阅读说明：本技术 一种检测碱性磷酸酶的生物传感器及其检测方法和应用 (Biosensor for detecting alkaline phosphatase, detection method and application thereof ) 是由 张春阳 王黎娟 刘昊 于 2021-07-27 设计创作，主要内容包括：本发明涉及检测分析技术领域,具体涉及一种检测碱性磷酸酶的生物传感器及其检测方法和应用,所述传感器包括DNA探针,末端脱氧核苷酸转移酶TdT和2'-脱氧胸苷-5'-三磷酸dTTPs。本发明的生物传感器及检测方法具有良好的特异性和高灵敏度,由于ALP催化去磷酸化和TdT介导的无模板聚合的高特异性,3'端修复驱动的poly-A MBs树突状自组装具有很高的扩增效率,以及无模板TdT辅助poly-A MBs延伸、组装和激活的方式非常容易,可以在等温条件下一步快速实现,只需一个DNA探针(即poly-A MB),且poly-A MB的延伸、组装和活化只需一个工具酶(即不依赖模板的TdT)即可完成；可以评价动力学参数,筛选潜在抑制剂,估算细胞抑制效应,甚至可以测定人类血清样本中的碱性磷酸酶,用于临床试验。(The invention relates to the technical field of detection and analysis, in particular to a biosensor for detecting alkaline phosphatase, a detection method and application thereof, wherein the biosensor comprises a DNA probe, terminal deoxynucleotidyl transferase TdT and 2 '-deoxythymidine-5' -triphosphate dTTPs. The biosensor and the detection method have good specificity and high sensitivity, because ALP catalyzes dephosphorylation and TdT mediated template-free polymerization has high specificity, the dendritic self-assembly of poly-A MBs driven by 3' end repair has high amplification efficiency, and the mode of template-free TdT assisted extension, assembly and activation of poly-A MBs is very easy, the biosensor and the detection method can be quickly realized in one step under isothermal conditions, only one DNA probe (namely poly-A MB) is needed, and the extension, assembly and activation of poly-A MB can be completed only by one tool enzyme (namely TdT independent of the template); kinetic parameters can be evaluated, potential inhibitors can be screened, cytostatic effects can be estimated, and even alkaline phosphatase in human serum samples can be assayed for clinical trials.)

1. A biosensor for detecting alkaline phosphatase, comprising a DNA probe, terminal deoxynucleotidyl transferase TdT and 2 '-deoxythymidine-5' -triphosphate dTTPs.

2. The biosensor for detecting alkaline phosphatase according to claim 1, wherein the nucleotide sequence of the DNA probe is: 5' -CGA TGC AGA AAA AAA AAA AAA AAA AAA ACT GCA TCG-P-3′。

3. The alkaline phosphatase-detecting biosensor according to claim 1, wherein the sensor further comprises CoCl₂And a buffer solution, wherein the buffer solution preferably comprises a CutSmart buffer solution and a TdT reaction buffer solution.

4. A method for detecting alkaline phosphatase, comprising the steps of:

the DNA probe poly-A MB was added to a solution containing different concentrations of ALP, TdT, dTTPs, CoCl₂And incubating at a certain temperature in a reaction system of a buffer solution, and carrying out ALP catalyzed dephosphorylation and subsequent template-free TdT assisted poly-A MBs dendritic self-assembly.

5. The method of detecting alkaline phosphatase as claimed in claim 4, wherein in one or more embodiments, the nucleotide sequence of the DNA probe is: 5' -CGA TGC AGA AAA AAA AAA AAA AAA AAA ACTGCA TCG-P-3' ("P" denotes modified phosphate group, italicized "C" and "T" denotes modified quenching molecule BHQ1 and fluorescent molecule FAM).

6. The method for detecting alkaline phosphatase according to claim 4, wherein the incubation is performed at 36-38 ℃ for 60-65 minutes, preferably at 37 ℃ for 60 minutes.

7. The method for detecting alkaline phosphatase according to claim 4, wherein the DNA probe is prepared by: diluting the synthesized oligonucleotide with Tris-EDTA buffer solution to prepare stock solution; diluting poly-A MBs by using hybridization buffer, incubating for 5-6 min at 90-95 ℃, and slowly cooling to room temperature to obtain a perfect folded hairpin structure.

