WO2023159532A1 - 一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法 - Google Patents

一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法 Download PDF

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WO2023159532A1
WO2023159532A1 PCT/CN2022/078143 CN2022078143W WO2023159532A1 WO 2023159532 A1 WO2023159532 A1 WO 2023159532A1 CN 2022078143 W CN2022078143 W CN 2022078143W WO 2023159532 A1 WO2023159532 A1 WO 2023159532A1
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channel
detection
solution
fluorescent sensing
microfluidic
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French (fr)
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胡雪桃
邹小波
蒋培
石吉勇
张新爱
黄晓玮
李志华
李亚惠
李文亭
张俊俊
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江苏大学
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
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    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of detection of organophosphorus pesticides, in particular to a method for detecting organophosphorus pesticides with a microfluidic chip based on a fluorescent sensing film.
  • Organophosphorus pesticides refer to organic compounds containing phosphorus elements, such as common organophosphorus pesticides trichlorfon, parathion, dimethoate and dichlorvos, etc., which have the effect of preventing and controlling crop diseases, insect pests and weeds.
  • the above methods can achieve accurate detection of organophosphorus pesticides, but there are still disadvantages such as cumbersome operation or dependence on large and heavy instruments, and cannot realize rapid and portable detection of pesticide residues on site.
  • fluorescent nanomaterials show great application potential in the fields of labeling, tracing, imaging and detection due to their stable morphological structure and high luminous efficiency.
  • detection of pesticide residues based on fluorescent nanomaterials is devoted to the preparation of specific nanomaterials, and the detection must be carried out in solution.
  • the commonly used specific molecules for detection are biological molecules such as aptamers and antigens/antibodies.
  • Fluorescent nanomaterials In the solution, due to poor solubility or poor stability, nanoparticle precipitation and fluorescence quenching will occur. The application of biomolecules leads to high detection cost and poor detection repeatability. The disadvantages of unstable solution and difficult portability limit the use of fluorescent nanomaterials. Application in field testing.
  • the combination of other materials or other detection methods can play a greater detection role, such as the preparation of fluorescent sensing films and the combination of microfluidic chips.
  • the disadvantages of poor stability in solution and easy photobleaching of fluorescent nanomaterials can be overcome by preparing fluorescent thin film sensors, and the sensors are endowed with characteristics such as easy portability and easy use.
  • the microfluidic chip technology is used to integrate various processes involved in sample pretreatment, mixing reaction and fluorescence detection into a chip of several square centimeters, which has multi-functional integration, small size and It is easy to carry and other advantages, and it is a commonly used instant detection device.
  • the present invention constructs a H 2 O 2 fluorescence
  • the sensing film provides an organophosphorus pesticide microfluidic chip system and detection method that integrates automatic sampling, pretreatment and detection functions, and intends to solve the problem that the microfluidic chip cannot be standardized and standardized to detect organophosphorus pesticides, and The problem of being unable to realize rapid on-site detection of organophosphorus pesticides.
  • a microfluidic chip system based on a fluorescent sensing film and an organophosphorus pesticide detection method constructed by the present invention are characterized in that they include the preparation of a specific fluorescent sensing film, the construction of a microfluidic chip system, the establishment of an on-site detection method, and the actual application method.
  • Step 1 Construction of porous fluorescent sensing film, including the synthesis of specific fluorescent materials and the construction of fluorescent sensing film, which can be used for the specific detection of acetylcholine hydrolyzate H2O2 :
  • molybdenum disulfide nanosheets, polystyrene polymers, and polydimethyldiallylammonium chloride were dissolved in ethanol at a certain ratio to obtain a mixed solution, and then the fluorescent sensing layer modified quartz substrate was mixed Soak in the liquid for M times, form M layers of macromolecular barrier layer on the surface of the fluorescent sensing layer, and finally obtain the organophosphorus pesticide fluorescent sensing film comprising the fluorescent sensing layer and the macromolecular barrier layer, which is recorded as a porous fluorescent sensing film;
  • Step 2 Design a microfluidic chip with automatic sample injection, sample pretreatment, sample and reagent mixing reaction, and detection functions;
  • the microfluidic chip includes a sample channel, a reaction tank, a detection tank, a microfluidic channel, and an optical fiber channel; there are n reaction tanks, which are sequentially recorded as the first reaction tank, the second reaction tank..., the n-1th reaction tank and the nth reaction tank;
  • One end of the microfluidic chip has a sample port, and one end of the first reaction tank is provided with a sample channel connected to the sample port; the other end is connected to the second reaction tank..., the n-1 reaction tank and the nth reaction tank through the microfluidic channel
  • the reaction tanks are connected sequentially; the other end of the nth reaction tank is connected to one end of the detection tank through a microfluidic channel; the other end of the detection tank is provided with a fiber optic channel, and is not connected to the fiber optic channel;
  • sampling channels on one side of the microfluidic chip, which are sequentially recorded as the first sampling channel, the second sampling channel..., the n-1th sampling channel and the nth sampling channel; the first The sampling channel communicates with the first reaction tank, the second sampling channel communicates with the second reaction tank..., the n-1th sampling channel communicates with the n-1th reaction tank; the nth sampling channel Connected with the nth reaction tank;
  • a fluorescent sensing thin film pick-and-release port and a visual detection port are opened above the detection groove; the n is a positive integer.
  • the preparation method of the microfluidic chip is:
  • the channel structure of the microfluidic chip First design the channel structure of the microfluidic chip, and print out the microfluidic channel model with the dissolving support material as the base material; then transfer the microfluidic channel model to the container, pour polydimethylsiloxane (PDMS) and The complex formed by mixing the curing agent is heated to a certain temperature to harden the PDMS to obtain a microfluidic platform template, and the microfluidic channel model in the microfluidic platform template is dissolved by an aqueous solution of an organic solvent to obtain a microfluidic chip platform; for microfluidic chips The upper surface of the platform is pretreated so that the microfluidic channel on the upper surface communicates with the external environment;
  • PDMS polydimethylsiloxane
  • Step 3 the establishment of organophosphorus pesticide fluorescence color card and quantitative detection model, the specific steps are:
  • microfluidic chip system for organophosphorus pesticides mainly including constant pressure injection pumps, syringes, connecting tubes, microfluidic chips, fluorescent optical fibers, lasers, spectrometers, signal transmitters, and signal displays;
  • the constant-pressure syringe pump is connected to the sampling port of the microfluidic chip through a connecting tube for sampling;
  • the syringe is connected to the sampling channel through the connecting tube, and the reaction reagent is added into the reaction tank; the reaction reagent is used to degrade acetylcholine to generate H 2 O 2 ;
  • One end of the fluorescent optical fiber is placed in the optical fiber channel to obtain the fluorescent signal of the porous fluorescent sensing film in the detection groove, and the other end is connected to the laser and the spectrometer; the spectrometer transmits the obtained porous fluorescent sensing film signal through the signal transmitter transmission to the signal display;
  • the amount ratio of europium nitrate, pyromellitic acid, oxalic acid and acetonitrile-ethanol solution in S1 of step 1 is 0.1 ⁇ 10mmol:0.05 ⁇ 0.5mmol:0.05 ⁇ 0.5mmol:50 ⁇ 100mL; acetonitrile-ethanol solution It is a mixture of acetonitrile and ethanol, wherein the mixed volume ratio of acetonitrile and ethanol is 6-10:1-4; the reaction temperature under the certain temperature conditions is 100-200°C, and the reaction time is 12-72h;
  • the purification step is to use an organic solvent to repeatedly wash 3 to 10 times, and the organic solvent includes any one of N,N-dimethylformamide, ethanol or acetone; the drying refers to drying at 40 to 60°C until constant weight;
  • the optimum excitation wavelengths of the organometallic framework powder and the organometallic framework material solution are consistent, ranging from 500 to 650 nm, and the optimum emission wavelengths are W 1 , W 2
  • the mass concentration of the oxalate solution in S1 of step 1 is 0.5-1 mg/mL; the mass concentration of the organometallic framework solution is 2-10 mg/mL; the oxalate solution and the organometallic framework
  • the volume ratio of the solution is 1:1; the oxalate solution is mixed with the organometallic framework material solution, and the temperature of oscillation under certain temperature conditions is 30-50°C, and the oscillation time is 12-48h; the oxalate-
  • the concentration of the organic metal framework material is 2-10 mg/mL, the concentration of the potassium chloroplatinite is 0.3-5 mg/mL, the concentration of the sodium borohydride is 0.1-1 mg/mL; the oxalate-organic
  • the volume ratio of metal frame material, potassium chloroplatinite and sodium borohydride is 1:0.1 ⁇ 2:0.5 ⁇ 1.5;
  • the particle size of the platinum nanoparticles in the Pt@TCPO-EuMOF solution does not exceed 50nm.
