A label-free fluorescent sensor and its preparation method and application

Abstract

The application belongs to the field of biological detection, and relates to a label-free fluorescent sensor, in particular to a label-free fluorescent sensor and a preparation method and application thereof. The application designs a plurality of molecular hairpins containing G-rich sequences by adding different non-G bases at the end, and constructs a label-free fluorescent sensor based on G-triplex (signal drop type) and G-quadruplex (signal rise type) by combining with ThT to detect the marker A beta DNA of Alzheimer's syndrome. + The sensor preparation based on G-triplex only needs a small amount of G-C base pair, ThT and K + , and the preparation cost is relatively low, but the output signal strength is weak, the detection limit is high, and it is not conducive to the detection of low-concentration target DNA; the sensor preparation based on G-quadruplex has a relatively high preparation cost, but the output signal strength is high and the detection limit is low, and it is more conducive to the detection of low-concentration samples.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, and relates to label-free fluorescent sensors, specifically a label-free fluorescent sensor, its preparation method, and its application. Background Technology

[0002] Single-stranded nucleic acid detection plays a crucial role in the early diagnosis and treatment of diseases. Recent studies have shown that certain nucleic acids can serve as biomarkers for some diseases, such as cancer, neurological disorders, and bacterial and viral infections. Detection of specific nucleic acid sequences can help prevent a variety of diseases. Currently, commonly used sensor methods for detecting single-stranded nucleic acids include colorimetry, electrochemical methods, and fluorescence methods. Among these, fluorescence methods are widely used due to their high sensitivity, good stability, and fast response speed.

[0003] The aggregation of β-amyloid protein (Aβ), a marker of Alzheimer's disease, leads to toxic oligomers that aggregate into insoluble β-amyloid plaques (Aβ plaques). This process ultimately results in the hyperphosphorylation of tau protein. Subsequently, these plaques form neurofibrillary tangles within neurons, triggering complex downstream reactions that ultimately lead to neuronal death. This neurodegenerative pathology caused by gene mutations is known as familial Alzheimer's disease. The roles of various mutations in familial Alzheimer's disease have been well understood; however, it accounts for less than 1-5% of all cases. The most common form of Alzheimer's disease is sporadic. This type of Alzheimer's is common in patients aged 65 and older, and the underlying genetic or molecular causes remain unknown. Although there are potential genetic differences between familial and sporadic forms of Alzheimer's disease, the two subtypes share overlap in various pathophysiological aspects of disease development, particularly the neuronal damage resulting from the gradual increase in Aβ accumulation. However, clinical studies have found that most Alzheimer's patients do not have mutations in APP or related genes. Furthermore, overexpression of normal APP protein in the mouse brain does not produce significant Aβ plaques. These studies indicate that Aβ produced by the enzymatic breakdown of APP is not the most important source of Aβ. Therefore, the origin of the most critical and pathogenic Aβ remains a significant mystery. Finding the true source of Aβ is crucial for the diagnosis, prevention, and treatment of Alzheimer's disease. However, currently, there is no efficient method for rapid detection of Aβ.

[0004] Patent CN 113234798 A discloses a fluorescence sensor based on in vitro transcription and G4-ThT, its preparation, and its application in HBV DNA detection. It uses two pairs of primers and two enzymes to transcribe an RNA G4 chain. The RNA G4 binds to thioflavin (ThT), enhancing the fluorescence signal, and collecting the fluorescence signal enables the detection of HBV DNA. This patent generates the G4 chain through an indirect enzymatic reaction, resulting in a complex sensor preparation process that cannot achieve rapid detection. In 2019, Wu et al. developed a method for detecting miRNA based on G-triplex molecular beacons and double-stranded specific nuclease signals. This method utilizes miRNA and enzymes to form a cyclic cleavage, continuously constructing a G-triplex structure, which binds to ThT to generate a strong fluorescence signal. Existing methods utilizing the luminescence properties of G3 and G4 chains binding to ThT mostly involve the indirect generation of G3 or G4 chains or the reaction of G3 or G4 chains with other substances, resulting in complex sensor systems and lengthy detection processes.

[0005] Our research group has conducted a comprehensive and in-depth exploration to address the two technical issues mentioned above. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a label-free fluorescent sensor, its preparation method, and its application.

[0007] The technical solution of this invention is implemented as follows: A label-free fluorescent sensor, wherein the working solution of the label-free fluorescent sensor is a buffer solution containing thiosulfate T and a G-rich sequence.

