Nucleic acid aptamer for recognizing sarcosine and application of nucleic acid aptamer

Abstract

The invention belongs to the field of medical detection, and discloses a nucleic acid aptamer for recognizing sarcosine and application of the nucleic acid aptamer. The aptamer is obtained by constructing an ssDNA initial library containing fixed primers at the two ends and 36 random basic groups in the middle, repeated incubation and screening are conducted through positive screening magnetic beads and negative screening magnetic beads, the positive screening magnetic beads are formed by coupling carboxyl functionalized magnetic beads with sarcosine, and the negative screening magnetic beads are formed by coupling carboxyl functionalized magnetic beads with glycine and alanine. Based on the nucleic acid aptamer, the invention also provides a nucleic acid aptamer-magnetic bead chemiluminescence kit, the kit comprises a reagent 1 and a reagent 2, the reagent 1 comprises a streptavidin magnetic bead coupled nucleic acid aptamer, and the reagent 2 comprises an acridinium ester labeled nucleic acid aptamer. The nucleic acid aptamer is high in stability and specificity, the detection method is easy and convenient to operate, the cost is low, detection is rapid, the nucleic acid aptamer is suitable for efficient detection of sarcosine in complex biological samples, and the nucleic acid aptamer has important application value in the fields of disease diagnosis and biomarker analysis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical detection, and particularly relates to a nucleic acid aptamer for recognizing sarcosine and application thereof. BACKGROUND

[0002] Sarcosine, as an important nitrogen-containing compound in the human body, plays a key role in muscle energy metabolism, neurotransmitter synthesis and cell oxidative stress regulation. Abnormal sarcosine concentration is closely related to many diseases such as prostate cancer, and its accurate detection in biological samples such as urine and blood is of great significance for early diagnosis, efficacy evaluation and prognosis of diseases. However, traditional detection methods such as spectrophotometry, capillary electrophoresis, fluorescence spectroscopy and chromatography-mass spectrometry have defects such as expensive instruments, complicated sample pretreatment, long detection period and high professional requirements for operation, which are difficult to meet the needs of large-scale rapid screening in clinical practice. Therefore, it is very important to design a rapid, sensitive and reliable diagnostic method to realize the detection of sarcosine in complex environment.

[0003] Nucleic acid aptamer is a single-stranded oligonucleotide obtained by screening with the method of systematic evolution of ligands by exponential enrichment, which can specifically bind to target molecules by folding to form a specific spatial structure. Compared with traditional antibodies, it has the advantages of high chemical stability, low synthesis cost and flexible modification, especially in the detection of small molecule compounds, which overcomes the bottleneck of low affinity and long preparation period of antibodies. However, there are still challenges in the screening of high-specificity nucleic acid aptamer for sarcosine and the detection technology based on aptamer: the existing screening method is easily interfered by structural analogues, resulting in insufficient specificity of aptamer; the detection platform mainly depends on fluorescence or electrochemical methods, which has limited sensitivity in complex biological matrix.

[0004] Therefore, it is urgent to develop a method for efficiently screening high-affinity sarcosine nucleic acid aptamer, and to construct a detection system with simple operation and high sensitivity, so as to realize the rapid, sensitive and stable detection of sarcosine content in complex sample environment, which has an urgent practical need to improve the clinical application value of sarcosine detection. SUMMARY

[0005] The application aims to provide a nucleic acid aptamer for efficiently recognizing sarcosine and application thereof.

[0006] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows: The nucleic acid aptamer for recognizing sarcosine comprises Sar-1 and Sar-8. Preferably, the Sar-1 is 5'-ATTGGCACTCCACGCATAGGGTCCGAGGTCGTAGGTTCTAAGGGCGCACTCCCATTCCTATGCGTGCTACCGTGAA-3', and the nucleotide sequence is shown as SEQ ID No. 1. Preferably, the Sar-8 is 5'-ATTGGCACTCCACGCATAGGGCTCCGAGGTCGTAGGTCAAGGTAGGGTTTGCATCCCCTATGCGTGCTACCGTGAA-3', the nucleotide sequence of which is shown as SEQ ID No. 2.

[0007] A screening method for identifying a nucleic acid aptamer of sarcosine, comprising: constructing an ssDNA initial library; repeatedly incubating and screening by positive screening magnetic beads and reverse screening magnetic beads, and taking the secondary library obtained in the previous operation as the starting nucleic acid library for each screening.

[0008] Preferably, the positive screening magnetic beads are carboxyl functionalized magnetic beads coupled with sarcosine.

[0009] Preferably, the reverse screening magnetic beads are carboxyl functionalized magnetic beads coupled with glycine and alanine.

[0010] Preferably, the ssDNA initial library is 5'-ATCCAGAGTGACGCAGCA-N36-TGGACACGGTGGCTTAGT-3', shown as SEQ ID No. 3, which is composed of a 20-base-length primer fragment fixed at both ends and a 36-base-length random fragment in the middle.

[0011] Preferably, the carboxyl functionalized magnetic beads comprise polystyrene modified nano-ferroferric oxide; or, 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane co-modified silicon-based functionalized magnetic beads, which comprise nano-ferroferric oxide modified by tetraethyl orthosilicate.

[0012] Preferably, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester and 3-aminopropyl trimethoxysilane is 5.75-57.5 g:10 mL, and the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane is 6.5-65 g:10 mL.

[0013] The silicon-based functionalized magnetic beads formed by nano-ferroferric oxide modified by tetraethyl orthosilicate have silicon hydroxyl groups on the surface, and the silicon-based functionalized magnetic beads are co-modified after the reaction of 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane, and carboxyl groups are formed after hydrolysis, thereby forming a composite functional layer through carboxyl groups with different spatial structures, improving the diversity and uniformity of active sites on the surface of the magnetic beads, adjusting the polarity of the surface of the magnetic beads, improving the coupling efficiency of the magnetic beads to sarcosine, and reducing non-specific adsorption.

[0014] Preferably, the mass-volume ratio of the silicon hydroxyl functionalized magnetic beads and 3-aminopropyl trimethoxysilane is 1-10 g:10 mL.

[0015] More preferably, 3-(trihydroxysilyl)-propane sulfonic acid can also be added in the modification of the silicon-based functionalized magnetic beads, and the mass ratio of 3-(trihydroxysilyl)-propane sulfonic acid to the silicon-hydroxyl functionalized magnetic beads is 2.5-25:2. 3-(trihydroxysilyl)-propane sulfonic acid can improve hydrophilicity, improve the dispersibility of the magnetic beads, and improve the intermolecular contact efficiency in the screening process; improve the negative charge density on the surface of the magnetic beads, and improve the screening specificity.

[0016] A nucleic acid aptamer-magnetic bead chemiluminescence kit, the chemiluminescence kit comprising reagent 1 and reagent 2, the reagent 1 comprising magnetic bead coupled nucleic acid aptamer, the reagent 2 comprising chemiluminescence label coupled nucleic acid aptamer, the nucleic acid aptamer comprising Sar-1 and Sar-8, the magnetic bead being modified with streptavidin, and the chemiluminescence label being acridinium ester.

[0017] A preparation method of a nucleic acid aptamer-magnetic bead chemiluminescence kit, comprising: coupling the nucleic acid aptamer with streptavidin modified magnetic beads to prepare reagent 1 after the modification of the magnetic beads with streptavidin, and diluting the reagent 1 with magnetic bead blocking solution; coupling acridinium ester with the nucleic acid aptamer to prepare reagent 2 after the coupling, and diluting the reagent 2 with acridinium ester buffer solution; the chemiluminescence detection kit comprising the reagent 1 and the reagent 2, and the nucleic acid aptamer comprising Sar-1 and Sar-8.

[0018] Preferably, the magnetic bead blocking solution comprises 0.5-1wt% of Tris, 0.5-2wt% of BSA, 0.1-0.2wt% of Tween20, and 96.8-98.9wt% of deionized water.

[0019] Preferably, the acridinium ester buffer solution comprises 1-2wt% of NaHPO4·12H2O, 0.1-0.5wt% of NaH2PO4·2H2O, 0.5-1.5wt% of NaCl, 0.2-1wt% of BSA, 0.02-0.1wt% of MgCl2·6H2O, 0.04-0.2wt% of Tween20, 0.04-0.2wt% of Proclin 300, and 94.5-98.1wt% of deionized water.

