CN114574553A - Carbon dot-nanogold spherical nucleic acid and preparation method and application thereof - Google Patents
Carbon dot-nanogold spherical nucleic acid and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a carbon dot-nanogold spherical nucleic acid and a preparation method and application thereof. When the carbon dot-nanogold spherical nucleic acid prepared by the invention is used for detecting exosome miRNA, the target miRNA is identified to trigger DNAzyme to walk to generate fluorescence under the condition of the existence of metal ions; and measuring the fluorescence signal of the CDs after walking by using a fluorescence spectrophotometer to realize the detection and analysis of miRNA. Meanwhile, the method is used for detecting two exosome miRNAs based on the bicolor CDs-SNA, and DNAzyme mediated CDs fluorescent signal amplification is adopted to realize high-sensitivity detection of the miRNAs; the simultaneous detection of miRNAs is realized by adopting the bicolor CDs-SNA, and a new method is provided for the precise detection of exosome miRNA.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to carbon dot-nanogold spherical nucleic acid and a preparation method and application thereof.
Background
Exosomes are extracellular vesicles actively secreted by cells and with the diameter of 30-150nm, exist in various body fluids, and research shows that microRNAs (miRNAs) carried by the exosomes can provide a lot of information about disease progression. miRNA is non-coding RNA composed of 19-25 nucleotides, can regulate protein expression after gene transcription by inhibiting expression of target mRNA, further regulate important physiological processes such as cell generation, cell differentiation, immune response and the like, and can also be used as protooncogene or cancer suppressor gene to be abnormally expressed in different cancer cells to regulate corresponding pathological processes. Due to the high stability of miRNAs in exosomes, the miRNAs are expected to be tumor markers with great potential in liquid biopsy, and specific exosome miRNAs such as bladder cancer serum exosome miRNAs have important clinical values in the aspects of disease diagnosis, disease course monitoring and the like.
The traditional exosome miRNA detection method is a qPCR method and the like. Primer design, temperature regulation and the like in the process of detecting the exosome miRNA by the qPCR method are relatively complex. Fluorescence detection has the advantages of simple preparation, high detection sensitivity and the like, and the nanomaterial represented by spherical nucleic acid combined with a fluorescence signal amplification strategy is used for detecting miRNA in multiple studies. The nanogold can realize efficient quenching of fluorescence, and has the characteristics of large specific surface area and easy surface modification. The carbon dots serving as the zero-dimensional spherical nano-particles have multicolor fluorescence, high stability, rich raw materials and simple preparation, become supplements and substitutes for organic dyes and quantum dots, and can improve the stability and safety of detection.
The exosome extraction steps are complex, the content is low, and the acquisition amount of exosome miRNA is extremely low. The characteristic of low content of exosome miRNA in body fluid puts higher requirements on the sensitivity of the detection method. Various isothermal amplification techniques such as chain hybridization reaction (HCR), Rolling Circle Amplification (RCA), chain displacement reaction (SDA), catalytic hairpin self-assembly (CHA), DNA nanomachines (DNAzyme walker) and the like, which are currently developed, can achieve effective signal amplification.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the carbon dot-nanogold spherical nucleic acid which uses the carbon dot as a fluorescent group, has the advantages of high fluorescence intensity, good light stability and the like, and further improves the detection sensitivity by using DNAzyme signal amplification.
The invention also provides a preparation method and application of the carbon dot-nano gold spherical nucleic acid.
The technical scheme is as follows: in order to achieve the purpose, the invention provides a carbon dot-nanogold spherical nucleic acid, which takes nanogold as a core, the surface of the carbon dot-nanogold spherical nucleic acid is coupled with a DNAzyme chain and a substrate with a terminal labeled carbon dot, the DNAzyme chain is a bifunctional nucleic acid of a target complementary sequence-DNAzyme, and the bifunctional nucleic acid comprises a target complementary sequence, two arms and an enzyme active center; the substrate with the end labeled carbon dots is a single-stranded nucleic acid containing DNAzyme cleavage sites and end-coupled carbon dots.