[0008] The aforementioned G-rich sequences exhibit a molecular hairpin structure, in which the G-rich sequence portion is locked within the molecular hairpin stem, while the remaining portion forms a G-triplex or G-quadruplex structure.

[0009] The nucleotide sequences of the G-rich sequences mentioned above are HP1 or HP9, wherein the sequence of HP1 is shown in SEQ ID No. 1 and the sequence of HP9 is shown in SEQ ID No. 2.

[0010] When the nucleotide sequence of the G-rich sequence is HP1, the buffer solution is 20 mmol / L Tris-HCl and 75 mmol / L KCl, and the final concentration of thiosulfate T in the working solution is 9 μmol / L and the final concentration of HP1 is 100 nmol / L.

[0011] When the nucleotide sequence of the G-rich sequence is HP9, the buffer solution is 20 mmol / L Tris-HCl and 300 mmol / L KCl, and the final concentration of thiosulfate T in the working solution is 21 μmol / L and the final concentration of HP1 is 100 nmol / L.

[0012] The above-mentioned method for preparing a label-free fluorescent sensor involves the following steps: adding thiosulfate T to a buffer solution containing a G-rich sequence to obtain the working solution of the label-free fluorescent sensor, wherein the buffer solution refers to a Tris-HCl solution containing KCl.

[0013] The above-mentioned method for using the label-free fluorescence sensor involves the following steps: adding the test solution to the working solution of the label-free fluorescence sensor, incubating it, and then using a fluorescence spectrophotometer to detect the emission spectrum of the mixed solution.

[0014] The volume ratio of the test solution to the working solution was 50:1, and the incubation temperature was 37℃ for 60 min.

[0015] Furthermore, the excitation wavelength for emission spectroscopy detection is 448 nm.

[0016] The above-mentioned label-free fluorescent sensor is used in the preparation of reagents or medical devices for detecting β-amyloid genes.

[0017] The present invention has the following beneficial effects: 1. This application uses the β-amyloid protein (Aβ), a biomarker of Alzheimer's disease, as the target DNA. Molecular hairpins containing G-rich sequences are designed, and the effects of different terminal bases on the formation of the G-rich sequence structure are analyzed. Label-free fluorescent sensors based on G-triplex and G-quadruplex structures are constructed using a one-step detection method. The HP1 sensor with a G-triplex structure detects the target DNA by relating the decrease in detected fluorescence signal to the target DNA concentration; the HP9 sensor with a G-quadruplex structure detects the target DNA concentration by relating the increase in detected fluorescence signal.

[0018] 2. In this application, it was found that when a non-G base is introduced at the end of a G-rich sequence, a G-quadruplex structure is gradually formed. As the number of bases increases, the G-quadruplex structure becomes more stable and has a stronger binding ability with ThT. The resulting G-quadruplex structure is more stable, indicating that adding a non-G base to the end of a G-rich sequence promotes the formation of a G-quadruplex structure.

[0019] 3. This application designs various molecular hairpins containing G-rich sequences by adding different non-G bases to the ends, and constructs label-free fluorescent sensors based on G-triplexes (signal downtrend) and G-quadruplexes (signal uptrend) to detect Aβ DNA, a biomarker for Alzheimer's disease, by combining them with ThT. Comparison of the preparation and detection results of the two sensors shows that the G-triplex-based sensor requires only a small number of GC base pairs and K... +Sensors based on ThT can be fabricated at a low cost, but their output signal strength is weak and their detection limit is high, which is not conducive to the detection of low-concentration target DNA. Sensors based on G-quadruplexes have a relatively high fabrication cost, but their output signal strength is high and their detection limit is low, which is more conducive to the detection of low-concentration samples.

[0020] 4. Analysis of the effect of terminal non-G bases on the structure of G-rich sequences revealed that the addition of terminal non-G bases promotes the formation of G-quadruplexes and increases the stability of the G-quadruplex structure. This study not only provides a new method for label-free fluorescent detection of single-stranded nucleic acids but also lays the research foundation for the further application of G-rich sequences in biosensors, which is of great significance in the early diagnosis and treatment of diseases. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the principle of a label-free fluorescent sensor based on G-rich sequences for detecting single-stranded nucleic acids.

[0023] Figure 2 The UV-Vis absorption spectra of (A) HP1, (B) HP3 and (C) HP9; and the circular dichroism spectra of (D) HP9, (E) HP3 and (F) HP9.