  • the cleaning of the quartz substrate in S2 of step 1 is specifically ultrasonic cleaning in an ethanol solution for 3 to 8 times, each cleaning for 1 to 10 minutes; the polydimethyldiallylammonium chloride (PDADMA) solution
  • PDADMA polydimethyldiallylammonium chloride
  • concentration is 0.5-5g/L
  • concentration of Pt@TCPO-EuMOF solution is 0.5-10mg/mL.
  • the soaking time in both PDADMA solution and Pt@TCPO-EuMOF solution is 5-30min.
  • the number of times N of repeated immersion is 5 ⁇ 100;
  • the consumption ratio of described molybdenum disulfide nanosheet, polystyrene polymer, polydimethyldiallylammonium chloride and ethanol is 0.1 ⁇ 5mg:0.2 ⁇ 2mg: 0.01-0.1mg: 1ml; the soaking times M is 2-10, and the soaking time is 3-10min; the macromolecule barrier layer on the surface of the porous fluorescent sensing film has a pore size of 2-15nm.
  • the channel structure of the microfluidic chip is designed as described in step 2.
  • the channel structure includes a microfluidic channel, a sample channel, a reaction tank, and a detection tank.
  • the specific design requirements are: the total length of the microfluidic channel is 5 to 20 cm, and the channel The depth and width are both 0.4-4mm; the lengths of the sample channel, the first sampling channel, the second sampling channel..., the n-1th sampling channel and the nth sampling channel are all 0.8-2cm, and the depth and The widths are all 0.4-5mm, the depths of the first reaction tank, the second reaction tank..., the n-1th reaction tank, the nth reaction tank and the detection tank are all 0.5-5mm; the area of the reaction tank is 0.5 ⁇ 3cm 2 ; the area of the detection groove is 0.3 ⁇ 5cm 2 ; the dimensions of the microfluidic chip and the quartz substrate are consistent, and the length, width and height are 6 ⁇ 15cm, 1 ⁇ 10cm and 0.5 ⁇ 2cm
  • the soluble support material described in step 2 is one of polyoxymethylene, polyvinyl alcohol or acrylic copolymer, or a mixture of multiple arbitrary proportions; the polydimethylsiloxane and its curing agent The mass ratio is 7-9:1-3; the curing agent is a type of silane coupling reagent, mainly composed of vinyltriamine, aminoethylpiperazine, diaminodiphenylmethane and m-phenylenediamine Or a mixture of several kinds; the organic solvent aqueous solution refers to a mixed solution of water and an organic solvent with a volume ratio of 1-4:6-9; the organic solvent includes one or more of acetone, isopropanol or nitric acid; The heating to a certain temperature is 65-80°C.
  • the aperture of the fluorescent sensing film access port and the visual detection port is 0.6-2.1 cm; the detection groove is not connected to the optical fiber channel and the distance is 0.5-1 cm, and the length, width, and height of the optical fiber channel are 1-5 cm respectively.
  • the reaction reagent in step three includes one or more of acetylcholinesterase, acetylcholine or choline oxidase; the fluorescent optical fiber is specifically a 1*2 type fluorescent optical fiber.
  • the reaction period in the detection tank described in step 3 is 2 to 10 minutes; the wavelength of the ultraviolet lamp and the excitation light is 301 to 399 nm; the concentration range of the standard organophosphorus pesticide is 0 to 1 g/mL, respectively record are 0, n 1 , n 2 , n 3 ,..., n h ; the pictures of the fluorescent sensing film are recorded as p 0 , p 1 , p 2 , p 3 ,..., ph respectively; the wavelength is W 1
  • the fluorescence signal intensities corresponding to different concentrations of organophosphorus pesticide standard substances are recorded as I 1,0 , I 1.1 , I 1.2 , I 1.3 , ..., I 1.h , and the wavelengths are determined by the different concentrations of organophosphorus pesticide standard substances at W 2
  • the corresponding fluorescent signal intensities are recorded as I 2.0 , I 2.1 , I 2.2 , I 2.3 , ..., I 2.h , ..., and the
  • the sum of the fluorescence signal intensity difference is y, which is respectively recorded as y 1 , y 2 , y 3 , ..., y h ;
  • the period of time for the reaction in the detection tank in step 4 is 2-10 minutes; the reaction reagent includes one or more of acetylcholinesterase, acetylcholine or choline oxidase.
  • the present invention constructs a fluorescent organometallic framework material with a cage structure, fixes specific molecular oxalate in the pore structure, and simultaneously introduces noble metal nanoparticles to form an oxalate-organometallic framework supported by platinum nanoparticles
  • Composite materials in which platinum nanoparticles can catalyze the specific reaction between oxalate and H 2 O 2 , and the chemiluminescence generated by the reaction can specifically enhance the fluorescence signal of the organometallic framework, and the oxalate-based
  • the new chemiluminescence resonance energy transfer system of H 2 O 2 reaction products and EuMOF the prepared fluorescent sensing material has dual functions of catalytic reaction and specific detection, which can improve the specificity and sensitivity of detecting organophosphorus pesticides.
  • the present invention assembles platinum nanoparticles@oxalate-organic metal framework composite material on the surface of the quartz substrate to prepare the fluorescent sensing layer.
  • Fluorescent materials are easy to use and portable.
  • the porous two-dimensional nanosheet material is wrapped on the outer surface of the fluorescent sensing layer, which can block macromolecules such as protease contained in the reaction solution from entering the fluorescent sensing layer, and reduce the impact of macromolecules such as protease on the fluorescent sensing layer. Unfavorable effects of fluorescent signal, improve detection accuracy and reduce detection limit.
  • the present invention builds a portable organophosphorus pesticide microfluidic chip system by designing a microfluidic chip with functions such as automatic sampling, sample pretreatment, and mixed reaction between samples and detection reagents, so that the detection equipment is integrated and miniaturized.
  • the standardization and standardization of pesticide testing will ultimately improve the stability and reproducibility of on-site testing.
  • the present invention combines the fluorescent nano-film with the microfluidic chip, and by designing the specific fluorescent film, the reaction between the organophosphorus pesticide and the specific detection reagent is sealed in the stable microfluidic chip, and the fluorescent signal can be obtained with the naked eye.
  • the change of organophosphorus pesticides can be quickly visualized, and the fluorescence intensity can be obtained by a portable spectrometer, so that the rapid quantitative detection of organophosphorus pesticides can be realized.
  • Figure 1 is a schematic diagram of the structure of the porous fluorescent sensing film, in which: 1-quartz substrate, 2-polydimethyldiallylammonium chloride layer, 3-platinum nanoparticles@oxalate-organic metal framework composite material layer, 4-molybdenum disulfide nanosheet-polystyrene polymer porous barrier layer;
  • Figure 2 is a schematic diagram of the microfluidic chip structure, wherein: 1-sample channel, 2-first reaction tank, 3-first microfluidic channel, 4-second reaction tank, 5-second microfluidic channel, 6-third Reaction tank, 7-third microfluidic channel, 8-detection tank, 9-fluorescent sensing film access port and visual detection port, 10-fiber optic channel, 11-quartz substrate, 12-first sampling channel, 13 -the second sampling channel, 14-the third sampling channel, 15-microfluidic platform;
  • Fig. 3 is a flow chart of organophosphorus pesticide detection; wherein 1-constant pressure injection pump, 2-first connecting pipe, 3-first syringe, 4-second syringe, 5-third syringe, 6-second connecting pipe, 7-third connecting tube, 8-fourth connecting tube, 9-microfluidic chip, 10-1*2 type fluorescent optical fiber, 11-laser, 12-spectrometer, 13-signal transmitter, 14-signal display.
