A microfluidic biosensing platform based on upconversion luminescence

Abstract

This invention relates to a microfluidic biosensing platform based on upconversion luminescence, comprising: an upconversion luminescence biosensor for specifically identifying EDCs; and a microfluidic chip for integrating the mixing, reaction, separation, and detection of the upconversion luminescence biosensor and a sample to be tested; the microfluidic chip comprising: a sample inlet for introducing the upconversion luminescence biosensor and the sample to be tested; an arc-shaped channel, the inlet of which is simultaneously connected to the sample inlet of the upconversion luminescence biosensor and the sample inlet of the sample to be tested, wherein the arc-shaped channel is used for mixing and reaction after the upconversion luminescence biosensor and the sample to be tested enter the arc-shaped channel; a separation channel connected to the outlet of the arc-shaped channel for magnetic separation of the upconversion luminescence biosensor after the reaction is completed; and a detection cell connected to the outlet of the separation channel for completing luminescence-enhanced quantitative detection of EDCs.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a microfluidic biosensing platform based on upconversion luminescence. Background Technology

[0002] Endocrine disrupting chemicals (EDCs) are defined as "exogenous agents that interfere with the synthesis, secretion, transport, metabolism, binding, or elimination of naturally occurring blood-derived hormones responsible for homeostasis, reproduction, and development within the human body." Humans may ingest hundreds of EDCs through food production (food additives, pesticides, food containers), industrial activities (air pollution, water pollutants, industrial chemicals), and medical treatment (medical products). Among these, exogenous agents with estrogenic effects, such as bisphenol A (BPA), diethylstilbestrol (DES), estradiol (E2), and nonylphenol, have attracted widespread attention. These EDCs may cause serious health hazards, including neurodevelopmental disorders, brain, liver, and lung damage, reproductive and endocrine disorders, and metabolic disturbances. Therefore, establishing effective assessment methods to determine the content of EDCs in media from which humans may be exposed is crucial.

[0003] Previous studies have relied on high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), electrochemical sensors, and photoelectrochemical immunoassay sensors to determine the content of EDCs. However, these methods often face challenges such as expensive equipment, strong background interference, cumbersome sample pretreatment steps, and high reagent consumption, making it difficult to achieve rapid on-site quantitative detection of EDCs. Therefore, developing novel detection platforms to overcome these shortcomings is crucial. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a microfluidic biosensing platform based on upconversion luminescence, which enables micro-sampling and simple, highly sensitive quantitative detection of EDCs.

[0005] Based on this, the purpose 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.

[0006] The microfluidic chip includes: a sample inlet for introducing the upconversion luminescent biosensor and the sample to be tested; an arc-shaped channel, the inlet of which is simultaneously connected to the sample inlet of the upconversion luminescent biosensor and the sample inlet of the sample to be tested, wherein the arc-shaped channel is used for mixing and reaction after the upconversion luminescent biosensor and the sample to be tested enter the arc-shaped channel; a separation channel, connected to the outlet of the arc-shaped channel, for magnetic separation of the upconversion luminescent biosensor after the reaction is completed; and a detection cell, connected to the outlet of the separation channel, for quantitative detection of light-enhanced luminescence (EDCs).

[0007] Furthermore, the width of all microchannels in the microfluidic chip is... The depth is .

[0008] According to the above technical solution, the upconversion luminescent biosensor and the sample to be tested are integrated on a microfluidic chip for mixing, reaction, separation, and detection. The specific process is as follows: the upconversion luminescent biosensor and the sample to be tested are injected into two sample cells of the microfluidic chip, respectively. The two microfluidics are fully mixed and reacted in the arc-shaped channel. Then, an external magnetic field is applied at the separation channel to separate the biosensor that has not reacted with EDCs. Finally, in the detection cell, the detached CSUCNPs complete bridging flocculation and sedimentation, and complete the acquisition of upconversion fluorescence signals, thereby enabling quantitative detection of EDCs.

[0009] Furthermore, the injection flow rate of the biosensor and the sample solution to be tested is... and .

[0010] Furthermore, the injection time between the biosensor and the sample solution to be tested is 8-12 minutes.

[0011] The second objective of this invention is to provide a method for preparing the aforementioned microfluidic biosensing platform based on upconversion luminescence.

[0012] The method for preparing the upconversion luminescence biosensor includes the following steps:

[0013] S1. Preparation of upconversion nanoparticle seeds (CUCNPs) containing rare earth elements;

[0014] S2. The outer layer of the CUCNPs prepared in step S1 is coated to prepare upconversion nanoparticles (CSUCNPs) with a core-shell structure.

[0015] S3. The CSUCNPs prepared in the modification step S2 are hydrophilic.

[0016] S4. Functionalize the hydrophilic CSUCNPs obtained in step S3 to obtain biomolecule-functionalized CSUCNPs.

[0017] S5. Preparation of magnetic nanoparticles (MNPs);

[0018] S6. Functionalize the MNPs obtained in step S5 to obtain biomolecularly functionalized MNPs.

[0019] The biomolecule-functionalized CSUCNPs obtained in step S7 and S4 are combined with the biomolecule-functionalized MNPs obtained in step S6 to prepare the upconversion luminescence biosensor.

[0020] Furthermore, the process of step S1 is as follows:

[0021] Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth elements hexahydrate were dissolved in methanol, respectively. Then, oleic acid and 1-octadecene were added, and the mixture was heated to 150-170℃ for 25-35 min. After the reaction was completed and cooled, a mixed solution of sodium hydroxide and ammonium fluoride was added dropwise, and the mixture was reacted at 125-135℃ for 25-35 min. Then, the temperature was raised to 290-310℃ and held for 50-60 min. After the reaction was completed, ethanol and ultrapure water were added, and the mixture was centrifuged to obtain upconversion nanoparticle seeds (CUCNPs).

[0022] Furthermore, the total amount of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth elements hexahydrate is 1-1.5 mmol.

[0023] Furthermore, when the rare earth element is erbium, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth element hexahydrate is 0.78:0.2:0.02.

[0024] Furthermore, when the rare earth element is thulium, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth element hexahydrate is 0.795:0.2:0.005.

[0025] Furthermore, the volume ratio of methanol, oleic acid, and 1-octadecene is 10:(5-7):(14-17).

[0026] Furthermore, the molar ratio of sodium hydroxide to ammonium fluoride is 5:(7-9).

[0027] Furthermore, the volume ratio of ethanol to ultrapure water is 1:(0.6-1).

[0028] Furthermore, the centrifugation parameters are: centrifugation at 8000-12000 rpm for 5-10 minutes.