[0024] Figure 3 The fluorescence spectra of sensors (A) HP1 and (B) HP9 are shown.

[0025] Figure 4 This describes the principle of label-free fluorescent sensors based on G-tristan and G-quadristan for detecting target DNA.

[0026] Figure 5 The effect of adding A / T bases to the ends of (A) G-rich sequences on fluorescence signal; (B) Line graph of fluorescence signal changes.

[0027] Figure 6 The effects of (A) the number of stem bases of HP1 and HP9 on fluorescence signal; (B) the effect of ThT concentration on fluorescence signal; (C) K + The effects of concentration on fluorescence signal; (D) The effects of pH on fluorescence signal; (E) The effects of temperature on fluorescence signal; (F) The effects of reaction time on fluorescence signal; ΔF for F -F 0 The absolute value of.

[0028] Figure 7 Fluorescence spectra of HP1 (A) and HP9 (B) sensors in the presence of different concentrations of target DNA; target DNA concentrations of HP1 (C) and HP9 (D) sensors versus... FF 0 A linear curve between them.

[0029] Figure 8 Specific to HP1 (A) and HP9 (B) sensors. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Instruments and reagents F-7000 fluorescence spectrophotometer (HITACHI, Japan); UV-2700 ultraviolet-visible spectrophotometer (SHIMADZU, Japan); MOS-500 circular dichroism spectrometer (BioLogic Science Instruments, France); ME204 electronic balance and FE-20K pH meter (METTLER TOLEDO, Switzerland); HZQ-F200 constant temperature vibrating screen (Beijing Donglian Haer Instrument Co., Ltd.); Eppendorf 5418 centrifuge (Eppendorf AG, Germany); PURELAB Option-R pure water system (ELGA, UK).

[0032] All oligonucleotide sequences and ThT were purchased from Shanghai Sangon Biotech Co., Ltd., and the oligonucleotide sequences are detailed in Table 1. Tris-hydroxymethylaminomethane (Tris-HCl), KCl, and other reagents were all of analytical grade and purchased from Zhengzhou Xinmaijie Biotechnology Co., Ltd. All solutions used in the experiments were prepared with ultrapure water (18.2 MΩ·cm).

[0033] Table 1 shows the oligonucleotide sequences used. Example 1: Fabrication of a label-free fluorescent sensor 6 μL of ThT was added to 12 μL of HP1 and HP9 solutions, respectively, and buffer solution was added to a total volume of 600 μL to form working solutions, which were then stored at 4°C for later use. In the HP1 sensor, the final concentrations of ThT and HP1 were 9 μmol / L and 100 nmol / L, respectively, and the buffer solutions were 20 mmol / L Tris-HCl and 75 mmol / L KCl. In the HP9 sensor, the final concentrations of ThT and HP9 were 21 μmol / L and 100 nmol / L, respectively, and the buffer solutions were 20 mmol / L Tris-HCl and 300 mmol / L KCl.

[0034] 2. Detection Principle During the experiment, the inventors designed various molecular hairpins containing G-rich sequences (nucleotide sequences shown in Table 1), each with different terminal non-G base sequences, to detect target DNA. The principle is explained below. Figure 1 as follows: (1) Design HP1 molecular hairpins. In the absence of target DNA, HP1 has a hairpin structure, with the G-rich sequence portion locked in the hairpin stem, and the remaining portion forming a G-triplex structure, which binds to ThT to generate a strong fluorescent signal; when target DNA is added, the stem-loop structure of HP1 is destroyed, and the remaining G-rich sequence is completely released, with only a small portion remaining in the K-loop. + Under induction, a G-quadruplex structure is formed, resulting in a significant decrease in fluorescence signal compared to before. For example... Figure 1 As shown in Figure A, the target DNA is detected based on the relationship between the decrease in fluorescence signal and the target DNA concentration.

[0035] (2) Add an A base to the 5' end of the G-rich sequence of the HP1 molecular hairpin to form the HP3 molecular hairpin, and add target DNA, such as Figure 1 The fluorescence intensity of the solution shown in B remains basically unchanged, indicating that after the molecular hairpin is opened, the destroyed G-triplex and the newly formed G-quadruplex are basically in equilibrium.