  • a microfluidic chip preparation and on-site detection method for organophosphorus pesticides comprising the following steps:
  • the organic metal framework material (EuMOF) is obtained and placed in a 45°C drying oven Dry to obtain powder, redissolve in water to obtain organometallic framework solution, determine the optimal excitation wavelength of the two by obtaining the fluorescence spectrum of the solution and powder is 560nm, the excitation wavelength range is 500-650nm, and the optimal emission wavelength is 520nm and 610nm, Under the irradiation of the best excitation light (560nm), both the organometallic framework solution and the powder show weak red fluorescence, because the ligand has a large number of carboxyl groups, so the organometallic framework material is negatively charged; 10mL of bis( 2,4,6-Trichlorophenyl)oxalate solution (TCPO) was stirred with 10mL organometallic framework solution with a concentration of 5mg/mL for 48h to obtain bis(2,4,6-trichlorophenyl)oxalate -Organometallic framework composite solution; the
  • the microfluidic chip includes a sample channel 1, a first reaction tank 2, a first microfluidic channel 3, a second reaction tank 4, a second microfluidic channel 5, a third reaction tank 6, and a third microfluidic channel 7.
  • Groove 8 Fluorescent sensing film access port and visual detection port 9, Fiber channel 10, Quartz substrate 11, First sampling channel 12, Second sampling channel 13, Third sampling channel 14 and Microfluidic platform 15 ;
  • One end of the microfluidic chip opens a sample port and communicates with the first reaction tank 2 through the sample channel 1; the other end of the first reaction tank 2 communicates with the second reaction tank 4 through the first microfluidic channel 3; the second reaction tank The other end of 4 communicates with the third reaction tank 6 through the second microfluidic channel 5; the other end of the third reaction tank 6 communicates with the detection tank 8 through the third microfluidic channel 7; the right side of the detection tank Fiber Channel is provided and is not connected to Fiber Channel;
  • One side of the microfluidic chip is also provided with 3 sampling channels, which are successively recorded as the first sampling channel 12, the second sampling channel 13 and the third sampling channel 14;
  • a reaction tank 2 is communicated, the second sampling channel 13 is communicated with the second reaction tank 4, and the third sampling channel 14 is communicated with the third reaction tank 6;
  • the preparation method of the microfluidic chip is:
  • microfluidic design diagram comprising 1 sample channel, 3 injection channels, 3 circular reaction slots, 1 circular detection slot and 3 U-shaped microfluidic channels), 3 U-shaped microfluidic channels
  • the total length and width of the fluid channel are 9cm and 3cm respectively, the depth of the channel is 500 ⁇ m, the width of the microfluidic channel itself is 1mm, and the length and width of the sample channel and the sampling channel are 1cm and 0.5cm respectively;
  • Alcohol composite materials are used as raw materials, and microfluidic channel templates are printed by 3D printing technology;
  • quartz substrate 11 is a transparent material, through which the quartz The substrate 11 can clearly see the substance and its flow in the pipeline;
  • a fluorescent sensing film pick-and-place port and a visual detection port 9 are provided for the pick-and-place of the fluorescent sensing film and the acquisition of the fluorescent visualization signal; a strip is processed on the right side of the detection groove 8
  • the optical fiber channel 10 that is not connected with the detection groove 8 is 0.1 cm away from the detection groove 8, and finally obtains a microfluidic chip;
