CN110408680A - Single quantum dot nanosensors based on ligase amplification reaction catalytic assembly for detection of alkaline phosphatase and its applications - Google Patents

Abstract

The present invention relates to a single quantum dot nano-sensor for detecting alkaline phosphatase, catalyzed and assembled based on ligase amplification reaction, and its application. For the first time, the ligation reaction was used to identify and amplify the active signal of alkaline phosphatase: in the presence of alkaline phosphatase, the 3'-phosphate end of the catalyzed detection probe was dephosphorylated to generate a 3'-hydroxyl end, and then a thermostable DNA ligase triggers the cyclic and repeated ligation of Cy5 probes and biotin probes to generate multiple Cy5-biotin dual-labeled signal probes; the signal probes pass through the specific interaction of streptavidin-biotin at 605 quanta The surface of the dots is self-assembled to realize efficient fluorescence resonance energy transfer (FRET) from 605 quantum dots to Cy5, and finally the single-molecule fluorescence detection technology is used for detection. In contrast, in the absence of alkaline phosphatase, no fluorescence resonance energy transfer signal was detected. Only one thermostable ligase is needed to achieve ultra-high detection sensitivity with detection limits as low as 5.63 × 10 ^‑7 units per milliliter.

Description

Ligase-based amplification reaction-catalyzed assembly of monoliths for detection of alkaline phosphatase Quantum dot nanosensors and their applications

technical field

The invention belongs to the field of enzyme activity detection, in particular to a single quantum dot nano-sensor catalyzed and assembled based on a ligase amplification reaction for detecting alkaline phosphatase.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Alkaline phosphatase (ALP) is a hydrolase widely present in prokaryotes and eukaryotes, which can catalyze the removal of phosphate groups on phosphorylated proteins, nucleic acids, sugars and other biomolecules under alkaline conditions. It plays a key role in normal physiological functions such as cell division and osteogenesis; meanwhile, the imbalance of intracellular alkaline phosphatase level is closely related to a variety of human diseases such as hypophosphatemia, axial spondylitis, bone disease and cancer. making it a potential biomarker for clinically relevant disease diagnosis. In addition, alkaline phosphatase is also one of the most commonly used biological beacons in biological analysis such as enzyme immunoassay, histochemical staining and DNA hybridization analysis. Clinical diagnostic and bioanalytical applications are of great significance.

Previously, various alkaline phosphatase activity assays based on colorimetric detection, chemiluminescence detection, surface-enhanced Raman scattering detection, and fluorescence detection have been established. The above methods typically use non-nucleic acid substrates such as p-nitrophenol phosphate (PNPP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), and 5-bromo-4-chloro-3-indole phosphate, etc., although there are also However, because these non-nucleic acid substrates cannot be amplified like nucleic acids to amplify the signal during the detection process, it is difficult to improve the analytical sensitivity of these methods. Currently, only a few methods employ nucleic acid amplification strategies to achieve high-sensitivity alkaline phosphatase detection. However, these methods all require the introduction of multiple enzymes to complete the signal amplification step; for example, the T7 exonuclease-mediated signal amplification method initiated by molecular beacons requires the use of KF polymerase and T7 exonuclease, cyclic exponential amplification The amplification method requires the use of Vent polymerase and Nt.BstNBI nickase, and the transcription reaction-mediated dual-signal amplification method requires the use of λ exonuclease and T7 RNA polymerase and double-strand-specific nuclease, which not only increases the cost of analysis , but also complicates the analysis process. In addition, the detection accuracy of these assays may also be compromised due to nonspecific exonuclease digestion and DNA background amplification.

DNA ligase catalyzes the joining of two independent DNA strands by forming a phosphodiester bond between the juxtaposed 5'-phosphate and 3'-hydroxyl termini. Due to the extremely high fidelity and ligation efficiency, DNA ligases are widely used in the field of biosensing; moreover, cyclic amplification of the signal is easily achieved by repeating the strand ligation reaction using a single thermostable ligase. For example, the ligase detection reaction can achieve linear signal amplification by connecting two target-specific probes, while the ligase chain reaction can achieve exponential signal amplification by repeatedly connecting two pairs of probes. These highly specific and highly sensitive ligation reactions are widely used in the detection of biomolecules such as DNA, RNA, microRNA, proteins, and active enzymes. However, there is no report on the detection of alkaline phosphatase activity by ligation reactions. .

