Single-molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis and its application

Abstract

The present invention provides a single-molecule fluorescent biosensor based on enzyme-free toehold-mediated chain displacement reaction catalysis and its application, belonging to the field of single-molecule fluorescence detection technology. The present invention constructs a single-molecule Förster resonance energy transfer biosensor based on enzyme-free toehold-mediated chain displacement reaction catalysis for in vitro detection and in vivo imaging of UDG activity. The target UDG signal is amplified by an enzyme-free catalytic reaction, and the entire reaction can be completed in one step without laborious washing and separation steps, which can greatly simplify the experimental process, save experimental time, reduce detection costs, and has excellent sensitivity and specificity. It provides a valuable platform for basic biomedical research, clinical diagnosis and treatment based on UDG, and therefore has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of single-molecule fluorescence detection, and specifically relates to a single-molecule fluorescence biosensor based on enzyme-free toehold-mediated chain displacement reaction catalysis and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Maintaining genome integrity is essential for organisms, but genomic DNA is constantly damaged by various endogenous and exogenous factors, resulting in multiple types of DNA damage in cells (e.g., base pair mismatch, oxidized bases, DNA alkylation, DNA deamination, and DNA chain breaks). Uracil is one of the most common forms of DNA damage, which is caused by the misincorporation of dUTP or the spontaneous hydrolysis and deamination of cytosine during DNA replication. The presence of uracil in the genome may cause G:C base pairs to be replaced by A:U base pairs in subsequent DNA replication, and A:U base pairs may hinder the recognition of DNA binding proteins, resulting in incorrect coding information and even carcinogenesis. Uracil DNA glycosylase (UDG) is the initiator of the cellular base excision repair pathway, which can catalyze the cleavage of the N-glycosyl bond between uracil and deoxyribose to generate AP sites, thereby removing uracil from DNA. Dysregulation of UDG may lead to dysfunction of uracil excision repair, which in turn causes various diseases such as Bloom's syndrome, human immunodeficiency, lymphoma, neurasthenia, and cancer. Therefore, sensitive detection of UDG activity is very important for understanding the DNA damage repair process and improving the accuracy of early clinical diagnosis.

[0004] Traditional methods for UDG detection include gel electrophoresis combined with radiolabeling and mass spectrometry. However, these methods are usually time-consuming and labor-intensive, with low sensitivity, and often involve hazardous radiation, complex instruments, and complicated procedures. In order to improve the detection sensitivity, various enzyme-mediated isothermal signal amplification methods have been developed for UDG detection, including rolling circle amplification (RCA), strand displacement amplification (SDA), exponential amplification reaction (EXPAR), and exonuclease-assisted signal amplification. However, the introduction of enzyme-mediated signal amplification methods inevitably increases experimental costs, complicates the detection design, and is not suitable for in vivo imaging of endogenous UDG activity. In particular, the visualization of intracellular UDG at the single-cell level is of great significance for understanding the DNA repair system and studying mutation-related diseases.

[0005] Single-molecule fluorescence detection has become one of the most advanced analytical techniques with advantages such as ultra-high sensitivity, low sample consumption, and high signal-to-noise ratio. Although single-molecule detection has achieved brilliant success in biosensing applications, it is still difficult to detect rare targets with extremely low concentrations (such as biomarkers in clinical samples). In order to solve this problem, a variety of nucleic acid amplification methods have been introduced to develop new single-molecule detection methods with higher sensitivity. However, the inventors found that different enzymes, including polymerases, nucleases, and ligases, are always required when implementing the amplification reaction, so the limitations of the enzyme-based detection methods introduced above cannot be eliminated. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a single molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis and its application. The present invention constructs a single molecule Förster resonance energy transfer (FRET) based on enzyme-free toehold-mediated strand displacement reaction catalysis. The target UDG signal is amplified by a non-enzymatic reaction, and the entire reaction can be completed in one step without laborious washing and separation steps, which can greatly simplify the experimental process, save experimental time, and reduce detection costs. It has excellent sensitivity and specificity, and provides a valuable platform for basic biomedical research, clinical diagnosis and treatment based on UDG, so it has good practical application value.

[0007] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a single-molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis, wherein the fluorescent biosensor comprises at least one detection probe and two hairpin probes;

[0009] Wherein, the detection probe is a hairpin detection probe, which contains at least one uracil site. When UDG is present, it specifically recognizes and removes the uracil on the detection probe, causing the detection probe to break and expose a toehold domain;

[0010] The hairpin probes are all modified with fluorescent groups at the sequence ends. When the detection probe exposes the toehold domain, the catalytic self-assembly of two hairpin probes modified with fluorescent groups is initiated through a toehold-mediated strand displacement reaction, thereby generating a large number of DNA duplexes with amplified FRET signals.

[0011] The fluorescent biosensor may also include other components such as a buffer, which can be selected and used by those skilled in the art according to actual conditions, and no specific limitation is made here. Of course, the fluorescent biosensor can be produced and sold as a UDG detection kit.

[0012] Therefore, the second aspect of the present invention provides a UDG detection kit, which comprises the above-mentioned fluorescent biosensor.

[0013] The third aspect of the present invention provides a method for detecting uracil DNA glycosylase, which comprises using the above-mentioned fluorescent biosensor or detection kit for detection.

[0014] A fourth aspect of the present invention provides the use of the above-mentioned fluorescent biosensor, detection kit and / or detection method in uracil DNA glycosylase-related drug screening and / or sample uracil DNA glycosylase analysis.

[0015] In addition, it should be noted that although the present invention takes the detection of uracil DNA glycosylase as an example and provides related fluorescent biosensors, detection kits and detection methods, based on the concept of the present invention, it is obviously easy to replace the specific recognition site in the detection probe and then use it to detect other DNA repair enzymes, and therefore it is also within the scope of protection of the present invention.

