Carboxymethyl lysine and carboxyethyl lysine aptamers screened based on efficient microfluidic SELEX (systematic evolution of ligands by exponential enrichment) platform

Abstract

The invention discloses CML and CEL aptamers screened based on an efficient microfluidic SELEX platform, and belongs to the technical field of food safety biology. Through the microfluidic SELEX platform integrated with library fixation, target and library incubation, magnetic separation, fluorescence monitoring and other functions, the capsule SELEX screening method with less magnetic bead consumption, higher library immobilization rate, shorter screening time and higher enrichment efficiency is realized; the requirements and limitation of methods such as qPCR (quantitative polymerase chain reaction) and fluorescence ratio determination on specific instruments in the prior art are broken through; finally, the aptamer S2 with excellent performance of CML and CEL is obtained for the first time through the screening method, truncation optimization is carried out based on the combination mechanism of the aptamer S2, the aptamer S2-40 with shorter sequence and more excellent performance is obtained, and the dissociation constants measured by an ITC method and an SGI method are 6.65 + / -3.07 mu M and 42.1 + / -19.34 nM.

Description

Technical Field

[0001] The invention relates to carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) aptamers screened based on a high-efficiency microfluidic SELEX platform, and belongs to the field of food safety biotechnology. Background Art

[0002] Aptamers are single-stranded oligonucleotides with a length of 20-90 nt. Aptamers are effective recognition molecules for constructing biosensors. They are generated by systematic evolution of ligands by exponential enrichment (SELEX) and have specific targeting capabilities for a variety of molecules such as metal ions, organic compounds, macromolecular proteins, and cells. Each round of SELEX process includes library or target immobilization, incubation binding, separation of target binding library, PCR, and secondary library preparation. It takes up to 12 to 20 rounds of screening to achieve a high enrichment rate and end the screening. These steps are cumbersome and costly, and it usually takes several months to obtain a high-performance aptamer. In addition, the open screening environment and manual operation often lead to library contamination or degradation, which also makes it difficult to increase the enrichment rate. More importantly, in order to end SELEX in the right round, the enrichment rate of each round must be monitored by ratiometric fluorescence or qPCR, which requires special reagents and expensive instruments. At present, some studies have integrated some steps of the SELEX process into microfluidic chips by introducing microfluidic technology to achieve a more integrated and efficient microfluidic screening method. However, most existing microfluidic SELEX schemes are based on the target-immobilized SELEX method, which is suitable for the screening of aptamers for macromolecules such as proteins, but is less practical for small molecule targets that are difficult to immobilize by chemical bonds. Capture-SELEX can achieve the screening of aptamers for macromolecules and small molecules by fixing the library, but it also faces the problem of low screening efficiency. The problem of using microfluidic technology to assist capture-SELEX to achieve efficient aptamer screening has not yet been solved.

[0003] Advanced glycation end products (AGEs) are a general term for a series of substances produced by the Maillard reaction during food processing, and are widely present in foods with high sugar and protein content. Existing studies have shown that AGEs can cause diseases such as diabetes, atherosclerosis, Alzheimer's disease and even cancer. Carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) are representatives of AGEs and are usually used to assess the content of AGEs. Therefore, obtaining recognition molecules with high binding to CML and CEL is the key to constructing excellent biosensors and assessing AGE levels. However, the recognition molecules for CML and CEL are mostly antibodies, and there is a lack of more stable and low-cross-reactive high-performance aptamers as recognition molecules. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides CML and CEL aptamers screened by an efficient microfluidic SELEX platform based on capture-SELEX, aiming to solve the technical problem that the recognition molecules for CML and CEL are mostly antibodies, and there is a lack of more stable and low-cross-reactive high-performance aptamers as recognition molecules.

[0005] The first technical solution provided by the present invention is an aptamer of CML and CEL, wherein the aptamer is a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0006] In certain embodiments, the aptamer may be connected to a functional group or molecule. Optionally, the functional group or molecule may be connected to the 5' end or the 3' end of the nucleic acid aptamer.

[0007] In certain embodiments, the functional group or molecule may be an isotope, a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, an enzyme marker, a magnetic substance, biotin, an affinity ligand and / or a thiol group.