  • Sample channel 1 is the entry channel of the organophosphorus pesticide sample
  • the first sampling channel 12, the second sampling channel 13, and the third sampling channel 14 are respectively the sampling channels of acetylcholinesterase inlet, acetylcholine inlet and choline oxidase .
  • Step 3 establishment of microfluidic chip detection method
  • the present invention first builds a standard detection system, as shown in Figure 3, including a constant pressure injection pump 1, a first connecting pipe 2, a first syringe 3, a second syringe 4, a third syringe 5, a second connecting pipe 6,
  • the constant pressure syringe pump 1 is connected to the sampling port of the microfluidic chip 9 through the first connecting pipe 2;
  • the first syringe 3, the second syringe 4, and the third syringe 5 pass through the second connecting pipe 6, the third connecting pipe 7, the fourth connecting pipe 8 and the first sample injection channel 12 and the second sample injection channel 12 of the microfluidic chip 9 respectively.
  • Channel 13 is connected to the third sampling channel 14;
  • the 1*2 type fluorescent optical fiber 10 is used to obtain the fluorescent signal of the porous fluorescent sensing film in the detection groove 8, and the other end is connected with the laser 11 and the spectrometer 12;
  • the sensitive film signal is transmitted to the signal display 14;
  • the steps to establish a detection method for organophosphorus pesticides mainly include:

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Abstract

一种基于荧光传感薄膜的微流体芯片（9）检测有机磷农药的方法，属于有机磷农药检测领域。首先构建多孔荧光传感薄膜和微流体芯片（9）；荧光传感薄膜通过层层自组装铂纳米颗粒@草酸酯-有机金属框架复合材料层（3）和多孔二维纳米片制备而成，具有特异性检测有机磷农药和阻隔大分子干扰物的功能；微流体芯片（9）由样品通道（1）、进样通道、反应槽、微流体通道、检测槽（8）和光纤通道（10）构成，将样品预处理和检测过程集成在芯片内；通过结合便携式恒压注射泵（1）、激光器（11）、光谱仪（12）、信号传输器（13）和信号显示器（14）搭建了有机磷农药检测系统，使检测设备微型化和一体化、有机磷农药检测规范化。将纳米薄膜和微流体芯片（9）耦合，实现有机磷农药的快速现场定性和定量检测。

Description

一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法 技术领域
本发明属于有机磷农药检测领域，具体涉及一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法。
背景技术
有机磷农药(Organophosphorus Pesticides，OPs)是指含磷元素的有机化合物，如常见的有机磷农药敌百虫、对硫磷、乐果和敌敌畏等，具有防治农作物病虫和草害功效，是农产品种植过程中应用最广泛的农药之一。其检测方法主要有仪器分析法(气相色谱法、液相色谱法、红外光谱法、气相色谱-质谱联用法、紫外光谱法等)和基于生物技术的检测法(如酶抑制法、生物传感器和免疫分析法)。以上方法可以实现有机磷农药的准确检测，但仍存在操作繁琐或者依赖大型笨重仪器等缺点，无法实现农药残留的现场快速、便携式检测。
随着新型纳米材料的迅速发展，荧光纳米材料以其稳定的形态结构及高效的发光效率，在标记、示踪、成像和检测等领域显示巨大的应用潜力。但目前基于荧光纳米材料的农药残留检测致力于特异性纳米材料的制备，且检测都须在溶液中进行，检测常用的特异性分子为适配体和抗原/抗体等生物性分子，荧光纳米材料在溶液中因溶解性差或稳定性差会出现纳米颗粒沉析和荧光猝灭等现象，生物性分子的应用导致检测成本高且检测重复性差，溶液不稳定、不易携带等缺点限制了荧光纳米材料在现场检测中的应用。
相对于荧光检测的单一方式，联合其他材料或其他检测方式能发挥更大的检测作用，如制备荧光传感薄膜和结合微流体芯片等。通过制备荧光薄膜传感器可以克服荧光纳米材料在溶液中稳定性差、易光漂白等缺点，且赋予了传感器易携带及使用简便等特性。为了更好的对有机磷农药进行检测，利用微流体芯片技术将涉及的样品前处理、混匀反应及荧光检测等多种过程集成到几平方厘米的芯片上，具有多功能集成、体积小及便于携带等优点，是一种常用的即时检测装置。对于目前有机磷农药的检测，研究尚未实现样品前处理、特异性试剂添加、有害物与特异性试剂反应、信号获取以及信号处理等必要步骤的自动化、连续化及系统化。同时，环境和仪器等因素变化会影响微流体芯片系统检测的稳定性。因此，亟需设计一种能快速、稳定现场检测有机磷农药的微流体芯片系统，建立一套相应的有机磷农药现场快速检测方法，满足严酷的工业和农业生产环境下快速、高稳定检测需求。
发明内容
针对目前有机磷农药检测存在的不足，基于有机磷农药能够抑制乙酰胆碱酯酶催化乙酰胆碱水解，且有机磷农药的浓度与水解产物H 2O 2浓度呈相关性，本发明构建了H 2O 2荧光传感薄膜，提供了一种集自动化进样、前处理和检测等功能于一体的有机磷农药微流体芯片系 统及检测方法，拟解决微流体芯片无法规范化和标准化检测有机磷农药的问题，以及无法实现有机磷农药现场快速检测的问题。
本发明构建的一种基于荧光传感薄膜的微流体芯片系统及有机磷农药检测方法，其特征在于包含特异性荧光传感薄膜的制备、微流体芯片系统的构建、现场检测方法的建立以及实际应用方式。
步骤一、多孔荧光传感薄膜构建，包含特异性荧光材料的合成及其荧光传感薄膜的构筑，可用于的乙酰胆碱水解产物H 2O 2的特异性检测：
S1.合成特异性草酸酯@有机金属框架复合材料：
将硝酸铕、均苯四甲酸和草酸溶解于乙腈-乙醇溶液中，搅拌后得到的混合溶液在一定温度条件下反应，纯化并干燥后获得有机金属框架粉末，复溶于水中获得有机金属框架材料(EuMOF)溶液；
将草酸酯(TCPO)溶液与有机金属框架材料溶液振荡混合一定时间，获得草酸酯-有机金属框架复合(TCPO-EuMOF)溶液，将草酸酯-有机金属框架复合溶液与氯亚铂酸钾溶液混合，在一定温度条件下振荡后添加硼氢化钠，得到铂纳米颗粒负载的草酸酯-有机金属框架复合材料(Pt@TCPO-EuMOF)溶液，记为Pt@TCPO-EuMOF溶液；
S2.构筑表面阻隔大分子的多孔荧光传感薄膜：
将石英基底清洗后置于聚二甲基二烯丙基氯化铵(PDADMA)溶液中浸泡，浸泡后取出石英基底再次浸泡于步骤S1得到的Pt@TCPO-EuMOF溶液中，蒸馏水冲洗干净后获得Pt@TCPO-EuMOF膜；随后将Pt@TCPO-EuMOF膜依次再浸泡在聚二甲基二烯丙基氯化铵(PDADMA)溶液和Pt@TCPO-EuMOF溶液中，如此重复浸泡N次，最终在石英基底表面形成N+1层的Pt@TCPO-EuMOF荧光传感层，记为荧光传感层修饰的石英基底；