[0029] Furthermore, the process of step S2 is as follows:

[0030] Yttrium chloride hexahydrate was dissolved in methanol, followed by the addition of oleic acid and 1-octadecene. The mixture was heated to 150-170℃ and reacted for 25-35 min. After the reaction was completed and cooled, the CUCNPs prepared in step S1 were added, and a mixed solution of sodium hydroxide and ammonium fluoride was added dropwise. The mixture was then reacted at 125-135℃ for 25-35 min, followed by a temperature increase to 290-310℃ and a holding temperature of 20-40 min. After the reaction was completed, ethanol and ultrapure water were added, and the mixture was centrifuged to obtain core-shell upconversion nanoparticles (CSUCNPs).

[0031] Furthermore, the amount of yttrium chloride hexahydrate used is 0.35-0.45 mmol.

[0032] Furthermore, the volume ratio of methanol, oleic acid, and 1-octadecene is 10:(2.5-3.5):(7-9).

[0033] Furthermore, the molar ratio of sodium hydroxide to ammonium fluoride is 2:(2.5-3.5).

[0034] Furthermore, the volume ratio of ethanol to ultrapure water is 1:(0.6-1).

[0035] Furthermore, the centrifugation parameters are: centrifugation at 8000-12000 rpm for 5-10 minutes.

[0036] Furthermore, the process of step S3 is as follows:

[0037] The CSUCNPs prepared in step S2 were added to a mixed solution of chloroform and toluene, followed by the addition of an aqueous solution of polyacrylic acid and the mixture was sealed and stirred vigorously. After the reaction was completed, the mixture was washed with ethanol and ultrapure water and centrifuged to obtain hydrophilic polyacrylic acid-modified CSUCNPs (PAA-CSUCNPs).

[0038] Furthermore, the ratio of CSUCNPs, chloroform, toluene and polyacrylic acid is 50 mg:(2-6 mL):(4-10 mL):(15-20 mL).

[0039] Furthermore, the concentration of the polyacrylic acid aqueous solution is 10-20 mg / mL.

[0040] Furthermore, the stirring reaction time is 24-48 hours.

[0041] Furthermore, the volume ratio of ethanol to ultrapure water is 1:(0.6-1).

[0042] Furthermore, the centrifugation parameters are: centrifugation at 8000-12000 rpm for 5-10 minutes.

[0043] Furthermore, the process of step S4 is as follows:

[0044] The PAA-CSUCNPs prepared in step S3 were added to a morpholine ethanesulfonic acid buffer solution containing carbodiimide and N-hydroxythiosuccinimide for the first incubation. After incubation, the activated PAA-CSUCNPs were obtained by centrifugation and dispersed in phosphate buffer. Streptavidin solution was added to the solution for the second incubation. After incubation, streptavidin-modified CSUCNPs were obtained by centrifugation and dispersed in phosphate buffer. Then, 5'-terminal biotin-modified EDCs aptamers were added for the third incubation. After incubation, the modified aptamers were obtained by centrifugation and redispersed in phosphate buffer. Then, bovine serum albumin solution was added for the fourth incubation. After incubation, the CSUCNPs were centrifuged and washed with phosphate buffer to prepare biomolecularly functionalized CSUCNPs.

[0045] Further, the ratio of PAA-CSUCNPs, carbodiimide, N-hydroxythiosuccinimide and morpholine ethanesulfonic acid buffer solution is (0.8-1.2 mg): 4 mg: 2 mg: (2.0-2.4 mL).

[0046] Furthermore, the initial incubation conditions are 20-40℃ for 2-4 hours.

[0047] Furthermore, the dosage of streptavidin is 0.8-1.2 mg.

[0048] Furthermore, the second incubation conditions are 30-40℃ for 10-14 hours.

[0049] Furthermore, the dosage of aptamers is... The concentration is .

[0050] Furthermore, the third incubation conditions are 30-40℃ for 10-14 hours.

[0051] Furthermore, the amount of bovine serum albumin used is 3-6 mL, with a mass fraction of 2%.

[0052] Furthermore, the fourth incubation conditions are 30-40℃ for 1.5-3 hours.

[0053] Furthermore, in step S4, the centrifugation parameters are 8000-12000 rpm for 5-10 min.

[0054] Furthermore, in step S4, the pH of the phosphate buffer is 7.2-7.4, and the volume is 5-15 mL.

[0055] Furthermore, the process of step S5 is as follows:

[0056] Ferric chloride hexahydrate, trisodium citrate dihydrate, and sodium acetate were added to an ethylene glycol solution, stirred vigorously until fully dissolved, and then transferred to a reaction vessel for high-temperature reaction. After the reaction was completed, the prepared magnetic nanoparticles (MNPs) were collected by a magnetic field and washed with ethanol and ultrapure water.

[0057] Furthermore, the ratio of ferric chloride hexahydrate, trisodium citrate dihydrate, sodium acetate, and ethylene glycol is 5 mmol: 0.4 mmol: (1.2-1.5 g): (15-25 mL).

[0058] Furthermore, the conditions for the high-temperature reaction are 190-210℃ for 8-12 hours.

[0059] Furthermore, the volume ratio of ethanol to ultrapure water is 1:(0.6-1).

[0060] Furthermore, the process of step S6 is as follows:

[0061] The MNPs prepared in step S5 were added to a morpholine ethanesulfonic acid buffer solution containing carbodiimide and N-hydroxythiosuccinimide for the first incubation. After incubation, the activated MNPs were obtained by magnetic separation and dispersed in phosphate buffer. Streptavidin solution was added to the solution for the second incubation. After incubation, streptavidin-modified MNPs were obtained by magnetic separation and dispersed in phosphate buffer. Subsequently, a complementary sequence of an EDC aptamer modified with biotin at the 5' end was added for the third incubation. After incubation, the modified aptamer MNPs were obtained by magnetic separation and redispersed in phosphate buffer. Subsequently, bovine serum albumin solution was added for the fourth incubation. After incubation, the MNPs were magnetically separated and washed with phosphate buffer to prepare biomolecularly functionalized MNPs.

[0062] Further, the ratio of the MNPs, carbodiimide, N-hydroxythiosuccinimide and morpholine ethanesulfonic acid buffer solution is (8-12 mg): 10 mg: 5 mg: (0.8-1.2 mL).

[0063] Furthermore, the initial incubation conditions are 20-40℃ for 2-4 hours.

[0064] Furthermore, the dosage of streptavidin is 0.8-1.2 mg.

[0065] Furthermore, the second incubation conditions are 30-40℃ for 10-14 hours.

