CN110699452A

CN110699452A - Silver nanocluster pair and G-triplex based ratio type fluorescent probe for detecting microRNA-21

Info

Publication number: CN110699452A
Application number: CN201910941365.7A
Authority: CN
Inventors: 蔡昌群; 向灵; 王双; 陈小明
Original assignee: Xiangtan University
Current assignee: Xiangtan University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2020-01-17
Anticipated expiration: 2039-09-30
Also published as: CN110699452B

Abstract

The invention provides a ratiometric fluorescent probe for detecting microRNA-21 based on silver nanocluster pairs and a G-triplex, belonging to the field of analysis and detection. The probe adopts silver nanocluster pairs and a G-triplex/THT complex as a fluorescence output signal. When no target exists, one probe sequence (G-triplex) is locked by the molecular beacon (MB1) and shows a weak fluorescence state, and the other probe sequence (MB2) can synthesize a silver nanocluster pair and show a strong fluorescence state. After adding microRNA-21, a target is used as a trigger chain to trigger a Hybrid Chain Reaction (HCR) of MB1 and MB2, two hairpins are continuously opened mutually to release a large number of G-triplex sequences and separate silver nanocluster pairs, the fluorescence of the silver nanocluster pairs is weakened, and after adding thioflavin (THT), the G-triplex and the THT are combined to emit strong fluorescence. The invention not only has high sensitivity and high selectivity, but also can effectively detect the target object in complex samples such as cell lysate and the like.

Description

Silver nanocluster pair and G-triplex based ratio type fluorescent probe for detecting microRNA-21

Technical Field

The invention belongs to the field of analysis and detection, and particularly relates to a ratiometric fluorescent probe for detecting microRNA-21 based on silver nanocluster pairs and a G-triplex.

Background

MicroRNA is an important marker in early diagnosis of tumors and can regulate the cell division and differentiation process. Therefore, it is very valuable to propose an accurate and sensitive detection strategy for the monitoring of microRNA. In recent years, many fluorescence detection strategies rely on the output of a single signal channel to detect micrornas. However, these methods may suffer from false positive signals and poor reproducibility due to the influence of light source stability, probe concentration and instrument factors. The ratiometric fluorescence strategy can be internally calibrated by dual fluorescence emission to avoid the effects of these interference factors. Therefore, it is needed to design a ratiometric fluorescence strategy that can accurately monitor micrornas and avoid potential interference.

At present, various ratiometric fluorescent probes have been tried to be designed for sensitive detection of low-level microRNA, however, these methods have certain defects. For example, the use of enzymes, which is a common amplification method at present, is highly efficient, but the expensive price and harsh environmental requirements of enzymes limit its use. In recent years, catalytic hairpin self-assembly (CHA) and Hybrid Chain Reaction (HCR) have attracted attention, and these two amplification methods have the advantages of low cost, good stability, mild operation conditions, enzyme immunity, etc., and have been successfully applied to high-sensitivity and high-selectivity detection of biomolecules. In addition, at present, the signal output of most fluorescent probes is still an expensive fluorescent chemical group modified on a DNA chain, and the modification process is complex and time-consuming, so that the development of a high-sensitivity label-free fluorescent probe still has strong scientific significance.

In conclusion, the high-sensitivity detection of the microRNA can provide important reference for early diagnosis of the tumor, so that the design of the high-sensitivity low-cost detection method has important scientific significance. The enzyme-free labeling-free ratio type fluorescent probe with low cost is applied to the detection of microRNA, so that not only can background signals be reduced, high-sensitivity detection be realized, but also the high cost brought by the use of enzyme and the modification of fluorescent chemical groups can be reduced.

Disclosure of Invention

The invention aims to design a ratiometric fluorescent probe for detecting microRNA-21 based on silver nanocluster pairs and a G-triplex, the probe is low in preparation cost and short in time, the problems of false positive signals and the like in the detection process of the traditional single-signal probe are expected to be solved, and the high-sensitivity detection of the microRNA-21 can be simply and quickly realized.

The invention can be realized by a silver nanocluster, a G-triplex and a hybrid chain reaction between two hairpins, and the detection principle is shown in figure 1:

(1) one (G-triplex) of the two probe sequences is locked by the stem of MB1 and shows a weak fluorescence state, and the other (silver nanocluster pair DNA synthesis template) is positioned at the 3 'end and the 5' end of MB2 and can synthesize a silver nanocluster pair which shows a strong fluorescence state.

(2) When the target microRNA-21 was added, the target opened MB1, releasing the probe sequence (G-triplex) and the partial complement of MB2, which in turn opened MB 2.

(3) After addition of the dye thioflavin t (THT), a G-triplex/THT complex is formed, with increased fluorescence at 490nm, the opening of MB2 leading to the separation of silver nanocluster pairs and a decrease in fluorescence at 594 nm.

