CN110243792A - A fluorescent chemical sensor based on quantum dot and tetrahedral DNA structure and its detection method and application - Google Patents

Abstract

The present invention provides a kind of fluorescence chemical sensor based on quantum dot and tetrahedron DNA structure and its detection method and application, the fluorescence chemical sensor includes a 3 D stereo DNA structure, tetrahedron DNA is constructed by four oligonucleotides, four oligonucleotides are marked with biotin and the fluorescent dye flower mountain valley with clumps of trees and bamboo 3 (Cy3), texas Red and the flower mountain valley with clumps of trees and bamboo 5 respectively.It is subsequently assembled on the quantum dot of Streptavidin modification, obtains a QD-Cy3-Texas Red-Cy5 tetrahedron DNA up to fluorescence chemical sensor.The fluorescence chemical sensor has space length between specific dyestuff, and the energy transfer between median receptor and terminal receptor has high controllability, the fluorescence chemical sensor can generate the multistep FRET between QD and three color dyestuffs, the detection of a variety of enzymes can be realized as multiple interactive paths FRET of donor using QD and screen inhibitor, the value with good practical application.

Description

A fluorescent chemical sensor based on quantum dots and tetrahedral DNA structure and its detection Measurement methods and applications

technical field

The invention belongs to the technical field of biological detection and molecular biology, in particular to a fluorescent chemical sensor based on a quantum dot and tetrahedral DNA structure and a detection method and application thereof.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily to be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

Complex 3D DNA origami nanostructures have been engineered for broad applications in biosensing, optics, drug delivery, and gene regulation. However, the inventors found that most molecular detection systems based on three-dimensional DNA nanostructures can only detect one type of molecule, and often involve complex logic operations.

Fluorescent organic dyes are commonly used in fluorescence resonance energy transfer (FRET) systems. However, fluorescent organic dyes are prone to photobleaching, have short fluorescence lifetimes, severe spectral crosstalk, and high background fluorescence in biological samples. In order to overcome some limitations of fluorescent organic dyes, a large number of luminescent nanomaterials have been widely used, such as: quantum dots (QDs), barcode microparticles, polymer nanoparticles and organic dye-coated silica nanoparticles and other materials. The inventors have found that semiconductor quantum dots have the characteristics of long fluorescence lifetime, wide absorption spectrum from ultraviolet to near infrared, narrow and adjustable emission spectrum, chemical corrosion resistance, and good luminescence. And under the excitation of a single light source, a single quantum dot can couple one or more fluorescent organic dyes as FRET acceptors. Therefore, QDs can serve as ideal donors to realize multiple FRET configurations.

Contents of the invention

Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a fluorescent chemical sensor based on quantum dots and tetrahedral DNA structure and its detection method and application. The fluorescent chemical sensor includes a novel three-dimensional DNA structure, and the tetrahedral DNA consists of Four oligonucleotides were constructed, and the four oligonucleotides were labeled with biotin and fluorescent dyes Cy3 (Cy3), Texas Red and Cy5, respectively. Then assembled on the streptavidin-modified quantum dot (QD), obtain a QD-Cy3-Texas Red-Cy5 tetrahedral DNA i.e. fluorescent chemical sensor. The fluorescent chemosensor has a well-defined spatial distance between the dyes, and the energy transfer between the intermediate acceptor and the terminal acceptor is highly controllable, and the fluorescent chemosensor can generate QDs and trichromatic dyes (Cy3, Texas Red, and The multi-step FRET between Cy5) can use multiple interactive FRET pathways of QD as the donor to realize the detection of various enzymes and screen inhibitors, which has good practical application value.

The present invention is achieved through the following technical solutions:

The first aspect of the present invention provides a fluorescent chemical sensor based on quantum dots and a tetrahedral DNA structure, the fluorescent chemical sensor at least includes quantum dots and a tetrahedral DNA structure assembled on the surface of the quantum dots, the tetrahedral The DNA is constructed from four oligonucleotides labeled with biotin and a different fluorescent dye.

The fluorescent dyes include but are not limited to Cylin 3 (Cy3), Texas Red (Texas Red) and Cylin 5 (Cy5).

At least one cleavage site of the enzyme to be tested exists on the one oligonucleotide chain.

A second aspect of the present invention provides a method for simultaneously detecting multiple enzymes based on the above-mentioned fluorescent chemical sensor, the method comprising:

S1. Construct the above-mentioned tetrahedral DNA structure;

S2, the above-mentioned tetrahedral DNA structure is added in the sample solution to be tested, carries out incubation reaction, then adds quantum dot, forms 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure;

S3. Single-molecule detection of fluorescent signals, and quantitative analysis of the contents of various enzymes to be tested.

A third aspect of the present invention provides the application of the above-mentioned fluorescent chemical sensor and/or detection method in quantitative detection of enzyme and/or screening of enzyme inhibitor.

Beneficial effects of the present invention:

(1) The present invention is based on the fact that the fluorescent chemical sensor does not require complex instruments or cumbersome procedures during the entire reaction process of detecting the enzyme to be tested; at the same time, the operation is extremely simple, the cost is low, and the operation is simple and convenient, and does not involve logical operations at the same time. Effective cost savings.