[0066] Furthermore, the amount of aptamer complementary sequence used is The concentration is .

[0067] Furthermore, the third incubation conditions are 30-40℃ for 10-14 hours.

[0068] Furthermore, the amount of bovine serum albumin used is 3-6 mL, with a mass fraction of 2%.

[0069] Furthermore, the fourth incubation conditions are 30-40℃ for 1.5-3 hours.

[0070] Furthermore, in step S4, the pH of the phosphate buffer is 7.2-7.4, and the volume is 5-15 mL.

[0071] Furthermore, the process of step S7 is as follows:

[0072] The biomolecular functionalized CSUCNPs prepared in step S4 and the biomolecular functionalized MNPs prepared in step S6 were added to phosphate buffer and heated at high temperature. After heating, the mixture was slowly annealed and transferred to a shaker for incubation. After incubation, the upconversion luminescent biosensor was obtained by magnetic separation and washed three times with phosphate buffer. Finally, it was dispersed in phosphate buffer.

[0073] Furthermore, the quality ratio of CSUCNPs modified with EDCs aptamers to MNPs modified with complementary sequences of EDCs aptamers is (1.5-2):5.

[0074] Furthermore, the high-temperature reaction conditions are 90-95℃ for 3-5 minutes.

[0075] Furthermore, the annealing conditions are 3-5℃ / min, up to 60-65℃.

[0076] Furthermore, the incubation conditions are 20-40℃ for 0.5-2 hours.

[0077] Furthermore, in step 7, the pH of the phosphate buffer solution is 7.2-7.4, and the volume is 5-10 mL.

[0078] According to the above technical solution, the process of achieving efficient luminescence enhancement of the upconversion luminescent biosensor by aptamer-mediated nanoparticle bridging flocculation is as follows: After mixing and reacting the upconversion luminescent biosensor prepared in step S7 with the sample solution to be tested, the aptamer specifically binds to EDCs, causing CSUCNPs to detach from the surface of MNPs; magnetic separation removes the biosensor that has not reacted with the EDC target, and the CSUCNPs detached in the solution reflect the number of target EDCs; the aptamers on the surface of the detached CSUCNPs that have not bound to the target EDCs undergo complementary base pairing, generating aptamer-mediated nanoparticle bridging flocculation, which further settles, achieving concentration enrichment of nanoparticles; by optimizing and adjusting the focal length, the signal of the settled CSUCNPs is acquired, realizing quantitative detection of EDCs with enhanced luminescence.

[0079] Furthermore, the volume ratio of the upconversion luminescent biosensor prepared in step S7 to the sample solution to be tested is 4:1.

[0080] Furthermore, the focal length for signal acquisition is 11.5mm.

[0081] Furthermore, the settling time of the detached CSUCNPs was 20-30 minutes.

[0082] A third objective of this invention is to provide a method for using the aforementioned microfluidic biosensing platform based on upconversion luminescence, specifically comprising the following steps:

[0083] A series of EDC standard solutions with concentrations ranging from 0 to 250 ng / mL were taken and injected together with the prepared upconversion luminescent biosensor into the microfluidic chip to complete the mixing, reaction, separation and detection steps of the biosensor and the target EDCs.

[0084] The sample was allowed to stand to complete the bridging flocculation and sedimentation of the detached CSUCNPs. The fluorescence spectrum of the CSUCNPs interface formed by sedimentation in the detection cell was collected by a fluorescence spectrometer. A standard curve for EDC content detection was established by linearly fitting the concentration logarithm of the EDCs standard solution and the fluorescence signal characteristic value. The fluorescence signal characteristic value is the characteristic fluorescence emission intensity of rare earth elements used in the biosensor that specifically identifies EDCs.

[0085] Take the sample solution to be tested, replace the above EDCs standard solution, and inject it into the microfluidic chip together with the biosensor. Substitute the collected fluorescence signal characteristic values ​​into the standard curve to calculate the content of EDCs in the sample to be tested.

[0086] In summary, the present invention has the following beneficial effects:

[0087] 1. This invention discloses a novel aptamer-mediated nanoparticle bridging and flocculation phenomenon. Based on this discovery, a significant enhancement of the fluorescence signal of the nanobiosensor is achieved, which greatly improves the sensitivity of target detection and raises the detection limit by at least one order of magnitude. In addition, by forming a stable interface through aptamer-mediated nanoparticle bridging and flocculation, the influence of the gravitational field on the nanoparticle dispersion is greatly reduced, effectively improving the signal stability of the nanobiosensor.

[0088] 2. This invention designs a novel microfluidic chip that integrates 126 semi-circular channels and 12 quarter-circular channels within a limited space. This increases the turbulence of the laminar microfluidic flow with a Reynolds number of approximately 0.79, enabling repeated mixing and reaction between the biosensor and the sample to be tested. With the assistance of an external magnetic field, the mixing, reaction, separation, and detection steps on the microfluidic chip are integrated into one, greatly improving the detection efficiency.

[0089] 3. This invention fabricates an upconversion luminescence microfluidic biosensor. The upconversion luminescence process effectively avoids background fluorescence interference from other matrices, while the biorecognition element aptamer exhibits good economy and specificity. Furthermore, after integration onto a chip as a microfluidic biosensor, micro-sampling is achieved. The rapid detection (10 min) provides a promising prospect for practical field applications.

[0090] 4. This invention enables the simultaneous detection of multiple EDCs. A single EDC component below its hazard threshold may pose a serious health hazard when mixed with other EDCs, i.e., a cumulative effect may exist. This invention prepares a biosensor for the simultaneous detection of BPA and DES by tuning multicolor CSUCNPs and modifying them with specific EDC aptamers, achieving ultrasensitive detection at levels as low as 0.0076 ng / mL and 0.0131 ng / mL, respectively. Attached Figure Description

[0091] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0092] Figure 1The images show the characterization of the prepared nanoparticles; where A is a transmission electron microscope (TEM) image of erbium-doped CUCNPs; B is a TEM image of erbium-doped CSUCNPs; C is a TEM image of thulium-doped CUCNPs; D is a TEM image of thulium-doped CSUCNPs; E is a TEM image of MNPs; and F is a scanning electron microscope (SEM) image of MNPs.

[0093] Figure 2 The images show the characterization of the prepared biosensors; where A is a transmission electron microscope (TEM) image of the erbium-doped biosensor; B is a high-magnification TEM image of the erbium-doped biosensor, where B1 is a high-magnification TEM image and B2-B4 are mapping images of iron and erbium, respectively; C is a TEM image of the thulium-doped biosensor; and D is a high-magnification TEM image of the thulium-doped biosensor, where D1 is a high-magnification TEM image and D2-D4 are mapping images of iron and thulium, respectively.

