Monolithic column fluorescence sensor for high-specificity on-line monitoring of microcystic toxins and preparation method of monolithic column fluorescence sensor

Abstract

The invention discloses a monolithic column fluorescence sensor for high-specificity online monitoring of microcystic toxins and a preparation method of the monolithic column fluorescence sensor. According to the sensor, alkenylated graphene oxide is adopted as an acidic reaction solution and a functional modification material at the same time, an alkenyl silane reagent, an auxiliary siloxane reagent, a pore-foaming agent and an initiator are combined, polymerization reaction is carried out in a quartz capillary tube through a sol-gel method, and a graphene functionalized porous silica gel hybrid monolithic column is formed; and modifying the surface of the monolithic column with a fluorophore-labeled microcystic toxin nucleic acid aptamer by using the pi-pi action between graphene and the nucleic acid aptamer, thereby preparing the monolithic column fluorescence sensor capable of specifically recognizing microcystic toxins on line. When the microcystin-LR exists in a detection sample, the aptamer falls off, and a fluorescence signal in a mobile phase is enhanced, so that high-sensitivity online monitoring is realized. The invention provides a rapid and accurate microcystic toxin detection scheme, and provides an effective means for water body monitoring and safety.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental monitoring and biosensing, and particularly relates to a high-specificity integral column fluorescence sensor for online monitoring of microcystin and a preparation method thereof. Background Art

[0002] Microcystin-LR (MC-LR) is a hepatotoxic toxin produced by cyanobacteria (specifically Microcystis). It is the most common and most widely studied toxin in the microcystin family and is widely present in freshwater environments. This toxin not only poses a serious threat to aquatic ecosystems, but also poses a potential risk to human health. The presence of MC-LR can lead to deterioration of water quality, thereby affecting the safety of drinking water and increasing the risk of human exposure to toxic substances. In addition, MC-LR may also affect aquatic organisms through the food chain, leading to ecological imbalance. Usually, the concentration of MC-LR in the actual environment is low, the background interference is complex, and MC-LR has no obvious characteristic spectrum of fluorescence signal and weak ultraviolet absorption, making it difficult to directly and sensitively analyze and detect it by spectral analysis. At present, the main analytical methods for MC-LR include mouse analysis, cytotoxicity detection, protein phosphatase inhibition and enzyme-linked immunosorbent assay, etc. These methods are mainly used for toxicity screening and total analysis of MCs, while the quantitative analysis and detection methods of MC-LR mainly include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), mass spectrometry, etc. As a classic analytical technique, high performance liquid chromatography has high sensitivity and separation ability, and can effectively detect the content of MC-LR in water. However, this method usually requires complex sample pretreatment steps and a long analysis time, which limits its application in on-site monitoring. Enzyme-linked immunosorbent assay (ELISA) is widely used for its simplicity and high sensitivity, but it may be affected by matrix effects in complex samples, resulting in unstable test results. Although mass spectrometry has extremely high sensitivity and accuracy, the equipment is expensive and the operation is complicated, making it difficult to achieve rapid on-site detection. Therefore, a new type of online monitoring technology is urgently needed to achieve rapid and highly sensitive detection of microcystins.

[0003] In recent years, fluorescence sensing technology has become a research hotspot due to its high sensitivity, rapid response and real-time detection capabilities. Fluorescence sensors can achieve specific recognition and quantitative analysis of target substances by designing suitable fluorescent probes. This technology shows great application potential in the fields of biosensors and environmental monitoring. However, existing fluorescence sensors often face problems of signal interference and insufficient selectivity in complex samples, especially in samples with complex background matrices. The background signal may affect the accurate detection of the target analysis, and the use of fluorescence methods to analyze MC-LR usually requires fluorescent derivatization. Therefore, how to effectively enrich and separate MC-LR in a complex environment and realize chemical label-free derivatization detection has become a key challenge in current research.

