Double-split aptamer probe and application thereof

Abstract

The invention relates to the field of molecular biology and clinical medicine, and relates to a double-split aptamer probe and application thereof. The double-split aptamer probe comprises two split aptamers, namely a split aptamer 1 and a split aptamer 2, the split aptamer 1 comprises an aptamer fragment 1 and a split initiator 1, the split aptamer 2 comprises an aptamer fragment 2 and a split initiator 2, and the two aptamer fragments are from a complete aptamer sequence A. The invention further discloses a preparation method of the double-split aptamer probe. The invention proves that DNA (deoxyribonucleic acid) can be successfully self-assembled in a solution to form a fluorescence tree with larger molecular weight under the triggering of a target, so that signal amplification is realized. The constructed sandwich-type fluorescence sensor can be successfully applied to Ade detection, in the concentration range of 0.01-100 ng / mL, the detected fluorescence intensity is enhanced along with increase of the concentration of Ade, LOD is not higher than 5.23 pg / mL, the detection time is not longer than 30 min, and the sandwich-type fluorescence sensor has the advantages of being high in sensitivity and short in detection time.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and clinical medicine, and relates to a double-fission aptamer probe and application thereof. Background Art

[0002] Sandwich immunoassay is a universal detection method with low detection limit and high specificity, and has a wide range of application prospects. However, this assay requires that the analyte has at least two epitopes that can bind to two antibodies simultaneously. However, due to their limited size, small molecules are usually considered to be monovalent and cannot bind to two antibodies simultaneously. Therefore, sandwich immunoassays are mostly used to detect large molecules such as proteins, while it is difficult to detect small targets with a molecular weight of less than 1000. Compared with antibodies, aptamers have the advantages of small molecular weight, flexible molecular modification, long-term storage at room temperature, and reusability after denaturation and renaturation, which provides the possibility of overcoming the limitations of antibodies. In the special case of small molecule detection, aptamers have greater advantages over antibodies and are considered to be a promising alternative to antibodies. Aptamer recognition is based on intermolecular forces, such as electrostatic interactions, hydrogen bonds, etc., and has strong affinity and selectivity for small molecules. However, due to their long nucleotide sequences, aptamers tend to fold into non-specific secondary structures, which may bind to other substances and produce false positive or non-specific signals, which is obviously not conducive to establishing a highly specific detection method. Secondly, traditional small molecule aptamers have only one binding epitope or even a partial epitope, which poses a challenge to the design of sandwich sensors with both capture function and signal reporting.

[0003] In order to solve the difficulties of the above-mentioned complete aptamers and improve the specificity and sensitivity of aptamers. Scientists have proposed the concept of split aptamers, that is, creatively dividing a complete aptamer into two fragments. Split aptamers are shorter than complete aptamer sequences and can avoid the disadvantage of steric hindrance. In addition, due to the lack of secondary structure, split aptamers will disperse when there is no target. Only when the target is present can the aptamer be reassembled into a complete complex that specifically binds to it. This technology can effectively avoid the generation of false positives or nonspecific signals. For example, Bai Yunfeng et al. first used the high specificity of split aptamers and the unique interaction between graphene oxide and the two states of the designed aptamer sequence to construct an adenosine on / off fluorescent aptamer sensor with a detection limit of 6μ (OSYPOVAA, THAKAR D, DEJEU J, et al. Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes [J]. Analytical Chemistry, 2015, 87 (15): 7566-7574). Although this method achieves simple detection, it suffers from low sensitivity due to the 2:1 ratio between target input and signal output.

[0004] Compared with linear HCR, nonlinear hybridization chain reaction (NHCR) has higher amplification efficiency due to the formation of branched nanostructures and simplifies enzyme-free operation. Yang Tiantian et al. successfully utilized the specific advantages of aptamer proximity hybridization and the high sensitivity of NHCR to develop a co-localization-triggered DNA nanoassembly strategy for amplified imaging (QI C, BINGT, MEI H, et al. G-quadruplex aptamers for zeatin recognizing [J]. Biosensors and Bioelectronics, 2013. 299 (13): 157-162.). This method ensures higher detection sensitivity while ensuring specificity.

