Electrochemiluminescence sensor based on multi-channel microfluidic chip and its application in detection of aflatoxin b1 in edible oil

Abstract

The application belongs to the field of electrochemiluminescence (ECL) sensing and analytical detection, and relates to an electrochemiluminescence sensor based on a multi-channel microfluidic chip and application thereof in detection of aflatoxin B1 (AFB1) in edible oil. The ECL sensor comprises a carbon ink patterned electrode layer, an insulating layer and a channel layer; a circular working area of the carbon ink patterned electrode layer overlaps with a first through hole of the insulating layer and a second through hole of the channel layer to form a reaction cell; four third through holes of the channel layer are respectively used for dropping mixed solution of a target probe-molecularly imprinted polymer, an eluent, a sample solution to be measured and an electrolyte, and all can be injected into the reaction cell through the channel. The target probe-molecularly imprinted polymer can specifically capture and identify AFB1 after eluting AFB1, causing a change in electrochemiluminescence signal. The ECL sensor constructed by the application can collect ECL signals using a portable electrochemiluminescence detector, thereby realizing quantitative analysis of AFB1, and the ECL sensor has high sensitivity and good selectivity.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence sensing and analysis technology, specifically relating to an electrochemiluminescence sensor based on a multi-channel microfluidic chip and its application in the detection of aflatoxin B1 (AFB1) contamination in edible oils. Background Technology

[0002] Currently, the main methods for quantifying AFB1 in edible oils include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), fluorescence spectroscopy, and electrochemiluminescence (ECL) sensing. HPLC determination of AFB1 in edible oils requires first extracting the toxin from the oil sample using an extractant, then purifying and enriching it using techniques such as immunoaffinity column extraction or magnetic solid-phase extraction, followed by separation and detection by HPLC. This method generally involves complex pretreatment and is time-consuming, and HPLC typically requires professional laboratory testing, which cannot meet the needs of rapid on-site detection of edible oils. ELISA determination of AFB1 in edible oils requires a series of pretreatment steps such as homogenization, vortexing, and centrifugation to obtain the supernatant and extract the toxin from the oil sample. Its detection principle utilizes horseradish peroxidase to label AFB1, which specifically binds to an anti-aflatoxin B1 antibody (Anti-AFB1) based on a competitive immune response, allowing for rapid quantification through colorimetric intensity. However, this method is susceptible to matrix interference leading to false positives, and Anti-AFB1 is difficult to prepare, costly, and requires strict storage. These factors together result in the overall low sensitivity of this method, making it difficult to meet the requirements for high-precision detection.

[0003] Fluorescence methods include traditional fluorescence spectroscopy and fluorescence immunochromatography. The core principle of fluorescence spectroscopy for detecting AFB1 in edible oils is utilizing the physical property of AFB1 molecules to produce fluorescence under excitation light of a specific wavelength. Quantitative detection of AFB1 is achieved by monitoring changes in fluorescence signal intensity. Fluorescence immunochromatography utilizes fluorescently labeled antibodies to bind to AFB1 in edible oils. The ratio of fluorescence signals at the test line (T line) and control line (C line) on the test strip is used to achieve rapid and accurate quantification of AFB1 in edible oils. Compared to HPLC and ELISA, while fluorescence methods allow for rapid on-site detection of AFB1 in edible oils, they still suffer from problems such as cumbersome pretreatment, high cost and difficulty in preserving fluorescently labeled antibodies, and inherent background fluorescence interference with the detection signal. Furthermore, fluorescence immunochromatography itself has technical limitations, including a tendency to produce false positive signals and relatively low detection sensitivity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an electrochemiluminescence sensor based on a multi-channel microfluidic chip to address the shortcomings of the prior art. This sensor can realize the quantitative analysis and detection of AFB1 in edible oil with low detection cost and high accuracy.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows: The electrochemiluminescence sensor based on a multi-channel microfluidic chip comprises three parts: a carbon-patterned electrode layer (i.e., a sensing layer), an insulating layer, and a channel layer. The carbon-patterned electrode layer includes a circular working area and three strip-shaped lead areas. An insulating layer containing a first through-hole is disposed above the circular working area. A channel layer is constructed above the insulating layer using an insulating material. The channel layer has a second through-hole and four third through-holes, all of which are connected to the second through-holes. The four third through-holes are used for adding a mixture of a target probe and a molecularly imprinted polymer, an eluent, a sample solution, and an electrolyte, respectively, with the outlet located on the sidewall of the second through-hole. The circular working area, the first through-hole, and the second through-hole overlap to form a reaction cell. The third through-holes and the channels between them and the second through-holes are isolated from the carbon-patterned electrode layer by the insulating layer; that is, neither of these two structural components contacts the carbon-patterned electrode layer.

[0006] According to the above scheme, in the mixture of target probe and molecularly imprinted polymer, the target probe is 1~3 mg / mL, the solvent is n-hexane, and the mass ratio of target probe to molecularly imprinted polymer is 1:(150~250).

[0007] The target probe is a composite of CsPbBr3 quantum dots and honeycomb porous carbon materials (HPCMs), abbreviated as CsPbBr3QDs@HPCMs. The theoretical mass ratio of HPCMs to CsPbBr3QDs in this target probe CsPbBr3QDs@HPCMs composite is (2~4):1. The molecularly imprinted polymer uses the target compound AFB1 as the imprinted molecule, and is generated through photopolymerization with functional monomers and crosslinking agents under the action of an initiator. The CsPbBr3 quantum dots have a size of 6~8 nm, with a UV characteristic absorption peak in the range of 512~530 nm, and the maximum emission wavelength is obtained at 315~320 nm, with the maximum emission peak located at 519~521 nm. The HPCMs material has a pore size of 30~50 nm and a specific surface area of ​​not less than 70 m². 2 / g.