最后将二硫化钼纳米片、聚苯乙烯类聚合物和聚二甲基二烯丙基氯化铵以一定比例溶解在乙醇中，得到混合液，然后将荧光传感层修饰的石英基底在混合液中浸泡M次，在荧光传感层表面形成M层大分子阻隔层，最终获得包括荧光传感层和大分子阻隔层的有机磷农药荧光传感薄膜，记为多孔荧光传感薄膜；
步骤二、设计具有自动进样、样品前处理、样品与试剂混合反应、检测功能的微流体芯片；
所述微流体芯片包括样品通道、反应槽、检测槽、微流体通道、光纤通道；所述反应槽设有n个，依次记为第一反应槽、第二反应槽…、第n-1反应槽和第n反应槽；
所述微流体芯片的一端开设样品口，所述第一反应槽的一端设有样品通道，连接样品口；另一端通过微流体通道与第二反应槽…、第n-1反应槽和第n反应槽依次相连通；所述第n 反应槽的另一端通过微流体通道与检测槽一端相连通；所述检测槽的另一端设有光纤通道，且与光纤通道不连通；
在微流体芯片的一侧还设有n个进样通道，依次记为第一进样通道、第二进样通道…、第n-1进样通道和第n进样通道；所述第一进样通道与第一反应槽连通、所述第二进样通道与第二反应槽连通…、所述第n-1进样通道与第n-1反应槽连通；所述第n进样通道与第n反应槽连通；
所述检测槽的上方开设有荧光传感薄膜取放口及可视化检测口；所述n为正整数。
所述微流体芯片的制备方法为：
首先设计微流体芯片的通道结构，并以溶解型支撑材料为基材打印出微流体通道模型；然后将微流体通道模型转移至容器中，固定后浇筑聚二甲基硅氧烷(PDMS)和其固化剂混合形成的复合物，加热至一定温度使PDMS硬化后，获得微流体平台模板，利用有机溶剂水溶液溶解微流体平台模板中的微流体通道模型，获得微流体芯片平台；对微流体芯片平台上表面进行预处理，使得上表面的微流体通道与外界环境相通；
最后，利用等离子体技术将PDMS表面甲基转化成羟基，然后再利用石英基片对微流体平台上表面进行密封，作为微流体平台的上盖；在检测槽的正上方石英基片区域开设荧光传感薄膜取放口及可视化检测口，用于荧光传感薄膜的取放和荧光可视化信号的获取，在检测槽靠近端部的一侧加工出光纤通道，最终获得有机磷农药检测微流体芯片；
步骤三、有机磷农药荧光比色卡及定量检测模型的建立，具体步骤为：
首先，搭建了有机磷农药微流体芯片系统，主要包括恒压注射泵、注射器、连接管、微流体芯片、荧光光纤、激光器、光谱仪、信号传输器、信号显示器；
所述恒压注射泵通过连接管连接微流体芯片的进样口，用于进样；
所述注射器通过连接管连通进样通道，将反应试剂加入反应槽中；所述反应试剂用于降解乙酰胆碱，产生H 2O 2；
所述荧光光纤一端置于光纤通道内，用于获取检测槽内多孔荧光传感薄膜的荧光信号，另一端与激光器和光谱仪相连；所述光谱仪通过信号传输器将获取的多孔荧光传感薄膜信号传输给信号显示器；
其次，配制h个不同浓度梯度的有机磷农药标准品，将步骤一制备的多孔荧光传感薄膜置于微流体芯片检测槽内，利用恒压注射泵通过进样口加入有机磷农药样品，使用注射器通过进样通道将反应试剂加入反应槽中，加入的有机磷农药标准品和反应试剂在微流体通道内混合混匀，并使通道内的有机磷农药降解成H 2O 2，混合反应后的反应液最终流入检测槽，反应液中的H 2O 2与检测槽内的多孔荧光传感薄膜反应一段时间；反应结束后，在紫外灯照射下 从检测槽上方的荧光传感薄膜取放口及可视化检测口观察荧光传感薄膜颜色变化情况，并获取不同有机磷浓度下荧光传感薄膜的图片，按照有机磷农药标准品的浓度由小到大组合不同浓度所对应的图片获得荧光检测比色卡；
将荧光光纤通入光纤通道，激发光照射后利用光谱仪获取不同有机磷浓度下荧光传感薄膜的荧光信号强度；根据有机磷农药标准品的浓度与荧光信号强度变化值之和的线性关系建立检测有机磷农药的校正曲线模型，记为y＝a+bx，其中x为有机磷农药标准品的浓度，y为荧光信号强度变化值之和，a和b分别为方程的常数项和系数；
步骤四、有机磷农药的检测：
参照国标中样品预处理方法制得样品提取液；按照步骤三中检测有机磷农药标准品的操作，将待测样品提取液通过进样口通入微流体芯片中，然后通过进样通道加入反应试剂，在检测槽反应结束后通过肉眼观察荧光传感薄膜的荧光颜色，根据步骤三所获得的荧光检测比色卡，初步判断样品中有机磷农药的浓度为n c，其中1≤c≤h，且c为正整数，完成有机磷农药的定性检测；
进一步通过光谱仪获取检测槽内多孔荧光传感薄膜的荧光信号，根据荧光信号强度差值之和y代入建立的校正曲线模型中，计算得到样品有机磷的浓度X，X＝(a-y)/b，a和b分别为方程的常数项和系数；完成有机磷农药的定量检测。
优选的，步骤一的S1中所述硝酸铕、均苯四甲酸、草酸和乙腈-乙醇溶液的用量比为0.1～10mmol:0.05～0.5mmol:0.05～0.5mmol:50～100mL；乙腈-乙醇溶液为乙腈和乙醇的混合液，其中乙腈和乙醇的混合的体积比为6～10:1～4；所述一定温度条件下反应的温度为100～200℃，反应时间为12～72h；所述纯化步骤是利用有机溶剂反复清洗3～10次，有机溶剂包括N,N-二甲基甲酰胺、乙醇或丙酮的任意一种；所述干燥是指在40～60℃下干燥直至恒重；所述有机金属框架粉末和有机金属框架材料溶液的最佳激发波长一致，范围为500～650nm，最佳发射波长为W 1、W 2、W 3、……、W e，e为大于零的正整数。
优选的，步骤一的S1中所述草酸酯溶液的质量浓度为0.5～1mg/mL；所述有机金属框架溶液的质量浓度为2～10mg/mL；所述草酸酯溶液与有机金属框架溶液的体积比为1:1；所述草酸酯溶液与有机金属框架材料溶液混合后在一定温度条件下振荡的温度为30～50℃，振荡时间为12～48h；所述草酸酯-有机金属框架材料的浓度为2～10mg/mL，所述氯亚铂酸钾的浓度为0.3～5mg/mL，所述硼氢化钠的浓度为0.1～1mg/mL；所述草酸酯-有机金属框架材料、氯亚铂酸钾和硼氢化钠的体积比为1：0.1～2：0.5～1.5；
所述Pt@TCPO-EuMOF溶液中铂纳米颗粒的粒径不超过50nm。
优选的，步骤一的S2中所述石英基底清洗具体是在乙醇溶液中超声清洗3～8次，每次清 洗1～10min；所述聚二甲基二烯丙基氯化铵(PDADMA)溶液浓度为0.5～5g/L，Pt@TCPO-EuMOF溶液的浓度为0.5～10mg/mL，重复浸泡N次过程中，在PDADMA溶液和Pt@TCPO-EuMOF溶液中浸泡的时间均为5～30min，重复浸泡的次数N为5～100；所述二硫化钼纳米片、聚苯乙烯类聚合物、聚二甲基二烯丙基氯化铵和乙醇的用量比为0.1～5mg:0.2～2mg:0.01～0.1mg：1ml；所述浸泡次数M为2～10，浸泡的时间均为3～10min；所述多孔荧光传感薄膜表面的大分子阻隔层的孔径为2～15nm。
优选的，步骤二中所述设计微流体芯片的通道结构，通道结构包含微流体通道、样品通道、反应槽、检测槽的，具体设计要求为：微流体通道的总长度为5～20cm，通道深度和宽度均为0.4～4mm；所述样品通道、第一进样通道、第二进样通道…、第n-1进样通道和第n进样通道的长度均为0.8～2cm，深度和宽度均为0.4～5mm，所述第一反应槽、第二反应槽…、第n-1反应槽、第n反应槽和检测槽的深度均为0.5～5mm；所述反应槽的面积为0.5～3cm 2；检测槽的面积为0.3～5cm 2；所述微流体芯片和石英基片的尺寸保持一致，长宽高为6～15cm、1～10cm和0.5～2cm。
优选的，步骤二中所述溶解型支撑材料为聚甲醛、聚乙烯醇或丙烯酸类共聚物的一种，或者多种任意比例的混合物；所述聚二甲基硅氧烷和其固化剂的质量比为7～9：1～3；所述固化剂为一类硅烷偶联试剂，主要由乙烯基三胺、氨乙基哌嗪、二氨基二苯基甲烷和间苯二胺的一种或者几种混合而成；所述有机溶剂水溶液是指水与有机溶剂以体积比为1～4：6～9的混合溶液；有机溶剂包括丙酮、异丙醇或硝酸的一种或多种；所述加热至一定温度为65～80℃。
优选的，步骤二中所述荧光传感薄膜取放口及可视化检测口的孔径为0.6～2.1cm；检测槽与光纤通道不连通且相距0.5～1cm，光纤通道长宽高分别为1～5cm、0.1～1cm、0.1～1cm；所述反应槽之间的微流体通道的形状为T个“U”叠加的“蛇形”通道；所述T为正整数。
优选的，步骤三中所述反应试剂包括乙酰胆碱酯酶、乙酰胆碱或胆碱氧化酶的一种或多种；所述荧光光纤具体为1*2型荧光光纤。
优选的，步骤三中所述在检测槽内的反应一段时间为2～10min；紫外灯和激发光的波长为301～399nm；有机磷农药标准品的浓度范围为0～1g/mL，分别记为0、n 1、n 2、n 3、……、n h；荧光传感薄膜的图片分别记为p 0、p 1、p 2、p 3、……、p h；波长为W 1处不同浓度有机磷农药标准品所对应的荧光信号强度记为I 1，0、I 1.1、I 1.2、I 1.3、……、I 1.h，波长为W 2处不同浓度有机磷农药标准品所对应的荧光信号强度记为I 2.0、I 2.1、I 2.2、I 2.3、……、I 2.h，……、波长为W e处不同浓度有机磷农药标准品所对应的荧光信号强度记为I e.0、I e.1、I e.2、I e.3、……、I e.h；I 1.0、I 2.0….I e.0为有机磷农药标准品的浓度为0时对应的荧光信号强度；