[0005] Extracellular adenosine triphosphate (ATP) and Ade derivatives such as Ade have become recognized biochemical markers of cancer due to their high concentration levels in tumor stroma. Under normal physiological conditions, extracellular ATP levels are at low nM levels, but increase to μM levels in tumor stroma and nearby areas. Cancer cells integrate activation (ATP) and inhibition (Ade) signaling pathways to produce the "final" Ade derivative concentration results. The difference in the content of Ade as a human tumor marker in normal people and cancer patients. For example, the normal content of Ade in the human body is 0.18μM-4.7μM, and the Ade concentration in colorectal cancer patients is nearly 3 times that of healthy people. However, the detection limit of the Ade derivative detection kit currently available on the market is only at the microgram level, and its detection sensitivity cannot meet the needs of clinical cancer monitoring. In addition, most of them are enzyme-linked immunosorbent assays, which have complex operation processes and require strict control of the experimental environment. Summary of the invention

[0006] The purpose of the present invention is to construct a high-sensitivity double-split aptamer probe and its application.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A double split aptamer probe comprises two split aptamers, namely split aptamer 1 and split aptamer 2. Split aptamer 1 comprises aptamer fragment 1 and split initiator 1, split aptamer 2 comprises aptamer fragment 2 and split initiator 2, and the two aptamer fragments are from a complete aptamer sequence A. The aptamer fragment 1 and the aptamer fragment 2 have the ability to recognize a target, and the split initiator 1 and the split initiator 2 have the function of initiating NHCR.

[0009] The probe can specifically recognize the target. In the presence of the target, the probe can form a sandwich structure, so that two independent initiators are close to each other, synergistically induce nonlinear hybridization chain reaction (NHCR), and realize the secondary or exponential growth of trace small molecule detection signals. Unlike the typical NHCR initiator, which is complete, the initiator in the present invention is divided into two parts and can be induced by Ade together. Therefore, in the absence of Ade, it can be effectively inhibited. First, the split aptamer can be constructed by cutting the complete aptamer sequence into two fragments, and then combined with the NHCR method to construct a simple and universal small molecule detection platform. The double split aptamer probe consists of two sequences, each of which can be subdivided into two fragments with different functions, one fragment is derived from the split aptamer and has the ability to recognize Ade, and the other fragment is derived from the split initiator to start NHCR. In the free state, the two sequences are separated from each other, and the split initiator alone cannot start NHCR, leaving a stable probe with quenched fluorescence. However, in the presence of Ade, two split aptamers can bind two molecules of Ade and reassemble into a specific configuration according to the “induced fit effect”. Then, the recognition-driven remodeling of the dual split aptamer probe can bring the two segments of split initiators closer together to trigger the NHCR process as a complete initiator.

[0010] In a preferred embodiment, the molar ratio of split aptamer 1 to split aptamer 2 is 1-2:1-2.

[0011] In a preferred embodiment, the sequence of aptamer fragment 2 is such as ACCTGGGGGAGTAT; the sequence of cleavage initiator 2 is GATCAAGCAGT; the sequence of aptamer fragment 1 is such as TGCGGAGGAAGGT; the sequence of cleavage initiator 1 is GTCGAAGTAGTT; and the sequence of aptamer sequence A is ACCTGGGGGAGTATTGCGGAGGAAGGT.

[0012] The cleavage initiator and the aptamer fragments should not interfere with each other.

[0013] The present invention also claims to protect the use of the double-split aptamer probe in preparing a reagent or a kit for detecting adenosine or adenosine derivatives.

[0014] The invention also claims to protect the use of the double-split aptamer probe in preparing a reagent or a kit for detecting colorectal cancer, prostate cancer or lung cancer.

