Use of borophene in preparation of potassium ion fluorescent detection reagent

Abstract

The application relates to a use of boronene in preparation of a potassium ion fluorescent detection reagent, a potassium ion fluorescent detection reagent and a method. The potassium ion fluorescent detection reagent is creatively prepared by combining boronene and a single-stranded nucleic acid fragment with a fluorescent group to form a fluorescent probe, the preparation method is simple, the production process only takes 10 minutes, the fluorescence quenching of the fluorescent probe reaches 90%, and the potassium ion concentration can be detected under a relatively low fluorescence background; the potassium ion fluorescent detection reagent has simple detection steps, the potassium ion detection time is only 1-4 hours, no dangerous and complicated instrument is needed, other common cations such as sodium ions, magnesium ions and calcium ions have a relatively small influence on the detection result, the potassium ion fluorescent detection reagent has the advantages of high safety, fast detection speed and high specificity, and the detection process is reversible.

Description

Technical Field

[0001] This application relates to the field of nanobiosensing technology, and more specifically, to the use of borone in the preparation of potassium ion fluorescence detection reagents. Background Technology

[0002] Potassium ions play a crucial role in maintaining fluid homeostasis, neurotransmission, enzyme activation, and many other physiological processes. Disruptions in potassium ion balance in body fluids can lead to diseases such as stroke, hypokalemia, hypertension, and cancer. Studies have shown that rapid tumor proliferation and subsequent necrosis of tumor cells cause them to secrete intracellular potassium ions extracellularly, resulting in potassium ion concentrations in the tumor microenvironment reaching approximately 45 mM, nearly nine times higher than normal serum potassium concentrations. High potassium ion concentrations lead to the inactivation of tumor-killing T cells and further tumor cell proliferation. Therefore, monitoring potassium ion concentration is beneficial for a deeper understanding and regulation of this process, and has potential value for early detection and treatment of tumors.

[0003] Traditional methods for potassium ion detection include ion chromatography, electrochemical sensing, and flame atomic absorption spectrometry. Ion chromatography relies on a closed mobile phase transport system and a high-performance separation column, with demanding detection conditions and lengthy startup and elution equilibrium times. Electrochemical sensing suffers from poor experimental repeatability, and electrode modification is cumbersome and time-consuming. Flame atomic absorption spectrometry involves the use of propane fuel, which is highly hazardous. These methods generally suffer from drawbacks such as complex detection processes, the need for specialized personnel, and high costs.

[0004] Therefore, providing a rapid, safe, and simple method for detecting potassium ions is a challenge in monitoring potassium ion concentration. Summary of the Invention

[0005] To address the aforementioned issues, the inventors of this application discovered that some nucleic acid sequences exhibit good specificity for recognizing potassium ions. Specifically, in the absence of potassium ions, the nucleic acid fragments are in a randomly coiled single-stranded state, while in the presence of potassium ions, the nucleic acid fragments rapidly fold into a G-quadruplex structure. Therefore, this property of the G-quadruplex is utilized in conjunction with borene to prepare a fluorescent probe. Through the structural changes of the single-stranded nucleic acid fragments, the adsorption and desorption of the single-stranded nucleic acid fragments by borene are achieved. Furthermore, the detection of potassium ions is realized based on the resulting change in fluorescence intensity in the solution. This detection method has the advantages of high safety, fast detection speed, and high specificity, and the detection process is reversible.

[0006] Based on this, the first objective of this application is to provide a use of borene in the preparation of a potassium ion fluorescent detection reagent, wherein the potassium ion reagent comprises a single-stranded nucleic acid fragment linked to a fluorescent group and borene, wherein the single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and borene is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group.

[0007] In one embodiment, the borene is a borene nanosheet.

[0008] In one embodiment, the boroene has a particle size of 50 nm to 200 nm;

[0009] In one embodiment, the thickness of the borophene is 2 nm to 4 nm.

[0010] The second objective of this application is to provide a potassium ion fluorescence detection reagent, comprising borene and a single-stranded nucleic acid fragment linked to a fluorescent group, wherein the single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and the borene is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group.

[0011] In one embodiment, single-stranded nucleic acid fragments can be dissociatively bound to the surface of borogenene.