[0004] In order to meet the above challenges, the present invention proposes a novel online monitoring scheme, which utilizes a graphene-functionalized porous silica hybrid monolithic column combined with a fluorescently labeled microcystin nucleic acid aptamer, so that the resulting monolithic column not only has excellent affinity and selectivity, but also can significantly improve the sensitivity in real-time detection. Among them, graphene, as a new type of nanomaterial, has excellent electrical conductivity, thermal conductivity and chemical stability, and plays a key role in the construction of the sensor. By loading the fluorescently labeled nucleic acid aptamer on the graphene silica hybrid monolithic column, efficient capture and detection of MC-LR can be achieved. Summary of the invention

[0005] Microcystin is a toxic substance produced in water bodies, which poses a potential threat to aquatic ecology and human health. Existing detection methods often have insufficient sensitivity and long response time, so it is necessary to develop a highly specific online monitoring technology. In response to this problem, the present invention provides a highly specific monolithic column fluorescence sensor for online monitoring of microcystin and a preparation method thereof, which not only helps to ensure the safety of drinking water and the ecological environment, but also provides new ideas and methods for research in related fields. The present invention provides an effective solution for the online monitoring of microcystin through innovative monolithic column design and fluorescence sensing technology, and has important scientific value and application prospects.

[0006] To achieve the above object, the present invention adopts the following technical solution: A high-specificity monolithic column fluorescence sensor for online monitoring of microcystin, which utilizes a "sol-gel" method, uses alkenyl graphene oxide as both an acidic reaction liquid and a functional modification material, uses alkenyl silane reagents and auxiliary siloxane reagents as reaction monomers, and carries out a "one-pot" polymerization reaction with a porogen and an initiator in a quartz capillary to prepare a graphene-functionalized porous silica hybrid monolithic column, and then loads a microcystin nucleic acid aptamer labeled with a fluorescent group on the surface of the obtained graphene silica hybrid monolithic column to prepare a microcystin nucleic acid aptamer-modified graphene silica hybrid affinity monolithic column, which is used as a fluorescence sensor for high-specificity online monitoring of microcystin.

[0007] In the monolithic column fluorescence sensor, alkenyl graphene oxide and alkenyl siloxane monomers are bonded by double bond addition reaction to achieve functionalization; the graphene on the filler and the DNA base of the nucleic acid aptamer are adsorbed and combined with the nucleic acid aptamer through adsorption to form an affinity layer modified with the microcystin nucleic acid aptamer.

[0008] Furthermore, the alkenylated graphene oxide is prepared by functionalizing the graphene oxide surface with alkenyl using methacryloxypropyltri(trimethylsiloxy)silane (γ-MAPS).

[0009] Furthermore, the alkenyl silane reagent is vinyltrimethoxysilane.

[0010] Furthermore, the auxiliary siloxane agent is tetramethoxysilane.

[0011] Furthermore, the porogen is a binary porogen composed of polyethylene glycol and urea.

[0012] Furthermore, the initiator is 2,2'-azobisisobutyronitrile.

[0013] Furthermore, the microcystin is specifically microcystin-LR (MC-LR).

[0014] Furthermore, the fluorescent group-labeled microcystin nucleic acid aptamer is specifically a tetramethylrhodamine (TAMRA)-labeled anti-MC-LR nucleic acid aptamer, and its base sequence is 5′- ATA CCA CCT CAT TAT GCC CCATCT CCG C-3′.

[0015] When there is no MC-LR in the environment, the aptamer sequence can be well adsorbed on the material based on the π-π interaction between graphene and the aptamer base; when there is MC-LR in the environment, the aptamer can bind to MC-LR with high specificity and fall off from the graphene, enhancing the fluorescence signal in the eluate. Based on the high-specific affinity of the aptamer, the monolithic column fluorescence sensor can form an affinity chromatography enrichment, identification and online detection, which is used to achieve high-specificity online fluorescence detection of microcystin.

