CN114713300A - Dithiothreitol detection method based on single particle up-conversion luminescence - Google Patents
Dithiothreitol detection method based on single particle up-conversion luminescence Download PDFInfo
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- CN114713300A CN114713300A CN202210181149.9A CN202210181149A CN114713300A CN 114713300 A CN114713300 A CN 114713300A CN 202210181149 A CN202210181149 A CN 202210181149A CN 114713300 A CN114713300 A CN 114713300A
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 55
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- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 description 1
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Abstract
The invention belongs to a microfluidic sensing technology, and particularly relates to a dithiothreitol detection method based on single particle up-conversion luminescence. The invention utilizes the photonic crystal effect and the local surface plasma resonance effect to cooperatively regulate and control the enhancement of the light-induced upconversion luminous intensity of single upconversion nano particles through MnO2The fluorescence intensity of the up-conversion nanoparticles is greatly reduced by wrapping, and when the up-conversion nanoparticles are used for in-vitro detection of dithiothreitol, the up-conversion luminescence is gradually recovered by gradually increasing dithiothreitol with the help of an atomic force microscope and an in-situ fluorescence lifetime imaging system, so that the high-sensitivity and high-selectivity in-vitro detection of dithiothreitol is realized. The invention provides a competition strategy for biomedical sensing, is suitable for detecting dithiothreitol, and has high universality for other detections.
Description
Technical Field
The invention belongs to a microfluidic sensing technology, and particularly relates to a dithiothreitol detection method based on single particle up-conversion luminescence.
Background
With the rapid development of single molecule/single particle measurement technology, single particle sensing technology provides a unique tool for detecting individual behaviors and processes, and has attracted wide interest in the detection fields of DNA, protein, gas and the like. The single particle sensing can realize ultrahigh sensitivity and extremely low detection limit by improving the contact surface with a target object and eliminating the phenomena of statistical effect, reabsorption, non-uniform broadening and the like of the aggregate nanoparticles. However, the method has the limitations of difficult detection of single luminescent particles, uneven shift and deformation of the spectrum, and flicker of the traditional fluorescent probe. In order to overcome these problems, it is urgently needed to develop a single particle sensor which has the advantages of stable brightness, high luminous intensity and the like, and can realize ultrahigh sensitivity and extremely low detection limit.
Lanthanide ion-doped upconversion nanoparticles (UCNPs) can absorb two or more low-energy photons and convert them to one high-energy photon. The large Stokes displacement and near infrared light are used as pumping sources, the lanthanide ion doped up-conversion nanoparticles have the advantages of no photobleaching and background fluorescence, are widely researched in aspects of biological imaging, sensing and the like, and are widely explored in aspects of detecting disease biomarkers, nerve agents and the like in vitro as fluorescent probes. With the development of preparation technology, UCNPs having a high uniformity in size and morphology are currently available. Meanwhile, up-conversion emission comes from 4f-4f orbital transitions, uneven spectral shifts and distortions and flickering of UCNPs rarely occur. Therefore, UCNPs are considered as the most promising single particle sensing luminescence probe. However, current UCNPs-based sensing exists primarily in solution, powder, or film form. Previous studies have shown that the emission phenomenon of UCNPs in ensemble spectroscopy and single particle spectroscopy measurements is significantly different. And the sensitivity of integrated nanoparticle-based sensors is susceptible to interference from statistical effects, which necessarily results in low sensitivity and high detection limits of the device. For the current single particle sensing system, the main limitation is that the fluorescence signal of a single upconversion nanoparticle is weak due to the nature of the UCNPs luminous efficiency, and a higher pumping threshold is required. It remains a challenge to be able to achieve fluorescence sensing based on single up-converting nanoparticles.
Dithiothreitol (DTT), as a small molecule, can completely reduce disulfides, protect sulfhydryls from oxidation, and play an important role in biology, biochemistry, and biomedicine [ Dunaway-Mariano, d.; holden, h.m.; raushel, F.M.W.W. "Mo" Cleland: A Catalytic Life. biochemistry2013,52, 9092-. However, excess DTT is toxic and causes irreversible oxidative damage to certain biomolecules [ Charrier, j.g.; (DTT) as a Measure of Oxidative Positive for organic Compounds: evaluation for the evaluation of solvent transfer metals, analytical chem.and Phys.2012,12, (19), 9321-. Therefore, it is necessary to develop an efficient and accurate method for detecting DTT in real time. Therefore, various different analytical methods have been used to achieve the selective detection of dithiothreitol, and the following are arranged:
(I) electrophoresis-electrochemical method: in 1993, Lunte et al at Kansas university in USA uses gold/mercury amalgam as a microelectrode and realizes the detection of free mercaptan (glutathione) by a capillary electrophoresis-electrochemical method, wherein the detection limit is as low as 0.53 fM. Although this method has a very low detection limit, the detection device is complicated in preparation process and can be used for further detection experiments after being stabilized in the environment for 12 hours after the preparation is completed [ Thomas J.O' Shea, Susan M. Lunte. Selective detection of free thiols by capillary electrophoresis-electrochemical use a gold/polymeric analyte. chemical. chem.1993,65, 247-;
(II) ratio fluorescence probe method: in 2010, Tan et al achieved highly selective detection of DTT using ratiometric fluorescent probes, with a detection limit of 5.0 mM. Such ratiometric fluorescent probes, while capable of reducing detection time, have a high detection limit and lack design strategies to achieve effective changes in fluorescence. Meanwhile, how to make the difference of the emission peak before and after the response as large as possible and reduce the overlapping between the emission spectrums so as to improveThe sensitivity of the probe response is also another disadvantage [ Baocun Zhu, Xiaolingg Zhuang, Hongying Jia, Yamin Li, Haipeng Liu, Weihong Tan. A high selectivity ratio metric fluorescent probe for 1,4-Dithio (DTT) detection. org. biomol. chem.2010,8,1650-](ii) a Luxiaquan et al discloses a ratiometric fluorescent probe for detecting 1,4-dithiothreitol, which is based on a metal organic framework, and Cu-TCPP and ZrCl are firstly required4Dissolving toluic acid in DMF by ultrasonic wave, reacting for 45-50h at 100-120 ℃, and then obtaining the ratiometric fluorescent probe PTA-NH based on the metal organic framework through a series of operation processes2@ PCN-224(Cu) [ Luxiaoquan, Wangni, Nyanqing, Jiaxiamao, Liujuan, Yangshuang, Jieyangqi, Bailei, Hanzhenggang]. As can be seen from the preparation process, the method is extremely tedious and complex, and is not suitable for rapidly detecting DTT;
(III) intensity variation type fluorescence probe method: the invention discloses a dithiothreitol fluorescent probe based on xanthene structure, which has the advantages of low price, easy obtaining, simple use, low detection sensitivity and obvious limitations in quantitative detection of object species [ Linweiying, Sinkiang, Dongbao, Chongqi, Wangsuper, Song dynasty ] and a preparation method and application thereof, namely, the invention patent CN 108003866B ]; in 2018, Sun et al prepared a 6- (methylsulfinyl) -2-phenyl-1H-benzo [ de ] iso-quinoline-1,3(2H) -dione (NC-DTT) fluorescent probe for detecting DTT, which has a low detection limit (140nM), and belongs to a fluorescence-enhanced probe. The enhanced fluorescent probe has no fluorescence or only emits weak fluorescence, and the signal change of the enhanced fluorescence is easier to effectively detect, so the sensitivity of the probe is higher. However, NC-DTT fluorescent probes employed by Sun et al exhibit poor specific detection of DTT [ Tong Sun, Lili Xia, Jinxin Huang, Yueqing Gu, Peng Wang. A high selective fluorescent probe for fast recognition of DTT and its application in one-and two-photon imaging. Talanta.2018,187,295-301 ].