(2) The fluorescent chemical sensor of the present invention uses tetrahedral DNA and 525QD in combination, and uses multiple FRET pathways, so that three or even multiple enzymes can be detected quantitatively at the same time with high sensitivity, and further, enzyme inhibitors can be screened at the same time, Therefore, it has a good prospect of practical application.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute improper limitations to the present invention.

Fig. 1 is the mechanism diagram of the detection of multiple different endonucleases by the fluorescent chemical sensor in Example 1 of the present invention;

Figure 2 (A) is the polyacrylamide gel electrophoresis image of biotin-Cy3-Texas Red-Cy5 tetrahedral DNA without SYBR Gold staining; Figure 2 (B) is the biotin-Cy3-Texas Red-Cy5 stained with SYBR Gold Tetrahedral DNA polyacrylamide gel electrophoresis; Figure 2 (C) is the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA agarose gel electrophoresis; where band 1 is biotin-Cy3-Texas Red-Cy5 tetrahedral DNA Electrophoretic imaging image, band 2 is the electrophoretic imaging image of 525QD wrapped by streptavidin, band 3 is the electrophoretic imaging image of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, band 4, band 5, and band 6 are the addition of HaeIII respectively, The electrophoretic image of EcoRV and PvuII; band 7 is the electrophoretic image of the presence of all three enzymes. Figure 3(A) is the fluorescence spectrum of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA; the insert part is the fluorescence spectrum in the range of acceptor emission spectrum; Figure 3(B) is the FRET efficiency between tetrahedral DNA composed of different acceptors Compare chart.

Figure 4(A) is the single-molecule imaging of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA; Figure 4(B) is the single-molecule imaging of tetrahedral DNA in the presence of HaeIII; Figure 4(C) is the single-molecule imaging of tetrahedral DNA in the presence of HaeIII, EcoRV and Single-molecule imaging of tetrahedral DNA in the presence of PvuII; Figure 4(D) is the fluorescence spectrum of the HaeIII enzyme cleavage reaction; Figure 4(E) is the change of the fluorescence intensity reduction value of Cy5 corresponding to different concentrations of HaeIII, and the inset is the fluorescence intensity reduction of Cy5 The linear relationship between the value and the logarithmic form of the concentration of HaeIII; Fig. 4 (F) is the fluorescence spectrum of EcoRV enzyme cleavage reaction; Fig. 4 (G) is the change graph of the fluorescence intensity reduction value corresponding to TexasRed for different concentrations of EcoRV, The inset is the linear relationship between the fluorescence intensity reduction value of Texas Red and the logarithmic form of EcoRV concentration; Figure 4(H) is the fluorescence spectrum of PvuII enzyme cleavage reaction; Figure 4(I) is the corresponding FRET efficiency of different concentrations of PvuII Changes in reduction values, inset shows the linear relationship between the reduction in FRET efficiency and the logarithmic form of the PvuII concentration.

Figure 5(A) is the FRET efficiency diagram corresponding to different specificity experimental groups; among them, (a) normal HaeIII+EcoRV+PvuII reaction group; (b) heat inactivated HaeIII+EcoRV+PvuII reaction group; Figure 5(B) is The fluorescence spectrum of HaeIII methyltransferase reaction, the concentration of HaeIII is fixed at 0.5 units per microliter; Figure 5 (C) is the change of the fluorescence intensity increase value of Cy5 corresponding to different concentrations of HaeIII methyltransferase, the illustration is Cy5 The linear relationship diagram between the fluorescence intensity increase value and the logarithmic form of the concentration of HaeIII methyltransferase; Figure 5(D) is the activity relationship diagram of HaeIII, EcoRV and PvuII under different EDTA concentrations.

Detailed ways

It should be pointed out that the following detailed description is exemplary and intended to provide further explanation to the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used here is only for describing specific implementations, and is not intended to limit the exemplary implementations according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprise" and/or "comprise" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

As mentioned earlier, complex 3D DNA origami nanostructures have been engineered for broad applications in biosensing, optics, drug delivery, and gene regulation. However, the inventors have found that most molecular detection systems based on three-dimensional DNA nanostructures can only detect one type of molecule, and often involve complex logic operations.

In view of this, the present invention has developed a novel three-dimensional DNA structure, tetrahedral DNA is constructed by four oligonucleotides, and the four oligonucleotides are respectively marked with biotin and fluorescent dye cyanine 3 ( Cy3), Texas Red (Texas Red) and Huaqing 5 (Cy5). Then assembled on streptavidin-modified quantum dots (QD), a QD-Cy3-Texas Red-Cy5 tetrahedral DNA is obtained, which is a fluorescent chemical sensor. The fluorescent chemical sensor has a well-defined spatial distance between the dyes, and the energy transfer between the intermediate acceptor and the terminal acceptor is highly controllable, and the tetrahedron can generate QDs and trichromatic dyes (Cy3, Texas Red, and Cy5 ), the multi-step FRET between QDs can be used to realize the detection of various enzymes and the screening of inhibitors by using multiple interactive FRET pathways with QDs as donors.