[0016] The preparation method of the monolithic column fluorescence sensor comprises the following steps: (1) Preparation of olefinic graphene oxide solution: accurately weigh 0.01 g of graphene oxide, add 10 mL of ultrapure water, and ultrasonicate for 60 min to disperse it evenly in the water to form a graphene oxide dispersion. Then, add 20 μL of methacryloxypropyl tris(trimethylsiloxy)silane and stir at room temperature (25 °C) for 24 h to fully olefinate the graphene oxide surface. Then, centrifuge the obtained reaction solution at 10,000 r / min for 10 min to remove unreacted substances. Wash the precipitate with ultrapure water three times and redisperse it in ultrapure water to obtain an olefinic graphene oxide solution. (2) Preparation of graphene functionalized porous silica hybrid monolithic column: the porogen is mixed with the alkenyl graphene oxide solution prepared in step (1), and stirred until the porogen is completely dissolved, then stirred continuously at 500 r / min in a 0°C ice bath, and a mixture of alkenyl silane reagent, auxiliary siloxane reagent and initiator is added dropwise at a uniform speed, and the sol-gel reaction is carried out in an ice bath at 0°C for 45 min. After the reaction is completed, the solution obtained by the reaction is ultrasonically degassed to form a clear, transparent and uniform prepolymer solution, which is then injected into a quartz capillary, sealed at both ends and placed in a 47°C water bath for constant temperature reaction for 24 h. The monolithic column after the reaction is taken out and placed in a 120°C gas phase heating box for heating for 3 h. Finally, the column is rinsed with water and methanol in turn to remove unreacted residues, thereby obtaining a graphene functionalized porous silica hybrid monolithic column. (3) Preparation of graphene-silica gel hybrid affinity monolith modified with microcystin nucleic acid aptamer: A fluorescent group is modified at the 5' end of the microcystin nucleic acid aptamer, and then the microcystin nucleic acid aptamer labeled with the fluorescent group is introduced into the prepared graphene functionalized porous silica gel hybrid monolith, and the nucleic acid aptamer is loaded on the surface of the monolith by utilizing the π-π interaction between the graphene on the monolith stationary phase and the nucleic acid aptamer base, thereby preparing the monolith fluorescent sensor.

[0017] Furthermore, the formula composition of the prepolymer solution in step (2) is, by mass percentage, 21.25% of auxiliary siloxane reagent, 6.73% of alkenyl silane reagent, 57.25% of alkenyl graphene oxide, 14.31% of porogen, and 0.46% of initiator.

[0018] Furthermore, the aptamer fragment selected in step (3) meets the following two conditions: 1) when there is no MC-LR in the detection environment, the aptamer sequence can be well adsorbed on the material based on the π-π interaction between graphene and the aptamer base; 2) when there is MC-LR in the detection environment, the aptamer can bind to MC-LR with high specificity and fall off from the graphene, thereby enhancing the fluorescence signal in the eluate.

[0019] Furthermore, the fluorescent group-labeled microcystin nucleic acid aptamer solution in step (3) is obtained by centrifuging the microcystin nucleic acid aptamer modified with a fluorescent group at the 5' end at 5000 r / min for 3 min, then adding Tris-HCl buffer solution for dissolution, and placing it in a constant temperature water bath at 90°C for 3 min for aptamer activation treatment, and then naturally cooling it to room temperature (25°C) to obtain.

[0020] Furthermore, in the monolithic column fluorescence sensor obtained in step (3), the loading amount of the microcystin nucleic acid aptamer is 4492.72 pmol / μL.

[0021] The present invention is based on the "sol-gel method" reaction of alkenyl graphene oxide, alkenyl silane reagent, auxiliary siloxane reagent, porogen and initiator. No other acid catalytic reagent (such as hydrochloric acid, acetic acid, etc.) needs to be added during the reaction process. The prepolymer obtained by sol-gel is uniformly injected into the pretreated quartz capillary through a booster pump, and a porous silica hybrid monolithic column polymer material with graphene oxide functionalization on the surface is formed in the capillary through a "one-pot method" polymerization reaction. Further, a fluorescent group recognition chain aptamer is loaded on the surface of the monolithic column to prepare a silica hybrid affinity monolithic column modified with microcystin nucleic acid aptamer, and it is used as a fluorescent sensor. When microcystin is present in the sample to be tested, the fluorescent labeled nucleic acid aptamer will specifically bind to the target and fall off the graphene of the monolithic column, so that the fluorescent signal in the eluent is significantly enhanced as the concentration of microcystin increases. The constructed integral column fluorescence sensor integrates affinity chromatography enrichment, recognition and online fluorescence detection, and can be used to achieve high-specificity online detection of microcystins in tap water and other water bodies. It is a trace MC-LR nucleic acid aptamer online affinity recognition and label-free fluorescence analysis technology.

[0022] Furthermore, the fluorescence detection excitation wavelength is 555 nm, and the emission wavelength scanning range is 560-650 nm.