[0020] The application further provides a preparation method of positive screening magnetic beads, comprising: Sarcosine is dissolved in PBS buffer, 5-15 mmol / L sodium acetate solution is added to obtain a sarcosine mixed solution; carboxyl functionalized magnetic beads are dispersed in deionized water, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, the carboxyl groups on the surface of the magnetic beads are activated by incubation at room temperature for 10-30 min, the magnetic beads are washed with DPBS buffer for 1-2 times, the sarcosine mixed solution is added, and the magnetic beads are incubated at room temperature for 1-3 h, then the supernatant is discarded, the magnetic beads are washed with DPBS buffer for 1-2 times, 0.5-1.5 mmol / L ethanolamine solution is added, and the magnetic beads are incubated at room temperature for 10-20 min to block the unreacted active sites on the surface of the magnetic beads, the blocking solution is discarded, the magnetic beads are washed with DPBS buffer for 3-5 times, and a positive screening magnetic bead is obtained.

[0021] Preferably, the molar volume ratio of sarcosine to PBS buffer is 0.5-5 µmoL:100 mL.

[0022] Preferably, the volume ratio of sodium acetate solution to PBS buffer is 50-500 µL:100 mL.

[0023] Preferably, the carboxyl functionalized magnetic beads are polystyrene modified nanometer ferroferric oxide, and the particle size of the carboxyl functionalized magnetic beads is 0.8-1.2 µm.

[0024] Preferably, the mass volume ratio of carboxyl functionalized magnetic beads to deionized water is 5-50 mg:1 mL.

[0025] Preferably, the mass ratio of N-hydroxysuccinimide to carboxyl functionalized magnetic beads is 1-10:10.

[0026] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl functionalized magnetic beads is 2.5-25:10.

[0027] Preferably, the molar mass ratio of sarcosine to carboxyl functionalized magnetic beads is 0.5-5 µmoL:10 mg.

[0028] Preferably, the volume mass ratio of ethanolamine solution to carboxyl functionalized magnetic beads is 0.5-5 mL:10 mg.

[0029] The application also provides a preparation method of a negative screening magnetic bead, comprising: Glycine and alanine are dissolved in PBS buffer, 5-15 mmol / L sodium acetate solution is added to obtain a glycine mixed solution; carboxyl functionalized magnetic beads are dispersed in deionized water, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride are added, the carboxyl on the surface of the magnetic beads is activated by incubation at room temperature for 10-30 min, the activated sites on the surface of the magnetic beads are blocked by washing with DPBS buffer for 1-2 times, adding sarcosine mixed solution, incubating on a shaking bed at room temperature for 1-3 h, discarding the supernatant, washing with DPBS buffer for 1-2 times, adding ethanolamine solution, incubating at room temperature for 10-20 min, and obtaining the reverse screening magnetic beads.

[0030] Preferably, the molar volume ratio of glycine to PBS buffer is 5-50 µmoL:100 mL.

[0031] Preferably, the molar volume ratio of alanine to PBS buffer is 5-50 µmoL:100 mL.

[0032] Preferably, the volume ratio of sodium acetate solution to PBS buffer is 50-500 µL:100 mL.

[0033] Preferably, the carboxyl functionalized magnetic beads are polystyrene modified nanometer ferroferric oxide, and the particle size of the carboxyl functionalized magnetic beads is 0.8-1.2 µm.

[0034] Preferably, the mass volume ratio of carboxyl functionalized magnetic beads to deionized water is 5-50 mg:1 mL.

[0035] Preferably, the mass ratio of N-hydroxysuccinimide to carboxyl functionalized magnetic beads is 1-10:10.

[0036] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to carboxyl functionalized magnetic beads is 2.5-25:10.

[0037] Preferably, the molar mass ratio of glycine to carboxyl functionalized magnetic beads is 0.5-5 µmoL:10 mg.

[0038] Preferably, the volume mass ratio of ethanolamine solution to carboxyl functionalized magnetic beads is 0.5-5 mL:10 mg.

[0039] The application also provides a construction method of a screening library, comprising: The ssDNA initial library used in the application has a full length of 76 bases, is composed of primer fragments of 20 base lengths at two ends and random fragments of 36 bases in the middle, and is synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and the primer is synthesized by Jinweizhi Biotechnology Co., Ltd.; the ssDNA initial library, the forward primer used for constructing the library, the reverse primer used for constructing the library, the biotin-labeled library, the poly-A library and the FAM-labeled library are used in the application.

[0040] Preferably, the ssDNA initial library is 5'-ATCCAGAGTGACGCAGCA-N36-TGGACACGGTGGCTTAGT-3', the nucleotide sequence is shown as SEQ ID No. 3, and N36 is a sequence of 36 arbitrary nucleotide bases connected.

[0041] Preferably, the forward primer used for constructing the library is 5'-ATTGGCACTCCACGCATAGG-3', the nucleotide sequence is shown as SEQ ID No. 4.

[0042] Preferably, the reverse primer used for constructing the library is 5'-TTCACGGTAGCACGCATA-3', the nucleotide sequence is shown as SEQ ID No. 5.

[0043] Preferably, the biotin-labeled library is 5'-GCGTGGAGTGCCAAT-Biotin-3', the nucleotide sequence is shown as SEQ ID No. 6.

[0044] Preferably, the poly-A library is 5'-AAAAAAAAAAAAAAAAAAAATTCACGGTAGCACGCATA-3', the nucleotide sequence is shown as SEQ ID No. 7.

[0045] Preferably, the FAM-labeled library is 5'-FAM-ATTGGCACTCCACGCATAGG-3', the nucleotide sequence is shown as SEQ ID No. 8.

[0046] The application further provides a screening method for a nucleic acid aptamer for recognizing sarcosine, comprising the following steps: S1, taking 0.5-1.5 OD random ssDNA initial library, centrifuging at 10000-15000 rpm for 1-3 min, adding DPBS buffer and 50-150 μmol / L Lib-biotin primer, vortexing for 2-5 min, performing PCR amplification, diluting the hybridization library to 0.5-1.5 mL with a diluent, and obtaining a pretreated ssDNA library.

[0047] Preferably, the volume ratio of the ssDNA initial library to the DPBS buffer is 1:10-50.

[0048] Preferably, the volume ratio of the ssDNA initial library to the Lib-biotin primer is 5:15-40.

[0049] Preferably, the diluent is the DPBS buffer.

[0050] Preferably, the PCR reaction program comprises denaturation at 94-96℃ for 9-11 min, annealing at 59-61℃ for 59-61 min, and extension at 24-26℃ for 19-21 min.

[0051] Preferably, in the subsequent screening rounds, the volume ratio of the ssDNA initial library to the DPBS buffer is 0.3:10-50.

[0052] Preferably, in the subsequent screening rounds, the volume ratio of the ssDNA initial library to the Lib-biotin primer is 1:0.5-3.

[0053] S2, add herring sperm DNA, BSA and sodium chloride in the PBS buffer, mix to obtain a mixed buffer, disperse the positive screening magnetic beads in the mixed buffer, add the pretreated ssDNA library, incubate on a shaker at room temperature for 60-100 min, collect the supernatant after magnetic separation, add double distilled water, 94-96℃ metal bath for 8-12 min, ice bath for 1-3 min, collect the supernatant after magnetic separation, and obtain the initial positive screening ssDNA library.

[0054] Preferably, the mass-volume ratio of the herring sperm DNA to the PBS buffer is 1 μg:5-20 μL.

[0055] Preferably, the mass-volume ratio of the BSA to the PBS buffer is 1 μg:0.5-3 μL.

[0056] Preferably, the molar-volume ratio of the sodium chloride to the PBS buffer is 1 μg:0.5-3 μL.

[0057] Preferably, the mass-volume ratio of the positive screening magnetic beads to the mixed buffer is 1 mg:20-60 μL.

[0058] Preferably, the volume ratio of the pretreated ssDNA library to the mixed buffer is 1:20-60.

[0059] Preferably, the mass-volume ratio of the positive screening magnetic beads to the double distilled water is 1 mg:10-20 μL.

[0060] S3, repeat the steps of S1 and S2 for 8-12 rounds to perform subsequent screening, and obtain the positive screening ssDNA library.