In a specific embodiment of the present invention, a fluorescent chemical sensor based on quantum dots and a tetrahedral DNA structure is provided, the fluorescent chemical sensor at least includes quantum dots and a tetrahedral DNA structure assembled on the surface of the quantum dots, the tetrahedral DNA structure Somatic DNA is constructed from four oligonucleotides labeled with biotin and different fluorescent dyes.

In yet another specific embodiment of the present invention, the fluorescent dyes include but are not limited to Cylin 3 (Cy3), Texas Red (Texas Red) and Cylin 5 (Cy5).

In yet another specific embodiment of the present invention, there is at least one cleavage site of the enzyme to be tested on the said oligonucleotide chain;

In yet another specific embodiment of the present invention, the quantum dots are coated with streptavidin, so as to be connected with biotin-labeled oligonucleotides so that the tetrahedral DNA structure is assembled on the quantum dots;

In yet another specific embodiment of the present invention, the quantum dot is 525QD;

In yet another specific embodiment of the present invention, when the endonuclease to be tested is HaeIII, EcoRV and PvuII;

In yet another specific embodiment of the present invention, the base sequences of the four oligonucleotides are as follows:

(1) DNA single strand labeled with biotin: 5'-CTA TGT GGC CAA TCA AAC GAG AGC AAG TGT ATGAGT AAG ATC GCG ACC AT–biotin-3' (SEQ ID NO.1);

(2) Cy3-labeled DNA single strand: 5'-CAT GGG ATA TCT ACG GAC ATA CAC TTG CTC TCGAAG ACT TCA GCT GGT TA-Cy3-3' (SEQ ID NO.2);

(3) DNA single strand labeled by Texas Red: 5'-Texas Red-CCG TAG ATA TCC CAT GAG TTGAGC CTG GAC AGG AAT GGT CGC GAT CTT AC-3' (SEQ ID NO.3);

(4) Cy5-labeled DNA single strand: 5'-Cy5-TTG ATT GGC CAC ATA GAC CTG TCC AGG CTCAAC ATA ACC AGC TGA AGT CT-3' (SEQ ID NO.4).

In yet another specific embodiment of the present invention, a method for simultaneously detecting multiple enzymes based on the above-mentioned fluorescent chemical sensor is provided, the method comprising:

S1. Construct the above-mentioned tetrahedral DNA structure;

S2, the above-mentioned tetrahedral DNA structure is added in the sample solution to be tested, carries out incubation reaction, then adds quantum dot, forms 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure;

S3. Single-molecule detection of fluorescent signals, and quantitative analysis of the contents of various enzymes to be tested.

In yet another specific embodiment of the present invention, in the step S1,

The specific method of constructing the tetrahedral DNA structure is as follows: preparing a variety of single-stranded DNAs, including DNA single-strands labeled with biotin of different lengths; Cy3-labeled DNA single-stranded; Texas Red-labeled DNA single-stranded; Cy5-labeled DNA single-stranded; Same DNA single-strand sequence as above, but the DNA single-strand (sp strand) is not labeled with dye. Add the desired DNA to 1×CutSmart buffer, and add the mixture to 92-96°C (preferably 95°C) for 5-15 minutes (preferably 10 minutes); then place it on ice for 0.5-1.5 hours (preferably 1 hour) ;

In yet another specific embodiment of the present invention, the tetrahedral DNA sequence self-assembled into 16 base pair side lengths is as follows:

(1) DNA single strand labeled with biotin: 5'-CTA TGT GGC CAA TCA AAC GAG AGC AAG TGT ATGAGT AAG ATC GCG ACC AT–biotin-3' (SEQ ID NO.1);

(2) Cy3-labeled DNA single strand: 5'-CAT GGG ATA TCT ACG GAC ATA CAC TTG CTC TCGAAG ACT TCA GCT GGT TA-Cy3-3' (SEQ ID NO.2);

(3) DNA single strand labeled by Texas Red: 5'-Texas Red-CCG TAG ATA TCC CAT GAG TTGAGC CTG GAC AGG AAT GGT CGC GAT CTT AC-3' (SEQ ID NO.3);

(4) Cy5-labeled DNA single strand: 5'-Cy5-TTG ATT GGC CAC ATA GAC CTG TCC AGG CTCAAC ATA ACC AGC TGA AGT CT-3' (SEQ ID NO.4).

In yet another specific embodiment of the present invention, in the step S2,

The incubation reaction conditions are specifically: 30-40°C (preferably 37°C) for 0.5-1.5 hours (preferably 1 hour);

In yet another specific embodiment of the present invention, in the step S3, a single molecule imaging system based on total internal angle reflection fluorescence (TIRF) is used for detection. Specifically, the excitation light source is a 405 nm laser, and the excitation intensity is 30 milliwatts. The 525QD uses a 500-550 nm filter, the Cy3/Texas Red uses a 573-617 nm filter and a 565-605 nm filter, and the Cy5 uses a 672-712 nm filter.

In yet another specific embodiment of the present invention, the application of the above-mentioned fluorescent chemical sensor and/or detection method in quantitative detection of enzyme and/or screening of enzyme inhibitor is provided.