[0023] The monolithic column fluorescent sensor prepared by the present invention combines graphene, silica-based polymer porous materials and nucleic acid aptamer affinity recognition, has excellent affinity specificity and high specific surface area, and can effectively enrich and selectively capture microcystins. Using fluorescently labeled nucleic acid aptamers as probes, through online fluorescence detection technology, when the microcystin to be tested is present, the fluorescently labeled nucleic acid aptamer specifically binds to the target and falls off the graphene, so that the fluorescent signal in the eluent is significantly enhanced as the concentration of microcystin increases, thereby ensuring high sensitivity and accuracy in complex water samples. The design of the sensor realizes rapid and real-time detection of microcystin and greatly reduces background interference, thereby improving the specificity and reliability of detection. Through this technology, the present invention can effectively respond to challenges in water quality monitoring and provide reliable technical support for water source safety and ecological environmental protection.

[0024] The beneficial effects of the present invention are: The invention discloses a high-specificity monolithic column fluorescence sensor for online monitoring of microcystin, which is composed of a functionalized graphene oxide monolithic column and a fluorescently labeled aptamer. The sensor introduces alkenyl graphene oxide as an acidic reaction solution and a functional modification material, combines an alkenyl silane reagent, an auxiliary siloxane reagent and an initiator, and performs a "one-pot" polymerization reaction in a quartz capillary to prepare a graphene silica hybrid monolithic column. Subsequently, a fluorescently labeled nucleic acid aptamer is loaded on the surface of the monolithic column to form a microcystin nucleic acid aptamer-modified silica hybrid affinity monolithic column, which is used as a fluorescent sensor material. The sensor makes full use of bonded graphene and can effectively adsorb MC-LR fluorescently labeled nucleic acid aptamers. Through online combined fluorescence detection technology, when MC-LR is present in the sample to be tested, the fluorescently labeled aptamer can specifically bind to the target and quickly fall off the monolithic column, so that a significantly enhanced fluorescence signal is generated in the eluent, thereby realizing efficient online detection of MC-LR in a complex environment. With its excellent selectivity and sensitivity, the minimum detection limit of the monolithic column fluorescence sensor can reach 5×10 -11 mol / L, which effectively realizes the rapid online monitoring of trace amounts of microcystins without the need for additional fluorescent labeling.

[0025] The preparation method of the monolithic column of the present invention is simple and easy, can be completed in a short time, and is suitable for on-site real-time monitoring applications. Compared with traditional detection methods, the fluorescence sensor of the present invention has significant advantages in sensitivity and selectivity, can effectively reduce background interference, and improve detection accuracy.

[0026] The invention provides a novel and effective technical means, which has important practical application value and has broad application prospects in water quality monitoring and environmental protection. The high sensitivity and selectivity of this sensor make it an important tool for microcystin detection and can provide strong protection for water safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a schematic diagram of the preparation process and affinity mechanism of the monolithic column fluorescence sensor of the present invention.

[0028] Figure 2 3 is an electron microscope morphology image of the integral column fluorescence sensor prepared in Example 1, wherein A is a cross-sectional scanning electron microscope image of the integral column fluorescence sensor, and B is a partial magnified image of the cross-sectional scanning electron microscope image.

[0029] Figure 3 This is a schematic diagram of the operation of online specific analysis and detection of MC-LR without fluorescence labeling in Example 2.

[0030] Figure 4 It is a graph of the specific analysis detection results of the integral column fluorescence sensor in Example 2, wherein A is the spectral analysis graph of different samples, B is the physical photograph of different sample eluates (a-100 nM OTA, b-100 nM MC-RR, c-100nM MC-YR, d-100 nM MC-LR, e: -100 nM mixed sample eluate), and C is the fluorescence response value of different samples.

[0031] Figure 5 The results of analyzing and detecting MC-LR of different concentrations using the integral column fluorescence sensor in Example 2 are shown in Figure 2, wherein A is the fluorescence spectra of MC-LR of different concentrations, and B is the logarithmic working curve of MC-LR concentration.

[0032] Figure 6 This is a graph showing the results of specific analysis and detection of MC-LR using different integral column fluorescence sensors in Example 2.

[0033] Figure 7 This is a result diagram of the detection and analysis of MC-LR in tap water samples with different spiked concentrations using the integral column fluorescence sensor in Example 3, wherein A is the fluorescence spectrum of water samples with different spiked concentrations, and B is the fluorescence intensity. DETAILED DESCRIPTION

[0034] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0035] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0036] The specifications of the quartz capillary used in the examples are 365 μm in outer diameter and 100 μm in inner diameter.

[0037] The Tris-HCl buffer solution used in the examples consists of 10 mM Tris-HCl, 120 mM NaCl and 5 mM KCl, with a pH of 7.4.