8. The method for detecting alkaline phosphatase according to claim 4, wherein the concentration of poly-A MB is: 400-500 nM; alternatively, the concentration of TdT is: 35-45U; alternatively, the concentration of dTTPs is: 164-; alternatively, CoCl₂The concentration of (A) is as follows: 0.2-0.3 mM.

9. The method for detecting alkaline phosphatase according to claim 4, wherein the buffer solutions comprise a CutSmart buffer solution and a TdT reaction buffer solution, and further wherein the CutSmart buffer solution has a concentration of: 1 × CutSmart buffer: 10mM Mg (AC)₂,50mM KAC,20mM Tris-Ac,0.1mg/mL BSA, pH 7.9; the concentration of the TdT reaction buffer solution is as follows: 1 × TdT reaction buffer: 10mM Mg (Ac)₂,50mM potassium acetate,20mM Tris-Ac,pH 7.9。

10. Use of the biosensor according to any one of claims 1 to 3 or the detection method according to any one of claims 4 to 9 for the detection of alkaline phosphatase.

Technical Field

The invention relates to the technical field of detection and analysis, in particular to a biosensor for detecting alkaline phosphatase, and a detection method and application thereof.

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.

Alkaline phosphatase (ALP) is an essential phosphohydrolase present in all tissues of the human body, and catalyzes the hydrolysis of phosphate groups in various biomolecules such as nucleic acids, proteins, carbohydrates, etc. Play an important role in the regulation of various biological processes such as cell division, proliferation, apoptosis and signal transduction. ALP levels in adult normal human serum are 40-190U/L, and above or below normal threshold levels can induce a variety of human diseases (e.g., bone disorders, cardiovascular disorders, inflammatory disorders), and cancers (e.g., oral cancer, prostate cancer, liver cancer). More importantly, because ALP has the advantages of high catalytic activity, wide substrate specificity, good stability and low cost, the ALP is widely applied to the fields of biomolecule monitoring, histochemical staining, gene expression analysis and the like. Therefore, the method for detecting the alkaline phosphatase activity is simple, convenient, specific, sensitive and reliable, and has important significance for biomedical research, clinical diagnosis and tumor treatment.

In view of the important role of alkaline phosphatase in biological and clinical research, analytical studies on alkaline phosphatase have been vigorously developed. Conventional methods for detecting ALP include colorimetry, electrochemistry, Surface Enhanced Resonance Raman Scattering (SERRS), and fluorescence. Colorimetric method using TiO₂Nanoparticles (NPs) or G20-cu (ii) catalyze the oxidation of 3,3', 5,5' -Tetramethylbenzidine (TMB) with hydrogen peroxide to produce colored oxTMB, enabling visual detection of ALP, but their nanomaterial preparation takes a long time. Electrochemical methods achieve ratiometric detection of ALP by the release of ferrocenylphosphate by dephosphorylation of ALP to release ferrocenylamine, but it requires laborious immobilization of the antibody and repeated washing steps. The SERRS method is to synthesize indigo dye derivatives by ALP-catalyzed 5-bromo-4-chloro-3-indolyl phosphate (BCIP), but inevitably requires complicated operations and expensive spectrometers. Fluorescence methods have recently attracted more and more attention because of their advantages of simple operation and high sensitivity, and are often used for the measurement of alkaline phosphatase. The fluorescence analysis method reported at present is mainly divided into nanometerMaterial method and synthetic chemical probe method. In nanomaterial-based fluorescence analysis, carbon dots, graphene quantum dots, copper nanoparticles, silver nanoclusters are commonly used to monitor ALP-induced fluorescence changes. In the fluorescence analysis based on the synthetic chemical probe, polyethyleneimine modified by trimethylamine and ethyl lactone, moc-K (FITC) FFYp, Aggregation Induced Emission (AIE) fluorescein, a two-photon fluorescent probe and a tetrapolydiene probe are all used for quantitative detection of ALP. However, these fluorescence analysis methods are complex to synthesize, laborious to characterize, and subject to interference from many factors (e.g., fluorescence photobleaching, excitation intensity, emission collection efficiency, and medium polarity), which limits their practical applications. It is worth noting that the currently developed methods for detecting ALP mostly rely only on the catalytic reaction of ALP for signal readout, and are simple but lack signal amplification strategies and have poor sensitivity. Therefore, it is necessary to establish a novel, powerful, simple, specific and sensitive system for detecting alkaline phosphatase for various diagnoses and treatments.