Preferably, the target complement sequence-DNAzyme bifunctional nucleic acid comprises DNAzyme-133b or DNAzyme-135b, which have the sequences of SEQ ID nos. 1 and 2:
HS-T(10)TAGCTGGTTGAAGGGGACCAAATTTTTTTTTTTTTTTCAACCAGCTCCGAGCCGGTCGAAATAGT
HS-T(10)TCACATAGGAATGAAAAGCCATATTTTTTTAAAAAATTCCTATGTCCGAGCCGGTCGAAATAGT。
preferably, the Substrate of the single-stranded nucleic acid comprising a DNAzyme cleavage site, optionally coupled to a carbon site at the terminus comprises Substrate-133b or Substrate-135b, the sequences of which are SEQ ID nos. 3 and 4:
HS-T(3)ACTAT rA GGCTGGTT-NH2，HS-T(3)ACTAT rA GCATAGGA-NH2；
preferably, the carbon dots include green carbon dots or blue carbon dots.
The preparation method of the carbon dot-nanogold spherical nucleic acid comprises the following steps of:
(1) preparing green carbon dots or blue carbon dots;
(2) preparing nano gold;
(3) preparing carbon dot-nanogold spherical nucleic acid: coupling nucleic acid on the surface of the nanogold, mixing two nucleic acid bifunctional nucleic acids and single-stranded nucleic acid, adding TCEP (trichloroacetic acid) for incubation, then adding nanogold solution for standing overnight, adding SDS (sodium dodecyl sulfate) for incubation, then adding sodium chloride solution for multiple times for standing overnight, and performing centrifugal washing to obtain AuNPs-DNA solution; and (3) taking the carbon dots, adding EDC and NHS to activate surface carboxyl, adding the carbon dots into AuNPs-DNA solution after incubation, and centrifuging, washing and resuspending to obtain CDs-SNA solution, namely the carbon dot-nanogold spherical nucleic acid.
Wherein the molar ratio of the DNAzyme chain to the substrate is 1-4: 6-9.
Preferably, the molar ratio of the DNAzyme strand to the substrate is 2: 8.
wherein the nucleic acid is a DNAzyme chain and a substrate, and the carbon dot is coupled with the amino terminal of the substrate.
The carbon dot-nanogold spherical nucleic acid disclosed by the invention is applied to exosome miRNA detection.
Wherein the exosome miRNA comprises exosome miRNA in conventional body fluid and special exosome miRNA such as bladder cancer serum exosome miRNA.
The method comprises the following steps: under the condition of the existence of metal ions, the target miRNA identifies and triggers DNAzyme in the carbon dot-nanogold spherical nucleic acid to walk to generate fluorescence; and (3) measuring a fluorescence signal of the carbon point after the walking is finished by adopting a fluorescence spectrophotometer, and realizing the detection and analysis of miRNA.
The carbon dot-nanogold spherical nucleic acid disclosed by the invention is applied to preparation of an exosome miRNA detection reagent or a kit.
The carbon dot-nanogold spherical nucleic acid (CDs-SNA) for detecting the exosome miRNA, which is prepared by the invention, comprises nanogold, DNAzyme coupled to the surface of the nanogold and a substrate of which the end is marked with the carbon dot; when the carbon dot-nanogold spherical nucleic acid is used for detecting exosome miRNA, under the condition of the existence of metal ions, the target miRNA recognition triggers DNAzyme on the carbon dot-nanogold spherical nucleic acid to automatically walk on the surface of nanogold to generate fluorescence, specifically, the target and a target complementary sequence-DNAzyme hybridization releases the upper and lower arms of the DNAzyme, recognizes adjacent substrates, activates the rA site on the DNAzyme activity specificity shearing substrate in combination with the metal ions, and releases the carbon dot after shearing; and continuously shearing and driving the DNAzyme to walk along the surface of the nanogold, so that a large number of carbon points are separated from the surface of the nanogold, and signal amplification is realized. And (3) measuring the fluorescence signals of CDs after DNAzyme walking is finished by adopting a fluorescence spectrophotometer to realize the detection and analysis of miRNA. The invention also provides a method for detecting two exosome miRNAs based on the bicolor CDs-SNA, DNAzyme mediated CDs fluorescent signal amplification is adopted, two target miRNAs are marked by the carbon point fluorescence emission wavelength, the number of the target miRNAs is marked by the carbon point fluorescence intensity, and the high-sensitivity detection of the miRNAs is realized; the simultaneous detection of miRNAs is realized by adopting the bicolor CDs-SNA, and a new method is provided for the precise detection of exosome miRNA.