(IV) electrochemical method: the invention discloses a method for detecting the concentration of dithiothreitol in a solution by using an electrochemical method, wherein DTT is detected by using an aminated graphene quantum dot modified electrode, the detection process is simple and convenient, but the detection sensitivity and the detection limit are low [ Shagchen, Huangshan, Von Germing, Wuzi Hua.
(V) colorimetric type fluorescent probe: zhao et al use two near-end thiol based on DTT to crosslink with monodisperse silver nanoparticles to form aggregated silver nanoparticles with larger particle size, and make their color and absorbance change correspondingly, to prepare a colorimetric fluorescent probe. The probe has the advantages of simple preparation method and low cost. However, the visual colorimetry has subjective errors, the measurement accuracy is difficult to grasp, the sensitivity is low, and the 20 th century 30-60 th century is a vigorous development period of the colorimetry, and then the colorimetry is gradually replaced by other fluorescent probes due to the limitation on detection.
Disclosure of Invention
In order to solve the problems, the invention provides a microfluidic sensing technology, which utilizes the photonic crystal effect and the local surface plasmon resonance effect to cooperatively regulate and control the photoinduced upconversion luminous intensity enhancement of single upconversion nanoparticles, and MnO is adopted2The fluorescence intensity of the up-conversion nanoparticles is greatly reduced by wrapping, and when the up-conversion nanoparticles are used for in-vitro detection of dithiothreitol, the up-conversion luminescence is gradually recovered by gradually increasing dithiothreitol with the help of an atomic force microscope and an in-situ fluorescence lifetime imaging system, so that the high-sensitivity and high-selectivity in-vitro detection of dithiothreitol is realized. The invention provides a competition strategy for biomedical sensing, is suitable for detecting dithiothreitol, and has high universality for other detections.
In order to achieve the purpose, the invention adopts the following technical scheme:
a dithiothreitol detection method based on single particle up-conversion luminescence comprises the following specific steps:
step 1: with methyl methacrylateEsters (MMA), styrene or Silica (SiO)2) The opal photonic crystal template A is prepared by a three-dimensional vertical self-assembly method as a microsphere for manufacturing photonic crystals.
Washing substance M with strongly corrosive strong alkaline solution sodium hydroxide solution to remove polymerization inhibitor, wherein substance M is MMA, styrene or SiO2Suspension, the concentration of the sodium hydroxide solution is 10-100mg/mL, and the volume ratio of the sodium hydroxide solution to the substance M is (40-90): 1; SiO 22Preparation of a suspension from SiO with a size of 200-600nm2Dissolving the microsphere particles in ethanol to obtain SiO with the volume fraction of 0.3-2%2A suspension;
then, mixing the cleaned substance M, deionized water and an initiator according to a volume ratio of (1-10): (20-80): 1, wherein the initiator plays a role in redox polymerization of the emulsion; slowly stirring the mixed solution at 90-220 deg.C for 1-4h to obtain polymethyl methacrylate (PMMA) microsphere solution, Polystyrene (PS) microsphere solution or silicon dioxide (SiO)2) The microsphere solution is referred to as A-microsphere solution.
The up-conversion nano-particles (which can be NaYF) related to the invention4:10-60％Yb3+,1-10％Er3+，KYF4:10-60％Yb3+,1-10％Er3+，LiYF4:10-60％Yb3+,1-10％Er3+One of them) has excitation light of 980nm, so the position of the photon forbidden band of the OPCs should match the excitation light wavelength. Therefore, the particle diameter of the microsphere for preparing the photonic crystal template is adjusted by using a coating method to obtain the opal photonic crystal with the photon forbidden band position near 400-1600 nm. And during coating, mixing the obtained A-microsphere solution with deionized water and a substance M, wherein the volume ratio of the A-microsphere solution to the deionized water to the substance M is (3-60): (3-60): 1, slowly stirring for 0.5-2h at the temperature of 90-220 ℃, and then obtaining the coated A-microsphere solution.
Diluting the wrapped A-microsphere solution with deionized water, wherein the volume ratio of the A-microsphere solution to the deionized water is 1: (20-80), vertically inserting the slide glass into the beaker, and transferring the whole beakerStanding in an oven at 25-70 deg.C for 24-144h to obtain opal photonic crystal template A, which is polymethyl methacrylate (PMMA) opal photonic crystal template, Polystyrene (PS) opal photonic crystal template and silicon dioxide (SiO)2) An opal photonic crystal template.
The initiator is potassium persulfate, sodium persulfate, aluminum persulfate, ammonium persulfate or sodium bisulfite.
Step 2: preparing a semiconductor plasma solution B containing cesium tungstate (Cs) having a localized surface plasmon resonance effectxWO3) Semiconductor plasma solution, tungsten oxide (WO)3) Semiconductor plasma solution or cuprous sulfide (Cu)2S) semiconductor plasma solution, which comprises the following steps:
CsxWO3the preparation method of the semiconductor plasma solution comprises the following steps: cesium chloride, tungsten chloride, oleic acid and oleylamine were mixed in a molar ratio (22-45): (1-3): (1-3): 1, fully stirring at the temperature of 300-660 ℃ until the mixture turns dark blue, and cooling the mixture to room temperature to obtain a cesium tungstate solution; adding toluene into the cesium tungstate solution, and carrying out centrifugal treatment for three times; during the first centrifugal treatment, the volume ratio of the cesium tungstate solution to the toluene is 1: (3-10); and (3) dissolving the centrifuged product in acetone, fully dissolving, adding toluene again for second centrifugation treatment, wherein the volume ratio of the cesium tungstate solution to the acetone to the toluene is 1: (3-10): (3-10); and (3) dissolving the product after the second centrifugal treatment in acetone again, adding toluene after full dissolution, and performing third centrifugal treatment, wherein the volume ratio of the cesium tungstate solution to the acetone to the toluene is 1: (3-10): (3-10); finally, the obtained cesium tungstate precipitate is dispersed in toluene to obtain cesium tungstate (Cs)xWO3) A semiconductor plasma solution.
