WO2024239352A1 - 一种基于上转换发光的微流体生物传感平台 - Google Patents

一种基于上转换发光的微流体生物传感平台 Download PDF

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WO2024239352A1
WO2024239352A1 PCT/CN2023/096734 CN2023096734W WO2024239352A1 WO 2024239352 A1 WO2024239352 A1 WO 2024239352A1 CN 2023096734 W CN2023096734 W CN 2023096734W WO 2024239352 A1 WO2024239352 A1 WO 2024239352A1
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csucnps
incubation
biosensor
upconversion
edcs
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French (fr)
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陈全胜
吴继忠
欧阳琴
魏文雅
赵松光
朱阿芳
王震
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Jiangsu University
Jimei University
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Jiangsu University
Jimei University
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    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • 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
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • 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 present invention relates to the technical field of food safety detection, and in particular to a microfluidic biosensing platform based on up-conversion luminescence.
  • Endocrine disrupting chemicals are defined as "exogenous agents that interfere with the synthesis, secretion, transport, metabolism, binding or elimination of natural blood-borne hormones present in the human body that are responsible for homeostasis, reproduction and developmental processes". Humans may ingest hundreds of EDCs through food production (food additives, pesticides, food containers), industrial activities (air pollution, water pollutants, industrial chemicals), medical treatment (medical products), etc. Among them, exogenous agents with estrogenic effects, such as bisphenol A (BPA), diethylstilbestrol (DES), estradiol (E2), nonylphenol, etc. have attracted widespread attention.
  • BPA bisphenol A
  • DES diethylstilbestrol
  • E2 estradiol
  • nonylphenol, etc. have attracted widespread attention.
  • EDCs may cause serious health hazards, including neurodevelopmental disorders, brain, liver and lung damage, reproductive and endocrine disorders, metabolic disorders, etc. Therefore, it is very important to establish an effective assessment method to determine the content of EDCs in media that humans may be exposed to.
  • the present invention provides a microfluidic biosensing platform based on upconversion luminescence to achieve microsampling and simple high-sensitivity quantitative detection of EDCs.
  • the object of the present invention is to provide a microfluidic biosensing platform based on upconversion luminescence, comprising: an upconversion luminescence biosensor for specifically identifying EDCs; and a microfluidic chip, as a reaction platform between the upconversion luminescence biosensor and the sample to be tested, for integrating the mixing, reaction, separation, and detection of the upconversion luminescence biosensor and the sample to be tested;
  • the microfluidic chip comprises: a sample injection pool for injecting the upconversion luminescent biosensor and the sample to be tested; an arc-shaped channel, the feed port of which is simultaneously connected to the sample injection pool of the upconversion luminescent biosensor and the sample to be tested, and after the upconversion luminescent biosensor and the sample to be tested enter the arc-shaped channel, the arc-shaped channel is used for mixing and reacting the two; a separation channel is connected to the discharge port of the arc-shaped channel, and is used for the separation after the reaction is completed.
  • the magnetic separation of the upconversion luminescent biosensor; a detection pool, connected to the discharge port of the separation channel, is used for luminescence enhanced quantitative detection of EDCs.
  • all microchannels of the microfluidic chip have a width of 400 ⁇ m and a depth of 200 ⁇ m.
  • the mixing, reaction, separation and detection of the upconversion luminescence biosensor and the sample to be tested are integrated on the microfluidic chip, and the specific process is: the upconversion luminescence biosensor and the sample to be tested are respectively injected from the two injection pools of the microfluidic chip, and the two microfluids are fully mixed and reacted in the arc channel; then, a magnetic field is applied at the separation channel to separate the biosensor that has not reacted with EDCs; finally, in the detection pool, the detached CSUCNPs complete the bridging flocculation and sedimentation, and the upconversion fluorescence signal is collected, so that EDCs can be quantitatively detected.
  • the injection flow rates of the biosensor and the sample solution to be tested are 12 ⁇ L/min and 3 ⁇ L/min.
  • the injection time of the biosensor and the sample solution to be tested is 8-12 minutes.
  • the second object of the present invention is to provide a method for preparing the above-mentioned microfluidic biosensing platform based on upconversion luminescence.
  • the preparation method of the upconversion luminescence biosensor comprises the following steps:
  • step S2 coating the outer layer of the CUCNPs prepared in step S1 to prepare upconversion nanoparticles (CSUCNPs) with a core-shell structure;
  • the CSUCNPs prepared in the modification step S2 are hydrophilic;
  • step S4 functionalizing the hydrophilic CSUCNPs obtained in step S3 with biomolecules to obtain biomolecule-functionalized CSUCNPs;
  • MNPs magnetic nanoparticles
  • step S6 functionalizing the MNPs obtained in step S5 with biomolecules to obtain biomolecule-functionalized MNPs
  • step S7 The biomolecule functionalized CSUCNPs obtained in step S4 are combined with the biomolecule functionalized CSUCNPs obtained in step S6 to prepare the upconversion luminescent biosensor.
  • step S1 is:
  • Yttrium chloride hexahydrate, ytterbium chloride hexahydrate and rare earth element hexahydrate are dissolved in methanol respectively, then oleic acid and 1-octadecene are added, mixed and heated to 150-170°C for reaction for 25-35 minutes, after the reaction is completed and cooled, a mixed solution of sodium hydroxide and ammonium fluoride is added dropwise, and reacted at 125-135°C for 25-35 minutes, then heated to 290-310°C and maintained for 50-60 minutes; after the reaction is completed, ethanol and ultrapure water are added and centrifuged to obtain upconversion nanoparticle seeds (CUCNPs).
  • CRCNPs upconversion nanoparticle seeds
  • the total amount of the yttrium chloride hexahydrate, the ytterbium chloride hexahydrate and the rare earth element hexahydrate is 1-1.5 mmol.
  • the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate and the rare earth element hexahydrate is 0.78:0.2:0.02.
  • the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate and the rare earth element hexahydrate is 0.795:0.2:0.005.
  • volume ratio of methanol, oleic acid and 1-octadecene is 10:(5-7):(14-17).
  • the molar ratio of the sodium hydroxide to the ammonium fluoride is 5:(7-9).
  • volume ratio of the ethanol to ultrapure water is 1:(0.6-1).
  • centrifugation parameter is 8000-12000 rpm for 5-10 min.
  • step S2 is:
  • Yttrium chloride hexahydrate is dissolved in methanol, and then oleic acid and 1-octadecene are added, mixed and heated to 150-170°C for reaction for 25-35 minutes; after the reaction is completed and cooled, the CUCNPs prepared in step S1 are added, and a mixed solution of sodium hydroxide and ammonium fluoride is added dropwise, and reacted at 125-135°C for 25-35 minutes, and then the temperature is raised to 290-310°C and maintained for 20-40 minutes; after the reaction is completed, ethanol and ultrapure water are added and centrifuged to obtain core-shell upconversion nanoparticles (CSUCNPs).
  • CSUCNPs core-shell upconversion nanoparticles
  • the dosage of the yttrium chloride hexahydrate is 0.35-0.45 mmol.
  • volume ratio of methanol, oleic acid and 1-octadecene is 10:(2.5-3.5):(7-9).
  • the molar ratio of the sodium hydroxide to the ammonium fluoride is 2:(2.5-3.5).
  • volume ratio of the ethanol to ultrapure water is 1:(0.6-1).
  • centrifugation parameter is 8000-12000 rpm for 5-10 min.
  • step S3 is:
  • CSUCNPs prepared in step S2 were added to a mixed solution of chloroform and toluene, and then a polyacrylic acid aqueous solution was added and sealed, and the reaction was vigorously stirred; after completion, ethanol and ultrapure water were used to wash and centrifuge to obtain hydrophilic polyacrylic acid-modified CSUCNPs (PAA-CSUCNPs).
  • the ratio of the CSUCNPs, chloroform, toluene and polyacrylic acid is 50 mg:(2-6 mL):(4-10 mL):(15-20 mL).
  • the concentration of the polyacrylic acid aqueous solution is 10-20 mg/mL.
  • step S4 is:
  • the ratio of the PAA-CSUCNPs, carbodiimide, N-hydroxysulfosuccinimide and morpholineethanesulfonic acid buffer solution is (0.8-1.2 mg): 4 mg: 2 mg: (2.0-2.4 mL).
  • the first incubation condition is 20-40°C, 2-4h.
  • the second incubation condition is 30-40°C, 10-14h.
  • the amount of the aptamer used is 200-300 ⁇ L, and the concentration is 10 ⁇ M.
  • bovine serum albumin used is 3-6 mL, and the mass fraction is 2%.
  • the fourth incubation condition is 30-40° C., 1.5-3 h.
  • the centrifugal separation parameter is centrifugation at a speed of 8000-12000 rpm for 5-10 min.
  • step S4 the pH of the phosphate buffer is 7.2-7.4, and the amount used is 5-15 mL.
  • step S5 is:
  • Ferric chloride hexahydrate, trisodium citrate dihydrate, and sodium acetate were respectively added to ethylene glycol solution, stirred vigorously to fully dissolve, and then transferred to a reactor for high-temperature reaction; after the reaction was completed, the prepared magnetic nanoparticles (MNPs) were collected through a magnetic field and washed with ethanol and ultrapure water.
  • ferric chloride hexahydrate trisodium citrate dihydrate, sodium acetate and ethylene glycol is 5mmol:0.4mmol:(1.2-1.5g):(15-25mL).
