A high-specificity microcystin detection whole column-sers / fluorescence dual-mode detection platform and a construction method thereof

Abstract

The application provides a high-specificity detection microcystin whole column-SERS / fluorescence dual-mode detection platform and a construction method thereof. The platform takes a three-dimensional porous amino silica gel monolithic column as a carrier, solidifies specific recognition probes through electrostatic adsorption, constructs the whole column-SERS / fluorescence dual-mode detection platform through base complementary pairing of cDNA and aptamers on the specific probe of Au@Ag@cDNA / Aptamer-Cy3, and utilizes the Au@Ag core-shell structure to generate SERS 'hot spot' enhancement effect. When target algae toxin molecules exist in the sample, the aptamers are specifically combined with the target substances and separated from the monolithic column, so that the base SERS signal is weakened and the fluorescence signal of the effluent is enhanced, high-sensitivity and high-specificity dual-mode qualitative or quantitative detection of microcystin is realized through the dual signal changes, and the detection sensitivity and accuracy are improved.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology for environmental pollutants, specifically relating to a monolithic column-SERS / fluorescence dual-mode detection platform for the detection of microcystin and its construction method. Background Technology

[0002] Microcystins (MCs) are a class of cyclic heptacapeptide hepatotoxins produced by cyanobacteria, with microcystin-LR (MC-LR) considered a representative subtype due to its high toxicity and widespread availability. These toxins exhibit extremely high chemical and thermal stability, making them difficult to completely degrade using conventional water treatment processes, thus posing a persistent threat to global freshwater ecosystems and human health. The World Health Organization (WHO) has set a guideline of 1.0 μg / L for MC-LR in drinking water, highlighting the necessity of strict monitoring. However, MC concentrations in natural water bodies are typically at trace levels (ng / L to μg / L), and the presence of coexisting organic matter and inorganic ions poses significant background interference, placing extremely high demands on the sensitivity and anti-interference capabilities of detection technologies.

[0003] Currently, conventional methods for detecting microorganisms (MCs) mainly rely on large-scale analytical instruments and bioidentification techniques. High-performance liquid chromatography (HPLC) and its coupling with mass spectrometry (LC-MS) are considered the "gold standard" for MC detection, possessing excellent separation and accurate quantification capabilities. However, these methods generally suffer from inherent drawbacks such as cumbersome sample pretreatment, long analysis cycles, expensive equipment, and the need for specialized operators, making it difficult to meet the urgent need for rapid, high-frequency screening in water sources, reservoirs, and other similar environments. While enzyme-linked immunosorbent assay (ELISA) offers advantages such as ease of operation and high throughput, and is widely used in environmental screening, its results are susceptible to matrix effects in water samples, posing a risk of false positives. Furthermore, it struggles to distinguish between different MC subtypes, and its accuracy and specificity in complex environmental samples need improvement.

[0004] To overcome the aforementioned limitations, optical signal-based sensing technologies, particularly surface-enhanced Raman scattering (SERS) and fluorescence analysis, have become research hotspots due to their high sensitivity, rapid response, and potential portability. SERS technology, through the localized surface plasmon resonance effect generated by noble metal nanostructures, can amplify the Raman signal of adsorbed molecules by millions of times, theoretically enabling single-molecule detection. However, existing SERS technologies face two major challenges when applied to the detection of molecular weight compounds (MCs): firstly, MC molecules themselves have small Raman scattering cross sections and lack strong affinity groups for noble metal surfaces, resulting in insufficient direct detection sensitivity; secondly, the uniformity and reproducibility of the SERS substrate are difficult to control, leading to significant signal fluctuations and affecting the reliability of quantitative analysis. On the other hand, while fluorescence analysis offers advantages such as ease of operation and intuitive signal delivery, MCs themselves do not possess fluorescence properties, typically requiring complex fluorescent labeling or derivatization reactions. This not only increases the detection steps and time but may also introduce additional interference factors. More importantly, both SERS and fluorescence methods, when applied alone in complex water samples, are susceptible to background matrix interference, leading to a decrease in the signal-to-noise ratio and detection limits that fail to meet practical requirements.

[0005] Another key bottleneck in existing technologies lies in the lack of efficient pretreatment and effective integration of specific recognition units. While traditional solid-phase extraction columns can achieve a certain degree of enrichment, they are often disconnected from subsequent detection steps and have limited selective adsorption capacity for MCs. Aptamers, as artificially synthesized oligonucleotides obtained through in vitro screening techniques, possess high affinity and specificity comparable to antibodies, and have unique advantages such as good stability and ease of synthesis and modification, making them ideal recognition elements for constructing biosensors. However, how to stably and densely immobilize aptamers on the sensing interface and achieve sensitive and reliable signal output after target binding remains a key challenge in current aptamer sensor research. Furthermore, single detection modes often struggle to guarantee the reliability and accuracy of results when dealing with samples from complex environments.

[0006] To address the systemic shortcomings of existing technologies, such as insufficient detection sensitivity, weak anti-interference ability, complex pretreatment, and lack of rapid on-site detection capabilities, this invention aims to provide an innovative solution. This invention proposes for the first time the construction of a "monolithic column-SERS / fluorescence dual-mode detection platform" integrating efficient enrichment, specific recognition, and dual-mode signal output. First, this platform prepares a three-dimensional porous amino-silica monolithic column through in-situ polymerization within a quartz capillary using a "one-pot" method. This monolithic column not only possesses a continuous, interconnected porous structure, enabling rapid mass transfer and efficient enrichment of samples, but its abundant amino groups provide high-density active sites for subsequent probe immobilization. Second, an Au@Ag core-shell nanostructure is designed and constructed as a SERS enhancement substrate. By precisely controlling the silver shell thickness, the "hot spot" effect is maximized, significantly enhancing the SERS signal intensity. Finally, highly stable recognition probes are constructed using base complementarity pairing between cDNA and Cy3-labeled MCs-specific aptamers, and these probes are immobilized on the surface of the monolithic column through electrostatic adsorption.

