccharide and the buffer solution is 1: (10-1000): (1-10): (10-1000): (10-200): (10-200): (100000-800000).
7. A method according to claim 3, wherein the conditions of the first incubation comprise: incubating at 35-40 ℃ for 8-12 hours;
the conditions of the centrifugal washing include: centrifugally washing for 3-5 times at 4000-5000rpm for 4-6 min;
the mixing conditions include: magnetically stirring at 25-40deg.C and 400-1000rpm for 10-14 hr.
8. The method of claim 6, wherein the first polysaccharide is chitosan; the second polysaccharide is any one selected from pectin, starch and cellulose.
9. A method of detecting intestinal probiotics, the method comprising:
the fluorescent nanoprobe according to any of claims 1 to 2 and/or the fluorescent nanoprobe prepared by the method according to any of claims 3 to 8 is subjected to a second incubation with a test solution possibly containing a target bacterium, and then the fluorescence intensity is measured.
10. The method of claim 9, wherein the conditions of the second incubation comprise: incubating for 1-3h at 35-40 ℃;
the intestinal probiotics comprise at least one of lactobacillus acidophilus, lactobacillus casei and lactobacillus plantarum.
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