[0036] (3) Adding the base AATT to the 5' end of the G-rich sequence of the HP1 hairpin forms the HP9 hairpin. Upon addition of target DNA, the HP9 hairpin opens, and the fluorescence intensity of the solution increases significantly, indicating a stronger change in the G-rich sequence structure, forming an antiparallel G-quadruplex structure. For example... Figure 1 As shown in C, the concentration of target DNA can be detected based on the magnitude of the fluorescence signal increase. In the above studies, HP1 and HP9 can detect target DNA with both signal decrease and signal increase patterns, so further studies will be conducted on them respectively.

[0037] To analyze the experimental principle, the HP1, HP3, and HP9 sequences were analyzed using ultraviolet-visible absorption spectroscopy, such as... Figure 2AC, the characteristic absorption peak of ThT is at 412 nm; the absorption peak position remains unchanged after ThT reacts with the target DNA, but the absorption peak shows an 8 nm red shift (412–420 nm) after reacting with the HP1 / HP3 / HP9 molecular hairpin, indicating that ThT can bind to the molecular hairpin but not to the target DNA; compared with ThT+ HP1 / HP3 / HP9, the absorption peak of the ThT+ HP1+Target sample did not show a red shift (420–420 nm). Figure 2 A), the absorption peak of ThT+ HP3+Target was red-shifted by 2 nm (420–422 nm). Figure 2 B), The absorption peak of ThT+ HP9+Target was red-shifted by 7 nm (420–427 nm). Figure 2 C) indicates that when a non-G base is introduced at the end of a G-rich sequence, a G-quadruplex structure is gradually formed, and the more bases are added, the more stable the G-quadruplex structure becomes and the stronger its binding ability with ThT becomes.

[0038] Circular dichroism spectroscopy is a commonly used method for detecting and characterizing the structures of G-triplexes and G-tetraplexes. To further analyze the structural changes of G-rich sequences, circular dichroism spectroscopy was performed on samples with the same concentrations of HP and HP+Target, respectively. The results are as follows: Figure 2 DF. HP1 shows positive peaks at approximately 276 nm and 291 nm, and a negative peak at 250 nm, indicating the formation of G-triplexes with parallel / antiparallel structures; after the addition of target DNA, a negative peak appears at 242 nm, and a positive peak appears at 281 nm. Figure 2 (D) indicates that the G-rich sequence was completely released, forming a few parallel G-quadruplexes. HP3 has a negative peak at approximately 254 nm and a positive peak near 285 nm. After the addition of target DNA, a negative peak appears at approximately 250 nm, and a positive peak appears near 279 nm. Figure 2 E), indicating that parallel G-quadruplex structures are formed after the addition of target DNA. HP9 has a negative peak at 248 nm and a positive peak near 275 nm. After the addition of target DNA, the circular dichroism characteristic peaks shift red to approximately 254 nm (negative peak) and 283 nm (positive peak), respectively. Figure 2 (F) This is a typical antiparallel structure, indicating that adding bases to G-rich sequences can promote the formation of numerous antiparallel G-quadruplex structures. Circular dichroism spectroscopy and UV-Vis spectroscopy results are consistent, verifying the correctness of the analysis that adding non-G bases to the ends of G-rich sequences promotes the formation of G-quadruplex structures.

[0039] To verify the feasibility of the HP1 and HP9 sensors, the fluorescence spectra of ThT, ThT+ HP1 / HP9, and ThT+ HP1 / HP9+Target samples were tested. The HP1 sensor is shown below. Figure 3A. ThT alone exhibits extremely low fluorescence signal. Adding HP1 forms a G-triplex structure, inducing an enhancement of the ThT fluorescence signal. Upon addition of target DNA, the G-triplex structure is disrupted, leading to a decrease in fluorescence. A schematic diagram of this principle is shown in 4A. The HP9 sensor is shown below. Figure 3 B. The addition of HP9 produces a weak fluorescent signal. After the addition of target DNA, an antiparallel G-quadruplex is formed, and the ThT fluorescent signal is significantly enhanced. The schematic diagram of the principle is shown in 4B.

[0040] 3. Condition Optimization To analyze the influencing factors in the sensor fabrication process, the following factors were considered: the number of non-G bases at the ends of G-rich sequences, the number of bases in the hairpin stem of the molecule, and K. + The concentration, ThT concentration, pH, temperature, and reaction time were optimized experimentally.