Quantum dots (QDs) have excellent photophysical properties such as high photostability, high quantum yield, broad absorption spectrum, narrow-size tunable emission spectrum, and large Stokes shift, making them attractive biosensor building blocks. . In addition, the single-molecule-based nanosensor constructed by introducing single-molecule detection has excellent analytical performance, such as short analysis time, high signal-to-noise ratio, low sample consumption, and ultra-high sensitivity. However, the current detection methods of alkaline phosphatase activity based on quantum dots still have the problems of low selectivity and low sensitivity.

SUMMARY OF THE INVENTION

In order to overcome the above problems, the present invention develops a single quantum dot nanosensor catalyzed and assembled based on ligase amplification reaction, which can realize simple and sensitive alkaline phosphatase activity analysis. This nanosensor only needs one thermostable ligase for signal amplification of target alkaline phosphatase activity, with high selectivity and ultra-high sensitivity, with a detection limit as low as 5.63×10 ^-7 units per milliliter, which can be further applied to enzymes Activity kinetic analysis, inhibitor screening and detection of endogenous alkaline phosphatase activity in tumor cells.

For realizing the above-mentioned technical purpose, the technical scheme adopted in the present invention is as follows:

A single quantum dot nanosensor for detecting alkaline phosphatase, catalyzed and assembled based on ligase amplification reaction, comprising: detection probe, identification probe, Cy5 probe, biotin probe and quantum dot.

The application does not limit the specific composition of the detection probe, as long as it can recognize the activity signal of alkaline phosphatase and convert it into a DNA signal. In some embodiments, the sequence of the detection probe from 5' to 3' is: ATCGAGTGCACCTGACTCCTG-P, wherein P is a phosphorylation modification, which can catalyze the 3'-phosphate of the detection probe in the presence of alkaline phosphatase The terminal (3'-P) is dephosphorylated to generate the 3'-hydroxy terminal (3'-OH).

In this application, after the secondary template is denatured, it is annealed with the biotin probe at the 5'-phosphate end and the Cy5 probe at the 3'-hydroxyl end, so that it is linked under the action of ligase to form biotin-Cy5 Dual-labeled signaling probes. Therefore, in some embodiments, the Cy5 probe has the nucleotide sequence of SEQ ID No. 1: Cy5-ACGGCAGACTTCTCC; under thermal denaturation conditions, the denatured secondary template can further trigger the biotin probe and the Cy5 probe The cyclic ligation of the needles generates a large number of Cy5-biotin dual-labeled signal probes that self-assemble on the surface of 605 QDs through efficient streptavidin-biotin interactions.

In some embodiments, the identification probe has the nucleotide sequence of SEQ ID No. 1: P-GGAGAAGTCTGCCGTATCGAG;

In some embodiments, the sequence of the biotin probe from 5' to 3' is: P-CAGGAGTCAGGTGCA-biotin;

The self-assembly of the double-labeled signal probe on the surface of 605 quantum dots can induce efficient fluorescence resonance energy transfer from 605 quantum dots to Cy5. Therefore, in some embodiments, the 605 quantum dots are: streptavidin-modified quantum dots, and the maximum emission wavelength is 605 nm, which effectively improves the detection sensitivity.

The present invention also provides a method for detecting alkaline phosphatase based on a single quantum dot nanosensor catalyzed and assembled by a ligase amplification reaction, comprising:

Mix and incubate alkaline phosphatase and detection probe in buffer I to form a dephosphorylation reaction product;

Mixing the above-mentioned phosphorylation reaction product, identification probe, DNA template, biotin probe, Cy5 probe, buffer II, and DNA ligase to carry out a thermal cycle ligation reaction to form a ligation product;

Mixing the ligation product with 605 quantum dots in buffer III to form 605 quantum dots/Cy5 nanocomponents;

Perform single-molecule fluorescence detection on 605 quantum dots/Cy5 nanocomponents. If fluorescence resonance energy transfer signal is detected, there is alkaline phosphatase, and the intensity of fluorescence resonance energy transfer signal determines the content of alkaline phosphatase; if no fluorescence resonance energy transfer signal is detected energy transfer signal, alkaline phosphatase is absent.