The carbon dot-nanogold spherical nucleic acid (CDs-SNA) comprises two carbon dot-nanogold spherical nucleic acids, specifically comprises two carbon dots with two colors, two targets, two DNAzymes, corresponding substrates and metal ions thereof; the carbon dots of the two colors are green and blue respectively; the two target molecules are two miRNAs in serum exosomes; the two DNAzymes comprise DNAzyme-133b and DNAzyme-135b, and the 5' ends of the two DNAzymes are modified with sulfydryl; the two substrates comprise Substrate-133b and Substrate-135b, wherein the 5 'end of the two substrates is modified with a sulfhydryl group, and the 3' end of the two substrates is modified with an amino group; the metal ions are magnesium ions.
The two exosome miRNAs are bladder cancer related exosomes miR-133b and exosomes miR-135 b.
Preferably, the carbon dot-nanogold spherical nucleic acid (CDs-SNA) comprises different target complementary sequences-DNAzyme hairpin structures which are designed based on green and blue carbon dots and used for specific recognition of different miRNAs. The two target molecules are two miRNAs in serum exosomes, such as bladder cancer related exosome miR-133b and exosome miR-135 b; the two target complementary sequences-DNAzyme and the corresponding Substrate sequences are DNAzyme-133b/Substrate-133b, DNAzyme-135b/Substrate-135b, the 5 ' end of DNAzyme is modified with sulfhydryl, and the 5 ' end and the 3 ' end of Substrate are modified with sulfhydryl and amino respectively. Wherein DNAzyme-133b/Substrate-133 b/green CDs are used to detect miR-133 b; DNAzyme-135b/Substrate-135 b/blue CDs were used to detect miR-135b, coupled to the nanogold surface, respectively. Since CDs are close to the nanogold surface, fluorescence is in a quenched state. In the detection process, the target miRNA recognition triggers DNAzyme to walk along the surface of the nanogold under the condition that metal ions exist. Under the condition of no miRNA, the target complementary sequence-DNAzyme is a hairpin structure and has no shearing activity. mutual interaction of miRNA and targetThe complementary sequences are hybridized to cause the conformation of the hairpin structure to be changed, so as to form a DNAzyme structure; further adding Mg2+DNAzyme cleavage activity is activated to cleave single-stranded nucleic acids; the DNAzyme is driven to walk along the surface of the nanogold by continuous shearing activity, so that a large number of carbon points are separated from the surface of the nanogold, and signal amplification is realized. The quantitative relation between the fluorescence intensity of the carbon dots and the concentration of the added target can be measured by adopting a fluorescence spectrophotometer, and the detection limit and the linear detection range are calculated.
The invention designs a specific DNAzyme chain, which comprises a target complementary sequence, two arms and an active center of an enzyme. The DNAzyme chain of the invention is designed for single molecule, and is different from the common DNAzyme in that a target complementary sequence is designed into the DNAzyme chain, intramolecular hybridization is carried out to form a hairpin structure, when no target exists, the hairpin structure is maintained, and the DNAzyme is inactive.
According to the invention, the carbon dots, the DNAzyme and the substrate are integrated on the surface of the nanogold, and the high-sensitivity detection of miRNA is realized by utilizing the high quenching performance of the nanogold, the high fluorescence intensity and the high flexibility light stability of the carbon dots and the DNAzyme signal amplification function. The invention adopts carbon dots as fluorescent groups to replace organic fluorescent dyes and traditional quantum dots in previous researches. The organic fluorescent dye is easy to quench, the traditional quantum dots are generally toxic, the environment is greatly harmed, and the particle size is large. The carbon dots are simple and convenient to synthesize, low in cost, high in fluorescence intensity and fluorescence stability, low in toxicity, small in particle size and beneficial to achieving detection accuracy, and can be excited by using the same excitation wavelength, so that simultaneous and multiple detection is more convenient.
The DNAzyme sequence of the invention is flexible in design, can be used for detecting any given miRNA corresponding to the design of the DNAzyme, and can realize the specific detection of the target miRNA.
In addition, the carbon dot-nanogold spherical nucleic acid detection exosome has single reaction condition in the experiment, can complete all reactions at 37 ℃, and does not need complex operation steps and higher reaction temperature.