The present invention is further explained and illustrated by the following examples, but does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, the molecular biology methods not described in detail in the examples are all conventional methods in the art, and for specific operations, please refer to the molecular biology guide or product instructions.

Example 1

1. Detection principle and method steps of the present invention:

The experimental principle of this technology is shown in Figure 1: four DNA oligonucleotides labeled with biotin, Cy3, Texas Red and Cy5 self-assemble to form biotin-Cy3-Texas Red-Cy5 tetrahedral DNA. Subsequently, the self-assembled biotin-Cy3-Texas Red-Cy5 tetrahedral DNA was linked to the streptavidin-coated QD surface through specific biotin and streptavidin to form 525QD-Cy3-Texas Red-Cy5 Tetrahedral DNA. When the excitation wavelength of 392 nm excites the tetrahedral DNA, the fluorescence signals of four fluorescent substances, 525QD, Cy3, Texas Red, and Cy5, can be observed simultaneously. The system includes FRET of QD-Cy3, QD-Texas Red, QD-Cy5, Cy3-Texas Red, Cy3-Cy5, Texas Red-Cy5. Among them, the FRET of QD-Cy3 is beneficial to the fluorescence emission signal of Cy3; the FRET of QD-Texas Red and Cy3-Texas Red is beneficial to the fluorescence emission signal of Texas Red; the FRET of QD-Cy5, Cy3-Cy5 and Texas Red-Cy5 is beneficial Fluorescent emission signal based on Cy5.

The 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA designed by this technology has double-stranded DNA with 16 base pairs on each side, and an enzyme catalytic substrate on each side. There is a catalytic site for PvuII cleavage enzyme at 7 base pairs from Cy3, a catalytic site for EcoRV cleavage enzyme at 8 base pairs from Texas Red, and a catalytic site at 8 base pairs from Cy5. Catalytic site of HaeIII cleavage enzyme. When only one endonuclease (HaeIII as an example) is present, HaeIII initiates the cleavage reaction resulting in the separation of the Cy5-labeled DNA fragment from the tetrahedron, and subsequently, due to the FRET pathway of QD-Cy5, Cy3-Cy5 and Texas Red-Cy5 The disappearance of Cy5 signal decreased or disappeared. When there are two endonucleases (take HaeIII and EcoRV as examples), HaeIII triggers the enzyme cleavage reaction to separate the Cy5-labeled DNA fragment from the tetrahedron, and EcoRV triggers the enzyme cleavage reaction to cause the Texas Red-labeled DNA fragment to separate from the tetrahedron. . Therefore, due to the disappearance of the FRET pathway of QD-Cy5, Cy3-Cy5 and Texas Red-Cy5, the signal of Cy5 is reduced or disappeared; due to the disappearance of the FRET pathway of QD-Texas Red, and Cy3-Texas Red, the signal of Texas Red is reduced Or disappear. When the three endonucleases (HaeIII, EcoRV and PvuII) exist, the enzyme cleavage reaction initiated by HaeIII, EcoRV and PvuII makes Cy3-labeled DNA fragments, Texas Red-labeled DNA fragments and Cy5-labeled DNA fragments from the tetrahedron For DNA isolation, due to the disappearance of the FRET pathway between QD-Cy3, QD-Texas Red, QD-Cy5Cy3-Texas Red, Cy3-Cy5 and Texas Red-Cy5, the Cy3 signal, Texas Red signal and Cy5 signal could no longer be observed. Cy5 indicates HaeIII, Texas Red indicates EcoRV, and Cy3 indicates PvuII. This technology can simultaneously detect three enzymes and screen multiple enzyme inhibitors.

Construction of tetrahedral DNA: prepare a variety of single-stranded DNAs, including biotin-labeled DNA single-strands of different lengths; Cy3-labeled DNA single-stranded; Texas Red-labeled DNA single-stranded; Cy5-labeled DNA single-stranded; A single strand of DNA (sp strand) with the same strand sequence but not labeled with a dye. The desired DNA (1 micromole per liter) was added to 1×CutSmart buffer, and the mixture was heated to 95°C for 10 minutes. Then place on ice for 1 hour.

Self-assemble into tetrahedral DNA sequences with side lengths of 16 base pairs:

(1) DNA single strand labeled with biotin: 5'-CTA TGT GGC CAA TCA AAC GAG AGC AAG TGT ATGAGT AAG ATC GCG ACC AT–biotin-3' (SEQ ID NO.1);

(2) Cy3-labeled DNA single strand: 5'-CAT GGG ATA TCT ACG GAC ATA CAC TTG CTC TCGAAG ACT TCA GCT GGT TA-Cy3-3' (SEQ ID NO.2);

(3) DNA single strand labeled by Texas Red: 5'-Texas Red-CCG TAG ATA TCC CAT GAG TTGAGC CTG GAC AGG AAT GGT CGC GAT CTT AC-3' (SEQ ID NO.3);

(4) Cy5-labeled DNA single strand: 5'-Cy5-TTG ATT GGC CAC ATA GAC CTG TCC AGG CTCAAC ATA ACC AGC TGAAGT CT-3' (SEQ ID NO.4).

