A fluorescence aptamer sensor with wide detection range and a preparation method and application thereof

Abstract

The application belongs to the field of sensors, and relates to detection of ATP and AMP, in particular to a wide-range fluorescence aptamer sensor and a preparation method and application thereof. Four kinds of fluorescence quenching type aptamer sensors based on FRET effect are designed by using the unique optical properties of AuNPs and the specificity of nucleic acid aptamer, and by controlling the distance between AuNPs and FAM fluorescence groups through the number of bases T, so that wide-range detection of ATP is realized. Through comparative analysis of the four kinds of sensors, it is found that the more the number of bases T is, the less the nucleic acid aptamer modified to the surface of AuNPs is, the detection range of the sensor gradually narrows, and the detection sensitivity gradually increases; the ATP nucleic acid aptamer is replaced by the AMP nucleic acid aptamer, the detection of AMP is successfully realized, the detection range reaches 1nM-50muM, and the universality of the sensor is verified.

Description

Technical Field

[0001] This invention belongs to the field of sensors and relates to the detection of ATP and AMP, specifically a fluorescent aptamer sensor with a wide detection range, its preparation method, and its application. Background Technology

[0002] Small biological molecules play a vital role in the normal life activities and growth and development of organisms, and abnormal concentrations of these molecules can cause a variety of diseases. Traditional detection of small biological molecules usually employs instrumental methods, mainly high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and micellar liquid chromatography (MLC). These methods offer advantages such as high accuracy and a wide detection range, but the instruments used are relatively expensive, complex to operate, and require specialized personnel, making them difficult to widely apply.

[0003] Fluorescent aptamer sensors utilize nucleic acid aptamers to specifically recognize targets and convert the reaction signal into a fluorescence intensity signal, thereby enabling the detection of the target analyte concentration. Due to their advantages such as simple preparation process, low cost, high sensitivity, and fast analysis speed, they have attracted widespread research attention in recent years. Fahmida Nasrin et al., based on the principle of localized surface plasmon resonance, used peptide chains to control the distance between quantum dots and AuNPs to enhance the fluorescence signal, and developed a highly sensitive biosensor for virus detection based on this principle, with a linear range of 10 for influenza virus. -14 ~10 - 9The detection limit was 17.02 fg / mL. Based on the same principle, Jin et al. used single-stranded nucleic acid sequences to adjust the distance between AuNPs and fluorescent groups, achieving highly sensitive detection of the proteolytic enzyme caspase-3, with a detection range of 0.01-10 ng / mL and a detection limit of 10 pg / mL. Li et al. developed a fluorescent aptamer sensor for detecting trace thrombin based on fluorescence resonance energy transfer between cadmium sulfide quantum dots and AuNPs. The energy donor, cadmium sulfide quantum dots, was modified with nucleic acid aptamers, and the energy acceptor, AuNPs, was modified with their complementary DNA. FRET between the two caused fluorescence quenching of the cadmium sulfide quantum dots. Upon addition of thrombin, the aptamers specifically bound to them to form G-quadruplexes, and the AuNPs moved away from the quantum dots, resulting in fluorescence recovery. The linear range was 35-54.0 nmol / L, and the detection limit was 0.38 nmol / L. Esmaelpourfarkhani et al. developed a pM-level ampicillin fluorescent aptamer sensor using 3,4,9,10-triphenyltetracarboxylic acid diimide (PTCDI) as a fluorescent carrier. The nucleic acid aptamer and complementary strand were modified onto AuNPs, at which point the PTCDI fluorescence signal was strong. After the analyte was added, the complementary strand detached from the AuNPs and became free in the solution, causing the PTCDI fluorescence intensity to decrease. The linear detection range of the sensor was 100–1000 pM, and the detection limit was 29.2 pM. The research on fluorescent aptamer sensors for small biological molecules has largely focused on achieving higher sensitivity and lower detection limits. However, in practical applications, the concentration ranges of various small biological molecules are extremely wide. For example, adenosine triphosphate (ATP) is present in normal human tissues at approximately 1-10 nmol / L, in lung cancer tissues at approximately 100-400 μmol / L, and in liver cancer tissues at 0.1-0.4 μmol / L; ampicillin (AMP) is present in meat at approximately 50 μg / kg and in milk at approximately 4 μg / kg. Therefore, improving the detection range of fluorescent aptamer sensors while maintaining low detection limits is crucial for their further application. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a fluorescent aptamer sensor with a wide detection range, its preparation method, and its application, thereby constructing a fluorescent aptamer sensing detection method with a wide range and low detection limit.