The ratiometric fluorescent probe realizes high-sensitivity and high-selectivity detection of the microRNA-21.

Furthermore, the feasibility of the probe on microRNA-21 ratio type analysis is verified by detecting through fluorescence spectroscopy.

Furthermore, the probe has stronger fluorescence feedback signals to microRNA-21 and no obvious fluorescence signals to other homologous microRNAs through representation by fluorescence spectrometry, and shows good selectivity of the probe.

Furthermore, the detection limit of the fluorescent probe to microRNA-21 is 67pM and the detection linear range is 0.1-300nM by fluorescence spectrometry.

Drawings

FIG. 1 is a schematic diagram of a ratiometric probe for detecting microRNA-21 according to the present invention;

FIG. 2 shows the DNA sequence used for experimental analysis;

FIG. 3 is a circular dichroism spectrum representation chart for feasibility verification;

FIG. 4 is a UV-VIS absorption spectrum (A) of AgNCs pairs; excitation and emission spectra (B); transmission electron micrograph (C); a stability effect graph (D);

FIG. 5 is a fluorescence spectrum chart of feasibility verification of detecting a target object by a ratio type fluorescent probe;

FIG. 6 is a diagram of the UV-VIS absorption spectra of AgNCs under different conditions;

FIG. 7 is a diagram showing the effect of polyacrylamide gel electrophoresis on the mechanism verification;

FIG. 8 is a graph of the concentration optimization effect of THT (A) and MB2 (B);

FIG. 9 shows K⁺(A) And Mg²⁺(B) The concentration optimization effect graph of (1);

FIG. 10 shows MB2: Ag⁺:NaBH₄Optimization effect graph of ratio (A) and pH (B);

FIG. 11 is a graph of the optimization of temperature (A) and reaction time (B);

FIG. 12 is a fluorescence spectrum of THT (A) and AgNCs (B) after adding different concentrations of microRNA-21; fluorescence intensity ratio (I)₄₉₀/I₅₉₄)/(I₄₉₀/I₅₉₄)₀And a linear graph (C) of microRNA-21;

FIG. 13 is a diagram showing the effect of specificity verification of a ratiometric fluorescent probe;

FIG. 14 is a diagram showing the detection effect of an actual sample;

detailed description of the invention

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

Preparation of ratiometric fluorescent probes

The DNA probe in this example had the following base sequence:

MB2:CCC TTA ATC CCC CAG ACT GAT GTT TGG GAT TAA CAT CAG TCT GCC CTAACT CCC C

the bold part is the G-triplex sequence and the italic part is the DNA template sequence for the synthetic AgNCs pairs.

MB is incubated and annealed at 195 ℃ to obtain a hairpin structure locked with a G-triplex sequence.

MB is incubated at 295 ℃ and annealed. Then, AgNO is added₃Adding the solution into the above solution, shaking vigorously for 1min, incubating at 4 deg.C in the dark for 30min, and adding newly prepared NaBH₄Adding the solution into the above solution, and shaking for 1min to obtain DNA: Ag⁺:NaBH₄In a ratio of 1:24:24, and finally, placing the sample in a dark room at 4 ℃ for 4h to obtain the molecular beacon with the silver nanocluster pairs at the 5 'end and the 3' end.

Example 2

Feasibility verification for ratio-type detection of microRNA-21 by fluorescent probe

(1) Qualitative analysis of chain structure

The conditions for the Circular Dichroism (CD) detection are as follows:

the instrument comprises the following steps: chirascan (TM) CD chiroptical spectrometer

The instrument parameters are as follows: path length: 0.1 cm; wavelength range: 200-500 nm; scanning speed: 200 nm/min; response time: 0.5 s; bandwidth: 1.0 nm.

Circular dichroism spectroscopy was used to confirm the formation of the G-triplex conformation. As shown in FIG. 3, MB1 has a positive peak at approximately 265nm and a negative peak at 240nm, which are characteristic peaks of ssDNA and dsDNA. In addition, the CD spectrum of the solution containing THT added to MB1 showed no significant change, indicating that the G-triplex sequence was effectively locked in MB1 and did not form a G-triplex structure. However, addition of microRNA-21 to the mixture solution of MB1 and THT resulted in a significant positive peak in the CD spectrum at about 265nm and a strong THT binding band at about 425 nm. When MB2 was added, a small amount of MB1 and MB2 continuously opened each other to form a self-assembled complex, resulting in an increased CD peak with a weaker THT binding band at about 425 nm. When the target was added to the mixed solution of MB1, MB2 and THT, the CD peak signals at 265nm and 425nm increased significantly. The result proves that the target miRNA can enable the two probes to generate self-assembly reaction, and the target miRNA can trigger signal amplification.

