Potassium ion fluorescence detection reagent and method

Abstract

This application relates to a potassium ion fluorescence detection reagent and method. The potassium ion fluorescence detection reagent of this application creatively uses molybdenum disulfide combined with a single-stranded nucleic acid fragment with a fluorescent group to make a fluorescent probe. The preparation method is simple, and the production process only takes 10 minutes. Moreover, the fluorescence quenching of the fluorescent probe reaches 85%, which can achieve the detection of the concentration of potassium ions under a low fluorescence background. The detection steps of the potassium ion fluorescence detection reagent of this application are simple. The potassium ion detection time only takes 1 to 4 hours, without the need to rely on dangerous and complex instruments. The influence of other common cations, such as sodium ions, magnesium ions, calcium ions, etc. on the detection results is small. It 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 specifically, to a potassium ion fluorescence detection reagent and method. Background Art

[0002] Potassium ions play an important role in maintaining fluid homeostasis, neurotransmission, enzyme activation and other physiological processes. Deviation of potassium ion balance in body fluids can lead to diseases such as stroke, hypokalemia, hypertension, cancer, etc. Research has pointed out that due to the rapid proliferation of tumors, necrotic tumor cells will secrete intracellular potassium ions into the extracellular space, resulting in an increase in the potassium ion concentration in the tumor microenvironment to about 45 mM, which is nearly 9 times higher than the potassium ion concentration in normal serum. The high potassium ion concentration causes the inactivation of T cells that can kill tumors and further proliferation of tumor cells. Therefore, monitoring the potassium ion concentration is beneficial to deeply understand and regulate this process, and has potential value for the early detection and treatment of tumors.

[0003] Traditional potassium ion detection methods include ion chromatography, electrochemical sensing, flame atomic absorption spectrometry, etc. Ion chromatography relies on a closed mobile phase transmission system and a well-performing separation column, with harsh detection conditions, and the start-up and elution condition equilibration of the chromatographic system takes a long time; the experimental repeatability of electrochemical sensing is poor, and the modification steps of the electrode are cumbersome and time-consuming; the use of fuel gas propane is involved in flame atomic absorption spectrometry, which has high danger. These methods generally have the disadvantages of complex detection processes, requiring professional personnel to operate, and high detection costs.

[0004] Therefore, how to provide a rapid, safe and simple potassium ion detection method is a difficult point in monitoring the potassium ion concentration. Summary of the Invention

[0005] To solve the above problems, the first object of this application is to provide the use of molybdenum disulfide in the preparation of a potassium ion fluorescence detection reagent. The potassium ion reagent includes a single-stranded nucleic acid fragment linked with a fluorescent group and molybdenum disulfide. The single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and molybdenum disulfide is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group.

[0006] The second object of this application is to provide a potassium ion fluorescence detection reagent, including a single-stranded nucleic acid fragment linked with a fluorescent group and molybdenum disulfide. The single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex, and molybdenum disulfide is used to bind the single-stranded nucleic acid fragment to quench the fluorescent group.

[0007] In one embodiment, the single-stranded nucleic acid fragment can dissociatively bind to the surface of molybdenum disulfide.

[0008] In one embodiment, the concentration ratio of the single-stranded nucleic acid fragment to the molybdenum disulfide nanosheets is 1:25000.

[0009] In one embodiment, the concentration of the single-stranded nucleic acid fragment is 0.05 μM to 0.5 μM.

[0010] In one embodiment, the length of the single-stranded nucleic acid fragment is 15 bp to 25 bp.

[0011] In one embodiment, the sequence of the single-stranded nucleic acid fragment is as shown in SEQ ID NO.1.

[0012] In one embodiment, the particle size of the molybdenum disulfide nanosheets is 50 nm to 200 nm.

[0013] In one embodiment, the concentration of molybdenum disulfide is 0.3 mg / mL to 0.6 mg / mL.

[0014] In one embodiment, the fluorescent group is at least one of 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.