[0008] The present invention provides a microfluidic SELEX platform for efficient screening of CML and CEL aptamers, which mainly includes the following contents:

[0009] (1) Based on the chip functions, the Rhino software was used to draw and construct a three-dimensional model of the microfluidic chip. The constructed microfluidic chip structure includes two sample inlets (A and B), two sample outlets (I and II), two fluorescence quantification chambers, one incubation and separation chamber, and four convenient push-pull valves;

[0010] (2) Using polymethyl methacrylate (PMMA) as the substrate, the CNC milling machine is used to mill the chamber and flow channel according to the size of the three-dimensional model;

[0011] (3) cleaning and drying the chip after milling, and sealing the chip with a polypropylene (PP) film by an ionic bonding method after passivation and hydrophilic and hydrophobic treatment;

[0012] (4) Use Rhino software to draw and construct a three-dimensional model of the microfluidic SELEX platform. The constructed microfluidic SELEX platform includes an incubation vibration module, a magnetic separation module, and a fluorescence monitoring module;

[0013] (5) Using 3D printing technology, a microfluidic SELEX platform is manufactured according to the constructed three-dimensional model, and each functional module and microfluidic chip are assembled;

[0014] (6) Design and develop microfluidic SELEX enrichment rate monitoring software REMS to monitor the enrichment rate of each round;

[0015] (7) The constructed microfluidic SELEX platform includes an empty state, a vibration incubation state, a magnetic separation state, and a fluorescence monitoring state, which can realize the functions of library fixation, target and library incubation, magnetic separation, and fluorescence monitoring in capture-SELEX.

[0016] The present invention also provides a method for efficient aptamer screening and truncation optimization based on the developed microfluidic SELEX platform, which mainly comprises the following steps:

[0017] (1) Inject streptavidin-labeled magnetic beads and biotinylated capture chain-library complexes into the incubation separation chamber from injection port B and injection port A, respectively, and incubate with vibration for 40 min to immobilize the five-segment library onto the magnetic beads. The unimmobilized library is introduced into the waste liquid pool from the sample outlet II through the fluorescence monitoring chamber;

[0018] (2) Inject the counter-screening target lysine from the injection port B into the incubation separation chamber for vibration incubation for 1 hour. After magnetic separation, the counter-screening target-library complex is introduced into the waste liquid pool through the sample outlet I;

[0019] (3) Inject the target CML and CEL from the injection port B into the incubation and separation chamber for vibration incubation for 1 h. After magnetic separation, the target-library complex is introduced into the collection pool from the sample outlet II through the fluorescence detection chamber;

[0020] (4) When the initial library, the library not immobilized by magnetic beads, and the library bound to the target pass through the fluorescence quantification chamber, fluorescence images are collected and analyzed by REMS software to obtain the immobilization rate and enrichment rate of each round;

[0021] (5) amplifying the library in the collection pool at an appropriate PCR annealing temperature and amplification round number, purifying the PCR product, and preparing a secondary library using Lambda exonuclease;

[0022] (6) When the enrichment rate reaches 63.9% in the eighth round of screening, the screening can be terminated, and the enriched products of the eighth round of screening are subjected to high-throughput sequencing. The candidate aptamers S1, S2, S3, S4, S10 and S17 are determined based on the sequencing results;

[0023] (7) The affinity and specificity of the candidate aptamers were analyzed based on isothermal titration calorimetry (ITC) and SYBR Green I fluorescence (SGI) and sequence S2 with strong affinity and best specificity was selected as the aptamer for the target;

[0024] (8) Validation of the binding of aptamer S2 to CML and CEL based on circular dichroism spectroscopy;

[0025] (9) Analysis of the binding sites of aptamer S2 with CML and CEL based on molecular docking simulation;

[0026] (10) Based on the docking results and binding sites, the non-binding regions of the original sequence that bind to the target were removed, and the binding regions were retained, and the affinity and specificity were further analyzed by ITC and SGI.

[0027] The second technical solution provided by the present invention is a probe, which is a substance obtained by labeling the aptamers of CML and CEL described in the first technical solution with a labeling substance.