Enzyme cleavage reaction: 0.12 micromole per liter of tetrahedral DNA was added to 1×CutSmart buffer, followed by different concentrations of cutting enzymes HaeIII, EcoRV and HaeIII. The mixture system was reacted at 37 degrees Celsius for 1 hour. Subsequently, 5 nmol per liter of streptavidin-coated 525QD was added to the reaction system. Thus forming 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure.

Methylase reaction: 0.12 μmol/L tetrahedral DNA was added to 1×CutSmart buffer, followed by 60 μmol/L S-adenosylmethionine (SAM) and different concentrations of HaeIII methyltransferase , and finally add 0.5 units per microliter of HaeIII. The mixture system was reacted at 37 degrees Celsius for 1 hour. Subsequently, spectroscopic measurements were performed after adding 5 nmol per liter of streptavidin-coated 525QD to the reaction system.

Biological fluid enzyme cleavage reaction: 0.12 micromole per liter of tetrahedral DNA was added to 1×CutSmart buffer, followed by different concentrations of cleavage enzymes and 10% volume fraction of fetal bovine serum, and the mixture was reacted at 37 degrees Celsius for 1 hour.

Inhibitor experiments: Prepare 525QD-Cy3-Texas Red-Cy5, 525QD-Cy3-Texas Red-sp, and 525QD-Cy3-sp-sp for HaeIII, EcoRV, and PvuII inhibitor experiments, respectively. Add 0.12 micromoles per liter of tetrahedral DNA to 1×CutSmart buffer, add different concentrations of inhibitors and cleavage enzymes corresponding to different types of tetrahedral DNA, and the concentration of cleavage enzyme is 0.5 units per microliter. The mixture system was reacted at 37 degrees Celsius for 1 hour.

Gel electrophoresis: 8% polyacrylamide gel electrophoresis was used to image the biotin-Cy3-Texas Red-Cy5tetrahedral DNA not stained with SYBR Gold DNA indicator and stained with SYBR Gold DNA indicator, and the reaction system was TBE buffer ( 44.5 mmol per liter of Tris-boric acid, 1 mmol per liter of EDTA), and the electrophoresis time was 45 minutes. Use 1% agarose gel electrophoresis to image 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, the reaction system is TAE buffer (40 mmol per liter of Tris-acetic acid, 2 mmol per liter of EDTA), and the electrophoresis time is 30 minutes. Finally, gel imaging was performed by a gel imaging system. 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA uses multiple excitation light sources. When the excitation light source is blue light (460-490 nm excitation wavelength), 525QD is excited, and 525QD uses a 518-546 nm filter. When the excitation light source is green light (520-545 nm excitation wavelength), the Cy3 or Texas Red fluorophore is excited, and the Cy3 or TexasRed fluorophore uses a 577-613 nm filter. When the excitation light source is red light (625-650 nm excitation wavelength), the Cy5 fluorescent group is excited, and the 525QD uses a 675-725 nm filter.

Fluorescence measurement: Fluorescence signal is measured and collected by a fluorometer, and the excitation wavelength is 392nm. The emission peak intensities at 530 nm, 565 nm, 613 nm and 670 nm were collected respectively, corresponding to the fluorescence intensities of 525QD, Cy3, Texas Red and Cy5.

Single-molecule detection: Single-molecule imaging was performed using a total internal reflection fluorescence microscope equipped with an oil objective and an inverted microscope. The excitation light source is a 405 nm laser, and the excitation intensity is 30 mW. 525QD uses a 500-550 nm filter, Cy3/Texas Red uses a 573-617 nm filter and a 565-605 nm filter, and Cy5 uses a 672-712 nm filter. Subsequently, data analysis was performed on a region of 70 × 70 pixels collected. Analysis of test results

1. Gel electrophoresis analysis

This example uses non-denaturing polyacrylamide gel (PAGE) electrophoresis to analyze self-assembled tetrahedral DNA. As shown in Figure 2, in Figure 2A and Figure 2B, single-stranded DNA (Figure 2A band 1, 2, 3; Figure 2B band 1) is the fastest in gel electrophoresis, and two-strand or three-strand self-assembled DNA (Zones 4-13 in FIG. 2A; Bands 2 and 3 in FIG. 2B) are slower than single-stranded DNA, and the self-assembly of four DNAs (Zone 14 in FIG. 2A; Band 4 in FIG. 2B) is the slowest. And each band has no miscellaneous bands, indicating that the biotin-Cy3-Texas Red-Cy5 tetrahedral DNA self-assembled successfully.

In this example, agarose gel electrophoresis was used to analyze 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA. In Figure 2C, the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA (Figure 2C band 3) and the biotin-Cy3-Texas Red-Cy5 tetrahedral DNA (Figure 2C band 1) have different positions and no bands, indicating that biotin- Cy3-Texas Red-Cy5 tetrahedral DNA was successfully assembled onto the 525QD surface.