[0061] S4, adding herring sperm DNA, BSA and sodium chloride in PBS buffer, mixing to obtain a mixed buffer, dispersing the negative screening magnetic beads in the mixed buffer, adding the positive screening ssDNA library, incubating on a shaker at room temperature for 20-40 min, magnetically separating to discard the supernatant, adding 50-150 μmol / L sarcosine solution, incubating on a shaker at room temperature for 40-80 min, magnetically separating to collect the supernatant, transferring to a dialysis bag, dialyzing in the dialysis bag with DPBS buffer at 2-5°C for 10-15 h to obtain the eluate after dialysis, adding the eluate to the e-PCR system, mixing with the e-PCR microdroplet generation oil on a vortex for 3-7 min to form an emulsion, performing e-PCR amplification, adding an equal volume of n-butanol after amplification, vortexing for 8-12 min, centrifuging at 5000-7000 rpm for 20-40 min, and aspirating the bottom aqueous phase to obtain the negative screening dsDNA.

[0062] Preferably, 0.5-2 mL of the e-PCR system comprises 50-200 μL of the eluate, 250-1000 μL of 1-3x TaqPCR Master Mix, 25-100 μL of 50-150 μM of Lib-polyA, 25-100 μL of 50-150 μM of Lib-FAM, and 150-600 μL of double-distilled water.

[0063] Preferably, the e-PCR amplification program comprises 94-96°C pre-denaturation for 2-4 min, 94-96°C denaturation for 55-65 s, 59-61°C annealing for 55-65 s, 71-73°C extension for 55-65 s, 12-18 cycles, and 71-73°C final extension for 4-6 min.

[0064] Preferably, the mass-volume ratio of herring sperm DNA to PBS buffer is 1 μg:5-20 μL.

[0065] Preferably, the mass-volume ratio of BSA to PBS buffer is 1 μg:0.5-3 μL.

[0066] Preferably, the molar-volume ratio of sodium chloride to PBS buffer is 1 μg:0.5-3 μL.

[0067] Preferably, the mass-volume ratio of the negative screening magnetic beads to the mixed buffer is 1 mg:20-80 μL.

[0068] Preferably, the volume-mass ratio of the initial positive screening ssDNA library to the mixed buffer is 1:20-80.

[0069] Preferably, the volume-mass ratio of the sarcosine solution to the negative screening magnetic beads is 1 mg:20-80 μL.

[0070] Preferably, the molecular weight cut-off of the dialysis bag is 3-4 KD.

[0071] S5, adding loading buffer to the anti-screen dsDNA, 94-96℃ metal bath for 4-6 min, then ice bath for 1-3 min, loading to Urea-PAGE gel, 100-140V constant voltage electrophoresis for 15-25 min; cutting the target fluorescent band under the ultraviolet transmission instrument, adding double deionized water, heating at 92-98℃ for 2-8 min, ice bath for 1-3 min, then centrifugation to obtain supernatant, repeating 1-2 times to combine the supernatant, concentrating with n-butanol, dialysis at 2-5℃ for 3-5h, to obtain the anti-screen ssDNA library.

[0072] S6, incubating, eluting, amplifying and purifying the anti-screen ssDNA library according to the steps of S2-S5 to obtain the positive screening ssDNA library for the next round of screening.

[0073] S7, after each round of screening, taking 1-2μL of the ssDNA library as a template for quantification using Q-PCR reaction system, calculating the nucleic acid concentration, and analyzing the melting curve; repeating the steps of S3-S5 for 10-13 rounds, and high-throughput sequencing the final library to obtain the nucleic acid aptamer recognizing sarcosine.

[0074] Preferably, the main peak Tm value of the melting curve is 83-87℃ for specific qualification.

[0075] Preferably, the screening is terminated when the Ct value changes ≤1.5 for 3 consecutive rounds and the melting curve is single.

[0076] Preferably, the nucleic acid aptamer includes Sar-1 and Sar-8.

[0077] Preferably, Sar-1 is 5'-ATTGGCACTCCACGCATAGGGTCCGAGGTCGTAGGTTCTAAGGGCGCACTCCCATTCCTATGCGTGCTACCGTGAA-3', and the nucleotide sequence is shown as SEQ ID No. 1.

[0078] Preferably, Sar-8 is 5'-ATTGGCACTCCACGCATAGGGCTCCGAGGTCGTAGGTCAAGGTAGGGTTTGCATCCCCTATGCGTGCTACCGTGAA-3', and the nucleotide sequence is shown as SEQ ID No. 2.

[0079] Preferably, 10-40μL of the Q-PCR reaction system includes 5-20μL of 1-3×Taq Q-PCR Mix, 0.5-2μL of 10-40μmol / L of Lib-F, 0.5-2μL of 10-40μmol / L of Lib-R, and 3.4-13.2μL of ddH2O.

[0080] The application further provides a preparation method of the streptavidin magnetic bead coupled nucleic acid aptamer, comprising the following steps: dispersing the nucleic acid aptamer in 5-15 mmol / L Tris hydrochloride buffer to obtain a nucleic acid aptamer solution; dispersing magnetic beads in deionized water, adding the nucleic acid aptamer solution, uniformly mixing at room temperature for 20-40 min, performing magnetic separation for 1-3 min, discarding the supernatant, adding 400-600 μL of magnetic bead blocking liquid for blocking for 20-40 min, and cleaning the magnetic beads with 400-600 μL of magnetic bead blocking liquid for 2-4 times to obtain the streptavidin magnetic bead coupled nucleic acid aptamer.

[0081] Preferably, the volume ratio of the nucleic acid aptamer to the Tris hydrochloride buffer is 5-50: 50-500.

[0082] Preferably, the mass-volume ratio of the magnetic beads to the deionized water is 2.5-25 mg: 250-2500 μL.

[0083] Preferably, the mass-volume ratio of the magnetic beads to the nucleic acid aptamer solution is 2.5-25 mg: 2.5-30 μL.

[0084] Preferably, the magnetic bead blocking liquid comprises 0.5-1 wt% of Tris, 0.5-2 wt% of BSA, 0.1-0.2 wt% of Tween 20 and 96.8-98.9 wt% of deionized water.

[0085] The application further provides a preparation method of the acridinium ester labeled nucleic acid aptamer, comprising the following steps: adding the nucleic acid aptamer and PBS buffer into 3-5 mmol / L acridinium ester solution, performing constant temperature oscillation bath reaction under light shielding conditions for 20-40 min, adding 90-110 mmol / L lysine solution, and neutralizing the excess acridinium ester for 20-40 min to obtain the acridinium ester labeled nucleic acid aptamer.

[0086] Preferably, the volume ratio of the acridinium ester solution to the nucleic acid aptamer is 1.5-17: 12-150.

[0087] Preferably, the volume ratio of the acridinium ester solution to the PBS buffer is 1.5-17: 70-850.

[0088] Preferably, the volume ratio of the acridinium ester solution to the lysine is 1.5-1: 1.5-17.

[0089] The application further provides a preparation method of the nucleic acid aptamer-magnetic bead chemiluminescence kit, comprising the following steps: The streptavidin magnetic beads coupled nucleic acid aptamer is diluted to the concentration of 0.5 mg / mL of the nucleic acid aptamer by using the magnetic bead blocking solution, and is mixed by using a three-dimensional rotary instrument for 5-15 min to obtain reagent 1; the acridinium ester labeled nucleic acid aptamer is diluted to the concentration of 0.1-0.2 µg / mL of the nucleic acid aptamer by using the acridinium ester buffer, and is mixed by using a three-dimensional rotary instrument for 5-15 min to obtain reagent 2.

[0090] Preferably, the composition of the acridinium ester buffer is: 1-2 wt% of NaHPO4·12H2O, 0.1-0.5 wt% of NaH2PO4·2H2O, 0.5-1.5 wt% of NaCl, 0.2-1 wt% of BSA, 0.02-0.1 wt% of MgCl2·6H2O, 0.04-0.2 wt% of Tween20, 0.04-0.2 wt% of Proclin 300, and 94.5-98.1 wt% of deionized water.

