Lithium ion aptamer and screening method and application thereof

Abstract

The application belongs to the technical field of lithium ion detection, and particularly relates to a lithium ion aptamer, a screening method and application thereof. The sequence of the lithium ion aptamer is shown as SEQ ID NO. 1. The application provides a 42nt single-stranded DNA aptamer, which has a specific base sequence, a single stem-loop secondary structure, high affinity to lithium ions and strong specificity. The application also provides application of the aptamer in preparation of a lithium ion detection probe, a detection reagent, a kit and a sensor, opens up a new way for lithium ion detection, can meet the detection requirements of lithium ions in different matrices in the medical, industrial and environmental fields, and has great potential for practical application.

Description

Technical Field

[0001] This invention belongs to the field of lithium ion detection technology, specifically relating to a lithium ion nucleic acid aptamer, its screening method, and its application. Background Technology

[0002] Lithium ions are key target ions in clinical medicine, environmental monitoring, and the new energy industry. Their accurate detection and monitoring are crucial for human health and environmental safety. Lithium carbonate is commonly used to treat bipolar disorder (BD), but its therapeutic window is extremely narrow (requiring a blood lithium concentration within the range of 0.6-1.2 mM): treatment efficacy is poor when blood lithium concentration is <0.6 mM, and symptoms such as mania and depression are difficult to control; blood lithium concentration >1.2 mM may cause symptoms of poisoning such as confusion and kidney damage; blood lithium concentration >2.0 mM can cause severe neurological damage (such as cognitive impairment and ataxia) and even endanger life. Therefore, blood lithium levels need to be monitored frequently during treatment to adjust medication, prevent poisoning, ensure patient safety, and improve treatment adherence. Furthermore, Lithium carbonate in industrial wastewater (such as battery manufacturing wastewater) is also a concern. + Excessive levels can interfere with the metabolism of aquatic organisms and affect human bone development. Real-time monitoring of Li-ion batteries is necessary for recycling waste lithium-ion batteries in the new energy sector. + Leachate concentration. Currently available detection technologies, traditional instrumental methods (such as atomic absorption spectrometry), require large equipment, specialized operation, and have long cycles. Rapid detection technologies, on the other hand, suffer from poor specificity and susceptibility to interference from other ions. Therefore, establishing a highly sensitive and easy-to-operate rapid lithium-ion detection system is urgently needed.

[0003] Nucleic acid aptamers, as oligonucleotide recognition molecules for in vitro screening, have advantages such as easy synthesis, modifiability, low immunogenicity, and high target binding specificity. Capture-SELEX technology, by immobilizing the target on a solid-phase carrier, can directionally screen for aptamers with higher affinity and stronger specificity for the target, providing a new approach to address the pain points of existing lithium-ion detection technologies.

[0004] For example, prior art CN114015694A discloses a nucleic acid aptamer for detecting copper ions and its application. This nucleic acid aptamer is a 42-nt single-stranded DNA, and its nucleic acid sequence contains the bases GGCAGGGTGAGGTTGAGGTCCG. This copper ion nucleic acid aptamer exhibits high affinity, good specificity, low synthesis cost, stable properties, and ease of modification; it is obtained through a magnetic bead-SELEX screening method and is compatible with Cu ions. 2+The established method for detecting copper ions using colloidal gold with nucleic acid aptamers exhibits high binding affinity and specificity. Characterized and detected using an enzyme-linked immunosorbent assay (ELISA) reader, it boasts a wide detection range, high sensitivity, and a detection limit as low as 1.99 ng / mL. The method is rapid and highly suitable for real-time on-site detection scenarios. Furthermore, the established method is convenient to operate, has simple steps, requires no labeling, is low-cost, and portable, providing a novel analytical method for copper ion detection in fields including food safety.