[0007] When the sample flows through the functionalized monolithic column, MCs molecules are specifically captured by the aptamer, causing a conformational change in the aptamer and its dissociation from the monolithic column. This process triggers two key signal changes: firstly, the number of probes remaining on the monolithic column decreases, resulting in a significant weakening of the intensity of its SERS characteristic peak (quantified at 1188 cm⁻¹); secondly, the dissociation of the Cy3-labeled aptamer-MCs complex into the effluent leads to a corresponding enhancement of the fluorescence signal. This "give and take" dual-mode signal response mechanism not only achieves internal self-verification of the detection results, greatly improving the reliability and accuracy of the detection, but also effectively overcomes the drawback of single detection modes being susceptible to environmental interference. This invention, by cleverly integrating the separation and enrichment advantages of the monolithic column, the high specificity of the aptamer's recognition capability, and the synergistic advantages of SERS / fluorescence dual-mode detection, successfully overcomes the bottlenecks of existing technologies in terms of sensitivity, specificity, and field applicability, providing a novel technical path for achieving highly sensitive, reliable, and rapid online monitoring of trace MCs in water. Summary of the Invention

[0008] The purpose of this invention is to provide a highly specific monolithic column-SERS / fluorescence dual-mode detection platform for the detection of microcystin and its construction method. The prepared detection platform combines an aminosilicone monolithic column, an Au@Ag core-shell nanostructure, and the specific recognition function of nucleic acid aptamers, exhibiting superior affinity selectivity and high specific surface area, enabling effective enrichment and selective capture of microcystin. Using Cy3-labeled nucleic acid aptamers as signal probes, and employing online SERS and fluorescence dual-mode detection technology, when the target microcystin is present, the aptamer specifically binds to the target and detaches from the monolithic column, resulting in a weakening of the substrate SERS signal. Simultaneously, the fluorescence signal in the eluent significantly increases with increasing microcystin concentration. This dual-signal variation mode enables mutual verification of detection results, ensuring high sensitivity and accuracy in complex water samples. The platform design achieves rapid, real-time dual-mode detection of microcystin, significantly reducing background interference through signal complementarity, thereby improving the specificity and reliability of the detection. This invention effectively addresses the challenges in water quality monitoring, providing reliable technical support for water source safety and ecological environmental protection.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A highly specific online monitoring platform for microcystin-based monolithic column-SERS / fluorescence dual-mode detection comprises three parts: a sample introduction system, a detection substrate, and a detection system. The sample introduction system utilizes a unidirectional syringe pump for precise sample control. The detection substrate is constructed using an amino-silicone monolithic column as the base, with Au@Ag@cDNA / Aptamer-Cy3 specific recognition probes immobilized via electrostatic adsorption. The amino-silicone monolithic column is formed by in-situ polymerization within a pretreated quartz capillary using a one-pot method, creating a three-dimensional porous structure. The abundant amino functional groups on the column surface provide high-density active sites for probe immobilization, and its continuous, interconnected porous structure ensures rapid mass transfer and efficient enrichment of the sample. Furthermore, the Au@Ag@cDNA / Aptamer-Cy3 probe is modified onto the porous packing surface within the monolithic column via electrostatic adsorption, forming a stable dual-mode sensing interface. The Au@Ag core-shell structure, through controlled silver deposition, creates a high-density SERS "hotspot," significantly enhancing detection sensitivity. Meanwhile, the Cy3-labeled aptamer binds to cDNA via complementary base pairing, achieving highly specific recognition of microcystins. When microcystins are present in the sample, the aptamer specifically binds to the target and detaches from the monolithic column, resulting in a weakening of the substrate SERS signal. Simultaneously, the Cy3 fluorescence signal in the eluent significantly increases with increasing target concentration. This "ebb and flow" dual-signal variation pattern enables internal self-verification of the detection results, effectively overcoming the susceptibility to environmental interference inherent in single-mode detection. The constructed platform integrates efficient enrichment, specific recognition, and dual-mode signal output, ensuring high sensitivity, high specificity, and high reliability in microcystin detection through the mutual verification mechanism of SERS and fluorescence signals.

[0011] The amino-silica hybrid monolithic column is a three-dimensional porous monolithic column prepared by in-situ polymerization in a quartz capillary via a one-pot method using tetraethoxysilane (TEOS) and 3-(2-aminoethylamino)propyltriethoxysilane (AEAPTES) as reactants and hexadecyltrimethylammonium bromide (CTAB) as a porogen.

[0012] The quartz capillary is a UV-transmitting quartz capillary with an inner diameter of 100 μm and an outer diameter of 360 μm.

[0013] The polymerization conditions for the monolithic column are as follows: first, react in a 40°C water bath for 24 hours, and then heat at 120°C for 3 hours to solidify and stabilize the skeleton structure.

[0014] The aptamer is a Cy3-labeled microcystin-specific nucleic acid aptamer, and its base sequence is as follows:

[0015] 5′- Cy3-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATTATG CCC CAT CTC CGC-3′;

[0016] The complementary strand cDNA sequence is: 5′- GTC CTG TTT GGC GAC TTT TTT TTT T-(CH2)3-SH-3′, and the microcystin is microcystin-LR (MC-LR).