[0008] According to the above scheme, the eluent is preferably ethanol, which is used to elute the imprinted molecules (i.e., target AFB1) in the molecularly imprinted polymer to form a cavity structure of the target, thereby realizing the identification of the target.

[0009] According to the above scheme, the electrolyte is a 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid ([Bmim][PF6]) solution, preferably dissolved in ethyl acetate (EA) to a concentration of 0.05~0.15 M.

[0010] According to the above scheme, the insulating layer is preferably a polydimethylsiloxane (PDMS) layer, which can be formed by coating and curing a mixed liquid composed of PDMS prepolymer and curing agent in a mass ratio of 10:1.

[0011] According to the above scheme, the sample solution to be tested is the eluent of edible oil after treatment with a humic acid solid-phase extraction column (HAS-SPE column). More preferably, the preparation method of the sample solution to be tested is as follows: 1 g of edible oil is diluted with 4-6 mL of n-hexane and vortexed, then added to the HAS-SPE column and washed with 4-6 mL of isopropanol, followed by washing with 10 mL of acetone / water (8 / 2, v / v Elution was performed, the eluent was collected, and filtered through a 0.22 μm filter membrane to obtain the test sample solution of edible oil.

[0012] Preferably, the diameters of the first through hole, the second through hole, and the circular working area are the same, ranging from 6 to 10 mm; the depth of the reaction tank is 1 to 1.5 mm; the diameter of the third through hole is ranging from 1 to 3 mm, and the depth is 1 to 1.5 mm; the channel between the third through hole and the second through hole has a width of 1 to 2 mm, a length of 2 to 2.5 mm, and a depth of 1 to 1.5 mm.

[0013] Based on the above, the present invention also provides a method for fabricating the above-mentioned electrochemiluminescence sensor based on a multi-channel microfluidic chip, comprising the following steps: (1) Using a plastic substrate (such as PET) as the electrode support material, a carbon ink pattern containing a circular working area and strip leads is formed on its surface to obtain a carbon ink patterned electrode (CPE). (2) A mixture of PDMS prepolymer and curing agent is coated on the surface of a glass slide. After curing, a PDMS film layer, i.e. an insulating layer, is obtained. A first through hole is opened on the insulating layer. The first through hole is overlapped with the circular working area of ​​the carbon ink patterned electrode. The diameter of the first through hole is the same as that of the circular working area, which can completely expose the circular working area. (3) A channel layer is constructed by an insulating material. The channel layer has one second through hole and four third through holes. All the third through holes are connected to the second through holes. The diameter of the second through hole is the same as that of the first through hole. The channel layer is stacked on top of the insulating layer, completely exposing the first through hole, so that the circular working area, the first through hole and the second through hole overlap to form a reaction pool. (4) Prepare a mixture of target probe-molecularly imprinted polymer, eluent and electrolyte respectively, and combine them with the device obtained in step (3) to form the electrochemiluminescence sensor based on multichannel microfluidic chip described in this invention.

[0014] According to the above scheme, a small amount of a mixture of PDMS prepolymer and curing agent is applied between the insulating layer and the carbon ink patterned electrode, and between the insulating layer and the channel layer. After curing, the two are fixedly connected.

[0015] The aforementioned electrochemiluminescence sensor based on a multi-channel microfluidic chip can be used to detect AFB1 in edible oils. The application method includes the following steps: 1) Using acetonitrile as solvent, accurately weigh AFB1 standard to prepare AFB1 standard solution, store at 4 ℃ protected from light for later use; 2) ① Inject the mixture of target probe and molecularly imprinted polymer into the reaction cell through any of the third through holes, and irradiate it under ultraviolet light with a wavelength of 360~370 nm for 8~10 min to immobilize the target probe and molecularly imprinted polymer on the surface of the circular working area of ​​the carbon ink patterned electrode; wherein, the amount of the target probe and molecularly imprinted polymer mixture added per unit area is 0.2~0.4 μL / mm 2 (Based on the bottom area of ​​the third through hole, the same applies below); ② Inject the eluent into the reaction vessel through another third through-hole and let it stand for 6-8 minutes (the amount added per unit area is 0.8-1.2 μL / mm). 2 This process achieves the elution of AFB1 from the molecularly imprinted polymer. After elution, the eluent is discharged from the outlet. ③ Add electrolyte dropwise into the reaction tank through a third through-hole, different from the first two (the amount of electrolyte added per unit area is 0.8~1.2 μL / mm). 2 The three strip lead areas of the carbon ink patterned electrode layer were connected to the three-electrode detection holes of the portable ECL instrument, and the ECL signal at this time was collected and recorded. I 0, and then the electrolyte is discharged from the outlet; ④ Add standard solution dropwise into the reaction chamber through the last remaining third through-hole (the amount of standard solution added per unit area is 0.8~1.2 μL / mm). 2 Incubate for 9-12 minutes, then drain the incubated liquid from the outlet. Add electrolyte as per step ②, and collect the ECL signal again, recording it as follows. I 1; with I 1 and I The difference Δ to 0 I ECL A standard curve is established with lg c, the logarithm of the concentration of the target analyte AFB1 in the standard solution, as the ordinate and lg c as the abscissa. 3) Replace the AFB1 standard solution with the sample solution to be tested. Under the same conditions as in step 2), follow steps ①, ②, and ④ in step 2) and then measure its ECL signal, subtracting... I After substituting the values ​​into the standard curve, the AFB1 content in the sample solution was calculated and converted into the AFB1 content in the edible oil sample.

[0016] According to the above scheme, in step 1), the concentration range of the standard solution is 1×10⁻⁶. 5 ng / mL ~ 1 ng / mL.