[0028] In certain embodiments, the label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radioactive labels, such as 32P; conjugated coupling groups, such as biotin; haptens, such as digoxigenin (DIG); chemiluminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight measurement, quantum dots, electrochemistry, X-ray diffraction or absorption, magnetism, immunoenzyme label reaction, immunoassay affinity precipitation of base filter paper, affinity chromatography, enzyme activity, microscopic projection or scanning imaging, super-resolution imaging, cell tracking, live nanoparticle tracking imaging of animals or humans, nanoflow, adjustable resistive pulse induction, fluorescence correlation spectroscopy surface plasmon resonance, fluorescence polarization, surface enhanced Raman spectroscopy, electrochemical sensing, microfluidics or microfluidics, chip analysis, proteomics, genomics, metabolomics, microbiome, RNA (mRNA, 1nRNA, SnRNA), miRNA, etc. The label can be a charged part (positive or negative charge) or can be selected as needed, and can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence). The label can also be used for targeted drug delivery.

[0029] In certain embodiments, the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click chemistry reactions, and initiator groups for polymer labeling), and can also be selected from polypeptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptoids), non-natural nucleic acids and their analogs (nucleotide mimics) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced emission molecules, rare earth ion ligand molecules, polymetallic oxygen clusters, etc.).

[0030] In certain embodiments, the fluorophore can be selected from fluorescein dyes, rhodamine dyes and other common dyes.

[0031] In certain embodiments, the fluorescein dye includes standard fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), fluorescein amylase (FAM), tetrachlorofluorescein (TET), cy5, cy3, Quasar670, Alexa Fluor 488 / 647, and the like.

[0032] In certain embodiments, the rhodamine dye includes R101, tetraethyl rhodamine (RB200) or carboxytetramethylrhodamine (TAMRA) and the like.

[0033] In certain embodiments, the general dye is mainly selected from two categories, one is thiazole orange (T0), oxazole orange (Y0) series and dimer dyes thereof, and the other is polymethine series cyanine dyes.

[0034] In certain embodiments, the fluorophore can also be selected from the following dyes: stilbene, naphthalimide, coumarins, acridines, pyrenes, and the like.

[0035] Fluorophores are usually labeled at the 5' end of the probe sequence, but can also be placed at the 3' end by changing the modification bond (eg, -OH or -NH bond).

[0036] The third technical solution provided by the present invention is a cell containing the aptamer described in the first technical solution or the probe described in the second technical solution.

[0037] In certain embodiments, the cell may be an animal cell, a plant cell, or a microbial cell.

[0038] The fourth technical solution provided by the present invention is a biosensor, which contains the aptamer described in the first technical solution or the probe described in the second technical solution.

[0039] The fifth technical solution provided by the present invention is the use of the aptamer described in the first technical solution, the probe described in the second technical solution or the cell described in the third technical solution in detecting CML and / or CEL.

[0040] The sixth technical solution provided by the present invention is a reagent or a kit for detecting CML and / or CEL, wherein the reagent or the kit contains the aptamer described in the first technical solution or the probe described in the second technical solution.

[0041] The seventh technical solution provided by the present invention is a method for detecting CML and / or CEL, which comprises labeling a reporter group on the aptamer described in the first technical solution, allowing the aptamer labeled with the reporter group to interact with the sample to be tested, and realizing the detection of CML and / or CEL by detecting the signal of the reporter group.

[0042] The eighth technical solution provided by the present invention is the application of the aptamer described in the first technical solution, the probe described in the second technical solution, the cell described in the third technical solution, the biosensor described in the fourth technical solution, the reagent or kit described in the sixth technical solution, and the method described in the seventh technical solution in the fields of food, biology or medicine.

