1. A QD-capture probe nanostructure is characterized in that the nanostructure comprises a single quantum dot QD and a capture probe, streptavidin is modified on the surface of the single quantum dot QD, and a phosphate group and biotin are respectively modified at two ends of the capture probe; the capture probe self-assembles to the surface of a single quantum dot QD using biotin-streptavidin.

2. The QD-capture probe nanostructure of claim 1, wherein the modified capture probe is 5' P-TTT TTT TTT T-biotin.

3. A kit for detecting PKA and/or PNK, comprising the QD-capture probe nanostructure of claim 1 or 2 and Zr ion⁴⁺。

4. The kit for detecting PKA and/or PNK according to claim 3, further comprising a Cy 5-modified probe; further, the probes include polypeptide probes and/or DNA probes; or, the kit further comprises adenosine 5' -triphosphate and a PKA and/or PNK reaction buffer.

5. A zirconium ion-mediated nanosensor, wherein the nanosensor comprises the QD-capture probe nanostructure of claim 1, and zirconium ion Zr in that order from inside to outside⁴⁺And Cy 5-modified Probe to construct QD-Zr⁴⁺-Cy5 nanostructures.

6. The zirconium ion-mediated nanosensor of claim 5, wherein said probe comprises a polypeptide probe and/or a DNA probe.

7. The zirconium ion-mediated nanosensor of claim 6, wherein said polypeptide probe is a polypeptide probe comprising a PKA recognition site, and a Cy5 fluorophore is labeled at the carboxyl terminal of the probe; further, the sequence of the polypeptide probe is Cy5- (COOH) -KLRRASL-(NH₂) (ii) a Further, the recognition site is serine; or, the sequence of the DNA probe is 5 '-GTT GAG C-Cy 5-3'.

8. The preparation method of the zirconium ion mediated nano sensor is characterized by comprising the steps of preparing a QD-capture probe nanostructure and QD-Zr⁴⁺-preparation of Cy5 nanostructures; further, the preparation method of the QD-capture probe nanostructure comprises the following steps of mixing the capture probe, the single quantum dot QD and the 10 XQD buffer solution, and incubating in a dark place; further, the incubation time is 10-30min, preferably 20 min;

or, the QD-Zr⁴⁺-preparation of Cy5 nanostructures comprising: mixing the probe, ATP, PKA and/or PNK reaction buffer solution, PKA and/or PNK reaction solution, reacting at 30-45 deg.C for 1-4 hr, and incubating at 55-75 deg.C.

9. A method of screening for inhibitors of PKA and/or PNK comprising co-reacting a model inhibitor with a zirconium ion mediated nanosensor of any of claims 5-7; further, the model inhibitor is H-89 model inhibitor and/or adenosine diphosphate and ammonium sulfate.

10. Use of a QD-capture probe nanostructure according to claim 1 or 2 and/or a kit for PKA and/or PNK detection according to claim 3 or 4 and/or a zirconium ion mediated nanosensor according to any one of claims 5-7 and/or a method of preparing a zirconium ion mediated nanosensor according to claim 8 for detection of PKA and/or PNK activity.

Background

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

Intracellular phosphorylation plays an important role in biological processes such as signal transduction, metabolism, gene expression, cell cycle, and the like. Kinases are responsible for regulating the phosphorylation process and can be classified into Protein Kinases (PKA), T4 polynucleotide kinases (PNK) and small molecule kinases. During a typical kinase-catalyzed phosphorylation process, the gamma-phosphate group is transferred from adenosine-5-triphosphate (ATP) to a specific site on the substrate. PKA and PNK are the two most widely studied classes of kinases. PKA can catalyze the phosphorylation of proteins by transferring gamma-phosphate from ATP to conserved tyrosine, threonine or serine residues in peptide/protein substrates. About 30% of human proteins are regulated by phosphorylation, and abnormal expression of PKA is closely related to various human diseases such as cancer, immunodeficiency, neurodegenerative diseases, endocrine disorders, and the like. PNK catalyzes the phosphorylation of the 5' -hydroxyl end of DNA, which is critical for repair of DNA strand breaks. Abnormal expression of PNK can induce a variety of human diseases, such as the Boolean (Loom) syndrome, the Warner (Werner) syndrome, and the pigmentation (Rothmund-Thomson) syndrome. Furthermore, PNK is a potential cancer therapeutic target due to the positive effects of PNK inhibitors. Therefore, the development of a general kinase detection biosensor is of great significance for the treatment and diagnosis of diseases.