[0005] The technical solution of this invention is implemented as follows:

[0006] The fabrication method of a fluorescent aptamer sensor with a wide detection range includes the following steps:

[0007] (1) AuNPs were synthesized by reducing chloroauric acid with sodium citrate. A 1% HAuCl4 solution was heated to 95°C, and then a 1% sodium citrate aqueous solution was added. The mixture was kept at this temperature for a period of time until the color turned wine red. After cooling at room temperature, the AuNPs solution was obtained. The prepared solution was stored at 4°C for later use.

[0008] AuNPs were stabilized and protected using dipotassium bis(p-sulfonylphenyl)phenylphosphine dihydrate (BSPP). A BSPP concentration of 10 mg / mL was added to the prepared AuNPs solution, and the mixture was reacted in a shaking incubator for 8 h to obtain a BSPP-stabilized AuNPs solution.

[0009] (2) Take the activated PolyT n Mix 150 μL each of -Apt solution and cDNA@FAM solution in a 1:1 ratio. Incubate the mixture in PBS buffer (pH 7.4) at 37°C for 3 hours to obtain a product containing PolyT. n A solution of FAM@dsDNA nanostructures, where n = 3-36;

[0010] The preferred value is n=6.

[0011] (3) Add PolyT to the PolyT-containing process in step (2) n Add 600 μL of the BSPP-stabilized AuNPs solution from step (1) to the FAM@dsDNA nanostructure solution, mix well, and react at 37°C for 16 h in a constant temperature shaking incubator. Then, add 30 μL of 2M NaCl solution three times every 3 h to bring the final NaCl concentration to 0.2M. Continue incubation at constant temperature for 12 h to obtain the AuNPs@PolyT nanostructure. n - Working solution for FAM@dsDNA nanostructures. Store at 4°C for later use; this is the working solution for the fluorescent aptamer sensor.

[0012] In step (1) above, the concentration of AuNPs solution is 1 nM and the concentration of BSPP solution is 10 mg / mL.

[0013] In step (2) above, PolyT nThe sequence of -Apt is shown in any one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12, and the sequence of cDNA@FAM is shown in SEQ ID No. 13 or SEQ ID No. 14.

[0014] Preferably, in step (2) above, PolyT n The sequence of -Apt is shown in SEQ ID No. 3 or SEQ ID No. 12, and the sequence of cDNA@FAM is shown in SEQ ID No. 13 or SEQ ID No. 14.

[0015] The above PolyT n The concentration of the Apt solution was 10 μM, and the concentration of the cDNA@FAM solution was 10 μM.

[0016] A fluorescent aptamer sensor was prepared using the above-described method.

[0017] The aforementioned fluorescent aptamer sensors are used in the detection of ATP or AMP.

[0018] ① PolyT in the fluorescent aptamer sensor for ATP detection n The sequence of -Apt is shown in any one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11;

[0019] Preferably, PolyT in the fluorescent aptamer sensor is used for ATP detection. n The sequence of -Apt is shown in SEQ ID No. 3, and the sequence of cDNA@FAM is shown in SEQ ID No. 13;

[0020] ② PolyT in the fluorescent aptamer sensor during AMP detection n The sequence of -Apt is shown in SEQ ID No. 12, and the sequence of cDNA@FAM is shown in SEQ ID No. 14.