[0017] The monolithic column-SERS / fluorescence dual-mode detection platform innovatively constructs a dual-mode signal response mechanism based on aptamer-specific recognition. When MC-LR is absent in the detection environment, the probe is stably immobilized on the surface of the amino silica monolithic column through electrostatic adsorption between the Au@Ag core-shell nanostructure and the Cy3-labeled aptamer, forming a high-density SERS "hot spot" and obtaining a significant Cy3 characteristic Raman signal. When MC-LR is present in the sample, the aptamer specifically binds to the target analyte, causing the probe to dissociate from the monolithic column, resulting in a weakening of the substrate SERS signal. Simultaneously, the Cy3-labeled aptamer-MC-LR complex dissociated into the mobile phase generates a significantly enhanced fluorescence signal. Based on the high specific affinity of nucleic acid aptamers, this platform integrates efficient enrichment, specific recognition, and dual-mode signal output. Through a mutual verification mechanism between SERS and fluorescence signals, it achieves high sensitivity, high specificity, and high reliability in the detection of microcystin toxins.

[0018] The method for constructing a monolithic column-SERS / fluorescence dual-mode detection platform for highly specific detection of microcystin includes the following steps:

[0019] Quartz capillary pretreatment: Rinse the capillary with 1.0 mol / L HCl for 30 min, then pass secondary water through it until the pH of the outflow is neutral (about 30 min). Continuously pass 1.0 mol / L NaOH into the capillary for 30 min, seal both ends of the capillary, and then heat at 100℃ under nitrogen protection for 3 h. Afterward, rinse the capillary with water for 30 min (rinse until neutral), rinse with 0.1 mol / L HCl for 30 min, then rinse with secondary water for 30 min, continuously pass methanol through it for 30 min, and then heat at 180℃ under nitrogen protection for 3 h. Dry and set aside for later use.

[0020] Preparation of the aminosilicone monolithic column: 11.1 mg CTAB, 200.0 μL ethanol, and 50.0 μL water were mixed and sonicated at 0 °C for 5 min. Then, 40.0 μL AEPETES and 160.0 μL TEOS were added to the above mixture and sonicated at 0 °C for 30 s to form a homogeneous solution. The resulting solution was pumped into a pretreated capillary. The capillary was then immersed in a constant temperature water bath at 40 °C for 24 h, and both ends were sealed with silicone rubber. Finally, the obtained capillary monolithic column was rinsed with ethanol and water for 30 min each using a high-pressure infusion pump to remove residual reagents, yielding the aminosilicone monolithic column.

[0021] The amounts of each component used in the preparation steps of the amino silica monolithic column, calculated as a sum of 100% by mass, are as follows: the mass percentages of each component are: tetraethoxysilane 41.08%, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane 10.68%, hexadecyltrimethylammonium bromide 2.44%, water 11.01%, and anhydrous ethanol 34.79%.

[0022] Preparation of Au NPs: 99 mL of ultrapure water and 1 mL of 1% tetrachloroauric acid solution were added to a 250 mL three-necked round-bottom flask to prepare a 0.01% HAuCl4 reaction solution. The mixture was heated to boiling under constant temperature and magnetic stirring at 130 °C. Then, 0.75 mL of 1% sodium citrate solution was quickly added. The color of the reaction system successively changed from pale yellow to grayish-black to purplish-red to wine-red. After maintaining boiling and stirring for 15 min, heating was stopped, and the mixture was allowed to cool naturally to room temperature. The resulting colloidal solution of gold nanoparticles was stored at 4 °C for later use. Dynamic light scattering characterization confirmed that the average particle size was 50 ± 3 nm.

[0023] Preparation of nucleic acid aptamer solution: 5' end-modified Cy3 fluorescent group-modified nucleic acid aptamers and their complementary strand cDNA lyophilized powders were placed in centrifuge tubes and centrifuged at 5000 r / min for 5 min to collect the raw materials at the bottom of the tube. Then, 10 mmol / L tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution was added to specifically cleave the disulfide bonds in the molecules through a reduction reaction to restore the active site. The dissolved nucleic acid solution was precisely diluted to a working concentration of 100 μmol / L using 10 mmol / L Tris-HCl buffer (pH=7.4). Before use, the solution was vortexed for 1 minute to ensure uniform molecular dispersion, followed by thermal activation in a 90℃ water bath for 3 minutes to promote the formation of standard secondary structures in the nucleic acid chains. Finally, the solution was cooled to room temperature (25℃) and stored at 4℃ for later use.

[0024] Preparation of Au@Ag@cDNA NPs: 10 mL of gold nanoparticle colloid with an average particle size of 50 nm was added, and 10 μL of activated cDNA solution with a concentration of 100 μmol / L was added. The mixture was incubated at 4℃ and 400 rpm for 24 hours. During the incubation, 20 μL of 1 mol / L NaCl solution was added three times at intervals to perform gradient salt aging, promoting the directional self-assembly of cDNA on the surface of gold nanoparticles through Au-S bonds. Subsequently, 2 mL of Au@cDNA NPs colloid and 13.4 mL of ultrapure water were added to a 50 mL three-necked flask and mixed thoroughly under a 40°C water bath and 800 rpm stirring conditions. Then, 180 μL of 1.0% ascorbic acid (AA) and 180 μL of 1.0% sodium citrate (CA) were added rapidly as reducing agent and stabilizer, respectively. After stirring for 15 min, 60 μL of 10 mmol / L AgNO3 solution was added dropwise at a precise flow rate of 10 μL / min using a microsyringe. The reaction was continued for 30 min at 40°C and 800 rpm. Finally, Au@Ag@cDNA core-shell nanoparticle colloid with uniform particle size was obtained by centrifugation purification.