[0015] In one embodiment, the potassium ion fluorescence detection reagent further includes a buffer reagent for dispersing molybdenum disulfide.

[0016] The third object of the present application is to provide a method for detecting potassium ions by fluorescence, including the following steps:

[0017] Mix the sample to be tested and the above-mentioned potassium ion fluorescence detection reagent for reaction;

[0018] Detect the fluorescence intensity of the reaction solution after the reaction ends to obtain the fluorescence intensity of the sample to be tested; and

[0019] Calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested.

[0020] In one embodiment, the step of calculating the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested includes:

[0021] Obtain the standard curve of the fluorescence intensity of the reaction solution and the potassium ion concentration, and calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested and the standard curve. The standard curve is:

[0022] F1 = 1.62 + 0.68 × c [K+] ;

[0023] wherein, F1 = F / F0 - 1, F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity of the reaction solution to the background fluorescence intensity, and c [K+]It represents the potassium ion concentration of the reaction solution.

[0024] In one embodiment, the potassium ion concentration of the sample to be measured is 0 mM to 150 mM.

[0025] In one embodiment, the fluorescence intensity of the reaction solution after the detection reaction is specifically:

[0026] The fluorescence intensity of the reaction solution after the detection reaction is measured at 510 nm to 640 nm.

[0027] In one embodiment, before mixing and reacting the sample to be measured with the above potassium ion fluorescence detection reagent, it further includes:

[0028] Mix and oscillate the single-stranded nucleic acid fragment solution linked with a fluorescent group and molybdenum disulfide so that the single-stranded nucleic acid fragment is adsorbed on molybdenum disulfide to prepare the potassium ion fluorescence detection reagent.

[0029] In one embodiment, the time of the mixing reaction is 1 h to 4 h.

[0030] In one embodiment, the time of the oscillation reaction is 10 minutes to 60 minutes.

[0031] The potassium ion fluorescence detection reagent of the present application creatively uses molybdenum disulfide combined with a single-stranded nucleic acid fragment with a fluorescent group to form a fluorescent probe. The preparation method is simple, and the production process only takes 10 minutes. Moreover, the fluorescence quenching of the fluorescent probe reaches 85%, which can realize the detection of the potassium ion concentration under a low fluorescence background; the detection steps of the potassium ion fluorescence detection reagent of the present application are simple, and the potassium ion detection time only needs 1 to 4 hours. It does not need to rely on dangerous and complex instruments, and the influence of other common cations, such as sodium ions, magnesium ions, calcium ions, etc. on the detection results is small. It has the advantages of high safety, fast detection speed and high specificity, and the detection process is reversible. Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the principle of the potassium ion fluorescence detection method in an embodiment of the present application;

[0033] Figure 2 It is a schematic diagram of the preparation process of the fluorescent probe and a schematic diagram of the fluorescence detection result in Example 1 of the present application;

[0034] In Figure 2 a represents a schematic flow chart of preparing a fluorescent probe by mixing molybdenum disulfide nanosheets with FAM-DNA; b represents the fluorescence spectra of FAM-DNA in solutions of different concentrations of molybdenum disulfide nanosheets; c represents the molybdenum disulfide nanosheet concentration-dependent fluorescence curve of FAM-DNA at 520 nm;

[0035] Figure 3 This is the fluorescence spectrum diagram after the reaction of the fluorescent probe and potassium ions with different concentrations in Example 2 of this application; in Figure 3 , a represents the fluorescence spectrum diagram measured by adding 2M potassium chloride solution to the fluorescent probe; b represents the fluorescence spectrum diagram of the fluorescent probe in potassium ion solutions with different concentrations; c represents the scatter plot of (F / F0 - 1) of FAM-DNA at 520nm against the potassium ion concentration;