[0012] In one embodiment, the molar ratio of the single-stranded nucleic acid fragment to the borene is 1:250000;

[0013] In one embodiment, the concentration range of the single-stranded nucleic acid fragment is 0.05 μM to 0.5 μM;

[0014] In one embodiment, the length of the single-stranded nucleic acid fragment is 15bp to 25bp.

[0015] In one embodiment, the sequence of the single-stranded nucleic acid fragment is shown in SEQ ID NO.1.

[0016] In one embodiment, the borene is a borene nanosheet.

[0017] In one embodiment, the boroene has a particle size of 50 nm to 200 nm;

[0018] In one embodiment, the thickness of the borophene is 2 nm to 4 nm.

[0019] In one embodiment, the concentration of borophene is 0.2 mg / mL to 0.5 mg / mL.

[0020] In one embodiment, the fluorescent group is at least one selected from FAM fluorescent group, TAMRA fluorescent group, Cy3 fluorescent group, Cy5 fluorescent group, Alexa Fluor 488 fluorescent group, Atto 590 fluorescent group and Texas red fluorescent group.

[0021] In one embodiment, the potassium ion fluorescence detection reagent further includes a buffer reagent for dispersing borophene.

[0022] In one embodiment, the pH of the buffer reagent is 6.0 to 8.0.

[0023] The third objective of this application is to provide a method for detecting potassium ion fluorescence, comprising: mixing and reacting a sample to be tested with the aforementioned potassium ion fluorescence detection reagent;

[0024] The fluorescence intensity of the reaction solution after the reaction is completed is detected to obtain the fluorescence intensity of the sample to be tested; and

[0025] The potassium ion concentration of the sample is calculated based on the fluorescence intensity of the sample.

[0026] In one embodiment, the step of calculating the potassium ion concentration of the test sample based on the fluorescence intensity of the test sample includes:

[0027] Obtain a standard curve of fluorescence intensity versus potassium ion concentration in the reaction solution. Calculate the potassium ion concentration of the sample based on the fluorescence intensity of the test sample and the standard curve. The standard curve is as follows:

[0028] F1 = -0.2182 + 1.047 × c [K+] ;

[0029] Where F1 = F / F0 - 1, F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity to the background fluorescence intensity of the reaction solution, and c [K+] This indicates the potassium ion concentration in the reaction solution.

[0030] In one embodiment, the potassium ion concentration is 0 mM to 100 mM.

[0031] In one embodiment, the reaction process further includes:

[0032] Boronene nanosheets were obtained by sequentially treating boron powder with probe ultrasound and water bath ultrasound.

[0033] A potassium ion fluorescent detection reagent is prepared by mixing and oscillating a single-stranded nucleic acid fragment with a fluorescent group attached to boronene nanosheets, thereby adsorbing the single-stranded nucleic acid fragment onto the boronene nanosheets.

[0034] In one embodiment, the oscillation reaction time is 10 to 60 minutes.

[0035] In one embodiment, the mixing reaction time is 1 hour to 4 hours.

[0036] The potassium ion fluorescent detection reagent of this application creatively uses borene and a single-stranded nucleic acid fragment with a fluorescent group to form a fluorescent probe. The preparation method is simple, and the process only takes 10 minutes. Moreover, the fluorescence quenching of the fluorescent probe reaches 90%, which can realize the detection of potassium ion concentration in a low fluorescence background. The detection steps of the potassium ion fluorescent detection reagent of this application are simple, and the potassium ion detection time is only 1 to 4 hours. It does not require dangerous and complicated instruments. Other common cations, such as sodium ions, magnesium ions, and calcium ions, have little impact on the detection results. It has the advantages of high safety, fast detection speed and high specificity, and the detection process is reversible. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the principle of a potassium ion fluorescence detection method in one embodiment of this application;

[0038] Figure 2 This is a schematic diagram illustrating the preparation and characterization of borophene nanosheets in Example 1 of this application. Figure 2 In the diagram, a represents a schematic diagram of the liquid phase exfoliation process for preparing boronene nanosheets; b represents the boronene nanosheet suspension; c represents the TEM characterization image of the boronene nanosheets; and d represents the UV-Vis-IR absorption spectrum of the boronene nanosheets.

[0039] Figure 3 This is a schematic diagram illustrating the preparation and detection of the fluorescent probe in Example 2 of this application; Figure 3 In the diagram, a represents a schematic diagram of the process of preparing a fluorescent probe by mixing boronene nanosheets with FAM-DNA; b represents the fluorescence spectra of FAM-DNA in solutions of different concentrations of boronene nanosheets; and c represents the concentration-dependent fluorescence curve of FAM-DNA at 520 nm using boronene nanosheets.