[0043] The technical effects of the present invention are as follows:

[0044] The microfluidic SELEX platform designed and constructed by the present invention integrates the capture-SELEX screening method based on fixed library, and integrates the functions of library fixation, target and library incubation, magnetic separation and fluorescence monitoring into the platform. The existing microfluidic SELEX methods are mostly screening methods for fixed macromolecular targets, and there are few microfluidic SELEX methods for small molecule target aptamers. The present invention firstly integrates a vibration incubation module and a magnetic separation module. Compared with the manual screening method, less magnetic beads are used, the library immobilization rate is higher, the screening time is shorter, and the enrichment efficiency is faster; secondly, the REMS software developed by the method can conveniently monitor each round of enrichment rate, breaking the requirements and restrictions on specific instruments by previous methods such as qPCR and fluorescence ratio determination; finally, the excellent aptamer S2 of CML and CEL is obtained for the first time through the screening method, and truncation optimization is performed based on its binding mechanism to obtain an aptamer S2-40 with a shorter sequence and better performance, and its dissociation constants determined by the ITC method and the SGI method are 6.65±3.07μM and 42.1±19.34nM, and the sequence information is: 5'-CCATCCGGGGGTGCACCCATTCTTGGAGCTTCCCGTCAGT-3' (SEQ ID NO: 6). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the principle of aptamer screening based on the microfluidic SELEX platform.

[0046] Figure 2 Figure 2 is the functional design and fabrication diagram of the microfluidic chip (a) and the functional design diagram of the microfluidic SELEX platform (b).

[0047] Figure 3 It is a schematic diagram of screening and trimming optimization.

[0048] Figure 4 These are images collected in the fluorescence detection area; (a) is the initial fluorescence image of the library; (b) is the fluorescence image of the library after immobilization; (c) is the fluorescence image of the library bound to the target.

[0049] Figure 5 are the secondary structures of the sequences in Table 2; (a) is the secondary structure of S1; (b) is the secondary structure of S2; (c) is the secondary structure of S3; (d) is the secondary structure of S4; (e) is the secondary structure of S10; and (f) is the secondary structure of S17.

[0050] Figure 6 It is the circular dichroism spectrum and molecular docking result diagram.

[0051] Figure 7 is a graph of candidate aptamer specificity results. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0053] The reagents and raw materials used in the present invention: carboxymethyl lysine and carboxyethyl lysine were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; ready-to-use O'RangeRuler 10bp DNA molecular weight standard kit, Dynabeads TMM-270 streptavidin magnetic beads were purchased from Thermo Fisher Scientific; acrylamide / bisacrylamide (30% solution) and N, N, N', N'-tetramethylethylenediamine (TEMED) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; urea was purchased from Maclean's Biochemical Technology Co. Lambda exonuclease (5000U / mL) and 10× Lambda exonuclease reaction buffer were purchased from New England BioLabs; streptavidin, Taq Plus DNA polymerase, 2×TBE-urea sampling buffer and dNTP mixture (25mM solution) were purchased from Shangyu Biotechnology (Shanghai) Co., Ltd. All DNA chains were synthesized and purified (HPLC) by Sangon Biotech Co. The above reagents and raw materials meet relevant regulations, meet national standards, and will not endanger human health.

[0054] Example 1 Aptamer Screening

[0055] 1. Design and construction process of the microfluidic SELEX platform

[0056] 1. Design of microfluidic chip

[0057] In order to realize the chip-based capture-SELEX aptamer screening method, an efficient microfluidic SELEX chip was designed, such as Figure 1 As shown. The structure includes an incubation separation chamber, two fluorescence detection chambers, two injection ports (A, B) and two outlet ports (Ⅰ, Ⅱ). Each chamber is connected by a flow channel, and 4 cylindrical pistons are set as push-pull valves. The injection port A and the outlet port Ⅱ are connected to the fluorescence detection chamber through the flow channel, and then communicated with the incubation separation chamber, which is convenient for fluorescence detection of the library content before and after immobilization and incubation elution in the capture-SELEX process. The injection port B and the outlet port Ⅰ are directly connected to the incubation separation chamber, which is convenient for injecting magnetic beads, counter-screening targets and targets into the incubation separation chamber for screening. The switch of the flow channel is controlled by lifting and pressing the push-pull valve. The chip can realize the library fixation, incubation, magnetic separation and enrichment rate monitoring in the capture-SELE aptamer screening process.