[0036] Figure 4 This is the schematic diagram of the conformational change of the fluorescent probe and the corresponding fluorescence intensity change in Example 3 of this application; in Figure 4 , a represents the schematic diagram of the conformational change of the fluorescent probe in Example 3 of this application; b represents the schematic diagram of the normalized fluorescence intensity change of the solution in Example 3 of this application under the conditions of adding potassium chloride and 18-crown-6;

[0037] Figure 5 This is the fluorescence spectrum diagram of the fluorescent probe in the presence of different metal ions in Example 4 of this application; in each small figure, the three spectral curves respectively correspond to the FAM-DNA solution, the solution after adding molybdenum disulfide nanosheets to FAM-DNA, and the solution after adding molybdenum disulfide nanosheets and different salt solutions to FAM-DNA;

[0038] Figure 6 This is the schematic diagram of the change of the fluorescence intensity of the fluorescent probe over time before and after adding potassium chloride solution in Example 5 of this application. Detailed Description of the Invention

[0039] Reference will now be provided in detail to embodiments of the present application, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0040] Accordingly, it is intended that the present application cover such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or are apparent from the following detailed description. Those of ordinary skill in the art should understand that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0041] As described above, traditional potassium ion detection methods have disadvantages such as complex detection processes, requiring professional operation, and high detection costs.

[0042] In order to at least partially solve at least one of the above technical problems, the first aspect of the present application provides a use of molybdenum disulfide in the preparation of a potassium ion fluorescence detection reagent, the potassium ion reagent comprising a single-stranded nucleic acid fragment connected to a fluorescent group and molybdenum disulfide, the single-stranded nucleic acid fragment can specifically bind to potassium ions to form a G-quadruplex, and the molybdenum disulfide is used to bind to the single-stranded nucleic acid fragment to quench the fluorescent group.

[0043] Specifically, molybdenum disulfide (MoS2) is a metallic luster mineral purified from molybdenite. It is in the form of a black solid powder with a melting point of 2375°C and a density of 4.80 g / cm 3 (14°C), with a Mohs hardness of 1.0-1.5. A single layer of molybdenum disulfide is structured like a sandwich, consisting of a central layer of Mo atoms and two layers of S atoms on either side. One Mo atom is connected to six S atoms, with the Mo-S bonds being covalent. The interlayer spacing is 0.65 nm, with only weak van der Waals forces acting between them.

[0044] As a transition metal dichalcogenide, molybdenum disulfide nanosheets have a large surface area, resulting in a relatively strong electrostatic attraction on their surface. Furthermore, because the electron clouds of molybdenum and sulfur atoms tend to be distributed in the center and at the ends, respectively, the surface of the molybdenum disulfide nanosheets carries a certain negative charge. Single-stranded nucleic acids, with their exposed bases, are easily adsorbed by the molybdenum disulfide nanosheets through electrostatic attraction.

[0045] G-quadruplexes are a type of specialized nucleic acid secondary structure formed by DNA or RNA sequences rich in guanine (G) repeats, which fold upon binding to metal cations. Through screening, the present applicant discovered that some DNA sequences exhibit good specificity for potassium ion recognition. In the absence of potassium ions, the DNA sequences are in a random coiled single-stranded state, but in the presence of potassium ions, they rapidly fold into a G-quadruplex structure.

[0046] This application creatively utilizes this property of G quadruplexes in combination with molybdenum disulfide to prepare a fluorescent probe. Through the structural changes of single-stranded nucleic acid fragments, the adsorption and desorption of single-stranded nucleic acid fragments by molybdenum disulfide are achieved, thereby realizing the detection of potassium ions. It has the advantages of high safety, fast detection speed and high specificity, and the detection process is reversible.