In order to distinguish two spherical nucleic acids, the spherical nucleic acids with two carbon dot colors are designed to be respectively used for detecting two targets, and the carbon dots with the two colors can mutually exchange corresponding DNAzyme and Substrate. Meanwhile, the CDs-SNA prepared by the invention can be used for simultaneously detecting two miRNAs.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention prepares the DNAzyme-loaded carbon dot-nanogold spherical nucleic acid (CDs-SNA), and realizes high sensitivity and simultaneous detection of exosome miRNA by triggering DNAzyme automatic walking mediated CDs fluorescence signal enhancement through target recognition.
The carbon dots are used as fluorescent groups to replace conventional organic fluorescent dyes, so that the detection accuracy is more favorably realized, and the multicolor carbon dots can be excited by using the same excitation wavelength, so that the simultaneous and multiple detection is more convenient.
Drawings
FIG. 1 is a schematic diagram of DNAzyme walking-based detection of exosome miRNA by CDs-SNA;
FIG. 2 is a characterization of the properties of CDs-SNA, wherein Panel A is a transmission electron micrograph; b picture is water power size of nano gold (AuNPs), nano gold surface coupling DNA (AuNP-DNA) and CDs-SNA; c picture is the UV-vis spectrogram of nano-gold, nano-gold surface coupling DNA and CDs-SNA;
FIG. 3 is a PAGE electrophoresis demonstrating the feasibility of DNAzyme walking;
FIG. 4 is a sensitivity and standard curve for CDs-SNA detection of miRNA;
FIG. 5 shows the specificity of detecting miRNA by CDs-SNA,***P<0.001,**P<0.01,*P<0.05 ns, no significant difference (P)>0.05)。
FIG. 6 is an assessment of the ability of CDs-SNA to detect two targets simultaneously;
FIG. 7 is a standard curve obtained by qPCR using miR-133b and miR-135b standard substances as templates;
FIG. 8 shows that CDs-SNA detects serum exosome miRNA, NC-SNA indicates that a normal control group is detected by a carbon dot-nanogold spherical nucleic acid method, NC-PCR indicates that a normal control group is detected by a qPCR method, BC-SNA indicates that a bladder cancer group is detected by a carbon dot-nanogold spherical nucleic acid method, BC-PCR indicates that a bladder cancer group is detected by a qPCR method,***P<0.001。
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1
Target complementary sequence-DNAzyme and sequence design of substrate
The target was selected as two exosome mirnas associated with bladder cancer: miR-133b and miR-135 b. As shown in FIG. 1, the corresponding DNAzyme-133b and DNAzyme-135b and the corresponding substrates (substrate-133b and substrate-135b) were designed and synthesized for these two targets, respectively, and the sequences are listed in Table 1. As shown in Table 1, the bold portions of DNAzyme-133b and DNAzyme-135b are the corresponding target complementary sequences, the underlined portions are the upper and lower arms of DNAzyme, and the italic portions are the active center sequences of DNAzyme; rA in both Substrate-133b and Substrate-135b is the corresponding DNAzyme cleavage site, and the italic portions are the complementary sequences of the upper and lower arms of the corresponding DNAzyme. miR-133b-1mut, miR-135b-1mut, miR-3 mut and miR-5 mut are respectively 1 base mutation sequence, 3 base mutation sequence and 5 base mutation sequence of target miRNA.
TABLE 1 target miRNA, DNAzyme, substrate and base mutation nucleic acid sequence design
Example 2
Preparation of carbon dots
Preparation of green carbon dots: first, 3g of citric acid and 3g of urea were added to 10mL of distilled water to form a transparent solution. The solution was then heated in a 750W microwave oven for 4-5min to change from a colorless liquid to brown and finally to a dark brown solid, indicating the formation of carbon dots. The aqueous solution of carbon dots was centrifuged (3000rpm, 20min) to remove large or agglomerated particles. Filtering the supernatant with a 0.45 mu m filter membrane, and dialyzing for 48h with a 100-D dialysis bag, wherein the obtained solution is the green fluorescent carbon dot solution.
Preparation of blue carbon dots: 0.5g of citric acid and 0.5g of thiourea were added to 25mL of water and stirred. Then heated in a 750W microwave oven for 7min until the solution turned from colorless to dark brown indicating that the reaction had carbonized. After cooling to room temperature, it was dissolved in 25mL of water. The obtained dark brown solution is centrifuged for 5min at 10000rpm, and the supernatant is filtered by a 0.45 mu m filter membrane and dialyzed for 48h by a 100-fold 500D dialysis bag to obtain a blue carbon dot solution.