[0091] The application further provides a preparation method of the carboxyl functionalized magnetic beads, comprising: Preparation of the nano four-iron oxide: (NH4)2Fe(SO4)2·6H2O and FeCl3·6H2O are dissolved in deionized water, stirred and dissolved under the protection of nitrogen and at 55-65 ℃, the pH is adjusted to 10.9-11.1, and the solution is statically aged for 1-3 h at 75-85 ℃, and then is cooled to room temperature, subjected to magnetic separation, washed with anhydrous ethanol and deionized water alternately until the pH of the washing solution is 6.9-7.1, and vacuum dried at 55-65 ℃ for 10-15 h to obtain the nano four-iron oxide.

[0092] Preferably, the mass-volume ratio of (NH4)2Fe(SO4)2·6H2O to deionized water is 1.9-19 g:100 mL.

[0093] Preferably, the mass-volume ratio of FeCl3·6H2O to deionized water is 2.3-23 g:100 mL.

[0094] Preferably, the stirring speed is 200-400 rpm.

[0095] Preparation of the silicon hydroxyl functionalized magnetic beads: the nano four-iron oxide is dispersed in anhydrous ethanol, deionized water is added, ultrasonic dispersion is performed for 20-40 min, ammonia water is added, stirring is performed for 5-15 min, tetraethyl orthosilicate is added, reaction is performed at 25-35 ℃ for 8-12 h, and then the solution is cooled to room temperature, subjected to magnetic separation, washed with anhydrous ethanol and deionized water alternately until the pH of the washing solution is 6.9-7.1, and vacuum dried at 55-65 ℃ for 10-15 h to obtain the silicon hydroxyl functionalized magnetic beads.

[0096] Preferably, the mass-volume ratio of the nanometer-sized ferriferrous oxide and the anhydrous ethanol is 1-10 g: 1200 mL.

[0097] Preferably, the mass-volume ratio of the nanometer-sized ferriferrous oxide and the deionized water is 1-10 g: 400 mL.

[0098] Preferably, the mass-volume ratio of the nanometer-sized ferriferrous oxide and the ammonia water is 1-10 g: 100 mL.

[0099] Preferably, the mass-volume ratio of the nanometer-sized ferriferrous oxide and the tetraethyl orthosilicate is 1-10 g: 10 mL.

[0100] Preferably, the stirring speed is 400-600 rpm.

[0101] Preparation of carboxyl-functionalized magnetic beads: 3-aminopropyltrimethoxysilane is dispersed in deionized water at 3-5℃, stirred for 0.5-1.5h, 4,4,4-trichlorobutyric acid methyl ester and 4,4,4-trichloroacetyl acetic acid ethyl ester are added, the pH is adjusted and maintained at 8.9-9.1 at 75-85℃, the reaction is carried out for 5-7h, the pH is adjusted to 2.9-3.1, silicon hydroxyl-functionalized magnetic beads are added, ultrasonic dispersion is carried out for 5-15min, the reaction is carried out at 90-100℃ for 1-3h, after cooling to room temperature, magnetic separation is carried out, and the washing liquid is washed alternately with anhydrous ethanol and deionized water until the pH of the washing liquid is 6.9-7.1, vacuum drying is carried out at 55-65℃ for 10-15h, and carboxyl-functionalized magnetic beads are obtained.

[0102] Preferably, the volume ratio of 3-aminopropyltrimethoxysilane and deionized water is 5-50:200.

[0103] Preferably, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester and 3-aminopropyltrimethoxysilane is 5.75-57.5 g: 10 mL.

[0104] Preferably, the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyltrimethoxysilane is 6.5-65 g: 10 mL.

[0105] Preferably, the mass-volume ratio of the silicon hydroxyl-functionalized magnetic beads and 3-aminopropyltrimethoxysilane is 1-10 g: 10 mL.

[0106] The application also provides a preparation method of carboxyl-functionalized magnetic beads, comprising: Disperse 3-aminopropyltrimethoxysilane in deionized water under the condition of 3-5 DEG C, stir for 0.5-1.5 h, add methyl 4,4,4-trichlorobutyrate, 4,4,4-trichloroacetylacetate, adjust and maintain pH to 8.9-9.1 under the condition of 75-85 DEG C, react for 5-7 h, adjust pH to 2.9-3.1, add silicon hydroxyl functionalized magnetic beads, 3-(trihydroxysilyl)-propane sulfonic acid, ultrasonic dispersion for 5-15 min, react for 1-3 h under the condition of 90-100 DEG C, after cooling to room temperature, magnetic separation is carried out, and the washing liquid is washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid is 6.9-7.1, vacuum drying at 55-65 DEG C for 10-15 h to obtain carboxyl functionalized magnetic beads.

[0107] Preferably, the volume ratio of 3-aminopropyltrimethoxysilane to deionized water is 5-50:100-1000.

[0108] Preferably, the mass-volume ratio of methyl 4,4,4-trichlorobutyrate to 3-aminopropyltrimethoxysilane is 5.75-57.5 g:10 mL.

[0109] Preferably, the mass-volume ratio of 4,4,4-trichloroacetylacetate to 3-aminopropyltrimethoxysilane is 6.5-65 g:10 mL.

[0110] Preferably, the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane is 1-10 g:10 mL.

[0111] Preferably, the mass ratio of 3-(trihydroxysilyl)-propane sulfonic acid to silicon hydroxyl functionalized magnetic beads is 2.5-25:2.

[0112] The present application has the following beneficial effects: the obtained nucleic acid aptamer has high affinity for sarcosine, and the nucleic acid aptamer-magnetic bead chemiluminescence kit has high sensitivity. Therefore, the present application is a nucleic acid aptamer for efficiently recognizing sarcosine and application thereof. BRIEF DESCRIPTION OF DRAWINGS

[0113] Figure 1 The schematic diagram of the sarcosine adsorption capacity test results of the carboxyl functionalized magnetic beads.

[0114] Figure 2 The schematic diagram of the sarcosine adsorption capacity test results of the carboxyl functionalized magnetic beads.

[0115] Figure 3Schematic diagram of the results of affinity test of Sar-1 with sarcosine.

[0116] Figure 4 Schematic diagram of the results of affinity test of Sar-8 with sarcosine.

[0117] Figure 5 Schematic diagram of the change curve of chemiluminescence value of sarcosine quality control product of different concentrations.

[0118] Figure 6 Schematic diagram of the linear range curve of sarcosine quality control product.

[0119] Figure 7 Schematic diagram of the detection of sarcosine by nucleic acid aptamer-magnetic bead chemiluminescence kit.

[0120] Figure 8 Schematic diagram of the standard curve of the detection of sarcosine by nucleic acid aptamer-magnetic bead chemiluminescence kit. DETAILED DESCRIPTION

[0121] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0122] The concepts involved in the present application will be described below in combination with the drawings. It should be pointed out here that the descriptions of the various concepts below are only to make the content of the present application easier to understand, and do not represent a limitation on the scope of protection of the present application. Meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0123] Example 1: Preparation of positive screening magnetic beads: sarcosine was dissolved in PBS buffer, 10 mmol / L sodium acetate solution was added to obtain a mixed solution of sarcosine; carboxyl functionalized magnetic beads were dispersed in deionized water, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, the carboxyl groups on the surface of the magnetic beads were activated by incubation at room temperature for 20 min, the activated carboxyl groups were washed once with DPBS buffer, the mixed solution of sarcosine was added, and the magnetic beads were incubated on a shaker at room temperature for 2 h, then the supernatant was discarded, the magnetic beads were washed once with DPBS buffer, 1 mmol / L ethanolamine solution was added, the unreacted activated sites on the surface of the magnetic beads were blocked by incubation at room temperature for 15 min, the blocking solution was discarded, the magnetic beads were washed three times with DPBS buffer to obtain positive screening magnetic beads. The molar volume ratio of sarcosine to PBS buffer was 1 µmol:100 mL; the volume ratio of sodium acetate solution to PBS buffer was 1 µL:1 mL; the carboxyl functionalized magnetic beads were polystyrene modified nanometer ferroferric oxide, purchased from Biyun Tian Biotechnology Co., Ltd., with a particle size of 1 µm; the mass volume ratio of carboxyl functionalized magnetic beads to deionized water was 10 mg:1 mL; the mass ratio of N-hydroxysuccinimide to carboxyl functionalized magnetic beads was 1:5; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl functionalized magnetic beads was 1:2; the molar mass ratio of sarcosine to carboxyl functionalized magnetic beads was 1 µmol:10 mg; and the volume mass ratio of ethanolamine solution to carboxyl functionalized magnetic beads was 1 mL:10 mg.