[0047] Therefore, the second object of the present application is to provide a potassium ion fluorescence detection reagent, which includes a single-stranded nucleic acid fragment linked with a fluorescent group and molybdenum disulfide. The single-stranded nucleic acid fragment can specifically bind to potassium ions to form a G-quadruplex, and molybdenum disulfide is used to bind to the single-stranded nucleic acid fragment to quench the fluorescent group. When the potassium ion fluorescence detection reagent of the present application detects potassium ions in a solution, if there are no potassium ions in the solution, the fluorescent group is in a fluorescence quenched state due to being close to molybdenum disulfide. 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 molybdenum disulfide, thereby generating fluorescence. Further, the concentration of potassium ions in the solution can be calculated based on the fluorescence intensity.

[0048] It should be noted that the single-stranded nucleic acid fragment and molybdenum disulfide in the above potassium ion fluorescence detection reagent can be independently packaged and used, or the two can be mixed to form a fluorescent probe by the electrostatic adsorption between the single-stranded nucleic acid fragment and molybdenum disulfide and directly used for detection.

[0049] In some embodiments, after the single-stranded nucleic acid fragment and molybdenum disulfide are mixed to form a fluorescent probe, the single-stranded nucleic acid fragment can be dissociably bound to the surface of molybdenum disulfide through electrostatic adsorption. After the single-stranded nucleic acid fragment binds to molybdenum disulfide, in the presence of potassium ions, it can desorb from molybdenum disulfide to form a G-quadruplex, and after forming the G-quadruplex, under the action of a potassium ion chelating reagent, the DNA sequence forming the G-quadruplex can be restored to an irregularly coiled single-stranded state and bind to molybdenum disulfide, thereby making the entire detection process reversible.

[0050] In some embodiments, the potassium ion fluorescence detection reagent further includes a buffer reagent for dispersing molybdenum disulfide, which is used to maintain the pH value of the fluorescent probe and ensure that the single-stranded nucleic acid fragment of the fluorescent probe is not affected by pH changes during the dissociation and adsorption processes of molybdenum disulfide.

[0051] In some embodiments, the buffer reagent for dispersing molybdenum disulfide is selected from at least one of Tris buffer reagent, MOPS buffer solution, HEPES buffer solution, sodium acetate-acetic acid buffer solution, and phosphate buffer solution. 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 specific embodiments, the Tris-hydrochloric acid buffer solution contains 2 mM magnesium chloride.

[0052] In some embodiments, the length of the single-stranded nucleic acid fragment is 15 bp - 25 bp, and further can be 20 bp - 25 bp to facilitate molybdenum disulfide to quench the fluorescent group. The single-stranded nucleic acid fragment of the present application is a DNA fragment. In some specific embodiments, the sequence of the single-stranded nucleic acid fragment is 5’-TTTTTGGTTGGTGTGGTTGG-3’.

[0053] On the one hand, the DNA fragment of the present application can be adsorbed on the surface of molybdenum disulfide through electrostatic interaction, and the quenching efficiency of the fluorescent group can reach 85%. At the same time, it can specifically bind to potassium ions and reversibly transform into a G-quadruplex conformation, so as to realize the detection of potassium ions through the adsorption and desorption process of molybdenum disulfide on single-stranded nucleic acid fragments.

[0054] In some specific embodiments, the fluorescent group linked to the single-stranded nucleic acid fragment is the FAM fluorescent group. When there is no potassium ion in the detection solution, the fluorescent group is close to molybdenum disulfide, and the fluorescent signal is quenched by molybdenum disulfide, and the quenching efficiency is more than 85%; when there is potassium ion in the detection solution, the single-stranded nucleic acid fragment binds to the potassium ion to form a G-quadruplex, so that the fluorescence emitted by the fluorescent group can be detected.

[0055] In order to achieve a better fluorescence quenching effect and further improve the sensitivity of potassium ion detection, the concentration ratio of the single-stranded nucleic acid fragment to molybdenum disulfide is 1:25000. In some specific embodiments, the concentration of the single-stranded nucleic acid fragment in the potassium ion fluorescence detection reagent is 0.05 μM to 0.5 μM, further 0.1 μM to 0.3 μM; the concentration of molybdenum disulfide is 0.3 mg / mL to 0.6 mg / mL, further 0.4 mg / mL to 0.5 mg / mL, so as to achieve a better fluorescence quenching effect and reduce the background signal.