[0017] According to the above scheme, in steps 2) and 3), when detecting the ECL signal, the high voltage of the photomultiplier tube (PMT) is set to 700~900 V, the number of amplification stages is set to 5~7, the cyclic voltammetric potential scanning range is 0 ~ +1.4 V, and the scanning rate is set to 50~150 mV / s.

[0018] The technical concept and principle of this invention are as follows: Figure 1 As shown, the electrochemiluminescence sensor based on a multi-channel microfluidic chip of this invention has four third through-holes, which are respectively used to inject a mixture of target probe and molecularly imprinted polymer, an eluent, a sample solution, and an electrolyte into the reaction cell. After the target probe-molecularly imprinted polymer mixture is injected into the reaction cell, it is cured into a film by ultraviolet light and fixed on the surface of the circular working area of ​​the carbon ink patterned electrode. Figure 2 As shown, CsPbBr3QDs@HPCMs, acting as an electrochemiluminescent probe, are oxidized on a carbon-patterned electrode at a scanning voltage of 0 to +1.4 V. CsPbBr3QDs lose electrons and are oxidized to CsPbBr3QDs. + The intramolecular co-reactant octadecyl diethanolamine (ODA) is oxidized to the cationic free radical ODA· + After undergoing deprotonation, intramolecular electron transfer occurs, generating excited state CsPbBr3QDs. During the process of returning to the ground state, photons are emitted. Before binding to the target, after the template molecule AFB1 is eluted by the eluent in the molecularly imprinted polymer, a large number of specific recognition cavities matching the shape and size of the target are left in the polymer film. These cavities constitute channels that promote the diffusion of charged ions and facilitate the effective transfer of electrons at the electrode interface, thus exhibiting a strong electrochemiluminescence signal. When the target molecule AFB1 is injected into the reaction cell, it can specifically bind to and fill these cavities, forming a physical barrier of steric hindrance and electron transfer on the electrode surface, which severely hinders the electron transfer process at the electrode interface. Therefore, the higher the concentration of the target molecule AFB1, the weaker the electrochemiluminescence signal generated by the sensor, and the two show a significant negative correlation. Accordingly, the sensor described in this invention can specifically capture and recognize the target molecule AFB1 in the sample solution, thereby causing corresponding changes in the ECL signal, and achieve quantitative detection of AFB1 by combining the standard curve method. Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes the integration of microfluidic channels and ECL chips to construct the electrochemiluminescence sensor based on the multi-channel microfluidic chip. It can be combined with a portable electrochemiluminescence detector to collect ECL signals to achieve quantitative detection of aflatoxin B1, exhibiting high sensitivity and excellent selectivity.

[0019] 2. This invention uses a hydrophobic ionic liquid as the electrolyte, which is suitable for non-polar (i.e., oil-based) environments and is easy to mix with the test sample solution of edible oil, which helps to reduce random errors in the system. Moreover, using a hydrophobic ionic liquid as the electrolyte during detection can apply a wider electrochemical window. Combined with the high conductivity of the hydrophobic ionic liquid, the ECL reaction efficiency on the electrode surface is greatly improved, which can improve the detection performance.

[0020] 3. This invention uses CsPbBr3QDs@HPCMs as an electrochemiluminescence probe, which is formed by encapsulating CsPbBr3QDs with biomass-derived porous carbon material HPCMs, and using octadecyl diethanolamine (ODA) as the end-capping ligand. This electrochemiluminescence probe can achieve ECL response without the addition of external co-reaction reagents, which improves the ECL signal, simplifies the operation steps, further reduces the random error of the detection method, and improves the detection sensitivity.

[0021] 4. When the electrochemiluminescence sensor of the present invention detects aflatoxin B1, it is simple to operate, avoids the use of large-scale instruments and equipment and the need for professional operators, and the device can be reused after cleaning, resulting in low detection and manufacturing costs. Attached Figure Description

[0022] Figure 1This is a schematic diagram of the detection principle of the electrochemiluminescence sensor based on a multi-channel microfluidic chip.

[0023] Figure 2 ECL potential diagram of CsPbBr3QDs@HPCMs.

[0024] Figure 3 In the diagram, A is the TEM characterization image of HPCMs, and B is the nitrogen adsorption-desorption isotherm of HPCMs.

[0025] Figure 4 The image shows the TEM characterization of CsPbBr3QDs.

[0026] Figure 5 In the image, A shows the TEM and HRTEM images of CsPbBr3QDs@HPCMs, while B and C show the fluorescence and UV absorption spectra of CsPbBr3QDs@HPCMs, respectively.

[0027] Figure 6 This is a schematic diagram of the assembly of the electrochemiluminescence sensor based on a multi-channel microfluidic chip according to the present invention; wherein, 1-carbon ink patterned electrode layer, 2-insulating layer, 3-channel layer, 4-circular working area, 5-lead area, 6-first through hole, 7-second through hole, 8-third through hole, 9-outlet.

[0028] Figure 7 In the diagram, A represents the fabrication process of the carbon-patterned electrode layer, B and C represent the water contact angle and SEM image of the bare plastic substrate and the carbon-patterned electrode layer, respectively, and D represents the electrochemical performance of the carbon-patterned electrode layer.

[0029] Figure 8 This is a flowchart illustrating the fabrication process of the channel layer in the electrochemiluminescence sensor described in this invention.

[0030] Figure 9 In the diagram, A represents the ECL signal intensity graphs of each standard solution in Example 2, and B represents the corresponding standard curve graphs.

[0031] Figure 10 Electrochemical impedance spectroscopy (A) and electrochemiluminescence spectroscopy (B) were performed on carbon-ink patterned electrodes and their modified target probes after molecular imprinting polymers, after elution of AFB1, and after addition of AFB1.

[0032] Figure 11 This is a selective experiment diagram for Example 3.

[0033] Figure 12 This is a diagram of the reproducibility test for Example 3.