[0016] Beneficial technical effects of one or more of the above technical solutions:

[0017] 1. The above technical scheme constructs a single molecule fluorescence resonance energy transfer biosensor based on enzyme-free toehold-mediated chain displacement reaction catalysis for in vitro detection and in vivo imaging of UDG activity.

[0018] When UDG is present, it specifically recognizes and removes uracil from the detection probe, causing the detection probe to break and expose a toehold domain. The exposed toehold will activate the strand displacement reaction, inducing the hairpin probes labeled with two fluorophores (Cy3 and Cy5) to self-assemble into a DNA double-stranded strand labeled with Cy3 and Cy5, in which efficient FRET occurs between Cy3 and Cy5.

[0019] 2. High sensitivity and good specificity: Due to the high efficiency of toehold-mediated strand displacement reaction and the high signal-to-noise ratio of single-molecule detection technology, this method has high sensitivity, with a detection limit of 0.00029 units per milliliter. The application of single-molecule detection further improves sensitivity and reduces sample consumption.

[0020] 3. Wide range of applications: This technical solution can be used to screen potential inhibitors and analyze kinetic parameters, and has potential application value in molecular diagnosis and point-of-care testing. Importantly, it can become a universal platform by designing appropriate DNA substrates.

[0021] 4. Can be used to image endogenous UDG activity in living cells.

[0022] 5. Simple operation: In this technical solution, the target UDG signal is amplified through an enzyme-free catalytic reaction, and the entire experiment can be completed in one step without laborious washing and separation steps, which can greatly simplify the experimental process, save experimental time, and reduce experimental costs, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 Schematic diagram of the principle of catalytic single-molecule FRET for UDG detection in the present invention; wherein, (A) Overview of one-step single-molecule UDG detection. (B) The principle of UDG inducing FRET complex catalytic assembly through toehold-mediated strand displacement reaction. Numbers represent functional domains, and domain x is complementary to domain x*. The molecular structure changes during the base excision repair reaction are shown in the dotted box.

[0025] Figure 2The following are the relevant diagrams for the principle verification of catalytic single-molecule FRET for UDG detection in the embodiments of the present invention, wherein (A) PAGE analysis with SYBR Gold as the indicator. Lane M: DNA marker; Lane 1: 100 nanomoles per liter detection probe; Lane 2: 200 nanomoles per liter Cy5-labeled hairpin probe 1; Lane 3: 200 nanomoles per liter Cy3-labeled hairpin probe 2; Lane 4: 100 nanomoles per liter detection probe + 200 nanomoles per liter Cy5-labeled hairpin probe 1 + 200 nanomoles per liter Cy3-labeled hairpin probe 2 + 2 units of APE1; Lane 5: 100 nanomoles per liter detection probe + 200 nanomoles per liter Cy5-labeled hairpin probe 1 + 200 nanomoles per liter Cy3-labeled hairpin probe 2 + 2 units of APE1 + 1 unit per milliliter of UDG. (B) PAGE analysis of direct excitation of the Cy3 fluorescent group. The signal of Cy3 is shown in green, the signal of Cy5 is shown in red, and the colocalization of Cy3 and Cy5 is indicated in yellow. (C) Fluorescence emission spectra of Cy3 and Cy5 in the absence (control) and presence of UDG. The concentration of UDG is 1 unit per ml. (D) The change curve of Cy5 fluorescence intensity as a function of reaction time in the presence or absence of UDG.

[0026] Figure 3 Figure 1 is a graph showing the activity of UDG detected by single-molecule detection based on total internal reflection fluorescence (TIRF) imaging in an embodiment of the present invention, wherein single-molecule fluorescence images of Cy3 and Cy5 in the absence (AC) and presence (DF) of UDG. The signal of Cy3 is shown in green (A and D), the signal of Cy5 is shown in red (B and E), and the co-localization signal of Cy3 and Cy5 is shown in yellow (C and F). The concentration of UDG is 1 unit per milliliter. The scale bar is 5 microns.

[0027] Figure 4 The single-molecule step-by-step photobleaching trajectories of Cy3 (A) and Cy5 (B) fluorescent dots in the present invention are shown in the left panel. The scale bar is 5 μm.

[0028] Figure 5 The following are the relevant diagrams for the screening and optimization of reaction conditions in the embodiments of the present invention, wherein (A) the change of F-F0 value with the reaction temperature. (B) the relationship between F-F0 value and the amount of APE1 used. The concentration of UDG is 1 unit per milliliter. The error bars show the standard deviation of three independent experiments.

[0029] Figure 6Figure 1 is a graph related to sensitivity detection in an embodiment of the present invention, wherein (A) the change of fluorescence emission spectrum with UDG concentration. (B) the linear relationship between Cy5 fluorescence intensity and the logarithm of UDG concentration in the range of 0.005 to 0.5 units per milliliter. (C) the change of Cy5 count with UDG concentration. (D) the linear relationship between Cy5 count and the logarithm of UDG concentration in the range of 0.0005 to 0.5 units per milliliter. Error bars show the standard deviation of three independent experiments.

[0030] Figure 7 The single molecule counts of Cy5 were measured in the presence of reaction buffer (control), 0.1 mg / ml BSA, 0.1 mg / ml IgG, 1 unit / ml Fpg, 1 unit / ml hAAG, 1 unit / ml UDG, and a mixture containing 1 unit / ml UDG + all the above interferences in the present embodiment. The error bar shows the standard deviation of three independent experiments.

[0031] Figure 8 Figures of kinetic and inhibitor-related experiments in the examples of the present invention, where (A) the change in initial velocity (V) with the concentration of the detection probe. The concentration of UDG is 1 unit per milliliter. (B) The inhibitory effect of different concentrations of UGI on the relative activity of UDG. The concentration of UDG is 0.2 units per milliliter. Error bars show the standard deviation of three independent experiments.