[0056] Therefore, the third aspect of the present application also provides a preparation method of a potassium ion fluorescence detection reagent, including:

[0057] Mix and oscillate the solution of the single-stranded nucleic acid fragment linked with the fluorescent group and molybdenum disulfide, so that the single-stranded nucleic acid fragment is adsorbed on molybdenum disulfide to prepare the potassium ion fluorescence detection reagent.

[0058] Specifically, the oscillation reaction time is 10 minutes to 60 minutes, further 10 minutes to 40 minutes, and still further 10 minutes to 20 minutes. It can be understood that in order to improve the sensitivity of potassium ion detection, a fluorescent probe is prepared by controlling the concentration ratio of the single-stranded nucleic acid fragment to molybdenum disulfide, so that the concentration of the single-stranded nucleic acid fragment is 0.05 μM to 0.5 μM and the concentration of molybdenum disulfide is 0.3 mg / mL to 0.6 mg / mL.

[0059] In some specific embodiments, in order to provide a pH-stable potassium ion detection environment, molybdenum disulfide is dispersed in a buffer solution and then mixed with the single-stranded nucleic acid fragment linked with the fluorescent group, thus completing the preparation of the potassium ion fluorescence detection reagent.

[0060] In addition, the fourth aspect of the present application also provides a potassium ion fluorescence detection method, including: mixing and reacting the sample to be tested with the above potassium ion fluorescence detection reagent;

[0061] Detect the fluorescence intensity of the reaction solution after the reaction ends to obtain the fluorescence intensity of the sample to be tested; and

[0062] Calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested.

[0063] In some specific embodiments, the mixing reaction time is 1 h to 4 h, further it can be 2 h to 4 h, and further it can be 3 h to 4 h. When detecting the fluorescence intensity, the original fluorescence intensity of the sample to be tested is collected at 520 nm to 640 nm. Further, the fluorescence intensity of the sample to be tested can be the fluorescence intensity detected by the instrument after the reaction between the sample to be tested and the fluorescent probe, that is, the original fluorescence intensity of the sample to be tested, or it can be the fluorescence intensity obtained after normalization processing according to the original fluorescence intensity of the sample to be tested, that is, the normalized fluorescence intensity of the sample to be tested.

[0064] It should be noted that in this application, by binding a single-stranded nucleic acid fragment that can specifically bind potassium ions to molybdenum disulfide, it is creatively found that during the desorption and adsorption process of molybdenum disulfide and the single-stranded nucleic acid fragment, there is a certain functional relationship between the potassium ion concentration in the solution and the fluorescence intensity of the solution, and according to this functional relationship, a linear relationship between the normalized fluorescence intensity of the solution and the potassium ion concentration of the solution can be obtained.

[0065] Therefore, in some embodiments, in order to calculate the potassium ion concentration in the solution, calculating the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested specifically includes:

[0066] Obtain the standard curve of the fluorescence intensity and potassium ion concentration of the reaction solution, and calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested and the standard curve.

[0067] It can be understood 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 the functional relationship between the original fluorescence intensity of the reaction solution and the potassium ion concentration of the reaction solution, or it can be the functional relationship between the normalized fluorescence intensity of the reaction solution and the potassium ion concentration of the reaction solution.

[0068] In some specific embodiments, the standard curve of the fluorescence intensity and potassium ion concentration of the reaction solution is:

[0069] F1 = 1.62 + 0.68×c [K+] ;

[0070] wherein, F1 = F / F0 - 1, F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity of the reaction solution to the background fluorescence intensity, and c [K+] represents the potassium ion concentration of the reaction solution.

[0071] Specifically, the potassium ion concentration calculated by the above standard curve and the normalized fluorescence intensity of the sample to be measured is the potassium ion concentration of the sample to be measured.