[0124] Preparation of the anti-screening magnetic beads: glycine and alanine were dissolved in PBS buffer, 10 mmol / L sodium acetate solution was added to obtain a glycine mixed solution; carboxyl functionalized magnetic beads were dispersed in deionized water, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride were added, the carboxyl groups on the surface of the magnetic beads were activated by incubation at room temperature for 20 min, the activated sites on the surface of the magnetic beads were blocked by adding ethanolamine solution and incubating at room temperature for 15 min, the unreacted activated sites on the surface of the magnetic beads were discarded, and the magnetic beads were washed with DPBS buffer three times to obtain the anti-screening magnetic beads. The molar volume ratio of glycine to PBS buffer was 1 µmol:10 mL; the molar volume ratio of alanine to PBS buffer was 1 µmol:10 mL; the volume ratio of sodium acetate solution to PBS buffer was 1 mL:1 µL; the carboxyl functionalized magnetic beads were polystyrene modified nanometer ferroferric oxide, purchased from Biyun Tian Biotechnology Co., Ltd., and the particle size was 1 µm; the mass volume ratio of carboxyl functionalized magnetic beads to deionized water was 10 mg:1 mL; the mass ratio of N-hydroxysuccinimide to carboxyl functionalized magnetic beads was 1:5; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to carboxyl functionalized magnetic beads was 1:2; the molar mass ratio of glycine to carboxyl functionalized magnetic beads was 1 µmol:10 mg; and the volume mass ratio of ethanolamine solution to carboxyl functionalized magnetic beads was 1 mL:10 mg.

[0125] Construction of screening library: The ssDNA initial library used in the present application is 76 bases in length, which is composed of 20 base length primer fragments at both ends and 36 base random fragments in the middle. The library is synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and the primer is synthesized by Jinweizhi Biotechnology Co., Ltd. The ssDNA initial library, the forward primer used for constructing the library, the reverse primer used for constructing the library, the biotin-labeled library, the poly-A library and the FAM-labeled library are used in the present application. The ssDNA initial library is 5'-ATTGGCACTCCACGCATAGG -N36-CCTATGCGTGCTACCGTGAA -3', the nucleotide sequence is shown as SEQ ID No. 3, N36 is a sequence of 36 arbitrary nucleotide bases; the forward primer used for constructing the library is 5'-ATTGGCACTCCACGCATAGG -3', the nucleotide sequence is shown as SEQ ID No. 4; the reverse primer used for constructing the library is 5'-TTCACGGTAGCACGCATA -3', the nucleotide sequence is shown as SEQ ID No. 5; the biotin-labeled library is 5'-GCGTGGAGTGCCAAT-Biotin-3', the nucleotide sequence is shown as SEQ ID No. 6; the poly-A library is 5'-AAAAAAAAAAAAAAAAAAAATTCACGGTAGCACGCATA-3', the nucleotide sequence is shown as SEQ ID No. 7; the FAM-labeled library is 5'-FAM-ATTGGCACTCCACGCATAGG-3', the nucleotide sequence is shown as SEQ ID No. 8.

[0126] Screening of nucleic acid aptamer recognizing sarcosine, comprising, S1, taking 1 OD of the ssDNA initial library, centrifuging at 14000 rpm for 5 min, discarding the supernatant, adding DPBS buffer and 100 μmol / L of Lib-biotin primer, vortexing for 3 min, and performing PCR amplification, and diluting the hybridization library to 1 mL with diluent to obtain a pretreated ssDNA library. The volume ratio of the ssDNA initial library to the DPBS buffer is 1:20, the volume ratio of the ssDNA initial library to the Lib-biotin primer is 5:26, and the diluent is the DPBS buffer; the PCR reaction program comprises denaturation at 95℃ for 10 min, annealing at 60℃ for 60 min, and extension at 25℃ for 20 min; in subsequent screening rounds, the volume ratio of the ssDNA initial library to the DPBS buffer is 0.3:20, and the volume ratio of the ssDNA initial library to the Lib-biotin primer is 1:1.

[0127] S2, add herring sperm DNA, BSA and sodium chloride in PBS buffer, mix to obtain a mixed buffer, disperse positive screening magnetic beads in the mixed buffer, add pretreated ssDNA library, incubate on a shaker at room temperature for 80 min, collect the supernatant after magnetic separation, add double distilled water, 10 min in a 95℃ metal bath followed by 2 min in an ice bath, collect the supernatant after magnetic separation, and obtain an initial positive screening ssDNA library. The mass-volume ratio of herring sperm DNA to PBS buffer is 1 μg:10 μL, the mass-volume ratio of BSA to PBS buffer is 1 μg:1 μL, the molar-volume ratio of sodium chloride to PBS buffer is 1 μg:1 μL, the mass-volume ratio of positive screening magnetic beads to mixed buffer is 1 mg:40 μL, the volume ratio of pretreated ssDNA library to mixed buffer is 1:40, and the mass-volume ratio of positive screening magnetic beads to double distilled water is 1 mg:20 μL.

[0128] S3, repeat steps S1 and S2 for 10 rounds to perform subsequent screening, and obtain a positive screening ssDNA library.

[0129] S4, add herring sperm DNA, BSA and sodium chloride in PBS buffer, mix to obtain a mixed buffer, disperse the negative screening magnetic beads in the mixed buffer, add the positive screening ssDNA library, incubate on a shaker at room temperature for 30 min, magnetically separate to discard the supernatant, add 100 μmol / L sarcosine solution, incubate on a shaker at room temperature for 60 min, magnetically separate to collect the supernatant, transfer to a dialysis bag, dialyze in DPBS buffer at 4°C for 12 h to obtain the eluate after dialysis, add the eluate to the e-PCR system, mix with the e-PCR microdroplet generation oil on a vortex for 5 min to form an emulsion, perform e-PCR amplification, add an equal volume of n-butanol after amplification, vortex for 10 min, centrifuge at 6000 rpm for 30 min, and absorb the bottom aqueous phase to obtain the negative screening dsDNA. 1 mL of the e-PCR system comprises 100 μL of the eluate, 500 μL of 2x Taq PCR Master Mix, 50 μL of 100 μM of Lib-polyA, 50 μL of 100 μM of Lib-FAM, and 300 μL of double deionized water; the e-PCR amplification procedure comprises pre-denaturation at 95°C for 3 min, denaturation at 95°C for 60 s, annealing at 60°C for 60 s, extension at 72°C for 60 s, 15 cycles, and terminal extension at 72°C for 5 min; the mass / volume ratio of herring sperm DNA to PBS buffer is 1 μg:10 μL, the mass / volume ratio of BSA to PBS buffer is 1 μg:1 μL, the molar / volume ratio of sodium chloride to PBS buffer is 1 μg:1 μL, the mass / volume ratio of the negative screening magnetic beads to the mixed buffer is 1 mg:40 μL, the volume ratio of the initial positive screening ssDNA library to the mixed buffer is 1:40, and the volume / mass ratio of the sarcosine solution to the negative screening magnetic beads is 1 mg:40 μL; the molecular weight cut-off of the dialysis bag is 3.5 KD.

[0130] S5, add the loading buffer to the negative screening dsDNA, heat at 95°C for 5 min, then ice bath for 2 min, load to the Urea-PAGE gel, and perform constant voltage electrophoresis at 120 V for 20 min; cut the target fluorescent band under the ultraviolet transmission instrument, add double deionized water, heat at 95°C for 5 min, then ice bath for 2 min, centrifuge to take the supernatant, repeat once, combine the supernatants, concentrate with n-butanol, dialyze at 4°C for 4 h to obtain the negative screening ssDNA library.

[0131] S6, incubate, elute, amplify and purify the negative screening ssDNA library according to the steps S2-S5 to obtain the positive screening ssDNA library for the next round of screening.