[0021] The above application involves the following steps: adding an equal volume of the test solution to 100 μL of the working solution of the fluorescent aptamer sensor, then adjusting the volume to 600 μL with PBS buffer (pH = 7.4), incubating at 37°C for 2 hours, and then performing fluorescence spectroscopy detection; substituting the fluorescence intensity x into the formula y = 14.9439 + 14.7453Logx, the concentration of ATP in the test sample is calculated; or substituting the fluorescence intensity x into the formula y = 45.7241 + 37.1901Logx, the concentration of AMP in the test sample is calculated; wherein the fluorescence spectroscopy detection temperature is 30°C, and the excitation wavelength is set to 495 nm.

[0022] The present invention has the following beneficial effects:

[0023] This application utilizes the unique optical properties of AuNPs and the specificity of nucleic acid aptamers to design four fluorescence quenching aptamer sensors based on the FRET effect, controlling the distance between AuNPs and the FAM fluorescent group by the number of base T, achieving a wide range of ATP detection. Comparative analysis of the four sensors revealed that a higher number of base Ts results in fewer nucleic acid aptamers modified onto the AuNP surface, leading to a gradually narrower detection range and a gradually increasing detection sensitivity. Based on AuNPs@PolyT... n The FAM@dsDNA fluorescence sensor (n=6, 12, 24, and 36) showed specificity in detecting ATP within detection ranges of 1nM-100μM, 1nM-5μM, 1nM-1μM, and 10nM-800nM, respectively. The sensor demonstrated the best detection performance when the number of modified T bases was 6, exhibiting good selectivity for ATP compared to similar interfering agents. Replacing the ATP aptamer with the AMP aptamer successfully enabled AMP detection within a range of 1nM-50μM, validating the sensor's versatility. By changing the type of aptamer, a universal method can be provided for the detection of other small biological molecules, metal ions, and proteins. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a schematic diagram illustrating the detection principle of a fluorescence sensor with a wide detection range and a composite nanostructure.

[0026] Figure 2 For AuNPs@PolyT n- Feasibility analysis diagram of FAM@dsDNA fluorescence sensor; where A, B, C, and D represent n values ​​of 6, 12, 24, and 36, respectively.

[0027] Figure 3 Figure A shows the TEM characterization of AuNPs; Figure B shows the UV-Vis absorption spectra of AuNPs solution with different concentrations of NaCl; Figure C shows the relationship between the UV-Vis absorption peaks of AuNPs and the concentration of BSPP after adding different concentrations of BSPP to AuNPs solution and then adding 50mM NaCl solution.

[0028] Figure 4 Fluorescence spectra of different DNA structures before and after adding Hoechst dye to DNA; Agarose gel electrophoresis images of DNA before and after modification with BAuNPs (lanes 1-6 are PolyT...). 24 -Apt,cDNA@FAM,PolyT 24 -FAM@dsDNA,AuNPs,AuNPs@PolyT 24 -Apt,AuNPs@PolyT 24 -FAM@dsDNA); UV-VIS absorption spectra of CAuNPs before and after dsDNA modification; D and E are fluorescence spectra of ATP detection by sensors prepared according to schemes one and two, respectively; F is a graph showing the relationship between ATP concentration and fluorescence intensity under the two preparation schemes.

[0029] Figure 5 A: Original spectrum and line graph showing the effect of pH on FAM fluorescent groups; B: Original spectrum and line graph showing the effect of reaction time after adding the analyte; C: Spectrum and line graph showing the effect of temperature on fluorescence intensity in the working solution; D: AuNPs@PolyT with different numbers of T bases. 24 -FAM@dsDNA complex fluorescence spectrum, E is the histogram of T base number and fluorescence peak; F is the fluorescence spectrum based on AuNPs@PolyT n - Gel electrophoresis characterization of the FAM@dsDNA fluorescence sensor.

[0030] Figure 6 In the diagram, A, B, C, and D represent AuNPs@PolyT. n The original spectra and line graphs of the FAM@dsDNA (n=6, 12, 24, 36) fluorescence sensors with different concentrations of ATP.