[0034] Figure 13 The image shows the ECL signal of the NIP-ECL sensor as a comparative example. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0036] In this invention, the target probe CsPbBr3QDs@HPCMs is a composite of CsPbBr3QDs and porous carbon material HPCMs, which can be obtained by the following methods, but is not limited to the following preparation methods.

[0037] 1. Preparation method of biomass-derived porous carbon HPCMs: Using tree branches as raw material, the slices are calcined at 800-1200℃ under a protective atmosphere, polished, and washed to obtain carbon slices. The carbon slices are then thoroughly soaked in an alkaline solution, dried, and calcined at 600-800℃ under a protective atmosphere to obtain activated lignocarbon. The activated lignocarbon is washed until neutral to obtain porous carbon material HPCMs. This porous carbon material contains uniform honeycomb-like mesopores with a pore size of 30-50 nm and a specific surface area of ​​not less than 70 m². 2 / g. Porous carbon materials made from biomass are abundant, inexpensive, and possess an ideal porous structure. In addition to their structural advantages, biomass itself is rich in C, H, O, and heteroatoms (N, P, S, etc.), resulting in a rich array of functional groups on the surface of its derived carbon materials. This synergistic effect of structure and chemical properties can effectively regulate the interaction between carbon materials and perovskite quantum dots and oil-phase matrix, thereby facilitating the realization of efficient electrochemiluminescence. Carbon materials with similar porous properties are also applicable to this invention.

[0038] 2. Synthesis of CsPbBr3QDs@HPCMs complex by hot injection method: Using Cs2CO3, 1-octadecene, and oleic acid as raw materials, a transparent CsOA solution was formed by heating under a nitrogen protective atmosphere. This solution was then mixed with an HPCMs dispersion to obtain a mixed precursor solution. Simultaneously, 1-octadecene, octadecyl diethanolamine, oleic acid, and PbBr2 were mixed to obtain a PbBr2 reaction solution. The mixed precursor solution was injected into the PbBr2 reaction solution, cooled in an ice-water bath, and then centrifuged and washed to obtain CsPbBr3QDs@HPCMs.

[0039] In the following examples, the target probe CsPbBr3QDs@HPCMs used is a composite of CsPbBr3QDs and porous carbon material HPCMs. The specific preparation method includes the following steps: (1) Preparation of biomass-derived porous carbon HPCMs: Dry poplar branches were cut into thin slices (5 mm thick) perpendicular to the growth direction and dried in an oven for 6 h to remove moisture. They were then transferred to a tube furnace and heated to 1000 °C at a rate of 5 °C / min, and maintained at this temperature for 6 h under an Ar atmosphere. After cooling to room temperature, the samples were removed from the tube furnace and sanded to remove debris and smooth the surface. The sanded samples were then ultrasonically washed with ultrapure water until the washing solution became transparent, and then dried in an oven at 120 °C for 4 h to obtain carbon slices.

[0040] The carbon slices were soaked in 5 M KOH solution for 30 h, then dried in an oven at 80 °C, and then transferred to a tube furnace for activation. The temperature was raised to 700 °C within 30 min and held at that temperature for 2 h under a continuous nitrogen flow. After cooling to room temperature, activated wood char was obtained. The activated wood char was washed with 20% HNO3 until slightly acidic, and then washed with deionized water until neutral to remove excess KOH and K2CO3 from the wood. After drying, porous carbon material HPCMs was obtained and stored in an oven for later use.

[0041] like Figure 3 As shown, this porous carbon material exhibits a uniform honeycomb structure, is mesoporous with a pore size of 30–50 nm, and has a specific surface area of ​​90.2451 m². 2 / g.

[0042] (2) Synthesis of CsPbBr3QDs by hot injection method: 10 mmol Cs2CO3, 20 mL 1-octadecene and 20 mL oleic acid (OA) were added to a N2-filled flask and evacuated at 120 °C for 1 h. The temperature was then continuously increased to 150 °C to form a transparent CsOA solution. After maintaining this temperature for 30 min, the solution was cooled to room temperature and stored for later use.

[0043] 20 mL of 1-octadecene, 40 mg of octadecyl diethanolamine (ODA), 5 mL of OA, and 2 mmol of PbBr2 were completely dissolved to obtain a PbBr2 reaction solution. 1 mL of CsOA solution preheated to 70 °C was rapidly injected into this PbBr2 reaction solution using a syringe, and then the solution was quickly placed in an ice-water bath and cooled over 5–10 s. After centrifugation, the cooled solution was subjected to reaction with n-hexane / ethyl acetate. v / v Wash three times with a 2:1 ratio, and centrifuge to obtain the precipitate CsPbBr3QDs, which can be dispersed in n-hexane.

[0044] like Figure 4 As shown, the CsPbBr3QDs exhibit a monodisperse, uniform cubic shape with a lattice parameter of 0.29 nm and an average size of 7 ± 1.8 nm.

[0045] (3) When synthesizing CsPbBr3QDs by the hot injection method, the CsOA solution is added to the HPCMs dispersion to prepare the target probe CsPbBr3QDs@HPCMs complex: First, prepare the CsOA solution and PbBr2 reaction solution according to step (2). Disperse 100 mg HPCMs in 10 mL DMF and stir for 1 h to ensure complete dispersion. Then, mix the HPCMs dispersion with the CsOA solution at a volume ratio of 3:2 to obtain a mixed precursor solution. Quickly inject 1 mL of the mixed precursor solution into the PbBr2 reaction solution, and then rapidly place it in an ice-water bath to cool within 5-10 s. After centrifuging the cooled solution system, use n-hexane / ethyl acetate ( v / v The mixture was washed three times (2:1), and the precipitate obtained by centrifugation was CsPbBr3QDs@HPCMs, which was dispersed in n-hexane at a concentration of 2 mg / mL. The theoretical specific mass ratio of HPCMs to CsPbBr3QDs in the target probe CsPbBr3QDs@HPCMs complex is approximately 29:10.