The inventors have found that the conventional method for detecting kinase activity is radiolabeling, which achieves detection of kinases using the principle that kinases catalyze transfer of radioactive phosphate groups (. gamma. -32P) on ATP to specific substrate peptides/DNA. Although the radiolabelling method is suitable for kinase detection of different substrates, the method has the defects of complicated operation steps, radioactive contamination and the like. In recent years, a series of highly sensitive, highly specific detection methods have been developed to detect PKA and PNK activity. Electrochemical and fluorescence assays based on phospho-specific antibody recognition of phosphorylated substrate peptides were developed for detection of PKA activity, but these methods require expensive antibody proteins and are complicated and time consuming to perform. In addition, fluorescence methods have also been developed to detect PKA using biotin-modified ATP as a phosphate donor, but these assays add complexity and cost to the experiment because of the need to label ATP. The newly developed PNK detection methods mainly realize quantitative analysis of PNK by detecting phosphorylated DNA, and can be combined with nucleic acid amplification technology to improve detection sensitivity. However, these methods require complicated DNA probe design and have inevitably high background signal due to non-specific amplification. In addition, since recognition substrates of PNK and PKA are different, no general biosensor for detecting kinase activity has been reported so far. Therefore, the development of a general biosensor for detecting kinase activity is of great significance for the treatment and diagnosis of diseases.

Disclosure of Invention

Since recognition substrates of PNK and PKA are different, no general biosensor for detecting kinase activity has been reported so far, and in order to solve the problems, the present disclosure provides a zirconium ion-mediated nanosensor, a preparation method and an application thereof, by developing a zirconium ion (Zr) -based biosensor⁴⁺) Mediated single Quantum Dot (QD) self-assembly universal nanosensors were used to detect PKA and PNK activities, for which Cy 5-modified substrate peptides and substrate DNA were designed, respectively. The biotin-modified capture probe is self-assembled on the QD surface to construct the nanosensor. In the presence of PKA/PNK, they catalyze phosphorylation of Cy 5-modified peptide/DNA and by Zr⁴⁺Coordination with phosphate groups assembles to the surface of individual QDs to form QD-Zr⁴⁺-Cy5 nanostructure and resulting in Fluorescence Resonance Energy Transfer (FRET) of QD and Cy 5. By single molecule detection, the FRET signal can be easily measured.

Specifically, the technical scheme of the present disclosure is as follows:

in a first aspect of the present disclosure, a QD-capture probe nanostructure includes a single quantum dot QD and a capture probe, streptavidin is modified on the surface of the single quantum dot QD, and a phosphate group and biotin are each modified at both ends of the capture probe; the capture probe self-assembles to the surface of a single quantum dot QD using biotin-streptavidin.

In a second aspect of the disclosure, a kit for PKA and/or PNK detection comprising one of the QD-capture probe nanostructures described above and zirconium ion Zr⁴⁺。

In a third aspect of the present disclosure, a zirconium ion-mediated nanosensor, which comprises a QD-capture probe nanostructure and zirconium ion Zr sequentially from inside to outside⁴⁺And Cy 5-modified Probe to construct QD-Zr⁴⁺-Cy5 nanostructures.

In a fourth aspect of the present disclosure, a method for preparing a zirconium ion-mediated nanosensor, the method for preparing comprising preparing a QD-capture probe nanostructure and QD-Zr⁴⁺-preparation of Cy5 nanostructures.

In a fifth aspect of the disclosure, a method of screening for a PKA and/or PNK inhibitor, the method comprising co-reacting a model inhibitor with said one zirconium ion-mediated nanosensor.

In a sixth aspect of the present disclosure, the use of a QD-capture probe nanostructure and/or a kit for detecting PKA and/or PNK and/or a zirconium ion-mediated nanosensor and/or a method of preparing a zirconium ion-mediated nanosensor for detecting PKA and/or PNK activity.

One or more technical schemes in the disclosure have the following beneficial effects:

(1) the QD-capture probe nanostructure can realize rapid identification of coordination ions, particularly can quickly combine zirconium ions, provides an optimal adaptation basis for the combination of metal ions such as zirconium ions and the like capable of being coordinated with phosphate groups and single Quantum Dots (QDs), and is convenient for further realizing the application of the QD-capture probe nanostructure in the field of detection of kinases such as PKA and PNK.

(2) The kit for detecting PKA and/or PNK disclosed by the disclosure can be conveniently and rapidly applied to the detection of the activity of PKA and PNK, zirconium ions and phosphoric acid are self-assembled on a single quantum dot QD, no matter PKA or PNK is detected, a phosphorylation probe is obtained when PKA/PNK exists, and P on the probe is self-assembled with the zirconium ions in the kit, so that the detection of PKA and/or PNK is realized. The kit is very convenient and efficient to apply, and only an adaptive probe is selected based on the type of the kinase to be detected.