  • the high temperature reaction conditions are 190-210° C., 8-12 h.
  • volume ratio of the ethanol to ultrapure water is 1:(0.6-1).
  • step S6 is:
  • the MNPs prepared in step S5 are added to a morpholineethanesulfonic acid buffer solution containing carbodiimide and N-hydroxysulfosuccinimide for a first incubation; after the incubation, the activated MNPs are obtained by magnetic separation and dispersed in a phosphate buffer; a streptavidin solution is added to the solution for a second incubation; after the incubation, the activated MNPs are obtained by magnetic separation.
  • MNPs modified with streptavidin and dispersed in phosphate buffer were added for a third incubation; after the incubation, the aptamer-modified MNPs were obtained by magnetic separation and redispersed in phosphate buffer; then, bovine serum albumin solution was added for a fourth incubation; after the incubation, the MNPs were magnetically separated and washed with phosphate buffer to prepare MNPs functionalized with biomolecules.
  • the ratio of the MNPs, carbodiimide, N-hydroxysulfosuccinimide and morpholineethanesulfonic acid buffer solution is (8-12 mg): 10 mg: 5 mg: (0.8-1.2 mL).
  • the first incubation condition is 20-40°C, 2-4h.
  • the amount of streptavidin used is 0.8-1.2 mg.
  • the second incubation condition is 30-40°C, 10-14h.
  • the amount of the aptamer complementary sequence is 200-300 ⁇ L, and the concentration is 10 ⁇ M.
  • the third incubation condition is 30-40°C, 10-14h.
  • bovine serum albumin used is 3-6 mL, and the mass fraction is 2%.
  • the fourth incubation condition is 30-40° C., 1.5-3 h.
  • step S4 the pH of the phosphate buffer is 7.2-7.4, and the amount used is 5-15 mL.
  • step S7 is:
  • biomolecule-functionalized CSUCNPs prepared in step S4 and the biomolecule-functionalized MNPs prepared in step S6 are added to a phosphate buffer and heated at high temperature; after heating, they are slowly annealed and transferred to a shaker for incubation; after incubation, upconversion luminescent biosensors are obtained by magnetic separation, washed three times with a phosphate buffer, and finally dispersed in a phosphate buffer.
  • the high temperature reaction conditions are 90-95°C, 3-5min.
  • the annealing condition is 3-5°C/min, to 60-65°C.
  • the incubation conditions are 20-40°C, 0.5-2h.
  • step 7 the pH of the phosphate buffer is 7.2-7.4, and the amount used is 5-10 mL.
  • the aptamer-mediated nanoparticle bridging flocculation is used to achieve the efficient luminescence enhancement process of the upconversion luminescence biosensor.
  • the aptamer specifically binds to EDCs, causing CSUCNPs to fall off from the surface of MNPs; magnetic separation is used to remove the biosensor that has not reacted with the EDCs target, and the CSUCNPs that fall off in the solution reflect the target EDCs.
  • the aptamers on the surface of the detached CSUCNPs that are not bound to the target EDCs undergo base complementary pairing, resulting in aptamer-mediated nanoparticle bridging flocculation, which further settles to achieve concentration enrichment of the nanoparticles; by optimizing and adjusting the focal length, the signals of the settled CSUCNPs are collected to achieve luminescence-enhanced quantitative detection of EDCs.
  • the volume ratio of the upconversion luminescent biosensor prepared in step S7 to the sample solution to be tested is 4:1.
  • the focal length of signal acquisition is 11.5 mm.
  • the sedimentation time of the detached CSUCNPs was 20–30 min.
  • the third object of the present invention is to provide a method for using the above-mentioned microfluidic biosensing platform based on upconversion luminescence, which specifically comprises the following steps:
  • a series of EDCs standard solutions with concentrations ranging from 0 to 250 ng/mL are taken and co-injected with the prepared upconversion luminescent biosensor into the microfluidic chip to complete the steps of mixing, reacting, separating and detecting the biosensor and the target EDCs;
  • the CSUCNPs were allowed to stand to complete the bridge flocculation and sedimentation of the detached CSUCNPs, and the fluorescence spectrum of the CSUCNPs interface formed by the sedimentation in the detection pool was collected by a fluorescence spectrometer.
  • the concentration logarithm of the EDCs standard solution and the characteristic value of the fluorescence signal were linearly fitted to establish a standard curve for EDCs content detection; the characteristic value of the fluorescence signal was the characteristic fluorescence emission intensity of the rare earth element used in the biosensor that specifically identified EDCs;
  • the sample solution to be tested is taken to replace the above EDCs standard solution and is injected into the microfluidic chip together with the biosensor.
  • the characteristic value of the collected fluorescence signal is substituted into the standard curve for calculation to obtain the content of EDCs in the sample to be tested.
  • the present invention has the following beneficial effects:
  • the present invention prepares an upconversion luminescence microfluidic biosensor.
  • the upconversion luminescence process effectively avoids the background fluorescence interference of other matrices.
  • the biorecognition element aptamer has good economy and specificity.
  • Figure 1 is a characterization diagram of the prepared nanoparticles; wherein A is a transmission electron microscopy image of CUCNPs doped with erbium; B is a transmission electron microscopy image of CSUCNPs doped with erbium; C is a transmission electron microscopy image of CUCNPs doped with thulium; D is a transmission electron microscopy image of CSUCNPs doped with thulium; E is a transmission electron microscopy image of MNPs; and F is a scanning electron microscopy image of MNPs.
  • Figure 2 is a characterization diagram of the prepared biosensor; wherein A is a transmission electron microscopy image of the biosensor doped with erbium; B is a high-magnification transmission electron microscopy characterization of the biosensor doped with erbium, wherein B1 is a high-magnification transmission electron microscopy image, and B2-B4 are mapping images of iron and erbium, respectively; C is a transmission electron microscopy image of the biosensor doped with thulium; D is a high-magnification transmission electron microscopy characterization of the biosensor doped with thulium, wherein D1 is a high-magnification transmission electron microscopy image, and D2-D4 are mapping images of iron and thulium, respectively.
  • Figure 3 is a schematic diagram of a microfluidic chip; wherein A is a schematic diagram of the overall structure of the microfluidic chip; B is a diagram of the microchannels of the microfluidic chip, wherein: 1. sample inlet; 2. biosensor inlet; 3. liquid inlet channel; 4. semicircular channel; 5. connecting channel; 6. 1/4 arc channel; 7. separation channel; 8. detection pool.
  • Figure 4 is an illustration and characterization of aptamer-mediated nanoparticle bridging flocculation
  • A is a schematic diagram of the secondary structure analysis of BPA and DES aptamers and the mediated nanoparticle bridging flocculation
  • B is a transmission electron micrograph of aptamer-mediated CSUCNPs bridging flocculation
  • C is a comparison of aptamer-mediated CSUCNPs bridging flocculation before and after under macroscopic conditions, wherein C1 is the state without laser excitation, and C2 is the state with 980nm laser excitation.
  • Figure 5 is an illustration and characterization of the luminescence enhancement of CSUCNPs; wherein A is a schematic diagram and spectrum of the luminescence enhancement obtained by constructing a core-shell structure at the single particle level; B is a schematic diagram of the interface formed by aptamer-mediated bridging flocculation of CSUCNPs; and C is a spectrum of the interface-enhanced luminescence formed by aptamer-mediated bridging flocculation of CSUCNPs.
  • Figure 6 is a graph showing the results of the biosensor detecting EDCs of different concentrations; wherein A is a fluorescence spectrum diagram for detecting EDCs of different concentrations; B is a graph showing the relationship between the characteristic value of the fluorescence signal at 450 nm and the DES concentration; C is a linear fitting curve and equation between the characteristic value of the fluorescence and the logarithmic value of the EDCs concentration; and D is a graph showing the relationship between the characteristic value of the fluorescence signal at 541 nm and the BPA concentration.
  • A is a fluorescence spectrum diagram for detecting EDCs of different concentrations
  • B is a graph showing the relationship between the characteristic value of the fluorescence signal at 450 nm and the DES concentration
  • C is a linear fitting curve and equation between the characteristic value of the fluorescence and the logarithmic value of the EDCs concentration
  • D is a graph showing the relationship between the characteristic value of the fluorescence signal at 541 nm and the BPA concentration
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the experimental materials and reagents used in the following examples are all commercially available unless otherwise specified.
  • the quantitative tests in the following examples were performed in triplicate, and the data are the average or average ⁇ standard deviation of the triplicate experiments.
  • the "and/or" in the full text includes three solutions. Taking A and/or B as an example, it includes technical solution A, technical solution B, and a technical solution that satisfies both A and B.
  • the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.
  • an erbium-doped upconversion luminescent biosensor is prepared, and the upconversion luminescent biosensor is synthesized under nitrogen protection and continuous magnetic stirring.
  • the preparation method of the up-conversion luminescent biosensor doped with erbium element comprises the following steps:
  • step S3 Take 50 mg of CSUCNPs prepared in step S2, add them to a mixed solution of 4 mL of chloroform and 6 mL of toluene, then add 20 mL of 10 mg/mL polyacrylic acid aqueous solution and seal, stir vigorously at room temperature for 48 h; after completion, wash with 10 mL of ethanol and 10 mL of ultrapure water, centrifuge at 9000 rpm for 8 min, and obtain hydrophilic polyacrylic acid.