The advantages of the above method are: the present invention only needs a thermostable ligase to complete the signal amplification, which simplifies the analysis procedure; at the same time, it avoids false positive results caused by non-specific digestion and non-specific DNA amplification, and improves the performance of the method. Detection sensitivity. The invention can also be further applied to the kinetic analysis of enzyme activity, the screening of enzyme activity inhibitors, and the detection of endogenous alkaline phosphatase activity in different cell lines, and provides the biomedical research and clinical application research related to alkaline phosphatase. Powerful technology platform.

In some embodiments, the specific conditions of the incubation are: incubation at 37-38 degrees Celsius for 28-30 minutes, and incubation at 65-67 degrees Celsius for 4-5 minutes to terminate the reaction.

In some embodiments, the specific conditions of the ligation reaction of the thermal cycle are to perform the ligation reaction of 80 thermal cycles according to the program of 95 degrees Celsius for 1 minute and 58 degrees Celsius for 2 minutes.

The invention also provides any of the above nanosensors in the alkaline phosphatase inhibitor screening experiment, the kinetic determination of the alkaline phosphatase enzyme activity, and the high-sensitivity and specific detection of the alkaline phosphatase activity in the actual sample with complex components. application.

The beneficial effects of the present invention are:

(1) Simple operation: Compared with other methods for detecting alkaline phosphatase by nucleic acid amplification, the present invention does not need to introduce multiple enzymes but only needs one ligase to complete signal amplification, which not only reduces the analysis cost, but also reduces the cost of analysis. The analysis process is simplified; in addition, due to the use of high-fidelity thermostable ligase, non-specific exonuclease digestion and non-specific DNA background amplification are avoided, so the detection accuracy of the present invention is also guaranteed.

(2) High efficiency and good effect: the ligation and amplification reaction in the present invention can achieve a good amplification effect within 80 cycles, and the introduction of the single-molecule detection method also greatly reduces the difficulty of data processing and improves data presentation. The intuitiveness and data processing efficiency enable efficient and simple determination of alkaline phosphatase activity.

(3) High sensitivity and huge application potential: In the present invention, due to the effective signal amplification generated by the ligase amplification reaction induced by alkaline phosphatase, multiple double-labeled signal probes are assembled on a single 605 quantum dot The resulting high fluorescence resonance energy transfer efficiency and the inherent high sensitivity of single-molecule detection result in a detection limit as low as 5.63 × 10 ^-7 units per milliliter, which is superior to most existing alkaline phosphatase activity detection methods; in addition, The invention can also realize quantitative detection of the catalytic activity of alkaline phosphatase, and can well carry out experiments on the screening of alkaline phosphatase inhibitors. The sensitive determination of alkaline phosphatase activity in actual samples (cancer cells) has a wide range of applications and great research potential.

(4) Good specificity: due to the specific recognition and conversion of the alkaline phosphatase activity signal by the high-fidelity thermostable ligase used in the present invention, the present invention has high detection specificity; The reaction conditions have also been carefully optimized, so the detection specificity is excellent.

Description of drawings

The accompanying drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application and do not constitute improper limitations on the present application.

Figure 1: Schematic diagram of the single quantum dot nanosensor catalyzed and assembled based on the ligase amplification reaction in Example 1 for alkaline phosphatase analysis.

Figure 2: Gel electrophoretic analysis of ligation products in Example 1 (A) in the absence (control) and presence of alkaline phosphatase (1 unit per milliliter). (B) Fluorescence emission spectra of 605 QDs and Cy5 in the absence (control, black line) and with alkaline phosphatase (1 unit per milliliter, red line). The inset zoom shows the fluorescence emission spectrum from 650 nm to 700 nm.

Figure 3: Single molecule fluorescence images of 605 quantum dots and Cy5 in the absence (A-C) and presence (D-F) of alkaline phosphatase (0.1 units per milliliter) in Example 1. The signal of 605 quantum dots is shown in green (A and D), the signal of Cy5 is shown in red (B and E), and the superimposed signal of 605 quantum dots and Cy5 is shown in yellow (C and F). Scale bar length is 2 μm.

Figure 4: Increase in Cy5 single molecule counts with increasing alkaline phosphatase concentration in the concentration range of 0-2 units per milliliter in Example 1. The inset shows the linear relationship between Cy5 single molecule counts and the logarithm of alkaline phosphatase concentration in the range of 1 × 10 ^-6 -0.1 units per milliliter. Error bars represent the standard deviation of triplicate experiments.