[0031] Figure 7 The results are based on the specific detection results of the AuNPs@PolyT6-FAM@dsDNA fluorescence sensor.

[0032] Figure 8In the middle, A and B are the original spectra and line graphs of AMP with different concentrations added based on the AuNPs@PolyT6-FAM@dsDNA fluorescence sensor, respectively; C is the detection result of AMP specificity based on the AuNPs@PolyT6-FAM@dsDNA fluorescence sensor. Detailed Implementation

[0033] 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.

[0034] Experimental materials:

[0035] Hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium citrate (C6H5Na3O7·2H2O), and chloroauric acid (HAuCl4·3H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. All of the above reagents are AR grade.

[0036] Urate-5'-triphosphate trisodium salt (UTP), guanosine-5'-triphosphate trisodium salt (GTP), cytidine-5'-triphosphate tetrasodium salt (CTP), adenosine-5'-triphosphate disodium salt (ATP), ampicillin (AMP), phosphate buffer (20×PBS), and sodium chloride (NaCl) were all purchased from Shanghai Sangon Biotech Co., Ltd.

[0037] Dipotassium di(p-sulfonylphenyl)phenylphosphine dihydrate (BSPP) was purchased from Beijing Bailingwei Technology Co., Ltd.

[0038] Nucleic acid aptamer sequence listing:

[0039] Table 1: DNA probe sequences

[0040]

[0041] Example 1

[0042] This embodiment provides a method for fabricating a fluorescent aptamer sensor with a wide detection range:

[0043] Preparation and stabilization protection of 1AuNPs

[0044] AuNPs were synthesized by reducing chloroauric acid with sodium citrate. A 1% HAuCl4 solution was heated to 95°C, followed by the addition of a 1% sodium citrate aqueous solution. The mixture was kept at this temperature for a period of time until the color turned wine-red. After cooling to room temperature, the AuNPs solution was obtained. The prepared solution was stored at 4°C for later use.

[0045] AuNPs were stabilized and protected using dipotassium bis(p-sulfonylphenyl)phenylphosphine dihydrate (BSPP). A BSPP concentration of 10 mg / mL was added to the prepared AuNPs solution, and the mixture was reacted in a shaking incubator for 8 h to obtain a BSPP-stabilized AuNPs solution.

[0046] 2. Sensor fabrication

[0047] Take the activated PolyT n Mix 150 μL each of -Apt solution and cDNA@FAM solution in a 1:1 ratio. Incubate the mixture in PBS buffer (pH 7.4) at 37°C for 3 hours to obtain a product containing PolyT. n A solution containing AuNPs@dsDNA nanostructures was prepared. 600 μL of AuNPs solution was added to the solution, mixed thoroughly, and reacted in a constant temperature shaking incubator at 37°C for 16 h. Subsequently, 30 μL of 2M NaCl solution was added to the solution three times every 3 h to achieve a final NaCl concentration of 0.2M. The mixture was then incubated at the same temperature for another 12 h to obtain the AuNPs@dsDNA nanostructure. n - Working solution of FAM@dsDNA nanostructures. Store at 4°C for later use.

[0048] 3. Detection of analytes

[0049] Take 100 μL of the working solution, add 100 μL of ATP and AMP solutions of different concentrations, and bring the volume to 600 μL with PBS buffer (pH 7.4). Incubate at 37°C for 2 hours before fluorescence spectroscopy. Add PBS buffer to 100 μL of the working solution to bring the volume to 600 μL as a blank control sample, incubate for 2 hours, and then perform fluorescence spectroscopy. The fluorescence spectroscopy test temperature is controlled at 30°C, and the excitation wavelength is set to 495 nm. Using the same method, different concentrations of AMP solution are prepared for detection, with AMP solution concentrations set as 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 50 μM, 100 μM, and 200 μM.