[0025] Preparation of Au@Ag@cDNA / Aptamer-Cy3 probe: 2 μL of a 100 μmol / L Cy3-labeled aptamer solution was precisely added to 2 mL of Au@cDNA@Ag NPs colloidal solution, and the mixture was vortexed for 2 min at room temperature to achieve initial mixing. The mixture was then transferred to a constant-temperature water bath and heat-treated at 95℃ for 5 min to induce double-stranded DNA dissociation and activate the aptamer binding sites. The solution was then slowly cooled to room temperature (25℃) to allow the aptamers to specifically hybridize and assemble with cDNA according to the base pairing principle. After the reaction, unbound nucleic acids were removed by centrifugation, and the probe was finally redispersed in ultrapure water to obtain a homogeneous Au@Ag@cDNA / Aptamer-Cy3 probe solution, which was stored at 4℃ for later use.

[0026] Preparation of a monolithic column-SERS / fluorescence dual-mode detection platform: Au@Ag@cDNA / Aptamer-Cy3 probe solution was passed into an amino silica monolithic column at a precise flow rate of 20 μL / min. Through electrostatic adsorption between the amino groups and the nanoprobes, a uniform and stable functionalized modification layer was formed on the inner surface of the column, thus preparing the monolithic column-SERS / fluorescence dual-mode detection substrate. This detection substrate was integrated with a quantitative injection pump, a Raman spectrometer, and a fluorescence spectrophotometer to construct a complete online detection platform. Using microcystin LR (MC-LR) as the research model, in the target analyte environment, the Cy3-labeled aptamer specifically binds to MC-LR, causing the probe to detach from the monolithic column. This results in a weakening of the Cy3 SERS signal on the monolithic column detection platform, while the Cy3 fluorescence signal in the elution buffer is enhanced. Highly sensitive and specific quantitative detection of microcystin is achieved through changes in both SERS and fluorescence signals.

[0027] The Raman spectroscopy detection uses the characteristic peak of Cy3 at 1188 cm⁻¹ as the quantitative analysis index; the fluorescence detection uses the fluorescence intensity in the range of 560-650 nm as the quantitative basis.

[0028] Furthermore, the aforementioned monolithic column-SERS / fluorescence dual-mode detection platform can be applied to environmental water quality monitoring, food safety testing, or biomedical diagnosis.

[0029] The construction and dual-mode detection schematic diagram of the above-mentioned monolithic column-SERS / fluorescence dual-mode detection platform are shown below. Figure 1 As shown.

[0030] The beneficial effects of this invention are as follows: The monolithic column-SERS / fluorescence dual-mode detection platform for highly specific detection of microcystin disclosed in this invention consists of three parts: an injection system, a detection substrate, and a detection system. The constructed monolithic column-SERS / fluorescence dual-mode detection platform achieves technological breakthroughs through multiple innovative mechanisms.

[0031] (1) Based on the synergistic effect of the three-dimensional porous structure of the monolithic amino silica column and the Au@Ag core-shell nanoprobe, a SERS / fluorescence dual-mode detection platform with a high-density "SERS hotspot" detection interface was constructed, enabling the SERS detection sensitivity to reach 3.16×10⁻⁶. −13 mol / L, while the fluorescence detection sensitivity reaches 1.47×10 mol / L. −12 mol / L.

[0032] (2) The innovative dual-mode self-validation mechanism of "SERS signal attenuation - fluorescence signal enhancement" exhibits excellent linearity (R² > 0.99) in the MC-LR concentration range of 0.5-100 nM, effectively overcoming the limitations of a single detection mode. Experimental results show that the platform achieves a recovery rate of 95.6%-103.7% in the SERS detection mode with a relative standard deviation of less than 4.5%, and a recovery rate of 96.8%-102.4% in the fluorescence mode with a relative standard deviation of less than 3.4%, demonstrating excellent accuracy and reproducibility.

[0033] (3) The platform detection time is shortened to within 10 minutes, which has significant advantages for on-site application. This invention is the first to organically combine whole column separation and enrichment, aptamer-specific recognition and dual-mode detection technology. The reliability of the detection results is ensured through the signal complementarity mechanism, providing an innovative solution for the safe monitoring of microcystin content. It has important application value in the fields of environmental monitoring and food safety analysis. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram illustrating the construction of a monolithic column-SERS / fluorescence dual-mode detection platform and its dual-mode detection capabilities.

[0036] Figure 2 Figure A shows the electron microscope (EM) morphology of the monolithic amino silicone column in Example 1. Figure B and C are magnified partial views of the cross-sectional SEM images of the monolithic amino silicone column.

[0037] Figure 3 This is a schematic diagram of the preparation of the monolithic column-SERS / fluorescence dual-mode detection substrate in Example 1.

[0038] Figure 4 Figure A shows the electron microscope morphology of the monolithic column-SERS / fluorescence dual-mode detection substrate in Example 1. Figure B and C are magnified partial views of the cross-sectional scanning electron microscope images of the monolithic column substrate.

[0039] Figure 5 This is a schematic diagram of the online SERS / fluorescence dual-mode detection process for microcystin samples.

[0040] Figure 6 This is a specific analysis of the overall column-SERS / fluorescence dual-mode detection platform for MC-LR detection. In the figure, the purple bars represent the SERS response of different samples, and the blue bars represent the fluorescence intensity of different samples (from left to right: OTA, MC-RR, MC-YR, MC-LR, and mixed samples).