[0046] like Figure 5 As shown in Figure A, after CsPbBr3QDs were grown in situ inside HPCMs, dark contrast spots appeared in some areas of the pore surface of the HPCMs, indicating that CsPbBr3QDs were successfully loaded into the pores of the HPCMs. Figure 5 The HRTEM inset in Figure A shows clear lattice fringes with a spacing of 0.29 nm, corresponding to the (200) crystal plane of CsPbBr3QDs. The optical properties of the composite material CsPbBr3QDs@HPCMs were analyzed, such as... Figure 5 As shown in B and 5C, the maximum emission wavelength of CsPbBr3QDs@HPCMs can be obtained in the fluorescence excitation wavelength range of 315~320nm, with its fluorescence emission peak located at 520 nm and the ultraviolet characteristic absorption peak in the range of 512~530 nm. This indicates that the prepared CsPbBr3QDs@HPCMs composite has excellent optical properties such as high purity and good monochromaticity.

[0047] The following embodiments illustrate the preparation method of the target probe-molecularly imprinted polymer mixture: First, a mixed solution of functional monomer MAA methacrylate, initiator AIBN, and crosslinking agent EDMA (the molar ratio of the functional monomer, initiator, and crosslinking agent is 8:1:3) is diluted with methanol at a volume ratio of 1:5, denoted as MIP polymerization solution. AFB1 is then added to this solution at a concentration of 1×10⁻⁶. 5The concentration of the target probe was ng / mL to 1 ng / mL. The MIP polymerization solution containing AFB1 was thoroughly mixed with the CsPbBr3QDs@HPCMs complex (concentration 2 mg / mL) at a volume ratio of 2:5 to obtain the target probe-molecularly imprinted polymer mixture, abbreviated as CsPbBr3QDs@HPCMs / MIP. The mass ratio of CsPbBr3QDs@HPCMs to MIP was approximately 1:214.

[0048] In the following examples, the electrolyte is 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid ([Bmim][PF6]), dissolved in ethyl acetate (EA) to 0.1 M for later use; the eluent is analytical grade ethanol.

[0049] In the following embodiments, the portable ECL instrument specifically adopts the RPEL-B type portable electrochemiluminescence detector. Example 1 Electrochemiluminescence sensors based on multi-channel microfluidic chips, such as Figure 6 As shown, the structure comprises three parts: a carbon-patterned electrode layer 1 (i.e., a sensing layer), an insulating layer 2, and a channel layer 3. The carbon-patterned electrode layer 1 includes a circular working area 4 and three strip-shaped lead areas 5. An insulating layer 2 containing a first through-hole 6 is disposed above the circular working area 1. A channel layer 3 is constructed above the insulating layer 2 using an insulating material. The channel layer 3 has a second through-hole 7, four third through-holes 8, and an outlet 9. All third through-holes 8 are connected to the second through-holes 7. The four third through-holes 8 are used to drop a mixture of target probe and molecularly imprinted polymer, an eluent, a sample solution, and an electrolyte, respectively. The outlet 9 is located on the sidewall of the second through-hole 7. The circular working area 1, the first through-hole 6, and the second through-hole 7 overlap to form a reaction cell. The third through-holes 8 and the channels between the third through-holes 8 and the second through-holes 7 are separated from the carbon-patterned electrode layer 1 by the insulating layer 2. The diameters of the first through hole 6, the second through hole 7, and the circular working area 4 are all 8 mm; the depth of the reaction tank is 1.25 mm; the diameter of the third through hole 8 is 2 mm and the depth is 1.25 mm; the width of the channel between the third through hole 8 and the second through hole 7 is 2 mm, the length is 2 mm, and the depth is 1.25 mm; the opening width of the outlet 9 is 1.5 mm and the height is 1.25 mm.

[0050] The above-mentioned method for fabricating the electrochemiluminescence sensor based on a multi-channel microfluidic chip includes the following steps: (1) such as Figure 7 As shown in Figure A, a flexible PET plastic substrate is used as the electrode support material. Carbon ink is coated on it with a patterned sticker. After removing the surface adhesive, an electrode chip including a circular working area and a lead area is obtained, which is called a carbon ink patterned electrode (CPE) and serves as the sensing layer of the device.

[0051] like Figure 7 As shown in Figure B, the surface of the bare PET plastic substrate is smooth, and the static water contact angle is 63.4°; Figure 7 As shown in Figure C, after coating with carbon ink, the water contact angle of the resulting carbon ink patterned electrode increased to 96.4°, providing an interface with enhanced hydrophobicity and conductivity. The electrochemical performance of the CPE was characterized by cyclic voltammetry (CV) in 5 mM K3[Fe(CN)6] containing 0.1 M KCl solution. Figure 7 As shown in Figure D, after 50 consecutive cycles, the peak current of the CPE redox peak exhibits high consistency and stability, providing excellent preconditions for subsequent ECL testing.

[0052] (2) A mixture of PDMS prepolymer and curing agent is coated on the surface of a glass slide. After curing, a PDMS film layer, i.e. an insulating layer, is obtained, which is a square sheet of 12.5 mm × 15 mm. A first through hole with a diameter of 8 mm is opened on the insulating layer. The first through hole is overlapped and assembled with the circular working area of ​​the carbon ink patterned electrode. The diameter of the first through hole is the same as that of the circular working area. During assembly, they are overlapped and aligned so that the circular working area can be completely exposed.