[0032] Fig. 9 Figure 1 is a graph showing the detection of cellular UDG in an embodiment of the present invention. (A) Cy5 counts were measured in the presence of lysis buffer, lysis buffer + UGI, cell extract + UGI, and cell extract. (B) Changes in Cy5 counts induced by different HeLa cell numbers. The inset shows the linear relationship between Cy5 counts and the logarithm of the HeLa cell number. Error bars show the standard deviation of three independent experiments.

[0033] Fig.10 The following are the imaging-related diagrams of UDG in cells in the embodiments of the present invention, respectively, using (A) Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2, (B) detection probe (T) + Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2, (C) detection probe + Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2 transfected HeLa cells for UDG activity imaging. (D) FRET signals in response to different conditions. Group a, Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2; Group b, detection probe (T) + Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2; Group c, detection probe + Cy5-labeled hairpin probe 1 + Cy3-labeled hairpin probe 2. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0036] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0037] As mentioned above, conventional UDG detection methods include gel electrophoresis combined with radiolabeling and mass spectrometry. However, these methods are usually time-consuming and labor-intensive, have low sensitivity, and often involve hazardous radiation, complex instruments, and complicated procedures. In order to improve the detection sensitivity, various enzyme-mediated isothermal signal amplification methods have been developed for UDG detection, as well as exonuclease-assisted signal amplification. However, the introduction of enzyme-mediated signal amplification methods inevitably increases experimental costs, complicates the detection design, and is not suitable for in vivo imaging of endogenous UDG activity.

[0038] In view of this, the present invention constructs a single molecule Förster resonance energy transfer biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis for in vitro detection and in vivo imaging of UDG activity. Target UDG can remove the uracil base on the detection probe, resulting in the breakage of the detection probe with the assistance of APE1. The cleaved detection probe exposes its toehold domain and initiates the catalytic self-assembly of two fluorescent group-labeled hairpin probes through a toehold-mediated strand displacement reaction to produce a large number of DNA duplexes with amplified FRET signals. Due to the high efficiency of the toehold-mediated strand displacement reaction and the high signal-to-noise ratio of the single molecule detection technology, this method has high sensitivity. The self-calibration property of the FRET method ensures that the proposed biosensor has high accuracy even in complex intracellular environments, providing a new platform for specific and sensitive detection and imaging of UDG activity in living cells.

[0039] The experimental principle (such as Figure 1): To illustrate the entire reaction process, each DNA strand is broken down into several functional domains, represented by different numbers. The experiment involves two fluorescently labeled hairpin probes - Cy5-labeled hairpin probe 1 (Cy5-HP1) and Cy3-labeled hairpin probe 2 (Cy3-HP2), and a hairpin detection probe modified with a uracil base (U) between domains 1* and 2*. In the presence of UDG, UDG specifically recognizes the uracil base in the detection probe and hydrolyzes the N-glycosidic bond between uracil and deoxyribose, releasing the uracil base and forming an apurinic / pyrimidinic (AP) site. The AP site is subsequently cleaved by APE1 to generate a single nucleotide gap, resulting in the automatic dissociation of the stem domain 1* of the detection probe. The cleaved detection probe exposes toehold domain 1, which can bind to domain 1* of Cy5-labeled hairpin probe 1, initiate a branch migration reaction, form a stable promoter / Cy5-labeled hairpin probe 1 duplex, and then expose domain 3 of Cy5-labeled hairpin probe 1. The exposed domain 3 will serve as another toehold to initiate the next branch migration reaction, generate a Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex, and release the cleaved detection probe. The free cleaved detection probe can bind to another Cy5-labeled hairpin probe 1, catalyze a cyclic toehold-mediated strand displacement reaction, and generate a large amount of Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex. The resulting duplex can bring the Cy3 donor and the Cy5 acceptor into close proximity, induce significant FRET from Cy3 to Cy5, and the FRET signal can be simply and sensitively quantified by total internal reflection fluorescence microscopy (TIRF). In contrast, in the absence of UDG, the uracil base cannot be excised, the toehold domain 1 is blocked in the stem region of the detection probe, the strand displacement reaction cannot occur, and no FRET signal can be detected.

[0040] Therefore, in a typical embodiment of the present invention, a single-molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis is provided, wherein the fluorescent biosensor comprises at least one detection probe and two hairpin probes;

[0041] Wherein, the detection probe is a hairpin detection probe, which contains at least one uracil site. When UDG is present, it specifically recognizes and removes the uracil on the detection probe, causing the detection probe to break and expose a toehold domain;

[0042] The hairpin probes are all modified with fluorescent groups at the sequence ends. When the detection probe exposes the toehold domain, the catalytic self-assembly of two hairpin probes modified with fluorescent groups is initiated through a toehold-mediated strand displacement reaction, thereby generating a large number of DNA duplexes with amplified FRET signals.