[0058] 2. Fabrication of microfluidic chips

[0059] like Figure 2As shown in (a), polymethyl methacrylate (PMMA) is used as the substrate, and a CNC milling machine is used to mill structures such as a fluorescence detection chamber, an incubation and separation chamber, and a flow channel according to the size of the three-dimensional model; the chip after milling is cleaned and dried, the flow channel and the chamber are passivated and hydrophilic and hydrophobic treatment is performed, the chip is sealed with a polypropylene (PP) sealing film by an ion bonding method, and the push-pull valve is assembled with the chip to complete the production of the microfluidic chip.

[0060] 3. Microfluidic SELEX platform design

[0061] In order to realize the highly integrated capture-SELEX aptamer screening method, the present invention designs and develops an efficient microfluidic SELEX platform based on microfluidic chips, such as Figure 2 (b) As shown. The platform further integrates the vibration incubation module, magnetic separation module and fluorescence monitoring module into the platform, which can realize capture-SELEX aptamer screening in a more integrated manner. The platform consists of a chip operation platform and a top cover. The chip operation platform consists of a microfluidic chip slot, a waste liquid pool positioning hole, a collection pool positioning hole, a microfluidic chip slot (with a magnet and a vibrator embedded at the bottom), a top cover (with an ultraviolet lamp connected to the inside), and an observation hole on the top of the top cover (which can collect the fluorescence of the chip fluorescence detection chamber). The vibrator and the platform side controller constitute the incubation vibration module, the magnet is the magnetic separation module, and the top cover observation hole, the ultraviolet lamp and its switch constitute the fluorescence monitoring module. The microfluidic chip is placed in the microfluidic chip slot, and the incubation separation chamber is tightly fitted with the magnet or vibrator by sliding, so that the magnetic beads in the incubation separation chamber are aggregated or dispersed, thereby realizing vibration incubation, magnetic separation and enrichment rate monitoring during capture-SELEX.

[0062] 4. 3D printing and construction of microfluidic SELEX platform

[0063] The microfluidic platform was obtained through 3D printing technology, and was further assembled and constructed by assembling components such as oscillators, ultraviolet lamps and magnets.

[0064] Table 1 Equipment parts model

[0065]

[0066] 2. Aptamer screening based on microfluidic SELEX platform

[0067] like Figure 3 The process shown is used to screen the aptamers. The specific steps are as follows:

[0068] 1. Library immobilization

[0069] After the magnetic beads are injected into the incubation separation chamber (ISC) from the B inlet, valves 1 and 2 are closed, and the chip is placed in the magnetic separation state and allowed to stand for 5 seconds. Valves 3 and 4 are opened, and the supernatant is discharged from outlet I into the waste pool under pressure. The heat-denatured capture probe-library complex is injected into the fluorescence monitoring chamber 1 (FMC1) from the A inlet, the lid is closed, the UV light is turned on, and the fluorescence image of FMC1 is captured through the information collection hole with a smartphone (a). Continue to pressurize the chip so that it completely enters the ISC, and then close valves 3 and 4. Set the chip to the vibration incubation state, turn on the vibration incubation switch, and incubate for 40 minutes. Set the chip to the magnetic separation state and let it stand for 5 seconds. Open valves 1 and 2, pressurize inlet B, allow the supernatant to enter the fluorescence monitoring chamber 2 (FMC2), and then capture the fluorescence image of FMC2 with a smartphone (b), and continue to pressurize it so that it is completely discharged into the waste pool.

[0070] 2. Counter-screening of target lysine incubation elution library

[0071] Lysine was injected into the ISC from injection port B, valves 1 and 2 were closed, the chip was placed in a vibration incubation state for 60 min, and then the chip was placed in a magnetic separation state and left to stand for 5 s. Valves 3 and 4 were opened, and the eluate was discharged into the waste liquid pool by pressurizing from outlet I.

[0072] 3. Positive screening of target CML and CEL incubation elution library

[0073] Inject the CML & CEL mixed solution into the ISC from injection port B, close valves 1 and 2, set the chip to the vibration incubation state, and incubate for 60 minutes. Open valves 1 and 2, pressurize through port A, and after the eluent enters FMC2, use a smartphone to collect the fluorescence image of FMC2 through the information collection port (c). Continue to pressurize to allow the eluent to drain into the collection tube.