[0053] (3) Constructing a channel layer using insulating material PDMS, such as Figure 8 As shown, the channel layer has one second through hole with a diameter of 8 mm and four third through holes with a diameter of 2 mm. All the third through holes are connected to the second through holes. The channel layer is stacked on top of the insulating layer, completely exposing the first through hole, so that the circular working area, the first through hole and the second through hole overlap to form a reaction cell. A small amount of mixed liquid of PDMS prepolymer and curing agent is applied between the insulating layer and the carbon ink patterned electrode, and between the insulating layer and the channel layer. After curing, the two are fixedly connected.

[0054] (4) Prepare a mixture of target probe-molecularly imprinted polymer, eluent and electrolyte respectively, and combine them with the device obtained in step (3) to form the electrochemiluminescence sensor based on multichannel microfluidic chip (abbreviated as MC-CPEECL sensor) of the present invention.

[0055] Example 2 The electrochemiluminescence sensor based on a multi-channel microfluidic chip constructed in Example 1 is used to detect AFB1. The application method includes the following steps: 1) Accurately weigh 5.0 mg (accurate to 0.1 mg) of AFB1 standard, then dilute to 5.0 mL with acetonitrile to prepare a standard stock solution with a mass concentration of 1.0 mg / mL. Store in a sealed brown bottle at 4°C protected from light. Transfer 0.1 mL of the above standard stock solution, add acetonitrile and dilute to 20 mL, mix well to prepare a standard solution with a mass concentration of 5.0 μg / mL. Then, serially dilute with acetonitrile to prepare a 1×10⁻⁶ standard solution. 5 Standard working solutions of different concentrations, ranging from ng / mL to 1 ng / mL, should be stored at 4 ℃ protected from light for future use.

[0056] 2) ① For example Figure 1 As shown, the mixture of target probe and molecularly imprinted polymer was injected into the reaction chamber through the third through-hole 1 (due to the large interfacial tension between the mixture and the electrode surface and the small droplet volume, the liquid could adhere stably without overflowing). After irradiation under ultraviolet light at a wavelength of 365 nm for 8 min, the target probe and molecularly imprinted polymer were immobilized on the circular working area surface of the carbon ink patterned electrode; the amount of the target probe and molecularly imprinted polymer mixture added was 0.314 μL / mm. 2 .

[0057] ② Inject eluent through the third through-hole 2, let stand for 6 min, the amount added is 0.995 μL / mm 2 This process elutes AFB1 from the molecularly imprinted polymer. After elution, the eluent is drawn out from the outlet using filter paper. Then, electrolyte is added dropwise into the reaction chamber through the third through-hole 4 (the amount of electrolyte added is approximately 0.995 μL / mm). 2 The three strip lead areas of the carbon ink patterned electrode layer were connected to the three-electrode detection holes of the portable ECL instrument, and then the entire assembly was inserted into the portable ECL instrument for ECL detection. The photomultiplier tube (PMT) high voltage was set to 800 V, the amplification stage was set to 6 stages, the cyclic voltammetric potential scan range was 0~+1.4V, and the scan rate was set to 100 mV / s. The ECL signal at this time was acquired and recorded. I 0. Use filter paper to draw the electrolyte out from the outlet; ③ After adding the standard solution dropwise into the reaction cell through the third through-hole (the amount of standard solution added per unit area is approximately 0.995 μL / mm), 2 After incubating for 9 minutes, the electrolyte was added according to step ②, and the ECL signal was collected and recorded. I 1; with I 1 and I The difference Δ to 0 I ECL A standard curve is constructed with the vertical axis representing the ordinate and the logarithm of the concentration of the target analyte AFB1 in the standard solution, lg c, representing the horizontal axis. For example... Figure 9 As shown, in 1×10 5 Within a linear range of ~1 ng / mL, the linear fit is good, and it can be used to achieve quantitative detection of the target analyte AFB1.

[0058] In addition, this embodiment also verified the successful construction of the sensor through electrochemical impedance spectroscopy and electrochemiluminescence tests during the acquisition of ECL signals. Figure 10 As shown in Figure A, due to the high conductivity of carbon ink, the bare ink electrode (i.e., the carbon ink patterned electrode) exhibits low impedance, thus enabling rapid charge transfer. According to the equivalent circuit of the Randle model, its impedance is 926.1 Ω. After immobilizing the target probe-molecularly imprinted polymer on the surface of the carbon ink patterned electrode (then labeled as the probe-molecularly imprinted ink electrode), due to its dense morphology and insulating properties, electron transfer on the electrode surface is severely hindered, resulting in a relatively high resistance. Figure 10 The green curve in A (25912 Ω) indicates this. When the target molecule AFB1 is eluted with ethanol, the stripping of AFB1 creates cavities of the same shape and size as the target substance. These cavities provide channels for electron transfer, reducing impedance. Figure 10 The purple curve in A represents 7659 Ω. After injecting a standard solution containing AFB1 into the sensor and incubating it for a period of time, the results showed that the impedance increased again. Figure 10 (Blue curve in A, 14407 Ω).

[0059] The ECL signals generated in each of the above stages were also recorded, such as Figure 10 As shown in Figure B, no ECL signal was recorded on the bare ink electrode. Figure 10 (Black curve in B). After modifying the electrode surface with CsPbBr3QDs@HPCMs / MIP (i.e., probe-molecularly imprinted ink electrode), there is a weak ECL peak ( Figure 10 (Green curve in B). When the target molecule AFB1 is eluted with ethanol, the ECL signal increases accordingly ( Figure 10 (Pink curve in B); After injecting a standard solution containing AFB1 into the sensor and incubating for a period of time, the ECL signal decreased sharply ( Figure 10 (Blue curve in B). This is because the target substance recombines with the cavity left after elution, which slows down electron transfer to some extent.

[0060] Example 3 Selectivity experiments and reproducibility tests were conducted on the electrochemiluminescence sensor constructed in this invention.