[0040] Figure 4 The images show the fluorescence spectra of the fluorescent probe and different concentrations of potassium ions after reaction in Example 3 of this application; Figure 4 In the diagram, a represents the fluorescence spectrum obtained after adding potassium chloride solution to the fluorescent probe; b represents the fluorescence spectrum of the fluorescent probe in potassium ion solutions of different concentrations; and c represents the scatter plot of normalized fluorescence intensity of FAM-DNA at 520 nm versus potassium ion concentration.

[0041] Figure 5 This is a schematic diagram illustrating the conformational changes of the fluorescent probe and the corresponding changes in fluorescence intensity in Example 4 of this application; Figure 5 In the diagram, a represents a schematic diagram of the conformational changes of the fluorescent probe; b represents a schematic diagram of the normalized fluorescence intensity changes of the reaction solution under the conditions of adding potassium chloride and 18-crown ether-6.

[0042] Figure 6The images show the fluorescence spectra of Example 5 in the presence of different metal ions; the three spectral curves in each small figure correspond to the FAM-DNA solution, the FAM-DNA solution with borene nanosheets, the FAM-DNA solution with borene nanosheets, and the solutions with different salts. Detailed Implementation

[0043] Reference will now be made to detailed embodiments of this application, one or more of which are described below. Each example is provided for explanation and not for limitation of this application. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to this application without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0044] Therefore, this application is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of this application are disclosed in or will be apparent from the following detailed description. It will be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of this application.

[0045] As mentioned above, traditional potassium ion detection methods have drawbacks such as complex detection processes, the need for professional personnel to operate them, and high detection costs.

[0046] To at least partially solve at least one of the above-mentioned technical problems, a first aspect of this application provides the use of borene in the preparation of a potassium ion fluorescent detection reagent, wherein the potassium ion reagent comprises a single-stranded nucleic acid fragment linked to a fluorescent group and borene, the single-stranded nucleic acid fragment specifically binding to potassium ions to form a G-quadruplex, and the borene being used to bind the single-stranded nucleic acid fragment to quench the fluorescent group.

[0047] Specifically, G quadruplexes are a special type of nucleic acid secondary structure formed by the folding of DNA or RNA sequences rich in guanine (G) repeats with metal cations. Through screening, some nucleic acid sequences have shown good specificity for recognizing potassium ions; that is, in the absence of potassium ions, the nucleic acid fragments are in a random coiled single-stranded state, while in the presence of potassium ions, the nucleic acid fragments rapidly fold into G quadruplex structures.

[0048] Boronene refers to a two-dimensional planar structure composed of boron, possessing extremely high electrical conductivity, with a conductivity of approximately 10⁻⁶. 2 Ω - 1 cm -1 Extremely high carrier migration rate, approximately 10² cm⁻¹ 2 V -1 s -1Furthermore, it possesses strong fluorescence emission properties, good thermal stability, and biocompatibility. Specifically, borene is borene nanosheets. The inventors of this application discovered that borene can adsorb single-stranded nucleic acid fragments through van der Waals forces and quench the fluorescent groups on the single-stranded nucleic acid fragments.

[0049] This application creatively utilizes the properties of borene combined with those of G-quadruplexes to prepare fluorescent probes. By changing the spatial structure of single-stranded nucleic acid fragments, borene can adsorb and desorb these fragments. Furthermore, potassium ions can be detected based on the resulting changes in fluorescence intensity in the solution. This method offers advantages such as high safety, fast detection speed, high specificity, and reversible detection process.

[0050] Therefore, a second aspect of this application provides a potassium ion fluorescent detection reagent, comprising borene and a single-stranded nucleic acid fragment linked to a fluorescent group. The single-stranded nucleic acid fragment can specifically bind to potassium ions to form a G-quadruplex, and the borene is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group. When the potassium ion fluorescent detection reagent of this application detects potassium ions in solution, if there are no potassium ions in the solution, the fluorescent group is in a fluorescence-quenched state due to its proximity to the borene. If there are no potassium ions in the solution, the single-stranded nucleic acid fragment forms a G-quadruplex under the action of potassium ions and desorbs from the borene, thereby generating fluorescence. The concentration of potassium ions in the solution can be calculated based on the fluorescence intensity.