In recent years, DNA encodes unique structural information (i.e., predictable structure, programmable sequence, and precise molecular length (0.34nm bp) in Watson-Crick double helix structure^-1) And becomes a marker biological material of a molecular building block. And are referred to in an emerging field as "supramolecular DNA assembly" to produce higher order functional nanostructures (e.g., DNA dendritic molecules, DNA tetrahedrons, and DNA nanowires). Because of its high degree of customizability, spatial addressability, and excellent biocompatibility, DNA can provide a versatile platform for devices designed for sensing, computing, and driving, and has wide applications in biological, chemical, and physical sciences. However, the construction of DNA nanostructures is typically based on the identification, assembly and consumption of large numbers of scaffolds, making probe design, conformational dynamics and sensing systems more complex, expensive and time consuming.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a biosensor for detecting alkaline phosphatase and a detection method and application thereof, which utilize the uniqueness of TdT and the inherent advantages of Molecular Beacons (MB) to firstly prove the feasibility of 3' -terminal repair-driven dendritic nano-assembly of poly-A MBs and use the feasibility for one-step quantification of ALP in human serum; in the invention, a 3' -phosphorylated molecular beacon is designed, and poly-A is embedded into a ring (namely poly-A MB), which is a catalytic substrate of ALP and a construction template of a dendritic DNA nanostructure. The presence of ALP can catalyze dephosphorylation of poly-A MBs, induce template-free polymerization mediated by TdT, generate long-chain poly-thymidine (T) sequences, a poly-T chain can be used as an anchoring template to hybridize with free poly-A MBs, so that FAM-BHQ1 pairs are dissociated, all 3' -hydroxylated poly-A MBs on the chain can be continuously extended through the TdT to generate branched poly-T chains, so that more free poly-A MBs are hybridized, more FAM-BHQ1 pairs are dissociated, and the poly-A MBs are subjected to multiple rounds of extension, assembly and activation to form dendritic DNA nanostructures, so that a large number of fluorophores are dissociated from FAM-BHQ1 pairs to generate exponentially amplified fluorescent signals. The biosensor has good specificity and high sensitivity for detecting alkaline phosphatase, can be quickly realized by only one DNA probe (namely poly-A MB) under an isothermal condition, can realize the extension, assembly and activation of the poly-A MB by only one tool enzyme (namely TdT independent of a template), and has huge application prospect in biochemical and clinical research.

In order to achieve the above object, the technical solution of the present invention is as follows:

in a first aspect of the present invention, there is provided a biosensor for detecting alkaline phosphatase, the biosensor comprising a DNA probe (i.e., poly-A MB), terminal deoxynucleotidyl transferase TdT and 2 '-deoxythymidine-5' -triphosphate dTTPs;

in one or more embodiments, the nucleotide sequence of the DNA probe is: 5' -CGA TGC AGA AAA AAA AAA AAA AAA AAA ACT GCA TCG-P-3' ("P" denotes a modified phosphate group, italicized "C" and "T" denotes modification of the quencher molecule BHQ1 and the fluorescent molecule FAM);

the DNA probe is poly-A MB, a poly-adenine (A) intercalating molecular beacon, which, like the common MB, is circular and labeled with a Fluorophore (FAM) and a quencher (BHQ1), but when modified to 3' -PO₄End tipAnd poly (A) is embedded in the cyclic structure, it can serve both as a catalytic substrate for ALP, a basic constituent of dendritic DNA nanostructure, and as a generator of an effective fluorescent signal.

Terminal deoxynucleotidyl transferase (TdT) is a template-independent repair polymerase that catalyzes the random incorporation of mononucleotides to the 3' -hydroxyl (OH) terminus of single-stranded dna (ssdna) fragments, facilitating the fabrication of multifunctional sensing strategies.