[0041] ① Optimization of non-G bases at the ends of G-rich sequences Adding a non-G base to the end of a G-rich sequence induces a conformational change in the G-rich sequence, thereby affecting the fluorescence signal generated by ThT binding. The changes in sensor output signal before and after the addition of the non-G base to the end of a G-rich sequence (T, A, AA, TT, AT, ATT, TTAA, AATT, ATAT) were examined. F- F 0 The relationship (where, F 0 This indicates the fluorescence intensity output by the sensor when there is no target DNA. F (This indicates the fluorescence intensity output by the sensor after the addition of target DNA). Results are as follows: Figure 5 When no base was added to the end of the G-rich sequence (HP1) or one T or A base was added (HP2, HP3), the fluorescence signal decreased; when multiple bases were added, the fluorescence signal increased (HP4-HP9), indicating that adding non-G bases to the end of the G-rich sequence promotes the formation of the G-quadriplex structure. The fluorescence signal decreased the most when no base was added (HP1), and increased the most when an AATT base was added (HP9). Therefore, HP1 and HP9 were subsequently selected to prepare label-free fluorescent sensors.

[0042] ② Optimization of stem length The length of the molecular hairpin stem affects the release of G-rich sequences, thus influencing the sensor output signal intensity. In this experiment, HP1 (HP1-4~HP1-9) and HP9 (HP9-8~HP9-11) with different stem lengths were prepared. The absolute value of the change in sensor output signal before and after the addition of target DNA was examined. ΔF (in, ΔF for FF 0 The absolute value, due to the HP1 sensor F < F 0 , F- F 0 To compare the performance of the two sensors, this paper expresses the relationship between the changes in the sensor's output signal in absolute value form, since the values ​​are negative. The results are as follows: Figure 6 In the HP1 sensor, when the stem length is 4, 6, 7, or 9, the fluorescence signal output by the molecular hairpin decreases with the increase of the number of bases in the stem. This is because as the stem length increases, the released G-rich sequence cannot form a G-triplex and its binding ability with ThT weakens. After the addition of target DNA, the fluorescence signal of different stem lengths remains basically unchanged, and the G-rich sequence is completely released. Among them, the stem length is 4 (HP1-4). ΔF Maximum; similarly, in the HP9 sensor, the longer the stem, the weaker the fluorescence signal of the molecular hairpin. After adding target DNA, an excessively long stem is not conducive to the opening of the hairpin. Therefore, as the number of bases in the hairpin stem increases, the G-quadruplex structure decreases, and the output fluorescence signal weakens. When the stem length is 8 (HP9-8). ΔF The largest. Therefore, HP1-4 and HP9-8 were subsequently selected for experiments.

[0043] ③ Optimization of ThT concentration ThT exhibits low autofluorescence but produces strong fluorescence upon binding to G-tristan and G-tetrastan. Experiments analyzed the changes in fluorescence signals before and after target addition in HP1 and HP9 sensors at different ThT concentrations. Figure 6 B. The fluorescence signal increases with the increase of ThT concentration. The ThT concentrations corresponding to the largest changes in fluorescence signal of HP1 and HP9 sensors are 9 μmol / L and 21 μmol / L, respectively.

[0044] ④ Buffer K + and pH concentration optimization K in solution + pH and other pH factors participate in and influence DNA structure formation. Different concentrations of K were prepared experimentally. + Examine K with buffer solutions of different pH values. + Concentration, solution pH and ΔF The relationship. The result is as follows: Figure 6 C and 6D, two types of sensors ΔF All follow K + The concentration initially increases and then decreases with increasing pH, and increases slowly with increasing pH; therefore, K is chosen. + Concentration 75 mmol / L, pH=8 (HP1) and K + Subsequent experiments were conducted at a concentration of 300 mmol / L and pH=9 (HP9).

[0045] ⑤ Optimization of reaction time and test temperature Target DNA was added to the HP1 and HP9 sensors, and fluorescence spectra were measured every 3 minutes. The fluorescence signal changes over time as shown in the curves. Figure 6 E. Within 0-60 min, the output signal strength of both HP1 and HP9 sensors varied significantly over time. After 60 min, the output of both sensors tended to stabilize; therefore, subsequent experiments were conducted at 60 min. The effects of different test temperatures and... ΔF The relationship, the result is as follows Figure 6 F, as temperature increases ΔF The temperature gradually decreased because rising temperatures can affect or destroy the spatial structure of DNA hybridization. Therefore, 15℃ and 10℃ were chosen as the test temperatures for the HP1 and HP9 sensors.