WO3The preparation method of the semiconductor plasma solution comprises the following steps: sodium tungstate, malonic acid and deionized water are mixed according to a molar ratio (10-50): (22.5-66.8): 1 for 0.5 to 6.5 hours to obtain a mixed solution; then adding nitric acid into the mixed solution, wherein the volume ratio of the mixed solution to the nitric acid is (2.5-62.8): 1, stir again 0.5-6.5h until a yellow suspension is formed. Pouring the yellow suspension into a reaction kettle, sealing and placing the reaction kettle in a drying box, setting the temperature to be 100-900 ℃, setting the reaction time to be 5-48h, and naturally cooling the reaction kettle to room temperature after the reaction is finished. Washing the dried mixed solution with deionized water and ethanol for 3 times to remove soluble impurities in the mixed solution, wherein the volume ratio of the mixed solution to the deionized water to the ethanol is 1: (1-10): (1-10), and dispersing the tungsten oxide precipitate obtained finally in ethanol to obtain tungsten oxide (WO)3) A semiconductor plasma solution.
Cu2The preparation method of the S semiconductor plasma solution comprises the following steps: preparing a copper precursor solution, namely mixing copper chloride, oleic acid and oleylamine according to a molar ratio of 1: (2.5-78.6): (1.8-66.8) and stirring for 0.5-5.5h under the protection of nitrogen. Dissolving sulfur powder in octadecene to prepare a sulfur precursor solution, wherein the molar ratio of the sulfur powder to the octadecene is 1: (2.5-78.9). Heating the sulfur precursor solution to 20-300 ℃, and then quickly injecting the copper precursor solution, wherein the volume ratio of the copper precursor solution to the sulfur precursor solution is (1-10): 1, stirring for 5-100min, stopping heating and cooling to room temperature. And washing the obtained mixed solution for 3 times by using acetone and toluene, wherein the volume ratio of the mixed solution to the acetone to the toluene is 1: (1-10): (1-10), dispersing the finally obtained cuprous sulfide precipitate in toluene to obtain cuprous sulfide (Cu)2S) a semiconductor plasma solution.
And step 3: synthesizing up-conversion nanoparticles by a solvothermal method, wherein the up-conversion nanoparticles are NaYF4:10-60％Yb3 +,1-10％Er3+，KYF4:10-60％Yb3+,1-10％Er3+，LiYF4:10-60％Yb3+,1-10％Er3+Is one of (1), denoted as CYF4:10-60％Yb3+,1-10％Er3+C ═ Na or K or Li; coating MnO on the surface of the nano-particles after the surface is processed without ligand2The film comprises the following specific steps:
cleaning of CYF Using hydrochloric acid solution4:10-60％Yb3+,1-10％Er3+Surface ligands on nanoparticles, CYF4:10-60％Yb3+,1-10％Er3+The volume ratio of the nanoparticles to the hydrochloric acid is 1: (0.8-4.8), and fully dissolving; and centrifuging the obtained mixed solution twice by using ethanol, wherein the volume ratio of the mixed solution to the ethanol is 1: (3-10); the product after centrifugation is CYF without ligand4:10-60％Yb3+,1-10％Er3+Nanoparticles and dissolved in deionized water, ligand-free NaYF4:Yb3+,Er3+The volume ratio of the nano particles to the deionized water is (0.3-2): 1, obtaining ligand-free CYF4:10-60％Yb3+,1-10％Er3+And (3) solution.
Will be ligand-free CYF4:10-60％Yb3+,1-10％Er3+The nano-particle solution is uniformly dispersed in the mixed solution of potassium permanganate and hydrochloric acid, wherein CYF4:10-60％Yb3+,1-10％Er3+The volume ratio of the solution to the potassium permanganate to the hydrochloric acid is (1-3): (1-4): 1, standing at room temperature for 12-96h, wherein the color of the mixture gradually changes from dark purple to red black to obtain CYF4:10-60％Yb3+,1-10％Er3+@MnO2The solution was centrifuged twice using deionized water, wherein CYF4:10-60％Yb3+,1-10％Er3+@MnO2The volume ratio of the solution to the deionized water is 1 (3-10); finally, the resulting product is diluted in deionized water, in which CYF4:10-60％Yb3+,1-10％Er3+@MnO2The volume ratio of the solution to the deionized water is 1: (300-1000) to obtain diluted CYF4:10-60％Yb3+,1-10％Er3+@MnO2And (3) solution. The quenching of the upconversion fluorescence occurs upon irradiation of the nanoparticles with a 980nm laser, indicating that CYF is obtained4:10-60％Yb3+,1-10％Er3+(C-Na or K or Li) @ MnO2And (3) solution.
And 4, step 4: preparation of microfluidic single particle sensor
Dripping any one semiconductor plasma solution B obtained in the step 2 along one side of any one opal photonic crystal template A obtained in the step 1 to prepare an A/B composite structure; diluting CYF obtained in step 34:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2Dropping the solution on the A/B composite structure to obtain A/B/CYF4:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2And (5) compounding the structure to obtain the microfluidic single particle sensor.
And 5: dithiothreitol detection
Tracking the microfluidic single particle sensor A/B/CYF prepared in the step 4 by using an atomic force microscope and an in-situ fluorescence lifetime imaging system4:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2The position of a single up-conversion nanoparticle is recorded, and the fluorescence spectrum and the service life information of the single up-conversion nanoparticle are recorded; injecting Dithiothreitol (DTT) to the microfluidic single particle sensor prepared in the step 4 through a micro-injection pump, and recording the fluorescence spectrum and the service life information of the Dithiothreitol (DTT) again; adjusting the microinjection pump so that as the concentration of DTT increases, MnO2The fluorescence spectrum of the coated up-conversion nano material is changed, and the concentration of dithiothreitol can be obtained.
The detection principle is as follows: DTT molecular pair MnO2The dissociation of the nano material has higher reactivity, so the A/B/CYF is added with DTT molecules4:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2Mn of composite structure4+Reduction to Mn2+So that MnO is2And (3) gradually decomposing, and gradually recovering the quenched up-conversion fluorescence to realize the in vitro high-sensitivity DTT detection. Single CYF cooperatively regulated by photonic crystal effect and semiconductor plasma resonance effect4:10-60％Yb3+,1-10％Er3+The up-conversion fluorescence of (C ═ Na or K or Li) nanoparticles contributes to the improvement of the sensitivity of microfluidic single particle sensors.
The invention has the beneficial effects that:
(1) the single particle sensor is based on A/B/single CYF4:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2A single particle sensor of composite construction. Compared with a sensor based on integrated nano particles, the single particle sensor can eliminate the statistical average effect of the integrated nano particles and obtain more accurate detectionAnd (6) measuring information.
(2) The fluorescence intensity of the up-conversion nano particles is cooperatively regulated and controlled by the opal photonic crystal and the semiconductor plasma effect, so that the up-conversion fluorescence intensity is enhanced by 1600 times, and the pumping threshold of laser is reduced. The higher fluorescence intensity is more beneficial to obtaining single particle fluorescence signals, and the detection sensitivity is improved.