[0005] However, lithium ions, as the smallest alkali metal ion in nature (atomic weight 6.941, much smaller than copper ions), present significant technical challenges in aptamer development due to their small molecular weight. On the one hand, the small molecular weight limits the interaction sites with nucleic acid molecules, making it difficult to form stable and specific binding conformations. In conventional nucleic acid library screening, weak binding signals often prevent the capture of target sequences. On the other hand, they are susceptible to interference from other ions, leading to cross-reactivity in traditional recognition elements (such as antibodies and chemical sensors), hindering high-specificity recognition. Therefore, existing nucleic acid aptamer screening methods are not suitable for lithium ions. Summary of the Invention

[0006] The purpose of this invention is to provide a highly specific lithium-ion nucleic acid aptamer, its screening method, and its application.

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

[0008] A lithium-ion nucleic acid aptamer, the sequence of which is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1:

[0010] GACGACCAGCGCGTTCCACGTAGGGTGTTCAATCAGGTCGTC.

[0011] The lithium-ion nucleic acid aptamer forms a characteristic stem-loop secondary structure, the structure of which is as follows: Figure 1 As shown, Li + The binding portion is more concentrated on the loop. Experimental testing showed that the equilibrium dissociation constant Kd of the lithium-ion nucleic acid aptamer, determined by ThT fluorescence spectroscopy, is 57.42 nmol / L.

[0012] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0013] Based on the same inventive concept, this invention also claims protection for a nucleic acid aptamer derivative obtained by modifying or labeling a lithium-ion nucleic acid aptamer.

[0014] In one preferred embodiment, the modification includes phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopization.

[0015] In one preferred embodiment, the label includes fluorescent markers, radioactive substances, therapeutic substances, biotin, digoxin, nanomaterials, small peptides, siRNA, or enzymes.

[0016] In one preferred embodiment, the fluorescent marker is thioflavone T; and the nanomaterial is graphene oxide.

[0017] The modified or labeled nucleic acid aptamer derivatives mentioned above still have the same or similar molecular structure, physicochemical properties and functions as the original nucleic acid aptamers, and can all be used for binding with lithium ions. The modified or labeled nucleic acid aptamer derivatives can maintain or improve the affinity of nucleic acid aptamers for lithium ions, or can improve the stability of nucleic acid aptamers.

[0018] Based on the same inventive concept, this invention also claims protection for the above-mentioned screening method for lithium-ion nucleic acid aptamers, comprising the following steps:

[0019] S1. First positive screening: Mix the single-stranded DNA library with the complementary strand to form double-stranded DNA; fix the double-stranded DNA to obtain a fixed library; incubate the fixed library with the first solution, elute, amplify, and denature to obtain the product; the first solution contains lithium ions but does not contain sodium ions;

[0020] S2, Second positive screening: The product of S1 is mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA is fixed to obtain a fixed library; the fixed library is co-incubated with the second solution, eluted, amplified, and denatured to obtain the product; the second solution contains lithium ions and sodium ions;

[0021] S3, First negative screening: The product of S2 is mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA is fixed to obtain a fixed library; the fixed library is co-incubated with the third solution and eluted to obtain the product; the third solution does not contain lithium ions but contains sodium ions;

[0022] S4, Third positive screening: The product of S3 is co-incubated with the fourth solution, eluted, amplified, and denatured to obtain the product; the fourth solution contains lithium ions and sodium ions;

[0023] S5, Second negative screening: Mix the product of S4 with the complementary strand to form double-stranded DNA; fix the double-stranded DNA to obtain a fixed library; incubate the fixed library with the fifth solution, elute, and obtain the product; the fifth solution does not contain lithium ions but contains sodium ions;

[0024] S6, Fourth positive screening: The product of S5 is co-incubated with the sixth solution, eluted, amplified, and denatured to obtain the product; the sixth solution contains lithium ions and sodium ions;

[0025] The eluent used for elution does not contain sodium ions.

[0026] In one preferred embodiment, the eluent contains 8-15 mM 2-(N-morpholine)ethanesulfonic acid (MES), 5-10 mM KCl, 2-5 mM MgCl2, and has a pH of 6-7.