HaeIII (Figure 2C, lane 4), EcoRV (Figure 2C, lane 5), PvuII (Figure 2C, lane 6), and HaeIII+EcoRV+PvuII (Figure 2C, lane 7) were subsequently added to the biotin-Cy3-Texas Red-Cy5 tetrahedral DNA. ), small fragments carrying Cy5, TexasRed, Cy3, and Cy5+Texas Red+Cy3, and 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can be observed to decrease. Three enzymes were shown to initiate cleavage of tetrahedral DNA.

2. Fluorescence spectral analysis

In this example, the self-assembled tetrahedral DNA was then analyzed using fluorescence spectroscopy, as shown in FIG. 3 . In FIG. 3A , the assembly of a single dye acceptor (Cy3) to the surface of a QD triggers a decrease in the fluorescence intensity of the QD. The assembly of dual-dye acceptors (Cy3 and Texas Red) on the QD surface induced a decrease in QD intensity, and the decrease was greater than that of single-dye acceptor (Cy3). The assembly of triple-dye acceptors to the QD surface triggered a reduction in QD intensity that was greater than that of single-dye acceptors (Cy3) and double-dye acceptors (Cy3 and TexasRed). Figure 3B shows that the FRET efficiency of QD-tetrahedral DNA varies with acceptor type and acceptor number, and reaches a maximum when there are three acceptor dyes on the surface of 525QD. This result also proves the formation of biotin-Cy3-Texas Red-Cy5 tetrahedral DNA and the successful assembly of the tetrahedron to the 525QD surface.

3. Single molecule detection analysis

As shown in Figure 4: when the 405 nanometer laser excites the 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA, the fluorescence imaging at 525 (525QD) and 595 (Cy3 or Texas Red) nanometers can be obtained at the same time, and the 585 ( Fluorescence imaging at Cy3 or Texas Red) and 692 (Cy5) nm. In the absence of the enzyme, distinct 525QD fluorescence signals (Fig. 5A, a) and Cy3/Texas Red fluorescence signals (Fig. 5A, b and d) and Cy5 signals (Fig. 5A, e) were observed. This result also indicated that biotin-Cy3-Texas Red-Cy5 tetrahedral DNA successfully self-assembled.

In the presence of HaeIII, the fluorescence signal of 525QD (Fig. 5B, a) and Cy3/Texas Red fluorescence signal (Fig. 5B, b and d) could be observed, but the Cy5 signal could not be observed (Fig. 5B, e). When HaeIII+EcoRV+PvuII existed at the same time, only the signal of 525QD (Fig. 5C, a) could be observed, but the fluorescence signal of Cy3 or Texas Red (Fig. 5C, b and d) or Cy5 (Fig. 5C, e ). This result also indicates that the enzyme initiates cleavage of tetrahedral DNA.

4. Minimum detection concentration of endonuclease

The signals generated by cleavage reactions initiated by HaeIII, EcoRV and PvuII can be expressed as a function of enzyme concentration.

The presence of HaeIII triggered a decrease in the Cy5 fluorescence signal intensity, accompanied by an increase in the QD fluorescence intensity signal (Fig. 4D). Meanwhile, the higher the concentration of HaeIII, the greater the reduction of Cy5 signal intensity (Fig. 4E). There was a good linear relationship between the reduction of Cy5 fluorescence intensity and the logarithm of the concentration of HaeIII from 0.025 to 0.5 units per microliter (Fig. 4E inset), and the correlation function was ΔI=107.96+51.85log ₁₀ C (R ² =0.990), Where C is the concentration of HaeIII, and ΔI is the reduction intensity of Cy5. A detection limit of 0.0112 units per microliter was obtained by calculating the mean plus three times the standard deviation.

Figure 4F shows the corresponding fluorescence spectra in the presence of a fixed concentration of HaeIII (0.5 units per microliter) and different concentrations of EcoRV. In the absence of EcoRV, a distinct Texas Red signal could be observed (Fig. 4F), indicating that FRET between the QDs and Texas Red occurred. In contrast, the presence of EcoRV triggered a decrease in Texas Red fluorescence signal intensity accompanied by an increase in QD fluorescence intensity signal (Fig. 4F), and at the same time, the higher the EcoRV concentration, the greater the decrease in Texas Red signal intensity (Fig. 4G). There was a good linear relationship between the reduction of TexasRed fluorescence intensity and the logarithm of the concentration of EcoRV from 0.025 to 0.5 units per microliter (Fig. 4G inset), and the correlation function was ΔI =78.13+42.43log ₁₀ C (R ² =0.998), Where C is the EcoRV concentration, ΔI is the reduced intensity of Texas Red, and the detection limit is 0.0233 units per microliter.

Figure 4H shows the corresponding fluorescence spectra in the presence of fixed concentrations of HaeIII (0.5 units per microliter) and fixed concentrations of EcoRV (0.5 units per microliter) and different concentrations of PvuII. In the absence of PvuII, a clear Cy3 signal could be observed (Fig. 4H), indicating FRET between QDs and Cy3. In contrast, the presence of PvuII induced a decrease in Cy3 fluorescence signal intensity accompanied by an increase in QD fluorescence intensity signal (Fig. 4H), and at the same time, FRET efficiency increased substantially with the increase in PvuII intensity (Fig. 4I). There was a good linear relationship between FRET efficiency and the logarithm of PvuII concentrations from 0.025 to 0.5 units per microliter (Fig. 4I inset), the correlation function was ΔE=7.69+3.92log ₁₀ C (R ² =0.997), where C was PvuII concentration, ΔE is the decrease in FRET intensity. The detection limit was 0.0233 units per microliter.