[0015] A reagent or kit for detecting adenosine or adenosine derivatives, each detection volume comprising 10 μL AT1 solution, 10 μL AT2 solution, 16.5 μL buffer, 1 μL FA solution, 1 μL FB solution, 1.5 μL QA solution, 3 μL QB solution, 2 μL AA solution and 4 μL AB solution;

[0016] The sequence of AT1 is shown in SEQ ID NO.1, the sequence of AT2 is shown in SEQ ID NO.2, the sequence of FA is shown in SEQ ID NO.3, the sequence of FB is shown in SEQ ID NO.4, the sequence of QA is shown in SEQ ID NO.5, the sequence of QB is shown in SEQ ID NO.6, the sequence of AA is shown in SEQ ID NO.7-11, and the sequence of AB is shown in SEQ ID NO.12.

[0017] A reagent or kit for detecting colorectal cancer, prostate cancer or lung cancer, each detection volume comprising 10 μL AT1 solution, 10 μL AT2 solution, 16.5 μL buffer, 1 μL FA solution, 1 μL FB solution, 1.5 μL QA solution, 3 μL QB solution, 2 μL AA solution and 4 μL AB solution;

[0018] The sequence of AT1 is shown in SEQ ID NO.1, the sequence of AT2 is shown in SEQ ID NO.2, the sequence of FA is shown in SEQ ID NO.3, the sequence of FB is shown in SEQ ID NO.4, the sequence of QA is shown in SEQ ID NO.5, the sequence of QB is shown in SEQ ID NO.6, the sequence of AA is shown in SEQ ID NO.7-11, and the sequence of AB is shown in SEQ ID NO.12.

[0019] In a preferred embodiment, the sequence of AA is shown as SEQ ID NO.8.

[0020] In a preferred embodiment, the solvents of the AT1 solution, AT2 solution, FA solution, FB solution, QA solution, QB solution, AA solution and AB solution are all buffer solutions.

[0021] In a preferred embodiment, the buffer solution includes: 1 mL 50×TAE solution, 0.625 mL Mg 2+ Solution, 48.375 mL water, pH 8.0.

[0022] In a preferred embodiment, the concentrations of AT1 solution, AT2 solution, FA solution, FB solution, QA solution, QB solution, AA solution and AB solution are 0.5-1.0 μM.

[0023] The present invention is further explained below:

[0024] The present invention uses adenosine (Ade) as a model to design a dual-split aptamer probe. First, a split aptamer can be constructed by cutting the complete aptamer sequence into two fragments, and then combined with the NHCR method to construct a simple and universal small molecule detection platform. The dual-split aptamer probe consists of two sequences, each of which can be subdivided into two fragments with different functions, one fragment is derived from the split aptamer and has the ability to recognize Ade, and the other is derived from the split initiator to initiate NHCR. In the free state, the two sequences are separated from each other, and the split initiator alone cannot initiate NHCR, leaving a stable probe with quenched fluorescence. However, in the presence of Ade, the two split aptamers can bind to two molecules of Ade and reassemble into a specific configuration according to the "induced fit effect". Then, the recognition-driven remodeling of the dual-split aptamer probe can bring the two segments of the split initiator closer to trigger the NHCR process as a complete initiator. Therefore, the fluorescent probe (FA and FB labeled by fluorophores) can be continuously turned on and produce a branched fluorescent tree with activated fluorescence, so that the detection signal of Ade is amplified, such as Figure 1 shown.

[0025] The present invention proves that under the triggering of the target, DNA can successfully self-assemble in the solution to form a fluorescent tree with a larger molecular weight, thereby achieving signal amplification. In the process of DNA self-assembly, by optimizing the length of the auxiliary chain A, it is found that the background signal of NHCR self-assembly can be reduced by reducing one base from the original one.

[0026] The constructed sandwich-type fluorescence sensor can be successfully applied to the detection of Ade. In the concentration range of 0.01-100 ng / mL, the measured fluorescence intensity increases with the increase of Ade concentration. The linear regression equation is: y = 499.77x-323.9 (R 2 =0.9951), good linearity, LOD no higher than 5.23pg / mL, and detection time no more than 30min. Compared with the recently reported method for detecting Ade, it still has the advantages of higher sensitivity and shorter detection time. And it has been confirmed that this method can accurately quantify Ade in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The detection principle of the dual-split aptamer-mediated immune NHCR biosensor;