Figure 5: TDG (0.1 units per ml), HhaI (0.1 units per ml), GOX (50 nanomoles per liter), BSA (50 nanomoles per liter) and alkaline phosphatase (0.1 units per liter) in Example 1 Cy5 single molecule counts determined in the presence of milliliters. Error bars represent the standard deviation of triplicate experiments.

Figure 6: Inhibitory effect of different concentrations of sodium vanadate on alkaline phosphatase activity in Example 1. The concentration of alkaline phosphatase was 0.1 units per milliliter. Error bars represent the standard deviation of triplicate experiments.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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 herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

As described in the background art, the current alkaline phosphatase activity detection methods have the problems of low selectivity, low sensitivity and complicated operation. Therefore, the present invention proposes to construct a novel, simple and ultra-sensitive alkaline phosphatase detection method based on ligase amplification reaction to catalyze the assembly of single quantum dot nanosensors.

For the first time, the alkaline phosphatase activity signal was accurately identified by ligation reaction and converted into DNA nucleic acid signal. A large number of Cy5-biotin double-labeled signal probes can be efficiently generated by ligase amplification reaction. Subsequently, the double-labeled signal probe completed self-assembly on the surface of 605 quantum dots, and then induced efficient fluorescence resonance energy transfer from 605 quantum dots to Cy5. Combined with single-molecule detection, the nanosensor has ultra-high sensitivity, and the detection limit is as low as 5.63×10 ^-7 units per milliliter. Compared with various enzyme-assisted amplification alkaline phosphatase activity analysis methods, the present invention only needs one A thermostable ligase can complete signal amplification, simplifying analysis procedures; at the same time, it avoids false positive results caused by non-specific digestion and non-specific DNA amplification, and improves detection sensitivity. The invention can also be further applied to the kinetic analysis of enzyme activity, the screening of enzyme activity inhibitors, and the detection of endogenous alkaline phosphatase activity in different cell lines, and provides the biomedical research and clinical application research related to alkaline phosphatase. Powerful technology platform.

The nanosensor constructed in the present invention is due to the efficient signal amplification generated by the alkaline phosphatase-induced ligase amplification reaction, and the high fluorescence resonance energy transfer caused by assembling multiple double-labeled signal probes on a single 605 quantum dot. The high sensitivity inherent in the efficiency and single molecule detection itself results in a detection limit as low as 5.63 x ^10-7 units per milliliter. Compared with the carbon quantum dot fluorescence method without signal amplification (1.4 × 10 ^-3 units per milliliter), the sensitivity is increased by 2222 times, compared with the Taqman probe fluorescence method based on transcription reaction-mediated double signal amplification (2 × 10 ^-5 units per milliliter). per milliliter) was increased by 35 times.

Previously reported assays for alkaline phosphatase activity are often limited in specificity due to their susceptibility to interference from unrelated biomolecules, or to false-positive results due to nonspecific digestion and nonspecific DNA amplification. The invention has higher detection specificity due to the specific recognition and conversion of the alkaline phosphatase activity signal by the high-fidelity thermostable ligase used.

The invention can not only realize the quantitative detection of alkaline phosphatase catalytic activity, but also can well carry out experiments on alkaline phosphatase inhibitor screening; in addition, the invention can also be used for the relative determination of alkaline phosphatase enzyme activity kinetics As well as sensitive determination of alkaline phosphatase activity in real samples (cancer cells) with complex components, it has a wide range of applications.

The technical solutions of the present application will be described below through specific examples. Among them, 605 quantum dots are quantum dots of 605 nanometers (Qdot 605ITK) purchased from Thermo Fisher (Eugene, Oregon, USA).