[0050] 4. Detection Principle and Feasibility Analysis

[0051] Sensor detection principle as follows Figure 1As shown, a nucleic acid aptamer (PolyTn-Apt) containing multiple T bases at its 3' end and a complementary strand (cDNA@FAM) modified with a FAM fluorescent group at its 5' end are first paired to form a double-stranded DNA structure (PolyTn-Apt). n -FAM@dsDNA), which is then combined with AuNPs via Au-S bonds to construct AuNPs@PolyT n In the FAM@dsDNA nanostructure, due to the close proximity of AuNPs and the FAM fluorescent group, fluorescence resonance energy transfer occurs, quenching the FAM fluorescence. Upon addition of the analyte, the nucleic acid aptamer preferentially and specifically binds to the analyte molecule, causing the double strand to open and the cDNA@FAM to become free in the solution, thus restoring the FAM fluorescence. By establishing a linear relationship between fluorescence intensity and analyte concentration before and after analyte addition, the concentration of the analyte can be indirectly determined.

[0052] Using ATP as the detection target, AuNPs@PolyT with different numbers of T bases were prepared. n - The feasibility of using the FAM@dsDNA nanostructure for sensor detection was analyzed, and the results are as follows: Figure 2 As shown in AD, AuNPs@PolyT 24 Taking the FAM@dsDNA fluorescence sensor as an example for analysis, such as... Figure 2 As shown in C, PolyT 24 -FAM@dsDNA is the fluorescence spectrum of functionalized double-stranded DNA, with high fluorescence intensity; AuNPs@PolyT 24 -FAM@dsDNA is the fluorescence spectrum of double-stranded DNA bound to AuNPs. Due to the close proximity of FAM and AuNPs, resonance energy transfer occurs, resulting in a low fluorescence intensity. The fluorescence intensity recovers after adding different concentrations of the analyte, with higher analyte concentrations leading to stronger recovered fluorescence. This result is consistent across different numbers of T bases in the double-stranded DNA, validating the feasibility of this method.

[0053] Example 2

[0054] To further investigate the impact of experimental conditions on the sensor:

[0055] (1) Dispersibility of gold nanoparticles

[0056] To improve the sensor's detection range, the amount of nucleic acid aptamer modification on individual AuNPs should be maximized. Therefore, the AuNPs should be kept dispersed in solution. The AuNPs were characterized using transmission electron microscopy (TEM) and ultraviolet spectrophotometry, and the results are as follows: Figure 3As shown in A and 3B, the AuNPs are relatively uniformly and rounded, with a size of approximately 20 nm, and exhibit a distinct characteristic absorption peak at 520 nm. Adding different concentrations of NaCl to AuNPs solutions of equal concentration yielded the following results: Figure 2 B. As the NaCl concentration increases, the peak value of the absorption peak of AuNPs gradually decreases, indicating that AuNPs gradually aggregate; when the NaCl concentration is 50 mM, AuNPs are completely aggregated, and the characteristic absorption peaks basically disappear. Adding different concentrations of BSPP before adding 50 mM NaCl causes AuNPs to disperse to varying degrees, such as... Figure 3 C. When the concentration of BSPP is 10 mg / mL, the characteristic absorption peak of AuNPs is relatively high. This indicates that using BSPP will improve the dispersibility of AuNPs in solution, thereby improving the binding efficiency of AuNPs with other nanomaterials. Therefore, 10 mg / mL BSPP was selected as the dispersant for AuNPs in subsequent experiments.

[0057] (2) Characterization of the binding effect of functionalized double-stranded DNA to AuNPs