(3)MnO2Has strong absorption spectrum between 200 and 1000nm, thus being a good fluorescence quenching material. MnO2As an energy acceptor, the compound can effectively quench the upconversion fluorescence of UCNPs, and can be decomposed into Mn after undergoing redox reaction with DTT2+Ions. A/B/Single CYF of the invention4:10-60％Yb3+,1-10％Er3+(C ═ Na or K or Li) @ MnO2The sensor of (a) is of the intensity-change type rather than the ratiometric or colorimetric type. The response to the target detection substance DTT comes from the change of the up-conversion luminescence intensity, and has the advantages of simple and convenient operation, easy preparation and the like. The single particle sensor does not have fluorescence or has extremely weak fluorescence, the fluorescence is enhanced after the single particle sensor acts with a target detection object, the signal change after the fluorescence enhancement is easier to effectively detect, the weak fluorescence of the device can reduce background signals, and the sensitivity of the device is improved.
Drawings
FIG. 1 is a scanning electron micrograph of an opal photonic crystal of the present invention.
FIG. 2 shows a semiconductor plasma CsxWO3Transmission electron micrograph of solution.
FIG. 3 shows NaYF4:20％Yb3+,2％Er3+@MnO2Transmission electron microscopy of nanoparticles.
FIG. 4 shows NaYF4:20％Yb3+,2％Er3+@MnO2Nanoparticle X-ray diffraction pattern.
FIG. 5 is a diagram of a single upconversion nanoparticle NaYF4:20％Yb3+,2％Er3+And single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+@MnO2Schematic diagram of the emission spectrum of (1).
FIG. 6 is a diagram of a single upconversion nanoparticle NaYF4:20％Yb3+,2％Er3+And OPCs/CsxWO3Single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+Schematic diagram of the emission spectrum of (1).
Fig. 7 is a schematic diagram of a single particle sensor structure.
In FIG. 8, (a) is OPCs/CsxWO3Single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+The (b) is an in-situ fluorescence lifetime imaging graph corresponding to the position of a single upconversion nanoparticle in the (a).
FIG. 9 is a schematic diagram of the emission spectrum of a single particle sensor under different concentrations of dithiothreitol.
FIG. 10 shows single particle sensor OPCs/CsxWO3Single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+Response range plots for DTT detection.
FIG. 11 is a schematic representation of the effect of interfering substances on dithiothreitol assays.
Detailed Description
The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
Example 1:
(1) dissolving 0.1g of NaOH in 10ml of deionized water to prepare a NaOH solution, adding 2.5ml of MMA into the NaOH solution, and violently stirring the mixed solution to play a role in cleaning a polymerization inhibitor on the surface of the MMA; taking 3ml of the mixed solution, mixing the mixed solution with 40mol of deionized water and 18mg of potassium persulfate, slowly stirring for 1 hour at the temperature of 90 ℃, and obtaining PMMA microspheres after the reaction is finished;
(2) and (3) adjusting the diameter of the PMMA microspheres by a coating method, taking 20ml of the PMMA microspheres obtained in the step (1), 20ml of deionized water and 3ml of MMA, and slowly stirring for 0.5h at 90 ℃ to obtain the coated PMMA microspheres.
(3) And (3) diluting the 3ml of PMMA microspheres wrapped in the step (2) with 60ml of deionized water, vertically inserting a clean glass slide into the solution, integrally moving the glass slide into an oven at 25 ℃, standing for 24h, and then obtaining the opal photonic crystal, wherein the process is used for enhancing the stability of the three-dimensional opal structure. As shown in fig. 1, scanning electron microscopy images show that a three-dimensional opal template was successfully prepared.
(4) Adding 22mol of cesium chloride, 1mol of tungsten chloride, 1mol of oleic acid and 1mol of oleylamine into a three-necked flask, transferring the three-necked flask into a heating sleeve, fully stirring at the temperature of 300 ℃ until the mixture turns into dark blue, and cooling the mixture to room temperature to obtain a cesium tungstate solution.
(5) Carrying out first centrifugal treatment on the cesium tungstate solution obtained in the step (4), adding 3mol of toluene, using 9000 r/min, and centrifuging for 15 min; fully dissolving the centrifuged precipitate in 3mol of toluene, adding 3mol of acetone, and performing secondary centrifugation treatment at 9000 r/min for 15 min; the conditions of the third centrifugation were the same as those of the second centrifugation. As shown in fig. 2, transmission electron microscopy images show that cesium tungstate was successfully prepared.
(6) Synthesis of upconversion nanoparticles NaYF by classical solvothermal method4:20％Yb3+,2％Er3+. 0.24g of yttrium chloride hexahydrate powder, 0.08g of ytterbium chloride hexahydrate powder and 0.01g of erbium chloride hexahydrate powder are put into a three-necked bottle, 6ml of oleic acid and 15ml of octadecene solution are added into the three-necked bottle, the mixture is fixed in an oil bath kettle, the mixture is stirred at the speed of 700-950rpm under the protection of nitrogen at the temperature of 160 ℃ until the medicines in the three-necked bottle are completely dissolved, and then the mixture is cooled to 30 ℃.
(7) 0.1g of sodium hydroxide and 0.148g of ammonium fluoride powder are dissolved in 6ml of methanol solution. The mixed solution of methanol was slowly added to the mixed solution obtained in step (6), and after sufficiently stirring (about 1 hour), the temperature was raised from room temperature to 125 ℃. And in the heating process, continuously bubbling foam in the three-necked bottle until no foam exists in the three-necked bottle, connecting the three-necked bottle into a condenser pipe, slowly heating the solution in the three-necked bottle to 300 ℃, fully stirring for 2h, stopping heating, and cooling to 30 ℃.
(8) Subpackaging the mixed solution obtained in the step (7) in a centrifuge tube, adding 15ml of ethanol solution into the centrifuge tube, centrifuging at 10000rpm for 20min, pouring out supernatant, adding 16ml of cyclohexane solutionUltrasonically treating, dissolving and precipitating, adding 20ml of ethanol solution, centrifuging again, and repeating the step twice to obtain NaYF4:20％Yb3+,2％Er3+Precipitating, collecting the precipitate in a centrifugal tube, adding 5ml of cyclohexane, and performing ultrasonic treatment to obtain NaYF4:Yb3+，Er3+Cyclohexane solution.
(9) NaYF cleaning by using hydrochloric acid solution4:20％Yb3+,2％Er3+Surface ligands on the nanoparticles. Taking 5ml of NaYF obtained in the step (8)4:Yb3+，Er3+Adding 0.8mol of hydrochloric acid into the cyclohexane solution, and carrying out ultrasonic treatment for 5 min.
(10) And (4) centrifuging the mixed solution obtained in the step (9) twice by using ethanol. Centrifuging for the first time, taking 5ml of the mixed solution obtained in the step (9), adding 15ml of ethanol, and centrifuging for 30min at 9000 r/min; the obtained product is centrifuged again under the same conditions, and the centrifuged product is NaYF4:20％Yb3+,2％Er3+And then dissolved in 5ml of deionized water to obtain ligand-free NaYF4:20％Yb3+,2％Er3+And (3) solution.