[0072] In some specific embodiments, the potassium ion concentration detected by the above detection method ranges from 0 mM to 150 mM. For the samples to be measured within this concentration range, the goodness of fit of the above standard curve can reach 0.9898, thereby achieving the detection effect of accurately detecting the potassium ion concentration.

[0073] The embodiments of the present application will be described in detail below in conjunction with examples, but the present application is not limited to these examples.

[0074] The specific information of the reagents and instruments used in the embodiments of the present application is as described below.

[0075] The FAM-modified DNA used in the embodiments of the present application was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the molybdenum disulfide nanosheet dispersion was purchased from Nanjing Xianfeng Nano Materials Technology Co., Ltd.; other reagents were all purchased from Shanghai Macklin Biochemical Co., Ltd. The model of the fluorescence spectrophotometer is SHIMADZU RF-6000.

[0076] The detection conditions of the samples in the embodiments of the present application are as follows: during detection, the total volume of the sample to be measured in the quartz cuvette is 100 μg; all fluorescence spectroscopy experiments are carried out at room temperature; the sample is thoroughly mixed with a vortex oscillator and allowed to stand for 5 minutes until the fluorescence is stable before performing fluorescence spectroscopy detection. The specific fluorescence test conditions are: the excitation and emission slit widths are set to 10 nm and 5 nm respectively, the integration time is 0.2 s, the excitation wavelength is 490 nm, and the emission wavelength scanning range is from 510 to 640 nm, with a step size of 1 nm.

[0077] Example 1 Preparation of Fluorescent Probe

[0078] The FAM-DNA sample synthesized by Sangon Biotech was prepared into a solution (20 μM) with DEPC water, and Tris-hydrochloric acid buffer (50 mL, 20 mM) was prepared with Tris, hydrochloric acid solution (1 M), and ultrapure water. Mixed solutions with final concentrations of molybdenum disulfide nanosheets of 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL and a final concentration of DNA of 0.1 μM were prepared in this Tris buffer. For example, 0.5 μg of 20 μM DNA and 10 μg of 4 mg / mL molybdenum disulfide nanosheets were added to 90 μg of Tris buffer (20 mM, pH 7.4), and the final concentration of molybdenum disulfide nanosheets was 400 μg / mL and the DNA concentration was 0.1 μM. The mixture was mixed and oscillated at room temperature in the dark for 10 minutes to allow the DNA to adsorb onto the molybdenum disulfide nanosheets, forming a fluorescent probe.

[0079] After placing these samples in quartz ultra-micro cuvettes, the adsorption and fluorescence quenching effects of molybdenum disulfide nanosheets on FAM-DNA were measured with a fluorescence spectrometer to verify that the fluorescent probe had a low background signal. The results are as Figure 2 shown, where Figure 2 (b) is the fluorescence spectrum of FAM-DNA in the presence of molybdenum disulfide nanosheets at different concentrations. It can be clearly seen that as the concentration of nanosheets increases, the detected fluorescence intensity decreases sharply. Figure 2 (c) is the concentration-dependent fluorescence curve of molybdenum disulfide nanosheets obtained from the fluorescence intensity at 520 nm. It can be seen that after the concentration of nanosheets reaches 400 μg / mL, the fluorescence intensity basically enters a plateau. The fluorescence quenching efficiency of molybdenum disulfide nanosheets reaches about 85% at 400 μg / mL, achieving a low background signal.

[0080] Example 2 Quantitative detection of potassium ions

[0081] Potassium chloride solution (2 M) was added to the fluorescent probe prepared in step (1) to make the final concentrations of potassium ions 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 125 mM, 150 mM and 180 mM respectively, and the mixture was reacted at room temperature for 4 h. Detection was carried out with a fluorescence spectrometer to observe the influence of potassium ions at different concentrations on the fluorescence recovery efficiency of FAM-DNA, and a fluorescence spectrum diagram related to the potassium ion concentration was measured.

