CN-116429745-B - Microfluidic biosensing platform based on up-conversion luminescence

CN116429745BCN 116429745 BCN116429745 BCN 116429745BCN-116429745-B

Abstract

The invention relates to a micro-fluid biological sensing platform based on up-conversion luminescence, which comprises an up-conversion luminescence biological sensor, a micro-fluid chip, a detection pond, an arc-shaped channel, a separation channel and a detection pond, wherein the up-conversion luminescence biological sensor is used for specifically identifying EDCs, the micro-fluid chip is used for integrating the up-conversion luminescence biological sensor with the mixture, reaction, separation and detection of a sample to be detected, the micro-fluid chip comprises the sample introduction pond, the sample introduction pond is used for the up-conversion luminescence biological sensor and the sample to be detected, the feeding inlet of the arc-shaped channel is simultaneously communicated with the sample introduction pond of the up-conversion luminescence biological sensor and the sample to be detected, the arc-shaped channel is used for mixing and reaction of the up-conversion luminescence biological sensor and the sample to be detected after the up-conversion luminescence biological sensor and the sample to be detected enter the arc-shaped channel, the separation channel is communicated with the discharge outlet of the arc-shaped channel, and the detection pond is communicated with the discharge outlet of the separation channel and is used for completing luminescence enhancement quantitative detection of EDCs.

Inventors

CHEN QUANSHENG
WU JIZHONG
OUYANG QIN
WEI WENYA
ZHAO SONGGUANG
Zhu Afang
WANG ZHEN

Assignees

集美大学
江苏大学

Dates

Publication Date: 20260505
Application Date: 20230524

Claims (12)

