A kind of preparation method and its recyclable immune detection application of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material
阅读说明：本技术 一种四氧化三铁/二氧化钛/银核壳纳米材料的制备方法及其可循环免疫检测应用 (A kind of preparation method and its recyclable immune detection application of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material ) 是由 姜涛 周骏 姜勇 杜远远 顾辰杰 王福艳 于 2019-07-11 设计创作，主要内容包括：本发明公开了一种四氧化三铁/二氧化钛/银核壳纳米材料的制备方法及其直接可循环免疫检测应用,特点是活化三重核壳纳米材料使其与待测抗原相结合,利用表面增强拉曼散射效应实现待测抗原的直接拉曼检测,在拉曼检测完成后,对上述免疫结合的纳米材料进行紫外光照降解抗原,磁力回收表面已无抗原存在的Fe<Sub>3</Sub>O<Sub>4</Sub>/TiO<Sub>2</Sub>/Ag核壳纳米材料,并将其用于下一次对待测抗原的检测中,重复这一拉曼检测-催化降解-拉曼检测过程,实现肿瘤标志物直接可循环免疫检测,优点是三层核壳结构既具有磁性分离和拉曼增强的作用,又具有催化降解肿瘤标志物的作用,同时检测限低且可检测大分子肿瘤标志物。(The invention discloses a kind of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material preparation method and its directly recyclable immune detection application, feature is that activation triple core-shell nano material combines it with determined antigen, the direct Raman detection of determined antigen is realized using surface enhanced Raman scattering effect, after the completion of Raman detection, ultraviolet lighting degradation antigen is carried out to the nano material of above-mentioned immune combination, magnetic force recycles the nonantigenic existing Fe in surface 3 O 4 /TiO 2 / Ag core-shell nano material, and it is used for next time in the detection of determined antigen, repeat this Raman detection-catalytic degradation-Raman detection process, realize the directly recyclable immune detection of tumor markers, advantage is that three-layer nuclear shell structure had both had the function of Magnetic Isolation and Raman enhancing, have the function of catalytic degradation tumor markers again, while detecting and limiting low and detectable macromolecular tumor markers.)
1. a kind of ferroso-ferric oxide/titanium dioxide/silver core-shell nano material preparation method, it is characterised in that including following step It is rapid:
(1) 1.35-6.75 grams of Iron(III) chloride hexahydrate, 3.6-18.0 grams of sodium acetate and 1.0-5.0 grams of polyethylene glycol are dissolved in 40- In 200 milliliters of ethylene glycol, after ultrasound is until be completely dissolved, it is transferred in hydrothermal reaction kettle, is reacted 10 hours at 200 DEG C, from The heart simultaneously collects precipitating after being cleaned with ethyl alcohol to get to Fe3O4Nano particle；
(2) Fe that 50 milligrams of steps (1) obtain is weighed3O4It is 0.1 mM of every milliliter of aqueous hydrochloric acid solution that nano particle, which is dissolved in concentration, Middle ultrasonic 15 minutes, Magnetic Isolation was simultaneously cleaned three times with deionized water；Again after Magnetic Isolation, by Fe3O4Nano particle is dissolved in 120 milliliters by ethyl alcohol and the acetonitrile mixed solution that 3:1 is mixed by volume, after ultrasound makes it completely dissolved, Xiang Qijia Enter 500 microlitres of ammonium hydroxide, ultrasound is uniformly mixed it；Then 1-6 milliliters of four fourths of metatitanic acid are added dropwise under agitation Ester, reaction 1.5 hours after, Magnetic Isolation and with ethanol washing three times after to get arrive Fe3O4/TiO2Nano particle；
(3) Fe for taking 81 milligrams of steps (2) to obtain3O4/TiO2Nano particle and 75.4 milligrams of ammonium fluorides be dissolved in 20.45 milliliters by In the second alcohol and water mixed solution that 13.5:6.95 is mixed by volume, ultrasound puts it by reaction solution after completely dissolution It is placed in and persistently stirs 1 hour at room temperature, mixing speed is 180-200 rpms, and after the completion of stirring, reaction solution is transferred to It in reaction kettle, is reacted 24 hours at 180 DEG C, after reaction kettle cooled to room temperature, Magnetic Isolation is simultaneously washed with deionized water 3 times are washed to get the Fe for arriving crystallization3O4/TiO2Nano particle；
(4) Fe for the crystallization for taking 81 milligrams of steps (3) to obtain3O4/TiO2Nano particle is dissolved in 33.4 milliliters of ethyl alcohol, is continued 25 microlitres of 3- aminopropyl trimethoxy siloxanes are added dropwise under stirring, reaction solution is placed at 80 DEG C and is condensed back 4 hours, After its cooled to room temperature, Magnetic Isolation and with ethanol washing three times to get arrive amidized Fe3O4/TiO2Nanometer Grain；
(5) the amidized Fe for obtaining 1 gram of polyvinylpyrrolidone and 28 milligrams of steps (4)3O4/TiO2Nano particle is dissolved in In 10 ml deionized waters after mixing, silver ammino solution is added dropwise under mechanical stirring and is transferred in hydrothermal reaction kettle, Sealing, which is placed at 120 DEG C, reacts 11 hours, and after reaction kettle cooled to room temperature, Magnetic Isolation simultaneously successively uses tetrahydro furan It mutters, ethyl alcohol and deionized water are washed to get Fe is arrived3O4/TiO2/ Ag core-shell nano material, be placed in deionized water storage to With,
A kind of ferroso-ferric oxide according to claim 1/titanium dioxide/silver core-shell nano material preparation method, it is special Sign is silver ammino solution described in step (5) the preparation method comprises the following steps: taking 2 milliliters of concentration is 0.01-0.08 milligrams every milliliter of nitre Excessive ammonium hydroxide is added until reaction solution becomes clarification and obtains silver ammino solution in sour silver aqueous solution.