[0041] Figure 7 The SERS mode detection of different concentrations of MC-LR was performed using a monolithic column-SERS / fluorescence dual-mode detection platform. In this paper, A shows the SERS spectra of different concentrations of MC-LR, and B shows the logarithmic working curve of MC-LR concentration.

[0042] Figure 8 The fluorescence mode detection of different concentrations of MC-LR is performed using a monolithic column-SERS / fluorescence dual-mode detection platform. In this diagram, A shows the fluorescence spectra of different concentrations of MC-LR, and B shows the logarithmic working curve of MC-LR concentration. Detailed Implementation

[0043] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0044] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0045] Example 1

[0046] A highly specific monolithic column-SERS / fluorescence dual-mode detection platform for detecting microcystin and its construction method:

[0047] Quartz capillary pretreatment: Select a quartz capillary with an inner diameter of 100 μm and perform the following steps in sequence:

[0048] 1) Impurity removal: Rinse with 1.0 mol / L HCl for 30 min; 2) Neutralization cleaning: Rinse with ultrapure water until the effluent pH is neutral; 3) Surface etching and activation: Rinse with 1.0 mol / L NaOH for 30 min, then seal both ends of the capillary and heat at 100 ℃ for 3 h to increase the density of silanol groups; 4) Secondary neutralization: Rinse with ultrapure water until neutral; 5) Deep cleaning: Rinse with 0.1 mol / L HCl for 30 min; 6) Final neutralization: Rinse with ultrapure water until neutral; 7) Organic solvent washing: Rinse with methanol for 30 min to remove residues; 8) Drying treatment: Purge with 0.4 MPa nitrogen gas at 180 ℃ for 3 h. Through alternating acid-base treatment and high-temperature activation, the silanol groups on the inner wall of the capillary were fully exposed and impurities were completely removed, ensuring a firm bond between the subsequent monolithic column packing and the tube wall, thus providing a guarantee for constructing a stable microcystin detection platform interface.

[0049] Preparation of monolithic amino silica columns:

[0050] First, 11.1 mg of cetyltrimethylammonium bromide (CTAB) was dissolved in a mixed solvent of 200.0 μL anhydrous ethanol and 50.0 μL ultrapure water, and sonicated in an ice bath at 0 °C for 5 min. Then, 40.0 μL of N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) and 160.0 μL of tetraethoxysilane (TEOS) were added sequentially, and sonication in an ice bath was continued for 30 s to form a homogeneous prepolymer. The prepolymer was injected into a pretreated UV-permeable quartz capillary tube, and both ends were sealed. The tube was reacted in a water bath at 40 °C for 24 h. Finally, the tube was washed sequentially with ethanol and ultrapure water for 30 min each to remove the pore-forming agent, yielding an amino silica monolithic column.

[0051] The amounts of each component used in the preparation steps of the amino silica monolithic column, calculated as a sum of 100% by mass, are as follows: the mass percentages of each component are: tetraethoxysilane 41.08%, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane 10.68%, hexadecyltrimethylammonium bromide 2.44%, water 11.01%, and anhydrous ethanol 34.79%.

[0052] Figure 2 The scanning electron microscope (SEM) morphology of the monolithic aminosilicone column is shown. Figure A shows the cross-sectional morphology of the monolithic column, while Figures B and C are magnified images of its local areas.

[0053] Depend on Figure 2As can be seen, the prepared monolithic column polymer filler is tightly bonded to the inner wall of the capillary, without any interface peeling or wall detachment. The internal pore structure of the column is uniformly distributed, exhibiting a rich double-pore interconnected structure, and the monolithic column is free from structural defects such as cracking or collapse. This structural feature effectively ensures the mechanical strength and structural stability of the monolithic column, indicating that its preparation process is reliable and the material properties are excellent, providing an ideal structural basis for the subsequent construction of a SERS / fluorescence dual-mode detection platform based on Au@Ag@cDNA / Aptamer-Cy3 probe modification.

[0054] Preparation of Au NPs: 99 mL of ultrapure water and 1 mL of 1% tetrachloroauric acid solution were added to a 250 mL three-necked round-bottom flask to prepare a 0.01% HAuCl4 reaction solution. The mixture was heated to boiling under constant temperature and magnetic stirring at 130 °C. Then, 0.75 mL of 1% sodium citrate solution was quickly added. The color of the reaction system successively changed from pale yellow to grayish-black to purplish-red to wine-red. The mixture was kept at boiling and stirred for 15 min before heating was stopped. The mixture was allowed to cool naturally to room temperature. The resulting colloidal solution of gold nanoparticles was stored at 4 °C for later use. Dynamic light scattering characterization confirmed that the average particle size was 50 ± 3 nm.

[0055] Preparation of nucleic acid aptamer solution: 5' end-modified Cy3 fluorescent group-modified nucleic acid aptamers and their complementary strand cDNA lyophilized powders were placed in centrifuge tubes and centrifuged at 5000 r / min for 5 min to collect the raw materials at the bottom of the tube. Then, 10 mmol / L tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution was added to specifically cleave the disulfide bonds in the molecules through a reduction reaction to restore the active site. The dissolved nucleic acid solution was precisely diluted to a working concentration of 100 μmol / L using 10 mmol / L Tris-HCl buffer (pH=7.4). Before use, the solution was vortexed for 1 minute to ensure uniform molecular dispersion, followed by thermal activation in a 90℃ water bath for 3 minutes to promote the formation of standard secondary structures in the nucleic acid chains. Finally, the solution was cooled to room temperature (25℃) and stored at 4℃ for later use.