(2) Qualitative analysis of silver nanocluster pairs

As shown in FIG. 4A, 430nm is the absorption of Ag nanoparticles, and the characteristic absorption peak of successfully synthesized AgNCs pairs is at 550 nm. Due to Stokes' shift, the AgNCs pair excited at 550nm had a maximum emission peak at 594nm (FIG. 4B). The morphology of the AgNCs was successfully characterized by TEM (FIG. 4C), showing that the average diameter of AgNCs is about 2 nm. Furthermore, as can be seen from fig. 4D, the pair of AgNCs was able to maintain a stable fluorescence signal in solution for 4 h.

(3) Fluorescence detection of microRNA-21

And performing fluorescence detection by using an RF-5301 fluorescence spectrophotometer to prove the feasibility of the enzyme-free labeling-free ratio type fluorescent probe for detecting the microRNA-21. The excitation wavelengths of the G-triplex and silver nanocluster pairs were 420nm and 550nm, respectively. FIG. 5 shows the fluorescence emission spectra of G-triplex/THT and AgNCs in different cases. The THT fluorescence signal was lowest for the solution containing only MB1 (FIG. 5A), since the G-triplex structure was locked in the stem of MB1 and was unable to form a G-triplex/THT complex to emit a fluorescence signal. Meanwhile, the probe had a strong fluorescence signal at 594nm due to the formation of AgNCs pairs in the MB2 structure (FIG. 5B). Due to non-specific hybridization of MB1 and MB2, the samples containing MB1 and MB2 showed a slight increase in fluorescence signal at 490nm (fig. 5A) and a slight decrease in fluorescence signal at 595nm (fig. 5B). microRNA-21 was further added to MB1, microRNA-21 hybridized to MB1, opening the hairpin structure of MB1, and fluorescence increased at 490nm (FIG. 5A). When the target microRNA-21 was incubated with MB1 and MB2, a significant increase in signal at 490nm (fig. 5A) and a significant decrease in fluorescence signal at 595nm (fig. 5B) was observed. These changes in fluorescence were attributed to the addition of primer microRNA-21 that caused HCR self-assembly of MB1 and MB2, resulting in the generation of large numbers of G-triplexes and the isolation of AgNCs pairs. Therefore, the results effectively show the feasibility of the ratio type fluorescent probe for sensitively detecting the microRNA-21.

(4) Ultraviolet-visible (UV-Vis) absorption spectrum determination of silver nanocluster pair property and structure change

The UV-Vis absorption spectrum was used to verify the changes in optical properties or structure of the MB2 probe/AgNCs resulting from the addition of the target. As shown in FIG. 6, in the absence of the target microRNA-21, the MB2 probe has two absorption peaks. The plasma absorption peak of Ag nanoparticles is at 430nm, and the characteristic absorption peak of AgNCs pairs is at 550 nm. The intensities of these two characteristic peaks decreased slightly due to non-specific hybridization between MB1 and MB 2. The microRNA-21 is added into MB1 and MB2 to initiate HCR reaction, so that one sequence of the synthetic silver nanocluster pair is partially locked, the sequences of the synthetic AgNCs pair are separated, the AgNCs pair is reduced, and the absorption peak intensity at 550nm is obviously reduced. These results provide evidence that the target initiated the HCR reaction, which in turn resulted in a change in the optical properties or structure of the MB2 probe/AgNCs.

(5) Gel electrophoresis

As shown in fig. 7, the product of HCR was confirmed by polyacrylamide gel electrophoresis experiments.

Lanes

1, 2, 3 show bands for MB1, MB1+ microRNA-21 and MB2, respectively. A new band distinct from MB1 appeared in lane 2, indicating that MB1 was opened, forming the MB1/microRNA-21 duplex. No HCR product was observed in the bands of the MB1, MB2 mixed solution (lane 4), however, when target was added, a distinct HCR band appeared in

lanes

5 and 6, while the intensity of the probe band weakened with increasing target concentration as more MB1 and MB2 were consumed to form a long HCR product.

Example 3

Optimization of the Experimental conditions

To achieve the best results of an analytical method, a series of reaction conditions need to be optimized to obtain an optimal experimental condition. Considering the concentrations of THT, MB2, potassium ions, magnesium ions and DNA Ag⁺:NaBH₄The influence of factors such as proportion, pH, temperature, time and the like on the detection effect is researched respectively aiming at the influencing factors, and the microRNA-21 is detected by only changing a research object without changing other conditions in the research process. When different factors are researched, the method is carried out on the basis of the obtained optimal conditions. The optimization results are shown in fig. 8-11.