[0043] Specifically, the hairpin probe 1 is labeled with Cy5, and the hairpin probe 2 is labeled with Cy3;

[0044] The detection probe is a hairpin detection probe having a stem-loop structure, wherein a uracil site is contained in the stem structure near the 5' end, thereby dividing the structural region into domain 1* and domain 2*, and its complementary region is correspondingly divided into domain 1 and domain 2, and the loop portion of the hairpin detection probe is named domain 3; when UDG is present, UDG specifically recognizes the uracil base in the detection probe, and hydrolyzes the N-glycosidic bond between uracil and deoxyribose, releasing the uracil base, and forming an apurinic / pyrimidinic (AP) site; the AP site is subsequently cleaved by human apurinic / apyrimidinic endonuclease 1 (humanapurinic / apyrimidinic endonuclease1, APE1), generating a single nucleotide gap, resulting in the automatic dissociation of domain 1* of the detection probe;

[0045] The cleaved detection probe exposes the toehold domain 1, which binds to the domain 1* of the Cy5-labeled hairpin probe 1, initiates the branch migration reaction, and forms a stable promoter / Cy5-labeled hairpin probe 1 double strand, and then the domain 3 of the Cy5-labeled hairpin probe 1 is exposed; the exposed domain 3 acts as another toehold, initiating the next branch migration reaction, generating the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 double strand, and releasing the cleaved detection probe; the free cleaved detection probe binds to another Cy5-labeled hairpin probe 1, catalyzing the cyclic toehold-mediated strand displacement reaction, and generating a large amount of Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 double strands; the resulting double strand can bring the Cy3 donor and the Cy5 acceptor close to each other, inducing significant FRET from Cy3 to Cy5.

[0046] Therefore, the fluorescent biosensor also includes human apurinic / apyrimidinic endonuclease 1, namely APE1.

[0047] In another specific embodiment of the present invention,

[0048] The nucleotide sequence of the detection probe is shown in SEQ ID NO.1.

[0049] The nucleotide sequence of the hairpin probe 1 is shown in SEQ ID NO.2.

[0050] The nucleotide sequence of the hairpin probe 2 is shown in SEQ ID NO.3.

[0051] In another specific embodiment of the present invention, the fluorescent biosensor may further include other components such as a buffer, which can be selected and used by those skilled in the art according to actual conditions, and no specific limitation is made here. Of course, the fluorescent biosensor can be produced and sold as a UDG detection kit.

[0052] Therefore, in another specific embodiment of the present invention, a UDG detection kit is provided, wherein the detection kit comprises the above-mentioned fluorescent biosensor.

[0053] In another specific embodiment of the present invention, a method for detecting UDG is provided, the method comprising using the above-mentioned fluorescent biosensor or detection kit for detection.

[0054] Specifically, the detection method includes:

[0055] The detection probe, two hairpin probes, APE1 and the sample to be tested are incubated to obtain the result. From the above specific detection method, it can be seen that the present invention can be carried out in a homogenous mode without laborious washing and separation steps, thereby greatly saving human and material resources.

[0056] The incubation conditions are: incubating at 30-40° C. (preferably 37° C.) for 50-100 minutes (preferably 80 minutes).

[0057] The above incubation reaction is carried out in a solution environment, and the solution can be a reaction buffer, such as a standard Taq reaction buffer (10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl), 50 mmol / L potassium chloride (KCl), 1.5 mmol / L magnesium chloride (MgCl2), pH 8.3).

[0058] In another specific embodiment of the present invention, the method further comprises performing fluorescence detection analysis on the above-mentioned incubation reaction product.

[0059] The fluorescence detection analysis can be based on measuring the fluorescence spectrum at an excitation wavelength of 532 nanometers using a fluorescence spectrophotometer and recording the fluorescence spectrum at wavelengths between 550 and 750 nanometers; it can also be based on total internal reflection fluorescence microscopy (TIRF) imaging for single molecule detection analysis.

[0060] The sample to be tested may be an environmental sample or a biological sample, and the biological sample includes in vitro blood, body fluids, tissues and cells. In particular, experiments have shown that the fluorescent biosensor of the present invention can be used for imaging endogenous UDG activity in living cells, thereby sensitively detecting UDG in cells at the cellular level.

[0061] In another specific embodiment of the present invention, there is provided application of the above-mentioned biosensor, detection kit and / or detection method in UDG-related drug screening and / or sample UDG analysis.

[0062] The drug may be a UDG inhibitor or activator.

[0063] The sample can be an environmental sample or a biological sample, and the biological sample includes ex vivo blood, body fluids, tissues and cells. Experiments have shown that the fluorescent biosensor of the present invention can be used for imaging endogenous UDG activity in living cells, thereby sensitively detecting UDG in cells at the cellular level, providing a simple and powerful tool for visualizing enzymes and related pathways related to DNA repair in living cells, and has great potential in biological research, clinical diagnosis and drug discovery.

[0064] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments. In the following embodiments, the nucleotide sequences of the relevant probes are as follows:

[0065]

[0066] “*” represents thio modification.

[0067] Example

[0068] Experimental methods

[0069] Preparation of hairpin probes: All hairpin probes were incubated at 95°C for 5 minutes in 1× standard Taq reaction buffer (10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl), 50 mmol / L potassium chloride (KCl), 1.5 mmol / L magnesium chloride (MgCl2), pH 8.3), and then slowly cooled to room temperature to allow the probes to fold into a hairpin structure. The obtained hairpin probes were stored at 4°C for further use.

[0070] UDG activity detection: The entire reaction was incubated at 37°C for 80 min in a 10 μl reaction mixture containing different concentrations of UDG, 100 nmol detection probe, 200 nmol Cy5-labeled hairpin probe 1, 200 nmol Cy3-labeled hairpin probe 2, 2 units of APE1, and 1× standard Taq reaction buffer (10 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl), 50 mM potassium chloride (KCl), 1.5 mM magnesium chloride (MgCl2), pH 8.3).