(3) The detection limit of the zirconium ion mediated nano sensor to PKA is 8.82 multiplied by 10^-4The detection limit of PNK per milliliter is 1.40 multiplied by 10^-5The unit per milliliter, the sensitivity is higher than that of the existing method. In addition, the nano sensor can be used for detecting the PKA and PNK activities in the HeLa cells, can be further applied to screening of inhibitors of the PKA and the PNK, and has great application potential in the aspects of drug discovery and clinical diagnosis.

(4) By phosphoric acid group with inorganic ion (Zr)⁴⁺) Coordination covalent interaction between, Zr⁴⁺Selectively combined with two phosphate functionalized molecules as a bridge for developing universal kinase nano-sensors. Semiconductor Quantum Dots (QDs) have unique optical and electronic properties (e.g., high quantum yield, narrow and size-tunable emission spectra, and broad excitation band, long fluorescence lifetime, and large chemically modified surface area, etc.), and are widely used as donors for fluorophore and fluorescence resonance energy transfer. Thus, a zirconium ion (Zr) -based alloy was developed⁴⁺) Mediated self-assembly of single Quantum Dots (QDs) universal nanosensors were used to detect PKA and PNK activity.

(5) The nano structure formed by assembling a plurality of Cy5 modified substrate probes on the surface of one QD improves the FRET efficiency; the single molecule detection has high signal-to-noise ratio, and has the remarkable advantages of high sensitivity, short analysis time, small sample consumption and the like.

(6) The nano sensor based on the present disclosure can efficiently, rapidly and accurately realize the screening of the PKA and/or PNK inhibitor, and the experimental result shows that when the nano sensor is used for screening the PKA inhibitor, the screening effect is consistent with the result of the double-signal readout fluorescence analysis method; screening for PNK, screening Effect and IC of ammonium sulfate measured by bioluminescence assay₅₀The results of the values are consistent, verifying the accuracy of PKA or PNK inhibitor screening with the nanosensors of the disclosure.

(7) The preparation method provided by the disclosure does not need expensive antibodies and specially labeled ATP, does not need any washing and separation steps, and is very simple to operate; also, complicated probe design and nucleic acid amplification steps are not required, and the method can be carried out under isothermal and homogeneous conditions.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1: is a schematic diagram of the mechanism of example 1, wherein FIG. 1a is a schematic diagram of the mechanism for detecting PKA and FIG. 1b is a schematic diagram of the mechanism for detecting PNK;

FIG. 2: measurement of QD and Cy5 fluorescence in the absence of PKA (control, blue line) and in the presence of PKA (red line).

FIG. 3: measurement of QD and Cy5 fluorescence in the absence of PNK (control, blue line) and in the presence of PMK (red line).

FIG. 4: PKA activity was detected by total internal reflection fluorescence microscopy based single molecule imaging. Single molecule fluorescence images in the absence of PKA (A-C) and in the presence of PKA (D-F). The fluorescence signals of QD are shown in green (A, D) and Cy5 in red (B, E), and the superposition of QD and Cy5 is shown in yellow (C, F). The PKA concentration is 100 units per ml. Scale bar 3 microns.

FIG. 5: PNK activity was detected by total internal reflection fluorescence microscopy based single molecule imaging. Single molecule fluorescence images in the absence of PNK (A-C) and in the presence of PNK (D-F). The fluorescence signals of QD are shown in green (A, D) and Cy5 in red (B, E), and the superposition of QD and Cy5 is shown in yellow (C, F). The PNK concentration is 10 units per ml. Scale bar 3 microns.

FIG. 6: (A) the number of Cy5 molecules varied with the concentration of PKA. (B) Linear relationship between Cy5 counts and log form of PKA concentration. Error bars represent standard deviations of three experiments.

FIG. 7: (A) the number of Cy5 molecules varied with the concentration of PNK. (B) Linear relationship between Cy5 counts and log form of PNK concentration. Error bars represent standard deviations of three experiments.

FIG. 8: (A) the number of Cy5 molecules varied in the presence of 100 units per ml PKA, 100 units per ml UDG, 100 units per ml PNK, 0.005 g per ml IgG, 0.005 g per ml BSA. (B) Inhibition of PKA activity by H-89. Error bars indicate the standard deviation of three experiments.