[0051] It should be noted that the single-stranded nucleic acid fragments and borene in the above-mentioned potassium ion fluorescent detection reagent can be packaged and used separately, or they can be mixed to form a fluorescent probe for direct detection by utilizing the electrostatic adsorption between the single-stranded nucleic acid fragments and borene.

[0052] In some implementation schemes, after single-stranded nucleic acid fragments and borene are mixed to form a fluorescent probe, the single-stranded nucleic acid fragments can be dissociatedly bound to the surface of borene through electrostatic adsorption. After the single-stranded nucleic acid fragments bind to borene, they can desorb from borene in the presence of potassium ions to form G-quadruplexes. Furthermore, after the formation of G-quadruplexes, under the action of potassium ion chelating reagents, the DNA sequence forming G-quadruplexes can be restored to a randomly coiled single-stranded state and bind to borene, thereby making the entire detection process reversible.

[0053] In some implementations, the potassium ion fluorescence detection reagent also includes a buffer reagent for dispersing borene, so as to better bind borene and single-stranded nucleic acid fragments and to ensure that the single-stranded nucleic acid fragments are not affected by pH changes during the dissociation and adsorption of borene.

[0054] In some embodiments, the buffer reagent used to disperse borophene is selected from at least one of Tris buffer, MOPS buffer, HEPES buffer, sodium acetate-acetic acid buffer, and phosphate buffer. Specifically, the concentration of the buffer reagent is 20 mM, and the pH value is 6.0–8.0, more specifically, the pH value is 7.4. In some embodiments, the Tris-hydrochloric acid buffer contains 2 mM magnesium chloride.

[0055] In some embodiments, the length of the single-stranded nucleic acid fragment is 15bp to 25bp, and more specifically, 20bp to 25bp, to facilitate molybdenum disulfide quenching of the fluorescent group. In some specific embodiments, the sequence of the single-stranded nucleic acid fragment is 5'-TTTTTGGTTGGTGTGGTTGG-3'. The DNA sequence of this application can be adsorbed onto the surface of borone via electrostatic interaction, achieving a quenching efficiency of up to 90% for the fluorescent group. Simultaneously, it can specifically and reversibly bind to potassium ions to form G-quadruplexes. Through the adsorption and desorption process of the single-stranded nucleic acid fragment by borone, the detection of potassium ions is achieved.

[0056] In some specific implementations, the fluorescent group linked to the single-stranded nucleic acid fragment is at least one of the following: FAM fluorescent group, TAMRA fluorescent group, Cy3 fluorescent group, Cy5 fluorescent group, Alexa Fluor 488 fluorescent group, Atto 590 fluorescent group, and Texas red fluorescent group. When potassium ions are absent in the detection solution, the fluorescent group is close to the borene, and the fluorescence signal is quenched by the borene with a quenching efficiency of over 90%. When potassium ions are present in the detection solution, the single-stranded nucleic acid fragment binds to the potassium ions to form a G-quadruplex, thereby enabling the detection of the fluorescence emitted by the fluorescent group.

[0057] To further improve the sensitivity of potassium ion detection, the concentration ratio of single-stranded nucleic acid fragment to molybdenum disulfide is 1:25000. In some specific implementations, the concentration of the single-stranded nucleic acid fragment in the potassium ion fluorescence detection reagent is 0.05 μM-0.5 μM, more specifically 0.1 μM-0.3 μM; the concentration of borogenene is 0.2 mg / mL-0.5 mg / mL, more specifically 0.3 mg / mL-0.4 mg / mL, thereby achieving better fluorescence quenching and reducing background signal.

[0058] Therefore, a third aspect of this application also provides a method for preparing a potassium ion fluorescence detection reagent, comprising:

[0059] Boronene nanosheets were obtained by sequentially treating boron powder with probe ultrasound and water bath ultrasound.

[0060] A potassium ion fluorescent detection reagent is prepared by mixing and oscillating a single-stranded nucleic acid fragment with a fluorescent group and borene, which adsorbs the single-stranded nucleic acid fragment onto borene nanosheets to form a fluorescent probe.