[0094] Figure 3 The diagram shows a microfluidic chip; where A is a schematic diagram of the overall structure of the microfluidic chip; B is a microchannel diagram of the microfluidic chip, where 1 is the sample inlet; 2 is the biosensor inlet; 3 is the liquid inlet channel; 4 is the semi-circular channel; 5 is the connecting channel; 6 is the 1 / 4 arc channel; 7 is the separation channel; and 8 is the detection cell.

[0095] Figure 4 The diagrams and characterizations of aptamer-mediated nanoparticle bridging flocculation are presented. A shows the secondary structure analysis of BPA and DES aptamers and the schematic diagram of nanoparticle bridging flocculation mediated by them. B is a transmission electron microscope image of aptamer-mediated CSUCNPs bridging flocculation. C is a comparison image of aptamer-mediated CSUCNPs bridging flocculation before and after macroscopic bridging, where C1 represents the unexcited state and C2 represents the state excited by a 980nm laser.

[0096] Figure 5 The diagrams and characterizations of CSUCNPs luminescence enhancement are shown below; where A is a schematic diagram and spectrum of 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 CSUCNPs bridging and flocculation; and C is the spectrum of luminescence enhancement at the interface formed by aptamer-mediated CSUCNPs bridging and flocculation.

[0097] Figure 6 The results of biosensor detection of different concentrations of EDCs are shown in the figure. Among them, A is the fluorescence spectrum of EDCs detected at different concentrations; B is the relationship between the fluorescence signal characteristic value at 450 nm and the DES concentration; C is the linear fitting curve and equation of the fluorescence characteristic value and the logarithm of the EDC concentration; D is the relationship between the fluorescence signal characteristic value at 541 nm and the BPA concentration. Detailed Implementation

[0098] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0099] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the data are the average of the three replicates or the average ± standard deviation.

[0100] Furthermore, the term "and / or" throughout the 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 simultaneously satisfies both A and B. In addition, the technical solutions of various embodiments can be combined with each other, but this must be based on the ability of a person skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0101] Example 1

[0102] In this embodiment, an upconversion luminescent biosensor doped with erbium was prepared, and the upconversion luminescent biosensor was synthesized under nitrogen protection and continuous magnetic stirring conditions.

[0103] A method for fabricating an erbium-doped upconversion luminescent biosensor includes the following steps:

[0104] S1: Dissolve 0.78 mmol of yttrium chloride hexahydrate, 0.2 mmol of ytterbium chloride hexahydrate, and 0.02 mmol of erbium chloride hexahydrate in 10 mL of methanol. Then add 8 mL of oleic acid and 15 mL of 1-octadecene, mix, and heat to 150 °C for 35 min. After the reaction is complete and cooled, add a mixed solution of 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride dropwise, and react at 125 °C for 35 min. Then raise the temperature to 300 °C and hold for 60 min. After the reaction is complete, add 10 mL of ethanol and 8 mL of ultrapure water, and centrifuge at 8500 rpm for 10 min to obtain erbium-doped upconversion nanoparticle seeds (CUCNPs). The transmission electron microscope image is shown below. Figure 1 As shown in Figure A.

[0105] S2: Dissolve 0.4 mmol of yttrium chloride hexahydrate in 10 mL of methanol, then add 3 mL of oleic acid and 8 mL of 1-octadecene. After mixing, heat to 170 °C and react for 25 min. After cooling, add the CUCNPs prepared in step S1, and dropwise add a mixed solution of 1 mmol of sodium hydroxide and 1.5 mmol of ammonium fluoride. React at 135 °C for 25 min, then raise the temperature to 290 °C and hold for 40 min. After the reaction is complete, add 10 mL of ethanol and 8 mL of ultrapure water, and centrifuge at 8500 rpm for 10 min to obtain erbium-doped core-shell upconversion nanoparticles (CSUCNPs). The transmission electron microscope image is shown below. Figure 1 As shown in B.

[0106] 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 20 mL of 10 mg / mL polyacrylic acid aqueous solution and seal. Stir vigorously at room temperature for 48 h. After the reaction, wash with 10 mL of ethanol and 10 mL of ultrapure water, and centrifuge at 9000 rpm for 8 min to obtain hydrophilic polyacrylic acid modified CSUCNPs (PAA-CSUCNPs).

[0107] S4: Take 5 mg of PAA-CSUCNPs prepared in step S3, add them to 10 mL of morpholine ethanesulfonic acid buffer containing 20 mg carbodiimide and 10 mg N-hydroxythiosuccinimide, and incubate at 25 °C for 3 h; after 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 incubation, centrifuge at 8500 rpm for 6 min to obtain streptavidin-modified CSUCNPs, and disperse them in 10 mL of phosphate buffer (pH 7.2); then add... Biotin-modified BPA aptamers were incubated at 37°C for 12 h. After incubation, the CSUCNPs modified with BPA aptamers were obtained by centrifugation at 9000 rpm for 5 min 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 2 h. After incubation, the mixture was centrifuged at 10000 rpm for 5 min and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecularly functionalized CSUCNPs. The sequence of the BPA aptamer is as follows:

[0108] 5'-Biotin-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-3' (SEQ ID NO: 1).

[0109] S5. Add 1.3515 g of ferric chloride hexahydrate, 0.1178 g of trisodium citrate dihydrate, and 1.2 g of sodium acetate to 20 mL of ethylene glycol solution. Stir vigorously for 30 min until fully dissolved, then transfer to a reaction vessel and react at 200 °C for 12 h. After the reaction is complete, collect the prepared magnetic nanoparticles (MNPs) using a magnetic field and wash with 10 mL of ethanol and 10 mL of ultrapure water. Transmission electron microscopy (TEM) images of the MNPs are shown below. Figure 1 As shown in E, the scanning electron microscope image is as follows: Figure 1 As shown in F.

[0110] S6. Take 100 mg of the MNPs prepared in step S5 and add them to 20 mL of morpholine ethanesulfonic acid buffer solution containing 200 mg of carbodiimide and 100 mg of N-hydroxythiosuccinimide. Incubate at 25 °C for 3 h. After incubation, magnetically separate the activated MNPs 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 incubation, magnetically separate the streptavidin-modified MNPs and disperse them in 10 mL of phosphate buffer (pH 7.2). Then add... The 5' end of the biotin-modified BPA aptamer complementary sequence was incubated at 37°C for 12 h. After incubation, the modified BPA aptamer complementary sequence MNPs were obtained by magnetic separation 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. After incubation, the MNPs were magnetically separated and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecularly functionalized MNPs. The complementary sequence of the BPA aptamer was 5'-Biotin-TTTTTTTGGTGCGAACCCGTGATG-3' (SEQ ID NO:2).