  • Modified CSUCNPs PAA-CSUCNPs
  • step S4 Take 5 mg of the PAA-CSUCNPs prepared in step S3, add it to a morpholineethanesulfonic acid buffer solution (10 mL) containing 20 mg of carbodiimide and 10 mg of N-hydroxysulfosuccinimide, and incubate at 25°C for 3 h; after the incubation, centrifuge at 8000 rpm for 10 min to obtain activated PAA-CSUCNPs, and disperse them in 10 mL of phosphate buffer (pH 7.2); add 1 mL of 1 mg/mL streptavidin solution to the solution, and incubate at 37°C for 12 h; after the incubation, centrifuge at 8500 rpm for 6 min to obtain CSUCNPs modified with streptavidin, and disperse them in 10 mL of phosphate buffer (pH 7.2); then add 200 ⁇ L of 10 ⁇ M BPA aptamer modified with biotin at the 5' end, 3 Incuba
  • step S6 Take 100 mg of the MNPs prepared in step S5, add them to 20 mL of morpholineethanesulfonic acid buffer solution containing 200 mg of carbodiimide and 100 mg of N-hydroxysulfosuccinimide, and incubate at 25°C for 3 h. After the incubation, magnetic separation is performed to obtain activated MNPs, which are dispersed in 10 mL of phosphate buffer (pH 7.2). 1 mL of 1 mg/mL streptavidin solution is added to the solution, and the solution is incubated at 37°C for 12 h.
  • phosphate buffer pH 7.2
  • MNPs modified with streptavidin which are dispersed in 10 mL of phosphate buffer (pH 7.2).
  • 200 ⁇ L of 10 ⁇ M 5 The ’-end of the MNPs was modified with biotin-containing BPA aptamer complementary sequence and incubated at 37°C for 12 h.
  • the MNPs modified with the BPA aptamer complementary sequence were magnetically separated and redispersed in 10 mL of phosphate buffer (pH 7.2).
  • 5 mL of 2% bovine serum albumin solution was added and the mixture was incubated at 37°C for 2 h.
  • the MNPs were magnetically separated and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecule-functionalized MNPs.
  • the complementary sequence of the BPA aptamer is 5'-Biotin-TTT TTT TGG TGC GAA CCC GTG ATG-3'.
  • step S7 take 400 ⁇ L 0.5 mg/mL biomolecule functionalized CSUCNPs prepared in step S4 and 500 ⁇ L 1 mg/mL biomolecule functionalized MNPs prepared in step S6, add them to 1 mL phosphate buffer, and heat at 95°C for 3 min; after heating, slowly anneal to 65°C, transfer to a shaker and incubate at 37°C for 1 h; after incubation, magnetic separation is performed to obtain the erbium-doped upconversion luminescent biosensor, and it is washed three times with 10 mL phosphate buffer, and finally separated.
  • an upconversion luminescent biosensor doped with thulium element is prepared, and the upconversion luminescent biosensor is synthesized under nitrogen protection and continuous magnetic stirring.
  • the preparation method of the up-conversion luminescent biosensor doped with thulium element comprises the following steps:
  • step S3 Take 50 mg of the CSUCNPs prepared in step S2, add them to a mixed solution of 4 mL of chloroform and 6 mL of toluene, then add 15 mL of 20 mg/mL polyacrylic acid aqueous solution and seal, and vigorously stir the reaction at room temperature for 36 hours; after completion, use 10 mL of ethanol and 6 mL of ultrapure water to wash, and centrifuge at 9500 rpm for 7 minutes to obtain hydrophilic polyacrylic acid modified CSUCNPs (PAA-CSUCNPs);
  • PAA-CSUCNPs hydrophilic polyacrylic acid modified CSUCNPs
  • the CSUCNPs modified with DES aptamer were obtained by centrifugation at 11500 rpm for 3 min and redispersed in 10 mL of phosphate buffer (pH 7.2). Then, 5 mL of 2% bovine serum albumin solution was added and incubated at 37°C for 2 h. The mixture was centrifuged at 10,000 rpm for 5 min and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecule-functionalized CSUCNPs; wherein the sequence of the DES aptamer was 5'-Biotin-GCC CTC TGA GGA TGC CGA AAA AGA AAA GAA ATT CTC TGG C-3'.
  • step S6 Take 120 mg of the MNPs prepared in step S5, add them to 20 mL of morpholineethanesulfonic acid buffer solution containing 200 mg of carbodiimide and 100 mg of N-hydroxysulfosuccinimide, and incubate at 30°C for 2 h. After the incubation, obtain the activated MNPs by magnetic separation, and disperse them in 10 mL of phosphate buffer (pH 7.2). Add 1 mL of 1 mg/mL streptavidin solution to the solution, and incubate at 37°C for 10 h.
  • the MNPs modified with streptavidin by magnetic separation After the incubation, obtain the MNPs modified with streptavidin by magnetic separation, and disperse them in 10 mL of phosphate buffer (pH 7.2). Then add 200 ⁇ L of 10 ⁇ M
  • the 5' end of the MNPs was modified with biotin and the complementary sequence of the DES aptamer was incubated at 37°C for 13 hours; after the incubation, the MNPs modified with the complementary sequence of the DES aptamer were magnetically separated and redispersed in 10 mL of phosphate buffer (pH 7.2); then 5 mL of 2% bovine serum albumin solution was added and the mixture was incubated at 37°C for 3 hours; after the incubation, the MNPs were magnetically separated and washed with 10 mL of phosphate buffer (pH 7.2) to prepare MNPs functionalized with biomolecules; wherein the complementary sequence of the DES aptamer is 5'-Biotin-
  • the microfluidic chip prepared by soft lithography has a structure as shown in FIG3A and FIG3B , including a sample inlet 1 and a biosensor inlet 2.
  • the cross-sections of the sample inlet 1 and the biosensor inlet 2 are circular with a diameter of 8 mm, and the depth of both is 1 cm.
  • the sample inlet 1 and the biosensor inlet 2 are respectively connected to a liquid inlet channel 3, and the other ends of the two liquid inlet channels 3 are connected to each other.
  • the two liquid inlet channels 3 are cross-connected, with an included angle of 90°, and the length of the two liquid inlet channels 3 is 9 mm.
  • 126 semicircular channels 4 (with a radius of 800 ⁇ m), 25 1/4 arc channels 6 (with a radius of 800 ⁇ m), and 11 connecting channels 5 (with a length of 500 ⁇ m) constitute an arc channel as shown in Figure 3B in this embodiment.
  • the feed port of the arc channel is connected to the intersection of the two liquid inlet channels 3, and the discharge port is connected to the separation channel 7.
  • the separation channel 7 is a straight channel with a length of 6.8 mm.
  • the other end of the separation channel 7 is connected to a detection pool 8, which is used for magnetic separation of the biosensor.
  • the detection pool 8 has a diameter of 1.2 cm and a depth of 1 cm, which is used for bridging flocculation, sedimentation, and fluorescence signal collection of the detached CSUCNPs; the width of all channels in this embodiment is 400 ⁇ m and the depth is 200 ⁇ m.
  • Aptamer-mediated nanoparticle bridging flocculation and luminescence enhancement comprising the following steps:
  • Example 1 and Example 2 core-shell CSUCNPs were prepared respectively, and the first luminescence enhancement was achieved at the single nanoparticle level, as shown in FIG5A , wherein the CSUCNPs doped with erbium achieved a 1.72-fold luminescence enhancement, and the CSUCNPs doped with thulium achieved a 2.28-fold luminescence enhancement; the secondary structure prediction of the DES and BPA aptamers is shown in FIG4A , and the presence of a stem-loop structure indicates that there are complementary paired base sequences in the aptamer sequence and that they can stably hybridize, thereby causing the CSUCNPs modified with the aptamer sequence to bind to each other to form bridge flocculation, as shown in FIG4A ; aptamer-mediated The microscopic transmission electron microscopy image of CSUCNPs bridge flocculation is shown in Figure 4B.
  • CSUCNPs are agglomerated into nanoaggregates from dispersed single nanoparticles ( Figures 1A-1D); this allows the surface biomolecule-functionalized CSUCNPs to settle from a dispersed solution state to form an interface ( Figure 4C, Figure 5B) from a macroscopic perspective, and adjust the fluorescence signal collection method to be parallel to the direction of the gravity field ( Figure 4C2), thereby achieving luminescence enhancement at the nanoparticle population level.
  • the luminescence of the biosensor doped with erbium was enhanced by 7.10 times
  • the luminescence of the biosensor doped with thulium was enhanced by 8.94 times.
  • the upconversion luminescence-based biosensor realizes the simultaneous detection of BPA and DES, including the following steps:
  • the upconversion luminescent biosensors prepared in Example 1 and Example 2 were mixed in equal volumes to form a mixed biosensor solution that can simultaneously identify BPA and DES; then the sample solution to be tested and the mixed biosensor solution were injected from the sample injection port 1 and the biosensor injection port 2 of the microfluidic chip, respectively, with injection flow rates of 12 ⁇ L/min and 3 ⁇ L/min, respectively, and the injection time was 10 min; the two microfluids were fully mixed and reacted in the microchannel of the microfluidic chip; at the same time, a magnetic field was applied at the separation channel 7 to separate the biosensors that did not react with BPA and DES; then the mixed liquid entered In detection pool 8, the detached CSUCNPs completed bridging flocculation and sedimentation, and the time for sedimentation to reach a steady state was 20 min, completing the collection of upconversion fluorescence signals.