所述荧光信号强度差值之和为y，分别记为y 1、y 2、y 3、……、y h；
y 1＝(I 1.1-I 1.0)+(I 2.1-I 2.0)+……+(I e.1-I e.0)、y 2＝(I 1.2-I 1.0)+(I 2.2-I 2.0)+……+(I e.2-I e.0)、y 3＝(I 1.3-I 1.0)+(I 2.3-I 2.0)+……+(I e.3-I e.0)、……、y h＝(I 1.h-I 1.0)+(I 2.h-I 2.0)+……+(I e.h-I e.0)；其中h、e为正整数。
优选的，步骤四中所述在检测槽内的反应一段时间为2～10min；所述反应试剂包括乙酰胆碱酯酶、乙酰胆碱或胆碱氧化酶的一种或多种。
根据定性检测的结果，可以在现场对待测样品中有机磷含量是否超过限量标准进行快速判断，根据定量的检测结果，可以实现待测样品的现场准确测定。
本发明的有益效果在于：
(1)本发明通过构建具有笼状结构的荧光有机金属框架材料，并将特异性分子草酸酯固定在孔隙结构中，同时引入贵金属纳米颗粒形成铂纳米颗粒负载的草酸酯-有机金属框架复合材料，在该材料中，铂纳米颗粒可以催化草酸酯和H 2O 2的特异性反应，且反应产生的化学荧光使有机金属框架的荧光信号特异性增强，构建了基于草酸酯-H 2O 2反应产物和EuMOF的化学发光共振能量转移新体系，制备的荧光传感材料具有催化反应和特异性检测双功能，可提高检测有机磷农药的特异性和灵敏性。
(2)本发明在石英基底表面层层组装铂纳米颗粒@草酸酯-有机金属框架复合材料制备荧光传感层，通过调节荧光传感材料的负载量，扩大有机磷农药检测范围，同时使荧光材料使用简便化、携带便携化，另外在荧光传感层外表面包裹多孔二维纳米片材料，可阻隔反应液中含有的蛋白酶等大分子进入荧光传感层，减小蛋白酶等大分子对荧光信号产生的不利影响，提高检测精度，降低检测限。
(3)本发明通过设计具有自动进样、样品前处理、样品与检测试剂混合反应等功能的微流体芯片，搭建有机磷农药微流体芯片便携式系统，使检测设备一体化、微型化，有机磷农药检测规范化和标准化，最终提高现场检测的稳定性和重现性。
(4)本发明将荧光纳米薄膜与微流体芯片相结合，通过设计特异性荧光薄膜，将有机磷农药与特异性检测试剂的反应封合在稳定的微流体芯片中，既可肉眼获得荧光信号的变化，实现有机磷农药的快速可视化检测，又可以利用便携式光谱仪获取荧光强度，实现有机磷农药的快速定量检测。
附图说明
图1为多孔荧光传感薄膜结构示意图，其中：1-石英基底，2-聚二甲基二烯丙基氯化铵层，3-铂纳米颗粒@草酸酯-有机金属框架复合材料层，4-二硫化钼纳米片-聚苯乙烯类聚合物多孔阻隔层；
图2为微流体芯片结构示意图，其中：1-样品通道，2-第一反应槽，3-第一微流体通道，4-第二反应槽，5-第二微流体通道，6-第三反应槽，7-第三微流体通道，8-检测槽，9-荧光传感薄膜取放口及可视化检测口，10-光纤通道，11-石英基片，12-第一进样通道，13-第二进样通道，14-第三进样通道，15-微流体平台；
图3为有机磷农药检测流程图；其中1-恒压注射泵，2-第一连接管，3-第一注射器，4-第二注射器，5-第三注射器，6-第二连接管，7-第三连接管，8-第四连接管，9-微流体芯片，10-1*2型荧光光纤，11-激光器，12-光谱仪，13-信号传输器，14-信号显示器。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步的说明。
实施例1：
一种有机磷农药的微流体芯片制备及现场检测方法建立，包括以下步骤：
步骤一、荧光传感薄膜的构建：
1)称量0.2mmol硝酸铕、0.8mmol均苯四甲酸、0.8mmol草酸溶解于100mL乙腈-乙醇溶液中，乙腈和乙醇溶液的体积比为6：4，搅拌30min后移入聚四氟乙烯内衬中，烘箱中160℃反应48h，反应后进行纯化，步骤是加入N,N-二甲基甲酰胺反复离心清洗5次，离心清洗后获得有机金属框架材料(EuMOF)置于45℃干燥箱中干燥获得粉末，再次溶解于水中获得有机金属框架溶液，通过获取溶液和粉末的荧光光谱确定两者的最佳激发波长为560nm，激发波长范围为500～650nm，最佳发射波长为520nm和610nm，在最佳激发光(560nm)照射下有机金属框架溶液和粉末都呈现微弱的红色荧光，因配体带有大量的羧基，所以有机金属框架材料带负电；将10mL浓度为1mg/mL的双(2，4，6-三氯苯基)草酸酯溶液(TCPO)与10mL浓度为5mg/mL的有机金属框架溶液搅拌48h，获得双(2，4，6-三氯苯基)草酸酯-有机金属框架复合溶液；有机金属框架显示出铕离子的特征荧光峰，嵌入的草酸酯与H 2O 2发生特异性反应产生激发态产物，激发态产物与有机金属框架构成化学荧光共振能量转移体系，由于有机金属框架的吸收能量正好与激发态产物发出的荧光能量相近，激发态产物发出的化学荧光能量被有机金属框架中的铕离子吸收，使有机金属框架荧光得到增强；由于草酸酯与H 2O 2之间的反应慢且反应不完全，将10mL草酸酯-有机金属框架复合溶液(3mg/mL)与10mL氯亚铂酸钾溶液(0.5mg/mL)混合，并逐滴添加硼氢化钠(0.2mg/mL)得到铂纳米颗粒负载的草酸酯-有机金属框架复合材料溶液，记为Pt@TCPO-EuMOF溶液；该复合材料引入贵金属纳米颗粒铂纳米颗粒作为草酸酯和H 2O 2反应的催化剂，加快了两者的反应速度和完成度，透射电镜表征结果表明铂纳米颗粒的粒径不超过10nm。
2)取直径为1cm的石英基底超声清洗3次，随后将石英基底浸泡在浓度为1g/L的聚二 甲基二烯丙基氯化铵(PDADMA)溶液中10min，使石英基底表面带正电，蒸馏水清洗干净后又浸泡在5mg/mL的铂纳米颗粒负载的草酸酯-有机金属框架复合溶液中10min，获得单层Pt@TCPO-EuMOF传感层；重复上述两次浸泡操作9次，使基底表面修饰了10层Pt@TCPO-EuMOF传感层；最后将15mg二硫化钼纳米片、5mg聚苯乙烯类聚合物和0.08mg聚二甲基二烯丙基氯化铵溶解在10mL乙醇中，并将修饰了10层Pt@TCPO-EuMOF传感层的石英基底浸泡在上述溶液中4次，每次浸泡3min；在传感层表面修饰上了大分子阻隔层，通过透射电镜表征发现二硫化钼纳米片-聚苯乙烯类聚合物阻隔层呈多孔状，平均孔径为8±0.9nm；
步骤二、微流体芯片的开发：
设计具有自动进样、样品前处理、样品与试剂混合反应、检测功能的微流体芯片；
所述微流体芯片包括样品通道1，第一反应槽2，第一微流体通道3，第二反应槽4，第二微流体通道5，第三反应槽6，第三微流体通道7，检测槽8，荧光传感薄膜取放口及可视化检测口9，光纤通道10，石英基片11，第一进样通道12，第二进样通道13，第三进样通道14和微流体平台15；
所述微流体芯片的一端开设样品口通过样品通道1与第一反应槽2相连通；第一反应槽2的另一端通过第一微流体通道3与第二反应槽4连通；第二反应槽4的另一端通过第二微流体通道5与第三反应槽6连通；所述第三反应槽6的另一端通过第三微流体通道7与检测槽8相连通；所述检测槽的右侧设有光纤通道，且与光纤通道不连通；
在微流体芯片的一侧还设有3个进样通道，依次记为第一进样通道12、第二进样通道13和第三进样通道14；所述第一进样通道12与第一反应槽2相连通、所述第二进样通道13与第二反应槽4相连通、所述第三进样通道14与第三反应槽6相连通；
所述检测槽8的上方开设有荧光传感薄膜取放口及可视化检测口9；
所述微流体芯片的制备方法为：
利用Auto CAD软件绘制微流体设计图(由1条样品通道、3条进样通道、3个圆形反应槽、1个圆形检测槽和3条U形微流体通道)，3条U形微流体通道的总长度和宽度分别为9cm和3cm，通道深度为500μm，微流体通道自身的宽度为1mm，样品通道和进样通道的长度和宽度分别为1cm和0.5cm；以聚甲醛和聚乙烯醇复合材料为原材料，采用3D打印技术打印出微流体通道模板；
将微流体通道模板转移至长为20cm的容器中，固定后浇筑聚二甲基硅氧烷(PDMS)液体(由聚二甲基硅氧烷和其固化剂以质量比1:9混合)，置于75℃烘箱加热使PDMS硬化后获得微流体平台模型；利用乙酸水溶液(水：乙酸＝1:9)溶解微流体通道模板，获得荧光微 流体平台，对微流体芯片平台上表面进行预处理，使得上表面的微流体通道与外界环境相通，即；
最后，利用等离子体技术将PDMS表面甲基转化成羟基，然后再利用石英基片11对微流体平台上表面进行密封，作为微流体平台的上盖；石英基片11为透明材质，透过石英基片11可以清楚看见管道内的物质及其流动情况；