[0028] Figure 2 The figure is the result of 12% PAGE electrophoresis; Lane M is Marker; Lanes 1 and 2 are SA and SB; Lanes 3 and 4 are byproduct A and byproduct B; Lanes 5, 6, and 7 are NHCR systems with A+B, 1μAT1+1μMAT2+A+B, and 1μM AT1+1μM AT2+A+B+1mg / mL Ade added respectively;

[0029] Figure 3 The fluorescence spectra of NHCR products in different groups;

[0030] Figure 4 Optimization of the number of AA base pairs and NHCR reaction time;

[0031] Figure 5 To optimize the concentration ratio of AT1 to AT2;

[0032] Figure 6 Results of quantitative detection of Ade by NHCR detection system, (A) fluorescence signals generated by different concentrations of Ade; (B) linear relationship between fluorescence intensity and logarithm of Ade concentration;

[0033] Figure 7 For the selective analysis of Ade and Ade analogs by immunoassay based on NHCR signal amplification;

[0034] Figure 8 Results of clinical sample testing; (A) The relationship between Ade concentration and fluorescence intensity in diluted urine; (B) The linear relationship between relative fluorescence intensity and added Ade concentration in normal human urine. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the examples. However, these examples are limited to illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified. The English abbreviations used in the present invention are described as follows:

[0036] Table 1 Description of English abbreviations

[0037]

[0038] The materials and reagents used in the present invention are as follows:

[0039] Table 2 Specifications and manufacturers of main materials and reagents

[0040]

[0041] Preparation of buffer I

[0042] Add 1mL 50×TAE solution, 0.625mL Mg2+ solution, add ultrapure water, and dilute to volume with a 50mL volumetric flask. Adjust pH to 8.0 with 1mol / L NaOH solution before use and store at room temperature.

[0043] Preparation of DNA oligonucleotide stock solution

[0044] Before adding the solution, in order to prevent the DNA powder in the test tube from diffusing out, centrifuge the test tube containing the oligonucleotide sequence with a high-speed refrigerated centrifuge before opening the lid, then open the lid and add ultrapure water, vortex mix, and fully dissolve the DNA sequence. For subsequent specific experiments, use buffer I solution to prepare other concentrations. For short-term use, store in a 4℃ refrigerator, and for long-term storage, store in a -20℃ refrigerator to avoid repeated freezing and thawing.

[0045] Preparation of Ade solution

[0046] Weigh 1.0 mg of Ade powder into a 2.0 mL epoxide tube, and then add 1 mL of PBS solution. Ultrasonicate for 3 min using an ultrasonic mixer to obtain a 1.0 mg / mL Ade solution. For subsequent specific experiments, use buffer I solution to prepare other concentrations, and store the solution in a refrigerator at 4°C.

[0047] Example 1

[0048] Nupack software was used to design the oligonucleotide sequences required for the reaction system. The specific sequences are shown in Table 3.

[0049] Table 3 NHCR system oligonucleotide sequences

[0050]

[0051]

[0052] 1.1 Synthesis of double-stranded substrates SA and SB

[0053] First, use bufferⅠ to prepare 10μM FA, 10μM QA, 10μM AA, 10μM FB, 10μM QB and 10μM AB solutions and store them at 4℃. Add bufferⅠ, 10μM FA and 10μM QA in a volume ratio of 5.5:1:1.5 to a 200μL ep tube, and add bufferⅠ, 10μM FB and 10μM QB in a volume ratio of twice that of the A mixed solution to another ep tube. Put the two ep tubes A and B into the PCR instrument, react at 95℃ for 5min, gradually cool down to 25℃, obtain double-stranded substrate SA and SB solutions, and store them temporarily in a 4℃ refrigerator.

[0054] 1.2 Synthesis of NHCR A and NHCR B reaction solutions

[0055] Add 2 times the volume of FA of 10 μM AA solution and 2 times the volume of FB of 10 μM AB to SA solution and SB solution respectively, and let them react in a refrigerator at 4°C for 45 minutes. After the reaction, NHCR A solution and NHCR B solution are obtained. In order to prevent further chemical changes, they need to be prepared and used immediately.