[0082] Figure 3 (b) is the quantitative detection of potassium ions by the fluorescent probe. When the concentration of potassium ions is in the range of 0 mM to 150 mM, the fluorescence intensity increases as the concentration of potassium ions increases. Figure 3(c) Scatter plot of fluorescence intensity at 520 nm against potassium ion concentration; Standard curve of potassium ion concentration and fluorescence intensity in the range of 0 mM to 150 mM: F1 = 1.62 + 0.68×c [K+] 。

[0083] Example 3 Verifying the reversibility of the fluorescence probe for detecting potassium ions

[0084] Theoretically, when the potassium ion concentration in the system increases, the signal of the fluorescence probe enhances; while when the potassium ion concentration in the system decreases, the signal of the probe should correspondingly weaken. 18-Crown-6 is a potassium ion chelator. As shown in Figure 4 a, 18-crown-6 binds potassium ions, causing the DNA to transform into a linear structure and thus bind to molybdenum disulfide. 18-crown-6 (2 M, 2.5 μg) was added to the fluorescence probe and 50 mM potassium ion system (total volume 100 μg), and the fluorescence spectrum was measured after standing at room temperature for 10 minutes; then potassium chloride solution (2 M, 2.5 μg) was added to this solution, and the fluorescence spectrum was measured after standing at room temperature for 10 minutes; 18-crown-6 and potassium chloride solution were added successively in this way for a total of 4 cycles. This experiment was repeated 3 times. Figure 4 b shows the normalized fluorescence intensity of the fluorescence probe under different conditions, and the fluorescence probe exhibits good reversibility for potassium ion detection.

[0085] Example 4 Verifying the specificity of the fluorescence probe for potassium ions

[0086] To avoid false positive signals, other metal cations (such as sodium ions, calcium ions, magnesium ions, etc.) in the sample to be tested except potassium ions should not affect the detection of potassium ions, that is, the fluorescence probe should have good specificity for potassium ions. To verify this specificity, potassium chloride solution (2.5 μg, 2 M), sodium chloride solution (2.5 μg, 2 M), magnesium chloride solution (0.5 μg, 200 mM), calcium chloride solution (0.5 μg, 200 mM), copper chloride solution (0.5 μg, 10 mM), manganese chloride solution (0.5 μg, 10 mM), zinc chloride solution (0.5 μg, 10 mM), tin tetrachloride solution (0.5 μg, 10 mM), chromium trichloride solution (0.5 μg, 10 mM) were respectively added to the fluorescence probe prepared in step (1). The final total volume of the system was 100 μg, and the final concentrations of metal ions were potassium ions (50 mM), sodium ions (50 mM), magnesium ions (1 mM), calcium ions (1 mM), copper ions (50 μM), manganese ions (50 μM), zinc ions (50 μM), tin ions (50 μM), chromium ions (50 μM). The fluorescence spectra of these samples were measured after standing at room temperature for 5 minutes. Figure 5It is a typical fluorescence spectrum under the condition of the presence of different metal ions. It can be seen that in the presence of other ions except potassium ions, the fluorescence probe only has a weak change in fluorescence intensity, showing good specificity for the detection of potassium ions.

[0087] Example 5 Kinetics of the Fluorescence Probe for Detecting Potassium Ions

[0088] An ideal detection probe should have rapid recognition and signal response to the target. To investigate the kinetics of the fluorescence probe in this application for potassium ions, potassium chloride solution (2.5 μg, 2 M) was rapidly added to the fluorescence probe solution (97.5 μg), and the change in fluorescence intensity of the fluorescence probe over time was detected using a fluorescence spectrometer. The results are as Figure 6 shown.

[0089] As Figure 6 can be seen, after the addition of potassium ions, the fluorescence increased rapidly, indicating that the fluorescence probe of this application can achieve rapid detection of potassium ions.

[0090] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as the scope described in this specification.