在检测槽8的正上方石英基片区域开设荧光传感薄膜取放口及可视化检测口9，用于荧光传感薄膜的取放和荧光可视化信号的获取；在检测槽8右侧加工出一条不与检测槽8连通的光纤通道10，其与检测槽8相距0.1cm，最后获得微流体芯片；
样品通道1为有机磷农药样品的进入通道，第一进样通道12、第二进样通道13、第三进样通道14分别为乙酰胆碱酯酶入口、乙酰胆碱入口和胆碱氧化酶的进样通道。
步骤三、微流体芯片检测方法的建立：
本发明首先搭建了标准的检测系统，如图3所示，包括恒压注射泵1、第一连接管2、第一注射器3、第二注射器4、第三注射器5、第二连接管6、第三连接管7、第四连接管8、微流体芯片9、1*2型荧光光纤10、激光器11、光谱仪12、信号传输器13和信号显示器14；
恒压注射泵1通过第一连接管2与微流体芯片9的进样口相连；
第一注射器3、第二注射器4、第三注射器5分别通过第二连接管6、第三连接管7、第四连接管8与微流体芯片9的第一进样通道12、第二进样通道13和第三进样通道14相连；
所述1*2型荧光光纤10用于获取检测槽8内多孔荧光传感薄膜的荧光信号，另一端与激光器11和光谱仪12相连；所述光谱仪12通过信号传输器13将获取的多孔荧光传感薄膜信号传输给信号显示器14；
利用搭建的检测系统，建立有机磷农药检测方法的步骤主要有：
(1)将制备的荧光传感薄膜置于检测槽8中，随后将0.5mL有机磷农药(0、0.1、0.5、1.0、3.0、5.0、10.0、15.0、20.0、25.0、30.0、40.0、50.0ng/mL)、0.2mL乙酰胆碱酯酶(200U/mL)、0.2mL乙酰胆碱(0.5g/mL)和0.2mL胆碱氧化酶(100U/mL)分别经进样口(有机磷农药入口)、第一进样通道12(乙酰胆碱酯酶入口)、第二进样通道13(乙酰胆碱入口)和第三进样通道14(胆碱氧化酶入口)进入微流体通道，在通道内进行混匀和反应，最后汇入检测槽8内继续孵育5min；
(2)在紫外灯(360nm)照射下肉眼观察检测槽8中的多孔荧光传感薄膜荧光颜色，利用智能手机获取不同浓度下检测槽8的荧光图片，根据浓度由低到高组合成荧光检测比色卡，通过观察发现随着有机磷农药浓度的增加红色荧光逐渐增强；
(3)同时利用便携式荧光光谱仪12获取检测槽8内荧光传感薄膜的荧光强度，发现520 nm和610nm处的荧光信号随着浓度的增加逐渐增强，根据有机磷农药浓度与荧光强度增值(520nm和610nm处的荧光差值之和，荧光差值为获取的荧光强度与有机磷浓度为0时的荧光强度之差)之间的关系检测有机磷农药的校正曲线，其曲线方程为y＝146.89x+64.52(R 2＝0.99)，其中x为有机磷农药的浓度，y为荧光信号强度变化值之和，a和b分别为方程的常数项和系数。
步骤四、有机磷农药的现场检测：
选取油菜、小麦、大白菜和自然水四种样品，称取3g(精确至0.1g)放入离心管中，加入5mL丙酮和5mL磷酸缓冲溶液，振摇50次，静置10min；按照步骤三中检测有机磷标准品的操作，将荧光传感薄膜放入检测槽8内，并将0.5mL待测样品提取液、0.2mL乙酰胆碱酯酶(200U/mL)、0.2mL乙酰胆碱(0.5g/mL)和0.2mL胆碱氧化酶(100U/mL)分别经有机磷农药入口、乙酰胆碱酯酶入口、乙酰胆碱入口和胆碱氧化酶入口进入微流体通道，在通道内进行混匀和反应，最后汇入检测槽内继续孵育5min，在紫外灯(360nm)照射下肉眼观察检测槽的荧光颜色，对比荧光检测比色卡，发现油菜样品提取液的荧光颜色与浓度为5ng/mL的荧光图片颜色接近，油菜样品提取液浓度大约为5ng/mL，同理，小麦样品提取液浓度大约为0.1ng/mL，大白菜样品提取液浓度大约为5ng/mL，自然水样品提取液浓度大约为0.1ng/mL，完成样品中有机磷农药的定性和半定量检测；将荧光光谱仪获得的520nm和610nm处的荧光强度差值之和代入建立的校正曲线中，计算得到油菜、小麦、大白菜和自然水样品中有机磷浓度分别为5.49、0.18、5.27和0.17ng/mL，完成有机磷农药的准确定量检测；除样品预处理外，该检测从进样到获取检测结果耗费的时间为15.5±1.5min。为了论证本发明方法的准确性和快速性，利用国标的方法对相同样品进行了检测，发现油菜、小麦、大白菜和自然水样品中有机磷浓度分别为6.29、0.58、7.25、0.24ng/mL，除样品预处理外检测耗费时间为5.1±0.3h。两种检测结果(表1)表明本发明的检测方法能够在现场实现对环境和农产品样本的准确检测，且检测速度快、设备携带简便。
表1本方法和国标法的检测结果对比
Figure PCTCN2022078143-appb-000001
Figure PCTCN2022078143-appb-000002
说明：以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此，尽管本说明书参照上述的各个实施例对本发明已进行了详细的说明，但是本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围内。

Claims (10)

  1. 一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，特征在于，包括以下步骤：
    步骤一、多孔荧光传感薄膜的制备：
    S1.将硝酸铕、均苯四甲酸和草酸溶解于乙腈-乙醇溶液中，搅拌后得到的混合溶液在一定温度条件下反应，纯化并干燥后获得有机金属框架粉末，复溶于水中获得有机金属框架材料溶液；
    将草酸酯溶液与有机金属框架材料溶液振荡混合一定时间，获得草酸酯-有机金属框架复合溶液；将草酸酯-有机金属框架复合溶液与氯亚铂酸钾溶液混合，在一定温度条件下振荡后添加硼氢化钠，得到铂纳米颗粒负载的草酸酯-有机金属框架复合材料溶液，记为Pt@TCPO-EuMOF溶液；
    S2.将石英基底清洗后置于聚二甲基二烯丙基氯化铵溶液中浸泡，浸泡后取出石英基底再次浸泡于步骤S1得到的Pt@TCPO-EuMOF溶液中，取出后蒸馏水冲洗干净，获得Pt@TCPO-EuMOF膜；
    随后将Pt@TCPO-EuMOF膜依次再浸泡在聚二甲基二烯丙基氯化铵溶液和Pt@TCPO-EuMOF溶液中，取出后蒸馏水冲洗干净，如此为一个浸泡循环；循环浸泡N次后，最终在石英基底表面形成N+1层的Pt@TCPO-EuMOF荧光传感层，记为荧光传感层修饰的石英基底；其中N为正整数；
    最后将二硫化钼纳米片、聚苯乙烯类聚合物和聚二甲基二烯丙基氯化铵以一定比例溶解在乙醇中，得到混合液，然后将荧光传感层修饰的石英基底浸泡其中M次，在荧光传感层表面形成M层大分子阻隔层，最终获得包括荧光传感层和大分子阻隔层的有机磷农药荧光传感薄膜，记为多孔荧光传感薄膜；其中M为正整数；
    步骤二、微流体芯片的制备；
    所述微流体芯片包括样品通道、反应槽、检测槽、微流体通道和光纤通道；
    所述反应槽设有n个，依次记为第一反应槽、第二反应槽…、第n-1反应槽和第n反应槽；
    所述微流体芯片的一端开设样品口，所述第一反应槽的一端设有样品通道，连接样品口；另一端通过微流体通道与第二反应槽…、第n-1反应槽和第n反应槽依次相连通；所述第n反应槽的另一端通过微流体通道与检测槽一端相连通；所述检测槽的另一端设有光纤通道，且与光纤通道不连通；
    在微流体芯片的一侧还设有n个进样通道，依次记为第一进样通道、第二进样通道…、第n-1进样通道和第n进样通道；所述第一进样通道与第一反应槽连通、所述第二进样通道与第二反应槽连通…、所述第n-1进样通道与第n-1反应槽连通；所述第n进样通道与第n反应槽连 通；
    所述检测槽的上方开设有荧光传感薄膜取放口及可视化检测口；所述n为正整数；
    所述微流体芯片的制备方法为：
    使用三维绘图软件设计微流体芯片的通道结构，并以溶解型支撑材料为基材，利用3D打印机打印出微流体通道模型；将微流体通道模型转移至容器中，固定后浇筑聚二甲基硅氧烷和其固化剂混合形成的复合物，加热至一定温度使聚二甲基硅氧烷硬化后，获得微流体平台模板，利用有机溶剂水溶液溶解微流体平台模板中的微流体通道模型，获得微流体芯片平台；对微流体芯片平台上表面进行预处理，使得上表面的微流体通道与外界环境相通；
    最后，利用等离子体技术将聚二甲基硅氧烷表面的甲基转化成羟基，然后再利用石英基片对微流体平台上表面进行密封，作为微流体平台的上盖；在检测槽的正上方石英基片区域开设荧光传感薄膜取放口及可视化检测口，用于荧光传感薄膜的取放和荧光可视化信号的获取，在检测槽靠近端部的一侧加工出光纤通道，最终获得有机磷农药检测微流体芯片；
    步骤三：有机磷农药荧光比色卡及定量检测模型的建立，具体步骤为：
    首先，搭建了有机磷农药微流体芯片系统，主要包括恒压注射泵、注射器、连接管、微流体芯片、荧光光纤、激光器、光谱仪、信号传输器和信号显示器；