[0056] Preparation of Au@Ag@cDNA NPs: 10 mL of gold nanoparticle colloid with an average particle size of 50 nm was added, and 10 μL of activated cDNA solution with a concentration of 100 μmol / L was added. The mixture was incubated at 4℃ and 400 rpm for 24 hours. During the incubation, 20 μL of 1 mol / L NaCl solution was added three times at intervals to perform gradient salt aging, promoting the directional self-assembly of cDNA on the surface of gold nanoparticles through Au-S bonds. Subsequently, 2 mL of Au@cDNA NPs colloid and 13.4 mL of ultrapure water were added to a 50 mL three-necked flask and mixed thoroughly under a 40°C water bath and 800 rpm stirring conditions. Then, 180 μL of 1.0% ascorbic acid (AA) and 180 μL of 1.0% sodium citrate (CA) were added rapidly as reducing agent and stabilizer, respectively. After stirring for 15 min, 60 μL of 10 mmol / L AgNO3 solution was added dropwise at a precise flow rate of 10 μL / min using a microsyringe. The reaction was continued for 30 min at 40°C and 800 rpm. Finally, Au@Ag@cDNA core-shell nanoparticle colloid with uniform particle size was obtained by centrifugation purification.

[0057] Preparation of Au@Ag@cDNA / Aptamer-Cy3 probe: 2 μL of a 100 μmol / L Cy3-labeled aptamer solution was precisely added to 2 mL of Au@cDNA@Ag NPs colloidal solution, and the mixture was vortexed for 2 min at room temperature to achieve initial mixing. The mixture was then transferred to a constant-temperature water bath and heat-treated at 95℃ for 5 min to induce double-stranded DNA dissociation and activate the aptamer binding sites. The solution was then slowly cooled to room temperature (25℃) to allow the aptamers to specifically hybridize and assemble with cDNA according to the base pairing principle. After the reaction, unbound nucleic acids were removed by centrifugation, and the probe was finally redispersed in ultrapure water to obtain a homogeneous Au@Ag@cDNA / Aptamer-Cy3 probe solution, which was stored at 4℃ for later use.

[0058] Preparation of the monolithic column-SERS / fluorescence dual-mode detection platform: Au@Ag@cDNA / Aptamer-Cy3 probe solution was passed into an amino silica monolithic column at a precise flow rate of 20 μL / min. Through electrostatic adsorption between the amino groups and the nanoprobes, a uniform and stable functionalized modification layer was formed on the inner surface of the column, thus preparing the monolithic column-SERS / fluorescence dual-mode detection substrate. This detection substrate was integrated with a quantitative injection pump, a Raman spectrometer, and a fluorescence spectrophotometer to construct a complete online detection platform. Using microcystin LR (MC-LR) as the research model, when a sample containing MC-LR flows through the detection substrate, the Cy3-labeled aptamer specifically binds to the target analyte, causing the probe to dissociate from the monolithic column surface. This triggers a dual-mode response: a decrease in the SERS signal on the substrate and an increase in the fluorescence signal in the elution buffer, thereby achieving highly sensitive quantitative detection of MC-LR.

[0059] Figure 3 This is a schematic diagram of the substrate preparation for detection. Figure 4 The scanning electron microscope (SEM) morphology of the substrate is shown. Figure A shows the cross-sectional morphology of the substrate, while Figures B and C are magnified images of its local areas.

[0060] Depend on Figure 4 As can be seen, the prepared monolithic column-SERS / fluorescence dual-mode detection substrate exhibits tight bonding with the capillary inner wall without detachment, and its surface is uniformly covered with probe nanoparticles of uniform particle size. This dense and ordered nanostructure significantly increases the specific surface area, providing an ideal interface for SERS "hot spot" formation and fluorescence signal enhancement, fully demonstrating the structural superiority and application reliability of this detection substrate.

[0061] Example 2

[0062] This embodiment specifically discloses the operation procedure of a monolithic column-SERS / fluorescence dual-mode detection platform for highly specific detection of microcystin. The platform consists of three parts: a sample introduction system, a detection substrate, and a detection system. Its dual-mode detection process includes the following steps:

[0063] 1) Pretreatment: Take the sample to be tested, extract and process it, filter it with a filter membrane, and then sonicate it at 25℃ to remove air bubbles;

[0064] 2) Sample Injection and Specific Identification: After equilibrating the detection system with Tris-HCl buffer (pH=7.4), 20 μL of sample was injected into the monolithic column substrate using a sample pump at a flow rate of 2 μL / min. The microcystin to be tested specifically bound to the Au@Ag@cDNA / Aptamer-Cy3 probe immobilized on the substrate surface, causing the probe to dissociate from the monolithic column. Subsequently, residual sample and free probe were washed with Tris-HCl buffer.

[0065] 3) Dual-mode signal acquisition:

[0066] SERS detection: The entire column substrate was scanned using a micro Raman spectrometer (20x objective lens, 785 nm excitation wavelength, 47.5 mW laser intensity, 6000 ms integration time), and the intensity change of the characteristic peak of Cy3 at 1188 cm⁻¹ was used as the quantitative basis.

[0067] Fluorescence detection: The effluent was analyzed simultaneously using a fluorescence spectrophotometer with an excitation wavelength of 555 nm and an emission wavelength of 560–650 nm. Cross-validation was achieved by measuring the fluorescence signal enhancement value.