[0071] Gel electrophoresis and fluorescence spectrum measurement: To verify the assembly of the Cy5-labeled hairpin probe 1-Cy3-labeled hairpin probe 2 double strands and the FRET between Cy3 and Cy5, the reaction products were analyzed by 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) in 1× TBE buffer (9 nanomoles per liter Tris-HCl, 9 millimoles per liter boric acid, 0.2 millimoles per liter ethylenediaminetetraacetic acid (EDTA), pH 7.9) at room temperature and a constant voltage of 120 volts for 50 minutes. The gel after electrophoresis was analyzed by ChemiDoc TM MP imaging system (Bio-Rad, Hercules, CA, USA) was used for imaging analysis. 10 μL of the reaction product was diluted to 50 μL with 1× standard Taq reaction buffer (10 mmol / L tris (hydroxymethyl) aminomethane-hydrochloride (Tris-HCl), 50 mmol / L potassium chloride (KCl), 1.5 mmol / L magnesium chloride (MgCl2), pH 8.3), and then the fluorescence spectrum was measured at an excitation wavelength of 532 nm using a Hitachi F-7000 fluorescence spectrophotometer, and the fluorescence spectrum was recorded between 550 and 750 nm. The fluorescence emission spectrum was measured by a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) equipped with a xenon lamp as an excitation source. The excitation wavelength of the Cy3 dye is 532 nm. The emission spectrum was recorded between 550 and 750 nm.

[0072] Single-molecule detection and data analysis: The reaction product was diluted 10,000-fold in imaging buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 1 mM water-soluble vitamin E, pH 8.0). 10 μL of the diluted reaction product was pipetted directly onto a coverslip for total internal reflection fluorescence microscopy (TIRF) imaging. A 561 nm laser was used to excite the Cy3 fluorescent molecules. Cy3 and Cy5 photons were collected by an oil-immersion 100x objective (Olympus, Japan), then separated and directed to the two halves of an Andor Ixon DU897 EMCCD with an exposure time of 500 ms. An 800 × 800 pixel region of interest (ROI) was selected for Cy5 molecule counting using Image J software, and the total Cy5 counts of 10 frames were used for data analysis. Single-molecule photobleaching experiments were performed as described above, with the diluted reaction mixtures excited continuously with either a 561 nm laser or a 640 nm laser, and live images recorded at high frequency with an exposure time of 400 ms.

[0073] Inhibition assay: Different concentrations of uracil glycosylase inhibitor (UGI) were incubated with the detection probe at 37 degrees Celsius for 15 minutes, and then other substrates and UDG were added to the solution and incubated at 37 degrees Celsius for 80 minutes. The relative activity (RA) of UDG was calculated according to the following equation:

[0074] RA(%)=C i / C t ×100%=10 (Ni-Nt) / 578.06 ×100%

[0075] Where N t is the Cy5 count in the presence of 0.2 units / mL UDG, and Ni is the Cy5 count in the presence of 0.2 units / mL UDG and different concentrations of UGI. IC was calculated by plotting the relationship between RA value and UGI concentration. 50 value.

[0076] Cell culture and preparation of cell extracts: Human hepatocytes (HL-7702 cells), human cervical cancer cells (HeLa cells), and human lung adenocarcinoma cells (A549 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody at 37 degrees Celsius and 5% carbon dioxide incubator for actual sample analysis. When the cells grew to the logarithmic growth phase, the cells were collected using trypsin and counted using the automatic cell counter IC1000 of Countstar Biotechnology Co., Ltd. Cell extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA).

[0077] Human cervical cancer cell line (HeLa cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 37°C, 5% carbon dioxide incubator. When the cells grew to the logarithmic growth phase, the cells were harvested using trypsin and counted using an automatic cell counter IC1000 from Countstar Biotechnology Co., Ltd. Nuclear extracts were collected using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. For cell inhibition experiments, 1 unit per ml of UGI was incubated with the detection probe at 37°C for 15 minutes before adding HeLa cell extracts or lysis buffer, and then the same procedure as above was performed.

[0078] Optical imaging of UDG in cancer cells: HeLa cells were seeded in glass-bottomed cell culture dishes and cultured overnight in a 37°C, 5% CO2 incubator. The original culture medium was then removed and the adherent cells were washed once with phosphate-buffered saline (PBS). Transfection experiments were performed using Lipofectamine 3000 reagent according to the manufacturer's instructions. 500 μL of a mixture of Opti-MEM (Invitrogen) containing 7.5 μL of Lipofectamine-3000, 20 μL of the detection probe or its control probe (5 μM), 20 μL of Cy5-labeled hairpin probe 1 (5 μM), 20 μL of Cy3-labeled hairpin probe 2 (5 μM), and 10 μL of P300 was added to each dish and incubated for 2 hours in a 37°C, 5% CO2 incubator. The cells were then washed several times with PBS to remove excess sample. Finally, fluorescent images of cells were acquired on an inverted Olympus IX71 fluorescence microscope (Olympus, Japan) using a 10× objective.

[0079] Experimental Results

[0080] 1. Experimental verification of the principle

[0081] First, 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to monitor the cleavage of the detection probe catalyzed by UDG and the subsequent strand displacement reaction ( Figure 2 A). In the presence of UDG, the characteristic bands of the cleaved detection probe and the double-stranded Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 appear simultaneously ( Figure 2 A, lane 5), accompanied by Cy5-labeled hairpin probe 1 ( Figure 2 A, lane 2) and Cy3-labeled hairpin probe 2 ( Figure 2 A, lane 3) disappeared, indicating that UDG can catalyze the cleavage of the detection probe to initiate the subsequent strand displacement reaction. In contrast, in the absence of UDG, neither the cleaved detection probe nor the characteristic band of the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 double strand was observed ( Figure 2 A, lane 4), indicating that no reaction occurred.