[0111] S7. Take 400µL of 0.5mg / mL biomolecularly functionalized CSUCNPs prepared in step S4 and 500µL of 1mg / mL biomolecularly functionalized MNPs prepared in step S6, add them to 1mL of phosphate buffer, and heat at 95℃ for 3min. After heating, slowly anneal to 65℃ and transfer to a shaker for incubation at 37℃ for 1h. After incubation, magnetic separation yields the erbium-doped upconversion luminescent biosensor, which is washed three times with 10mL of phosphate buffer and finally dispersed in 5mL of phosphate buffer. The transmission electron microscope image of the erbium-doped upconversion luminescent biosensor is shown below. Figure 2 As shown in Figure A, the high-magnification transmission electron microscope image and elemental mapping diagram are as follows: Figure 2 As shown in Figure B, this demonstrates the successful fabrication of an upconversion luminescent biosensor doped with erbium.

[0112] Example 2

[0113] In this embodiment, an upconversion luminescent biosensor doped with thulium was prepared, and the upconversion luminescent biosensor was synthesized under nitrogen protection and continuous magnetic stirring conditions.

[0114] A method for preparing a thulium-doped upconversion luminescent biosensor includes the following steps;

[0115] S1: Dissolve 0.795 mmol of yttrium chloride hexahydrate, 0.2 mmol of ytterbium chloride hexahydrate, and 0.005 mmol of thulium chloride hexahydrate in 10 mL of methanol. Then add 7 mL of oleic acid and 14 mL of 1-octadecene, mix, and heat to 170 °C for 25 min. After the reaction is complete and cooled, add a mixed solution of 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride dropwise, and react at 135 °C for 25 min. Then raise the temperature to 295 °C and hold for 55 min. After the reaction is complete, add 10 mL of ethanol and 10 mL of ultrapure water, and centrifuge at 9500 rpm for 5 min to obtain thulium-doped upconversion nanoparticle seeds (CUCNPs). Transmission electron microscopy image is shown below. Figure 1 As shown in C.

[0116] S2: Dissolve 0.45 mmol of yttrium chloride hexahydrate in 10 mL of methanol, then add 2.5 mL of oleic acid and 8 mL of 1-octadecene. After mixing, heat to 150 °C and react for 35 min. After cooling, add the CUCNPs prepared in step S1, and dropwise add a mixed solution of 1 mmol of sodium hydroxide and 1.5 mmol of ammonium fluoride. React at 125 °C for 35 min, then raise the temperature to 300 °C and hold for 20 min. After the reaction is complete, add 10 mL of ethanol and 10 mL of ultrapure water, and centrifuge at 12000 rpm for 4 min to obtain thulium-doped core-shell upconversion nanoparticles (CSUCNPs). The transmission electron microscope image is shown below. Figure 1 As shown in D.

[0117] 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. Stir vigorously at room temperature for 36 h. After the reaction, wash with 10 mL of ethanol and 6 mL of ultrapure water and centrifuge at 9500 rpm for 7 min to obtain hydrophilic polyacrylic acid modified CSUCNPs (PAA-CSUCNPs).

[0118] S4: Take 5 mg of PAA-CSUCNPs prepared in step S3, add them to 12 mL of morpholine ethanesulfonic acid buffer solution containing 20 mg carbodiimide and 10 mg N-hydroxythiosuccinimide, and incubate at 30 °C for 2 h; after incubation, centrifuge at 9500 rpm for 6 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 10 h; after incubation, centrifuge at 11000 rpm for 5 min to obtain streptavidin-modified CSUCNPs, and disperse them in 10 mL of phosphate buffer (pH 7.2); then add... The 5' end of the biotin-modified DES aptamer was incubated at 37°C for 13 h. After incubation, the DES-modified CSUCNPs 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 the mixture was incubated at 37°C for 2 h. After incubation, the mixture was centrifuged at 10000 rpm for 5 min and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecularly functionalized CSUCNPs. The sequence of the DES aptamer is as follows:

[0119] 5'-Biotin-GCCCTCTGAGGATGCCGAAAAAGAAAAGAAATTCTCTGGC-3' (SEQ ID NO: 3).

[0120] S5. Take 1.3515 g of ferric chloride hexahydrate, 0.1178 g of trisodium citrate dihydrate, and 1.2 g of sodium acetate and add them to 25 mL of ethylene glycol solution. Stir vigorously for 30 min to dissolve completely, then transfer to a reaction vessel and react at 200 °C for 10 h. After the reaction is completed, collect the prepared magnetic nanoparticles (MNPs) using a magnetic field and wash them with 10 mL of ethanol and 10 mL of ultrapure water.

[0121] S6. Take 120 mg of the MNPs prepared in step S5 and add them to 20 mL of morpholine ethanesulfonic acid buffer solution containing 200 mg of carbodiimide and 100 mg of N-hydroxythiosuccinimide. Incubate at 30 °C for 2 h. After incubation, magnetically separate the activated MNPs 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. After incubation, magnetically separate the streptavidin-modified MNPs and disperse them in 10 mL of phosphate buffer (pH 7.2). Then add... The 5' end of the biotin-modified DES aptamer complementary sequence was incubated at 37°C for 13 h. After incubation, the modified DES aptamer complementary sequence MNPs were obtained by magnetic separation 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 3 h. After incubation, the MNPs were magnetically separated and washed with 10 mL of phosphate buffer (pH 7.2) to prepare biomolecularly functionalized MNPs. The complementary sequence of the DES aptamer was 5'-Biotin-TTTTTTGCCAGAGAATTTCTT-3' (SEQ ID NO:4).

[0122] S7. Take 300 µL of 0.5 mg / mL biomolecularly functionalized CSUCNPs prepared in step S4 and 500 µL of 1 mg / mL biomolecularly functionalized MNPs prepared in step S6, add them to 1 mL of phosphate buffer, and heat at 90 °C for 5 min. After heating, slowly anneal to 65 °C and transfer to a shaker for incubation at 37 °C for 2 h. After incubation, magnetically separate the thulium-doped upconversion luminescent biosensor, wash it three times with 10 mL of phosphate buffer, and finally disperse it in 5 mL of phosphate buffer. The transmission electron microscope image of the thulium-doped upconversion luminescent biosensor is shown below. Figure 2 As shown in C, the high-magnification transmission electron microscope image and elemental mapping are as follows: Figure 2 As shown in Figure D, this indicates the successful fabrication of an upconversion luminescent biosensor doped with thulium.