FIG. 9: the number of Cy5 molecules varied in the presence of 10 units per ml PNK, 100 units per ml UDG, 100 units per ml PKA, 0.005 g per ml IgG, 0.005 g per ml BSA. Error bars represent standard deviations of three experiments.

FIG. 10: (A) adenosine diphosphate inhibition of PNK activity. (B) Inhibition of PNK activity by ammonium sulphate. Error bars represent standard deviations of three experiments.

Detailed Description

The disclosure is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

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. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

At present, the existing methods for detecting kinases have the problems of complex operation steps, radioactive pollution, low detection sensitivity and the like, and particularly, because different identified substrates are different for different kinases, no biosensor capable of universally detecting the activity of the kinases exists, so that the disclosure provides a zirconium ion mediated nano sensor, a preparation method and application thereof.

In one embodiment of the present disclosure, a QD-capture probe nanostructure includes a single quantum dot QD and a capture probe, streptavidin is modified on the surface of the single quantum dot QD, and a phosphate group and biotin are each modified at both ends of the capture probe; the capture probe self-assembles to the surface of a single quantum dot QD using biotin-streptavidin.

In the QD-capture probe nanostructure, the phosphate group and the biotin are respectively modified at two ends of the capture probe, and the special structural design is favorable for the QD-capture probe nanostructure to quickly realize the detection of the kinase, is favorable for the metal ions to quickly and accurately locate the phosphate group, realizes the self-assembly process, and improves the efficiency of the QD-capture probe nanostructure in the kinase detection. Particularly, in the process of detecting the kinase, different kinases and substrate probes are different, and when phosphate groups and biotin are respectively modified at two ends of the capture probe, the detection of different kinases is convenient to realize, and the application range of the QD-capture probe nanostructure in the kinase detection is widened.

However, the traditional invention conception is that the phosphate group and the biotin are directly connected and modified at the same end of the capture probe, and the structure design can only detect one kinase, but cannot detect the kinase with different substrate probes.

Further, the modified capture probe is 5' P-TTT TTT TTT T-biotin. The capture probe can improve the sensitivity of kinase detection.

In one embodiment of the present disclosure, a kit for detecting PKA and/or PNK, comprising said one QD-capture probe nanostructure and zirconium ion Zr⁴⁺. With Zr⁴⁺For recognition of elements for phosphoric acidRealizes effective differentiation of phosphorylated and non-phosphorylated substrate probes, and simultaneously Zr⁴⁺The fluorescent probe can also be a bridge for connecting a phosphorylation substrate and a capture probe, and the universal detection of PKA and PNK is realized by combining with a single-molecule detection technology.

Further, the kit also comprises a Cy5 modified probe, so that the detection of the kinase can be conveniently realized.

Further, the probe includes a polypeptide probe and/or a DNA probe, wherein the polypeptide probe is used when PKA is detected and the DNA probe is used when PNK is detected.

Further, the kit also comprises adenosine 5' -triphosphate (ATP) and PKA and/or PNK reaction buffer solution, and the buffer solution provides a proper detection environment for kinase detection.

In one embodiment of the present disclosure, a zirconium ion-mediated nanosensor, which comprises the QD-capture probe nanostructure and zirconium ion Zr sequentially from inside to outside⁴⁺And Cy 5-modified Probe to construct QD-Zr^{4 +}-Cy5 nanostructures. The nano structure that a plurality of Cy5 modified substrate probes are assembled on the surface of one QD improves the FRET efficiency, and by using the nano sensor, when kinase to be detected exists, the phosphorylation of Cy5 modified probes can be catalyzed, and Zr is passed through⁴⁺Coordination with phosphate groups assembles to the surface of individual QDs to form QD-Zr⁴⁺-Cy5 nanostructure and resulting in FRET for QD and Cy 5. By single molecule detection, the FRET signal can be easily measured.

The nano sensor can detect different kinases, and the obtained phosphate group can be detected by the nano sensor as long as the kinase to be detected can catalyze the probe to realize phosphorylation. This is not possible with conventional kinase detection methods. The nano sensor has high signal-to-noise ratio in single molecule detection, and has the remarkable advantages of high sensitivity, short analysis time, small sample consumption and the like.

Further, the probe comprises a polypeptide probe and/or a DNA probe, the polypeptide probe is a polypeptide probe containing a PKA recognition site, a Cy5 fluorescent group is marked at the carboxyl terminal of the probe,the structure can realize the detection of PKA, and has high detection sensitivity and simple detection method; further, the sequence of the polypeptide probe is Cy5- (COOH) -KLRRASL- (NH)₂) (ii) a Furthermore, the recognition site is serine, which is more favorable for improving the detection sensitivity. In addition, when PNK is detected, the DNA probe has a sequence of 5 '-GTT GAG C-Cy 5-3'.