[0061] Understandably, in order to improve the sensitivity of potassium ion detection, this application prepares fluorescent probes using the above-mentioned preparation method, such that the concentration of single-stranded nucleic acid fragments is 0.05 μM-0.5 μM and the concentration of borone is 0.2 mg / mL-0.5 mg / mL.

[0062] In some specific implementations, to better adsorb single-stranded nucleic acid fragments onto borene nanosheets to prepare fluorescent probes and to provide a pH-stable potassium ion detection environment for the fluorescent probes, borene is dispersed in a buffer reagent and then mixed with single-stranded nucleic acid fragments linked to fluorescent groups, thereby completing the preparation of the potassium ion fluorescent detection reagent. The type, pH, and concentration of the buffer reagent have been detailed above and will not be repeated here.

[0063] Furthermore, such as Figure 1 As shown, the fourth aspect of this application provides a method for detecting potassium ion fluorescence, comprising: mixing and reacting a sample to be tested with the above-mentioned potassium ion fluorescence detection reagent;

[0064] The fluorescence intensity of the reaction solution after the reaction is completed is detected to obtain the fluorescence intensity of the sample to be tested; and

[0065] The potassium ion concentration of the sample is calculated based on the fluorescence intensity of the sample.

[0066] Specifically, the mixing reaction is carried out under light-protected conditions for 1 to 4 hours, or more specifically, 2 to 4 hours. When detecting fluorescence intensity, the raw fluorescence intensity of the sample is collected at 520 nm to 640 nm. Furthermore, the fluorescence intensity of the sample can be the fluorescence intensity detected after the reaction between the sample and the fluorescent probe, i.e., the raw fluorescence intensity of the sample, or it can be the fluorescence intensity obtained after normalization of the raw fluorescence intensity, i.e., the normalized fluorescence intensity of the sample.

[0067] It should be noted that this application creatively combines single-stranded nucleic acid fragments that specifically bind potassium ions with borene. It was found that during the desorption and adsorption of borene and single-stranded nucleic acid fragments, there is a certain functional relationship between the potassium ion concentration in the solution and the fluorescence intensity of the solution. Furthermore, the potassium ion concentration in the solution can be calculated based on this functional relationship.

[0068] Therefore, in some implementation schemes, calculating the potassium ion concentration of the test sample based on the fluorescence intensity of the test sample in order to calculate the potassium ion concentration in the solution specifically includes:

[0069] Obtain a standard curve of fluorescence intensity versus potassium ion concentration in the reaction solution, and calculate the potassium ion concentration of the sample based on the fluorescence intensity of the sample and the standard curve.

[0070] It is understandable that the standard curve refers to the functional relationship between the fluorescence intensity of the reaction solution and the potassium ion concentration of the reaction solution. This functional relationship can be a function of the original fluorescence intensity of the reaction solution and the potassium ion concentration of the reaction solution, or it can be a function of the normalized fluorescence intensity of the reaction solution and the potassium ion concentration of the reaction solution.

[0071] In some specific implementation schemes, the standard curve of potassium ion concentration versus fluorescence intensity in the reaction solution is as follows:

[0072] F1 = -0.2182 + 1.047 × c [K+] ;

[0073] Where F1 = F / F0 - 1, F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity to the background fluorescence intensity of the reaction solution, and c [K+] This indicates the potassium ion concentration in the reaction solution.

[0074] Specifically, the potassium ion concentration calculated from the normalized fluorescence intensity of the sample using the aforementioned standard curve is the potassium ion concentration of the sample.

[0075] In some specific implementation schemes, the potassium ion concentration range detected by the above detection method is 0 mM to 100 mM. For the test sample within this concentration range, the goodness of fit of the above standard curve can reach 0.9985, thereby achieving an accurate detection effect for potassium ion concentration.

[0076] The implementation schemes of this application will be described in detail below with reference to the embodiments, but this application is not limited to these embodiments.

[0077] The specific information on the reagents and instruments used in the embodiments of this application is as follows.

[0078] The boron powder and isopropanol used in the embodiments of this application were purchased from Shanghai Maclean Biotechnology Co., Ltd. The oligonucleotide chains used in the embodiments of this application were all synthesized by Shanghai Sangon Biotech Co., Ltd. The ultrapure water used in the experiments was provided by the Milli-Q system, and all other solutions and reagents were of analytical grade. The fluorescence spectrophotometer was a SHIMADZU RF-6000.