[0123] Example 3

[0124] The structure of the microfluidic chip fabricated by soft photolithography is as follows: Figure 3 A and Figure 3 As shown in Figure B, the sample inlet 1 and the biosensor inlet 2 are both circular in cross-sections with a diameter of 8 mm and a depth of 1 cm.

[0125] The sample inlet 1 and the biosensor inlet 2 are respectively connected to the liquid inlet channel 3. The other ends of the two liquid inlet channels 3 intersect and are connected at an angle of 90°. The two liquid inlet channels 3 are 9mm long.

[0126] The microfluidic chip also includes a semi-circular channel 4 and a quarter-circular channel 6, and the connection points of each channel are tangentially connected to achieve a smooth transition of the microfluidic fluid within the microchannel. The microfluidic chip also includes a connecting channel 5, with its two ends tangentially connected to the semi-circular channel 4 and the quarter-circular channel 6, respectively, to change the direction of the arc-shaped channel.

[0127] 126 semicircular channels 4 (radius is ), 25 quarter-circular arc channels 6 (radius is ), 11 connection channels 5 (length is ), constitutes the embodiment as follows Figure 3 The arc-shaped channel B has its inlet connected to the intersection of two liquid inlet channels 3, and its outlet connected to a 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 cell 8, which is used for magnetic separation of the biosensor. The detection cell 8 has a diameter of 1.2 cm and a depth of 1 cm, and is used for bridging flocculation, sedimentation, and fluorescence signal acquisition of detached CSUCNPs. In this embodiment, the width of all channels is... The depth is .

[0128] Aptamer-mediated nanoparticle bridging flocculation and luminescence enhancement include the following steps:

[0129] Examples 1 and 2 respectively prepared core-shell structured CSUCNPs, achieving the first enhancement of luminescence at the single nanoparticle level, such as... Figure 5 As shown in Figure A, erbium-doped CSUCNPs achieved a 1.72-fold increase in luminescence, while thulium-doped CSUCNPs achieved a 2.28-fold increase in luminescence. The secondary structure predictions for the DES and BPA aptamers are as follows: Figure 4 As shown in Figure A, the presence of a stem-loop structure indicates the existence of complementary base pairs within the aptamer sequence, enabling stable hybridization. This leads to the binding of modified CSUCNPs in the aptamer sequence to form bridged flocculation, as shown in Figure A. Figure 4 As shown in Figure A; the microscopic transmission electron microscope image of aptamer-mediated CSUCNP bridging flocculation is shown in Figure A. Figure 4 As shown in Figure B, it can be clearly observed that CSUCNPs consist of dispersed single nanoparticles ( Figure 1 A-1D) aggregates into nano-aggregates; this allows surface-functionalized CSUCNPs to macroscopically transition from a dispersed solution state to a sedimentation interface ( Figure 4 C Figure 5 B), adjust the fluorescence signal acquisition method to be parallel to the direction of the gravitational field ( Figure 4 C2), to achieve luminescence enhancement at the nanoparticle population level, such as Figure 5 As shown in Figure C, the luminescence of the erbium-doped biosensor was enhanced by 7.10 times, and the luminescence of the thulium-doped biosensor was enhanced by 8.94 times.

[0130] Example 4

[0131] Simultaneous detection of BPA and DES using an upconversion luminescence biosensor includes the following steps:

[0132] A. Equal volumes of the upconversion luminescent biosensors prepared in Examples 1 and 2 were mixed uniformly to form a hybrid biosensor solution capable of simultaneously recognizing BPA and DES. Then, the sample solution to be tested and the hybrid biosensor solution were injected into the microfluidic chip's sample inlet 1 and biosensor inlet 2, respectively, at injection flow rates of [missing information]. and The injection time is 10 min; the two microfluidics are fully mixed and reacted in the microchannel of the microfluidic chip; at the same time, an external magnetic field is applied at the separation channel 7 to separate the biosensors that have not reacted with BPA and DES; then the mixed liquid enters the detection cell 8, and the detached CSUCNPs complete bridging flocculation and sedimentation. The sedimentation reaches steady state in 20 min, and the upconversion fluorescence signal is collected.

[0133] B. Take a series of mixed standard solutions of BPA and DES with concentrations ranging from 0-250 ng / mL, and inject them together with the prepared upconversion luminescent biosensor into the microfluidic chip according to step S1, completing the mixing, reaction, separation, and detection steps of the biosensor and the target EDCs; the upconversion fluorescence spectra collected by detecting mixed standard solutions of different concentrations are as follows: Figure 6 As shown in Figure A, with increasing BPA and DES concentrations, the fluorescence intensity at 541 nm and 450 nm gradually increases; the characteristic value of the fluorescence signal at 450 nm... The relationship with DES concentration is as follows: Figure 6 As shown in B; fluorescence signal characteristic value at 541 nm. The relationship between BPA concentration and BPA concentration is shown in the graph below. Figure 6 As shown in D.

[0134] C. Fit the fluorescence signal characteristic values ​​to the target EDCs concentration to obtain a linear mathematical formula for detection, such as... Figure 6 As shown in C, the standard curve fitting for BPA detection is... , The detection range is 0.025-100 ng / mL, and the detection limit is 0.0076 ng / mL. This is the logarithm of BPA concentration. The characteristic value of the fluorescence signal at 450 nm The normalized fluorescence intensity; the standard curve fitting for DES detection is as follows: , The detection range is 0.025-250 ng / mL, and the detection limit is 0.0131 ng / mL. This is the logarithm of the DES concentration. The characteristic value of the fluorescence signal at 541 nm The normalized fluorescence intensity.

[0135] Application Example 1

[0136] In this embodiment, the sample to be tested is seawater.

[0137] Raw seawater samples were collected from the coastal area. After centrifugation at 4000 rpm for 10 min, the supernatant was retained. The seawater supernatant was then further processed using... Membrane filtration is used to remove impurities and for detection.

[0138] The fluorescence signal characteristic values ​​were measured using an upconversion luminescence microfluidic biosensor platform. and fluorescence signal characteristic values The values ​​were 0.3592 and 0.6720, respectively. Substituting these values ​​into the standard curve, the calculated DES and BPA contents in the seawater sample were 8.24 ng / mL and 9.18 ng / mL, respectively.