By phosphoric acid groups with inorganic ions (Zr)⁴⁺) Coordination covalent interaction between, Zr⁴⁺Selectively combines with two phosphate functionalized molecules, is used as a bridge for developing a universal kinase nano sensor, and has important significance for the treatment and diagnosis of diseases.

In one embodiment of the present disclosure, a method for preparing a zirconium ion-mediated nanosensor, the method for preparing comprising preparing a QD-capture probe nanostructure and QD-Zr⁴⁺-preparation of Cy5 nanostructures; further, the preparation method of the QD-capture probe nanostructure comprises the following steps of mixing the capture probe, the single quantum dot QD and the 10 XQD buffer solution, and incubating in a dark place; further, the incubation time is 10-30min, preferably 20 min;

or, the QD-Zr⁴⁺-preparation of Cy5 nanostructures comprising: mixing the probe, ATP, PKA and/or PNK reaction buffer solution, PKA and/or PNK reaction solution, reacting at 30-45 deg.C for 1-4 hr, and incubating at 55-75 deg.C.

The preparation method has simple process and short time consumption, does not need expensive antibody and specially labeled ATP, does not need any washing and separating steps, and has very simple operation. Meanwhile, complex probe design and nucleic acid amplification steps are not needed, and the method can be carried out under isothermal and homogeneous conditions.

In one embodiment of the present disclosure, a method of screening for a PKA and/or PNK inhibitor, the method comprising co-reacting a model inhibitor with the one zirconium ion-mediated nanosensor; further, the model inhibitor is H-89 model inhibitor and/or adenosine diphosphate and ammonium sulfate. Because the zirconium ion-mediated nano sensor has the characteristic of high detection sensitivity, the sensitivity and the accuracy of screening the inhibitor can be greatly improved by utilizing the nano sensor and the inhibitor to carry out competitive reaction. The experimental result shows that the nano sensor is used for screening the inhibitor, so that the accuracy is higher, and the application of the nano sensor is further expanded.

In one embodiment of the present disclosure, the use of said QD-capture probe nanostructure and/or said kit for PKA and/or PNK detection and/or said zirconium ion-mediated nanosensor and/or said method of preparation of a zirconium ion-mediated nanosensor for PKA and/or PNK activity detection.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

Example 1

Preparation of QD-capture probe nanostructures:

150 nmol per liter capture probe (5' P-TTT TTT TTT T-biotin), 2.5 nmol per liter QD, 2. mu.l 10 XQD buffer (1 mol per liter Tris-HCl),30 mmol per liter magnesium chloride (MgCl)₂) 100 mmoles of ammonium sulphate (NH) per liter₄)₂SO₄) pH 8.0) was added to a 20 μ l reaction system and incubated for 20 minutes at room temperature under exclusion of light. The QD-capture probe nanostructure resulting from the reaction was stored at 4 ℃ for further use.

2.QD-Zr⁴⁺-preparation of Cy5 nanostructures and fluorescence spectroscopy:

reaction for PKA:

PKA was diluted with PKA dilution buffer (20 mmol per liter Tris-HCl,50 mmol per liter sodium chloride (NaCl),1 mmol per liter ethylenediaminetetraacetic acid (EDTA), 50% glycerol, pH 7.5) for its activity assay. The course of the PKA-catalyzed phosphorylation reaction was 20. mu.l of a reaction solution containing 2.5. mu.mol per liter of polypeptide probe (Peptide probe: Cy5-KLRRASL), 10. mu.mol per liter of adenosine 5' -triphosphate (ATP), 2. mu.l of 10 XPKA reaction buffer (500 mM Tris-HCl,100 mM magnesium chloride, 0.01% Brij-35, pH 7.5) and varying concentrations of PKAAfter mixing, the reaction was terminated by reaction at 37 ℃ for 2 hours and incubation at 65 ℃ for 20 minutes. To construct QD-Zr⁴⁺Cy5 nanostructures, 1.8. mu.l of 1 mmol per liter Zr⁴⁺Add to 20 microliters of the prepared QD-capture probe nanostructure solution to give 30 microliters of mixed solution, and incubate at room temperature for 1 hour. The PKA phosphorylation reaction product was then mixed with the above reaction solution to give 50 μ l of mixed solution and incubated at room temperature for 1 hour. The fluorescence signal of the reaction product was measured by an FLS-1000 fluorescence spectrometer (Edinburgh instruments, Inc., Liwenston, UK). Emission spectra in the range 550 nm-750 nm were recorded at an excitation wavelength of 405 nm, and data analysis was performed by reading fluorescence intensities at 608 nm (maximum emission wavelength of quantum dots) and 670 nm (maximum emission wavelength of Cy 5).