[0061] Interference tests were performed using other typical mycotoxins and mixtures thereof, following the procedure described in Example 2. When the interfering substances (DON, ZEN, FB1, AFB2, AFM1, AFM2, AFG1, and AFG2) were tested individually at a concentration of 1 ng / mL (approximately 10 times the target AFB1 concentration), Δ... I ECL The values ​​showed negligible changes. Furthermore, when a mixture of each interfering substance at a concentration 10 times higher than AFB1 and a 0.1 ng / mL AFB1 sample was tested, the resulting ECL signal was comparable to that of the standard AFB1 solution, indicating that the electrochemiluminescence sensor constructed in this invention exhibits excellent anti-interference capability for AFB1 detection (e.g., Figure 11 (As shown).

[0062] An electrochemiluminescence sensor was constructed according to Example 1, with multiple sensors prepared from the same batch. Five sensors were then randomly selected for testing. A standard solution of 0.01 ng / mL AFB1 was used for detection. The detection was performed according to Example 2. No significant fluctuations were observed in the ECL intensity. Figure 12 As shown in Figure A. Similarly, after a week of reproducibility testing on a certain electrochemiluminescence sensor, the ECL intensity did not show significant fluctuations, as shown in Figure A. Figure 12 As shown in B. Therefore, the electrochemiluminescence sensor constructed in this invention exhibits good reproducibility for AFB1 detection.

[0063] Example 4 This embodiment focuses on verifying the application capability of the electrochemiluminescence sensor constructed by the present invention in complex matrices. Commercially available corn oil was selected as a typical complex matrix sample. Three different concentrations of AFB1 standard solutions were added to the corn oil sample, and the sample solution to be tested was prepared according to step (1) in Example 4, followed by a spiked recovery experiment. Commercially available corn oil was selected as the actual sample to be tested, and the electrochemiluminescence sensor constructed in Example 1 was used to detect AFB1 in the actual sample. The specific steps included: (1) Weigh 1 g of corn oil into a 10 mL centrifuge tube, dilute with 5 mL of n-hexane, and vortex for 5 s to obtain a diluted oil sample. Add three different concentrations (1 pg / mL, 5 pg / mL, 10 pg / mL) of AFB1. The HAS-SPE extraction column is sequentially filled with 5 mL of acetone / water (8 / 2, v / v Activate with 5 mL of n-hexane, then add the spiked oil sample into the HAS-SPE column. After the liquid is drained, wash with 5 mL of isopropanol, followed by 10 mL of acetone / water (8 / 2). v / v Elution was performed. The eluent was collected and filtered through a 0.22 μm filter membrane to obtain the loading solution of the corn oil sample (i.e., the sample solution to be tested).

[0064] (2) Referring to step 2 of Example 2), the sample solution was injected into the reaction cell through the third through hole 1 and irradiated under ultraviolet light at a wavelength of 365 nm for 8 min; then, the eluent was injected through the third through hole 2, and AFB1 was eluted after standing for 6 min. After elution, the eluent was aspirated from the outlet with filter paper; then, the sample solution was added dropwise through the third through hole 3 and injected into the reaction cell, and incubated for 9 min. After incubation, the liquid was aspirated from the outlet, electrolyte was added through the third through hole 4, and the ECL signal was collected again and subtracted. I After 0, the standard curve of Example 2 was substituted to calculate the AFB1 in the sample solution and converted it into the AFB1 concentration in the oil sample.

[0065] In addition, the sample solution was detected by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS 8050), and the AFB1 concentration in the oil sample was calculated for comparison and verification with this example.

[0066] As shown in Table 1, the recovery rate of AFB1 in corn oil constructed by the present invention was 102.0%~110.0%, with a relative standard deviation of 1.1%~5.9%. In contrast, the recovery rate of AFB1 in corn oil detected by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) was 91.7%~116.7%, with a relative standard deviation (RSD) of 1.7%~7.5%. Therefore, the electrochemiluminescence sensor constructed by the present invention has higher reliability for the detection of AFB1 in complex corn oil samples.

[0067] Table 1

[0068] Comparative Example This comparative example is a non-molecularly imprinted polymer (NIP)-ECL sensor without template molecules. Its preparation method differs from Example 1 only in that the target molecule AFB1 was not added during the preparation of the target probe-molecularly imprinted polymer mixture. Referring to Example 2, the ECL signals generated by the NIP-ECL sensor at each stage were detected, such as... Figure 13 As shown, after modifying the surface of the bare ink electrode with CsPbBr3QDs@HPCMs / MIP without the target molecule AFB1 (i.e., probe-non-molecularly imprinted ink electrode), the ECL signal fluctuated after elution with ethanol and incubation with a standard solution containing AFB1, but the fluctuation was not comparable to... Figure 10 Compared to phase B, this NIP-ECL sensor cannot be used for the quantitative detection of AFB1.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An electrochemiluminescence sensor based on a multi-channel microfluidic chip, characterized in that, The device comprises three parts: a carbon-patterned electrode layer, an insulating layer, and a channel layer. The carbon-patterned electrode layer includes a circular working area and a strip-shaped lead area. An insulating layer containing a first through-hole is disposed above the circular working area. A channel layer is constructed above the insulating layer using an insulating material. The channel layer has a second through-hole and four third through-holes, all of which are connected to the second through-holes. The four third through-holes are used to drop a mixture of target probe and molecularly imprinted polymer, an eluent, a sample solution, and an electrolyte, respectively, with the outlet located on the sidewall of the second through-hole. The circular working area, the first through-hole, and the second through-hole overlap to form a reaction cell. The third through-holes and the channels between them and the second through-holes are isolated from the carbon-patterned electrode layer by the insulating layer. Among them, the target probe-molecular imprinted polymer uses the target as the imprinted molecule. After elution, the cavity formed can specifically capture and identify the target in the sample solution, causing changes in the electrochemiluminescence signal, thereby realizing the detection of the target.