(11) The ligand-free NaYF obtained in the step (10) is treated4:20％Yb3+,2％Er3+The solution is uniformly dispersed in 5ml of potassium permanganate and 5ml of hydrochloric acid, the obtained mixed solution is stood for 12 hours at room temperature, the color of the mixture gradually changes from dark purple to red black during the standing period, and NaYF is obtained4:20％Yb3+,2％Er3+@MnO2And (3) solution. As shown in FIG. 3, transmission electron microscopy images show that NaYF was successfully prepared4:20％Yb3+,2％Er3+@MnO2Upconversion nanoparticles, average size about 44.2 ± 9.2 nm. And (3) transmission electron microscope determination: transmission electron microscopy was tested using a Hitachi H-8100IV transmission electron microscope at an accelerating voltage of 200 kV. As shown in FIG. 4, the X-ray diffraction pattern shows that hexagonal phase NaYF was successfully prepared4:20％Yb3+,2％Er3+@MnO2The nanoparticles were upconverted and two distinct peaks were observed at 36.5 ° and 66.7 ° 2 θ, respectively, to be MnO 2110 and 020 crystal planes. Measurement of X-ray diffraction patternsTest: the X-ray diffraction pattern was recorded as a thin film on a Bruker AXS D8 diffractometer using alpha radiation (λ ═ 1.54178). The film is prepared by covering the surface of a silicon wafer substrate in a spin coating mode.
(12) Taking 5ml of NaYF obtained in the step (11)4:20％Yb3+,2％Er3+@MnO2Adding 15ml of deionized water into the solution, centrifuging at 9000 r/min for 15 min; the resulting product was redissolved in 15ml of deionized water and centrifuged once more under the same conditions.
(13) Diluting the precipitate obtained in the step (12) into 300ml of deionized water to prepare single particle NaYF4:20％Yb3 +,2％Er3+. And (3) sampling. When the nano particles are irradiated by laser of 980nm, the converted fluorescence on the nano particles is quenched, which shows that NaYF is obtained4:20％Yb3+,2％Er3+@MnO2And (3) solution. As shown in fig. 5, is a single upconversion nanoparticle NaYF4:20％Yb3+,2％Er3+With single up-conversion nanoparticles NaYF4:20％Yb3+,2％Er3+@MnO2Fluorescence intensity contrast plot of (a). It can be seen that MnO is being encapsulated2Then, single up-conversion nano-particle NaYF4:20％Yb3+,2％Er3+@MnO2The fluorescence of (a) is significantly quenched. Measurement of fluorescence spectrum: the emission spectra of all samples were recorded using a Princeton SP2300 spectrometer under continuous 980nm light source at room temperature.
(14) Heightening one side of the photonic crystals (OPCs) obtained in the step (3) by 2mm (heightening one end of the whole glass sheet), and heightening cesium tungstate (Cs) obtained in the step (5)xWO3) The solution is dripped along one end of the photonic crystal to obtain OPCs/CsxWO3And (3) a composite structure.
(15) Diluting the NaYF obtained in the step (13)4:20％Yb3+,2％Er3+@MnO2Dropping the solution to the OPCs/Cs obtained in step (14)xWO3On the composite structure, preparing OPCs/CsxWO3/NaYF4:20％Yb3+,2％Er3+@MnO2And (4) a composite structure, and the preparation of the microfluidic single particle sensor is completed. As shown in fig. 6, for a single up-conversion nanoparticle NaYF4:20％Yb3 +,2％Er3+And OPCs photonic crystal effect and CsxWO3Single up-conversion nano-particle NaYF cooperatively regulated and controlled by semiconductor plasma resonance effect4:20％Yb3+,2％Er3+Fluorescence intensity contrast plot of (a). It can be seen that benefit from OPCs/CsxWO3Cascade light field amplification effect of composite structure and single up-conversion nano particle NaYF4:20％Yb3+,2％Er3+The up-conversion fluorescence of (2) is improved by 1600 times.
(16) The microfluidic single-particle sensor is used for detecting DTT in vitro, and the working process comprises the following steps (the structure is shown in figure 7): and tracking the position of the single particle by using an atomic force microscope and an in-situ fluorescence lifetime imaging system, and recording the fluorescence spectrum and the lifetime information of the single particle. FIG. 8 shows the OPCs/Cs scanned by an atomic force microscope probexWO3Single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+@MnO2And an in situ fluorescence lifetime image (b). It can be seen that the atomic force scan and fluorescence lifetime imaging can be used to accurately confirm the position of a single upconversion nanoparticle, and it can also be proved that a sensor related to the present invention is built on the scale of a single particle.
DTT molecular pair MnO2The dissociation of the nanomaterial has high reactivity, so that OPCs/Cs are added with DTT moleculesxWO3/NaYF4:20％Yb3+,2％Er3+@MnO2Mn of composite structure4+Reduction to Mn2+So that MnO is2And (3) gradually decomposing, and gradually recovering the quenched up-conversion fluorescence to realize the in vitro high-sensitivity DTT detection. As shown in FIG. 9, the emission spectrum gradually became stronger with increasing dithiothreitol until recovery. Single NaYF for cascade light field regulation4:20％Yb3+,2％Er3+The enhancement of the upconversion fluorescence of the nanoparticles contributes to the improvement of the sensitivity of the device. As shown in FIG. 10, is a single particleSensor OPCs/CsxWO3Single upconversion nanoparticle NaYF4:20％Yb3+,2％Er3+For the linear corresponding range diagram of DTT detection, it can be seen that the single particle sensor has good linear response to DTT at 0-5nMOl, and the lowest detection limit is 0.05 nMOl.
The method of the invention has good specificity detection capability, as shown in FIG. 11, and is based on OPCs/CsxWO3Single up-conversion nanoparticle NaYF4:20％Yb3+,2％Er3+@MnO2After adding glucose, fructose, lactose, glutamic acid, glycine, homocysteine, potassium chloride, magnesium sulfate and manganese chloride, the single particle sensor up-converts the nano particle NaYF4:20％Yb3+,2％Er3+@MnO2Does not significantly increase the fluorescence intensity of (c).
Example 2:
(1) dissolving 0.1g of NaOH in 10ml of deionized water to prepare NaOH solution, adding 30ml of PS into the NaOH solution, and violently stirring the mixed solution to play a role in cleaning a polymerization inhibitor on the surface of the PS; mixing 6ml of the mixed solution with 70mol of deionized water and 30mg of potassium persulfate, slowly stirring for 2h at the temperature of 150 ℃, and obtaining PS microspheres after the reaction is finished;
(2) and (3) adjusting the diameter of the PS microspheres by a coating method, taking 6ml of the PS microsphere solution obtained in the step (1), 50mol of deionized water and 50ml of PS, and slowly stirring for 1.2h at the temperature of 150 ℃ to obtain the coated PS microspheres.
(3) And (3) taking 4ml of PS microspheres wrapped in the step (2), diluting with 50mol of deionized water, vertically inserting a cleaned glass slide into the solution, integrally moving the glass slide into a 45 ℃ oven, standing for 72h, and then obtaining the opal photonic crystal template.