[0027] MES is chemically stable and has minimal interference with the non-specific binding of nucleic acids and targets. Potassium ions maintain the normal secondary structure of nucleic acid aptamers, while magnesium ions stabilize the nucleic acid structure with minimal interference. However, other forms of potassium and magnesium ions, such as sulfate and nitrate ions, introduce negative charges that can lead to electrostatic interactions and coordination reactions, affecting the stability of nucleic acids.

[0028] In one preferred embodiment, the first solution contains 500-600 µM lithium ions.

[0029] In one preferred embodiment, the second solution contains 500-600 µM lithium ions and 100-200 µM sodium ions.

[0030] In one preferred embodiment, the third solution contains 1000-1500 µM sodium ions.

[0031] In one preferred embodiment, the fourth solution contains 500-600 µM lithium ions and 500-600 µM sodium ions.

[0032] In one preferred embodiment, the fifth solution contains 1000-1500 µM sodium ions.

[0033] In one preferred embodiment, the sixth solution contains 500-600 µM lithium ions and 1000-1500 µM sodium ions.

[0034] In one preferred embodiment, the sequence of the complementary strand is shown in SEQ ID NO.2.

[0035] SEQ ID NO. 2: 5'-GTC GTC CCG AGA GCC ATA / 3BioTEG / -3'.

[0036] In one preferred embodiment, the sequences of the amplification primers are as follows:

[0037] SEQ ID NO.3: 5'-GGA GGC TCT CGG GAC GAC-3';

[0038] SEQ ID NO.4: 5'- / 5Biosg / TTA CGA TTG CAG CAT CGG GAC G -3'.

[0039] In one preferred embodiment, each amplification system comprises: 38 µL of 50 μM upstream primer, 38 µL of 50 μM downstream primer, 40 µL of 10 mM dNTP, 200 µL of 10× buffer, and 10× Mg... 2+ 120µL, Taq DNA polymerase 10µL, ultrapure water 1474µL.

[0040] In one preferred embodiment, the denaturation step includes: mixing the PCR product with streptavidin-labeled agarose beads and immobilizing it in a gravity column, adding alkali and incubating for 15-30 minutes, then neutralizing to obtain single-stranded DNA.

[0041] In one preferred embodiment, the alkali is NaOH with a concentration of 0.2-0.3M and an addition amount of 600-800µL.

[0042] In one preferred embodiment, the neutralization step is as follows: 0.2-0.3M acid is added and mixed, and the amount added is 400-600µL.

[0043] Based on the same inventive concept, this invention also claims protection for the use of the above-mentioned lithium-ion nucleic acid aptamers or nucleic acid aptamer derivatives in the separation, enrichment or detection of lithium ions.

[0044] Based on the same inventive concept, the present invention also claims protection for the use of the above-mentioned lithium-ion nucleic acid aptamers or nucleic acid aptamer derivatives in the preparation of lithium-ion detection reagents, molecular probes, kits or sensors.

[0045] In one preferred embodiment, the lithium ions are derived from electrolyte, battery recycling leachate, blood, body fluids, or cerebrospinal fluid.

[0046] Based on the same inventive concept, the present invention also claims a kit for separating, enriching or detecting lithium ions, which includes the above-mentioned lithium ion nucleic acid aptamer or nucleic acid aptamer derivative.

[0047] Based on the same inventive concept, the present invention also claims protection for a molecular probe comprising the above-mentioned lithium-ion nucleic acid aptamer or nucleic acid aptamer derivative.

[0048] Based on the same inventive concept, this invention also claims a fluorescence spectroscopy method for detecting lithium ions, which involves fully reacting the aforementioned lithium ion nucleic acid aptamer or nucleic acid aptamer derivative with a fluorescent probe and with a sample to be tested, detecting the fluorescence intensity at wavelengths of 440 nm and 490 nm, and calculating the lithium ion concentration of the sample to be tested based on a standard curve.