[0056] Afterwards, native polyacrylamide gel (PAGE) electrophoresis was used to verify the feasibility of NHCR. The specific operation is as follows:

[0057] 1.3 Synthesis of NHCR-related sequences and products

[0058] Use a PCR instrument to synthesize the SA, SB, By-product A and By-product B sequences in Table 4 with a final concentration of 1 μM. Ensure that the PCR reaction conditions are correct to ensure the purity and integrity of the product. Next, prepare the NHCR system with or without Ade according to the composition ratio in Table 4. React at 37°C for 60 minutes to obtain NHCR products of different combinations. After the reaction is completed, mix the synthesized NHCR-related sequences and products with the loading buffer according to the ratio to ensure that the electrophoresis process is indicated in the PAGE electrophoresis analysis and to prevent the sample from floating out of the spotting well.

[0059] Table 4 Composition ratio of NHCR related sequences and NHCR products

[0060]

[0061] 1.4 Preparation of 12% PAGE

[0062] Mix 3.3mL 30% Acr-Bis and 6.6mL ultrapure water by pipetting, then add 0.1mL 10% APS and 0.004mL TEMED to ensure uniform mixing. Add the prepared gel between the two electrophoresis plates, and then insert the electrophoresis comb. Avoid the generation of bubbles during this process to avoid affecting the subsequent electrophoresis analysis. Let it stand at 37°C for 15-30 minutes to wait for the gel to solidify.

[0063] 1.5PAGE electrophoresis analysis

[0064] Pour 1×TBE electrophoresis solution into the electrophoresis tank, and carefully pull out the comb to ensure that the gel is not damaged. Then, add 3μL Marker and 10μL NHCR mixture in Table 4 to the sample wells in turn. When performing electrophoresis, maintain a constant current of 14mA, and the electrophoresis time is approximately 1.5h (adjust according to actual needs to achieve the best separation effect). After the electrophoresis, peel the gel off the plate, first shake and wash it with the electrophoresis solution, then stain it with Gel-red for 30min, and shake and wash it again. Finally, place the stained gel in a chemiluminescence imaging system for photography to obtain the electrophoresis result diagram. Observe the separation of NHCR-related sequences and products to evaluate whether the system can be successfully used for Ade detection. The results are shown in Figure 2 shown.

[0065] The results showed that Lane 6 and 7 were the NHCR products of adding AT1+AT2 and AT1+AT2+Ade, respectively. Lane 6 and 7 showed that when Ade was added, a band with lower mobility appeared near the sample well, indicating that in the presence of Ade, NHCR could be successfully induced to form a dendritic DNA structure with a larger molecular weight, indicating that the NHCR strategy has the potential to amplify the Ade detection signal and can be used to detect Ade.

[0066] 1.6 Fluorescence spectrophotometer analysis to verify the feasibility of NHCR detection of Ade

[0067] As shown in Table 5, mixture groups containing different NHCR sequence compositions were set up, (1) and (2) were SA and SB solutions, respectively; (3) was an A+B mixed solution; (4) and (5) were mixed solutions with only one recognition probe, respectively; (6) was an NHCR system without Ade; and (7) was an NHCR system with Ade added. These mixtures were placed at 37°C for 60 min and then detected by a fluorescence spectrophotometer. ex is 490nm, λ emThe fluorescence intensity of each mixture was measured under the conditions of a wavelength of 518 nm, a slit width of 5 nm, and a voltage of 700 V. The changes in fluorescence intensity of solutions containing different samples were analyzed by a fluorescence spectrophotometer to verify the feasibility of the NHCR strategy in detecting Ade.

[0068] Table 5 Composition and volume of different NHCR mixtures

[0069]

[0070] Because when NHCR does not occur, since the double-stranded substrates SA and SB maintain a double-stranded structure, the fluorescent group and the quenching group are close to each other and no fluorescent signal is generated. When NHCR occurs, the double strand opens, and the DNA self-assembles to form a fluorescent tree, which will carry a large amount of fluorescent signal. Therefore, the feasibility of quantitative detection of Ade by the NHCR strategy can be verified based on the changes in fluorescence intensity of different groups.