[0074] 4. REMS software monitors fluorescence enrichment rate

[0075] The acquired images (a), (b) and (c) (see Appendix Figure 4 ) were imported into the Enrichment Rate of Microfluidic SELEX (ERMS) software to analyze the immobilization rate and enrichment rate of this round.

[0076] 5. PCR and product purification

[0077] The PCR reaction was performed in 50 μL reaction buffer containing 5 μL ssDNA, 0.5 μL Taq DNA polymerase (5U / uL), 5 μL 10× polymerase buffer, 0.5 μL of each primer and 37.5 μL ddH2O. The amplification conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 2 min, a total of 13 cycles, and storage at 4°C. The amplified product was purified using a PCR product purification kit to remove impurities such as primers, buffer solutions, and enzymes in the PCR system.

[0078] 6. Secondary library preparation

[0079] Secondary libraries were prepared using Lambda exoribonuclease to specifically detect downstream primers of phosphorylated markers. Lambda enzyme and 10× Lambda digestion buffer were added to the PCR purified product for digestion reaction, and the reaction was terminated at 75°C. Finally, ssDNA was purified by ethanol precipitation and collected as secondary libraries for the next round of screening.

[0080] 7. High-throughput sequencing and sequence analysis

[0081] High-throughput sequencing was performed on the products of the 8th round of screening. According to the secondary structure, free energy (ΔG) and repetition frequency, S1 (SEQ ID NO: 7), S2 (SEQ ID NO: 1), S3 (SEQ ID NO: 8), S4 (SEQ ID NO: 9), S10 (SEQ ID NO: 10) and S17 (SEQ ID NO: 11) were selected as candidate aptamer sequences, and their secondary structures, free energies and repetition frequencies are shown in Table 2.

[0082] Table 2 Basic information of sequences

[0083] sequence Secondary structure Free energy kcal / mol Repeat frequency S1 Figure 5 (a) -2.85 2045 S2 Figure 5 (b) -3.23 2013 S3 Figure 5 (c) -3.82 1603 S4 Figure 5 (d) -3.52 1325 S10 Figure 5 (e) -2.87 529 S17 Figure 5 (f) -5.75 421

[0084] 8. Affinity and specificity analysis of candidate aptamers

[0085] (1) The affinity of candidate aptamers for the target was determined based on isothermal titration calorimetry (ITC).

[0086] The binding heat of candidate aptamers to CML&CEL was determined by PEAQ-ITC (Malvin, UK). The experimental conditions for binding heat determination were an initial delay of 60 s, an initial injection of 0.4 μL, a delay of 150 s, and then 12 drops of 4 μL were added every 150 s. (2) The affinity and specificity of candidate aptamers to the target were determined based on SYBR Green I fluorescence (SGI) method.

[0087] The candidate aptamers with different concentration gradients were reacted with SYBR Green I fluorescent dye, and CML&CEL was further added, with blank as control. The relative fluorescence intensity ΔF (ΔF=F0-F1, where F1 is the fluorescence value of the experimental group and F0 is the fluorescence value of the blank group) was measured at 527nm, with an excitation wavelength of 490nm, and the dissociation constant was obtained by processing using GraphPad Prism 7 software.

[0088] Glutamic acid, proline, arginine, serine and glycine were used to replace CML&CEL, and the relative fluorescence ratio (relative fluorescence ratio = ΔF1 / ΔF0 × 100%, where ΔF0 is the relative fluorescence intensity generated by the binding of CML&CEL to the aptamer, and ΔF1 is the glutamate, proline, arginine, serine and glycine generated by the binding of the aptamer) was calculated for analyzing the affinity of the candidate aptamers.

[0089] (3) Select candidate sequences with high affinity and good specificity as the best aptamer for the target

[0090] The affinity results determined by ITC and SGI methods are consistent as shown in Table 3. S1 and S2 have lower dissociation constants and have higher affinity for the target than other candidate aptamers. S1 has a higher cross-reaction rate to other targets such as glutamate and poor specificity ( Figure 7 a), thus S2 with high affinity and good specificity was selected as the best aptamer, and its dissociation constants determined by ITC and SGI methods were 10.5±14.7μM and 56.5±10.3nM.