[0049] In one preferred embodiment, the fluorescent probe is thioflavone T.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] This invention provides a 42nt single-stranded DNA aptamer with a specific base sequence and a single stem-loop secondary structure, exhibiting high affinity and specificity for lithium ions. The invention also provides applications of this aptamer in the preparation of lithium ion detection probes, reagents, kits, and sensors, opening up new avenues for lithium ion detection. This approach can meet the detection needs of lithium ions in different matrices in the medical, industrial, and environmental fields, and has great potential for practical applications.

[0052] This invention, through extensive experimental research, discovered that the hydration radius of lithium ions (3.82 Å) is highly similar to that of common coexisting ions such as sodium ions (3.58 Å). The small differences in their small molecular structures lead to cross-reactivity in traditional recognition elements (such as antibodies and chemical sensors), making it difficult to achieve high-specificity recognition. This invention, by completely removing sodium ions from the eluent system and using the MES-KOH system, fundamentally eliminates the sodium ion problem. + Competition interference for Li + Specific binding creates a pure screening environment, which is a key prerequisite for successfully obtaining highly specific lithium-ion aptamers.

[0053] Meanwhile, this invention further optimizes the screening strategy by introducing and gradually increasing the concentration of competing ions (sodium ions) during the screening process, implementing dynamic competitive screening. Compared with traditional single reverse screening, this strategy simulates the application environment of aptamers in complex matrices (such as blood), forcing the library to evolve sequences capable of accurately recognizing lithium ions in a sodium ion background, thus fundamentally solving the technical bottleneck of lithium / sodium ion differentiation. Specifically, this invention provides a strategy for screening nucleic acid aptamers that combines dynamic competition with functional negative screening. This strategy employs a combination of streptavidin-labeled agarose beads and biotin-labeled nucleic acid libraries. The core of this strategy lies in periodically inserting functional negative screening rounds and a dynamic competition mechanism between conventional positive screening rounds. In the negative screening rounds, the library is incubated with a solution containing only high concentrations of sodium ions and free of lithium ions, and all eluted sequences are collected and discarded. This step actively and efficiently physically removes sequences with affinity for sodium ions from the library, purifying the screening library. In subsequent positive screening rounds, solutions containing lithium ions and progressively increasing concentrations of sodium ions are used to further apply evolutionary pressure. The combination of these two methods fundamentally solves the problem of specific recognition between lithium and sodium ions. This invention overcomes the technical bottleneck caused by small molecular weight, and has screened out a lithium ion recognition tool that combines high affinity and specificity, which has significant practical implications for promoting technological upgrades in related fields. Attached Figure Description

[0054] Figure 1 This is a predicted secondary structure diagram of the Li-2 lithium-ion nucleic acid aptamer.

[0055] Figure 2 This is the ThT fluorescence spectrum of the Li-2 lithium-ion nucleic acid aptamer.

[0056] Figure 3 This is a linear fitting curve of the dissociation constant Kd value of the Li-2 lithium-ion nucleic acid aptamer.

[0057] Figure 4 This is a linear graph of the lithium-ion working principle of the Li-2 lithium-ion nucleic acid aptamer.

[0058] Figure 5 The graph shows the Ct values ​​of traditional sodium-containing ion buffers screened up to the 4th round.

[0059] Figure 6 This is a graph showing the Ct values ​​of the buffer solution screened up to the fourth round of the present invention.

[0060] Figure 7 The aptamers obtained by the present invention using dynamic competition and functional negative screening strategies are Li + Na + K + The specificity detection bar chart.

[0061] Figure 8 The aptamers obtained by traditional screening methods for Li + Na + K + The specificity detection bar chart. Detailed Implementation

[0062] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0063] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Unless otherwise specified, the instruments, reagents, materials, etc., involved in the following embodiments are all conventional instruments, reagents, materials, etc., already existing in the prior art and obtainable through legitimate commercial channels. Unless otherwise specified, the experimental methods, detection methods, etc., involved in the following embodiments are all conventional experimental methods, detection methods, etc., already existing in the prior art.