[0071] The results are as follows Figure 3 As shown in the figure, the fluorescence intensity after adding Ade was significantly stronger than that of the group with only the double-split aptamer probe, indicating that the NHCR strategy is feasible for quantitative detection of Ade. In addition, the results that the fluorescence signal of the NHCR mixture with only AT1 and AT2 added is similar to the signal generated by the NHCR mixed solution alone also provide key experimental verification, indicating that NHCR does require a complete system to occur. The results of this experiment will provide strong support for further verification of the feasibility of the NHCR strategy in Ade detection. In addition, the SA solution and the SB solution basically do not produce fluorescent signals, which shows that preservation in the form of a double-stranded structure is stable and reliable.

[0072] Example 2

[0073] Optimization of experimental conditions

[0074] 2.1 Optimization of the number of AA base pairs and NHCR reaction time

[0075] Due to the self-assembly phenomenon of the NHCR system, the reaction signal will be leaked and fluorescence will be emitted. The length of AA is crucial to the NHCR self-assembly phenomenon. If the length of the chain is too long, the reaction time will be prolonged, and even the primer will not be able to cause the NHCR reaction. At the same time, if the length of the auxiliary chain is too short, the background signal will be too high and false positive results will be caused. In addition, the NHCR reaction time will affect the amplification efficiency of the method. Therefore, the length of the AA chain is adjusted and the reaction time is optimized to solve the above problems. On the basis of the original, it is reduced by 1-4nt to obtain four chains of AA-1, AA-2, AA-3, and AA-4. According to the group, the NHCR A and NHCR B reaction solutions of different groups are synthesized, the same volume of buffer solution is added, the two recognition probes are mixed in the same volume (2μL), 2μL Ade solution (1ng / mL) is added, and then mixed with the NHCR system of different groups, and reacted at 37℃ for 0min, 15min, 30min, 45min, and 60min, respectively. The fluorescence is detected by fluorescence spectrophotometer, and the fluorescence difference between the experimental group and the control group is compared to select the optimal number of probes and the optimal reaction time.

[0076] like Figure 4 As shown in the figure, AA-1 caused the largest F-F0. In addition, as time went on, AT1 and AT2 contacted each other in the solution and could induce partial NHCR, and F-F0 became smaller. Therefore, the reaction time was optimal at 30 min, and the F-F0 difference was the largest.

[0077] 2.2 Optimization of AT1 and AT2 concentration ratio

[0078] The two recognition probes will form a sandwich structure in the presence of Ade. At the same time, since each aptamer is connected to a part of the initiator, as the reaction time increases, it may cause partial NHCR reaction, affecting the detection sensitivity of the reaction. Therefore, we optimized the concentration ratio of AT1 and AT2 to investigate the effect of the concentration ratio on the fluorescence intensity. The experiment was divided into 4 groups, and AT1 / AT2 reaction solutions with concentration ratios of 0.5μM:0.5μM, 0.5μM:1μM, 1μM:1μM, and 1μM:0.5μM were added to each ep tube. 2μL Ade solution (1ng / mL) and NHCR system were added, mixed evenly, and the ep tube was placed in a rotating mixer for reaction at 37℃ for 30min, and then fluorescence detection was performed using a fluorescence spectrophotometer.

[0079] like Figure 5 The results were consistent with the theory. When the concentration ratio of AT1 to AT2 was 1, the background intensity was lower than that of other groups, and when the concentrations of both were 1 μM, the fluorescence difference between the experimental group and the control group was the largest. Therefore, AT1:AT2=1 μM:1 μM was selected as the optimal reaction concentration ratio.

[0080] 2.3NHCR detection system quantitative detection of Ade

[0081] When performing sensitivity analysis on the immune biosensor, it is first necessary to determine the analytical sensitivity or detection limit of the detection system of the strategy. This parameter is usually determined experimentally and is defined as the lowest analyte concentration that the system can reliably detect. A series of samples of concentration (0, 0.01, 0.1, 1, 10, 100 ng / mL) were prepared using the Ade standard. Standard samples of different concentrations were added to the immunoassay system for detection, and the fluorescence intensity of the corresponding concentration was finally measured using a fluorescence spectrophotometer. The fluorescence intensity was matched to the concentration of the Ade standard sample, and the standard curve was fitted to investigate the analytical performance of the immunoassay strategy.