5. Tetrahedral specificity analysis

In order to further verify the specificity of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA to endonucleases, this example uses enzymes XhoI and KpnI as negative control groups. XhoI can recognize and cut 5'-CTCGAG-3' sequence, and KpnI can recognize and cut 5'-GGTACC-3' sequence. Neither enzyme can recognize and cleave 525QD-Cy3-Texas RedCy5 tetrahedral DNA. As expected, there was no significant decrease in FRET signal in the XhoI and KpnI controls (Fig. 5A). At the same time, the heating endonuclease HaeIII+EcoRV+PvuII experimental group also had a significant signal decrease (Figure 5A b), because heating can inactivate HaeIII and EcoRV, but PvuII has a certain resistance to high temperature. Contrary to the above phenomenon, when the active HaeIII+ EcoRV+PvuII was added, a significant decrease in FRET could be observed (Fig. 5A a). This result strongly indicates the specificity of 525QD-Cy3-Texas Red-Cy5 tetrahedral DNA to endonucleases.

6. Methyltransferase Activity Analysis

525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can also further detect HaeIII methyltransferase activity. HaeIII methyltransferase can catalyze the methylation of 5'-GGCC-3' sequence to generate 5'-GG ^m CC-3' sequence. However, HaeIII cannot recognize and cut the 5'-GG ^m CC-3' sequence. When there is no HaeIII methyltransferase, the 5'-GGCC-3' sequence cannot be methylated, so it is recognized and cleaved by HaeIII, so the Cy5 signal cannot be observed (Fig. 5B)

In the presence of HaeIII methyltransferase, 525QD fluorescence intensity decreased and Cy5 fluorescence intensity increased (Fig. 5B). The higher the concentration of HaeIII methyltransferase, the stronger the Cy5 fluorescence signal (Figure 5C), and there is a good linear relationship between the Cy5 fluorescence intensity and the logarithm of the concentration of HaeIII methyltransferase (Figure 5C inset), and the correlation function is ΔI= 70.47 +31.45 log ₁₀ C (R ² =0.993), where C is the concentration of HaeIII methyltransferase and ΔI is the intensity of Cy5. The limit of detection was 0.01 units per microliter. This result indicates that the tetrahedron can detect other kinds of enzyme activities as well.

7. Inhibitor experiments

525QD-Cy3-Texas Red-Cy5 tetrahedral DNA can also be further used to screen enzyme inhibitors. This example uses dehydrated ethylenediaminetetraacetic acid disodium salt (EDTA) as a model inhibitor. The inhibition of HaeIII by EDTA can be verified by measuring the intensity of Cy5 in the 525QD-Cy3-TexasRed-Cy5 tetrahedral DNA. The inhibition of EcoRV by EDTA can be verified by measuring the intensity of Texas Red in 525QD-Cy3-Texas Red-sp. The inhibition of PvuII by EDTA can be verified by measuring the intensity of Cy3 in 525QD-Cy3-sp-sp. The activity intensity of HaeIII, EcoRV and PvuII can be used as a function of EDTA concentration (FIG. 5D). In this example, the value of IC ₅₀ (half inhibitor) was used as an index to measure the inhibitory effect. The _IC50 of EDTA inhibitors for different enzymes exhibited the following trends: PvuII (26.02 mmol per L) < HaeIII (33.23 mmol per L) < EcoRV (49.65 mmol per L).

It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Shandong Normal University

<120> A fluorescent chemical sensor based on quantum dots and tetrahedral DNA structure and its detection method and application

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 50

<212>DNA

<213> Synthetic

<400> 1

ctatgtggcc aatcaaacga gagcaagtgt atgagtaaga tcgcgaccat 50

<210> 2

<211> 50

<212>DNA

<213> Synthetic

<400> 2

catgggatat ctacggacat acacttgctc tcgaagactt cagctggtta 50

<210> 3

<211> 50

<212>DNA

<213> Synthetic

<400> 3

ccgtagatat cccatgagtt gagcctggac aggaatggtc gcgatcttac 50

<210> 4

<211> 50

<212>DNA

<213> Synthetic

<400> 4

ttgattggcc acatagacct gtccaggctc aacataacca gctgaagtct 50

Claims (10)

1. a kind of fluorescence chemical sensor based on quantum dot and tetrahedron DNA structure, which is characterized in that the fluorescence chemical passes Sensor includes at least quantum dot and the tetrahedron DNA structure for being assembled in the quantum dot surface, and the tetrahedron DNA is by four widows Nucleotide construction, four oligonucleotides are marked with biotin and different fluorescent dyes respectively；

The fluorescent dye includes but is not limited to the flower mountain valley with clumps of trees and bamboo 3, texas Red and the flower mountain valley with clumps of trees and bamboo 5；

At least there is a kind of restriction enzyme site of enzyme to be measured on one oligonucleotide chain.