[0058] The binding effect of Hoechst dye on double-stranded DNA was analyzed. Hoechst dye is a dye that binds to the minor groove region of the double helix of dsDNA. In its free state, it emits weak fluorescence. Once it binds to double-stranded DNA, the molecular conformation of the complex changes, absorbing light energy at a wavelength of 360 nm and emitting fluorescence at a wavelength of 460 nm. Figure 4 In line A, the emission peak of the dye is around 460 nm, and the emission peak of the FAM fluorescent group is around 521 nm. Comparing lines d, e, and f, due to the entanglement of a small amount of single-stranded DNA, the emission peaks of lines e and f at 460 nm are higher than that of line d. Comparing the emission peaks of lines c, d, and g at 460 nm, due to the entanglement of a small amount of single-stranded DNA, the emission peaks of lines e and f are higher than those of line d. 24 -Apt and cDNA@FAM form a dsDNA double helix structure, and the fluorescence at 460 nm increases significantly, indicating that PolyT 24 -FAM@dsDNA nanostructure formation. Figure 4 B is an agarose gel electrophoresis characterization image. Comparing lanes 4 to 6, it can be seen that as the surface load of AuNPs increases, the lane distance gradually shortens, indicating that dsDNA is successfully modified on the surface of AuNPs. Figure 4 In the UV-Vis absorption spectrum, the absorption peak of AuNPs after DNA modification shows a significant red shift. This is because the dielectric constant of AuNPs changes after DNA modification, leading to a change in electronic resonance. Furthermore, AuNPs exhibit a distinct absorption peak at 260 nm after DNA modification, which is a typical characteristic absorption peak of DNA, indicating that AuNPs@PolyT... 24The FAM@dsDNA structure was successfully prepared, and the results were consistent with those obtained by gel electrophoresis.

[0059] To optimize AuNPs@PolyT n The preparation process of the -FAM@dsDNA complex structure was investigated using two methods. The sensing properties were then assessed by adding the analyte ATP. Method 1 involved first modifying the surface of AuNPs with single-stranded DNA, then adding a complementary strand to form a double strand through base pairing. Method 2 involved first combining two DNA strands to form a double strand, then modifying the AuNP surface. The detection results are shown below. Figure 4 D, E. Figure 4 F is a graph showing the relationship between ATP concentration and fluorescence intensity under the two preparation schemes. As can be seen from the graph, the linearity of the sensor is better under scheme two, so scheme two will be used for sensor preparation in the future.

[0060] (3) pH, reaction time and temperature

[0061] pH has a significant impact on the fluorescence signal intensity of FAM. To optimize the working solution pH and enhance the sensor output signal intensity, NaOH and HCl were used to adjust the solution pH in the experiment, and the effect of pH on FAM fluorescence signal intensity was studied within the range of 3–10. Figure 5 As shown in Figure A, the fluorescence intensity increases with increasing pH. FAM activity is low and fluorescence intensity is low when pH is between 3 and 6. When pH is between 7 and 11, the fluorescence intensity gradually increases with increasing pH. The pH value of human blood is between 7.0 and 7.4. In order to closely approximate the real blood environment, a buffer solution with pH = 7.4 was selected for subsequent experiments. Figure 5 To optimize the reaction time after adding the analyte, ATP was added to the working solution, and the fluorescence intensity was measured at regular intervals. The fluorescence intensity of the working solution increased slowly with increasing detection time and stabilized after 50 minutes. To reduce detection time and ensure sensor stability, 50 minutes was selected as the subsequent detection time. Figure 5 C represents the effect of ambient temperature on the fluorescence intensity of FAM in the working solution. As the temperature increases, the fluorescence intensity gradually increases, reaching its maximum value at 30℃. After 30℃, the fluorescence intensity does not change much with increasing temperature, so 30℃ was chosen as the detection temperature.

[0062] (4) Number of bases T

[0063] Figure 5 D represents the fluorescence spectrum of the AuNPs@PolyTn-FAM@dsDNA complex structure with different numbers of T bases. Figure 5 E is a bar chart showing the relationship between the number of T bases and the fluorescence peak. The chart shows that the fluorescence intensity is highest when there is no T base in the composite structure, and the fluorescence intensity decreases significantly when there is a T base. The number of T bases has little effect on the fluorescence intensity of the composite structure. Figure 5 E represents the gel electrophoresis characterization results of the complex structure. Without the base T, the complex structure migrates the farthest. With the base T, the migration distance decreases with increasing T number, due to its increasing mass. In summary, modifying double-stranded DNA with the base T can induce fluorescence quenching between AuNPs and FAM, which is beneficial for sensing; however, the effect of the number of T bases remains unclear.