Reaction for PNK:

the process of PNK-catalyzed phosphorylation reaction was carried out by mixing 20. mu.l of a reaction solution containing 2.5. mu.mol/l of a DNA probe (DNA probe: GTT GAG C-Cy5), 0.8 mmol/l of ATP, 2. mu.l of 10 XPNK reaction buffer (700 mmol/l Tris-HCl,100 mmol/l magnesium chloride, pH 7.5) and PNK at different concentrations, reacting at 37 ℃ for 1.5 hours, and then incubating at 65 ℃ for 20 minutes to terminate the reaction. Subsequent experiments were performed according to the PKA detection method described above.

3. Single molecule detection and data analysis:

the reaction product was diluted 50-fold with 1 × QD buffer (100 mmol per liter Tris-HCl,3 mmol per liter magnesium chloride, 10 mmol per liter ammonium sulfate, pH 8.0) for single molecule detection. 10 microliter of sample was pipetted directly onto the cover slip and imaged with a total internal reflection fluorescence microscope. A 405 nm laser is used to excite the quantum dots. The fluorescent signal was passed through a 100 × objective lens (Olympus, Japan) to obtain the emission signals of the quantum dots and Cy 5. The exposure time for imaging was 500 milliseconds. Image J software was used for Cy5 molecular counting, and data analysis was performed using the number of Cy5 molecules obtained from 10 pictures, with an Image area of 500 × 500 pixels selected.

4. Inhibitor screening:

the PKA inhibitor assay was performed by adding different concentrations of H-89 to the PKA reaction mixture, and the following reaction procedure and single molecule detection followed the procedure described above. PNK inhibitor experiments Adenosine Diphosphate (ADP) and ammonium sulfate were chosen as model inhibitors and added separately to the PNK reaction mixture, and the subsequent reaction process and single molecule detection followed the procedure described above.

5. Cell culture and preparation of cell extracts:

PKA cell assay:

cervical cancer cells (HeLa) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, maintaining a humid atmosphere containing 5% carbon dioxide at a temperature of 37 ℃. The cell culture medium was changed to serum-free medium for 4 hours before addition of forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX). Fsk and IBMX dissolved in dimethyl sulfoxide (DMSO) were added to the medium and cultured for 30 minutes to activate intracellular PKA. An equal volume of DMSO was added to the medium to culture the cells of the control group. Fsk, IBMX and H-89 were added together to the medium and incubated for 30 minutes for intracellular PKA inhibitor experiments. In cell extraction experiments, cells were harvested by trypsinization and washed twice with 1 × Phosphate Buffer (PBS) (pH 7.4). The cells were then collected in a centrifuge tube and centrifuged at 800 revolutions per minute (rpm) for 5 minutes. Cell number was measured by a Countstar cytometer (IC1000, wilminton, talawa, usa). Approximately 1X 106 cells were suspended in 100. mu.l of RIPA lysis buffer (50 mmoles per liter of Tris (pH7.4), 150 mmoles per liter of sodium chloride (NaCl), 1% of polyethylene glycol octylphenyl ether (Triton X-100), 1% of sodium deoxycholate (sodium deoxycholate), 0.1% of Sodium Dodecyl Sulfate (SDS)) and incubated on ice for 10 minutes, followed by sonication. The lysate was centrifuged at 14000 Xg for 5 minutes at 4 ℃ and the supernatant was collected to determine PKA activity. Protein concentration was quantified using an improved Lowry protein assay kit (C504041, biotechnology, shanghai, china).

PNK cell assay:

HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin, maintaining a humid atmosphere containing 5% carbon dioxide at a temperature of 37 ℃. Mature cells were cultured and counted by a Countstar cytometer. Then, cell lysates were prepared using a nucleoprotein extraction kit (Biotechnology, Shanghai, China) according to the manufacturer's instructions. The collected cell lysates were immediately used for PNK activity assay or stored at-80 ℃ for use.