[0079] The detection conditions for the samples in this embodiment are as follows: the total volume of the test solution in the quartz cuvette is 100 μg; all fluorescence spectroscopy experiments are performed at room temperature; the sample is thoroughly mixed with a vortex mixer before injection and allowed to stand for 5 minutes until the fluorescence is stable before fluorescence spectroscopy detection. Specific fluorescence testing conditions are as follows: the excitation spectrum is fixed at 490 nm, the emission spectrum collection range is 510 nm-640 nm, the excitation and emission slit widths are set to 5 nm and 5 nm respectively, the instrument data acquisition interval is 1.0 nm, the scanning speed is 2000 nm / min, and the step size is 1 nm.

[0080] Example 1: Preparation of boronene nanosheets

[0081] 300 mg of bulk two-dimensional material powder (Macklin Inc., 99.9% pure) was directly added to 300 mL of IPA solvent to form a suspension with an initial concentration of 1 mg / mL. The suspension was then first sonicated with a probe at 780 W for 1 h, followed by sonication in a water bath at 1050 W for 3 h. Note that both probe sonication and water bath sonication were performed at a constant temperature of 10 °C. To obtain ultrathin two-dimensional material nanosheets, the prepared two-dimensional material / IPA solution was first centrifuged at 7000 rpm for 15 min to obtain the supernatant, and then centrifuged at 10000 rpm for 15 min to obtain the product. The prepared precipitate was diluted, sonicated, and then subjected to further fluorescence studies.

[0082] This embodiment uses an improved liquid-phase exfoliation method to prepare ultrathin two-dimensional nanosheets, such as... Figure 2 As shown in Figure a, the unique anisotropic characteristics of probe ultrasound and the isotropic characteristics of water bath ultrasound are utilized. The process and principle are as follows: During probe ultrasound processing, the vertically oriented ultrasonic waves emitted by the probe cut the powdered material into two-dimensional sheets with large transverse dimensions. In the subsequent water bath ultrasound processing, the size of the two-dimensional sheets can be greatly reduced to obtain two-dimensional nanosheets. The direction of ultrasonic wave transmission is isotropic during this process, ensuring uniform size and thickness of the sheet-like product. The boronene nanosheets prepared in this embodiment have a particle size of 50 nm to 200 nm and a thickness of 2 nm to 4 nm. Furthermore, uniformly sized ultrathin boronene two-dimensional nanosheets can be obtained through differential centrifugation. The absorption spectrum of the boronene nanosheets is as follows: Figure 2 As shown in d. From Figure 2 The absorption spectrum shows that boronene nanosheets have obvious absorption peaks in the ultraviolet-visible-near-infrared region.

[0083] Example 2: Preparation of Fluorescent Probes

[0084] 1. Prepare FAM-DNA sample solution: Prepare a 20μM solution of FAM-DNA sample using DEPC water.

[0085] 2. Prepare Tris-hydrochloric acid buffer: Prepare Tris-hydrochloric acid buffer (40 mL, 20 mM) with Tris and ultrapure water, and adjust the pH to 7.4 with hydrochloric acid solution (concentration of 1 M).

[0086] 3. Optimal fluorescence quenching concentration of borophene nanosheets: Add appropriate volumes (0.5 μL or 1 μL) of borophene nanosheets (default volume remains unchanged, 100 μL) sequentially to Tris buffer containing DNA at a final concentration of 0.1 μM, so that the concentrations of borophene nanosheets are 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, respectively. For example, adding 0.5 μL of 10 mg / mL borophene nanosheets to 100 μL of Tris buffer (20 mM, pH 7.4, containing 0.1 μM DNA) yields a final concentration of 50 μg / mL borophene nanosheets in a 100 μL fluorescent probe system. Then, adding another 0.5 μL of 10 mg / mL borophene nanosheets to this 100 μL Tris buffer solution (containing 50 μg / mL borophene nanosheets) brings the final concentration to 100 μg / mL. Subsequent additions of 1 μL yield borophene nanosheet concentrations of 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL. The degree of fluorescence quenching is observed, and fluorescence intensity spectra corresponding to different concentrations of borophene nanosheets are collected using a fluorescence spectrometer.