    所述恒压注射泵通过连接管连接微流体芯片的进样口，用于进样；
    所述注射器通过连接管连通进样通道，将反应试剂加入反应槽中；所述反应试剂用于降解乙酰胆碱，产生H 2O 2；
    所述荧光光纤一端置于光纤通道内，用于获取检测槽内多孔荧光传感薄膜的荧光信号，另一端与激光器和光谱仪相连；所述光谱仪通过信号传输器将获取的多孔荧光传感薄膜信号传输给信号显示器；
    其次，配制h个不同浓度梯度的有机磷农药标准品，将步骤一制备的多孔荧光传感薄膜置于微流体芯片检测槽内，利用恒压注射泵通过进样口加入有机磷农药样品，使用注射器通过进样通道将反应试剂加入反应槽中，加入的有机磷农药标准品和反应试剂在微流体通道内混合混匀，并使通道内的有机磷农药降解成H 2O 2，混合反应后的反应液最终流入检测槽，反应液中的H 2O 2与检测槽内的多孔荧光传感薄膜反应一段时间；反应结束后，在紫外灯照射下从检测槽上方的荧光传感薄膜取放口及可视化检测口观察荧光传感薄膜颜色变化情况，并获取不同有机磷浓度下荧光传感薄膜的图片，按照有机磷农药标准品的浓度由小到大组合不同浓度所对应的图片获得荧光检测比色卡；
    将荧光光纤通入光纤通道，激发光照射后利用光谱仪获取不同有机磷浓度下荧光传感薄膜的荧光信号强度；根据有机磷农药标准品的浓度与荧光信号强度变化值之和的线性关系建 立检测有机磷农药的校正曲线模型，记为y＝a+bx，其中x为有机磷农药标准品的浓度，y为荧光信号强度变化值之和，a和b分别为方程的常数项和系数；
    步骤四、有机磷农药的检测：
    参照国标中样品预处理方法制得样品提取液；按照步骤三中检测有机磷农药标准品的操作，将待测样品提取液通过进样口通入微流体芯片中，然后通过进样通道加入反应试剂，在检测槽反应结束后通过肉眼观察荧光传感薄膜的荧光颜色，根据步骤三所获得的荧光检测比色卡，初步判断样品中有机磷农药的浓度为n c，其中1≤c≤h，且c为正整数，完成有机磷农药的定性检测；
    进一步通过光谱仪获取检测槽内多孔荧光传感薄膜的荧光信号，根据荧光信号强度差值之和y代入建立的校正曲线模型中，计算得到样品有机磷的浓度X，X＝(a-y)/b，a和b分别为方程的常数项和系数，完成有机磷农药的定量检测。
  2. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤一的S1中所述硝酸铕、均苯四甲酸、草酸和乙腈-乙醇溶液的用量比为0.1～10mmol:0.05～0.5mmol:0.05～0.5mmol:50～100mL；乙腈-乙醇溶液为乙腈和乙醇的混合液，其中乙腈和乙醇的混合的体积比为6～10:1～4；所述一定温度条件下反应的温度为100～200℃，反应时间为12～72h；所述纯化步骤是利用有机溶剂反复清洗3～10次，有机溶剂包括N,N-二甲基甲酰胺、乙醇或丙酮的任意一种；所述干燥是指在40～60℃下干燥直至恒重；所述有机金属框架粉末和有机金属框架材料溶液的最佳激发波长一致，范围为500～650nm，最佳发射波长为W 1、W 2、W 3、……、W e，e为大于零的正整数。
  3. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤一的S1中所述草酸酯溶液的质量浓度为0.5～1mg/mL；所述有机金属框架溶液的质量浓度为2～10mg/mL；所述草酸酯溶液与有机金属框架溶液的体积比为1:1；所述草酸酯溶液与有机金属框架材料溶液混合后在一定温度条件下振荡的温度为30～50℃，振荡时间为12～48h；所述草酸酯-有机金属框架材料的浓度为2～10mg/mL，所述氯亚铂酸钾的浓度为0.3～5mg/mL，所述硼氢化钠的浓度为0.1～1mg/mL；所述草酸酯-有机金属框架材料、氯亚铂酸钾和硼氢化钠的体积比为1：0.1～2：0.5～1.5；
    所述Pt@TCPO-EuMOF溶液中铂纳米颗粒的粒径不超过50nm。
  4. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤一的S2中所述石英基底清洗具体是在乙醇溶液中超声清洗3～8次，每次清洗1～10min；所述聚二甲基二烯丙基氯化铵溶液浓度为0.5～5g/L，Pt@TCPO-EuMOF溶液的浓度为0.5～10mg/mL，重复浸泡N次过程中，在PDADMA溶液和Pt@TCPO-EuMOF溶液 中浸泡的时间均为5～30min，重复浸泡的次数N为5～100；所述二硫化钼纳米片、聚苯乙烯类聚合物、聚二甲基二烯丙基氯化铵和乙醇的用量比为0.1～5mg:0.2～2mg:0.01～0.1mg：1ml；所述浸泡次数M为2～10，浸泡的时间均为3～10min；所述多孔荧光传感薄膜表面的大分子阻隔层的孔径为2～15nm。
  5. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤二中所述设计微流体芯片的通道结构，通道结构包含微流体通道、样品通道、反应槽、检测槽的，具体设计要求为：微流体通道的总长度为5～20cm，通道深度和宽度均为0.4～4mm；所述样品通道、第一进样通道、第二进样通道…、第n-1进样通道和第n进样通道的长度均为0.8～2cm，深度和宽度均为0.4～5mm，所述第一反应槽、第二反应槽…、第n-1反应槽、第n反应槽和检测槽的深度均为0.5～5mm；所述反应槽的面积为0.5～3cm 2；检测槽的面积为0.3～5cm 2；所述微流体芯片和石英基片的尺寸保持一致，长宽高为6～15cm、1～10cm和0.5～2cm。
  6. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤二中所述溶解型支撑材料为聚甲醛、聚乙烯醇或丙烯酸类共聚物的一种，或者多种任意比例的混合物；所述聚二甲基硅氧烷和其固化剂的质量比为7～9：1～3；所述固化剂为一类硅烷偶联试剂，主要由乙烯基三胺、氨乙基哌嗪、二氨基二苯基甲烷和间苯二胺的一种或者几种混合而成；所述有机溶剂水溶液是指水与有机溶剂以体积比为1～4：6～9的混合溶液；有机溶剂包括丙酮、异丙醇或硝酸的一种或多种；所述加热至一定温度为65～80℃。
  7. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤二中所述荧光传感薄膜取放口及可视化检测口的孔径为0.6～2.1cm；检测槽与光纤通道不连通且相距0.5～1cm，光纤通道长宽高分别为1～5cm、0.1～1cm、0.1～1cm；所述反应槽之间的微流体通道的形状为T个“U”叠加的“蛇形”通道；所述T为正整数。
  8. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤三中所述反应试剂包括乙酰胆碱酯酶、乙酰胆碱或胆碱氧化酶的一种或多种；所述荧光光纤具体为1*2型荧光光纤。
  9. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤三中所述在检测槽内的反应一段时间为2～10min；紫外灯和激发光的波长为301～399nm；有机磷农药标准品的浓度范围为0～1g/mL，分别记为0、n 1、n 2、n 3、……、n h；荧光传感薄膜的图片分别记为p 0、p 1、p 2、p 3、……、p h；波长为W 1处不同浓度有机磷农药标准品所对应的荧光信号强度记为I 1，0、I 1.1、I 1.2、I 1.3、……、I 1.h，波长为W 2处不同浓度有机磷农药标准品所对应的荧光信号强度记为I 2.0、I 2.1、I 2.2、I 2.3、……、I 2.h，……、波长为W e处不同浓度有机磷农药标准品所对应的荧光信号强度记为I e.0、I e.1、I e.2、I e.3、……、I e.h； I 1.0、I 2.0….I e.0为有机磷农药标准品的浓度为0时对应的荧光信号强度；
    所述荧光信号强度差值之和为y，分别记为y 1、y 2、y 3、……、y h；
    y 1＝(I 1.1-I 1.0)+(I 2.1-I 2.0)+……+(I e.1-I e.0)、y 2＝(I 1.2-I 1.0)+(I 2.2-I 2.0)+……+(I e.2-I e.0)、y 3＝(I 1.3-I 1.0)+(I 2.3-I 2.0)+……+(I e.3-I e.0)、……、y h＝(I 1.h-I 1.0)+(I 2.h-I 2.0)+……+(I e.h-I e.0)；其中h、e为正整数。
  10. 根据权利要求1所述的一种基于荧光传感薄膜的微流体芯片检测有机磷农药的方法，其特征在于，步骤四中所述在检测槽内的反应一段时间为2～10min；所述反应试剂包括乙酰胆碱酯酶、乙酰胆碱或胆碱氧化酶的一种或多种。
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