[0132] S7, after each round of screening, 1.2 μL of ssDNA library was taken as a template, and a Q-PCR reaction system was used for quantification, and the nucleic acid concentration was calculated; the steps of S3-S5 were repeated for 11 rounds, and the final library was subjected to high-throughput sequencing to obtain nucleic acid aptamers recognizing sarcosine, the nucleic acid aptamers including Sar-1 and Sar-8, Sar-1 is 5'-ATTGGCACTCCACGCATAGGGTCCGAGGTCGTAGGTTCTAAGGGCGCACTCCCATTCCTATGCGTGCTACCGTGAA-3', the nucleotide sequence is shown as SEQ ID No. 1; Sar-8 is 5'-ATTGGCACTCCACGCATAGGGCTCCGAGGTCGTAGGTCAAGGTAGGGTTTGCATCCCCTATGCGTGCTACCGTGAA-3', the nucleotide sequence is shown as SEQ ID No. 2; 20 μL of Q-PCR reaction system includes 10 μL of 2x Taq Q-PCR Mix, 1 μL of 20 μmol / L of Lib-F, 1 μL of 20 μmol / L of Lib-R and 6.8 μL of double-distilled water.

[0133] Preparation of streptavidin magnetic beads coupled nucleic acid aptamer: the materials in Table 1 were added in a beaker, stirred until the materials were completely dissolved, the pH was adjusted to 7.4 and the volume was adjusted to 100 mL to obtain a magnetic bead blocking solution; the nucleic acid aptamer was dispersed in 10 mmol / L Tris hydrochloride buffer to obtain a nucleic acid aptamer solution; the streptavidin magnetic beads were dispersed in deionized water, the nucleic acid aptamer solution was added, and the mixture was uniformly mixed at room temperature for 30 min, and then subjected to magnetic separation for 2 min, the supernatant was discarded, 500 μL of magnetic bead blocking solution was added for blocking for 30 min, and the magnetic beads were washed with 500 μL of magnetic bead blocking solution for three times to obtain streptavidin magnetic beads coupled nucleic acid aptamer. The streptavidin magnetic beads were purchased from Biyun Tian Biotechnology Co., Ltd., and the particle size was 1 μm; the volume ratio of nucleic acid aptamer to Tris hydrochloride buffer was 1:10; the mass volume ratio of streptavidin magnetic beads to deionized water was 1 mg:100 μL; the mass volume ratio of streptavidin magnetic beads to nucleic acid aptamer solution was 5 mg:5.97 μL.

[0134] Table 1 Preparation amount of magnetic bead blocking solution

[0135] Preparation of acridinium ester-labeled nucleic acid aptamer: add nucleic acid aptamer, PBS buffer solution in 4 mmol / L acridinium ester solution, under the condition of avoiding light, constant temperature oscillation bath reaction for 30 min, add 100 mmol / L lysine solution, neutralize the excess acridinium ester for 30 min, get acridinium ester-labeled nucleic acid aptamer. The volume ratio of acridinium ester solution to nucleic acid aptamer is 3.34:26.1, the volume ratio of acridinium ester solution to PBS buffer solution is 3.34:165, and the volume ratio of acridinium ester solution to lysine is 1:1.

[0136] Preparation of nucleic acid aptamer-magnetic bead chemiluminescence kit: dilute the streptavidin magnetic beads coupled nucleic acid aptamer to the concentration of 0.5 mg / mL with magnetic bead blocking solution, and mix with three-dimensional rotary instrument for 10 min to get reagent 1; add materials in beaker according to table 2, stir until the materials are completely dissolved, adjust pH to 7.4 to get acridinium ester buffer solution; dilute acridinium ester-labeled nucleic acid aptamer to the concentration of 0.125 µg / mL with acridinium ester buffer solution, and mix with three-dimensional rotary instrument for 10 min to get reagent 2. Table 2 Preparation of acridinium ester buffer solution

[0137] Example 2: Compared with example 1, the only difference is the preparation of carboxyl functionalized magnetic beads.

[0138] Preparation of nano-magnetic iron oxide: dissolve (NH4)2Fe(SO4)2·6H2O and FeCl3·6H2O in deionized water, under the condition of nitrogen protection and 60℃, stir and dissolve, adjust pH to 11, stand and age for 2h under the condition of 80℃, cool to room temperature, then perform magnetic separation, wash with anhydrous ethanol and deionized water alternately until the pH of washing liquid is 7, vacuum dry at 60℃ for 12h to get nano-magnetic iron oxide. The mass-volume ratio of (NH4)2Fe(SO4)2·6H2O to deionized water is 3.8g:100mL, the mass-volume ratio of FeCl3·6H2O to deionized water is 4.6g:100mL, and the stirring speed is 300rpm.

[0139] Preparation of the carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester were added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads were added, ultrasonic dispersion was performed for 10 min, reacted for 2 h at 95°C, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester to 3-aminopropyltrimethoxysilane was 13 g:10 mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL.

[0140] Preparation of the carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester were added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads were added, ultrasonic dispersion was performed for 10 min, reacted for 2 h at 95°C, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester to 3-aminopropyltrimethoxysilane was 13 g:10 mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL.

[0141] Example 3: The difference between this example and Example 2 is only in the preparation of the carboxyl functionalized magnetic beads.

[0142] Preparation of carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester were added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads were added, ultrasonic dispersion was performed for 10 min, reacted for 2 h at 95°C, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester to 3-aminopropyltrimethoxysilane was 26 g:10 mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL.

[0143] Example 4: The difference between this example and Example 2 is only in the preparation of the carboxyl functionalized magnetic beads.

[0144] Preparation of carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester were added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads, 3-(trihydroxysilyl)-propane sulfonic acid were added, ultrasonic dispersion was performed for 10 min, reacted for 2 h at 95°C, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl acetic acid ethyl ester to 3-aminopropyltrimethoxysilane was 13 g:10 mL, the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL, and the mass ratio of 3-(trihydroxysilyl)-propane sulfonic acid to silicon hydroxyl functionalized magnetic beads was 5:2.

[0145] Example 5: The difference between this example and Example 2 is only in the preparation of the carboxyl functionalized magnetic beads.

[0146] Preparation of carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester was added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads and 3-(trihydroxysilyl)-propane sulfonic acid were added, ultrasonic dispersion was performed for 10 min, reacted at 95°C for 2 h, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl ethyl acetate to 3-aminopropyltrimethoxysilane was 13 g:10 mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL. The mass ratio of 3-(trihydroxysilyl)-propane sulfonic acid to silicon hydroxyl functionalized magnetic beads was 10:2.

[0147] Comparative Example 1: The comparative example is compared with Example 2, and the only difference is the preparation of carboxyl functionalized magnetic beads.

[0148] Preparation of carboxyl functionalized magnetic beads: 3-aminopropyltrimethoxysilane was dispersed in deionized water at 4°C, stirred for 1 h, 4,4,4-trichlorobutyric acid methyl ester was added, pH was adjusted and maintained to 9 at 80°C, reacted for 6 h, pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads and 3-(trihydroxysilyl)-propane sulfonic acid were added, ultrasonic dispersion was performed for 10 min, reacted at 95°C for 2 h, magnetic separation was performed after cooling to room temperature, washed with anhydrous ethanol and deionized water alternately until the pH of the washing liquid was 7, vacuum dried at 60°C for 12 h to obtain the carboxyl functionalized magnetic beads. The volume ratio of 3-aminopropyltrimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichlorobutyric acid methyl ester to 3-aminopropyltrimethoxysilane was 11.5 g:10 mL, the mass-volume ratio of 4,4,4-trichloroacetyl ethyl acetate to 3-aminopropyltrimethoxysilane was 13 g:10 mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyltrimethoxysilane was 2 g:10 mL. The mass ratio of 3-(trihydroxysilyl)-propane sulfonic acid to silicon hydroxyl functionalized magnetic beads was 10:2.

[0149] Comparative Example 2: The comparative example is compared with Example 2, and the only difference is the preparation of carboxyl functionalized magnetic beads.

[0150] Preparation of carboxyl functionalized magnetic beads: 3-aminopropyl trimethoxysilane was dispersed in deionized water at 4℃, stirred for 1h, 4,4,4-trichloroacetyl ethyl acetate was added, the pH was adjusted and maintained to 9 at 80℃, the reaction was carried out for 6h, the pH was adjusted to 3, silicon hydroxyl functionalized magnetic beads were added, ultrasonic dispersion was carried out for 10min, the reaction was carried out at 95℃ for 2h, after cooling to room temperature, magnetic separation was carried out, deionized water and anhydrous ethanol were alternately washed until the pH of the washing liquid was 7, vacuum drying was carried out at 60℃ for 12h, and carboxyl functionalized magnetic beads were obtained. The volume ratio of 3-aminopropyl trimethoxysilane to deionized water was 10:200, the mass-volume ratio of 4,4,4-trichloroacetyl ethyl acetate to 3-aminopropyl trimethoxysilane was 13g:10mL, and the mass-volume ratio of silicon hydroxyl functionalized magnetic beads to 3-aminopropyl trimethoxysilane was 2g:10mL.