[0046] 4. Sensitivity Analysis The above two methods were used to detect different concentrations of target DNA at HP1 stem lengths of 4 mm. C ThT =9μmol / L C K+ The HP1 sensor was prepared under optimized conditions of 75 mmol / L and pH=8. Different concentrations of target DNA were added, and the mixture was incubated at 37°C for 60 min. The fluorescence spectra were measured at 15°C as follows: Figure 7 A. As the target DNA concentration increases, the HP1 hairpin opens, the G-trimolecular structure is disrupted, THT is released, and the sensor output signal gradually decreases. For example... Figure 7 C, within the range of 1-100 nmol / L, shows a good linear correlation between the change in the sensor's output signal and the target DNA concentration, with the linear equation being y = -3.02x - 2.62 (where, y The difference in fluorescence intensity FF 0 , x (target DNA concentration), R 2 =0.989, detection limit (S / N=3) is 1 nmol / L. At HP9 stem length of 8, C ThT =21μmol / L C K+ The HP9 sensor was prepared under optimized conditions of 300 mmol / L and pH 9. Different concentrations of target DNA were added, and the mixture was incubated at 37°C for 60 min. Fluorescence spectra were measured at 10°C as follows: Figure 7 B. As the concentration of target DNA increases, the HP9 hairpin opens, resulting in more G-quadruplex structures. Upon binding with THT, the fluorescence signal in the detection system continuously increases, such as... Figure 7D, within the range of 0.1-100 nmol / L, showed a good linear correlation between the change in the sensor's fluorescence signal and the target DNA concentration. The linear equation was y = 4.52x + 2.44 (R²). 2 =0.989). The limit of detection (S / N=3) is 0.1 nmol / L.

[0047] 5. Specificity analysis The above two sensors were used to detect target DNA (T), single-base mismatched DNA (MT1), triple-base mismatched DNA (MT3), random sequence DNA (MTR), and Tau protein DNA (Tau), another marker of AD, at a concentration of 100 nmol / L. The detection results are as follows: Figure 8 The A and 8B sensors showed that the HP1 sensor detected 77.6%, 13.7%, -16.6%, and 6.7% of the target DNA detection signal, respectively, while the HP9 sensor detected 81.4%, 25.7%, 6.1%, and 7.4% of the target DNA detection signal, respectively, indicating that both sensors have good specificity.

[0048] Example 2: Detection of target DNA 12 μL of target DNA of different concentrations were added to 600 μL HP1 and HP9 sensors respectively, and incubated at 37℃ for 60 min. The emission spectra of the mixed solutions were detected by fluorescence spectrophotometer, with the excitation wavelength set to 448 nm.

[0049] Application example: Analysis of actual samples Two sensors were used to detect target DNA in human serum samples to verify the practicality of the method. The human serum samples were purchased from Beijing Solarbio Science & Technology Co., Ltd., and were diluted 10-fold with ultrapure water for spiked recovery experiments. The results are shown in Table 2. Table 2 Results of target DNA detection in human serum samples As shown in Table 2, the recovery rates of the HP1 sensor were in the range of 96.2%-102.2%, and those of the HP9 sensor were in the range of 98.8%-107.3%. The relative standard deviation (RSD) of the sample recovery rates for both sensors (n=5) was less than 4.3%, indicating that the two label-free fluorescent sensors are practical for detecting AβDNA in human serum.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A label-free fluorescent sensor, characterized in that, The working solution of the label-free fluorescent sensor is a buffer solution containing thiosulfate T and G-rich sequences; The nucleotide sequence of the G-rich sequence is HP1, and the sequence of HP1 is shown in SEQ ID No. 1; the buffer solution is 20 mmol / L Tris-HCl and 75 mmol / L KCl, and the final concentration of thiosulfate T in the working solution is 9 μmol / L and the final concentration of HP1 is 100 nmol / L.

2. The method for preparing the label-free fluorescent sensor according to claim 1, characterized in that, The steps are as follows: thiosulfate T is added to a buffer solution containing a G-rich sequence to obtain the working solution of the label-free fluorescent sensor, wherein the buffer solution is the buffer solution described in claim 1; the nucleotide sequence of the G-rich sequence is shown in SEQ ID No.

1.

3. The use of the label-free fluorescent sensor of claim 1 in the preparation of reagents for detecting β-amyloid genes.

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