1. The microfluidic biosensing platform based on up-conversion luminescence is characterized by comprising an up-conversion luminescence biosensor and a microfluidic chip, wherein the up-conversion luminescence biosensor is used for specifically identifying Endocrine Disruptors (EDCs), and the microfluidic chip is used as a reaction platform of the up-conversion luminescence biosensor and a sample to be detected and is used for integrating mixing, reaction, separation and detection of the up-conversion luminescence biosensor and the sample to be detected; The up-conversion luminescence biosensor comprises a combined up-conversion nanoparticle with a core-shell structure and a magnetic nanoparticle, wherein the surface of the up-conversion nanoparticle with the core-shell structure is modified with an aptamer sequence; The microfluidic chip comprises: the sample injection pool is used for injecting samples into the up-conversion luminescence biosensor and the sample to be detected; The feeding port of the arc-shaped channel is simultaneously communicated with the sample injection pool of the up-conversion luminescence biosensor and the sample injection pool of the sample to be tested, and the arc-shaped channel is used for mixing and reacting the up-conversion luminescence biosensor and the sample to be tested after the up-conversion luminescence biosensor and the sample to be tested enter the arc-shaped channel; the separation channel is communicated with the discharge port of the arc-shaped channel and is used for removing the up-conversion luminescence biosensor which does not react with the EDCs target through magnetic separation after the reaction is finished; And the detection pool is communicated with the discharge port of the separation channel and is used for receiving the dropped up-conversion nano particles with the core-shell structure, so that the dropped up-conversion nano particles with the core-shell structure complete aptamer-mediated bridging flocculation and sedimentation to form an interface, and the up-conversion fluorescent signals are collected to complete the luminescence enhancement quantitative detection of EDCs.
2. The up-conversion luminescence based micro-fluidic biosensing platform of claim 1, wherein when the endocrine disruptors EDCs are bisphenol A, the sequence of the aptamer is shown as SEQ ID NO. 1, and the complementary sequence of the aptamer is shown as SEQ ID NO. 2.
3. The up-conversion luminescence based micro-fluidic biosensing platform of claim 1, wherein when the endocrine disruptor EDCs is diethylstilbestrol, the sequence of an aptamer is shown as SEQ ID NO. 3, and the complementary sequence of the aptamer is shown as SEQ ID NO. 4.
4. A method of manufacturing an up-conversion luminescence biosensor according to any of claims 1-3, wherein the method of manufacturing an up-conversion luminescence biosensor comprises the steps of: s1, preparing up-conversion nanoparticle seeds containing rare earth elements; S2, coating the outer layer of the rare earth element-containing up-conversion nanoparticle seed prepared in the step S1 to prepare up-conversion nanoparticle with a core-shell structure; s3, modifying the up-conversion nano particles with the core-shell structure prepared in the step S2 to be hydrophilic; S4, performing biomolecular functionalization on the hydrophilic up-conversion nanoparticle with the core-shell structure obtained in the step S3 to obtain the biomolecular functionalized up-conversion nanoparticle with the core-shell structure; s5, preparing magnetic nano particles; S6, performing biomolecular functionalization on the magnetic nanoparticles obtained in the step S5 to obtain biomolecular functionalized magnetic nanoparticles; S7, combining the up-conversion nano particles with the core-shell structure and the magnetic nano particles to obtain the up-conversion luminescence biosensor.
5. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the step S1 is performed by, Respectively dissolving yttrium chloride hexahydrate, ytterbium chloride hexahydrate and rare earth element hexahydrate in methanol, adding oleic acid and 1-octadecene, heating to 150-170 ℃ for reaction for 25-35min after mixing, cooling after the reaction is finished, dropwise adding a mixed solution of sodium hydroxide and ammonium fluoride, reacting for 25-35min at 125-135 ℃, then heating to 290-310 ℃ and keeping for 50-60min, adding ethanol and ultrapure water after the reaction is finished, and centrifugally separating to obtain up-conversion 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 an up-conversion luminescence biosensor according to claim 4, wherein the step S2 is performed by, Dissolving yttrium chloride hexahydrate in methanol, adding oleic acid and 1-octadecene, mixing, heating to 150-170 ℃ for reaction for 25-35min, cooling after the reaction, adding the up-conversion nanoparticle seeds prepared in the step S1, dropwise adding a mixed solution of sodium hydroxide and ammonium fluoride, reacting for 25-35min at 125-135 ℃, heating to 290-310 ℃ and keeping for 20-40min, adding ethanol and ultrapure water after the reaction is finished, and centrifugally separating to obtain the up-conversion nanoparticle with a core-shell structure.
7. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the step S3 is performed by, And (2) adding the up-conversion nano particles with the core-shell structure prepared in the step (S2) into a mixed solution of chloroform and toluene, then adding a polyacrylic acid aqueous solution, sealing, vigorously stirring for reaction, and washing and centrifuging by using ethanol and ultrapure water after the reaction is finished to obtain the hydrophilic polyacrylic acid modified up-conversion nano particles with the core-shell structure.
8. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the step S4 is performed by, Adding the polyacrylic acid modified upconversion nano particles with a core-shell structure obtained in the step S3 into morpholine ethanesulfonic acid buffer solution containing carbodiimide and N-hydroxy sulfosuccinimide for first incubation, centrifugally separating to obtain activated polyacrylic acid modified upconversion nano particles with a core-shell structure after incubation is finished and dispersing the activated polyacrylic acid modified upconversion nano particles in phosphate buffer solution, adding streptavidin solution into the solution for second incubation, centrifugally separating to obtain modified streptavidin upconversion nano particles with a core-shell structure after incubation is finished and dispersing the modified streptavidin upconversion nano particles in phosphate buffer solution, then adding endocrine disruptor EDCs aptamer with 5' -end modified biotin for third incubation, centrifugally separating to obtain modified aptamer upconversion nano particles with a core-shell structure after incubation is finished and redispersing the modified aptamer in phosphate buffer solution, then adding bovine serum albumin solution for fourth incubation, centrifugally separating after incubation and washing by using phosphate buffer solution to obtain biomolecule functionalized upconversion nano particles with a core-shell structure.
9. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the process of step S5 is, Respectively adding ferric chloride hexahydrate, trisodium citrate dihydrate and sodium acetate into glycol solution, stirring vigorously to dissolve, transferring into a reaction kettle, reacting at high temperature, collecting the prepared magnetic nano particles by a magnetic field after the reaction is finished, and cleaning by using ethanol and ultrapure water.
10. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the process of step S6 is, Adding the magnetic nanoparticles prepared in the step S5 into morpholine ethane sulfonic acid buffer solution containing carbodiimide and N-hydroxy sulfosuccinimide for first incubation, magnetically separating to obtain activated magnetic nanoparticles after incubation is finished and dispersing the activated magnetic nanoparticles in phosphate buffer solution, adding streptavidin solution into the solution for second incubation, magnetically separating to obtain magnetic nanoparticles modified with streptavidin after incubation is finished and dispersing the magnetic nanoparticles in the phosphate buffer solution, then adding endocrine disruptor EDCs aptamer complementary sequence of 5' -end modified biotin for third incubation, magnetically separating to obtain magnetic nanoparticles modified with aptamer after incubation is finished and dispersing the magnetic nanoparticles in phosphate buffer solution again, then adding bovine serum albumin solution for fourth incubation, magnetically separating after incubation is finished and washing the magnetic nanoparticles with phosphate buffer solution to prepare the magnetic nanoparticles functionalized with biomolecules.
11. The method for preparing an up-conversion luminescence biosensor according to claim 4, wherein the step S7 is performed by, Adding the endocrinopathy EDCs aptamer modified biomolecule functionalized upconversion nano particles with core-shell structures prepared in the step S4 and the endocrinopathy EDCs aptamer complementary sequence modified biomolecule functionalized magnetic nano particles prepared in the step S6 into a phosphate buffer solution, heating at high temperature, slowly annealing after heating, transferring to a shaking table for incubation, magnetically separating to obtain the upconversion luminescence biosensor after incubation, cleaning three times by using the phosphate buffer solution, and finally dispersing in the phosphate buffer solution.
12. A method of using the up-conversion luminescence based micro-fluidic biosensing platform according to any of claims 1-3, comprising the steps of: Taking 0-250ng/mL of a series of endocrine disruptor EDCs standard solutions, and injecting the standard solutions and the prepared up-conversion luminescence biosensor into the microfluidic chip together to complete the steps of mixing, reacting, separating and detecting the biosensor and the target endocrine disruptor EDCs; Standing to complete bridging flocculation and sedimentation of the fallen up-conversion nano particles CSUCNPs with the core-shell structure, collecting fluorescence spectra of the interface of the magnetic nano particles CSUCNPs with the core-shell structure formed by sedimentation in a detection pool through a fluorescence spectrometer, and linearly fitting the concentration logarithmic value of an endocrine disruptor EDCs standard solution and a fluorescence signal characteristic value to establish a standard curve for detecting the content of the endocrine disruptor EDCs, wherein the fluorescence signal characteristic value is characteristic fluorescence emission intensity of rare earth elements used by a biosensor for specifically identifying the endocrine disruptor EDCs; Taking a sample solution to be detected, replacing the endocrine disruptor EDCs standard solution, injecting the sample solution and the biosensor into a microfluidic chip together, substituting the acquired fluorescent signal characteristic value into a standard curve for calculation, and obtaining the content of the endocrine disruptor EDCs in the sample to be detected.