Example 1:

1. Fluorescence detection experiment

Preparation of cell extracts: Human breast cancer cells (MCF-7) and human embryonic kidney cells (HEK293T) were modified with Dulbecco's containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibody, respectively. Eagle's medium (DMEM) was grown at 37 degrees Celsius in a 5% carbon dioxide incubator and used for actual sample analysis. When the cells grew to the logarithmic growth phase, the cells were harvested with trypsin and counted using an automatic cell counter IC1000 from Countstar Biotechnology Co., Ltd. The resulting cells were washed twice with ice-cold PBS (pH 7.4), detached by centrifugation at 1000 rpm for 5 min, and then suspended in 50 μl of cell lysate (containing 10 mM pH 8.0 Tris-HCl, 150 mmol/L NaCl, 1% NP-40, 0.25 mmol/L sodium deoxycholate, 1% glycerol and 0.1 mmol/L 4 -(2-aminoethyl)benzenesulfonyl fluoride hydrochloride), incubated on ice for 30 minutes and centrifuged at 12,000 rpm for 20 minutes at 4 degrees Celsius. Transfer the supernatant to a new pre-chilled EP tube and store in a -80 °C freezer until use. The total protein concentration in the extracts was determined using a NanoDrop 2000c spectrophotometer.

Alkaline phosphatase activity detection: In order to form a dephosphorylated detection probe, a 20-microliter reaction system was prepared, which contained 2 microliters of alkaline phosphatase at different concentrations, 0.15 microliters of 10 micromolar detection probes per liter and 2 µl of 10x Buffer (500 mmol/L potassium acetate, 200 mmol/L Tris-acetic acid, 100 mmol/L magnesium acetate, 1000 μg/mL BSA, pH 7.9) at 37°C Incubate for 30 min and stop the reaction by incubating at 65°C for 5 min. Thereafter, the dephosphorylation reaction product was mixed with 0.15 μl 5 μmol per liter identification probe, 0.15 μl 5 μmol per liter DNA template, 1.2 μl 5 μmol per liter biotin probe, 1.2 μl 5 μM Cy5 probe per liter, 3 μL 10× reaction buffer (containing 200 mM Tris-HCl, pH 8.3, 250 mM KCl, 100 mM pH 8.3) Magnesium chloride per liter, 5 mmol per liter NAD and 0.1% X-100), 10 units of The DNA ligase was mixed and the reaction volume was adjusted to 30 μl, and the ligation reaction was performed for 80 thermal cycles according to the program of 95 °C for 1 min and 58 °C for 2 min. Finally, the ligation product was mixed with 8.3 nmol/L of 605 QDs in 60 µL of buffer (containing 1.5 mM MgCl, 50 mM Tris-HCl, 5 mM) at room temperature ammonium sulfate per liter, pH 8.0) for 10 minutes to form 605 quantum dots/Cy5 nanoassemblies.

Fluorescence measurement: Measure 50 microliters of reaction product on a Hitachi F-7000 spectrofluorometer using a microquartz dish. The excitation wavelength was set to 488 nm, and the emission spectra in the wavelength range of 500–720 nm were recorded simultaneously with a slit width of 5 nm for both excitation and emission.

Single-molecule fluorescence assay and data analysis: The reaction products were mixed in imaging buffer (containing 1 mg/mL glucose oxidase, 0.4% (mass volume ratio) D-glucose, 0.04% mg/mL catalase, 50 Micrograms per ml of bovine serum albumin, 67 mmol per liter of glycine-potassium hydroxide, 1 mg per ml of water-soluble vitamin E, 2.5 mg per ml of magnesium chloride, pH 9.4) diluted 100-fold. Use a pipette to pipette 10 μl of the above sample directly onto a coverslip for TIRF imaging, use a sapphire 488 nm laser (50 mW) to excite 605 quantum dots, and the luminescence signals of 605 quantum dots and Cy5 are determined by 100 × Å. The collection was performed with a Limbus objective and imaged by an Andor IxonDU897EMCCD with an exposure time of 500 ms, and Cy5 molecules were counted using Image J software to select an image area of 900 × 900 pixels.

Gel electrophoresis: The products of the ligase amplification reactions were analyzed by 12% denaturing polyacrylamide gel electrophoresis at room temperature and a constant voltage of 120 volts, using 1× TBE buffer (containing 9 mM pH per liter). 7.9 Tris-hydrochloric acid, 9 mmol/L boric acid and 0.2 mmol/L EDTA), electrophoresis time was 50 minutes. Thereafter, the gel was stained using a silver staining kit and photographed by the Bio-Rad ChemiDoc MP imaging system.