[0091] Table 3 Affinity determination results of candidate aptamers

[0092] sequence ITC method to determine affinity (μM) Affinity determination by SGI method (nM) S1 5.17±12.6 30.9±6.67 S2 10.5±14.7 56.5±10.3 S3 13.9±21.3 64.3±17.7 S4 16.5±17.4 100.3±24.1 S10 33.5±154 154.6±42.3 S17 46.3±379 175.1±50.0

[0093] 9. Analysis of the binding mechanism between the best aptamer and the target

[0094] (1) Analyze the binding mechanism between aptamers and targets based on circular dichroism spectroscopy.

[0095] To further verify the binding interactions between the aptamers and CML and CEL, circular dichroism spectroscopy was used to analyze conformational changes and. Figure 6 As shown in (a), S2 exhibits negative and positive peaks around 245 nm and 275 nm, indicating that both peaks represent typical B-type DNA structures and represent the helicity of DNA and the accumulation of bases in DNA. When S2 binds to CML and CEL, the intensity of the negative peak increases, indicating that the DNA helicity of the aptamer is enhanced, which is attributed to the conformational change of S2 when it binds to the target.

[0096] (2) Analysis of the binding sites between aptamers and targets based on molecular docking

[0097] Molecular docking was used to further explore the interaction sites between the aptamer and CML and CEL. Figure 6 (b) As shown. S2 interacts with CML and CEL mainly through hydrogen bonds, van der Waals forces and electrostatic interactions. Bases G27, T28, G29, C30, A31, C33, C34, T40, G41, G42, A43, G44, C45 and T46 interact with CML, among which G41, G42, A43, G44 and C45 have hydrogen bond interactions with CML. G27, T28, G29, C30, A31, G41, G42, G43, G44, C45 and T46 bases in S2 interact with CEL. Among them, C30, A31, G42, G43 and C45 have hydrogen bond interactions with CEL. In summary, the action sites of S2 and the target are relatively concentrated on the stem-loop structure.

[0098] Example 2: Optimization of aptamer truncation

[0099] 1. Truncate and optimize the original aptamer based on the binding site between the original aptamer and the target

[0100] The stem-loop structure of S2 was kept unchanged, and base pairs were gradually added on both sides of the stem-loop structure to improve the stability of the aptamer, thereby obtaining S2-32, S2-34, S2-36, S2-38 and S2-40, whose nucleotide sequences are shown in SEQ ID NOs: 2 to 6, respectively.

[0101] 2. Affinity and specificity analysis of different aptamers obtained by truncation optimization.

[0102] The affinity (Table 4) and specificity ( Figure 7 b).

[0103] Table 4 Affinity determination of truncated aptamers

[0104] sequence ITC method to determine affinity (μM) Affinity determination by SGI method (nM) S2-32 28.1±34.4 138.9±43.0 S2-34 21.3±135 115.6±32.7 S2-36 14.5±13.9 84.2±16.9 S2-38 7.83±4.32 48.2±11.7 S2-40 6.65±3.07 42.1±19.34

[0105] 3. Select truncated sequences with high affinity and good specificity as aptamers for the target

[0106] With the increase of base number, the stability of aptamer is improved, and the affinity is continuously improved. The affinity of S2-40 is no longer significantly improved on the basis of S2-38, and the specificity is further improved than the original sequence S2. Therefore, S2-40 is selected as the best aptamer. The dissociation constants determined by ITC and SGI methods are 6.65±3.07μM and 42.1±19.34nM, respectively. The specificity is good, and the sequence information is 5'-CCATCCGGGGGTGCACCCATTCTTGGAGCTTCCCGTCAGT-3'.

[0107] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An aptamer of carboxymethyllysine and carboxyethyllysine, characterized in that: The aptamer is a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

2. The aptamer according to claim 1, characterized in that The 5' end or 3' end of the aptamer is connected with a functional group or molecule, and the functional group or molecule is an isotope, a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, an enzyme marker, a magnetic substance, biotin, an affinity ligand and / or a thiol group.