Example 3
Preparation of nano gold
The gold nanoparticles are synthesized by a citrate reduction method, all glassware is soaked by chromic acid and washed by ultrapure water for multiple times, and the glassware is dried for use. First, 1% (g/mL) sodium citrate solution is prepared, 1g trisodium citrate is weighed and dissolved in 100mL H2O, shaking up before use. To a round bottom flask was added 96mL of H2O with 1mL HAuCl4(25mM) solution, heated to boiling and stirred vigorously, followed by the addition of 3mL of freshly prepared sodium citrate solution for 30 min. During the process, the solution started to be dark blue and then rapidly changed to red. After the heating is finished, the solution is continuously stirred vigorously until the temperature of the solution is recovered to the room temperature, and the solution is stored at 4 ℃ for standby.
Example 4
Preparation of carbon dot-Nanogold spherical nucleic acids (CDs-SNA)
Nucleic acid was first coupled to the surface of the nanogold, and DNAzyme-133b (1. mu.M, 20. mu.L) and Substrate-133b (1. mu.M, 80. mu.L) from example 1 were mixed, followed by addition of 100. mu.L of TCEP (2.5. mu.M) and incubation for 1 h. Subsequently, 150. mu.L of nanogold solution (prepared in example 3) was added, and PBS (pH 7.4) was added to make up the volume to 400. mu.L, and left to stand overnight. Adding 4 mu L of 1% SDS the next day, incubating for 20min, adding 2M sodium chloride solution, adding once every 30min, performing ultrasonic treatment for 10s after each addition to prevent sodium chloride from caking, increasing the final concentration of sodium chloride in the system by 0.1M each time until the final concentration of sodium chloride in the system reaches 0.7M, and standing overnight. The next day, centrifugation was carried out, and washing was carried out three times with pure water to obtain AuNPs-DNA solution. The green carbon dot prepared in example 2 was taken to be 8. mu.L, EDC and NHS (final concentration: 0.02M) were added to activate surface carboxyl, incubation was carried out for 30min, and the mixture was added to the AuNPs-DNA solution and incubated at room temperature for 4 h. The solution was washed three times with centrifugal water and resuspended in 100. mu.L Tris-HCl (10mM, pH 7.4) to give a CDs-SNA solution.
The prepared CDs-SNA is characterized by the shape, the size and the optical property, and the result is shown in figure 2. FIG. 2a is a transmission electron microscope picture of CDs-SNA, and the result shows that a layer of carbon dots is covered on the surface of the nano-gold, the nano-gold is in a satellite structure, and the monodispersity is good. FIG. 2b is a hydrodynamic size comparison of AuNPs, AuNPs-DNA and CDs-SNA, showing an increase of about 14nm after coupling nucleic acids, further coupling carbon dots, and a further increase in size of about 24nm compared to nanogold size. FIG. 2c is a comparison graph of ultraviolet-visible absorption spectra, and the result shows that the absorbance is red-shifted by 5nm after the nanogold is coupled with the nucleic acid, thereby indicating that the high-density nucleic acid is successfully coupled, the carbon dots are further coupled, and the absorbance is further red-shifted by 17nm, indicating that the carbon dots are successfully coupled. The above shows the successful preparation of carbon dot-nanogold spherical nucleic acid (CDs-SNA).
The preparation process was the same as that described above using DNAzyme-135b and Substrate-135b and blue spots, and the results were similar.
Example 5
Feasibility of DNAzymes as Signal amplifiers
Validation of the feasibility of DNAzyme amplification was performed in liquid phase, and electrophoresis was performed on 20% PAGE gels in TBE buffer. The sample is divided into 8 groups, and the sample loading amount of each group is 10 mu L. The sample was DNAzyme-133b (2. mu.M, 12. mu.L), target miRNA-133b (2. mu.M, 12. mu.L), Substrate-133b (2. mu.M, 3. mu.L) and Mg2+(5mM, 3.5. mu.L) and make up to 35. mu.L with Tris-HCl. Sample loading after reaction for 3h at 37 ℃: lane 1: DNAzyme chain (D); lane 2: target (t); lane 3: substrate (S); lane 4: d + T; lane 5: d + S; lane 6: d + T + S; lane 7: d + S + Zn2+(ii) a Lane 8: d + T + S + Mg2+. The PAGE results (figure 3) show that when the target is hybridized to the DNAzyme strand, the hairpin structure opens, resulting in a change in the conformation of the DNAzyme strand, with the DNAzyme/miRNA band (lane 4) being significantly different from the DNAzyme strand band alone (lane 1). When targeting miRNA and Mg2+In the absence of either or both, DNAzymes are inactive and do not trigger the cleavage reaction, so the substrate band remains intact ( lanes 5, 6, 7). When the target ismiRNA and Mg2+In the presence of the same, DNAzyme hybridised to the target miRNA, the substrate was sheared and the substrate band disappeared (lane 8). The PAGE results confirmed the feasibility of DNAzyme-133b as a signal amplifier.