Alkaline phosphatase activity inhibition assay: To assess the effect of alkaline phosphatase inhibitors, 2 microliters of sodium vanadate at various concentrations were premixed with 2 microliters of 1 unit per milliliter of alkaline phosphatase at 37 degrees Celsius for 20 min; after this, add 2 μl of 10× buffer and 0.15 microliters of 10 micromolar detection probe per liter and adjust the reaction volume to 20 microliters with ultrapure water. The subsequent process is the same as the detection of alkaline phosphatase activity.

Experimental principle (as shown in Figure 1): Using 3'-terminal phosphorylated single-stranded DNA as a detection probe, the activity signal of alkaline phosphatase is recognized and converted into a DNA signal. In the presence of alkaline phosphatase, it catalyzes the dephosphorylation of the 3'-phosphate end (3'-P) of the detection probe to generate the 3'-hydroxyl end (3'-OH). The 3'-hydroxyl end probe and the 5'-phosphate end helper probe can be annealed with the template probe to form a detection probe/helper probe/template sandwich structure. A thermostable ligase is added, and the detection probe and the auxiliary probe are ligated into a DNA strand to form a secondary template. After the secondary template is denatured, it is annealed with the biotin probe at the 5'-phosphate end and the Cy5 probe at the 3'-hydroxyl end to promote its ligation under the action of ligase to form a biotin-Cy5 double-labeled signal probe. Needle. In addition, under thermal denaturation conditions, the denatured secondary template can further trigger the cyclic ligation of biotin probes and Cy5 probes, resulting in a large number of Cy5-biotin dual-labeled signal probes that pass through highly efficient streptavidin-biotin The prime interaction self-assembles on the surface of 605 quantum dots. Thus, 605 quantum dots/dual-labeled signal probe/Cy5 nano-assemblies are formed, and the distance between 605 quantum dots and Cy5 is very close, so efficient fluorescence resonance energy transfer from 605 quantum dots to Cy5 can occur. Single-molecule fluorescence detection can simply and sensitively detect the fluorescence resonance energy transfer signal of each quantum dot nanoassembly. On the contrary, in the absence of alkaline phosphatase, the 3'-phosphate end of the detection probe is retained and cannot be linked with the helper probe to form a secondary template. Therefore, the Cy5-biotin double-labeled signal probe cannot be generated, 605 quantum dots Only the biotin probe is assembled on it, which cannot cause the phenomenon of fluorescence resonance energy transfer. It is worth noting that in the present invention, the high-fidelity thermostable ligase can catalyze the efficient amplification of the alkaline phosphatase activity signal, which will greatly improve the sensitivity of the analysis, with high operability and accuracy.

2. Experimental verification of the principle

The present invention uses 12% denaturing polyacrylamide gel electrophoresis silver staining to analyze the connection product induced by alkaline phosphatase. As shown in Figure 2A, in the presence of alkaline phosphatase, the connection between the detection probe and the auxiliary probe can be triggered to form a secondary template, and the connection between the Cy5 probe and the biotin probe can also be triggered to form a dual-labeled signal probe. pin, which is reflected on the gel map by the corresponding bands of the secondary template and signal probes. In contrast, in the control group without alkaline phosphatase, the bands of the secondary template and the signal probe disappeared, indicating that the ligase-catalyzed amplification reaction could not take place in the absence of alkaline phosphatase.

The present invention further verifies the assembly of the 605 quantum dot/signal probe/Cy5 nano-assembly under the induction of alkaline phosphatase by fluorescence assay. As shown in Figure 2B, in the absence of alkaline phosphatase, the Cy5-biotin double-labeled signal probe could not be generated, so only the biotin probe was assembled on the surface of 605 QDs, so only the 605 QDs were observed Fluorescence emission signal (Fig. 2B, blue line). On the contrary, alkaline phosphatase can induce the ligation reaction between the biotin probe and the Cy5 probe to form a complete dual-labeled signal probe and assemble on the surface of 605 quantum dots, resulting in efficient fluorescence resonance energy transfer. Therefore, it is observed that A decrease in the fluorescence emission signal of 605 quantum dots and an increase in the fluorescence emission signal of Cy5 (Fig. 2B, red line). The fluorescence resonance was calculated according to the equation E(%) = 1 - F _ALP /F (F _ALP is the fluorescence intensity of 605 QDs in the presence of alkaline phosphatase, F is the fluorescence intensity of 605 QDs in the absence of alkaline phosphatase) The efficiency of energy transfer is 51.06%.