[0082] Theoretically, based on the assumption that the distance between adjacent bases in double-stranded DNA is about 0.34 nm, the distance from Cy3 to Cy5 in the synthesized Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 double strand is about 2.72 nm, which is within the effective distance of FRET (for the Cy3 / Cy5 pair, 2R0 = 11.4 nm). We further used PAGE to verify the FRET between Cy3 and Cy5 by directly exciting the Cy3 fluorophore ( Figure 2 B). In the absence of UDG, only the Cy3-labeled hairpin probe 2 band was observed in the Cy3 channel, while no band was observed in the Cy5 channel, indicating that FRET from Cy3 to Cy5 did not occur in the absence of UDG. After the addition of UDG, the characteristic band of the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex appeared in the Cy3 channel, while the intensity of the Cy3-labeled hairpin probe 2 characteristic band decreased, and the characteristic band of the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex appeared in the Cy5 channel, completely colocalizing with the characteristic band in the Cy3 channel, proving that efficient FRET is the result of UDG-induced assembly of the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex.

[0083] We also used ensemble fluorescence measurements to investigate FRET between Cy3 and Cy5 ( Figure 2 C). In the control experiment without UDG ( Figure 2C), no significant Cy5 fluorescence signal was detected, indicating that in the absence of UDG, no FRET occurred between Cy3 and Cy5. In the presence of UDG, a decrease in the Cy3 fluorescence signal and an increase in the Cy5 fluorescence signal can be detected simultaneously ( Figure 2 C), indicating efficient FRET of target UDG-induced Cy3 to Cy5. Time course experiments showed that the Cy5 fluorescence signal increased with reaction time from 5 to 80 minutes and reached a plateau at 80 minutes ( Figure 2 D), while no significant signal increase was observed in the control group without UDG ( Figure 2 D) These results clearly demonstrate the targeted UDG-induced catalytic assembly.

[0084] We further used single-molecule detection based on total internal reflection fluorescence (TIRF) imaging to detect UDG activity ( Figure 3 In the absence of UDG, only Cy3 signal was observed ( Figure 3 A), no Cy5 signal was observed ( Figure 3 B), indicating that no Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 double-stranded structure was formed, and no FRET occurred. In contrast, when UDG was present, Cy3 ( Figure 3 D) and Cy5( Figure 3 E) signals can be detected simultaneously, Cy3 and Cy5 ( Figure 3 F) The signals are completely colocalized, indicating efficient FRET from Cy3 to Cy5. It is noteworthy that the presence of UDG leads to a significant decrease in the fluorescence signal of Cy3 ( Figure 3 D), which is due to the effective FRET from Cy3 to Cy5, which is consistent with the gel electrophoresis ( Figure 2 B) and fluorescence measurements ( Figure 2 C). Since a single molecule is irreversibly photobleached in one step, each step in the trajectory represents a bleached dye molecule. We tracked the photobleaching steps and found that both Cy3 and Cy5 fluorescent spots showed single-step photobleaching ( Figure 4 ), indicating that the observed spots represent single molecules. These results clearly demonstrate that Cy5 counting can be used for accurate measurement of UDG activity.

[0085] 2. Sensitivity experiment

[0086] Under the best experimental conditions ( Figure 5 ), the detection sensitivity of the UDG detection method was studied. Figure 6As shown in Figure 2, when the UDG concentration increased from 0.001 to 0.5 units per milliliter, a gradually decreasing Cy3 signal and an enhanced Cy5 signal were generated. When the UDG concentration exceeded 0.5 units per milliliter, the signal reached a plateau. In addition, the Cy5 fluorescence intensity was linearly correlated with the logarithm of the UDG concentration between 0.005 and 0.5 units per milliliter ( Figure 6 B), the regression equation is F = 39.54log 10 C+130.13(R 2 =0.9950), where F is the Cy5 fluorescence intensity and C is the UDG concentration (unit per milliliter). By evaluating the mean Cy5 fluorescence intensity of the control group plus three times the standard deviation, the detection limit was calculated to be 0.00367 units per milliliter. We further monitored the effect of different UDG concentrations on Cy5 counts by single molecule detection. Figure 6 As shown in C, Cy5 counts increased with increasing UDG concentration and were linearly correlated with the logarithm of UDG concentration in the range of 0.0005 to 0.5 units per milliliter ( Figure 6 D). The regression equation is N = 578.06log 10 C+2200.41(R 2 =0.9984), where N is the Cy5 count and C is the UDG concentration (unit per milliliter). The detection limit was calculated to be 0.00029 units per milliliter, which is higher than the ensemble fluorescence measurement ( Figure 6 B) The detection limit obtained was 10-fold lower. This high sensitivity can be attributed to the following three factors: (1) the efficient transduction of the UDG activity signal into an amplifiable DNA signal by the specific cleavage of the target-induced detection probe; (2) the high signal amplification efficiency of the toehold-mediated strand displacement reaction; and (3) the inherent high sensitivity and high signal-to-noise ratio of the single-molecule detection technology.

[0087] 3. Specificity Experiment

[0088] The selectivity of this technical scheme was studied using bovine serum albumin (BSA), immunoglobulin G (IgG), formamidopyrimidine-DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) as negative controls. BSA and IgG are unrelated proteins that cannot remove uracil from DNA substrates. Fpg, as a bifunctional DNA glycosylase, can specifically remove the damaged 8-oxo-7,8-dihydroguanine (8-oxoG) base from the 8-oxoG:C base pair and hydrolyze the 3'-phosphodiester bond of the AP site. TDG is a monofunctional DNA glycosylase that can remove T from G:T mismatches. Figure 7 As shown, under the same conditions, only in UDG ( Figure 7 ) was detected in the presence of Cy5, while in the reaction buffer ( Figure 7 )、BSA( Figure 7 )、IgG( Figure 7 )、Fpg( Figure 7 ) and hAAG( Figure 7 ) was not observed. In addition, when UDG was present, a significant Cy5 signal was observed even in the presence of all the above interferences ( Figure 7 ). These results indicate that the proposed method has good specificity for the target UDG.