  • B Take a series of BPA and DES mixed standard solutions with concentrations ranging from 0 to 250 ng/mL, and co-inject them with the prepared upconversion luminescent biosensor into the microfluidic chip according to step S1 to complete the mixing, reaction, separation and detection steps of the biosensor and the target EDCs; the upconversion fluorescence spectra collected by the detection of mixed standard solutions with different concentrations are shown in Figure 6A.
  • the fluorescence intensity at 541 nm and 450 nm gradually increases; the relationship between the characteristic value I 450 of the fluorescence signal at 450 nm and the DES concentration is shown in Figure 6B; the relationship between the characteristic value I 541 of the fluorescence signal at 541 nm and the BPA concentration is shown in Figure 6D.
  • the sample to be tested is seawater.
  • the fluorescence signal characteristic value I 450 and the fluorescence signal characteristic value I 541 were measured to be 0.3592 and 0.6720, respectively.
  • the DES and BPA contents in the seawater samples were calculated to be 8.24 ng/mL and 9.18 ng/mL, respectively, by substituting them into the standard curve.
  • GC-MS gas chromatography-mass spectrometry
  • the peak areas corresponding to the characteristic peaks of DES and BPA were measured to be 5585 and 7575, respectively.
  • Substituting the standard curve into the calculated contents of DES and BPA in the seawater samples were 8.16 ng/mL and 7.64 ng/mL, respectively.
  • the sample to be tested is shrimp.
  • the upconversion luminescence microfluidic biosensor platform was used for detection, and the fluorescence signal characteristic value I 450 and the fluorescence signal characteristic value I 541 were measured to be 0.3535 and 0.6801, respectively.
  • the DES and BPA contents in the shrimp samples were calculated to be 7.42 ng/mL and 9.90 ng/mL, respectively, by substituting them into the standard curve.
  • the peak areas corresponding to the characteristic peaks of DES and BPA were measured to be 5315 and 11480, respectively.
  • Substituting the standard curve into the calculated content of DES and BPA in the shrimp samples were 7.79 ng/mL and 9.71 ng/mL, respectively.
  • the sample to be tested is fish meat.
  • the upconversion luminescence microfluidic biosensor platform was used for detection, and the fluorescence signal characteristic value I 450 and the fluorescence signal characteristic value I 541 were measured to be 0.4462 and 0.6749, respectively.
  • the DES and BPA contents in the fish samples were calculated to be 40.00 ng/mL and 9.43 ng/mL, respectively, by substituting them into the standard curve.
  • the peak areas corresponding to the characteristic peaks of DES and BPA were measured to be 31959 and 10527, respectively.
  • Substituting the standard curve into the calculated content of DES and BPA in the fish samples were 36.01 ng/mL and 9.15 ng/mL, respectively.

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Abstract

一种基于上转换发光的微流体生物传感平台，包括：上转换发光的生物传感器，用于特异性识别EDCs；以及微流体芯片，用于集成上转换发光的生物传感器与待测样本的混合、反应、分离、检测；微流体芯片包括：进样池，用于上转换发光的生物传感器和待测样本进样；弧形通道，弧形通道的进料口同时连通于上转换发光的生物传感器的进样池和待测样本的进样池，待上转换发光的生物传感器和待测样本进入弧形通道后，弧形通道用于二者的混合和反应；分离通道（7），与弧形通道的出料口连通，用于反应结束后的上转换发光的生物传感器的磁分离；检测池（8），与分离通道的出料口连通，用于完成发光增强定量检测EDCs。

Description

一种基于上转换发光的微流体生物传感平台 技术领域
本发明涉及食品安全检测技术领域，特别涉及一种基于上转换发光的微流体生物传感平台。
背景技术
内分泌干扰化学物(EDCs)被定义为“干扰存在于人体内部负责平衡、繁殖和发育过程的天然血源性激素的合成、分泌、运输、代谢、结合或消除的外源性制剂”。人类可能通过食品生产(食品添加剂、杀虫剂、食品容器)、工业活动(空气污染、水污染物、工业化学品)、医疗(医疗产品)等摄入数百种EDCs。其中，具有雌激素作用的外源性制剂，例如双酚A(BPA)、己烯雌酚(DES)、雌二醇(E2)、壬基酚等吸引了广泛的关注，这类EDCs可能会导致严重的健康危害，包括神经发育障碍，脑、肝和肺损伤，生殖和内分泌失调，代谢紊乱等。因此，建立有效的评估方法来确定人类可能接触媒介中的EDCs含量是非常重要的。
以往的研究中，对于EDCs含量的测定是基于高效液相色谱、气质联用色谱、电化学传感器、光电化学免疫传感器等方法，但这些测定方法往往面临着检测仪器设备昂贵、背景干扰强、样品预处理步骤繁琐、试剂消耗量大等困难，难以实现EDCs的现场快速定量检测。因此，开发新型的检测平台用以克服上述缺陷是至关重要的。
发明内容
针对现有技术的不足，本发明提供了一种基于上转换发光的微流体生物传感平台，用以实现对EDCs的微量取样和简便的高灵敏定量检测。
基于此，本发明的目的在于提供一种基于上转换发光的微流体生物传感平台，包括：上转换发光的生物传感器，用于特异性识别EDCs；以及微流体芯片，作为所述上转换发光的生物传感器与待测样本反应平台，用于集成所述上转换发光的生物传感器与待测样本的混合、反应、分离、检测；
所述微流体芯片，包括：进样池，用于所述上转换发光的生物传感器和待测样本进样；弧形通道，所述弧形通道的进料口同时连通于所述上转换发光的生物传感器的进样池和待测样本的进样池，待所述上转换发光的生物传感器和待测样本进入弧形通道后，弧形通道用于二者的混合和反应；分离通道，与所述弧形通道的出料口连通，用于反应结束后的所 述上转换发光的生物传感器的磁分离；检测池，与所述分离通道的出料口连通，用于发光增强定量检测EDCs。