[0064] Example 1

[0065] Screening of lithium-ion nucleic acid aptamers

[0066] I. Experimental Methods

[0067] (I) Library fixation: The ssDNA library screened in the laboratory in the early stage was paired with the biotin-modified complementary strand. The library was fixed in the chromatography column by the specific binding of biotin to streptavidin agarose beads. The sequence information of the ssDNA library and the biotin-modified complementary strand is shown in Table 1. The ssDNA library sequence was obtained from a large number of experiments designed in the early stage.

[0068] The above ssDNA library and biotin-modified complementary strand were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0069] N30 represents 30 random nucleotides.

[0070] The specific steps for fixing the document are as follows:

[0071] The ssDNA library and biotin-modified complementary strand were mixed at a molar ratio of 1:5, denatured at 95°C for 10 min, and slowly cooled to 25°C for annealing for 30 min to form a double-stranded complex. The double-stranded complex was incubated with streptavidin agarose beads (MedChemExpress, catalog number: HY-K0218A) at room temperature for 1 h, and the double-stranded complex was fixed by biotin-streptavidin reaction. The fixation efficiency (>90%) was calculated by UV detection (260 nm).

[0072] (II) Screening for lithium-ion specific nucleic acid aptamers

[0073] The magnetic beads were washed six times with SELEX buffer to remove unfixed library material. Then, lithium-ion specific nucleic acid aptamers were screened, involving multiple screening cycles. Each cycle included library fixation, contacting the library with the target solution, eluting the binding sequence, and amplifying the eluted sequence. Specifically, the process included: S1, First positive screening: The single-stranded DNA library was mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA was fixed to obtain a fixed library; the fixed library was co-incubated with the first solution, eluted, amplified, and denatured to obtain the product; the first solution contained lithium ions but no sodium ions; S2, Second positive screening: The product from S1 was mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA was fixed to obtain a fixed library; the fixed library was co-incubated with the second solution, eluted, amplified, and denatured to obtain the product; the second solution contained lithium ions and sodium ions; S3, First negative screening: The product from S2 was mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA was fixed to obtain a fixed library; the fixed library was co-incubated with the target solution, eluted, amplified, and denatured to obtain the product; the second solution contained lithium ions and sodium ions; The library is co-incubated with the third solution, eluted, and the product is obtained; the third solution does not contain lithium ions but contains sodium ions; S4, third positive screening: the product of S3 is co-incubated with the fourth solution, eluted, amplified, and denatured to obtain the product; the fourth solution contains lithium ions and sodium ions; S5, second negative screening: the product of S4 is mixed with the complementary strand to form double-stranded DNA; the double-stranded DNA is fixed to obtain a fixed library; the fixed library is co-incubated with the fifth solution, eluted, and the product is obtained; the fifth solution does not contain lithium ions but contains sodium ions; S6, fourth positive screening: the product of S5 is co-incubated with the sixth solution, eluted, amplified, and denatured to obtain the product; the sixth solution contains lithium ions and sodium ions; the library fixation steps are the same as above.

[0074] The target solution that comes into contact with the library in each round is different. The specific solution composition and purpose are shown in the table below.

[0075] In the solution, lithium ions and sodium ions exist in the form of NaCl solution and LiCl solution, respectively.

[0076] During the screening round, agarose beads immobilized with the library were washed with SELEX buffer. Then, a specific concentration of Li was added... + and Na + The target solution was incubated at room temperature for 45 minutes. Elution was then performed using SELEX buffer, and the eluent (this is for use with Li) was collected. + (Sequences that specifically bind to and are dissociated by conformational changes) are used for subsequent PCR amplification.