PAGE electrophoresis was performed using DNAzyme-135b and Substrate-135b and the target miRNA-135b, and the results were consistent.
In the embodiment, firstly, the designed DNAzyme chain is verified to be capable of shearing a substrate in the presence of a target and metal ions in a liquid phase (the result can be presented by PAGE electrophoresis), and a foundation is laid for further coupling the DNAzyme chain and the substrate on the surface of the nanogold and realizing signal amplification.
Example 6
Sensitive detection of miRNAs
The CDs-SNA solution resuspended in example 4 was incubated with different concentrations of target miRNA (final concentrations were 0fM, 10fM, 50fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM and 100nM), and after sufficient reaction at 37 ℃ for 3h, the fluorescence spectrum was measured.
Example 4 CDs-SNA prepared by two different color carbon dots are respectively used for sensitivity detection of target miRNA-133b and miR-135b, wherein miR-135b corresponds to blue, and miR-133b corresponds to green.
The result is shown in fig. 4, as the concentration of the target miRNA is increased, the fluorescence of the CDs is increased, and the fluorescence intensity has a linear relationship with the logarithm of the corresponding target miRNA concentration.
The detection limit of the two miRNAs is about 10fM, the linear range is 50fM-10nM, and the correlation coefficients are 0.997 and 0.996 respectively.
Example 7
Specific detection of miRNAs
After incubating the two CDs-SNA solutions with the fluorescence color obtained in the resuspension in example 4 with miR-133b and miR-135b with the final concentrations of 1nM, and the single base mutation sequences (1mut), three base mutation sequences (3mut), five base mutation sequences (5mut) and random sequences (miR-133 b is used as a random sequence when miR-135b is detected and miR-135b is used as a random sequence when miR-133b is detected) at 37 ℃ for 3h, a blank solution without miRNA is used as a negative control, and the fluorescence spectrum is measured.
The result is shown in fig. 5, and the fluorescence intensity gradually decreases with the increase of the number of the mutant bases, which indicates that the method has higher specificity of detecting the miRNA.
Example 8
CDs-SNA for simultaneous detection of two miRNAs
Based on the characteristic that the multicolor fluorescent carbon dots can use the same excitation wavelength and generate different emission wavelengths, the two CDs-SNAs prepared in example 4 are mixed with two targets for incubation, and compared with single detection, and a blank solution without miRNA added is used as a negative control to measure the fluorescence spectrum.
Specifically, the method comprises the following steps: the two CDs-SNA solutions prepared in example 4 are mixed in equal volume, two target miRNAs (the final concentration is 1nM) are added respectively or simultaneously, a blank solution without the addition of the miRNAs is used as a negative control, the blank solution is subjected to water bath at 37 ℃ for 3 hours, and the fluorescence spectrum is measured.
The results are shown in fig. 6, and the results of the single detection and the simultaneous detection are similar, which shows that the method can realize simultaneous detection of the miRNAs and the analysis of the results is more convenient and intuitive.
Example 9
CDs-SNA detection of serum exosome miRNAs
Serum samples were bladder cancer patient (BC) serum and normal control group (NC) serum, and Total Exosome Isolation (from serum) was extracted as a serum Exosome kit. The step of extracting exosomes was performed according to the instructions in the kit, and total RNA of serum exosomes was extracted with TRIzol, dissolved in 10 μ L of DEPC water, and divided into two equal volumes. The two CDs-SNA solutions prepared in example 4 were mixed in equal volume, and the total RNA solution extracted from the serum exosomes was added by the method of example 6 and reacted at 37 ℃ for 3 hours. The fluorescence spectrum was measured with a fluorescence spectrophotometer. And calculating the concentrations of two miRNAs in the extracted serum exosomes according to the relation between the fluorescence intensity and the target concentration. Meanwhile, Ct values of miR-133b and miR-135b in another total RNA are determined by qPCR. And (3) carrying out qPCR (quantitative polymerase chain reaction) experiments after miR-133b and miR-135b standard substances are diluted according to gradient, and obtaining a standard curve according to the relation between the copy number and the Ct value (figure 7). Thereby calculating the concentrations of both mirnas.