(4) 10mol of sodium tungstate, 22.5mol of malonic acid and 1mol of deionized water are added into a three-necked bottle, the mixture is fully stirred for 0.5h at the temperature of 30 ℃, 5mol of nitric acid is added into the mixed solution, and the mixture is fully stirred for 0.5h again at the temperature of 30 ℃ until yellow suspension is generated.
(5) Pouring the yellow suspension obtained in the step (4) into a reaction kettle, sealing and placing the reaction kettle in a drying box, setting the temperature to be 200 ℃, setting the reaction time to be 5 hours, and naturally cooling to 30 ℃ after the reaction is finished. Washing the obtained mixed solution with deionized water and ethanol for 3 times to remove soluble impurities in the mixed solution, wherein the volume ratio of the mixed solution to the deionized water to the ethanol is 1: 3: 3, tungsten oxide obtained finally (WO)3) The precipitate is dispersed in ethanol to obtain WO3A semiconductor plasma solution.
(6) Synthesis of upconverting nanoparticles KYF by classical solvothermal method4:40％Yb3+,5％Er3+. 0.167g of yttrium chloride hexahydrate powder, 0.155g of ytterbium chloride hexahydrate powder and 0.019g of erbium chloride hexahydrate powder are put into a three-necked bottle, 9ml of oleic acid and 22.5ml of octadecene solution are added, the mixture is fixed in an oil bath pot, stirred at 700 plus 950rpm under the protection of nitrogen at 160 ℃ until the drugs in the three-necked bottle are completely dissolved, and then cooled to 30 ℃.
(7) 0.15g of potassium hydroxide and 0.222g of ammonium fluoride powder were dissolved in 9ml of methanol solution. The mixed solution of methanol was slowly added to the mixed solution obtained in step (6), and after sufficiently stirring (about 1 hour), the temperature was raised from room temperature to 125 ℃. And in the heating process, continuously bubbling foam in the three-necked bottle until no foam exists in the three-necked bottle, connecting the three-necked bottle into a condenser pipe, slowly heating the solution in the three-necked bottle to 300 ℃, fully stirring for 2h, stopping heating, and cooling to 30 ℃.
(8) Subpackaging the mixed solution obtained in the step (7) in a centrifuge tube, adding 27ml of ethanol solution into the centrifuge tube, centrifuging at 10000rpm for 20min, pouring out supernatant, adding 27ml of cyclohexane solution, performing ultrasonic treatment to dissolve precipitate, adding 27ml of ethanol solution, centrifuging again, repeating the step twice to obtain KYF4:40％Yb3+，5％Er3+Precipitating, collecting the precipitate in a centrifugal tube, adding 7.5ml of cyclohexane, and performing ultrasonic treatment to obtain KYF4:Yb3+，Er3+Cyclohexane solution.
(9) Cleaning of KYF Using hydrochloric acid solution4:40％Yb3+,5％Er3+Surface ligands on the nanoparticles. Taking 5ml of KYF obtained in step (8)4:40％Yb3+,5％Er3+Adding 8mol of hydrochloric acid into the cyclohexane solution, and carrying out ultrasonic treatment for 5 min.
(10) And (4) centrifuging the mixed solution obtained in the step (9) twice by using ethanol. Centrifuging for the first time, namely taking 7.5ml of the mixed solution obtained in the step (9), adding 30ml of ethanol, and centrifuging for 30min at 9000 r/min; the obtained product is centrifuged once again under the same conditions, and the centrifuged product is KYF4:40％Yb3+,5％Er3+Then dissolved in 11.25ml deionized water to give ligand-free KYF4:40％Yb3+,5％Er3+And (3) solution.
(11) KYF without ligand obtained in step (10)4:40％Yb3+,5％Er3+Uniformly dispersing the solution in 13.5ml of potassium permanganate and 9ml of hydrochloric acid, standing the obtained mixed solution at room temperature for 36 hours, gradually changing the color of the mixture from dark purple to red black during the standing period, and obtaining KYF4:40％Yb3+,5％Er3+@MnO2And (3) solution.
(12) Taking 7.5ml of KYF obtained in step (11)4:40％Yb3+,5％Er3+@MnO2Adding 22.5ml deionized water into the solution, centrifuging at 9000 r/min for 15 min; the resulting product was redissolved in 30ml of deionized water and centrifuged once more under the same conditions. When the nano particles are irradiated by laser of 980nm, the upconversion fluorescence is quenched, which shows that KYF is obtained4:40％Yb3+,5％Er3+@MnO2And (3) solution.
(13) Diluting the precipitate obtained in step (12) in 600ml of deionized water, and reserving the precipitate as a single particle sample.
(14) Heightening one side of the photonic crystal (PS) obtained in the step (3) by 2mm (heightening one end of the whole glass sheet), and heightening one end of the WO obtained in the step (5)3The solution is dripped along one end of the photonic crystal to obtain PS/WO3And (3) a composite structure.
(15) Carrying out KYF on the diluted product obtained in the step (13)4:40％Yb3+,5％Er3+@MnO2Dropping the solution to the PS/WO obtained in step (14)3On the composite structure, preparing PS/WO3/KYF4:40％Yb3+,5％Er3+@MnO2And (4) a composite structure, namely, the preparation of the microfluidic single particle sensor is completed, and then the composite structure can be used for DTT detection.
Example 3:
(1) 0.1g of NaOH is dissolved in 10ml of deionized water to prepare a NaOH solution, and SiO with the volume fraction of 1 percent is added into the NaOH solution2Suspending liquid, stirring the mixed solution vigorously to clean SiO2The action of a polymerization inhibitor on the surface of the suspension; mixing 8ml of the mixed solution with 80mol of deionized water and 46mg of potassium persulfate, slowly stirring for 4 hours at 220 ℃, and obtaining SiO after the reaction is finished2Microspheres;
(2) SiO conditioning by cladding2Taking 7ml of SiO in the step (1) according to the diameter of the microspheres2Microspheres, 80ml of deionized water, and 80ml of SiO2Slowly stirring for 2h at 220 ℃ to obtain coated SiO2And (4) microspheres.
(3) Taking 7ml of SiO coated in the step (2)2And (3) diluting the microspheres with 80ml of deionized water, vertically inserting a cleaned glass slide into the solution, moving the whole body into an oven at 70 ℃, standing for 144h, and then obtaining the opal photonic crystal template.
(4) Preparing a copper precursor, adding 1mol of copper chloride, 2.5mol of oleic acid and 1.8mol of oleylamine into a three-necked bottle, and mixing and stirring for 0.5h under the protection of nitrogen; preparing a sulfur precursor, mixing 1mol of sulfur powder with 2.5mol of octadecene, heating a sulfur precursor solution to 200 ℃, then quickly injecting a copper precursor solution, keeping the volume ratio of the sulfur precursor solution to the copper precursor solution at 1:1, stirring for 30min, stopping heating, and cooling to 30 ℃.