In one or more embodiments, the sensor further comprises CoCl₂And a buffer solution, wherein the buffer solution preferably comprises a CutSmart buffer solution and a TdT reaction buffer solution.

In a second aspect of the present invention, there is provided a method for detecting alkaline phosphatase, comprising the steps of:

the DNA probe poly-A MB was added to a solution containing different concentrations of ALP, TdT, dTTPs, CoCl₂And incubating at a certain temperature in a reaction system of a buffer solution, and carrying out ALP catalyzed dephosphorylation and subsequent template-free TdT assisted poly-A MBs dendritic self-assembly.

In one or more embodiments, the incubation is at 36-38 ℃ for 60-65 minutes, preferably at 37 ℃ for 60 minutes; the detection method only needs to be quickly realized in one step under isothermal conditions, and the detection of the alkaline phosphatase is more convenient and quicker.

In one or more embodiments, the nucleotide sequence of the DNA probe is: 5' -CGA TGC AGA AAA AAA AAA AAA AAA AAAACT GCA TCG-P-3' ("P" denotes a modified phosphate group, italicized "C" and "T" denotes modification of the quencher molecule BHQ1 and the fluorescent molecule FAM);

in one or more embodiments, the DNA probe is prepared by: diluting the synthesized oligonucleotide with 1 × Tris-EDTA buffer solution to prepare stock solution; with hybridization buffer (10mM Tris-HCl, 1.5mM MgCl)₂pH 8.0) to 10. mu.M, incubated at 95 ℃ for 5min and slowly cooled to room temperature to obtain a perfect folded hairpin structure.

In one or more embodiments, the concentration of poly-A MB is: 400-500 nM;

in one or more embodiments, the concentration of TdT is: 35-45U;

in one or more embodiments, the concentration of dTTPs is: 164-;

in one or more embodiments, CoCl₂The concentration of (A) is as follows: 0.2-0.3 mM;

in one or more embodiments, the buffer comprises a CutSmart buffer and a TdT reaction buffer, and further the CutSmart buffer has a concentration of: 1 XCutSmart buffer (10mM Mg (AC)₂50mM KAC,20mM Tris-Ac,0.1mg/mL BSA, pH 7.9); the concentration of the TdT reaction buffer solution is as follows: 1 XTdT reaction buffer (10mM Mg (Ac)₂,50mM potassium acetate,20mM Tris-Ac,pH 7.9)。

In a third aspect of the invention, there is provided a use of the biosensor of the first aspect or the detection method of the second aspect in alkaline phosphatase detection.

The specific embodiment of the invention has the following beneficial effects:

the biosensor and the detection method have good specificity and high sensitivity, and have good specificity and high sensitivity due to the high specificity of ALP catalytic dephosphorylation and TdT mediated template-free polymerization, the dendritic self-assembly of poly-A MBs driven by 3' end repair has high amplification efficiency, and the mode of template-free TdT assisted extension, assembly and activation of the poly-A MBs is very easy, so that the method has good specificity and high sensitivity, and the detection limit is 3.2 multiplied by 10^-7U/μ L, ALP can be quantified even at the single cell level;

the DNA probe is skillfully designed, can realize simple and convenient detection of ALP, provides a simple, convenient and stable platform for monitoring various phosphatases, and has huge application potential in the aspects of biochemical research, clinical diagnosis, drug discovery, cancer treatment and the like; the high specificity of ALP catalyzed dephosphorylation and TdT mediated template-free polymerization, the high amplification efficiency of 3' end repair driven dendritic self-assembly of poly-A MBs and the very easy extension, assembly and activation mode of template-free TdT assisted poly-A MBs;

the method can be quickly realized in one step under the isothermal condition, only one DNA probe (namely poly-A MB) is needed, and the extension, assembly and activation of the poly-A MB can be completed only by one tool enzyme (namely the TdT independent of a template);

kinetic parameters can be evaluated, potential inhibitors can be screened, cytostatic effects can be estimated, and even alkaline phosphatase in human serum samples can be assayed for clinical trials.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic representation of dendritic nano-assembly of 3' end repair driven poly-A molecular beacons for one-step quantification of ALP in human serum.