[0068] 4) Results evaluation: Based on the synergistic response of SERS signal attenuation and fluorescence signal enhancement, a dual standard curve was established to achieve highly sensitive and specific quantitative analysis of microcystin.

[0069] Figure 5 This diagram illustrates the use of a monolithic column-SERS / fluorescence dual-mode detection platform for MCs specific analysis.

[0070] Figure 6 This study demonstrates the specificity analysis of the monolithic column-SERS / fluorescence dual-mode detection platform for MC-LR detection. The purple bars in the figure represent the SERS responses of different samples, while the blue bars represent the fluorescence intensities of different samples (from left to right: OTA, MC-RR, MC-YR, MC-LR, and a mixed sample). Experiments were performed on samples containing 100 nM OTA, 100 nM MC-RR, 100 nM MC-YR, 100 nM MC-LR, and a mixed sample containing 100 nM OTA, 100 nM MC-RR, 100 nM MC-YR, and 100 nM MC-LR. Figure 6 As shown, in SERS detection mode, the response signals of OTA, MC-RR, and MC-YR remained stable at a high level (purple bars), while MC-LR and mixed samples caused a significant decrease in signal. In fluorescence detection mode, only MC-LR and mixed samples produced obvious fluorescence responses (blue bars). The dual-mode data together indicate that this platform can effectively eliminate interference from other toxins and exhibits high specificity for MC-LR.

[0071] Figure 7 The SERS mode detection of different concentrations of MC-LR was performed using a monolithic column-SERS / fluorescence dual-mode detection platform. In the figure, A is the SERS spectrum of different concentrations of MC-LR, and B is the logarithmic working curve of MC-LR concentration. Figure 8This section describes the fluorescence detection of different concentrations of MC-LR using a monolithic column-SERS / fluorescence dual-mode detection platform. A shows the fluorescence spectra of MC-LR at different concentrations, and B shows the logarithmic working curves for MC-LR concentrations. Figure 7 As shown, at 1.0×10 -12 ~ 1.0×10 -6 Within the MC-LR concentration range of mol / L, the SERS signal decreases sharply with increasing MC-LR concentration in the injected sample (e.g., Figure 7 As shown in Figure A), a good linear response was obtained between the changes in the SERS signal and the MC-LR content, with a correlation coefficient (R0). 2 The value is 0.9914 (e.g.) Figure 7 As shown in B), the SERS detection limit LOD (3σ / m) reached 3.16×10⁻⁶. −13 mol / L. The intensity of the fluorescence peak detected online gradually increases (e.g., ...). Figure 8 (As shown in A). The fluorescence intensity measured by the fluorescence detection system is positively correlated with the MC-LR concentration in the sample, and the MC-LR concentration at 1.0 × 10⁻⁶ is also positively correlated. -11 ~ 1.0×10 -6 It exhibits good linearity within the mol / L range, with a correlation coefficient R0. 2 =0.9924 (e.g.) Figure 8 As shown in B), the limit of detection (LOD) for fluorescence (3σ / m) reached 1.47 × 10⁻⁶. −12 mol / L. The two detection modes are cross-validated through signal verification, effectively improving the reliability of the detection results and making this method an effective platform for the ultrasensitive detection of microcystin toxins.

[0072] Example 3

[0073] Practical application verification of the monolithic column-SERS / fluorescence dual-mode detection platform in fish samples:

[0074] The dual-mode detection platform prepared in this invention was used to specifically identify and quantitatively analyze fish samples with different MC-LR spike concentrations, thereby verifying the feasibility of this detection method in the analysis of actual fish samples. Relevant recovery rate data are detailed in Table 1. Specifically, the monolithic column-based SERS / fluorescence dual-mode online detection platform demonstrated excellent performance in fish samples with MC-LR spike concentrations of 0.5 nM, 1.0 nM, and 5.0 nM. For MC-LR identification and capture, the recovery rate of SERS mode detection ranged from 95.6 ± 3.3% to 103.7 ± 4.5%, with relative standard deviations (RSDs) of less than 4.5% for all results; while the recovery rate of online fluorescence mode detection ranged from 96.8 ± 3.4% to 102.4 ± 2.7%, with RSDs of less than 3.4% for all results. The above experimental results fully demonstrate that the monolithic column-SERS / fluorescence dual-mode online detection platform can be efficiently and reliably applied to the analysis and detection of MC-LR in fish meat samples with complex actual matrices. It not only exhibits excellent recovery rate but also good reproducibility.

[0075] The results show that the MCs monolithic column-SERS / fluorescence dual-mode online detection platform constructed in this experiment can be used to achieve highly sensitive and specific quantitative SERS / fluorescence dual-mode analysis and detection of algal toxins.

[0076] Table 1 Recovery rate of MC-LR in fish meat samples

[0077]

[0078] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A highly specific monolithic column-SERS / fluorescence dual-mode detection platform for detecting microcystin, characterized in that: The platform consists of three parts: a sample introduction system, a detection substrate, and a detection system. The injection system uses a quantitative injection pump; The detection substrate is a dual-mode sensing interface constructed by electrostatic adsorption and immobilization of Au@Ag@cDNA / Aptamer-Cy3 specific recognition probes on the surface of an amino-silica hybrid monolithic column polymerized in situ within a capillary. The detection system integrates a micro Raman spectrometer and a fluorescence spectrophotometer to simultaneously acquire the SERS characteristic signal of Cy3 on the monolithic column and the fluorescence signal of Cy3 in the elution. When microcystin is present in the sample, the modified Cy3 aptamer specifically binds to the target, causing the probe to dissociate from the monolithic column. This triggers a dual-mode response of weakened SERS signal on the substrate and enhanced fluorescence signal in the elution. Through signal complementarity and mutual verification mechanisms, microcystin is quantitatively detected.