进一步地，所述微流体芯片的所有微通道的宽度均为400μm，深度均为200μm。
根据上述技术方案，上转换发光的生物传感器与待测样本在微流体芯片上的混合、反应、分离、检测集成，其具体过程为：将上转换发光的生物传感器与待测样本分别从微流体芯片的两个进样池注入，两种微流体在弧形通道内充分混合、反应；之后在分离通道处外加磁场，分离未与EDCs反应的生物传感器；最后在检测池中，脱落的CSUCNPs完成桥连絮凝和沉降，并完成上转换荧光信号的采集，从而可以定量检测EDCs。
进一步地，生物传感器与待测样本溶液的注射流速为12μL/min和3μL/min。
进一步地，生物传感器与待测样本溶液的注射时间为8-12min。
本发明的第二目的在于提供上述一种基于上转换发光的微流体生物传感平台的制备方法。
所述上转换发光的生物传感器的制备方法包括如下步骤：
S1、制备含有稀土元素的上转换纳米粒子种子(CUCNPs)；
S2、步骤S1制备得到的CUCNPs外层进行包覆处理，制备得到具有核壳结构的上转换纳米粒子(CSUCNPs)；
S3、改性步骤S2制备得到的CSUCNPs为亲水性；
S4、将步骤S3得到的亲水性CSUCNPs生物分子功能化，得到生物分子功能化CSUCNPs；
S5、制备磁性纳米粒子(MNPs)；
S6、将步骤S5得到的MNPs生物分子功能化，得到生物分子功能化MNPs；
S7、步骤S4得到生物分子功能化CSUCNPs和步骤S6得到生物分子功能化CSUCNPs相结合，制备得到所述上转换发光的生物传感器。
进一步地，步骤S1的过程为，
分别取六水合氯化钇、六水合氯化镱和六水合稀土元素溶解于甲醇中，之后加入油酸和1-十八稀，混合后加热至150-170℃反应25-35min，反应结束冷却后，滴加氢氧化钠和氟化铵的混合溶液，并于125-135℃反应25-35min，随后升温至290-310℃保持50-60min；反应结束后，加入乙醇和超纯水离心分离得到上转换纳米粒子种子(CUCNPs)。
进一步地，所述六水合氯化钇、六水合氯化镱和六水合稀土元素的总用量为1-1.5mmol。
进一步地，所述稀土元素为铒时，六水合氯化钇、六水合氯化镱和六水合稀土元素的比例为0.78:0.2:0.02。
进一步地，所述稀土元素为铥时，六水合氯化钇、六水合氯化镱和六水合稀土元素的比例为0.795:0.2:0.005。
进一步地，所述甲醇、油酸、1-十八稀的体积比例为10:(5-7):(14-17)。
进一步地，所述氢氧化钠和氟化铵的摩尔比为5:(7-9)。
进一步地，所述乙醇和超纯水的体积比为1:(0.6-1)。
进一步的，所述离心参数为8000-12000rpm转速下离心5-10min。
进一步地，步骤S2的过程为，
取六水合氯化钇溶解于甲醇中，之后加入油酸和1-十八稀，混合后加热至150-170℃反应25-35min；反应结束冷却后，加入步骤S1制备得到的CUCNPs，并滴加氢氧化钠和氟化铵的混合溶液，并于125-135℃反应25-35min，随后升温至290-310℃保持20-40min；反应结束后，加入乙醇和超纯水离心分离得到核壳上转换纳米粒子(CSUCNPs)。
进一步地，所述六水合氯化钇的用量为0.35-0.45mmol。
进一步地，所述甲醇、油酸、1-十八稀的体积比例为10:(2.5-3.5):(7-9)。
进一步地，所述氢氧化钠和氟化铵的摩尔比为2:(2.5-3.5)。
进一步地，所述乙醇和超纯水的体积比为1:(0.6-1)。
进一步的，所述离心参数为8000-12000rpm转速下离心5-10min。
进一步地，步骤S3的过程为，
取步骤S2制备得到的CSUCNPs加入三氯甲烷和甲苯的混合溶液中，随后加入聚丙烯酸水溶液并密封，剧烈搅拌反应；结束后使用乙醇和超纯水清洗并离心，得到具有亲水性的聚丙烯酸修饰的CSUCNPs(PAA-CSUCNPs)。
进一步地，所述CSUCNPs、三氯甲烷、甲苯和聚丙烯酸的比例为50mg:(2-6mL):(4-10mL):(15-20mL)。
进一步地，所述聚丙烯酸水溶液浓度为10-20mg/mL。
进一步地，所述搅拌反应时间为24-48h。
进一步地，所述乙醇和超纯水的体积比为1:(0.6-1)。
进一步的，所述离心参数为8000-12000rpm转速下离心5-10min。
进一步地，步骤S4的过程为，
取步骤S3制备得到的PAA-CSUCNPs，加入含有碳酰二亚胺和N-羟基硫代琥珀酰亚胺 的吗啉乙磺酸缓冲溶液中，进行第一次孵育；孵育结束后，离心分离得到活化的PAA-CSUCNPs，并分散在磷酸盐缓冲液中；向溶液中加入链霉亲和素溶液，进行第二次孵育；孵育结束后，离心分离得到修饰链霉亲和素的CSUCNPs，并分散在磷酸盐缓冲液中；随后加入5’端修饰生物素的EDCs适配体，进行第三次孵育；孵育结束后离心分离得到修饰适配体的CSUCNPs，并重新分散在磷酸盐缓冲液中；随后加入牛血清白蛋白溶液，进行第四次孵育；孵育结束后离心分离，并使用磷酸盐缓冲液清洗，制备得到生物分子功能化的CSUCNPs。
进一步地，所述PAA-CSUCNPs、碳酰二亚胺、N-羟基硫代琥珀酰亚胺和吗啉乙磺酸缓冲溶液的比例为(0.8-1.2mg):4mg:2mg:(2.0-2.4mL)。
进一步地，第一次孵育条件为20-40℃，2-4h。
进一步地，链霉亲和素的用量为0.8-1.2mg。
进一步地，第二次孵育条件为30-40℃，10-14h。
进一步地，适配体的用量为200-300μL，浓度为10μM。
进一步地，第三次孵育条件为30-40℃，10-14h。
进一步地，牛血清白蛋白的用量为3-6mL，质量分数为2％。
进一步地，第四次孵育条件为30-40℃，1.5-3h。
进一步地，步骤S4中，离心分离参数为8000-12000rpm转速下离心5-10min。
进一步地，步骤S4中，磷酸盐缓冲液的pH为7.2-7.4，用量为5-15mL。
进一步地，步骤S5的过程为，
分别取六水合氯化铁、二水合柠檬酸三钠、醋酸钠加入乙二醇溶液中，剧烈搅拌充分溶解后，转移到反应釜中，高温反应；反应结束通过磁场收集制备得到的磁性纳米粒子(MNPs)，并使用乙醇和超纯水清洗。
进一步地，六水合氯化铁、二水合柠檬酸三钠、醋酸钠和乙二醇用量比例为5mmol:0.4mmol:(1.2-1.5g):(15-25mL)。
进一步地，高温反应的条件为190-210℃，8-12h。
进一步地，所述乙醇和超纯水的体积比为1:(0.6-1)。
进一步地，步骤S6的过程为，
取步骤S5制备得到的MNPs，加入含有碳酰二亚胺和N-羟基硫代琥珀酰亚胺的吗啉乙磺酸缓冲溶液中，进行第一次孵育；孵育结束后，磁分离得到活化的MNPs，并分散在磷酸盐缓冲液中；向溶液中加入链霉亲和素溶液，进行第二次孵育；孵育结束后，磁分离得 到修饰链霉亲和素的MNPs，并分散在磷酸盐缓冲液中；随后加入5’端修饰生物素的EDCs适配体互补序列，进行第三次孵育；孵育结束后磁分离得到修饰适配体的MNPs，并重新分散在磷酸盐缓冲液中；随后加入牛血清白蛋白溶液，进行第四次孵育；孵育结束后磁分离，并使用磷酸盐缓冲液清洗，制备得到生物分子功能化的MNPs。
进一步地，所述MNPs、碳酰二亚胺、N-羟基硫代琥珀酰亚胺和吗啉乙磺酸缓冲溶液的比例为(8-12mg):10mg:5mg:(0.8-1.2mL)。
进一步地，第一次孵育条件为20-40℃，2-4h。
进一步地，链霉亲和素的用量为0.8-1.2mg。
进一步地，第二次孵育条件为30-40℃，10-14h。
进一步地，适配体互补序列的用量为200-300μL，浓度为10μM。
进一步地，第三次孵育条件为30-40℃，10-14h。
进一步地，牛血清白蛋白的用量为3-6mL，质量分数为2％。
进一步地，第四次孵育条件为30-40℃，1.5-3h。
进一步地，步骤S4中，磷酸盐缓冲液pH为7.2-7.4，用量为5-15mL。
进一步地，步骤S7的过程为，
取步骤S4制备得到的生物分子功能化的CSUCNPs和步骤S6制备得到的生物分子功能化的MNPs加入磷酸盐缓冲液中，高温加热；加热结束后缓慢退火，并转移至摇床孵育；孵育结束后，磁分离得到上转换发光的生物传感器，并使用磷酸盐缓冲液清洗三次，最后分散于磷酸盐缓冲液中。
进一步地，EDCs适配体修饰的CSUCNPs和EDCs适配体互补序列修饰的MNPs质量比为(1.5-2):5。
进一步地，高温反应条件为90-95℃，3-5min。
进一步地，退火条件为3-5℃/min，至60-65℃。
进一步地，孵育条件为20-40℃，0.5-2h。
进一步地，步骤7中，磷酸盐缓冲液pH为7.2-7.4，用量为5-10mL。
根据上述技术方案，适配体介导的纳米粒子桥连絮凝，用以实现所述上转换发光的生物传感器的高效发光增强过程为，将步骤S7制备的上转换发光的生物传感器与待测样本溶液混合反应后，适配体与EDCs特异性的结合，导致CSUCNPs从MNPs表面脱落；磁分离去除未与EDCs靶标反应的生物传感器，脱落在溶液中的CSUCNPs反映了靶标EDCs的 数量；脱落的CSUCNPs表面未结合靶标EDCs的适配体发生碱基的互补配对，产生适配体介导的纳米粒子桥连絮凝，进一步沉降，实现纳米粒子的浓度富集；通过优化调节焦距，对沉降的CSUCNPs信号采集，实现发光增强定量检测EDCs。
进一步地，步骤S7制备的上转换发光的生物传感器与待测样本溶液体积比4:1。
进一步地，信号采集的焦距为11.5mm。
进一步地，脱落的CSUCNPs的沉降时间为20-30min。
本发明的第三目的在于提供上述的一种基于上转换发光的微流体生物传感平台的使用方法，具体包括以下步骤：
取0-250ng/mL一系列浓度的EDCs标准溶液，与制备的上转换发光生物传感器共同注射进入所述微流体芯片中，完成生物传感器与靶标EDCs的混合、反应、分离与检测步骤；
静置以完成脱落的CSUCNPs的桥连絮凝和沉降，通过荧光光谱仪采集检测池中沉降形成的CSUCNPs界面的荧光光谱，以EDCs标准溶液的浓度对数值和荧光信号特征值进行线性拟合，建立EDCs含量检测的标准曲线；所述荧光信号特征值为特异性识别EDCs的生物传感器使用的稀土元素的特征荧光发射光强；