Principle of experiment (as shown in FIG. 1a and FIG. 1b)

A polypeptide probe (Peptide probe, FIG. 1a) containing a PKA recognition site (serine) is designed, and a Cy5 fluorescent group is marked at the carboxyl (C-) end of the probe. And catalyzing the transfer of the gamma-phosphate group on the ATP to the hydroxyl group of serine in the polypeptide probe in the presence of PKA to generate a phosphorylated polypeptide probe. Adding Zr⁴⁺Then, through-PO₃ ^2--Zr⁴⁺-PO₃ ^2-Interaction of phosphorylated polypeptide probe to QD-capture probe nanostructure to form QD-Zr⁴⁺-Cy5 nanostructure, resulting in FRET for QD and Cy 5. Therefore, detection of Cy5 signal at the single molecule level enables detection of PKA activity. In the absence of PKA, the Cy 5-modified polypeptide probe cannot be phosphorylated and thus cannot be assembled onto the QD surface, so FRET does not occur between QD and Cy 5.

To demonstrate the versatility of this nanosensor, it was further used to analyze PNK activity. Cy 5-labeled DNA probe (DNAprobe, FIG. 1b) was designed as a substrate for PNK analysis. When PNK is present, it can catalyze the transfer of the gamma-phosphate group from ATP to the 5' -hydroxyl of the oligonucleotide or nucleic acid, resulting in a phosphorylated DNA probe. Adding Zr⁴⁺Then, through-PO₃ ^2--Zr⁴⁺-PO₃ ^2-Interaction of phosphorylated DNA probe to QD-capture probe nanostructure to form QD-Zr⁴⁺-Cy5 nanostructure, resulting in FRET for QD and Cy 5. Therefore, detection of Cy5 signal at the single molecule level enables detection of PNK activity. When PNK is not present, the Cy 5-modified DNA probe cannot be phosphorylated and thus cannot be assembled to the QD surface, so FRET does not occur between QD and Cy 5.

Example 2

1. Feasibility test

The feasibility of the nanosensor obtained in example 1 for PKA detection (fig. 2) and PNK detection (fig. 3) was verified using fluorescence spectroscopy. No Cy5 signal was detected in the absence of PKA/PNK (FIG. 2/FIG. 3, upper blue line), indicating that QD-Zr could not be formed in the absence of PKA/PNK⁴⁺-Cy5 nanostructures. In contrast, with the addition of PKA/PNK, a significant Cy5 fluorescence signal was observed, accompanied by a decrease in QD fluorescence signal (fig. 2/fig. 3, lower red line), indicating that phosphorylation reactions by PKA can induce QD-Zr⁴⁺-Cy5 nanostructure formation and resulted in efficient FRET between QD and Cy 5.

The feasibility of the sensor in detecting PKA/PNK activity is further verified by single-molecule detection. As shown in fig. 4, in the absence of PKA, only QD fluorescence signal was detected (fig. 4A, green), and no Cy5 fluorescence signal was detected (fig. 4B), indicating that FRET would not occur without PKA. In contrast, fluorescence signals for QD (fig. 4D, green) and Cy5 (fig. 4E, red) were observed simultaneously in the presence of PKA, and both signals had distinct co-localization regions (fig. 4F, yellow), indicating that the presence of PKA can initiate FRET. As shown in fig. 5, in the absence of PNK, only QD fluorescence signal (fig. 5A, green) was detected, and Cy5 fluorescence signal (fig. 5B) was not detected, indicating that FRET would not occur without PNK. In contrast, fluorescence signals for QD (fig. 5D, green) and Cy5 (fig. 5E, red) were observed simultaneously in the presence of PNK, and both signals had a distinct co-localization zone (fig. 5F, yellow), indicating that FRET can be initiated by the presence of PNK.

2. Sensitivity detection

Under the optimal reaction conditions, the sensitivity of the nanosensor for detecting PKA was evaluated by measuring the variation of the number of Cy5 with different concentrations of PKA. As shown in fig. 6A, as PKA concentration increased from 0.003 units per ml to 100 units per ml, the Cy5 count increased accordingly. The Cy5 counts were linearly related to the log form of PKA concentration at concentrations ranging from 0.003 units per ml to 3 units per ml (fig. 6B). The regression equation is that N is 218.79+71.63log₁₀ C(R²0.9944) where N represents Cy5 molecules and C represents PKA concentration (units per milli-gram)Liters). According to the principle of adding 3 times of standard deviation to blank signal, the detection limit of the method can be calculated to be 8.82 multiplied by 10^-4Units per milliliter. The sensitivity of the method is improved by 3 orders of magnitude compared with an electrochemical analysis method (0.15 unit per milliliter) based on phosphorylation labels and enzyme signal amplification, and is improved by 2 orders of magnitude compared with an electrochemical biosensor (0.083 unit per milliliter) based on carboxypeptidase Y auxiliary peptide cracking.