[0064] Implementation Results Example

[0065] 1. Based on AuNPs@PolyT n -FAM@dsDNA fluorescent aptamer sensor for ATP detection

[0066] To analyze the effect of the number of T bases on the sensor detection performance, four sensors with T bases of 6, 12, 24, and 36 were prepared and their detection performance was analyzed. The results are as follows: Figure 6 As shown, fluorescence intensity gradually increased with increasing ATP concentration, and then tended to stabilize. When the T value was 6, ATP concentration and fluorescence intensity showed a logarithmic linear relationship in the range of 1 nM-100 μM, with the fitting formula being y = 14.9439 + 14.7453Logx, and the correlation coefficient R0. 2 The detection limit was 0.97 and the limit of detection was 0.41 nM. When the number of T bases was 12, 24, and 36, the sensor detection ranges were 1 nM-5 μM, 1 nM-1 μM, and 10 nM-800 nM, respectively, and the detection limits were 0.42 nM, 0.44 nM, and 4.35 nM, respectively. The comparison revealed that when the number of T bases was less than 24, the lower the number of T bases, the wider the sensor detection range, while the detection limit remained almost unchanged. This is because a small number of T bases helps AuNPs bind to double-stranded DNA, increasing the amount of double-stranded DNA modification on the AuNPs, thereby increasing the sensor detection range. However, when the number of T bases was too high, the functionalized double-stranded DNA strand length became too long, making it difficult to control the conformation with AuNPs, resulting in a narrower detection range and a higher detection limit. Therefore, the sensor performance was best when the number of T bases was 6.

[0067] A fluorescence sensor with 6 T bases was selected to detect ATP analogs at the same concentration (500 nM) under optimized experimental conditions. The results are as follows: Figure 7 As shown, the ATP output signal is significantly higher than that of ATP analogs (UTP, GTP, CTP), indicating that the sensor has good selectivity for ATP.

[0068] 2. Detection of AMP based on AuNPs@PolyT6-FAM@dsDNA fluorescent aptamer sensor

[0069] To analyze the universality of this sensing method, the aptamers and complementary strands in the functionalized DNA were replaced with the aptamer and complementary strand sequences of AMP. Using AMP as the detection target, the sensor's detection performance was analyzed. Figure 8 AMP concentration and fluorescence intensity showed a logarithmic linear relationship in the range of 1 nM to 50 μM, with the fitting formula being y = 45.7241 + 37.1901Logx and the correlation coefficient being R. 2 =0.99, the detection limit is 0.42 nM, and the specificity is obvious.

[0070] 3. Detection Performance Comparison

[0071] Compared with other methods for detecting ATP in other literature (Table 2) and other sensors constructed using the same FRET principle (Table 3), the method used in this paper has a wider detection range, spanning 5 orders of magnitude, and a lower detection limit of 0.41 nM for the same detection range.

[0072] Table 2 Comparison of ATP detection performance of different methods

[0073]

[0074] Table 3 Performance Comparison of Fluorescent Aptamer Sensors Based on FRET Principle

[0075]

[0076] Utilizing the unique optical properties of AuNPs and the specificity of nucleic acid aptamers, four fluorescence quenching aptamer sensors based on the FRET effect were designed by controlling the distance between AuNPs and the FAM fluorescent group by adjusting the number of T bases, achieving a wide range of ATP detection. Comparative analysis of the four sensors revealed that a higher number of T bases resulted in fewer nucleic acid aptamers modified onto the AuNP surface, leading to a gradually narrower detection range and a gradually increasing detection sensitivity. Under optimal experimental conditions, the AuNPs@PolyT sensor achieved… n The FAM@dsDNA fluorescence sensor (n=6, 12, 24, and 36) showed specificity in detecting ATP within detection ranges of 1nM-100μM, 1nM-5μM, 1nM-1μM, and 10nM-800nM, respectively. The sensor demonstrated the best detection performance when the number of modified T bases was 6, exhibiting good selectivity for ATP compared to similar interfering agents. Replacing the ATP aptamer with the AMP aptamer successfully enabled AMP detection within a range of 1nM-50μM, validating the sensor's versatility. By changing the type of aptamer, a universal method can be provided for the detection of other small biological molecules, metal ions, and proteins.