FIG. 2 is a diagram showing the feasibility verification of gel electrophoresis and fluorescence experiments in the feasibility experiment of the present invention;

wherein, FIG. 2A is agarose gel electrophoresis analysis, Lane M, DNA marker; lane 1, synthesis of poly-A MB; lane 2, poly-A MB + TdT; lane 3, poly-A MB + TdT + ALP. The concentration of ALP was 5X 10^-3U/μL；

FIG. 2B is a graph of the effect of fluorescence experiments by measuring the response of FAM emission spectra to ALP;

the three curves in fig. 2B are from top to bottom: fluorescence effect in the presence of poly-A MB + TdT + ALP, poly-A MB + ALP, and poly-A MB + TdT, the concentration of ALP being 2.5X 10^-3U/μL；

In 2A and 2B, the concentration of TdT was 40U and the concentration of poly-A MB was 600 nM.

FIG. 3 is a graph of experimental data for sensitivity detection according to the present invention;

FIG. 3A is a graph showing the response change of fluorescence emission spectra to different concentrations of ALP;

in fig. 3A, the curves are from top to bottom: 5X 10^-3U/μL；2.5×10^-3U/μL；1.5×10^-3U/μL；1×10^{- 3}U/μL；5×10^-4U/μL；2.5×10^-4U/μL；1×10^-4U/μL；5×10^-5U/μL；2.5×10^-5U/μL；1×10^-5U/μL；5×10^-6U/μL；2.5×10^-6U/μL；1×10^-6U/μL；5×10^-7U/μL；control；

FIG. 3B is a graph showing the change in fluorescence intensity with ALP concentration; at 5X 10^-7-1×10^-3In the U/mu L range, the fluorescence intensity and the ALP concentration logarithm value are in a linear relation; the panels in FIG. 3B are: plot of fluorescence intensity versus log of ALP concentration.

FIG. 4 is a graph showing the data of the specificity detection experiment, and the fluorescence intensities measured for different enzymes in each histogram in FIG. 4 are respectively control group (reaction buffer solution only), 0.1g/L BSA, 0.1g/L PP, and 2.5X 10^-3U/. mu.L trypsin, 2.5X 10^-3U/μL，2.5×10³U/μL hOGG1，2.5×10^-3U/. mu.L M.SssI and 2.5X 10^-3U/μLALP。

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 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 application. 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 stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

ALP catalyzed dephosphorylation and subsequent template independent TdT assisted dendritic self-assembly of Poly-A MBs.

All oligonucleotides were diluted with 1 × Tris-EDTA buffer to prepare stock solutions. With hybridization buffer (10mM Tris-HCl, 1.5mM MgCl)₂pH 8.0) to 10. mu.M, incubated at 95 ℃ for 5min and slowly cooled to room temperature to obtain a perfect folded hairpin structure. Then 0.9. mu.L of 450nM poly-A MBs was added to a solution containing different concentrations of ALP, 3. mu.L of 10 XTSmart buffer, 40U TdT, 3. mu.L of 10 XTdT reaction buffer, 3. mu.L of 10 XCOCL₂And 166 u M dTTPs 30 u L reaction system, at 37 degrees C were incubated for 60 minutes, dephosphorylation control template free TdT assisted poly-A MBs dendritic self assembly.

Gel electrophoresis analysis and fluorescence measurement: to investigate the reaction products of dephosphorylation and templateless TdT-assisted dendritic self-assembly of poly-A MBs. We used a 3% agarose gel and analyzed 30. mu.L of the reaction product in 1 XTAE buffer (40mM Tris-Ac,1mM EDTA, pH 8.0). After electrophoresis at 110V for 40 minutes, the gel was directly visualized by a ChemiDoc MP imaging system (Hercules, California, USA). For fluorescence measurement, 30. mu.L of the reaction product was diluted with ultrapure water to a final volume of 60. mu.L. FAM fluorescence was measured by HitachiF-7000 spectrofluorometer (Tokyo, Japan), excitation wavelength was 488nm, emission spectrum was measured in the wavelength range of 500-650nm, slit width was 5nm, and fluorescence intensity at 520nm emission wavelength was recorded for data analysis.