[0087] To obtain a fluorescent probe with a low background signal, facilitating the preparation of the optimal probe concentration, a sample containing only FAM-DNA was placed in a Shi Yingchao microcuvette and detected using a fluorescence spectrometer. Subsequently, the concentration of borene nanosheets in the system was gradually increased, and the mixture was stirred and reacted for 10 minutes at room temperature in the dark to allow DNA adsorption onto the borene nanosheets. The degree of fluorescence quenching was observed, and fluorescence spectra at different concentrations were obtained. Figure 3 (b) It can be clearly seen that as the concentration of boronene nanosheets increases, the fluorescence intensity of the detected FAM-DNA decreases sharply. When the concentration of boronene nanosheets increases to 300 μg / mL, we found that the fluorescence quenching degree has reached 90%. Furthermore, further increasing the concentration of boronene nanosheets did not change much, and a low background signal was achieved. Therefore, the optimal fluorescent probe concentration obtained in this embodiment is 0.1 μM DNA and 300 μg / mL boronene nanosheets.

[0088] Example 3: Quantitative Detection of Potassium Ions

[0089] To obtain a linear relationship between potassium ion concentration and the fluorescence intensity of the fluorescent probe, we added potassium chloride solutions of different concentrations to the prepared fluorescent probe (0.1 μM DNA, 300 μg / mL boronene nanosheets) to achieve final potassium ion concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM. The mixture was reacted at room temperature in the dark for 4 h. Fluorescence spectrometry was used to detect the fluorescence intensity spectra corresponding to different potassium ion concentrations.

[0090] Figure 4 (b) is the quantitative detection of potassium ions by a fluorescent probe. The concentration of potassium ions is between 0 mM and 100 mM, and the fluorescence intensity increases with the increase of potassium ion concentration. Figure 4 (c) is a scatter plot of fluorescence intensity at 520 nm versus potassium ion concentration; the linear relationship between potassium ion concentration and fluorescence intensity in the range of 0 mM to 100 mM is: F1 = -0.2182 + 1.047c [K+] F1 represents the normalized fluorescence intensity of the sample under test, c [K+] This indicates the potassium ion concentration in the reaction solution.

[0091] Example 4 verifies the reversibility of potassium ion detection using a fluorescent probe.

[0092] To verify the reversibility of potassium ion concentration, this embodiment used a potassium ion chelating agent (18-crown ether-6). The addition of 18-crown ether-6 is equivalent to the absence of potassium ions in the solution. Theoretically, as the potassium ion concentration in the system increases, the signal of the fluorescent probe should strengthen; conversely, as the potassium ion concentration decreases, the probe signal should weaken accordingly. 18-crown ether-6 (2M, 2.5μL) was added to a system of fluorescent probe and 50mM potassium ions (total volume 100μL), and the solution was allowed to stand at room temperature for 10 minutes before measuring the fluorescence spectrum. Then, potassium chloride solution (2M, 2.5μL) was added to this solution, and the solution was allowed to stand at room temperature for 10 minutes before measuring the fluorescence spectrum. This process of adding 18-crown ether-6 and potassium chloride solution was repeated for a total of 5 cycles. This experiment was repeated 3 times. Figure 5 (a) shows the normalized fluorescence intensity under different conditions. This fluorescent probe exhibits good reversibility in potassium ion detection.

[0093] Example 5 verifies the specificity of the fluorescent probe for potassium ions.

[0094] This embodiment selected nine other metal cations besides potassium ions (such as sodium ions, calcium ions, magnesium ions, etc.) to eliminate specific interference. In this experiment, potassium chloride solution (2.5 μL, 2M), magnesium chloride solution (0.5 μL, 200 mM), sodium chloride solution (2.5 μL, 2M), copper chloride solution (0.5 μL, 10 mM), ferric chloride solution (0.5 μL, 10 mM), calcium chloride solution (0.5 μL, 200 mM), zinc chloride solution (0.5 μL, 10 mM), and tin tetrachloride solution (0.5 μL) were used. 10 mM manganese chloride solution (0.5 μL, 10 mM) and 10 mM chromium trichloride solution (0.5 μL, 10 mM) were added to the fluorescent probe prepared in step (2), respectively, so that the final total volume of the system was 100 μL, and the final concentrations of the nine metal ions were potassium ion (50 mM), magnesium ion (5 mM), sodium ion (50 mM), copper ion (50 μM), calcium ion (5 mM), iron ion (50 μM), zinc ion (50 μM), tin ion (50 μM), manganese ion (50 μM), and chromium ion (50 μM). The fluorescence spectrum of the above samples was measured after standing at room temperature for 10 minutes. The fluorescence spectrum detection results of the fluorescent probe under the condition of 10 metal ions (potassium ion and 9 other metal ions) are as follows. Figure 6 As shown. According to Figure 6 It can be seen that the fluorescence intensity response of the fluorescent probe is very obvious before and after adding potassium ions, while there is only a slight change in fluorescence intensity for the other 9 ions. This indicates that the fluorescent probe has good specificity for potassium ion detection, thus effectively reducing the occurrence of false positives.