[0077] In the functional negative screening round, agarose beads were washed with SELEX buffer. Then, a mixture containing only a high concentration of Na+ was added. + (1000μM), Li-free + The negative screening solution was incubated at room temperature for 45 minutes. Elution was then performed using SELEX buffer, and the eluent from this step was collected and discarded (this is the Na⁺-binding sequence). The remaining libraries, still immobilized on agarose beads after negative screening, were used for the next round of positive screening.

[0078] SELEX buffer consists of 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 5 mM KCl, and 2 mM MgCl2, with the pH adjusted to 6 using KOH.

[0079] The steps for PCR amplification are as follows:

[0080] The solutions eluted from each round of positive screening were amplified by PCR. The primer sequence information used for PCR is shown in Table 3. The PCR amplification solution system and reaction parameters are shown in Tables 4 and 5.

[0081] The steps for preparing single-stranded DNA are as follows:

[0082] All PCR products were collected, mixed with streptavidin-labeled agarose beads, and fixed in a gravity column. 600 μL of 0.2 M NaOH was added, and the mixture was incubated for 15 min. Then, 400 μL of 0.2 M HCl was added to neutralize the mixture and obtain single-stranded DNA.

[0083] (iv) Screening Termination Judgment

[0084] Monitor the eluent circulation threshold for each round. When the circulation threshold of a certain round is significantly lower than that of the first round, and the positive sieve (Li) is used... + The value is lower than that of the reverse sieve (Na). + A value of ) indicates that the specific sequence has been enriched, and the screening is terminated.

[0085] II. Experimental Results

[0086] This invention achieves efficient screening of lithium-ion aptamers using a systematically optimized Capture-SELEX method. During the screening process, a combined dynamic competition and functional negative screening strategy is employed. This proactively and efficiently removes sequences with affinity for sodium ions from the library, purifying the screening library. Dynamic competition ensures that only aptamers exhibiting significant specific binding to free lithium ions are eluted and recovered, creating a rigorous anti-screening pressure and effectively reducing interference from non-specific sequences. After six rounds of efficient and simplified screening, the screened products are subjected to high-throughput sequencing using the Illumina platform to comprehensively obtain sequence information. Subsequently, multi-dimensional bioinformatics analysis is conducted using Geneious software: ① Statistical analysis of sequence repetition frequency to screen high-abundance sequences as key research targets; ② Prediction of secondary structures (such as stem-loop and hairpin structures) and analysis of potential lithium-ion binding regions; ③ Homology comparison to classify different sequence families to narrow the activity detection range. The top six most abundant lithium-ion aptamers are shown in Table 6.

[0087] Table 6 shows that the first two sequences had the highest enrichment during the screening process (accounting for 62.0% and 25.2% of the total sequencing volume, respectively), and possessed the potential to be candidate aptamers. To determine the optimal sequence, ThT fluorescence spectroscopy was used to verify the systematic activity of these six intermediate-abundance sequences. The ThT fluorescence spectroscopy procedure was as follows: the target aptamer with a final concentration of 0.5 μM was mixed with ThT with a final concentration of 2.5 μM and incubated for 10 min. Then, lithium ions were gradually titrated, with the final lithium ion concentration increasing from 50 nM to 1000 nM. The fluorescence intensity after the addition of the target was measured using HORIBA fluorescence spectrophotometry. Based on the experimental results of the fluorescence intensity, a linear fit was performed to create the function y = y0 + (a•Kd) / (Kd + x) (where x is the lithium ion concentration [Li...]). + [where y is fluorescence intensity, y0 is initial fluorescence intensity, a is the maximum fluorescence change under saturation, and Kd is the dissociation constant]. The fitting results for the lithium-ion aptamer Li⁻² show a dissociation constant Kd = 57.42 nM and a coefficient of determination R = 0.846, indicating that the lithium-ion aptamer Li⁻² has a strong binding affinity. The fitting curve is shown below. Figure 3 As shown in the diagram, the equilibrium dissociation constant of the lithium-ion aptamer Li-2 (Kd = 57.42 nM) is significantly lower than that of the first sequence (Kd = 1386.85 nM) and the other four sequences, ultimately determining this single-stranded DNA as the optimal lithium-ion aptamer. A schematic diagram of the ThT fluorescence spectroscopy determination of the lithium-ion aptamer Li-2 is shown below. Figure 2 As shown.