2. The detection platform according to claim 1, characterized in that: The amino-silicone hybrid monolithic column is a three-dimensional porous monolithic column prepared by in-situ polymerization in a quartz capillary via a one-pot method using tetraethoxysilane (TEOS) and 3-(2-aminoethylamino)propyltriethoxysilane as reactants and hexadecyltrimethylammonium bromide as a porogen.

3. The detection platform according to claim 2, characterized in that: The quartz capillary is a UV-transparent quartz capillary with an inner diameter of 100 μm and an outer diameter of 360 μm. The in-situ polymerization conditions are: first react in a 40℃ water bath for 24 h, and then heat at 120℃ for 3 h to cure.

4. The detection platform according to claim 1, characterized in that: The preparation method of the Au@Ag@cDNA / Aptamer-Cy3 specific recognition probe includes the following steps: (1) Au NPs with a particle size of 50 nm were prepared by sodium citrate reduction method; (2) Thiolized cDNA is modified onto the surface of AuNPs via Au-S bonds to form Au@cDNA NPs; (3) Using ascorbic acid as a reducing agent, AgNO3 was reduced in situ on the surface of Au@cDNA NPs to form a silver shell, thus obtaining the Au@Ag@cDNA core-shell structure; (4) Construct the complete Au@Ag@cDNA / Aptamer-Cy3 probe by base complementary pairing of cDNA and Cy3-labeled aptamer.

5. The detection platform according to claim 4, characterized in that: The Cy3-labeled aptamer is a Cy3-labeled microcystin LR-specific nucleic acid aptamer, and its base sequence is as follows: 5′- Cy3-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATGCCC CAT CTC CGC-3′; The cDNA sequence is: 5′- GTC CTG TTT GGC GAC TTT TTT TTT T-(CH2)3-SH-3′.

6. The method for preparing the monolithic column-SERS / fluorescence dual-mode detection platform as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Pretreatment of quartz capillary: The capillary was rinsed with 1 mol / L NaOH solution, ultrapure water and methanol in sequence, and then activated by heating at 120℃; (2) Preparation of monolithic amino silica column: CTAB, ethanol and water were mixed and sonicated at 0 °C for 5 min; then, AEPETES and TEOS were added to the above mixed solution and sonicated at 0 °C for 30 s to form a homogeneous prepolymer solution. The resulting solution was pumped into the pretreated capillary tube; then the capillary tube was immersed in a constant temperature water bath at 40 °C for 24 h and both ends were sealed with silicone rubber; finally, the obtained monolithic capillary column was rinsed with ethanol and water for 30 min respectively to remove the residual reagents. (3) Preparation of Au@Ag@cDNA / Aptamer-Cy3 probe; (4) Preparation of monolithic column-SERS / fluorescence dual-mode detection platform: The probe solution is passed into an amino silica monolithic column, and the inner surface of the column is modified by the electrostatic adsorption between the monolithic column surface and the probe. Au@Ag@cDNA / Aptamer-Cy3 probe is functionalized on the surface of the monolithic column to prepare the monolithic column-SERS / fluorescence dual-mode detection substrate. It is further connected with a quantitative injection pump and a detection system to construct a monolithic column-SERS / fluorescence dual-mode detection platform. Using microcystin LR as the research model, in the target environment, the Cy3-labeled aptamer specifically binds to microcystin LR, and the probe detaches from the monolithic column, resulting in a weakening of the Cy3 SERS signal on the monolithic column detection platform. At the same time, the Cy3 fluorescence signal in the elution solution is enhanced. The high sensitivity and high specificity of microcystin LR can be achieved by the changes in SERS and fluorescence signals.

7. The preparation method according to claim 6, characterized in that: The composition of the prepolymer solution in step (2) is as follows: based on the sum of mass percentages of 100%, the mass percentages of each component are: tetraethoxysilane 41.08%, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane 10.68%, hexadecyltrimethylammonium bromide 2.44%, water 11.01%, and anhydrous ethanol 34.79%.

8. A method for detecting microcystin using the detection platform according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Sample pretreatment: After the sample to be tested is extracted, it is filtered with an aqueous membrane and the air bubbles are removed by ultrasonication at 25°C; (2) Sample injection: Equilibrate the detection platform with Tris-HCl buffer, and introduce 20 μL of sample into the detection platform at a flow rate of 2 μL / min; (3) Dual-mode detection: The SERS signal of Cy3 on the whole column was detected by Raman spectrometer with an excitation wavelength of 785 nm, a laser intensity of 47.5 mW, and an integration time of 6000 ms; at the same time, the fluorescence signal of Cy3 in the effluent was detected by fluorescence spectrophotometer with an excitation wavelength of 555 nm and an emission wavelength of 560-650 nm. (4) Results analysis: A dual standard curve was established by the degree of SERS signal intensity reduction and fluorescence signal enhancement to achieve accurate quantification of microcystin.

9. The detection method according to claim 8, characterized in that: In step (3), the characteristic peak of Cy3 at 1188 cm⁻¹ is used as the quantitative analysis index for Raman spectroscopy detection; the fluorescence intensity in the range of 560-650 nm is used as the quantitative basis for fluorescence detection.

10. The application of the monolithic column-SERS / fluorescence dual-mode detection platform as described in any one of claims 1-5 in environmental water quality monitoring, food safety testing, or biomedical diagnosis.

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