The concentration of 2 miRNAs in serum exosomes was calculated from fluorescence spectra and standard curves, and the results are shown in FIG. 8. in the method of example 6 of the present invention (NC-SNA, BC-SNA), miR-135b expression in serum exosomes of bladder cancer patients (BC) was increased and miR-133b expression was decreased compared to Normal Controls (NC), which is similar to the concentrations calculated by qPCR methods (NC-PCR, BC-PCR). Therefore, the multicolor CDs-SNA-based exosome miRNA detection method is suitable for quantitative detection of miRNA in serum exosomes. Meanwhile, compared with qPCR, the method does not need complex operation steps such as reverse transcription and amplification, and can realize simultaneous and multiple detection under the same system.
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Claims (10)
1. The carbon dot-nanogold spherical nucleic acid is characterized in that nanogold is used as a core, the surface of the carbon dot-nanogold spherical nucleic acid is coupled with a DNAzyme chain and a substrate with a terminal labeled with a carbon dot, the DNAzyme chain is a target complementary sequence-DNAzyme bifunctional nucleic acid and comprises a target complementary sequence, two arms and an enzyme active center; the substrate with the end labeled carbon dots is a single-stranded nucleic acid containing DNAzyme cleavage sites and end-coupled carbon dots.
2. The carbon dot-nanogold spherical nucleic acid according to claim 1, wherein the DNAzyme chain comprises DNAzyme-133b or DNAzyme-135b, which preferably have the sequences of:
HS-T(10)TAGCTGGTTGAAGGGGACCAAATTTTTTTTTTTTTTTCAACCAGCTCCGAGCCGGTCGAAATAGT
HS-T(10)TCACATAGGAATGAAAAGCCATATTTTTTTAAAAAATTCCTATGTCCGAGCCGGTCGAAATAGT。
3. the carbon dot-nanogold spherical nucleic acid according to claim 1, wherein the Substrate comprises Substrate-133b or Substrate-135b, the sequences of which are:
HS-T(3)ACTAT rA GGCTGGTT-NH2，HS-T(3)ACTAT rA GCATAGGA-NH2。
4. the carbon dot-nanogold spherical nucleic acid according to claim 1, wherein the carbon dot comprises a green carbon dot or a blue carbon dot.
5. The method for preparing the carbon dot-nanogold spherical nucleic acid according to claim 1, comprising the steps of:
(1) preparing green carbon dots or blue carbon dots;
(2) preparing nano gold;
(3) preparing carbon dot-nanogold spherical nucleic acid: coupling nucleic acid on the surface of the nanogold, mixing a DNAzyme chain and a substrate, adding TCEP (trichloromethane) for incubation, then adding a nanogold solution for standing overnight, adding SDS (sodium dodecyl sulfate) for incubation, then adding a sodium chloride solution for multiple times for standing overnight, and performing centrifugal washing to obtain an AuNPs-DNA solution; and (3) taking the carbon dots, adding EDC and NHS to activate surface carboxyl, adding the carbon dots into AuNPs-DNA solution after incubation, and centrifuging, washing and resuspending to obtain CDs-SNA solution, namely the carbon dot-nanogold spherical nucleic acid.
6. The method of claim 5, wherein the nucleic acid is a DNAzyme strand and a substrate, and the carbon site is coupled to the amino terminus of the substrate.
7. The method of claim 5, wherein the molar ratio of the DNAzyme strand to the substrate is 1-4: 6-9.
8. The use of the carbon dot-nanogold spherical nucleic acid of claim 1 in exosome miRNA detection.
9. Use according to claim 1, characterized in that it comprises the following steps: under the condition of the existence of metal ions, the target miRNA identifies and triggers DNAzyme in the carbon dot-nanogold spherical nucleic acid to walk to generate fluorescence; and (3) measuring a fluorescence signal of the carbon point after the walking is finished by adopting a fluorescence spectrophotometer, and realizing the detection and analysis of miRNA.
10. The application of the carbon dot-nanogold spherical nucleic acid as claimed in claim 1 in preparation of exosome miRNA detection reagent or kit.
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