取待测样本溶液，代替上述EDCs标准溶液，与生物传感器共同注射进入微流体芯片，将采集得到的荧光信号特征值代入标准曲线计算，得出待测样本中EDCs的含量。
综上所述，本发明具有以下有益效果：
1、本发明公开了一种新型的适配体介导的纳米粒子桥连絮凝现象，并基于此发现，实现了纳米生物传感器荧光信号的大幅度增强，这极大的提高了对靶标检测的灵敏度，将检测限提升至少一个数量级；此外，通过适配体介导纳米粒子桥连絮凝形成稳定界面，大大减少了重力场对纳米粒子分散液的影响，有效提高纳米生物传感器的信号稳定性。
2、本发明设计了一种新型的微流体芯片，在有限的空间内集成了126个半圆通道和12个四分之一圆弧通道，为雷诺数约为0.79的层流态微流体增加湍流程度，实现了生物传感器与待测样本的重复混合与反应，通过外加磁场的辅助，实现了再微流体芯片上的混合、反应、分离、检测步骤的一体化集成，大大提高了检测效率。
3、本发明制备了一种上转换发光的微流体生物传感器，上转换发光过程有效的避免了其它基质的背景荧光干扰，同时生物识别元件适配体具有良好的经济性和特异性；进一步在芯片上集成为微流体生物传感器后，实现了微量取样(30μL)和快速检测(10min)，这为实际现场应用提供了良好的前景。
4、本发明实现了多种EDCs的同时检测。低于危害阈值的单一EDCs成分，在多种EDCs混合后，可能呈现严重的健康危害，即可能存在累计效应。本发明通过调谐制备多色CSUCNPs并分别修饰特异性EDCs适配体，制备了同时检测BPA和DES的生物传感器，并实现了低至0.0076ng/mL和0.0131ng/mL的超灵敏检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为制备的纳米粒子的表征图；其中，A为掺杂铒元素的CUCNPs透射电镜图；B为掺杂铒元素的CSUCNPs透射电镜图；C为掺杂铥元素的CUCNPs透射电镜图；D为掺杂铥元素的CSUCNPs透射电镜图；E为MNPs的透射电镜图；F为MNPs的扫描电镜图。
图2为制备的生物传感器的表征图；其中，A为掺杂铒元素的生物传感器的透射电镜图；B为掺杂铒元素的生物传感器的高倍透射电镜表征，其中B1为高倍透射电镜图，B2-B4分别为铁和铒元素的映射图像；C为掺杂铥元素的生物传感器的透射电镜图；D为掺杂铥元素的生物传感器的高倍透射电镜表征，其中D1为高倍透射电镜图，D2-D4分别为铁和铥元素的映射图像。
图3为微流体芯片示意图；其中，A为微流体芯片总体结构示意图；B为微流体芯片微通道图，其中，1、样本进样口；2、生物传感器进样口；3、进液通道；4、半圆通道；5、连接通道；6、1/4圆弧通道；7、分离通道；8、检测池。
图4为适配体介导纳米粒子桥连絮凝的图解与表征；其中，A为BPA和DES适配体二级结构分析及介导纳米粒子桥连絮凝示意图；B为适配体介导的CSUCNPs桥连絮凝的透射电镜图；C为宏观状态下适配体介导CSUCNPs桥连絮凝前后对比图，其中C1为未被激光激发状态，C2为被980nm激光激发状态。
图5为CSUCNPs发光增强图解与表征；其中，A为单粒子水平构建核壳结构得到的发光增强示意图及光谱；B为适配体介导的CSUCNPs桥连絮凝形成界面示意图；C为适配体介导的CSUCNPs桥连絮凝形成界面增强发光的光谱。
图6为生物传感器检测不同浓度EDCs结果图；其中，A为检测不同浓度EDCs的荧光光谱图；B为450nm处荧光信号特征值与DES浓度关系图；C为荧光特征值与EDCs浓度对数值的线性拟合曲线和方程；D为541nm处荧光信号特征值与BPA浓度关系图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料和试剂等，如无特殊说明，均可从商业途径获得。以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。
另外，全文中的“和/或”包括三个方案，以A和/或B为例，包括A技术方案、B技术方案，以及A和B同时满足的技术方案；另外，各个实施例之间的技术方案可以相互结合，但是必须是以本领域普通技术人员能够实现为基础，当技术方案的结合出现相互矛盾或无法实现应当认为这种技术方案的结合不存在，也不在本发明要求的保护范围之内。
实施例1
本实施例制备掺杂铒元素的上转换发光生物传感器，且上转换发光的生物传感器是在氮气保护、持续磁力搅拌条件下合成的。
掺杂铒元素的上转换发光生物传感器的制备方法，包括以下步骤：
S1：取0.78mmol六水合氯化钇、0.2mmol六水合氯化镱和0.02mmol六水合氯化铒溶解于10mL甲醇中，之后加入8mL油酸和15mL 1-十八稀，混合后加热至150℃反应35min，反应结束冷却后，滴加2.5mmol氢氧化钠和4mmol氟化铵的混合溶液，并于125℃反应35min，随后升温至300℃保持60min；反应结束后，加入10mL乙醇和8mL超纯水，8500rpm离心分离10min得到掺杂铒元素的上转换纳米粒子种子(CUCNPs)，其透射电镜图如图1A所示。
S2：取0.4mmol六水合氯化钇溶解于10mL甲醇中，之后加入3mL油酸和8mL1-十八稀，混合后加热至170℃反应25min；反应结束冷却后，加入步骤S1制备得到的CUCNPs，并滴加1mmol氢氧化钠和1.5mmol氟化铵的混合溶液，并于135℃反应25min，随后升温至290℃保持40min；反应结束后，加入10mL乙醇和8mL超纯水，8500rpm离心分离10min，得到掺杂铒元素的核壳上转换纳米粒子(CSUCNPs)，其透射电镜图如图1B所示。
S3：取50mg步骤S2制备得到的CSUCNPs，加入4mL三氯甲烷和6mL甲苯的混合溶液中，随后加入20mL 10mg/mL的聚丙烯酸水溶液并密封，室温下剧烈搅拌反应48h；结束后使用10mL乙醇和10mL超纯水清洗后，9000rpm离心8min，得到具有亲水性的聚丙烯酸 修饰的CSUCNPs(PAA-CSUCNPs)。
S4：取5mg步骤S3制备得到的PAA-CSUCNPs，加入含有20mg碳酰二亚胺和10mg N-羟基硫代琥珀酰亚胺的吗啉乙磺酸缓冲溶液(10mL)中，25℃孵育3h；孵育结束后，8000rpm离心分离10min得到活化的PAA-CSUCNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；向溶液中加入1mL 1mg/mL的链霉亲和素溶液，37℃孵育12h；孵育结束后，8500rpm离心分离6min得到修饰链霉亲和素的CSUCNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入200μL 10μM的5`端修饰生物素的BPA适配体，37℃孵育12h；孵育结束后9000rpm离心分离5min得到修饰BPA适配体的CSUCNPs，并重新分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入5mL 2％的牛血清白蛋白溶液，37℃孵育2h；孵育结束后10000rpm离心分离5min，并使用10mL磷酸盐缓冲液(pH为7.2)清洗，制备得到生物分子功能化的CSUCNPs；其中，BPA适配体的序列为5'-Biotin-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'。
S5、取1.3515g六水合氯化铁、0.1178g二水合柠檬酸三钠、1.2g醋酸钠加入20mL乙二醇溶液中，剧烈搅拌30min充分溶解后，转移到反应釜中，200℃高温反应12h；反应结束通过磁场收集制备得到的磁性纳米粒子(MNPs)，并使用10mL乙醇和10mL超纯水清洗。MNPs的透射电镜图如图1E所示，扫描电镜图如图1F所示。
S6、取100mg步骤S5制备得到的MNPs，加入含有200mg碳酰二亚胺和100mgN-羟基硫代琥珀酰亚胺的20mL吗啉乙磺酸缓冲溶液中，25℃孵育3h；孵育结束后，磁分离得到活化的MNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；向溶液中加入1mL 1mg/mL的链霉亲和素溶液，37℃孵育12h；孵育结束后，磁分离得到修饰链霉亲和素的MNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入200μL 10μM的5’端修饰生物素的BPA适配体互补序列，37℃孵育12h；孵育结束后磁分离得到修饰BPA适配体互补序列的MNPs，并重新分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入5mL 2％的牛血清白蛋白溶液，37℃孵育2h；孵育结束后磁分离，并使用10mL磷酸盐缓冲液(pH为7.2)清洗，制备得到生物分子功能化的MNPs；其中，BPA适配体的互补序列为5'-Biotin-TTT TTT TGG TGC GAA CCC GTG ATG-3'。
S7、取400μL 0.5mg/mL步骤S4制备得到的生物分子功能化的CSUCNPs和500μL1mg/mL步骤S6制备得到的生物分子功能化的MNPs加入1mL磷酸盐缓冲液中，95℃高温加热3min；加热结束后缓慢退火至65℃，并转移至摇床37℃孵育1h；孵育结束后，磁分离得到掺杂铒元素的上转换发光生物传感器，并使用10mL磷酸盐缓冲液清洗三次，最后分 散于5mL磷酸盐缓冲液中；铒元素掺杂的上转换发光生物传感器透射电镜图如图2A所示，高倍透射电镜图和元素映射图如图2B所示，表明成功制备掺杂铒元素的上转换发光生物传感器。
实施例2
本实施例制备掺杂铥元素的上转换发光生物传感器，且上转换发光的生物传感器是在氮气保护、持续磁力搅拌条件下合成的。
掺杂铥元素的上转换发光生物传感器的制备方法，包括以下步骤；