[0038] Example 1 A high-specificity monolithic column fluorescence sensor for online monitoring of microcystin, the preparation method of which comprises the following specific steps: 1. Before preparing the monolithic column fluorescence sensor, in order to ensure the close bonding between the column filler and the capillary column wall and improve the fixation of the column bed, the inner surface of the capillary needs to be pretreated. The specific implementation process is as follows: 1) Flushing the capillary: The empty capillary column is first rinsed with 1.0 mol / L hydrochloric acid solution (HCl) for 30 minutes to remove impurities in the capillary; 2) Neutral treatment: The capillary column is rinsed twice with ultrapure water until its pH value reaches neutral; 3) Alkaline etching: The capillary column is rinsed with 1.0 mol / L sodium hydroxide solution (NaOH) for 30 minutes to etch the inner wall of the capillary to increase the number of exposed silanol groups. 4) High temperature treatment: After the treatment, the two ends of the capillary column are sealed with rubber stoppers, and then placed in a gas phase heating box at 100 °C and heated for 3 hours to promote etching reaction and drying. 5) Neutral treatment again: Repeat the use of ultrapure water to rinse the capillary twice to ensure that the pH value of the inner wall is neutral again. 6) Acid washing: Use 0.1 M hydrochloric acid solution to rinse the capillary for another 30 minutes to further clean the inner wall. 7) Final neutral treatment: Rinse with ultrapure water again to ensure that the pH value of the inner wall remains in the neutral range. 8) Methanol washing: Finally rinse the capillary with methanol for 30 minutes to remove residues and ensure that the inner wall is dry. 9) Nitrogen drying: Connect the pretreated capillary to the gas phase heating box, pass nitrogen, set the gas pressure to 0.4 MPa, set the furnace temperature to 180 ℃, and blow nitrogen for 3 hours to fully dry the inner wall of the capillary to obtain a pretreated capillary column.

[0039] 2. Preparation of olefinic graphene oxide solution: Accurately weigh 0.01 g of graphene oxide in a reaction bottle, add 10 mL of ultrapure water, and ultrasonicate for 60 min to disperse it evenly in the water to form a graphene oxide aqueous solution with a concentration of 1 mg / mL. Then, add 20 μL of methacryloxypropyl tris(trimethylsiloxy)silane (γ-MAPS) to the reaction bottle and stir at room temperature (25 °C) for 24 h to allow the olefinic silanization reagent to be fully hydrolyzed and fully react with the graphene oxide surface. Subsequently, the obtained reaction solution was centrifuged at 10000 r / min for 10 min to remove unreacted substances, and the precipitate was washed 3 times with ultrapure water and redispersed in ultrapure water to obtain olefinic graphene oxide solution.

[0040] 3. Preparation of graphene functionalized porous silica hybrid monolithic column: Weigh the pore-forming agent urea and polyethylene glycol in proportion in a 10 mL round-bottom flask, then weigh the prepared olefinic graphene oxide solution and add it to the above round-bottom flask, stir until the pore-forming agent is completely dissolved, and then place it in a 0 ℃ ice bath and continue stirring at a speed of 500 r / min; separately weigh the olefinic silane reagent vinyltrimethoxysilane, the silanization reagent tetramethoxysilane and the initiator 2,2'-azobisisobutyronitrile in a centrifuge tube according to the ratio, vortex and shake until the initiator is completely dissolved, then uniformly add the mixed reaction reagents to the above round-bottom flask, and place the round-bottom flask in a 0 ℃ ice bath for sol-gel reaction for 45 min; after the reaction, place the obtained solution in an ultrasonicator for ultrasonic degassing to form a clear and uniform prepolymer solution, and then inject it into the pretreated capillary, seal both ends and place it in a 47 ℃ water bath for constant temperature reaction for 24 h; then the reacted monolithic column was taken out and placed in a 120 ℃ gas phase heating box for heating for 3 h; finally, the column was flushed with water and methanol in turn through a high-pressure infusion pump to remove unreacted residues, thereby obtaining a graphene-functionalized porous silica hybrid monolithic column.

[0041] Among them, calculated by mass percentage, the contents of each component in the prepolymer solution are: 21.25% tetramethoxysilane, 6.73% vinyltrimethoxysilane, 57.25% olefinated graphene oxide, 8.08% urea, 6.23% polyethylene, and 0.46% 2,2'-azobisisobutyronitrile.