[0151] Test Example 1: Sarcosine adsorption capacity test of carboxyl functionalized magnetic beads.

[0152] Test sample: carboxyl functionalized magnetic beads prepared in each example and comparative example.

[0153] Test method: 1µmoL of sarcosine was dissolved in 100mL of PBS buffer, 100μL of 10mmol / L sodium acetate solution was added to obtain a sarcosine mixed solution; the carboxyl functionalized magnetic beads were dispersed in deionized water, 2mg of N-hydroxysuccinimide and 5mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, the carboxyl on the surface of the magnetic beads was activated by incubation at room temperature for 20min, the magnetic beads were washed once with DPBS buffer, the sarcosine mixed solution was added, and the magnetic beads were incubated on a shaking table at room temperature for 2h, then the supernatant was collected by magnetic separation, and the concentration of residual sarcosine in the supernatant was detected by ultraviolet spectrophotometry to calculate the sarcosine adsorption capacity of the carboxyl functionalized magnetic beads.

[0154] The sarcosine adsorption capacity was calculated as follows:

[0155] In the formula: C 0 is the initial sarcosine concentration, C e is the residual sarcosine concentration in the supernatant after equilibrium, V is the solution volume, m is the mass of the carboxyl functionalized magnetic beads.

[0156] The test results of the sarcosine adsorption capacity of the carboxyl functionalized magnetic beads prepared in the present application are shown in Table 1. Figure 1

[0157] ​Example 1 uses basic carboxyl functionalized magnetic beads, Example 2 is modified by silicon hydroxyl functionalization of nano-magnetic iron oxide, and then modified with 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane, compared with Example 1, the sarcosine adsorption capacity is improved, which proves that this composite modification method can optimize the polarity of the surface of the magnetic beads and improve the coupling efficiency of sarcosine; Example 3 adjusts the amount of 4,4,4-trichloroacetyl acetic acid ethyl ester based on Example 2, and the sarcosine adsorption capacity is further improved; Example 4 adds 3-(trihydroxysilyl)-propane sulfonic acid for modification, and the sarcosine adsorption capacity is significantly improved, indicating that the introduction of this substance helps to adjust the charge distribution and hydrophilicity of the surface of the magnetic beads, promotes the ordered arrangement of the magnetic material, and thus enhances the interaction between the carboxyl functionalized magnetic beads and sarcosine and improves the adsorption capacity; Example 5 further increases the amount of 3-(trihydroxysilyl)-propane sulfonic acid, and the sarcosine adsorption capacity reaches a high level, indicating that increasing the amount of this substance within a certain range can continuously optimize the surface structure of the magnetic beads; Comparative Example 1 only uses 4,4,4-trichlorobutyric acid methyl ester for modification, and the sarcosine adsorption capacity is significantly lower than that of Examples 2-5, which shows that the use of a single reagent cannot form a stable surface structure and cannot effectively improve the sarcosine adsorption capacity; Comparative Example 2 only uses 4,4,4-trichloroacetyl acetic acid ethyl ester for modification, and the sarcosine adsorption capacity is also low, which again proves that single reagent modification cannot achieve the effect of multiple reagent modification, and cannot meet the demand for sarcosine adsorption capacity.

[0158] Test Example 2: Sarcosine adsorption capacity test of carboxyl functionalized magnetic beads coupled with sarcosine.

[0159] Test sample: carboxyl functionalized magnetic beads prepared in each example and comparative example.

[0160] Test method: 1 μmol sarcosine was dissolved in 100 mL PBS buffer, 100 μL of 10 mmol / L sodium acetate solution was added to obtain a mixed solution of sarcosine; the carboxyl functionalized magnetic beads were dispersed in deionized water, 2 mg of N-hydroxysuccinimide and 5 mg of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride were added, the carboxyl on the surface of the magnetic beads was activated at room temperature for 20 min, and then washed once with DPBS buffer, then the mixed solution of sarcosine was added, and incubated at room temperature for 2 h, then the supernatant was discarded, and then washed once with DPBS buffer, then 1 mL of 1 mmol / L ethanolamine solution was added, and incubated at room temperature for 15 min, then separated by magnetism, then 50 μL of 0.1 mol / L glycine-HCl buffer was added, and oscillated at room temperature for 10 min to destroy the amide bond and elute the sarcosine, and then the concentration C1 of sarcosine in the eluent was detected by ultraviolet spectrophotometry; after magnetic separation, the magnetic beads were washed twice with PBS, 50 μL of the EDC / NHS solution was added, and incubated at room temperature for 15 min to re-activate the carboxyl on the surface of the magnetic beads; after magnetic separation, the magnetic beads were washed twice with PBS to remove residual reagents, and then the above steps were repeated for 5 cycles, and then the concentration C2 of sarcosine in the eluent after the cycles was detected by ultraviolet spectrophotometry, and then the cycle stability was calculated by C2 / C1 x 100%.

[0161] The cycle stability test results of the carboxyl functionalized magnetic beads coupled with sarcosine prepared in the application are shown in Table 1. Figure 2 As shown in Table 1, the sarcosine coupling efficiency of the basic polystyrene modified magnetic beads of Example 1 is low due to the simple surface modification and the easy damage of the carboxyl active site during repeated activation. The magnetic beads of Example 2 are modified by 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane, and a more stable carboxyl functional layer is formed, which reduces the damage of the surface structure during activation, and the cycle stability is improved compared with Example 1. The amount of trichloroacetyl acetic acid ethyl ester is adjusted in Example 3 based on Example 2, and the complex carboxyl functional layer formed by the synergistic modification of the two reagents further enhances the stability of the coupling bond and reduces the non-specific shedding during elution. The 3-(trihydroxysilyl)-propane sulfonic acid is introduced in Examples 4-5, which increases the hydrophilicity and negative charge density on the surface of the magnetic beads, reduces non-specific adsorption and optimizes dispersibility, so that the sarcosine coupling efficiency is more stable, and the increase of the amount of the hydrophilic modifier within a certain range can further improve the stability. In comparison, the single trichlorobutyric acid methyl ester modified comparative example 1 and the single trichloroacetyl acetic acid ethyl ester modified comparative example 2 have a significantly lower cycle stability due to the single active site on the surface and the easy breakage of the coupling bond.

[0162] Test example 3: affinity test of the nucleic acid aptamer recognizing sarcosine and sarcosine.

[0163] Test sample: nucleic acid aptamer prepared in Example 1.

[0164] Test method: CM5 chip was selected, 0.4 mol / L 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride solution and 0.1 mol / L N-hydroxysuccinimide solution were mixed uniformly, and the chip surface was activated at a flow rate of 5 μL / min and a sample volume of 50 μL; sarcosine was diluted with 10 mmol / L sodium acetate solution at pH 4.5 to a final concentration of 50 μg / mL, and was injected at a flow rate of 5 μL / min and a sample volume of 50 μL to complete the coupling of sarcosine on the surface of the 2nd channel of the chip, and the final coupling amount of sarcosine was 2000 Ru; after the coupling of sarcosine was completed, ethanolamine was used to block the chip at a flow rate of 5 μL / min and a sample volume of 50 μL. For the 1st channel of the chip, the same activation and blocking steps as the 2nd channel were performed, but no sarcosine coupling operation was performed, which was used as a control channel; the surface plasmon resonance instrument was used for detection, and the kinetic detection parameters were set. The diluted nucleic acid aptamer sample obtained in Example 1 was sequentially flowed through the 1st, 2nd, 3rd and 4th channels of the chip; for each nucleic acid aptamer, the specific program setting was as follows: 30 μL / min for 2 min, 30 μL / min for 3 min, and finally 1 mol / L NaCl solution at a flow rate of 30 μL / min for 0.5 min, and the diluted nucleic acid aptamer was sequentially injected and detected according to the above program.