(5) And (3) washing the cuprous sulfide solution obtained in the step (4) with acetone and toluene for 3 times, wherein the volume ratio of the mixed solution to the acetone to the toluene is 1: 6: 6, using 11000 r/min, and centrifuging for 30 min; fully dissolving the centrifuged precipitate in 10mol of toluene, adding 10mol of acetone, and performing secondary centrifugation treatment again at 11000 r/min for 30 min; the conditions of the third centrifugation are the same as those of the second centrifugation, and the final product is obtainedThe cuprous sulfide precipitate is dispersed in toluene to obtain cuprous sulfide (Cu)2S) a semiconductor plasma solution.
(6) Synthesis of upconversion nanoparticles LiYF by classical solvothermal method4:60％Yb3+,10％Er3+. 0.038g of yttrium chloride hexahydrate powder, 0.232g of ytterbium chloride hexahydrate powder and 0.091g of erbium chloride hexahydrate powder are put into a three-necked bottle, 12ml of oleic acid and 30ml of octadecene solution are added into the three-necked bottle, the mixture is fixed in an oil bath pot, stirred at the speed of 700 plus 950rpm under the protection of nitrogen at 160 ℃ until the drugs in the three-necked bottle are completely dissolved, and then cooled to 30 ℃.
(7) 0.2g of lithium hydroxide and 0.296g of ammonium fluoride powder were dissolved in 12ml of methanol solution. The mixed solution of methanol was slowly added to the mixed solution obtained in step (6), and after sufficiently stirring (about 1 hour), the temperature was raised from room temperature to 125 ℃. And in the heating process, continuously bubbling foam in the three-necked bottle until no foam exists in the three-necked bottle, connecting the three-necked bottle into a condenser pipe, slowly heating the solution in the three-necked bottle to 300 ℃, fully stirring for 2h, stopping heating, and cooling to 30 ℃.
(8) Subpackaging the mixed solution obtained in the step (7) in a centrifuge tube, adding 40ml of ethanol solution into the centrifuge tube, centrifuging for 20min at 10000rpm, pouring out supernatant, adding 40ml of cyclohexane solution, performing ultrasonic treatment, dissolving and precipitating, adding 20ml of ethanol solution, centrifuging again, repeating the step twice to obtain LiYF4:60％Yb3+,10％Er3+Precipitating, collecting the precipitate in a centrifugal tube, adding 10ml of cyclohexane, and performing ultrasonic treatment to obtain LiYF4:Yb3+，Er3+Cyclohexane solution.
(9) Cleaning LiYF by using hydrochloric acid solution4:60％Yb3+,10％Er3+Surface ligands on the nanoparticles. Taking 8ml of LiYF obtained in the step (8)4:Yb3+，Er3+Adding 4.8mol of hydrochloric acid into the cyclohexane solution, and carrying out ultrasonic treatment for 5 min.
(10) And (4) centrifuging the mixed solution obtained in the step (9) twice by using ethanol. Performing first centrifugation, namely taking 16ml of the mixed solution obtained in the step (9), adding 80ml of ethanol, and centrifuging for 30min at 9000 r/min; the obtained product has the same stripThe piece is centrifuged once again, and the centrifuged product is LiYF4:20％Yb3+,2％Er3+Then dissolved in 30ml of deionized water to obtain ligand-free LiYF4:60％Yb3+,10％Er3+And (3) solution.
(11) The ligand-free LiYF obtained in the step (10) is used4:60％Yb3+,10％Er3+Uniformly dispersing the solution in 30ml of potassium permanganate and 20ml of hydrochloric acid, standing the obtained mixed solution at room temperature for 48 hours, gradually changing the color of the mixture from dark purple to red black during standing, and obtaining LiYF4:60％Yb3+,10％Er3+@MnO2And (3) solution.
(12) Taking 5ml of LiYF obtained in the step (11)4:20％Yb3+,2％Er3+@MnO2Adding 80ml deionized water into the solution, centrifuging at 9000 r/min for 15 min; the resulting product was redissolved in 80ml of deionized water and centrifuged once more under the same conditions. When the nanoparticles are irradiated by laser of 980nm, the converted fluorescence on the nanoparticles is quenched, which shows that LiYF is obtained4:60％Yb3+,10％Er3+@MnO2And (3) solution.
(13) Diluting the precipitate obtained in step (12) in 1000ml of deionized water, and reserving the precipitate as a single particle sample.
(14) The photonic crystal (SiO) obtained in the step (3)2) Raising one side by 2mm (raising one end of the whole glass sheet), and obtaining Cu in the step (5)2Dripping the S solution along one end of the photonic crystal to obtain SiO2/Cu2And (5) an S composite structure.
(15) Diluting the diluted LiYF obtained in the step (13)4:60％Yb3+,10％Er3+@MnO2Dropping the solution to the SiO obtained in step (14)2/Cu2On S composite structure, preparing SiO2/Cu2S/LiYF4:60％Yb3+,10％Er3+@MnO2And (4) a composite structure, namely, the preparation of the microfluidic single particle sensor is completed, and then the composite structure can be used for DTT detection.
Claims (1)
1. A dithiothreitol detection method based on single particle up-conversion luminescence is characterized by comprising the following specific steps:
step 1: with methyl methacrylate MMA, styrene or silica SiO2Preparing an opal photonic crystal template A as a microsphere for manufacturing photonic crystals by a three-dimensional vertical self-assembly method;
washing the material M with sodium hydroxide solution to remove the polymerization inhibitor, wherein the material M is MMA, styrene or SiO2Suspension, the concentration of the sodium hydroxide solution is 10-100mg/mL, and the volume ratio of the sodium hydroxide solution to the substance M is (40-90): 1; SiO 22Preparation of a suspension from SiO with a size of 200-600nm2Dissolving the microsphere particles in ethanol to obtain SiO with the volume fraction of 0.3-2%2A suspension;
then, mixing the cleaned substance M, deionized water and an initiator according to a volume ratio of (1-10): (20-80): 1, mixing; slowly stirring the mixed solution for 1-4h at the temperature of 90-220 ℃ to prepare polymethyl methacrylate (PMMA) microsphere solution, Polystyrene (PS) microsphere solution or silicon dioxide (SiO)2Microsphere solution, denoted as a-microsphere solution;
the size of the diameter of the microsphere for preparing the photonic crystal template is adjusted by using a coating method to obtain the opal photonic crystal with the photonic band gap position being near 400-1600 nm; and during coating, mixing the obtained A-microsphere solution with deionized water and a substance M, wherein the volume ratio of the A-microsphere solution to the deionized water to the substance M is (3-60): (3-60): 1, slowly stirring for 0.5-2h at the temperature of 90-220 ℃, and then obtaining a coated A-microsphere solution;
diluting the wrapped A-microsphere solution with deionized water, wherein the volume ratio of the A-microsphere solution to the deionized water is 1: (20-80), vertically inserting the glass slide into a beaker, integrally transferring the beaker into an oven at the temperature of 25-70 ℃ and standing for 24-144h to obtain an opal photonic crystal template A which is a polymethyl methacrylate opal photonic crystal template, a polystyrene opal photonic crystal template and a silicon dioxide opal photonic crystal template respectively;