[0082] like Figure 6 The experimental results shown in the figure show that within the concentration range of 0.01-100 ng / mL, the fluorescence intensity is linearly related to the Ade concentration, the linear regression equation is y=499.77x-323.9, and the LOD is 5.23 pg / mL. These data verify the sensitivity of the NHCR technology based on the double-split aptamer in the quantitative detection of Ade.

[0083] Table 6 shows the recently developed methods for detecting Ade. Compared with them, the method developed in this paper has obvious sensitivity advantages and a response time of only 30 minutes, which further highlights the practicality of this method and can be used for Ade instant detection.

[0084] Table 6 Comparison of Ade detection methods

[0085]

[0086] The experimental operation of the electrochemical sensor based on PdCu@MWCNTs dual enzyme label refers to the prior art LAUHON CT, SZOSTAK J W. RNA aptamers that bind flavin and nicotinamide redox cofactors [J]. Journal of the American Chemical Society, 1995, 117 (4): 1246-1257.

[0087] The experimental operation of the dual-function electrochemical aptamer sensor based on AuNPs coated ERGO nanosheets refers to the existing technology KLUG SJ, FAMULOK M. All you wanted to know about SELEX [J]. Molecular Biology Reports, 1994, 20 (2): 97-107.

[0088] The experimental operation of carbon quantum dot catalytic functionalized aptamer luminescent biosensor refers to the prior art CRAMERIA, STEMMER W P. 10(20)-fold aptamer library amplification without gel purification [J]. Nucleic Acids Research, 1993, 21(18): 10-19.

[0089] The experimental operation of chemiluminescent biosensing based on targeted responsive DNA hydrogel refers to the prior art KANAGAWAT. Bias and artifacts in multitemplate polymerase chain reactions (PCR) [J]. Journal of Bioscience and Bioengineering, 2003, 96 (4): 317-323.

[0090] The experimental operation of the separation aptamer labeling ratio fluorescence biosensor refers to the existing technology DU Z, BEHRENS SF. Effect of target gene sequence evenness and dominance on real-time PCR quantification of artificial sulfate-reducing microbial communities[J]. The Public Library of Science, 2024, 19(3): e0299930.

[0091] Experimental operation of the chaperone copolymer accelerated hybridization non-enzyme signal amplification strategy refers to the existing technology ZUKER M. Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction [J]. Nucleic Acids Research, 2003, 31 (13): 3406-3415.

[0092] The double-split aptamer-mediated NHCR method is the method of the present invention.

[0093] 2.4 Selectivity analysis

[0094] For aptamer sensors, the detection specificity is determined by the natural properties of the aptamer. It is reported that the aptamer we used can bind to Ade derivatives such as ATP, ADP, AMP, but not to other nucleosides. Therefore, in order to evaluate the selectivity, three Ade analogs, uridine, guanosine and cytidine, were selected as the control group to evaluate the selectivity of the NHCR detection system. The concentration of Ade was 1 ng / mL, and the concentration of the other three analogs was 10 ng / mL. Under the optimal experimental conditions, this immunoassay strategy was used to detect Ade and Ade analogs to study the selectivity of the method.

[0095] The results are as follows Figure 7 As shown, the results showed that the fluorescence intensity generated by the three Ade analogs was much smaller than that of Ade, indicating that this dual-split aptamer-based assay has excellent selectivity for the target Ade and has the ability to accurately detect Ade in biological samples.

[0096] 2.5 Detection of Ade in urine

[0097] To demonstrate the applicability of the proposed probe for Ade detection in actual samples, the recovery of the probe for Ade in human urine samples was examined. Healthy urine was first selected as a blank to draw a working curve. Ade was then added to the blank urine and diluted 100 times with buffer to obtain clinical sample solutions with final concentrations of 0, 0.5, 1, 3, 5, 8, and 10 ng / mL. A linear relationship between fluorescence intensity and Ade concentration (0-10 ng / mL) was obtained. Urine samples with three spiked concentrations of Ade (1, 5, and 10 ng / mL) were measured, the measurement results were quantified using the working curve, and the recovery was calculated.