Finally, the optimal detection conditions are obtained: c. C_THT＝1μM，c_MB2＝500nM，c_K+＝10mM，c_Mg2+＝5mM，DNA:Ag⁺:NaBH₄＝1:24:24，pH＝7.2，T＝37℃，t＝60min。

Example 5

Sensitivity analysis of ratiometric probes

To evaluate the ratioThe sensitivity of the fluorescent probe detects the intensity of fluorescent signals when different concentrations of target were added under optimal conditions, and the results are shown in FIG. 12. The target microRNA-21 triggered HCR reaction resulted in an increase in fluorescence at 490nm (FIG. 12A) and a decrease in fluorescence at 594nm (FIG. 12B). As shown in FIG. 12C, (I)₄₉₀/I₅₉₄)/(I₄₉₀/I₅₉₄)₀The ratio has a good linear relation with the concentration of the microRNA-21. Between 0.1-300nM, the linear equation can be expressed as Y-0.02261X +1.052 (R)²0.9949). By the 3 sigma calculation method, a detection limit of 67pM can be obtained. The ratio type probe has a wide linear range and has excellent sensitivity for quantitatively detecting microRNA-21.

Example 4

Specificity analysis of ratiometric probes

Under the optimal condition, the specificity of the ratiometric probe is verified by selecting microRNA-21 and other homologous microRNAs. The result is shown in FIG. 13, only in the presence of microRNA-21, the probe shows strong fluorescent signal response, and the response signal of the microRNA mixture containing the target is similar to that of the single microRNA-21, so that the prepared ratiometric fluorescent probe is proved to have good specificity for detecting the microRNA-21.

Example 5

Actual sample detection

In order to prove that the ratiometric fluorescent probe can realize the sensitive detection of microRNA-21 in a complex sample, normal liver cells (L-02), liver cancer cells (SMMC-7721) and human breast cancer cells (MCF-7) are selected as target cells, and the probe is applied to detect the microRNA-21 in a cell lysate. The results are shown in FIG. 14, where lysates from three different cells all produced progressively higher rate fluorescence responses as the number of cells increased. In addition, compared with LO2 and SMMC-7721, the lysate from MCF-7 generates stronger signal response, which indicates that the content of microRNA-21 in MCF-7 cells is higher than that of other two cells, and the result is consistent with previous reports, thereby proving that the probe has certain application value in the aspects of monitoring the content and change of microRNA-21 in actual samples.

Claims

1. A ratio-type fluorescent probe for detecting microRNA-21 based on silver nanocluster pairs and G-triplexes is characterized in that:

(1) one probe sequence (G-triplex) is locked by a molecular beacon (MB1) and shows a weak fluorescence state, and the other probe sequence (MB2) can synthesize a silver nanocluster pair and show a strong fluorescence state.

(2) The microRNA-21 was added and the target served as the trigger strand to trigger the Hybrid Chain Reaction (HCR) of MB1 and MB2, and the two hairpins were opened continuously to each other, resulting in the release of a large number of G-triplex sequences and the separation of silver nanocluster pairs.

(3) The separation of silver nanocluster pair leads to the decrease of fluorescence at 594nm, and after thioflavin (THT) is added, G-triplex and THT are combined to form a G-triplex/THT compound, and the fluorescence at 490nm is increased.

2. The ratiometric fluorescent probe of claim 1, wherein a signal transduction probe of the probe is a guanine-rich DNA strand that is released after binding of the target microRNA-21 to the molecular beacon (MB1) to form a G-triplex structure, and the fluorescent dye thioflavin t (THT) is added to form a G-triplex/THT complex that fluoresces strongly at 490nm as a fluorescent signal to detect microRNA-21.

3. The ratiometric fluorescent probe of claim 1, wherein the other signal transduction probe of the probe is a pair of silver nanoclusters formed at the 5 'and 3' ends of the molecular beacon (MB2), the addition of the target microRNA-21 triggers the hybridization chain reaction between the molecular beacon MB1 and MB2, and the silver nanoclusters are separated, and the fluorescence intensity at 594nm is reduced to detect the microRNA-21 as the other fluorescent signal.

4. A ratiometric fluorescent probe according to claim 1, wherein the two fluorescent output signals at 490nm and 594nm are internally calibrated to avoid false positive signals and poor reproducibility due to light source stability, probe concentration and instrumental factors.

5. The ratiometric fluorescent probe of claim 1, which is used for highly sensitive detection of microRNA-21, with a detection limit of 67 pM.

6. The ratiometric fluorescent probe of claim 1, which has strong fluorescence feedback on the target microRNA-21 and no obvious signal feedback on other homologous microRNAs, and shows high specificity to microRNA-21, as verified by fluorescence spectroscopy.

7. The ratiometric fluorescent probe of claim 1, which can be used to detect microRNA-21 in real biological samples (lysates of normal liver cells, liver cancer cells and human breast cancer cells) and to distinguish between normal and cancer cells.