the initiator is potassium persulfate, sodium persulfate, aluminum persulfate, ammonium persulfate or sodium bisulfite;
step 2: preparing a semiconductor plasma solution B with localized surface plasmon resonance effect comprising cesium tungstate CsxWO3Semiconductor plasma solution, tungsten oxide WO3Semiconductor plasma solution or cuprous sulfide Cu2S semiconductor plasma solution, the concrete steps are as follows:
CsxWO3the preparation method of the semiconductor plasma solution comprises the following steps: cesium chloride, tungsten chloride, oleic acid and oleylamine were mixed in a molar ratio (22-45): (1-3): (1-3): 1, fully stirring at the temperature of 300-660 ℃ until the mixture turns dark blue, and cooling the mixture to room temperature to obtain a cesium tungstate solution; adding toluene into the cesium tungstate solution, and carrying out centrifugal treatment for three times; during the first centrifugal treatment, the volume ratio of the cesium tungstate solution to the toluene is 1: (3-10); and (3) dissolving the centrifuged product in acetone, fully dissolving, adding toluene again for second centrifugation, wherein the volume ratio of the cesium tungstate solution to the acetone to the toluene is 1: (3-10): (3-10); and (3) dissolving the product after the second centrifugal treatment in acetone again, adding toluene after full dissolution, and performing third centrifugal treatment, wherein the volume ratio of the cesium tungstate solution to the acetone to the toluene is 1: (3-10): (3-10); finally, the obtained cesium tungstate precipitate is dispersed in toluene to obtain CsxWO3A semiconductor plasma solution;
WO3the preparation method of the semiconductor plasma solution comprises the following steps: sodium tungstate, malonic acid and deionized water are mixed according to a molar ratio (10-50): (22.5-66.8): 1 for 0.5 to 6.5 hours to obtain a mixed solution; then adding nitric acid into the mixed solution, wherein the volume ratio of the mixed solution to the nitric acid is (2.5-62.8): 1, stirring again for 0.5-6.5h until a yellow suspension is generated; pouring the yellow suspension into a reaction kettle, sealing and placing the reaction kettle in a drying box, setting the temperature to be 100-900 ℃, setting the reaction time to be 5-48h, and naturally cooling the reaction kettle to room temperature after the reaction is finished; washing the dried mixed solution with deionized water and ethanol for 3 times to remove soluble impurities in the mixed solution, wherein the volume ratio of the mixed solution to the deionized water to the ethanol is 1: (1-10): (1-10), oxygen obtained finallyDispersing tungsten oxide precipitate in ethanol to obtain WO3A semiconductor plasma solution;
Cu2the preparation method of the S semiconductor plasma solution comprises the following steps: preparing a copper precursor solution, namely mixing copper chloride, oleic acid and oleylamine according to a molar ratio of 1: (2.5-78.6): (1.8-66.8) and stirring for 0.5-5.5h under the protection of nitrogen; dissolving sulfur powder in octadecylene to prepare a sulfur precursor solution, wherein the molar ratio of the sulfur powder to the octadecylene is 1: (2.5-78.9); heating the sulfur precursor solution to 20-300 ℃, and then quickly injecting the copper precursor solution, wherein the volume ratio of the copper precursor solution to the sulfur precursor solution is (1-10): 1, stirring for 5-100min, stopping heating and cooling to room temperature; and washing the obtained mixed solution for 3 times by using acetone and toluene, wherein the volume ratio of the mixed solution to the acetone to the toluene is 1: (1-10): (1-10), dispersing the finally obtained cuprous sulfide precipitate in toluene to obtain Cu2S, semiconductor plasma solution;
and step 3: synthesizing up-conversion nanoparticles by a solvothermal method, wherein the up-conversion nanoparticles are NaYF4:10-60％Yb3+,1-10％Er3+，KYF4:10-60％Yb3+,1-10％Er3+，LiYF4:10-60％Yb3+,1-10％Er3+Is one of (1), denoted as CYF4:10-60％Yb3+,1-10％Er3+C ═ Na or K or Li; coating MnO on the surface of the nano-particles after the surface is processed without ligand2The film comprises the following specific steps:
cleaning of CYF Using hydrochloric acid solution4:10-60％Yb3+,1-10％Er3+Surface ligands on nanoparticles, CYF4:10-60％Yb3+,1-10％Er3+The volume ratio of the nanoparticles to the hydrochloric acid is 1: (0.8-4.8), and fully dissolving; and centrifuging the obtained mixed solution twice by using ethanol, wherein the volume ratio of the mixed solution to the ethanol is 1: (3-10); the product after centrifugation is CYF without ligand4:10-60％Yb3+,1-10％Er3+Nanoparticles and dissolved in deionized water, ligand-free NaYF4:Yb3+,Er3+Volume ratio of nanoparticles to deionized waterIs (0.3-2): 1, obtaining ligand-free CYF4:10-60％Yb3+,1-10％Er3+A solution;
will be ligand-free CYF4:10-60％Yb3+,1-10％Er3+The nano-particle solution is uniformly dispersed in the mixed solution of potassium permanganate and hydrochloric acid, wherein CYF4:10-60％Yb3+,1-10％Er3+The volume ratio of the solution to the potassium permanganate to the hydrochloric acid is (1-3): (1-4): 1, standing at room temperature for 12-96h, wherein the color of the mixture gradually changes from dark purple to red black to obtain CYF4:10-60％Yb3+,1-10％Er3+@MnO2The solution was centrifuged twice using deionized water, wherein CYF4:10-60％Yb3+,1-10％Er3+@MnO2The volume ratio of the solution to the deionized water is 1 (3-10); finally, the resulting product is diluted in deionized water, in which CYF4:10-60％Yb3+,1-10％Er3+@MnO2The volume ratio of the solution to the deionized water is 1: (300-1000) to obtain diluted CYF4:10-60％Yb3+,1-10％Er3+@MnO2A solution;
and 4, step 4: preparation of microfluidic single particle sensor
Dripping any one semiconductor plasma solution B obtained in the step 2 along one side of any one opal photonic crystal template A obtained in the step 1 to prepare an A/B composite structure; diluting CYF obtained in step 34:10-60％Yb3+,1-10％Er3+@MnO2Dropping the solution on the A/B composite structure to obtain A/B/CYF4:10-60％Yb3+,1-10％Er3+@MnO2Obtaining a microfluidic single particle sensor by a composite structure;
and 5: dithiothreitol detection
Tracking the microfluidic single particle sensor A/B/CYF prepared in the step 4 by using an atomic force microscope and an in-situ fluorescence lifetime imaging system4:10-60％Yb3+,1-10％Er3+@MnO2The position of a single upconversion nanoparticle is recorded, and the fluorescence spectrum and the service life information of the nanoparticle are recorded; injecting dithiothreitol into the step by a micro-syringe pump4, preparing the micro-fluidic single particle sensor, and recording the fluorescence spectrum and the service life information of the micro-fluidic single particle sensor again; adjusting the microinjection pump so that as the concentration of DTT increases, MnO2The fluorescence spectrum of the coated up-conversion nano material is changed, and the concentration of dithiothreitol can be obtained.
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