[0088] The secondary structure of the Li-2 aptamer was predicted using mFold software, and the results are as follows: Figure 1 As shown, the results indicate that it forms a typical stem-ring structure.

[0089] Example 2

[0090] Establishment of a method for quantitative determination of lithium ions using nucleic acid aptamers based on ThT fluorescence spectroscopy

[0091] I. Experimental Principle

[0092] ThT (thioflavin T) is a specific fluorescent probe. Its fluorescence signal is extremely weak in aqueous solution, but it can specifically bind to nucleic acid aptamers with specific secondary structures (such as stem-loop structures) through "hydrophobic interaction + hydrogen bond". After binding, the planar structure of the ThT molecule is more rigid and the fluorescence quantum yield is significantly improved, which is manifested as a strong enhancement of the fluorescence signal.

[0093] When lithium ions (the target) are present in the system, they can coordinate with specific binding sites (such as stem-loop structures) of the aptamer, forming an aptamer-lithium ion complex. This binding leads to a conformational change in the secondary structure of the aptamer, causing the ThT molecules originally bound to the aptamer to competitively dissociate and be released into the aqueous solution. As the lithium ion concentration increases, the amount of aptamer-lithium ion complex formed increases, the number of dissociated ThT molecules increases, and the fluorescence intensity of the system gradually decreases. Furthermore, the fluorescence intensity tends to stabilize after the lithium ion concentration reaches aptamer binding saturation.

[0094] II. Experimental Methods

[0095] Take a 96-well fluorescent plate, add 30 μL of aptamer diluent (1 μM) and 30 μL of ThT working solution (10 μM) to each well, and then add 40 μL of lithium ion standard solutions of different concentrations to make the total volume of the system 100 μL. The final concentrations of each component are: aptamer 0.3 μM, ThT 3 μM, and lithium ion 0~1000 nM. After incubation at room temperature for 15 min, the fluorescence intensity of each well is measured (denoted as F) under the conditions of Ex=440 nm and Em=490 nm. At the same time, the fluorescence intensity of the blank group (without lithium ions, denoted as F0) is measured. With "lithium ion concentration (x, nM)" as the abscissa and "fluorescence intensity change rate ((F0-F) / F0×100%)" as the ordinate, a standard curve is established by linear regression, and the regression equation and correlation coefficient (R2) are obtained.

[0096] Take the test sample (serum), filter it through a 0.22μm filter membrane to remove impurities, add aptamer and ThT as described above, measure the fluorescence intensity, and calculate the lithium ion concentration by substituting it into the standard curve; at the same time, perform a spiked recovery experiment (adding lithium ion standards at low, medium and high concentration levels) and calculate the recovery rate to verify the accuracy.

[0097] III. Experimental Results

[0098] The results are as follows Figure 4 As shown, the working equation is y = 2.972 × 10 -4 x + 0.06469, where x represents the lithium-ion concentration and y represents (F0 - F) / F0, shows a good linear relationship with an R² of 0.912. The method detection limit reaches 216.69 nM, thus establishing the detection method. Each group was repeated three times, and the results are shown below.

[0099] The results showed that the recoveries of the three groups of samples were 119.0%, 106.1%, and 98.56%, respectively.

[0100] Comparative Example 1

[0101] Screening efficacy verification of traditional sodium-containing buffer system

[0102] To demonstrate the necessity of the sodium-free buffer system, a parallel screening experiment was conducted against the traditional sodium-containing buffer system for comparison.

[0103] I. Experimental Methods and Materials

[0104] Using the same initial ssDNA library as in Example 1, screening was performed using conventional sodium-containing buffer (20mM Tris-HCl, 100mM NaCl, 5mM KCl, 2mM MgCl2, pH=7.5). Except for the use of the sodium-containing buffer, all other steps (library fixation, incubation, elution, PCR, etc.) were the same as in Example 1.