Compared with traditional fluorescence assay methods, single-molecule detection has the significant advantages of less sample consumption, shorter analysis time, and higher sensitivity. Therefore, the present invention further adopts the total internal reflection fluorescence imaging technology to measure the fluorescence signal of a single quantum dot. As shown in Figure 3, in the absence of alkaline phosphatase, only the fluorescence signal of 605 quantum dots was observed (Figure 3A), but no fluorescence signal of Cy5 (Figure 3B) was observed, indicating that when the alkaline phosphatase was not When present, the 605 quantum dot/dual-labeled signaling probe/Cy5 nanoassemblies are not formed, so no fluorescence resonance energy transfer occurs. In contrast, after the addition of alkaline phosphatase, the fluorescence signals of 605 QDs (Fig. 3D) and Cy5 (Fig. 3E) were simultaneously observed and superimposable (Fig. 3F), indicating that due to the 605 QDs/dual-labeled signal probe The formation of /Cy5 nanoassemblies resulted in efficient fluorescence resonance energy transfer. These results clearly demonstrate that the single quantum dot-based nanosensor proposed in the present invention can be used for quantitative detection of alkaline phosphatase activity.

3. Sensitivity experiment

Under the optimized experimental conditions, the present invention measures the Cy5 single molecule counts corresponding to different concentrations of alkaline phosphatase to evaluate the analytical sensitivity of the present invention. As shown in Figure 4, as the alkaline phosphatase concentration increased from 0 to 2 units per milliliter, the single molecule count of Cy5 increased gradually. Meanwhile, the single molecule counts of Cy5 were linearly correlated with the logarithm of the alkaline phosphatase concentration in the concentration range of 1×10 ⁻⁶ to 0.1 units per milliliter, with a linear correlation coefficient (R ² ) of 0.9987. The linear regression equation is N = 278.20 + 36.87 log ₁₀ C, where N is the single molecule count of Cy5 and C is the concentration of alkaline phosphatase (units per milliliter). The limit of detection determined by the mean signal of the control group plus three times the standard deviation was 5.63 x ^10-7 units per milliliter. The sensitivity of the nanosensor constructed in the present invention is 2222 times higher than that of the carbon quantum dots fluorescence method without signal amplification (1.4×10 ^-3 units per milliliter), and compared with the Taqman probe fluorescence method based on double signal amplification mediated by transcription reaction (2 x ^10-5 units per milliliter) 35 times higher. The ultra-high sensitivity of the present invention can be attributed to: (1) efficient signal amplification by the alkaline phosphatase-induced ligase amplification reaction; (2) by assembling multiple dual-labeled signal probes into a single 605 quantum The high fluorescence resonance energy transfer efficiency caused by the spot; (3) the inherent high sensitivity of single molecule detection itself.

4. Specificity Experiments

Previously reported assays for alkaline phosphatase activity are often limited in specificity due to their susceptibility to interference from unrelated biomolecules, or to false-positive results due to nonspecific digestion and nonspecific DNA amplification. In order to test the specific analysis effect of the present invention, the present invention measured Cy5 single-molecule count signal on thymidine DNA glycosylase (TDG), HhaI restriction nuclease (HhaI), glucose oxidase (GOX) and bovine serum albumin (BSA) and other biological interferences. TDG can remove the thymine moiety from G/T mismatch, HhaI can recognize and cleave 5'-GCGC-3' double-stranded DNA, GOX can catalyze the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone , BSA is a common unrelated protein. Theoretically, neither of these enzymes nor proteins can induce the dephosphorylation of the detection probe, so the fluorescent signal of Cy5 cannot be detected. As shown in Figure 5, a high Cy5 signal was detected in the presence of alkaline phosphatase, while a very low Cy5 signal was observed in the presence of TDG, Hhal, GOX, and BSA. The response signals of alkaline phosphatase were 13.22-fold, 12.13-fold, 14.41-fold and 13.16-fold higher than those of TDG, HhaI, GOX and BSA, respectively. The present invention is thus highly specific, which can be attributed to the specific recognition and conversion of alkaline phosphatase activity signals using a high-fidelity thermostable ligase.