Alkaline phosphatase determination principle: the principle of dendritic nano-assembly of 3' end repair driven poly-A molecular beacons is shown in FIG. 1. In the present invention, only one functional oligonucleotide probe, i.e., a DNA probe, called a poly-adenine (A) intercalating molecular beacon (i.e., poly-A MB), was ingeniously designed. Like the common MB, poly-A MB is cyclic and the labeled Fluorophore (FAM) and quencher (BHQ1) are blocked with respect to each other, but modified as 3' -PO₄The terminal and poly (A) are embedded in the loop structure, which can serve both as a catalytic substrate for ALP, a basic constituent of the dendritic DNA nanostructure, and as a generator of an effective fluorescent signal. Due to Fluorescence Resonance Energy Transfer (FRET) between FAM and BHQ1, poly-A MB is inactive and self-quenched. The biosensor and the detection method for detecting ALP comprise two partsThe method comprises the following steps: (1) ALP catalyzed dephosphorylation of the 3 '-terminus of poly-A MB to yield 3' -PO₄The group is converted into a 3' -OH group; (2) self-assembly of templaterless TdT-assisted dendritic poly-a MBs was accompanied by efficient fluorescence recovery. In the presence of ALP, poly-A MB is dephosphorylated at the 3 '-end from 3' -PO₄The group is hydrolyzed into a 3' -OH group; the resulting 3 '-hydroxylated poly-A MB will serve as an effective primer for TdT by repeated addition of 2' -deoxythymidine-5 '-triphosphates (dTTPs) at the 3' -OH terminus to induce template-free polymerization extension, thereby generating a long poly-thymidine (T) sequence. Importantly, since poly-A MB and poly-A are designed by cycle, the long poly-T chain can be used as an anchoring template to hybridize with a plurality of free poly-A MBs, so that a ring structure is opened and the FAM-BHQ1 pair is dissociated, and a fluorescent signal is recovered (the first signal amplification stage); in addition, all 3' -hydroxylated poly-A MBs on this strand can be extended by successive polymerization without template TdT, generating a large number of branched-length poly-T strands, resulting in more free poly-A MBs hybridization and more dissociation of the FAM-BHQ1 FRET pair, thereby inducing recovery of the high fluorescence signal (second amplification stage); through multiple rounds of extension, assembly and activation of poly-A MBs, dendritic DNA nanostructures are automatically formed, resulting in dissociation of a large number of fluorophore molecules (i.e., FAM) from the FAM-BHQ1 FRET pair, resulting in an exponentially amplified fluorescent signal (nth amplification stage). In contrast, in the absence of ALP, no poly-A MBs were dephosphorylated at the 3' end and could not induce dendritic self-assembly of template-free TdT-assisted poly-AMBs, so no fluorophore molecules were dissociated from the FAM-BHQ1 FRET pair, and no fluorescent signal was observed.

This method has distinct advantages over previously reported ALP detection methods: (1) the dendritic nano assembly of poly-A MBs driven by 3' end repair can induce exponential signal amplification, and has higher sensitivity; (2) the specific nucleic acid dephosphorylation catalyzed by ALP and template-free polymerization mediated by TdT can inhibit nonspecific amplification, and has higher specificity; (3) the method can be quickly and isothermally completed by only one poly-A MB probe, the poly-A MB is easy to extend, assemble and activate, only one template-independent TdT enzyme is needed, and the experimental operation is simplified.

(1) Feasibility experiments:

to investigate the feasibility of this strategy, we performed gel electrophoresis and fluorescence experiments. As shown in FIG. 2A, the reaction products under different conditions were analyzed using a 3% agarose gel. In the absence of ALP, only one 36-nt band was observed (FIG. 2A, lane 2), which is consistent with the size of the synthesized poly-A MB (FIG. 2A, lane 1), indicating that dephosphorylation of poly-AMB and subsequent TdT-mediated template-free polymerization did not occur. While in the presence of ALP, multiple bands (>36nt) were clearly observed to be stepped (fig. 2A, lane 3), suggesting that the presence of ALP dephosphorylates poly-a MBs and successfully initiates TdT-mediated templateless polymerization, inducing dendritic self-assembled poly-a MBs to generate DNA nanostructures of different sizes (i.e., >36nt bands). Furthermore, we performed fluorescence experiments by measuring the response of FAM emission spectra to ALP. As shown in FIG. 2B, in the presence of poly-A MB + TdT and poly-A MB + ALP, only very low background fluorescence signals were detected (FIG. 2B). In the presence of poly-A MB + TdT + ALP, a significant fluorescence signal was detected (FIG. 2B), 15.2-fold and 12.5-fold higher than the control without ALP or TdT. This indicates that only the presence of ALP initiates dephosphorylation-controlled, templatedependent TdT-a MBs dendritic self-assembly, thereby dissociating a large number of fluorophore molecules from the FAM-BHQ1 FRET pair, resulting in a significant fluorescent signal. These results clearly show that the proposed strategy can be used for highly sensitive detection of ALP.

(2) Sensitivity detection

Sensitivity and selectivity of alkaline phosphatase assay. For the detection sensitivity with the proposed method, we optimized the reaction conditions, including reaction time, amount of poly-A MB, TdT and dTTPs, and under optimal experimental conditions we examined the sensitivity of this strategy by measuring the fluorescence intensity of the response of different concentrations of ALP (FIG. 3). As shown in FIG. 3A, as the ALP concentration is increased from 5X 10^-7Increase to 5 × 10^-3U/uL, the fluorescence intensity is gradually increased, and the concentration is 2.5X 10^-3The plateau is reached when the concentration is higher than U/. mu.L. Notably, at 5 × 10^-7To 1X 10^-3U/uL over a dynamic range of 4 orders of magnitude, fluorescenceThere is a good linear relationship between light intensity and the logarithm of ALP concentration (fig. 3B). The correlation equation is F-1688.5 +248.2 log₁₀C, correlation coefficient 0.9996, where F is fluorescence intensity and C is ALP concentration. The detection limit is calculated by adding three times of standard deviation to the average control value^-7U/. mu.L. The sensitivity of the method is 312.5 times (1 × 10) higher than that of a fluorescence method based on betaine modified polyethyleneimine by excimers/monomer conversion^-4U/. mu.l) 187.5 times higher (6X 10) than the fluorescence method based on the sol-gel transition induced by enzymatic hydrolysis of the gel^-5U/. mu.L) comparable to the fluorescent method based on dephosphorylation, directed aggregation and depolymerization of carbon spots (1.4X 10)^-6U/. mu.L). The increase in sensitivity can be attributed to three factors: (1) ALP catalyzes the dephosphorylation and the high specificity of TdT mediated template-free polymerization, so that poly-A MBs are efficiently hydroxylated and then polymerized to form long poly-T chains; (2) the high amplification efficiency of 3' end repair driven poly-A MBs dendritic self-assembly enables a small amount of ALP to be converted into exponentially amplified FAM fluorescent signals; (3) the template-free polymerization mode facilitates extension, assembly and activation of poly-AMBs.

(3) Specificity detection

ALP is homologous to many enzymes, with certain overlapping catalytic properties and substrate specificity. The selectivity of this strategy was evaluated with Bovine Serum Albumin (BSA), Protein Phosphatase (PP), trypsin, Glucose Oxidase (GO), human 8-oxoguanine-DNA glycosylase (hgg 1) and CpG methyltransferase (m.sssi) as negative controls. As shown in fig. 4, under the same conditions, only very low fluorescence signals were observed in the presence of BSA, PP, trypsin, GO, hcogg 1 and m.sssi. There was no significant difference in the fluorescence signal obtained from the control using the reaction buffer alone. In contrast, the fluorescence signal in the presence of ALP was 11.2, 10.7, 12.4, 12.2 and 17.0 fold higher for BSA, trypsin, GO, hOGG1, M.SssI, PP and control reactions, respectively. This suggests that only ALP can specifically dephosphorize at the 3' end, thereby initiating the templatedependent TdT-assisted dendritic self-assembly of poly-AMBs while efficiently recovering the fluorescent signal. The results show that this strategy has good selectivity and can distinguish ALP from interfering enzymes, including other hydrolases (like trypsin and PP).

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. 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

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