2. fluorescence chemical sensor as described in claim 1, which is characterized in that the quantum dot is coated with Streptavidin, from And it is connect with the oligonucleotides for being marked with biotin and then makes the assembling of tetrahedron DNA structure over the qds.

3. fluorescence chemical sensor as described in claim 1, which is characterized in that the quantum dot is 525QD.

4. fluorescence chemical sensor as described in claim 1, which is characterized in that when restriction endonuclease to be measured be HaeIII, EcoRV and When PvuII；

The base sequence of four oligonucleotides is as follows:

The DNA of biotin labeling is single-stranded: 5 '-CTA TGT GGC CAA TCA AAC GAG AGC AAG TGT ATG AGT AAG ATC GCG ACC AT–biotin-3'(SEQ ID NO.1)；

The DNA of Cy3 label is single-stranded: 5 '-CAT GGG ATA TCT ACG GAC ATA CAC TTG CTC TCG AAG ACT TCA GCT GGT TA-Cy3-3'(SEQ ID NO.2)；

The DNA of Texas Red label is single-stranded: 5 '-Texas Red-CCG TAG ATA TCC CAT GAG TTG AGC CTG GAC AGG AAT GGT CGC GAT CTT AC-3'(SEQ ID NO.3)；

The DNA of Cy5 label is single-stranded: 5 '-Cy5-TTG ATT GGC CAC ATA GAC CTG TCC AGG CTC AAC ATA ACC AGC TGA AGT CT-3’(SEQ ID NO.4)。

5. the method that any one of the claim 1-4 fluorescence chemical sensor detects a variety of enzymes simultaneously, which comprises

S1, building tetrahedron DNA structure；

S2, tetrahedron DNA structure is added in testing sample solution, carries out incubation reaction, quantum dot is then added, formed 525QD-Cy3-Texas Red-Cy5 tetrahedron DNA nanostructure, that is, fluorescence chemical sensor；

S3, Single Molecule Detection fluorescence signal, a variety of enzyme contents to be measured of quantitative analysis.

6. the method as claimed in claim 5 for detecting a variety of enzymes simultaneously, which is characterized in that in the step S1,

Construct tetrahedron DNA structure method particularly includes: prepare a variety of single stranded DNAs, the DNA including different length biotin labeling is mono- Chain；The DNA of Cy3 label is single-stranded；The DNA of Texas Red label is single-stranded；The DNA of Cy5 label is single-stranded；And it is single-stranded with above-mentioned DNA Sequence is identical, but the DNA of unmarked dyestuff single-stranded (sp chain)；Required DNA is added in 1 × CutSmart buffer, is mixed Object is added to 92~96 DEG C (preferably 95 DEG C) 5~15 minutes (preferably 10 minutes)；Be subsequently placed on ice 0.5~1.5 hour it is (excellent It is selected as 1 hour).

7. the method as claimed in claim 6 for detecting a variety of enzymes simultaneously, which is characterized in that

The tetrahedron DNA sequence dna for being self-assembled into 16 base-pair side lengths is as follows:

The DNA of biotin labeling is single-stranded: 5 '-CTA TGT GGC CAA TCA AAC GAG AGC AAG TGT ATG AGT AAG ATC GCG ACC AT–biotin-3'(SEQ ID NO.1)；

The DNA of Cy3 label is single-stranded: 5 '-CAT GGG ATA TCT ACG GAC ATA CAC TTG CTC TCG AAG ACT TCA GCT GGT TA-Cy3-3'(SEQ ID NO.2)；

The DNA of Texas Red label is single-stranded: 5 '-Texas Red-CCG TAG ATA TCC CAT GAG TTG AGC CTG GAC AGG AAT GGT CGC GAT CTT AC-3'(SEQ ID NO.3)；

The DNA of Cy5 label is single-stranded: 5 '-Cy5-TTG ATT GGC CAC ATA GAC CTG TCC AGG CTC AAC ATA ACC AGC TGA AGT CT-3’(SEQ ID NO.4)。

8. the method as claimed in claim 5 for detecting a variety of enzymes simultaneously, which is characterized in that in the step S2,

Incubation reaction condition specifically: 30~40 DEG C (preferably 37 DEG C) reactions, 0.5~1.5 hour (preferably 1 hour).

9. the method as claimed in claim 5 for detecting a variety of enzymes simultaneously, which is characterized in that in the step S3, in complete The single molecular imaging system of corner reflection fluorescent technique is detected；

Preferably, excitation light source is 405 nano lasers, and excitation intensity is 30 milliwatts；525QD uses 500-550 nanometers of filter Mating plate, Cy3/Texas Red use 573-617 nanometers of optical filter and 565-605 nanometers of optical filter, and Cy5 uses 672-712 The optical filter of nanometer.

10. being detected simultaneously described in any one of the claim 1-4 fluorescence chemical sensor and/or claim any one of 5-9 Application of the method for a variety of enzymes in enzyme quantitative detection and/or inhibitor sifting.