[0042] 4. Preparation of fluorescent group-labeled microcystin nucleic acid aptamer solution: Place the microcystin nucleic acid aptamer modified with a fluorescent group at the 5' end (purchased from Sangon Biotech (Shanghai) Co., Ltd.) in a centrifuge and centrifuge at 5000 r / min for 3 min. Then gently open the centrifuge tube cover and use a pipette to transfer a certain volume of Tris-HCl buffer solution for dissolution. Place the solution in a 90℃ constant temperature water bath and heat for 3 min to activate the aptamer. Cool the solution naturally to room temperature (25℃) to obtain the nucleic acid aptamer solution, and place it in a refrigerator at 4℃ for later use.

[0043] 5. Preparation of monolithic column fluorescence sensor: The fluorescent group-labeled microcystin nucleic acid aptamer solution was introduced into the prepared graphene functionalized porous silica hybrid monolithic column through a high-pressure infusion pump. The nucleic acid aptamer was loaded on the surface of the monolithic column by utilizing the π-π interaction between the graphene on the monolithic column stationary phase and the nucleic acid aptamer base, and a monolithic column fluorescence sensor with a nucleic acid aptamer loading of 4492.72 pmol / μL was obtained.

[0044] Figure 2 The electron microscope morphology of the prepared monolithic column fluorescence sensor is shown in Figure 1, where A is a cross-sectional scanning electron microscope image of the monolithic column fluorescence sensor and B is a partial magnified image of the cross-sectional scanning electron microscope image. Figure 2 It can be seen that the polymer filler of the prepared monolithic column fluorescence sensor column is tightly combined with the inner wall of the capillary, and there is no wall detachment. At the same time, the pore structure of the column filler is evenly distributed, with a rich double-pore structure, and there is no cracking or collapse in the column structure, indicating that the column is not damaged during the modification of the nucleic acid aptamer. The prepared graphene nucleic acid aptamer affinity monolith still has a relatively uniform morphology and structure, without shrinkage or cracking. These basic structural characteristics can provide a strong guarantee for the mechanical strength and rigidity of the monolithic column, which can be further used for subsequent specific analysis and detection of target analytes.

[0045] Example 2 like Figure 3 , a high-specificity online monitoring of microcystin monolithic column fluorescence sensor for MC-LR fluorescence label-free analysis and detection, which includes four processes: online balancing, online sample enrichment, elution and online detection. The specific operations of each process are as follows: 1. Online balancing: Connect the monolithic column fluorescence sensor to a high-pressure infusion pump, and use the high-pressure infusion pump to pass the Tris-HCl binding buffer into the affinity monolithic column through the injection module, and balance and activate the affinity monolithic column for 10 minutes; 2. Online sample injection and enrichment: The sample solution is injected into the sensor through a high-pressure infusion pump, and the nucleic acid aptamer on the sensor column is used for on-column recognition and enrichment. During the sample injection process, the pump flow rate is 0.05 mL / min, and a 250 psi back pressure valve is used; 3. Online elution: After sample enrichment, the binding buffer is switched to the washing buffer to clean the sensor, and the fluorescently labeled nucleic acid aptamer that has been detached from the graphene after binding to the algae toxin is washed off. Under the action of the high-pressure pump, the eluted liquid is brought into the fluorescence detection system; 4. Online detection: The eluted liquid is quantitatively analyzed and detected by combining fluorescence detection technology.

[0046] The monolithic column fluorescence sensor prepared in Example 1 makes full use of bonded graphene and can effectively adsorb MC-LR fluorescently labeled nucleic acid aptamers. When MC-LR is present in the sample to be tested, the fluorescently labeled aptamer can specifically bind to the target and quickly fall off from the monolithic column, resulting in a significantly enhanced fluorescence signal in the eluent, thus realizing efficient online quantitative analysis and detection of MC-LR in a complex environment. The detection excitation wavelength is 555 nm, and the emission wavelength scanning range is 560~650nm.

[0047] Different types of microcystins (MC-LR, MC-YR and MC-RR), ochratoxin A (OTA) and mixed samples of the above toxins were selected to evaluate the specificity of the monolithic column fluorescence sensor prepared in Example 1. The concentration of all samples was 100 nM. The results are as follows: Figure 4 .like Figure 4 As shown in the figure, when the injected samples are MC-YR, MC-RR and OTA, no or only very weak fluorescence signals are detected; when the injected samples are MC-LR or mixed samples, obvious fluorescence signals are detected. The above shows that only when MC-LR exists in the sample solution can the corresponding fluorescence signal be detected, indicating that the monolithic column fluorescence sensor has good specificity and can be used for MC-LR specific online analysis and detection.