[0105] II. Experimental Results and Discussion

[0106] The products from the four rounds of screening and the products from the four rounds of screening in Example 1 were simultaneously subjected to qPCR amplification to see if the selected aptamers were enriched. The results are as follows: Figure 5 and Figure 6 As shown, where Figure 5 Ct value curves for traditional sodium-containing ion buffer solutions screened up to the 4th round; Figure 6 This is a graph showing the Ct values ​​of the buffer solution after the fourth round of screening according to the present invention. In the fourth round of screening, the Ct values ​​of the library eluted with target lithium ions were almost identical to the background Ct values, both being 16. However, the Ct values ​​of the library after four rounds of screening in Example 1 showed a very significant difference from the background Ct values. This indicates that under conventional sodium-containing buffer conditions, the specific binding of Li... + The sequence cannot be effectively enriched.

[0107] III. Conclusion

[0108] The above comparative experiments fully demonstrate that in traditional sodium-containing buffer systems, due to Na... + Competitive interference makes it impossible to screen for Li + Nucleic acid aptamers that combine high affinity and high specificity.

[0109] Comparative Example 2

[0110] Verification of the necessity of dynamic competition and functional negative screening strategies

[0111] This study aims to verify whether the "dynamic competition and functional negative screening" strategy described in this invention is indispensable for eliminating sodium ion-specific sequences, purifying the screening library, and obtaining highly specific lithium ion aptamers.

[0112] (I) Experimental Methods

[0113] Experimental group: The complete process of Embodiment 1 of the present invention is adopted, which includes two rounds of functional negative screening in the 3rd and 5th rounds and dynamic competitive screening starting from the 2nd round.

[0114] Control group: In the first round of screening, Li was added + The solution was incubated. Starting from the second round, negative sieve material was added, using 500 μM sodium ions for negative sieve treatment, initially with Na... + The solution was incubated with the library, and the eluted sequences were collected and discarded (i.e., the traditional reverse screening step). Dynamic competitive screening and high-concentration negative screening were not used, as detailed in Table 8.

[0115] In the solution, lithium ions and sodium ions exist in the form of NaCl solution and LiCl solution, respectively.

[0116] All other conditions (library, buffer, immobilization vector, etc.) were identical for both groups.

[0117] (II) Results and Discussion

[0118] Two groups of screened aptamers were specifically detected using ThT fluorescence spectroscopy with solutions containing 200 μM of LiCl, NaCl, and KCl. The results are as follows: Figure 7 and Figure 8 As shown, where Figure 7 The aptamers obtained using dynamic competition and functional negative screening strategies for Li + Na + K + Specific detection bar chart; Figure 8 The aptamers obtained by traditional screening methods for Li + Na + K +The results showed that the aptamers obtained in Example 1 of this invention and the control group had significant differences in their fluorescence responses. The aptamer obtained in Example 1 of this invention had a significantly higher response value to lithium ions than to other ions, while the response values ​​of the aptamer in the control group to the three ions were similar.

[0119] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A lithium ion aptamer, characterized in that, The sequence is shown as SEQ ID NO.

1.

2. The lithium ion aptamer according to claim 1 is applied to detecting lithium ion for the purpose of separation, enrichment or non-disease diagnosis.

3. The lithium ion aptamer according to claim 1 is applied to preparing a lithium ion detection reagent, a molecular probe, a kit or a sensor.

4. A method for detecting lithium ions by fluorescence spectroscopy, characterized by, The lithium ion aptamer according to claim 1 is fully reacted with a fluorescent probe and a sample to be detected, the fluorescence intensity at 440 nm and 490 nm is detected, the lithium ion concentration of the sample to be detected is calculated according to a standard curve, and the method is not directly used for the diagnosis of diseases.

Images