[0091] The above embodiments only represent several implementation manners of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limitations on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of the patent of this application should be subject to the appended claims.

Claims

1. Use of molybdenum disulfide in the preparation of a potassium ion fluorescence detection reagent, characterized in that, The potassium ion reagent includes a single-stranded nucleic acid fragment linked with a fluorescent group and molybdenum disulfide. The single-stranded nucleic acid fragment can specifically bind potassium ions to form a G-quadruplex. The molybdenum disulfide is used to bind to the single-stranded nucleic acid fragment to quench the fluorescent group. The sequence of the single-stranded nucleic acid fragment is shown as SEQ ID NO.

1. The molybdenum disulfide is molybdenum disulfide nanosheets, and the particle size of the molybdenum disulfide nanosheets is 50 nm to 200 nm. The molar concentration ratio of the single-stranded nucleic acid fragment to the molybdenum disulfide nanosheets is 1:25000. The fluorescent group is selected from at least one of FAM fluorescent group, TAMRA fluorescent group, Cy3 fluorescent group, Cy5 fluorescent group, Alexa Fluor488 fluorescent group, Atto 590 fluorescent group, and Texas red fluorescent group.

2. The use according to claim 1, characterized in that, The fluorescent group is selected from FAM fluorescent group.

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

1. The molybdenum disulfide is molybdenum disulfide nanosheets, and the particle size of the molybdenum disulfide nanosheets is 50 nm to 200 nm. The molar concentration ratio of the single-stranded nucleic acid fragment to the molybdenum disulfide nanosheets is 1:25000. The fluorescent group is selected from at least one of 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.

4. The potassium ion fluorescence detection reagent according to claim 3, wherein, The single-stranded nucleic acid fragment satisfies at least one of the following characteristics: (1) The single-stranded nucleic acid fragment can dissociatively bind to the surface of the molybdenum disulfide; (2) The concentration of the single-stranded nucleic acid fragment is 0.05 μM to 0.5 μM.

5. The potassium ion fluorescence detection reagent according to claim 3 or 4, characterized in that, The fluorescent group is selected from FAM fluorescent group.

6. A potassium ion fluorescence detection method, characterized in that, It includes the following steps: Mix and react the sample to be tested and the potassium ion fluorescence detection reagent according to any one of claims 3 to 5; Detect the fluorescence intensity of the reaction solution after the reaction ends to obtain the fluorescence intensity of the sample to be tested; and Calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested.

7. The potassium ion fluorescence detection method according to claim 6, wherein The step of calculating the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested includes: Obtain the standard curve of the fluorescence intensity of the reaction solution and the potassium ion concentration, and calculate the potassium ion concentration of the sample to be tested according to the fluorescence intensity of the sample to be tested and the standard curve. The standard curve is: F1 = 1.62 + 0.68×c [K+] ; Among them, F1 = F / F0 - 1, where F1 represents the normalized fluorescence intensity of the reaction solution, F / F0 represents the ratio of the original fluorescence intensity of the reaction solution to the background fluorescence intensity, and c [K+] represents the potassium ion concentration of the reaction solution.

8. The potassium ion fluorescence detection method according to claim 6 or 7, characterized in that The potassium ion concentration of the sample to be tested is 0 mM to 150 mM.

9. The potassium ion fluorescence detection method according to claim 6, wherein, Before mixing and reacting the sample to be tested and the potassium ion fluorescence detection reagent according to any one of claims 3 to 5, it further includes: Mix and oscillate the solution of the single-stranded nucleic acid fragment linked with the fluorescent group and the molybdenum disulfide so that the single-stranded nucleic acid fragment is adsorbed on the molybdenum disulfide to prepare the potassium ion fluorescence detection reagent.

10. The potassium ion fluorescence detection method according to claim 9, characterized in that, The time of the mixing reaction is 1 h to 4 h; and / or The time of the mixed oscillation is 10 minutes to 60 minutes.

Images