[0089] 4. Kinetic Experiments

[0090] To quantify the kinetic parameters of UDG, we incubated 1 unit / mL of UDG with different concentrations of the detection probe (0-120 nmol / mL) at 37°C for 20 min and measured the initial velocity of UDG. Figure 8 As shown in A, the initial velocity of UDG increases with the increase of the detection probe concentration. The kinetic parameters of UDG were calculated by fitting the experimental data to the following Michaelis-Menten equation:

[0091] V=V max [S] / (K m +[S])

[0092] Where V max represents the maximum initial velocity, [S] represents the concentration of the detection probe, and the Michaelis constant (K m ) represents the substrate concentration at half the maximum reaction rate. max The value was 3.04±0.36 per minute, K m The value was 59.50 ± 13.51 nmol / L. The K m The value is consistent with the value obtained by the fluorescence method based on Endo IV-assisted signal amplification (52.75 nanomoles per liter), indicating that the technical solution can be used to analyze the Michaelis-Menten kinetic parameters of UDG.

[0093] 5. Inhibitor Experiment

[0094] To demonstrate the capability of the proposed technique for UDG inhibition analysis, we used uracil glycosylase inhibitor (UGI) as a model inhibitor. UGI specifically binds to UDG in a 1:1 stoichiometric ratio to form an irreversible protein complex. Figure 8 As shown in B, the relative activity of UDG gradually decreased with the increase of UGI concentration. The half inhibitory concentration (IC 50 ) was 0.07 units per milliliter. These results indicate that the proposed technical scheme can be used for the screening of UDG inhibitors and thus has great application potential in the diagnosis of UDG-related diseases and drug discovery.

[0095] 6. Detection of Cellular UDG

[0096] Accurate quantification of intracellular UDG activity is of great significance for clinical diagnosis and treatment. To demonstrate the potential application of this technical solution in complex biological samples, we measured UDG activity in extracts of human cervical cancer cells (HeLa cells). Fig. 9 As shown in A, no obvious Cy5 signal was observed in the control group ( Fig. 9 A), while adding nuclear extract ( Fig. 9 A) A significantly enhanced Cy5 signal can be observed. To further verify that the enhanced Cy5 signal is induced by endogenous UDG in the cells rather than by other components of the nuclear extract, UGI was used to inhibit the activity of UDG in the nuclear extract. After adding 1 unit / ml of UGI, the Cy5 signal ( Fig. 9 A) compared to the Cy5 signal decreased by 73.88% ( Fig. 9 A), which indicates that UGI has an inhibitory effect on UDG activity. We further monitored the changes in Cy5 counts and HeLa cell numbers. Fig. 9 As shown in B, Cy5 counts increased with the increase in the number of HeLa cells, and the Cy5 counts were linearly correlated with the logarithm of the cell number in the range of 3 to 10,000 cells. The regression equation was N = 467.88log 10 X–67.47(R 2 =0.9899), where N represents the Cy5 count and X represents the number of HeLa cells. The detection limit was calculated to be as low as 2.7 cells, which is comparable to the bioluminescence analysis based on enzyme-mediated three-loop cascade signal amplification (3 cells). These results show that the present technical solution can be used to measure the activity of UDG in complex systems, thus paving a broad road for imaging endogenous UDG in living cells using the present technical solution.

[0097] 7. Imaging of Intracellular UDG

[0098] We further used this technology to monitor endogenous UDG activity in living HeLa cells. For cell imaging, we replaced uracil in the detection probe with thymine as a control probe, called detection probe (T). Fig.10 As shown in A, when only Cy5-labeled hairpin probe 1 and Cy3-labeled hairpin probe 2 were transfected into HeLa cells, bright Cy3 fluorescence was observed, but Cy5 fluorescence was relatively weak, indicating that there was no FRET signal. Similar results were also detected when the detection probe (T), Cy5-labeled hairpin probe 1, and Cy3-labeled hairpin probe 2 were transfected into HeLa cells together ( Fig.10B). In contrast, when the detection probe, Cy5-labeled hairpin probe 1, and Cy3-labeled hairpin probe 2 were transfected into HeLa cells, a decrease in the Cy3 fluorescence signal and a significant increase in the Cy5 fluorescence signal were simultaneously observed with perfect colocalization ( Fig.10 C), indicating that FRET signals were generated. In addition, we also quantified the fluorescence signals of live cell imaging. Considering the complexity of the intracellular microenvironment, the FRET signal (F A / F D , the fluorescence intensity ratio of Cy5 acceptor to Cy3 donor) rather than just the Cy5 fluorescence signal to evaluate the intracellular UDG activity. Fig.10 As shown in D, the average values ​​of FRET signals decreased in the order of group c > group b > group a, indicating that this technical solution can be used to monitor and image endogenous UDG activity in living cells.

[0099] It can be seen from the above embodiments that the technical solution of the present application has at least the following advantages:

[0100] 1. The cleavage detection probe generated by the uracil excision repair reaction acts as an effective catalyst and can amplify the signal without any enzyme involvement.

[0101] 2. The technical solution can be carried out in a homogenous mode without the need for laborious washing and separation steps.

[0102] 3. Since the UDG-induced base excision repair reaction has good compatibility with the toehold-mediated strand displacement reaction, the method can be completed in one step at a constant temperature, which greatly simplifies the detection procedure and ensures that the analysis time of the proposed method is shorter than or equivalent to the reported UDG detection method. The introduction of the single-molecule detection method also greatly reduces the difficulty of data processing, improves the intuitiveness of data presentation and data processing efficiency, and realizes efficient and simple determination of UDG activity.

[0103] 4. Excellent detection performance and huge application potential.