[0048] Figure 5 The results of analyzing and detecting different concentrations of MC-LR using the monolithic column fluorescence sensor prepared in Example 1 are shown in FIG. Figure 5As shown in the figure, within the MC-LR concentration range of 0.05~100 nmol / L, as the MC-LR concentration in the sample increases, the fluorescence peak intensity of the online detection gradually increases, that is, the number of target MC-LR binding to the fluorescent labeled aptamer increases, causing more fluorescent aptamers to fall off the graphene monolith, resulting in an increase in the fluorescence signal of the eluate (A); the fluorescence intensity measured by the fluorescence detection system is positively correlated with the MC-LR concentration in the sample, and has a good linear relationship within the MC-LR concentration range of 0.05~100 nM, with a correlation coefficient R 2 =0.9903(B).

[0049] At the same time, the blank silica gel monolith (unmodified graphene) and the unolefinated graphene monolith were loaded with fluorescent group-labeled microcystin nucleic acid aptamers to prepare blank monolith fluorescence sensors and control monolith fluorescence sensors, and compared with the monolith fluorescence sensor prepared in Example 1 to evaluate the specificity and sensitivity of the monolith fluorescence sensor prepared in Example 1. The results are shown in FIG. Figure 6 . When a 100 nM concentration of MC-LR sample was used for detection, the fluorescence sensor constructed using a blank silica gel monolith almost failed to detect a fluorescence signal, and the control monolith fluorescence sensor could detect a fluorescence signal, but the fluorescence signal was much lower than that of the monolith fluorescence sensor prepared in Example 1. This shows that the olefination treatment of graphene can not only make it more firmly bonded and modified on the monolith, but also the formed fluorescent monolith sensor can effectively adsorb the microcystin nucleic acid aptamer labeled with a fluorescent group to produce moderate fluorescence. Therefore, the present invention provides a highly sensitive and specific online monitoring method that can effectively detect the presence of microcystin-LR.

[0050] Example 3: On-line analysis and detection of MC-LR in water samples by monolithic column fluorescence sensor The integrated column fluorescence sensor prepared in Example 1 was used to perform specific recognition and online fluorescence analysis on tap water samples with different MC-LR spike concentrations. The results are as follows: Figure 7 shown. Figure 7 It shows that when the MC-LR in the tap water sample is 0 nM, no fluorescence signal is detected in the eluent. As the concentration of MC-LR spiked in the tap water sample increases, the fluorescence signal of the aptamer fluorescent marker in the corresponding eluent gradually increases, indicating that the monolithic column fluorescence sensor can be well applied to the analysis and detection of MC-LR in water environment. When the concentration of MC-LR spiked in the water sample is 0.05 nM, the concentration at this time reaches the minimum detection limit (LOD) of the MC-LR standard solution. The results show that in actual samples with complex background matrices, the prepared monolithic column fluorescence sensor can still achieve good specific recognition ability for MC-LR and maintain high detection sensitivity.

[0051] At the same time, the recoveries of water samples with different MC-LR spiked concentrations were calculated to further verify the feasibility in actual samples. The results are shown in Table 1.

[0052] Table 1 Recovery of MC-LR in tap water samples

[0053] As can be seen from Table 1, in tap water samples with MC-LR spiked concentrations of 0.5, 1.0 and 5.0 nM, the recognition and capture recovery rates of the monolithic column fluorescence sensor for MC-LR were 103.1±2.7%, 98.9±1.4% and 98.2±2.4%, respectively, and the detection relative standard deviations (RSDs) were all less than 2.7%. The above results indicate that the monolithic column fluorescence sensor can be well applied to the reliable analysis and detection of MC-LR in tap water samples, and has good recovery and good reproducibility.