The sensitivity of the nanosensor to detect PNK was evaluated by measuring the variation of the number of Cy5 with different concentrations of PNK. As shown in fig. 7A, as the PNK concentration increased from 0.0001 units per ml to 10 units per ml, the Cy5 count increased accordingly. The Cy5 counts were linearly related to the log form of PNK concentration at PNK concentrations ranging from 0.0001 units per milliliter to 0.03 units per milliliter (fig. 7B). The regression equation is that N is 455.41+93.85log₁₀ C(R²0.9956) where N represents Cy5 molecules and C represents PNK concentration (units per ml). According to the principle of adding 3 times of standard deviation to blank signal, the detection limit of the method can be calculated to be 1.40 multiplied by 10^-5Units per milliliter. The sensitivity of the method is improved by 2 orders of magnitude compared with a fluorescence method (0.0067 unit per milliliter) and a proportional fluorescence analysis method (0.0037 unit per milliliter) based on a three-dimensional DNA nano machine. These results clearly demonstrate that the single quantum dot nanosensor can be used for highly sensitive detection of PNK.

The increase in sensitivity of the method can be attributed to:

(1)Zr⁴⁺(ii) capable of distinguishing between phosphorylated and non-phosphorylated polypeptide probes and specifically self-assembling phosphorylated polypeptide probes onto the QD surface;

(2) the nano structure formed by assembling a plurality of Cy5 modified polypeptide probes on the surface of one QD can improve the FRET efficiency;

(3) single molecule detection has a high signal-to-noise ratio.

3. Experiment of specificity

To investigate the specificity of this nanosensor for PKA detection, Bovine Serum Albumin (BSA), immunoglobulin g (igg), T4 polynucleotide kinase (PNK) and Uracil DNA Glycosylase (UDG) were used as interfering proteins. As shown in fig. 8(a), no significant Cy5 signal was detected in the presence of interfering proteins, indicating that no FRET occurred between QD and Cy 5. In contrast, in the presence of PKA, a very high Cy5 signal was detected, indicating that there is efficient FRET between QD and Cy 5. These results clearly demonstrate the better selectivity of the method when applied to the detection of PKA.

To investigate the specificity of this nanosensor for PNK detection, we used Bovine Serum Albumin (BSA), immunoglobulin g (igg), PKA and Uracil DNA Glycosylase (UDG) as interfering proteins. As shown in fig. 9, in the presence of interfering proteins, no significant Cy5 signal was detected, indicating that no FRET occurred between QD and Cy 5. In contrast, when PNK is present, a very high Cy5 signal can be detected, indicating that there is efficient FRET between QD and Cy 5. These results clearly demonstrate the better selectivity of the method when applied to the detection of PKA.

4. Inhibitor assay

To verify that the nanosensor can be used for screening of PKA inhibitors, H-89 was selected as a model inhibitor for validation. H-89 can competitively bind to the ATP binding site on the PKA catalytic subunit to inhibit PKA activity. The number of Cy5 molecules was measured in the presence of different concentrations of H-89. As shown in FIG. 8(B), the relative activity of PKA decreased with increasing H-89 concentration. The inhibition of PKA enzyme by H-89 was assessed by the IC50 value (maximum half inhibitory concentration). The IC50 value measured by this method was 41.57 nanomoles per liter, consistent with the results of the dual signal read fluorescence assay (39.5 nanomoles per liter). This result indicates that the method can be used to screen for inhibitors of PKA.

In order to verify the feasibility of the nanosensor for screening PNK inhibitors, adenosine diphosphate and ammonium sulfate are selected as model inhibitors for verification. Adenosine diphosphate is a byproduct of PNK catalytic phosphorylation reaction, and because adenosine diphosphate and 5' -phosphorylated DNA coexist to reverse phosphorylation reaction, PNK activity is severely inhibited. In addition, high concentrations of salts (e.g., ammonium sulfate) can induce conformational changes in the PNK enzyme and effectively inhibit PNK activity. As the concentrations of adenosine diphosphate (fig. 10A) and ammonium sulfate (fig. 10B) increased, the relative activity of PNK decreased correspondingly. The IC50 values for adenosine diphosphate and ammonium sulfate measured by this method were 0.96 mmol per liter and 9.21 mmol per liter, respectively, consistent with the IC50 value for adenosine diphosphate (0.8 mmol per liter) and the IC50 value for ammonium sulfate (9.88 mmol per liter) measured by bioluminescence analysis based on molecular beacons, respectively. This indicates that the nanosensor can be used to screen for PNK inhibitors.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> zirconium ion mediated nano sensor, preparation method and application

<130> 202112601

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<170> PatentIn version 3.3

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