[0104] Combining signal-amplified FRET with single-molecule detection gives this assay high accuracy and sensitivity, allowing for direct and accurate detection of low-abundance UDG even in complex intracellular environments. The proposed biosensor has been successfully applied to kinetic analysis of UDG, inhibitor screening, and real-time imaging of UDG activity in living cells. Importantly, it can be easily extended to the detection of other DNA repair-related enzymes through the rational design of appropriate DNA substrates, providing a simple and powerful tool for the visualization of DNA repair-related enzymes and related pathways in living cells, with great potential in biological research, clinical diagnosis, and drug discovery.

[0105] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. SEQUENCE LISTING <110> Qingdao University of Science and Technology <120> Single-molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis and its application <130> <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 40 <212> DNA <213> Artificial sequence <400> 1 agaggcauca atgggagatc ccattcccat tgatgcctct 40 <210> 2 <211> 56 <212> DNA <213> Artificial sequence <400> 2 agaggcatca atgggaatgg gatcatgcct ctaacctagc gatcccattc ccattg 56 <210> 3 <211> 56 <212> DNA <213> Artificial sequence <400> 3 atgggatcgc taggttagag gcatgatccc attcccatac atgcctctaa cctagc 56 <210> 4 <211> 40 <212> DNA <213> Artificial sequence <400> 4 agaggcatca atgggagatc ccattcccat tgatgcctct 40

Claims

1. A single-molecule fluorescent biosensor based on enzyme-free toehold-mediated strand displacement reaction catalysis, characterized in that: The fluorescent biosensor comprises at least one detection probe and two hairpin probes; Wherein, the detection probe is a hairpin detection probe, which contains at least one uracil site. When UDG is present, it specifically recognizes and removes the uracil on the detection probe, causing the detection probe to break and expose a toehold domain; The hairpin probes are all modified with fluorescent groups at the end of the sequence, and when the detection probe exposes the toehold domain, the catalytic self-assembly of two hairpin probes modified with fluorescent groups is initiated through a toehold-mediated strand displacement reaction, thereby generating a large number of DNA duplexes with amplified FRET signals; The nucleotide sequence of the detection probe is: A*G*A*GGC AUC AAT GGG AGA TCC CAT TCC CAT TGA TGCC*T*C*T; The nucleotide sequence of Cy5-HP1 is: Cy5-A*G*A*GGC ATC AAT GGG AAT GGG ATC ATG CCT CTAACC TAGCGA TCC CAT TCC CA*T*T*G; The nucleotide sequence of Cy3-HP2 is: A*T*G*GGA TCG CTA GGT TAG AGG CAT GAT CCC ATT CCCATA CAT GCC TCT AAC CT*A*G*C-Cy3; Among them, "*" represents thio modification; The UDG is uracil DNA glycosylase.

2. The single-molecule fluorescent biosensor according to claim 1, characterized in that: The detection probe is a hairpin detection probe having a stem-loop structure; wherein a uracil site is contained in the stem structure near the 5' end, thereby dividing the structural region into domain 1* and domain 2*, and its complementary region is correspondingly divided into domain 1 and domain 2, and the loop portion of the hairpin detection probe is named domain 3; when UDG is present, UDG specifically recognizes the uracil base in the detection probe, and hydrolyzes the N-glycosidic bond between uracil and deoxyribose, releasing the uracil base, and forming an apurinic / pyrimidinic site; the apurinic / pyrimidinic site is subsequently cleaved by human apurinic / apyrimidinic nuclease 1, generating a single nucleotide gap, resulting in the automatic dissociation of domain 1* of the detection probe.

3. The single-molecule fluorescent biosensor according to claim 2, characterized in that: The cleaved detection probe exposes the toehold domain 1, which binds to the domain 1* of the Cy5-labeled hairpin probe 1, initiates the branch migration reaction, and forms the promoter / Cy5-labeled hairpin probe 1 duplex, and then the domain 3 of the Cy5-labeled hairpin probe 1 is exposed; the exposed domain 3 acts as another toehold, initiating the next branch migration reaction, generating the Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex, and releasing the cleaved detection probe; the free cleaved detection probe binds to another Cy5-labeled hairpin probe 1, catalyzing the cyclic toehold-mediated strand displacement reaction, and producing a large amount of Cy5-labeled hairpin probe 1 / Cy3-labeled hairpin probe 2 duplex; the resulting duplex brings the Cy3 donor and the Cy5 acceptor close to each other, inducing significant FRET from Cy3 to Cy5.

4. The single-molecule fluorescent biosensor according to claim 1, characterized in that: The fluorescent biosensor further comprises a buffer.

5. A UDG detection kit, characterized in that: The detection kit comprises the single-molecule fluorescent biosensor according to any one of claims 1 to 4.

6. A method for detecting UDG, characterized in that: The method comprises using the single-molecule fluorescent biosensor according to any one of claims 1 to 4 or the detection kit according to claim 5 to detect the sample to be detected.

7. The method according to claim 6, characterized in that Detection methods include: The detection probe, two hairpin probes, APE1 and the sample to be tested are incubated to obtain the result; The APE1 is a human apurinic / apyrimidinic endonuclease.

8. The method according to claim 7, characterized in that The incubation conditions are: incubation at 30-40° C. for 50-100 minutes.

9. The method according to claim 8, characterized in that The incubation conditions are: incubation at 37° C. for 80 minutes.

10. The method according to claim 8, characterized in that The incubation reaction is carried out in a solution environment, and the solution is a reaction buffer.

11. The method according to claim 10, characterized in that The reaction buffer is a standard Taq reaction buffer.

12. The method according to claim 7, characterized in that The method further comprises performing fluorescence detection analysis on the incubation reaction product.

13. The method according to claim 7, characterized in that The sample to be tested is an environmental sample or a biological sample, and the biological sample includes ex vivo blood, body fluid, tissue and cells.

14. Use of the fluorescent biosensor according to any one of claims 1 to 4, the detection kit according to claim 5 and / or the detection method according to any one of claims 6 to 13 in sample UDG analysis.

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