[0139] To verify the accuracy of the detection method of this invention, the same experimental samples were analyzed using gas chromatography-mass spectrometry (GC-MS) according to the Chinese national standard GB31660.2-2019. A BPA detection standard curve was established using GC-MS. , ,in BPA concentration, The peak area corresponding to the characteristic peak of BPA; the DES detection standard curve is... , ,in DES concentration, This represents the peak area corresponding to the characteristic peak of DES.

[0140] The GC-MS method was used for detection. The peak areas corresponding to the characteristic peaks of DES and BPA were 5585 and 7575, respectively. The DES and BPA contents in the seawater sample were calculated to be 8.16 ng / mL and 7.64 ng / mL, respectively, by substituting them into the standard curve.

[0141] Application Example 2

[0142] In this embodiment, the sample to be tested is a shrimp.

[0143] Fresh shrimp samples purchased from supermarkets were homogenized (5g of edible portion) and DES and BPA standard solutions of unknown concentrations were randomly added. The shrimp samples were then placed in 50mL centrifuge tubes with 3mL sodium carbonate solution and 20mL ethyl acetate, vortexed, and extracted ultrasonically for 10min. The mixture was then centrifuged at 4000r / min for 10min, and the supernatant was transferred to a 100mL pear-shaped flask. The residue was extracted once more with 10mL ethyl acetate. The supernatants from both centrifugations were combined and evaporated to dryness at 40℃. The residue was dissolved in 5mL of 50% cyclohexane-ethyl acetate solution, and DES and BPA were extracted using a 60mg / mL solid-phase extraction column. Finally, the eluent from the solid-phase extraction column was used for detection.

[0144] The fluorescence signal characteristic values ​​were measured using an upconversion luminescence microfluidic biosensor platform. and fluorescence signal characteristic values The values ​​were 0.3535 and 0.6801, respectively. Substituting these values ​​into the standard curve, the calculated DES and BPA contents in the shrimp samples were 7.42 ng / mL and 9.90 ng / mL, respectively.

[0145] The GC-MS method was used for detection. The peak areas corresponding to the characteristic peaks of DES and BPA were 5315 and 11480, respectively. The DES and BPA contents in the shrimp samples were calculated to be 7.79 ng / mL and 9.71 ng / mL, respectively, by substituting them into the standard curve.

[0146] Application Example 3

[0147] In this embodiment, the sample to be tested is fish meat.

[0148] Fresh fish samples purchased from supermarkets were homogenized (5g of edible portion) and DES and BPA standard solutions of unknown concentrations were randomly added. The fish samples were then placed in a 50mL centrifuge tube with 3mL sodium carbonate solution and 20mL ethyl acetate, vortexed, and extracted by sonication for 10min. The mixture was then centrifuged at 4000r / min for 10min, and the supernatant was transferred to a 100mL pear-shaped flask. The residue was extracted once more with 10mL ethyl acetate. The supernatants from both centrifugations were combined and evaporated to dryness at 40℃. The residue was dissolved in 5mL of 50% cyclohexane-ethyl acetate solution, and DES and BPA were extracted using a 60mg / mL solid-phase extraction column. Finally, the eluent from the solid-phase extraction column was used for detection.

[0149] The fluorescence signal characteristic values ​​were measured using an upconversion luminescence microfluidic biosensor platform. and fluorescence signal characteristic values The values ​​were 0.4462 and 0.6749, respectively. Substituting these values ​​into the standard curve, the DES and BPA contents in the fish sample were calculated to be 40.00 ng / mL and 9.43 ng / mL, respectively.

[0150] The GC-MS method was used for detection. The peak areas corresponding to the characteristic peaks of DES and BPA were 31959 and 10527, respectively. The DES and BPA contents in the fish meat sample were calculated to be 36.01 ng / mL and 9.15 ng / mL, respectively, by substituting them into the standard curve.

[0151] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A microfluidic biosensing platform based on upconversion luminescence, characterized in that, include: An upconversion luminescent biosensor for the specific identification of endocrine disruptors (EDCs); And a microfluidic chip, serving as the reaction platform for 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 upconversion luminescent biosensor comprises combined upconversion nanoparticles with a core-shell structure and magnetic nanoparticles; wherein the surface of the upconversion nanoparticles with the core-shell structure is modified with an aptamer sequence; and the surface of the magnetic nanoparticles is modified with an aptamer complementary sequence. The microfluidic chip includes: The sample injection cell is used for the upconversion luminescence biosensor and the sample to be tested. An arc-shaped channel is provided, with its inlet connected to both the sample inlet of the upconversion luminescent biosensor and the sample inlet of the test sample. After the upconversion luminescent biosensor and the test sample enter the arc-shaped channel, the arc-shaped channel is used for mixing and reaction between the two. A separation channel, connected to the outlet of the arc-shaped channel, is used to remove the upconversion luminescent biosensor that did not react with the EDCs target by magnetic separation after the reaction is completed; The detection cell, connected to the outlet of the separation channel, is used to receive the detached upconversion nanoparticles with core-shell structure, allowing the detached upconversion nanoparticles with core-shell structure to complete aptamer-mediated bridging flocculation and sedimentation, forming an interface, and collecting upconversion fluorescence signals to complete the luminescence-enhanced quantitative detection of EDCs.

2. The microfluidic biosensing platform based on upconversion luminescence according to claim 1, characterized in that: When the endocrine disruptor EDCs are bisphenol A, the sequence of its aptamer is shown in SEQ ID NO:1; the complementary sequence of the aptamer is shown in SEQ ID NO:

2.

3. The microfluidic biosensing platform based on upconversion luminescence according to claim 1, characterized in that: When the endocrine disruptor EDCs are diethylstilbestrol, the sequence of its aptamer is shown in SEQ ID NO:3; the complementary sequence of the aptamer is shown in SEQ ID NO:

4.

4. A method for preparing an upconversion luminescence biosensor as described in any one of claims 1-3, characterized in that, The method for preparing the upconversion luminescence biosensor includes the following steps: S1. Preparation of upconversion nanoparticle seeds containing rare earth elements; S2. The outer layer of the upconversion nanoparticle seed containing rare earth elements prepared in step S1 is coated to prepare upconversion nanoparticles with a core-shell structure. S3. The upconversion nanoparticles with a core-shell structure prepared in modification step S2 are hydrophilic. S4. The hydrophilic upconversion nanoparticles with core-shell structure obtained in step S3 are functionalized with biomolecules to obtain biomolecule-functionalized upconversion nanoparticles with core-shell structure. S5. Preparation of magnetic nanoparticles; S6. The magnetic nanoparticles obtained in step S5 are functionalized with biomolecules to obtain biomolecule-functionalized magnetic nanoparticles. S7 and S4 combine the biomolecule-functionalized upconversion nanoparticles with a core-shell structure obtained in step S4 with the biomolecule-functionalized magnetic nanoparticles obtained in step S6 to prepare the upconversion luminescent biosensor.

5. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S1 is as follows: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth elements hexahydrate were dissolved in methanol, respectively. Then, oleic acid and 1-octadecene were added, and the mixture was heated to 150-170℃ for 25-35 min. After the reaction was completed and cooled, a mixed solution of sodium hydroxide and ammonium fluoride was added dropwise, and the mixture was reacted at 125-135℃ for 25-35 min. Then, the temperature was raised to 290-310℃ and held for 50-60 min. After the reaction was completed, ethanol and ultrapure water were added, and the mixture was centrifuged to obtain upconversion nanoparticle seeds. When the rare earth element is erbium, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth element hexahydrate is 0.78:0.2:0.02; or When the rare earth element is thulium, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and rare earth element hexahydrate is 0.795:0.2:0.

005.

6. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S2 is as follows: Yttrium chloride hexahydrate was dissolved in methanol, followed by the addition of oleic acid and 1-octadecene. The mixture was heated to 150-170℃ and reacted for 25-35 min. After the reaction was completed and cooled, the upconversion nanoparticle seeds prepared in step S1 were added, and a mixed solution of sodium hydroxide and ammonium fluoride was added dropwise. The mixture was then reacted at 125-135℃ for 25-35 min, followed by a temperature increase to 290-310℃ and a holding temperature of 20-40 min. After the reaction was completed, ethanol and ultrapure water were added, and the mixture was centrifuged to obtain upconversion nanoparticles with a core-shell structure.

7. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S3 is as follows: The upconversion nanoparticles with a core-shell structure prepared in step S2 were added to a mixed solution of chloroform and toluene, followed by the addition of an aqueous solution of polyacrylic acid and the mixture was sealed and stirred vigorously. After the reaction was completed, the nanoparticles were washed with ethanol and ultrapure water and centrifuged to obtain hydrophilic polyacrylic acid-modified upconversion nanoparticles with a core-shell structure.

8. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S4 is as follows: The polyacrylic acid-modified upconversion nanoparticles with a core-shell structure prepared in step S3 were added to a morpholine ethanesulfonic acid buffer solution containing carbodiimide and N-hydroxythiosuccinimide for the first incubation. After the incubation, the activated polyacrylic acid-modified upconversion nanoparticles with a core-shell structure were obtained by centrifugation and dispersed in phosphate buffer. Streptavidin solution was added to the solution for a second incubation. After incubation, streptavidin-modified upconversion nanoparticles with a core-shell structure were obtained by centrifugation and dispersed in phosphate buffer. Then, 5'-terminal biotin-modified endocrine disruptor EDC aptamers were added for a third incubation. After incubation, the modified aptamers were centrifuged to obtain core-shell structured upconversion nanoparticles and redispersed in phosphate buffer. Then, bovine serum albumin solution was added for a fourth incubation. After incubation, the nanoparticles were centrifuged and washed with phosphate buffer to prepare biomolecularly functionalized core-shell structured upconversion nanoparticles.

9. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S5 is as follows: Ferric chloride hexahydrate, trisodium citrate dihydrate, and sodium acetate were added to an ethylene glycol solution, stirred vigorously until fully dissolved, and then transferred to a reaction vessel for high-temperature reaction. After the reaction was completed, the prepared magnetic nanoparticles were collected by a magnetic field and washed with ethanol and ultrapure water.

10. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S6 is as follows: The magnetic nanoparticles prepared in step S5 were added to a morpholine ethanesulfonic acid buffer solution containing carbodiimide and N-hydroxythiosuccinimide for the first incubation. After incubation, the activated magnetic nanoparticles were obtained by magnetic separation and dispersed in phosphate buffer. Streptavidin solution was added to the solution for the second incubation. After incubation, the streptavidin-modified magnetic nanoparticles were obtained by magnetic separation and dispersed in phosphate buffer. Subsequently, the 5' end-biotin-modified aptamer complementary sequence of endocrine disruptors (EDCs) was added for the third incubation. After incubation, the modified aptamer magnetic nanoparticles were obtained by magnetic separation and redispersed in phosphate buffer. Subsequently, bovine serum albumin solution was added for the fourth incubation. After incubation, the nanoparticles were magnetically separated and washed with phosphate buffer to prepare biomolecularly functionalized magnetic nanoparticles.

11. The method for preparing the upconversion luminescence biosensor according to claim 4, characterized in that: The process of step S7 is as follows: The endocrine disruptor EDCs aptamer modified biomolecule-functionalized upconversion nanoparticles with a core-shell structure prepared in step S4 and the endocrine disruptor EDCs aptamer-complementary sequence modified biomolecule-functionalized magnetic nanoparticles prepared in step S6 were added to phosphate buffer and heated at high temperature. After heating, the nanoparticles were slowly annealed and transferred to a shaker for incubation. After incubation, the upconversion luminescent biosensor was obtained by magnetic separation and washed three times with phosphate buffer. Finally, the nanoparticles were dispersed in phosphate buffer.

12. A method of using the microfluidic biosensing platform based on upconversion luminescence as described in any one of claims 1-3, characterized in that, Specifically, the following steps are included: A series of endocrine disruptor (EDC) standard solutions with concentrations ranging from 0 to 250 ng / mL were taken and co-injected into the microfluidic chip with the prepared upconversion luminescent biosensor to complete the mixing, reaction, separation and detection steps of the biosensor and the target endocrine disruptor (EDC). The particles were allowed to stand to complete the bridging flocculation and sedimentation of the detached core-shell upconversion nanoparticles (CSUCNPs). Fluorescence spectra of the interfaces of the settled core-shell upconversion nanoparticles (CSUCNPs) in the detection cell were collected using a fluorescence spectrometer. A standard curve for detecting endocrine disruptor (EDC) content was established by linearly fitting the concentration logarithmic value of the standard solution with the fluorescence signal characteristic value. The fluorescence signal characteristic value is the characteristic fluorescence emission intensity of rare earth elements used in biosensors that specifically identify endocrine disruptor EDCs. Take the sample solution to be tested, replace the above-mentioned endocrine disruptor EDCs standard solution, and inject it together with the biosensor into the microfluidic chip. Substitute the collected fluorescence signal characteristic values ​​into the standard curve to calculate the content of endocrine disruptor EDCs in the sample to be tested.

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