S1：取0.795mmol六水合氯化钇、0.2mmol六水合氯化镱和0.005mmol六水合氯化铥溶解于10mL甲醇中，之后加入7mL油酸和14mL 1-十八稀，混合后加热至170℃反应25min，反应结束冷却后，滴加2.5mmol氢氧化钠和4mmol氟化铵的混合溶液，并于135℃反应25min，随后升温至295℃保持55min；反应结束后，加入10mL乙醇和10mL超纯水，9500rpm离心分离5min得到掺杂铥元素的上转换纳米粒子种子(CUCNPs)，透射电镜图如图1C所示。
S2：取0.45mmol六水合氯化钇溶解于10mL甲醇中，之后加入2.5mL油酸和8mL1-十八稀，混合后加热至150℃反应35min；反应结束冷却后，加入步骤S1制备得到的CUCNPs，并滴加1mmol氢氧化钠和1.5mmol氟化铵的混合溶液，并于125℃反应35min，随后升温至300℃保持20min；反应结束后，加入10mL乙醇和10mL超纯水，12000rpm离心分离4min，得到掺杂铥元素的核壳上转换纳米粒子(CSUCNPs)，其透射电镜图如图1D所示。
S3：取50mg步骤S2制备得到的CSUCNPs，加入4mL三氯甲烷和6mL甲苯的混合溶液中，随后加入15mL 20mg/mL的聚丙烯酸水溶液并密封，室温下剧烈搅拌反应36h；结束后使用10mL乙醇和6mL超纯水清洗，并9500rpm离心7min，得到具有亲水性的聚丙烯酸修饰的CSUCNPs(PAA-CSUCNPs)；
S4：取5mg步骤S3制备得到的PAA-CSUCNPs，加入含有20mg碳酰二亚胺和10mgN-羟基硫代琥珀酰亚胺的12mL吗啉乙磺酸缓冲溶液中，30℃孵育2h；孵育结束后，9500rpm离心分离6min得到活化的PAA-CSUCNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；向溶液中加入1mL 1mg/mL的链霉亲和素溶液，37℃孵育10h；孵育结束后，11000rpm离心分离5min得到修饰链霉亲和素的CSUCNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入200μL 10μM的5’端修饰生物素的DES适配体，37℃孵育13h；孵育结束后11500rpm离心分离3min得到修饰DES适配体的CSUCNPs，并重新分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入5mL 2％的牛血清白蛋白溶液，37℃孵育2h；；孵育结束后 10000rpm离心分离5min，并使用10mL磷酸盐缓冲液(pH为7.2)清洗，制备得到生物分子功能化的CSUCNPs；其中，DES适配体的序列为5'-Biotin-GCC CTC TGA GGA TGC CGA AAA AGA AAA GAA ATT CTC TGG C-3'。
S5、取1.3515g六水合氯化铁、0.1178g二水合柠檬酸三钠、1.2g醋酸钠加入25mL乙二醇溶液中，剧烈搅拌30min充分溶解后，转移到反应釜中，200℃高温反应10h；反应结束通过磁场收集制备得到的磁性纳米粒子(MNPs)，并使用10mL乙醇和10mL超纯水清洗。
S6、取120mg步骤S5制备得到的MNPs，加入含有200mg碳酰二亚胺和100mgN-羟基硫代琥珀酰亚胺的20mL吗啉乙磺酸缓冲溶液中，30℃孵育2h；孵育结束后，磁分离得到活化的MNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；向溶液中加入1mL 1mg/mL的链霉亲和素溶液，37℃孵育10h；孵育结束后，磁分离得到修饰链霉亲和素的MNPs，并分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入200μL 10μM的5’端修饰生物素的DES适配体互补序列，37℃孵育13h；孵育结束后磁分离得到修饰DES适配体互补序列的MNPs，并重新分散在10mL磷酸盐缓冲液(pH为7.2)中；随后加入5mL 2％的牛血清白蛋白溶液，37℃孵育3h；；孵育结束后磁分离，并使用10mL磷酸盐缓冲液(pH为7.2)清洗，制备得到生物分子功能化的MNPs；其中，DES适配体的互补序列为5'-Biotin-TTTTTT GCC AGA GAA TTT CTT-3'。
S7、取300μL 0.5mg/mL步骤S4制备得到的生物分子功能化的CSUCNPs和500μL 1mg/mL步骤S6制备得到的生物分子功能化的MNPs加入1mL磷酸盐缓冲液中，90℃高温加热5min；加热结束后缓慢退火至65℃，并转移至摇床37℃孵育2h；孵育结束后，磁分离得到掺杂铥元素的上转换发光生物传感器，并使用10mL磷酸盐缓冲液清洗三次，最后分散在5mL磷酸盐缓冲液中；掺杂铥元素的上转换发光生物传感器透射电镜图如图2C所示，高倍透射电镜图和元素映射图如图2D所示，表明成功制备掺杂铥元素的上转换发光生物传感器。
实施例3
通过软光刻法制备的微流体芯片，其结构如图3A和图3B所示，包括样本进样口1、以及生物传感器进样口2，样本进样口1和生物传感器进样口2的截面呈圆形，直径为8mm，且二者的深度为1cm。
样本进样口1和生物传感器进样口2分别连通有进液通道3，两个进液通道3的另一端相 交连通，其夹角为90°，两个进液通道3长9mm。
微流体芯片还包含有半圆通道4和1/4圆弧通道6，且各个通道的连接处均为相切连接，以实现微流体在微通道内的平稳过渡；微流体芯片还包括有连接通道5，连接通道5的两端分别切向连接半圆通道4和1/4圆弧通道6，用以改变弧形通道的方向。
126个半圆通道4(半径为800μm)、25个1/4圆弧通道6(半径为800μm)、11个连接通道5(长度为500μm)，组成本实施例中如图3B的弧形通道，弧形通道的进料口与两个进液通道3的相交处连通，且出料口连通有分离通道7。分离通道7为长度6.8mm的直形通道，分离通道7的另一端连通有检测池8，检测池8用于生物传感器的磁分离。检测池8的直径为1.2cm，深度为1cm，用于脱落的CSUCNPs的桥连絮凝、沉降、荧光信号采集；本实施例中所有通道的宽度均为400μm，深度均为200μm。
适配体介导的纳米粒子桥连絮凝和发光增强，包括以下步骤：
实施例1和实施例2分别制备了核壳结构的CSUCNPs，从单纳米粒子水平实现了第一次发光增强，如图5A所示，其中，掺杂铒元素的CSUCNPs实现了1.72倍的发光增强，掺杂铥元素的CSUCNPs实现了2.28倍的发光增强；DES和BPA适配体的二级结构预测如图4A所示，存在茎环结构，表明适配体序列中存在互补配对的碱基序列并可以稳定的杂交，从而导致修饰了适配体序列的CSUCNPs相互结合形成桥连絮凝，如图4A所示；适配体介导的CSUCNPs桥连絮凝的微观透射电镜图如图4B所示，可以明显观察到CSUCNPs由分散的单纳米粒子(图1A-1D)团聚成纳米聚集体；这使得表面生物分子功能化的CSUCNPs从宏观上，由分散的溶液状态沉降形成界面(图4C、图5B)，调整荧光信号采集方式为平行于重力场方向(图4C2)，实现在纳米粒子群体水平上的发光增强，如图5C所示，掺杂铒元素的生物传感器发光得到7.10倍增强，掺杂铥元素的生物传感器发光实现了8.94倍的增强。
实施例4
基于上转换发光的生物传感器实现BPA和DES的同时检测，包括以下步骤：
A、将实施例1和实施例2制备得到的上转换发光生物传感器等体积混合均匀形成可同时识别BPA和DES的混合生物传感器溶液；之后将待测样本溶液与混合生物传感器溶液分别从微流体芯片的样本进样口1和生物传感器进样口2注入，注射流速分别为12μL/min和3μL/min，注射时间为10min；两种微流体在微流体芯片的微通道内充分混合、反应；同时在分离通道7处外加磁场，分离未与BPA和DES反应的生物传感器；之后混合液体进入 检测池8中，脱落的CSUCNPs完成桥连絮凝和沉降，沉降达到稳态的时间为20min，完成上转换荧光信号的采集。
B、取0-250ng/mL一系列浓度的BPA和DES混合标准溶液，与制备得到的上转换发光生物传感器按照步骤S1共同注射进入微流体芯片中，完成生物传感器与靶标EDCs的混合、反应、分离与检测步骤；不同浓度混合标准溶液检测采集的上转荧光光谱如图6A所示，随着的BPA和DES浓度的升高，541nm和450nm处的荧光强度也逐渐增强；450nm处荧光信号特征值I450与DES浓度的关系如图6B所示；541nm处的荧光信号特征值I541与BPA浓度的关系图如图6D所示。
C、将荧光信号特征值与靶标EDCs浓度进行拟合，得到检测的线性数学公式，如图6C所示，对BPA检测的标准曲线拟合为y1＝0.2475x1+0.4337，R2＝0.9962，检测范围为0.025-100ng/mL，检测限为0.0076ng/mL，其中x1为BPA浓度对数值，y1为450nm处荧光信号特征值I450的归一化荧光强度；对DES检测的标准曲线拟合为y2＝0.1267x2+0.2432，R2＝0.9958，检测范围为0.025-250ng/mL，检测限为0.0131ng/mL，其中x2为DES浓度对数值，y2为541nm处荧光信号特征值I541的归一化荧光强度。
应用实施例1
本实施例中，待测样本为海水。
采集海域沿岸的原始海水样本，在4000rpm离心10min后保留上清液，进一步将海水上清液用0.45μm滤膜过滤以去除杂质，并用于检测。
使用上转换发光的微流体生物传感平台进行检测，测定得到的荧光信号特征值I450和荧光信号特征值I541分别为0.3592和0.6720，带入标准曲线计算得到海水样本中DES和BPA含量分别为8.24ng/mL和9.18ng/mL。
为验证本发明检测方法的准确性，按照中国国家标准GB31660.2-2019规定，对相同的实验样本使用气相色谱-质谱联用方法(GC-MS)进行测定。使用GC-MS建立BPA检测标准曲线为y3＝721.83x3-6218.20，R2＝0.9947，其中x3为BPA浓度，y3为BPA特征峰对应的峰面积；DES检测标准曲线为y4＝372.07x4-2004.95，R2＝0.9953，其中x4为DES浓度，y4为DES特征峰对应的峰面积。