3. A microfluidic SELEX platform for screening the aptamer according to claim 1 or 2, characterized in that: The microfluidic SELEX platform was constructed by the following steps: (1) The Rhino software was used to draw and construct a three-dimensional model of the microfluidic chip. The constructed microfluidic chip structure includes inlet A, inlet B, outlet I, outlet II, two fluorescence quantification chambers, one incubation and separation chamber, and four convenient push-pull valves; (2) Using polymethyl methacrylate as the substrate, the CNC milling machine is used to mill the chamber and flow channel according to the size of the three-dimensional model; (3) cleaning and drying the milled chip, and sealing the chip with a polypropylene sealing film by an ionic bonding method after passivation and hydrophilic and hydrophobic treatment; (4) Use Rhino software to draw and construct a three-dimensional model of the microfluidic SELEX platform. The constructed microfluidic SELEX platform includes an incubation vibration module, a magnetic separation module, and a fluorescence monitoring module; (5) Using 3D printing technology, a microfluidic SELEX platform is manufactured according to the constructed three-dimensional model, and each functional module and microfluidic chip of step (4) are assembled; (6) Installed with microfluidic SELEX enrichment rate monitoring software REMS to monitor the enrichment rate of each round; The microfluidic SELEX platform integrates the following screening steps: library fixation, incubation of target and library, magnetic separation and fluorescence monitoring.

4. A method for screening aptamers of carboxymethyllysine and carboxyethyllysine, characterized in that: The main steps include: (1) Inject streptavidin-labeled magnetic beads and biotinylated capture chain-library complexes into the incubation separation chamber from injection port B and injection port A, respectively, and incubate with vibration for 40 min to immobilize the five-segment library onto the magnetic beads. The unimmobilized library is introduced into the waste liquid pool from the sample outlet II through the fluorescence monitoring chamber; (2) Inject the counter-screening target lysine from the injection port B into the incubation separation chamber for vibration incubation for 1 hour. After magnetic separation, the counter-screening target-library complex is introduced into the waste liquid pool through the sample outlet I; (3) Injecting the target carboxymethyl lysine and carboxyethyl lysine from the injection port B into the incubation separation chamber for vibration incubation for 1 hour. After magnetic separation, the target-library complex is introduced into the collection pool from the sample outlet II through the fluorescence detection chamber; (4) When the initial library, the library not immobilized by magnetic beads, and the library bound to the target pass through the fluorescence quantification chamber, fluorescence images are collected and analyzed by REMS software to obtain the immobilization rate and enrichment rate of each round; (5) amplifying the library in the collection pool at an appropriate PCR annealing temperature and amplification round number, purifying the PCR product, and preparing a secondary library using Lambda exonuclease; (6) After multiple rounds of screening, the enriched products are subjected to high-throughput sequencing, and candidate aptamers are determined based on the sequencing results; (7) The affinity and specificity of the candidate aptamers were analyzed based on isothermal titration calorimetry and SYBR Green I fluorescence assay, and the sequences with the strongest affinity and best specificity were selected as the aptamers for the target.

5. A probe, characterized in that: The probe is a substance obtained by labeling the aptamer according to claim 1 or 2 with a labeling substance.

6. A cell containing the aptamer according to claim 1 or 2 or the probe according to claim 5.

7. A biosensor comprising the aptamer according to claim 1 or 2 or the probe according to claim 5.

8. Use of the aptamer according to claim 1 or 2, the probe according to claim 5, the cell according to claim 6 or the biosensor according to claim 7 in detecting carboxymethyllysine and / or carboxyethyllysine.

9. A reagent or kit for detecting carboxymethyllysine and / or carboxyethyllysine, characterized in that: The reagent or kit contains the aptamer according to claim 1 or 2 or the probe according to claim 5.

10. A method for detecting carboxymethyllysine and / or carboxyethyllysine, characterized in that: The method comprises labeling a reporter group on the aptamer of claim 1 or 2, allowing the aptamer labeled with the reporter group to interact with a sample to be tested, and detecting carboxymethyl lysine and / or carboxyethyl lysine by detecting a signal of the reporter group.

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