[0077] 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 method for fabricating a fluorescent aptamer sensor with a wide detection range, characterized in that, The steps are as follows: (1) AuNPs solution was prepared by reducing chloroauric acid with sodium citrate, and then BSPP solution was added. The reaction was shaken and cultured to obtain AuNPs solution stabilized by BSPP. (2) Take PolyT n A mixture of Apt solution and cDNA@FAM solution in equal proportions was used to prepare a mixed solution, which was then reacted in PBS buffer to obtain PolyT. n -FAM@dsDNA solution, where n=0-36; (3) To the PolyT in step (2) n Add the BSPP-stabilized AuNPs solution from step (1) to the FAM@dsDNA solution, mix well, and shake to react. Then add NaCl solution and continue incubation at a constant temperature to obtain AuNPs@PolyT n -FAM@dsDNA nanostructure sensor working solution, i.e. fluorescent aptamer sensor working solution; In step (2), PolyT n The sequence of -Apt is shown in any one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12, and the sequence of cDNA@FAM is shown in SEQ ID No. 13 or SEQ ID No. 14; The PolyT n The concentration of the Apt solution was 10 μM, the concentration of the cDNA@FAM solution was 10 μM, and the pH of the PBS buffer was 7.0-7.

4. The reaction temperature in the PBS buffer was 37℃ and the reaction time was 3 h.

2. The method for preparing a wide detection range fluorescent aptamer sensor according to claim 1, characterized in that: In step (1), the concentration of AuNPs solution is 1 nM and the concentration of BSPP solution is 10 mg / mL.

3. The method for preparing a wide detection range fluorescent aptamer sensor according to claim 2, characterized in that: The shaking culture reaction time was 8 hours.

4. The method for preparing a wide detection range fluorescent aptamer sensor according to claim 1, characterized in that: In step (3), PolyT n The volume ratio of FAM@dsDNA solution to BSPP-stabilized AuNPs solution was 1:2; after adding NaCl solution, the final concentration of NaCl in the entire reaction system was 0.2M.

5. The method for preparing a wide detection range fluorescent aptamer sensor according to claim 4, characterized in that: The oscillation reaction was carried out at a temperature of 37°C for 16 hours; the isothermal incubation was carried out at a temperature of 37°C for 12 hours.

6. A fluorescent aptamer sensor prepared by the preparation method according to any one of claims 1-5.

7. The application of the fluorescent aptamer sensor according to claim 6 in the detection of ATP or AMP, characterized in that: PolyT in the fluorescent aptamer sensor for ATP detection n The sequence of -Apt is shown in any one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11, and the sequence of cDNA@FAM is shown in SEQ ID No. 13; PolyT in the fluorescent aptamer sensor during AMP detection n The sequence of -Apt is shown in SEQ ID No. 12, and the sequence of cDNA@FAM is shown in SEQ ID No.

14.

8. The application according to claim 7, characterized in that, The steps are as follows: Add an equal volume of the test solution to 100 μL of the working solution of the fluorescent aptamer sensor, then adjust the volume to 600 μL with PBS buffer (pH=7.4), incubate at 37℃ for 2 h, and then perform fluorescence spectroscopy detection; substitute the fluorescence intensity x into the formula y=14.9439+14.7453Logx to calculate the concentration of ATP in the test sample; or substitute the fluorescence intensity x into the formula y=45.7241+37.1901Logx to calculate the concentration of AMP in the test sample; the fluorescence spectroscopy detection temperature is 30℃, and the excitation wavelength is set to 495 nm.

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