[0095] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. The use of borone in the preparation of potassium ion fluorescent detection reagents, characterized in that, The potassium ion fluorescence detection reagent comprises a single-stranded nucleic acid fragment linked to a fluorescent group and borene. The single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and the borene is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group. The sequence of the single-stranded nucleic acid fragment is shown in SEQ ID NO.

1. The borene is a borene nanosheet; the particle size of the borene is 50 nm to 200 nm; the thickness of the borene is 2 nm to 4 nm; the molar ratio of the single-stranded nucleic acid fragment to the borene is 1:250000.

2. A potassium ion fluorescence detection reagent, characterized in that, The invention includes borene and a single-stranded nucleic acid fragment linked to a fluorescent group, wherein the single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and the borene is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group. The sequence of the single-stranded nucleic acid fragment is shown in SEQ ID NO.

1. The borene is a borene nanosheet; the particle size of the borene is 50 nm to 200 nm; the thickness of the borene is 2 nm to 4 nm; the molar ratio of the single-stranded nucleic acid fragment to the borene is 1:250000.

3. The potassium ion fluorescence detection reagent according to claim 2, characterized in that, The single-stranded nucleic acid fragment satisfies at least one of the following characteristics: (1) The single-stranded nucleic acid fragment can be dissociatively bound to the surface of the boronene; (2) The concentration range of the single-stranded nucleic acid fragment is 0.05μM-0.5μM.

4. The potassium ion fluorescence detection reagent according to claim 2 or 3, characterized in that, The fluorescent group is at least one of the following: FAM fluorescent group, TAMRA fluorescent group, Cy3 fluorescent group, Cy5 fluorescent group, Alexa Fluor 488 fluorescent group, Atto 590 fluorescent group, and Texas red fluorescent group.

5. A method for detecting potassium ions using fluorescence, characterized in that, Includes the following steps: The sample to be tested is mixed with the potassium ion fluorescence detection reagent according to any one of claims 2 to 4 and reacted. The fluorescence intensity of the reaction solution after the reaction is completed is detected to obtain the fluorescence intensity of the sample to be tested; and The potassium ion concentration of the sample to be tested is calculated based on the fluorescence intensity of the sample.

6. The potassium ion fluorescence detection method according to claim 5, characterized in that, The steps for calculating the potassium ion concentration of the test sample based on the fluorescence intensity of the test sample include: Obtain a standard curve of fluorescence intensity versus potassium ion concentration in the reaction solution. Calculate the potassium ion concentration of the sample based on the fluorescence intensity of the test sample and the standard curve. The standard curve is as follows: F1=-0.2182+1.047×c [K+] ; Where F1 = F / F0 - 1, F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity to the background fluorescence intensity of the reaction solution, and c [K+] This indicates the potassium ion concentration in the reaction solution.

7. The potassium ion fluorescence detection method according to claim 5 or 6, characterized in that, The potassium ion concentration ranges from 0 mM to 100 mM.

8. The potassium ion fluorescence detection method according to claim 7, characterized in that, Before mixing and reacting the sample to be tested with the potassium ion fluorescent detection reagent according to any one of claims 2 to 7, the following steps are also included: Boronene nanosheets were obtained by sequentially treating boron powder with probe ultrasound and water bath ultrasound. The potassium ion fluorescent detection reagent is prepared by mixing and oscillating a single-stranded nucleic acid fragment with a fluorescent group and borene, thereby adsorbing the single-stranded nucleic acid fragment onto borene nanosheets.

9. The potassium ion fluorescence detection method according to claim 8, characterized in that, The mixing reaction takes 1 to 4 hours.

10. The potassium ion fluorescence detection method according to claim 8, characterized in that, The duration of the oscillation reaction is 10 to 60 minutes.

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