使用GC-MS方法进行检测，测定得到的DES特征峰对应的峰面积和BPA特征峰对应的峰面积分别为5585和7575，带入标准曲线计算得到海水样本中DES和BPA含量分别为8.16ng/mL和7.64ng/mL。
应用实施例2
本实施例中，待测样本为对虾。
将从超市购买得到的新鲜对虾样本，取5g可食用部分均质处理，并随机添加未知浓度的DES和BPA标准溶液；将对虾样本于50mL离心管中加碳酸钠溶液3mL、乙酸乙酯20mL，涡旋混匀，超声提取10min；随后于4000r/min离心10min，取上清液至100mL梨形瓶中；残渣用乙酸乙酯10mL重复提取一次，合并两次离心的上清液，于40℃旋转蒸发至干，用体积分数为50％环已烷乙酸乙酯溶液5mL溶解残留物，并使用60mg/mL的固相萃取柱提取DES和BPA；最后将固相萃取柱的淋洗液用于检测。
使用上转换发光的微流体生物传感平台进行检测，测定得到的荧光信号特征值I450和荧光信号特征值I541分别为0.3535和0.6801，带入标准曲线计算得到对虾样本中DES和BPA含量分别为7.42ng/mL和9.90ng/mL。
使用GC-MS方法进行检测，测定得到的DES特征峰对应的峰面积和BPA特征峰对应的峰面积分别为5315和11480，带入标准曲线计算得到对虾样本中DES和BPA含量分别为7.79ng/mL和9.71ng/mL。
应用实施例3
本实施例中，待测样本为鱼肉。
将从超市购买得到的新鲜鱼肉样本，取5g可食用部分均质处理，并随机添加未知浓度的DES和BPA标准溶液；将鱼肉样本于50mL离心管中加碳酸钠溶液3mL、乙酸乙酯20mL，涡旋混匀，超声提取10min；随后于4000r/min离心10min，取上清液至100mL梨形瓶中；残渣用乙酸乙酯10mL重复提取一次，合并两次离心的上清液，于40℃旋转蒸发至干，用体积分数为50％环已烷乙酸乙酯溶液5mL溶解残留物，并使用60mg/mL的固相萃取柱提取DES和BPA；最后将固相萃取柱的淋洗液用于检测。
使用上转换发光的微流体生物传感平台进行检测，测定得到的荧光信号特征值I450和荧光信号特征值I541分别为0.4462和0.6749，带入标准曲线计算得到鱼肉样本中DES和BPA含量分别为40.00ng/mL和9.43ng/mL。
使用GC-MS方法进行检测，测定得到的DES特征峰对应的峰面积和BPA特征峰对应的峰面积分别为31959和10527，带入标准曲线计算得到鱼肉样本中DES和BPA含量分别为36.01ng/mL和9.15ng/mL。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例 中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种基于上转换发光的微流体生物传感平台，其特征在于，包括：上转换发光的生物传感器，用于特异性识别EDCs；以及微流体芯片，作为所述上转换发光的生物传感器与待测样本反应平台，用于集成所述上转换发光的生物传感器与待测样本的混合、反应、分离、检测；
    所述微流体芯片，包括：进样池，用于所述上转换发光的生物传感器和待测样本进样；弧形通道，所述弧形通道的进料口同时连通于所述上转换发光的生物传感器的进样池和待测样本的进样池，待所述上转换发光的生物传感器和待测样本进入弧形通道后，弧形通道用于二者的混合和反应；分离通道，与所述弧形通道的出料口连通，用于反应结束后的所述上转换发光的生物传感器的磁分离；检测池，与所述分离通道的出料口连通，用于完成发光增强定量检测EDCs。
  2. 一种如权利要求1所述上转换发光的生物传感器的制备方法，其特征在于，所述上转换发光的生物传感器的制备方法包括如下步骤：
    S1、制备含有稀土元素的上转换纳米粒子种子(CUCNPs)；
    S2、步骤S1制备得到的CUCNPs外层进行包覆处理，制备得到具有核壳结构的上转换纳米粒子(CSUCNPs)；
    S3、改性步骤S2制备得到的CSUCNPs为亲水性；
    S4、将步骤S3得到的亲水性CSUCNPs生物分子功能化，得到生物分子功能化CSUCNPs；
    S5、制备磁性纳米粒子(MNPs)；
    S6、将步骤S5得到的MNPs生物分子功能化，得到生物分子功能化MNPs；
    S7、步骤S4得到生物分子功能化CSUCNPs和步骤S6得到生物分子功能化CSUCNPs相结合，制备得到所述上转换发光的生物传感器。
  3. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S1的过程为，
    分别取六水合氯化钇、六水合氯化镱和六水合稀土元素溶解于甲醇中，之后加入油酸和1-十八稀，混合后加热至150-170℃反应25-35min，反应结束冷却后，滴加氢氧化钠和氟化铵的混合溶液，并于125-135℃反应25-35min，随后升温至290-310℃保持50-60min；反应结束后，加入乙醇和超纯水离心分离得到上转换纳米粒子种子(CUCNPs)；
    所述稀土元素为铒时，六水合氯化钇、六水合氯化镱和六水合稀土元素的比例为 0.78:0.2:0.02；或
    所述稀土元素为铥时，六水合氯化钇、六水合氯化镱和六水合稀土元素的比例为0.795:0.2:0.005。
  4. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S2的过程为，
    取六水合氯化钇溶解于甲醇中，之后加入油酸和1-十八稀，混合后加热至150-170℃反应25-35min；反应结束冷却后，加入步骤S1制备得到的CUCNPs，并滴加氢氧化钠和氟化铵的混合溶液，并于125-135℃反应25-35min，随后升温至290-310℃保持20-40min；反应结束后，加入乙醇和超纯水离心分离得到核壳上转换纳米粒子(CSUCNPs)。
  5. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S3的过程为，
    取步骤S2制备得到的CSUCNPs加入三氯甲烷和甲苯的混合溶液中，随后加入聚丙烯酸水溶液并密封，剧烈搅拌反应；结束后使用乙醇和超纯水清洗并离心，得到具有亲水性的聚丙烯酸修饰的CSUCNPs(PAA-CSUCNPs)。
  6. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S4的过程为，
    取步骤S3制备得到的PAA-CSUCNPs，加入含有碳酰二亚胺和N-羟基硫代琥珀酰亚胺的吗啉乙磺酸缓冲溶液中，进行第一次孵育；孵育结束后，离心分离得到活化的PAA-CSUCNPs，并分散在磷酸盐缓冲液中；向溶液中加入链霉亲和素溶液，进行第二次孵育；孵育结束后，离心分离得到修饰链霉亲和素的CSUCNPs，并分散在磷酸盐缓冲液中；随后加入5’端修饰生物素的EDCs适配体，进行第三次孵育；孵育结束后离心分离得到修饰适配体的CSUCNPs，并重新分散在磷酸盐缓冲液中；随后加入牛血清白蛋白溶液，进行第四次孵育；孵育结束后离心分离，并使用磷酸盐缓冲液清洗，制备得到生物分子功能化的CSUCNPs。
  7. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S5的过程为，
    分别取六水合氯化铁、二水合柠檬酸三钠、醋酸钠加入乙二醇溶液中，剧烈搅拌充分溶解后，转移到反应釜中，高温反应；反应结束通过磁场收集制备得到的磁性纳米粒子(MNPs)，并使用乙醇和超纯水清洗。
  8. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S6的过程 为，
    取步骤S5制备得到的MNPs，加入含有碳酰二亚胺和N-羟基硫代琥珀酰亚胺的吗啉乙磺酸缓冲溶液中，进行第一次孵育；孵育结束后，磁分离得到活化的MNPs，并分散在磷酸盐缓冲液中；向溶液中加入链霉亲和素溶液，进行第二次孵育；孵育结束后，磁分离得到修饰链霉亲和素的MNPs，并分散在磷酸盐缓冲液中；随后加入5’端修饰生物素的EDCs适配体互补序列，进行第三次孵育；孵育结束后磁分离得到修饰适配体的MNPs，并重新分散在磷酸盐缓冲液中；随后加入牛血清白蛋白溶液，进行第四次孵育；孵育结束后磁分离，并使用磷酸盐缓冲液清洗，制备得到生物分子功能化的MNPs。
  9. 根据权利要求2所述上转换发光的生物传感器的制备方法，其特征在于：步骤S7的过程为，
    取步骤S4制备得到的EDCs适配体修饰的CSUCNPs和步骤S6制备得到的EDCs适配体互补序列修饰的MNPs加入磷酸盐缓冲液中，高温加热；加热结束后缓慢退火，并转移至摇床孵育；孵育结束后，磁分离得到上转换发光的生物传感器，并使用磷酸盐缓冲液清洗三次，最后分散于磷酸盐缓冲液中。
  10. 一种如权利要求1所述基于上转换发光的微流体生物传感平台的使用方法，其特征在于，具体包括以下步骤：
    取0-250ng/mL一系列浓度的EDCs标准溶液，与制备的上转换发光生物传感器共同注射进入所述微流体芯片中，完成生物传感器与靶标EDCs的混合、反应、分离与检测步骤；
    静置以完成脱落的CSUCNPs的桥连絮凝和沉降，通过荧光光谱仪采集检测池中沉降形成的CSUCNPs界面的荧光光谱，以EDCs标准溶液的浓度对数值和荧光信号特征值进行线性拟合，建立EDCs含量检测的标准曲线；所述荧光信号特征值为特异性识别EDCs的生物传感器使用的稀土元素的特征荧光发射光强；
    取待测样本溶液，代替上述EDCs标准溶液，与生物传感器共同注射进入微流体芯片，将采集得到的荧光信号特征值代入标准曲线计算，得出待测样本中EDCs的含量。
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