[0054] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A high-specificity monolithic column fluorescence sensor for online monitoring of microcystins, characterized in that: The method utilizes the "sol-gel" method, uses alkenyl graphene oxide as both an acidic reaction liquid and a functional modification material, uses alkenyl silane reagents and auxiliary siloxane reagents as reaction monomers, and carries out polymerization reaction with porogens and initiators in a quartz capillary to prepare a graphene functionalized porous silica hybrid monolithic column, and then loads a microcystin nucleic acid aptamer labeled with a fluorescent group on the surface of the obtained graphene silica hybrid monolithic column to prepare a microcystin nucleic acid aptamer-modified graphene silica hybrid affinity monolithic column, which is used as a fluorescent sensor for high-specificity online monitoring of microcystin.

2. The integral column fluorescence sensor according to claim 1, characterized in that: The alkenylated graphene oxide is prepared by using methacryloxypropyl tris(trimethylsiloxy)silane to alkenylate the graphene oxide surface; the alkenyl silane reagent is vinyl trimethoxysilane; the auxiliary siloxane reagent is tetramethoxysilane; the porogen is a binary porogen composed of polyethylene glycol and urea; and the initiator is 2,2'-azobisisobutyronitrile.

3. The monolithic column fluorescence sensor for online monitoring of microcystins with high specificity and its preparation method according to claim 1, characterized in that: The fluorescent group-labeled microcystin nucleic acid aptamer is specifically a tetramethylrhodamine-labeled anti-microcystin-LR nucleic acid aptamer, and its base sequence is 5′- ATA CCA CCT CAT TAT GCCCCA TCT CCG C-3′.

4. A method for preparing a monolithic column fluorescence sensor as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of olefinic graphene oxide solution: accurately weigh 0.01 g of graphene oxide, add 10 mL of ultrapure water, and disperse it evenly in the water by ultrasonication for 60 min to form a graphene oxide dispersion. Then, add 20 μL of methacryloxypropyl tris(trimethylsiloxy)silane and stir at room temperature for 24 h to fully olefinate the graphene oxide surface. Then, centrifuge the obtained reaction solution at 10,000 r / min for 10 min to remove unreacted substances. Wash the precipitate with ultrapure water three times and redisperse it in ultrapure water to obtain an olefinic graphene oxide solution. (2) Preparation of graphene functionalized porous silica hybrid monolithic column: the porogen is mixed with the alkenyl graphene oxide solution prepared in step (1), and stirred until the porogen is completely dissolved, then stirred continuously at 500 r / min in a 0°C ice bath, and a mixture of alkenyl silane reagent, auxiliary siloxane reagent and initiator is added at a uniform rate, and reacted in an ice bath at 0°C for 45 min; after the reaction, the obtained solution is ultrasonically degassed to form a clear, transparent and uniform prepolymer solution, which is then injected into a quartz capillary, sealed at both ends and placed in a 47°C water bath for constant temperature reaction for 24 h, then the reacted monolithic column is taken out and placed in a 120°C gas phase heating box for heating for 3 h, and finally the column is rinsed with water and methanol in turn to remove unreacted residues, thereby obtaining a graphene functionalized porous silica hybrid monolithic column; (3) Preparation of graphene-silica gel hybrid affinity monolith modified with microcystin nucleic acid aptamer: Based on the π-π interaction between graphene and nucleic acid aptamer bases on the monolithic column stationary phase, a solution of microcystin nucleic acid aptamer labeled with a fluorescent group is passed into the prepared graphene functionalized porous silica gel hybrid monolithic column, and the nucleic acid aptamer is loaded on the surface of the monolithic column, thereby preparing the monolithic column fluorescent sensor.

5. The preparation method according to claim 4, characterized in that: Calculated by mass percentage, the formula composition of the prepolymer solution in step (2) is: 21.25% of auxiliary siloxane reagent, 6.73% of alkenyl silane reagent, 57.25% of alkenyl graphene oxide, 14.31% of porogen, and 0.46% of initiator.

6. The preparation method according to claim 4, characterized in that: The fluorescent group-labeled microcystin nucleic acid aptamer solution in step (3) is obtained by centrifuging the fluorescent group-labeled nucleic acid aptamer at 5000 r / min for 3 min, then adding Tris-HCl buffer solution to dissolve it, heating it in a 90°C constant temperature water bath for 3 min to activate the aptamer, and then naturally cooling it to room temperature.

7. The preparation method according to claim 4, characterized in that: In the monolithic column fluorescence sensor obtained in step (3), the loading amount of the microcystin nucleic acid aptamer is 4492.72 pmol / μL.

8. Use of the monolithic column fluorescence sensor as claimed in claim 1 in high-specificity online monitoring of microcystins in water.

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