2. The electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 1, characterized in that, The target substance is aflatoxin B1; the target probe is a complex of CsPbBr3 quantum dots and honeycomb porous carbon material; the molecularly imprinted polymer is generated by photopolymerization of aflatoxin B1 as the imprinted molecule with functional monomers and crosslinking agents under the action of an initiator.

3. The electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 2, characterized in that, CsPbBr3 quantum dots have a size of 6–8 nm, with characteristic UV absorption peaks in the range of 512–530 nm and fluorescence excitation wavelengths in the range of 315–320 nm, with a maximum emission peak at 519–521 nm; the honeycomb porous carbon material has a pore size of 30–50 nm and a specific surface area of ​​not less than 70 m². 2 / g.

4. The electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 1, characterized in that, The eluent is ethanol, used to elute the imprinted molecules in the molecularly imprinted polymer to form the cavity structure of the target analyte; the electrolyte is a 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid solution; the sample solution to be tested is the eluent obtained by diluting edible oil with acetone aqueous solution as the eluent and treating it with a humic acid solid-phase extraction column.

5. The electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 1, characterized in that, The diameters of the first through hole, the second through hole, and the circular working area are the same, ranging from 6 to 10 mm; the depth of the reaction tank is 1 to 1.5 mm; the diameter of the third through hole is ranging from 1 to 3 mm, and the depth is 1 to 1.5 mm; the channel between the third through hole and the second through hole has a width of 1 to 2 mm, a length of 2 to 2.5 mm, and a depth of 1 to 1.5 mm.

6. The method for fabricating the electrochemiluminescence sensor based on a multi-channel microfluidic chip according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Using a plastic substrate as the electrode support material, a carbon ink pattern containing a circular working area and strip leads is formed on its surface to obtain a carbon ink patterned electrode. (2) A mixture of PDMS prepolymer and curing agent is coated on the surface of a glass slide. After curing, a PDMS film layer, i.e. an insulating layer, is obtained. A first through hole is opened on the insulating layer. The first through hole is overlapped with the circular working area of ​​the carbon ink patterned electrode. The diameter of the first through hole is the same as that of the circular working area, which can completely expose the circular working area. (3) A channel layer is constructed by an insulating material. The channel layer has one second through hole and four third through holes. All the third through holes are connected to the second through holes. The diameter of the second through hole is the same as that of the first through hole. The channel layer is stacked on top of the insulating layer, completely exposing the first through hole, so that the circular working area, the first through hole and the second through hole overlap to form a reaction pool. (4) Prepare a mixture of target probe and molecularly imprinted polymer, eluent and electrolyte respectively, and combine them with the device obtained in step (3) to form the electrochemiluminescence sensor based on the multi-channel microfluidic chip.

7. The application of the electrochemiluminescence sensor based on a multi-channel microfluidic chip as described in any one of claims 1 to 5 for the detection of aflatoxin B1 in edible oil, characterized in that, The application method includes the following steps: 1) Using acetonitrile as solvent, accurately weigh aflatoxin B1 standard, prepare a standard solution of aflatoxin B1, and store it at 4 ℃ protected from light for later use. 2) ① Inject the mixture of target probe and molecularly imprinted polymer into the reaction cell through any of the third through holes and irradiate it under ultraviolet light to achieve the immobilization of target probe and molecularly imprinted polymer on the surface of the circular working area of ​​carbon ink patterned electrode. ② The eluent is injected into the reaction cell through another third through hole to elute aflatoxin B1 in the molecularly imprinted polymer. After elution, the eluent is discharged from the outlet. ③ Electrolyte is added dropwise into the reaction cell through a third through-hole, different from the previous two. The three strip lead areas of the carbon-patterned electrode layer are then connected to the three-electrode detection holes of the portable electrochemiluminescence instrument. The ECL signal at this point is collected and recorded. I 0, and then the electrolyte is discharged from the outlet; ④ After adding aflatoxin B1 standard solution dropwise into the reaction tank through the last remaining third through-hole for incubation, drain the incubated liquid from the outlet. Then, add electrolyte as in step ②, collect the ECL signal again, and record it. I 1; with I 1 and I The difference Δ to 0 I ECL A standard curve is established with lg c, the logarithm of the aflatoxin B1 concentration in the standard solution, as the ordinate and lg c as the abscissa. 3) Replace the standard solution of aflatoxin B1 with the sample solution to be tested. Under the same conditions as in step 2), follow steps ①, ②, and ④ in step 2) and then measure its ECL signal, subtracting... I After substituting the values ​​into the standard curve, the concentration of aflatoxin B1 in the sample solution was obtained and converted into the aflatoxin B1 content in the edible oil sample.

8. The method for fabricating an electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 7, characterized in that, The electrolyte is a 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid solution, using ethyl acetate as the solvent, with a concentration of 0.05~0.15 M; The method for preparing the test sample solution is as follows: the edible oil is diluted with n-hexane and vortexed, then added to a humic acid solid phase extraction column and washed with isopropanol. After elution with an acetone-water mixture, the eluent is collected and filtered through a filter membrane to obtain the test sample solution of the edible oil.

9. The application of the electrochemiluminescence sensor based on a multi-channel microfluidic chip according to claim 7 for the detection of aflatoxin B1 in edible oil, wherein in step 1), the concentration range of the standard solution is 1×10⁻⁶. 5 ng / mL ~ 1 ng / mL.

10. The application of the electrochemiluminescence sensor based on a multi-channel microfluidic chip for detecting aflatoxin B1 in edible oil according to claim 7, in steps 2) and 3), when detecting the ECL signal, the photomultiplier tube high voltage is set to 700~900 V, the amplification stage is set to 5~7 stages, the cyclic voltammetric potential scanning range is 0~+1.4 V, and the scanning rate is set to 50~150 mV / s.

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