Description

Microfluidic biosensing platform based on up-conversion luminescence Technical Field The invention relates to the technical field of food safety detection, in particular to a micro-fluid biological sensing platform based on up-conversion luminescence. Background Endocrine Disrupting Chemicals (EDCs) are defined as "exogenous agents that disrupt the synthesis, secretion, transport, metabolism, binding or elimination of natural blood-borne sex hormones present inside the human body responsible for the processes of balance, reproduction and development. Humans may ingest hundreds of EDCs through food production (food additives, pesticides, food containers), industrial activities (air pollution, water pollutants, industrial chemicals), medical (medical products), etc. Among them, exogenous agents having estrogenic effects, such as bisphenol a (BPA), diethylstilbestrol (DES), estradiol (E2), nonylphenol, etc., have attracted much attention, and such EDCs may cause serious health hazards including neurodevelopmental disorders, brain, liver and lung injuries, reproduction and endocrine disorders, metabolic disorders, etc. Therefore, it is important to establish an effective assessment method to determine the level of EDCs in a human likely contact medium. In the prior researches, the measurement of the content of the EDCs is based on methods such as high performance liquid chromatography, gas chromatography, electrochemical sensors, photoelectrochemical immunosensors and the like, but the measurement methods often face the difficulties of expensive detection instruments and equipment, strong background interference, complicated sample pretreatment steps, large reagent consumption and the like, and the on-site rapid quantitative detection of the EDCs is difficult to realize. Therefore, it is important to develop a novel detection platform to overcome the above-mentioned drawbacks. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a micro-fluid biological sensing platform based on up-conversion luminescence, which is used for realizing micro-sampling and simple and convenient high-sensitivity quantitative detection of EDCs. Based on the above, the invention aims to provide a micro-fluid biological sensing platform based on up-conversion luminescence, which comprises an up-conversion luminescence biological sensor, a micro-fluid chip and a micro-fluid sample detection platform, wherein the up-conversion luminescence biological sensor is used for specifically identifying EDCs; The microfluidic chip comprises a sample injection pool, an arc-shaped channel, a separation channel, a detection pool and an EDCs (electronic device manufacturing) detection device, wherein the sample injection pool is used for injecting samples of the up-conversion luminescence biosensor and a sample to be detected, the feed inlet of the arc-shaped channel is simultaneously communicated with the sample injection pool of the up-conversion luminescence biosensor and the sample injection pool of the sample to be detected, the arc-shaped channel is used for mixing and reacting the up-conversion luminescence biosensor and the sample to be detected after the up-conversion luminescence biosensor and the sample to be detected enter the arc-shaped channel, the separation channel is communicated with the discharge outlet of the arc-shaped channel and is used for magnetically separating the up-conversion luminescence biosensor after the reaction is finished, and the detection pool is communicated with the discharge outlet of the separation channel and is used for luminescence enhancement and quantitative detection. Further, the width of all the micro-channels of the micro-fluidic chip isDepth is all。 According to the technical scheme, the up-conversion luminescence biosensor and the sample to be detected are integrated in a microfluidic chip through mixing, reaction, separation and detection, and the method comprises the specific processes of injecting the up-conversion luminescence biosensor and the sample to be detected into two sample injection pools of the microfluidic chip respectively, fully mixing and reacting the two microfluids in an arc-shaped channel, then adding a magnetic field at a separation channel to separate the biosensor which does not react with the EDCs, and finally completing bridging flocculation and sedimentation in a detection pool through falling CSUCNPs, and completing collection of up-conversion fluorescence signals, so that the EDCs can be quantitatively detected. Further, the injection flow rate of the biosensor and the sample solution to be tested isAnd。 Further, the injection time of the biosensor and the sample solution to be measured is 8-12min. The second objective of the present invention is to provide a method for preparing a microfluidic biosensing platform based on up-conversion luminescence. The preparation method of the up-conversion luminescence biosensor comprises the followi