[0098] The results are as follows Figure 8 And as shown in Table 7.

[0099] Table 7 Determination of Ade in urine samples using this method

[0100]

[0101] The results showed that there was a good linear relationship between the fluorescence intensity and the concentration of Ade in urine. In order to test the applicability of Ade detection, we performed a recovery study by adding different concentrations of Ade (1, 5, and 10 ng / mL) to diluted urine samples. The recovery of Ade in diluted urine samples was 93.0% to 97.4%, and the relative standard deviation (RSD) was 3.33% to 9.12%. These data indicate that the sensor has good anti-interference and detection performance in complex environments.

[0102] It should be noted that, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A double-split aptamer probe, characterized in that: The invention comprises two split aptamers, namely split aptamer 1 and split aptamer 2. Split aptamer 1 comprises aptamer fragment 1 and split initiator 1, and split aptamer 2 comprises aptamer fragment 2 and split initiator 2. The two aptamer fragments are from a complete aptamer sequence A. The aptamer fragment 1 and the aptamer fragment 2 have the ability to recognize the target, and the split initiator 1 and the split initiator 2 have the function of initiating NHCR.

2. The double-split aptamer probe according to claim 1, characterized in that: The molar ratio of the split aptamer 1 to the split aptamer 2 is 1-2:1-2.

3. The double-split aptamer probe according to claim 1, characterized in that: The sequence of the aptamer fragment 2 is such as ACCTGGGGGAGTAT; the sequence of the cleavage initiator 2 is GATCAAGCAGT; the sequence of the aptamer fragment 1 is such as TGCGGAGGAAGGT; the sequence of the cleavage initiator 1 is GTCGAAGTAGTT; The sequence of aptamer sequence A is ACCTGGGGGAGTATTGCGGAGGAAGGT.

4. Use of the dual-split aptamer probe according to claims 1-3 in preparing a reagent or a kit for detecting adenosine or adenosine derivatives.

5. Use of the dual-split aptamer probe according to claims 1-3 in the preparation of a reagent or a kit for detecting colorectal cancer, prostate cancer or lung cancer.

6. A reagent or kit for detecting adenosine or adenosine derivatives, characterized in that: Each test volume includes 10μL AT1 solution, 10μL AT2 solution, 16.5μL buffer, 1μL FA solution, 1μL FB solution, 1.5μL QA solution, 3μL QB solution, 2μL AA solution and 4μL AB solution; the sequence of the AT1 is shown in SEQ ID NO.1, the sequence of the AT2 is shown in SEQ ID NO.2, the sequence of the FA is shown in SEQ ID NO.3, the sequence of the FB is shown in SEQ ID NO.4, the sequence of the QA is shown in SEQ ID NO.5, the sequence of the QB is shown in SEQ ID NO.6, the sequence of the AA is shown in SEQ ID NO.7-11, and the sequence of the AB is shown in SEQ ID NO.

12.

7. A reagent or kit for detecting colorectal cancer, prostate cancer or lung cancer, characterized in that: Each detection amount includes 10μL AT1 solution, 10μL AT2 solution, 16.5μL buffer, 1μL FA solution, 1μL FB solution, 1.5μL QA solution, 3μL QB solution, 2μL AA solution and 4μL AB solution; the sequence of the AT1 is shown in SEQ ID NO.1, the sequence of the AT2 is shown in SEQ ID NO.2, the sequence of the FA is shown in SEQ ID NO.3, the sequence of the FB is shown in SEQ ID NO.4, the sequence of the QA is shown in SEQ ID NO.5, the sequence of the QB is shown in SEQ ID NO.6, the sequence of the AA is shown in SEQ ID NO.7-11, and the sequence of the AB is shown in SEQ ID NO.

12.

8. The kit according to claim 6 or 7, characterized in that The sequence of the AA is shown in SEQ ID NO.8.

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