[0165] The affinity test results of Sar-1 and sarcosine are shown in Figure 3 The affinity test results of Sar-8 and sarcosine are shown in Figure 4 The KD values of the nucleic acid aptamer recognizing sarcosine are shown in Table 3.

[0166] Table 3 KD values of the nucleic acid aptamer recognizing sarcosine

[0167] The nucleic acid aptamer was detected by the SPR instrument to have strong binding with sarcosine, and the binding curve of the nucleic acid aptamer and sarcosine rapidly reached the response peak after injection, and the signal decayed slowly in the dissociation stage, indicating that the complex formed by the two had high stability. The high specificity sequence was effectively enriched through multiple rounds of optimization by positive screening and reverse screening in the screening process, which indicated that the corresponding nucleic acid aptamer and sarcosine showed strong specific binding.

[0168] Test example 4: Sensitivity test of nucleic acid aptamer-magnetic bead chemiluminescence kit.

[0169] Test sample: nucleic acid aptamer-magnetic bead chemiluminescence kit prepared in Example 1.

[0170] Test method: When the creatine nucleic acid aptamer-magnetic bead chemiluminescence kit is first loaded on the SMART 6500S system, in order to re-suspend the magnetic beads that may precipitate during transportation, the reagent 1 needs to be mixed, the reagent 1 is placed on a three-dimensional rotator for 10 minutes of turning, the speed should be controlled during turning to avoid air bubbles, and the reagent bottle is visually inspected until the microparticles are completely suspended. After mixing the reagent 1, add it to the a cavity of the reagent bottle A, and mix the reagent 2 before use by gently turning it up and down for 10 times, then add it to the b cavity of the reagent bottle A. Put the reagent bottle A into the reagent bin; put the sample into the sample bin and check the sample amount in the sample tube, ensure that the sample amount in the sample tube before each test is more than 200 μL, if necessary, execute the calibration command, see the SMART 6500S system operation manual for the calibration command information; perform the running parameter setting, including sample injection amount, reagent 1 component suction amount, reagent 2 component suction amount, reaction mode, incubation time, etc., and apply for testing. Press the run, the SMART 6500S system will perform the following operations: (1) move the sample transfer device to the sampling point; (2) load the reaction cup into the reaction disc; (3) suck the sample and transfer it to the reaction cup; (4) move the reaction cup to the front of a position, and add reagent 1 and reagent 2 to the reaction cup; (5) mix, incubate and wash the reaction mixture; (6) add substrate liquid and excitation liquid; (7) detect the relative luminescence intensity; (8) suck the contents in the reaction cup into the waste liquid, and put the reaction cup into the solid waste barrel.

[0171] The variation curve of the chemiluminescence value of creatine quality control at different concentrations is shown in Figure 5 The linear range curve of the creatine quality control is shown in Figure 6 The detection of creatine by the nucleic acid aptamer-magnetic bead chemiluminescence kit is shown in Figure 7 The standard curve of the detection of creatine by the nucleic acid aptamer-magnetic bead chemiluminescence kit is shown in Figure 8 The luminescence intensity is linearly related to the target molecule concentration in a wide dynamic range, and the minimum detection limit indicates that the nucleic acid aptamer-magnetic bead chemiluminescence kit has extremely strong sensitivity.

[0172] The above-described embodiments and / or implementations are only used to illustrate the preferred embodiments and / or implementations of the present technology, and do not limit the embodiments of the present technology in any form. Any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the scope of the technology disclosed in the present disclosure, but should be considered as substantially the same technology or embodiment.

[0173] The principles and implementation manners of the present application are described herein by using specific examples, and the above example descriptions are only used to help understand the method of the present application and its core idea. The above descriptions are only preferred embodiments of the present application, and it should be pointed out that, due to the limited nature of the language expression, there are objectively infinite specific structures, and for ordinary skilled persons in the technical field, some improvements, refinements or changes can be made without departing from the principles of the present application, and the above technical features can also be combined in an appropriate manner; these improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, shall be regarded as the protection scope of the present application.

Claims

1. A nucleic acid aptamer for identifying sarcosine, the nucleic acid aptamer comprising Sar-1 and Sar-8, the Sar-1 being 5'-ATTGGCACTCCACGCATAGGGTCCGAGGTCGTAGGTTCTAAGGGCGCACTCCCATTCCTATGCGTGCTACCGTGAA-3', the nucleotide sequence of which being shown as SEQ ID No.1, and the Sar-8 being 5'-ATTGGCACTCCACGCATAGGGCTCCGAGGTCGTAGGTCAAGGTAGGGTTTGCATCCCCTATGCGTGCTACCGTGAA-3', the nucleotide sequence of which being shown as SEQ ID No.

2.

2. The method of claim 1, wherein the method comprises: constructing an ssDNA initial library; the positive screening magnetic beads being carboxyl-functionalized magnetic beads coupled with sarcosine, and the negative screening magnetic beads being carboxyl-functionalized magnetic beads coupled with glycine and alanine.

3. The screening method for nucleic acid aptamers that recognize sarcosine according to claim 2, characterized in that, The ssDNA initial library is 5'-ATCCAGAGTGACGCAGCA-N36-TGGACACGGTGGCTTAGT-3', as shown in SEQ ID No.3, and is composed of a 20-base-length primer fragment fixed at both ends and a 36-base-length random fragment in the middle.

4. The screening method for nucleic acid aptamers that recognize sarcosine according to claim 2, characterized in that, The carboxyl-functionalized magnetic beads comprise polystyrene-modified nano-ferriferrous oxide, or 4,4,4-trichlorobutyric acid methyl ester, 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane co-modified silicon-based functionalized magnetic beads, the silicon-based functionalized magnetic beads comprising nano-ferriferrous oxide modified with tetraethyl orthosilicate.

5. The screening method for nucleic acid aptamers that recognize sarcosine according to claim 4, characterized in that, The mass-volume ratio of the 4,4,4-trichlorobutyric acid methyl ester and 3-aminopropyl trimethoxysilane is 5.75-57.5 g:10 mL, and the mass-volume ratio of the 4,4,4-trichloroacetyl acetic acid ethyl ester and 3-aminopropyl trimethoxysilane is 6.5-65 g:10 mL.

6. The screening method for nucleic acid aptamers that recognize sarcosine according to claim 4, characterized in that, The mass-volume ratio of the silicon hydroxyl-functionalized magnetic beads and 3-aminopropyl trimethoxysilane is 1-10 g:10 mL. 7.A nucleic acid aptamer-magnetic bead chemiluminescence kit, the chemiluminescence kit comprising reagent 1 and reagent 2, the reagent 1 comprising magnetic bead-coupled nucleic acid aptamer, and the reagent 2 comprising chemiluminescence marker-coupled nucleic acid aptamer, the nucleic acid aptamer comprising Sar-1 and Sar-8, the magnetic beads being modified with streptavidin, and the chemiluminescence marker being acridinium ester.

8. The method for preparing the nucleic acid aptamer-magnetic bead chemiluminescence kit of claim 7, comprising: After the magnetic beads are modified with streptavidin, the nucleic acid aptamer is coupled thereto, and the reagent 1 is prepared by dilution with a magnetic bead blocking solution; After the acridinium ester is coupled with the nucleic acid aptamer, the reagent 2 is prepared by dilution with an acridinium ester buffer solution; the chemiluminescence detection kit comprises the reagent 1 and the reagent 2, and the nucleic acid aptamer comprises Sar-1 and Sar-8.

9. A method for preparing a nucleic acid aptamer-magnetic bead chemiluminescence reagent kit according to claim 8, characterized in that, The composition of the magnetic bead sealing solution comprises 0.5-1 wt% of Tris, 0.5-2 wt% of BSA, 0.1-0.2 wt% of Tween20, 96.8-98.9 wt% of deionized water.

10. A method for preparing a nucleic acid aptamer-magnetic bead chemiluminescence reagent kit according to claim 8, characterized in that, The composition of the acridine ester buffer solution comprises 1-2 wt% of NaHPO4·12H2O, 0.1-0.5 wt% of NaH2PO4·2H2O, 0.5-1.5 wt% of NaCl, 0.2-1 wt% of BSA, 0.02-0.1 wt% of MgCl2·6H2O, 0.04-0.2 wt% of Tween20, 0.04-0.2 wt% of Proclin 300, 94.5-98.1 wt% of deionized water.

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