5. Inhibitor Experiment

Inhibitors of alkaline phosphatase are a potential disease treatment drug that can inhibit vascular smooth muscle cell calcification for the treatment of cardiovascular disease. The present invention uses a well-known alkaline phosphatase inhibitor, sodium vanadate (Na ₃ VO ₄ ), to test the ability of the present invention for alkaline phosphatase activity inhibition assays. As shown in Figure 6, the relative activity of alkaline phosphatase decreased in a dose-dependent manner with the increase of sodium vanadate concentration. After calculation, the _IC50 of the half inhibitory concentration is 87.69 micromoles per liter, which is similar to the result using the method based on the near-infrared fluorescent probe (141.9 micromoles per liter), which indicates that the present invention can be used for the screening of alkaline phosphatase inhibitors, and high selectivity. It shows great potential in drug development and disease treatment.

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will still Modifications may be made to the technical solutions described in the foregoing embodiments, or equivalent replacements may be made to some of them. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Although the specific embodiments of the present invention are described above, they do not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solutions of the present invention, those skilled in the art can make Various modifications or deformations made are still within the protection scope of the present invention.

Claims (10)

1. a single quantum dot nano-sensor for detecting alkaline phosphatase, based on ligase amplification reaction catalysis and assembling, is characterized in that, comprising: detection probe, identification probe, Cy5 probe, biotin probe and 605 quantum dots. 2. The single quantum dot nanosensor for detecting alkaline phosphatase based on ligase amplification reaction catalyzed assembly according to claim 1, wherein the detection probe is composed of a sequence from 5' to 3' It is: ATCGAGTGCACCTGACTCCTG-P, where P is phosphorylation modification. 3. The single quantum dot nanosensor for detecting alkaline phosphatase, catalyzed and assembled based on ligase amplification reaction as claimed in claim 1, wherein the Cy5 probe has the nucleus of SEQ ID No.1 nucleotide sequence: Cy5-ACGGCAGACTTCTCC. 4. The single quantum dot nanosensor for detecting alkaline phosphatase, catalyzed and assembled based on ligase amplification reaction as claimed in claim 1, wherein the identification probe has the nucleus of SEQ ID No.1 nucleotide sequence: P-GGAGAAGTCTGCCGTATCGAG. 5. The single quantum dot nanosensor for detecting alkaline phosphatase based on ligase amplification reaction catalyzed assembly according to claim 1, wherein the biotin probe is composed of 5' to 3'. The sequence is: P-CAGGAGTCAGGTGCA-biotin. 6. The single quantum dot nanosensor for detecting alkaline phosphatase, catalyzed and assembled based on ligase amplification reaction as claimed in claim 1, wherein 605 quantum dots are: streptavidin-modified quantum dots point, the maximum emission wavelength is 605nm. 7. A method for detecting alkaline phosphatase based on a single quantum dot nanosensor assembled by ligase amplification reaction catalysis, comprising: Mix and incubate alkaline phosphatase and detection probe in buffer I to form a dephosphorylation reaction product; Mixing the above-mentioned phosphorylation reaction product, identification probe, DNA template, biotin probe, Cy5 probe, buffer II, and DNA ligase to carry out a thermal cycle ligation reaction to form a ligation product; Mixing the ligation product with 605 quantum dots in buffer III to form 605 quantum dots/Cy5 nanocomponents; Perform single-molecule fluorescence detection on 605 quantum dots/Cy5 nanocomponents. If fluorescence resonance energy transfer signal is detected, there is alkaline phosphatase, and the intensity of fluorescence resonance energy transfer signal determines the content of alkaline phosphatase; if no fluorescence resonance energy transfer signal is detected energy transfer signal, alkaline phosphatase is absent. 8. The method of claim 7, wherein the specific conditions of the incubation are: incubation at 37-38 degrees Celsius for 28-30 minutes, and incubation at 65-67 degrees Celsius for 4-5 minutes to terminate the reaction. 9 . The method of claim 7 , wherein the specific conditions for the ligation reaction of the thermal cycle are to perform the ligation reaction of 80 thermal cycles according to a program of 95 degrees Celsius for 1 minute and 58 degrees Celsius for 2 minutes. 10 . 10. The nanosensor of claim 1-6 is used in alkaline phosphatase inhibitor screening experiments, alkaline phosphatase enzyme activity kinetic assays and high-sensitivity and specific detection of alkaline phosphatase activity in actual samples with complex components Applications.