2. a kind of based on the resulting ferroso-ferric oxide of claims 1 or 2 preparation method/titanium dioxide/silver core-shell nano material Tumor markers are recycled immunologic detection method, it is characterised in that the following steps are included:
(1) by 15 milligrams of Fe3O4/TiO2/ Ag core-shell nano material is dissolved in 2 milliliters of dimethyl formamide solutions, is then added 1 The DMF solution of milliliter succinic anhydride, after hatching being slowly shaken at 70 DEG C 24 hours, Magnetic Isolation is simultaneously washed with deionized 2 It is secondary to get arrive carboxylated Fe3O4/TiO2/ Ag core-shell nano material, and be dissolved in 1 milliliter of water, 1 milliliter of EDC/ is then added The PBS solution of NHS is washed with deionized 1 time after slowly shaking hatching at 30 DEG C 1 hour, then by the Fe after washing3O4/ TiO2/ Ag core-shell nano material, which is dissolved in 1 milliliter, to be compared in the mixed solution mixed by PBS buffer solution and water as isometric, to The tumor markers solution of 100 micrograms per millilitres is wherein added, after 30 DEG C slowly shake hatching 3.5 hours, uses deionized water It washed once, Magnetic Isolation obtains the Fe for being marked with tumor markers3O4/TiO2/ Ag core-shell nano material, utilizes Raman spectrum Instrument, which carries out spectral measurement to it, can detect the content of tumor markers to be measured；
(2) after detecting, the Fe of tumor markers will be marked with3O4/TiO2/ Ag core-shell nano material is dissolved in deionized water, And be transferred into Clear glass bottles and jars, assemble Fe by the way of small magnet attraction3O4/TiO2/ Ag core-shell nano, is adopted It is irradiated with the ultraviolet lamp that wavelength is 265 nanometers, until tumor markers degradation completely, then will urge under photocatalysis Fe after change3O4/TiO2/ Ag core-shell nano material repeats step (1) method and links new tumor markers, can be realized to swollen The directly recyclable immune detection of tumor markers.
3. according to claim 3 can based on ferroso-ferric oxide/titanium dioxide/silver core-shell nano material tumor markers Circulation immunity detection method, it is characterised in that: the concentration of succinic anhydride in the DMF solution of succinic anhydride as described in step (1) It is 10.3 milligrams every milliliter；The concentration of EDC is 10 milligrams every milliliter in the PBS solution of the EDC/NHS, and the concentration of NHS is 10 milligrams every milliliter.
4. according to claim 3 can based on ferroso-ferric oxide/titanium dioxide/silver core-shell nano material tumor markers Circulation immunity detection method, it is characterised in that: the tumor markers include prostate-specific antigen PSA, alpha-fetoprotein AFP, immune globulin IGg, sugar antigen CA199 and Carcinoembryonic Antigen CEA.
Technical field
The present invention relates to material engineering and field of nanometer technology, more particularly, to a kind of ferroso-ferric oxide/titanium dioxide/silver (Fe3O4/TiO2/ Ag) core-shell nano material preparation method and its recyclable immune detection application.
Background technique
In recent years, with the aggravation of environmental pollution, the lasting change of global climate, the generation of crisis in food and people are raw The increasing of pressure living, cancer take place frequently, and high mortality has become seriously threatening for human health.Therefore, it is developed one The advanced immunological technique of series, such as chemoluminescence method and enzyme-linked immunosorbent assay biochemistry detection technology visit cancer sufferer It surveys and treats.But the patient for being in cancer early stage, cancer markers content is lower in blood, is difficult to be sent out early Now and treated.Surface enhanced Raman scattering immunological technique developed in recent years makes full use of Surface enhanced Raman scattering The higher Electromagnetic enhancement degree that has and up to unimolecule rank highly sensitive advantage, it can be achieved that tumor markers trace Amount detection.But Surface enhanced Raman scattering technology based on material be mostly that noble metal nanometer material preparation cost is higher, it is unfavorable In its practical application in clinical immunization detection.Therefore, it is necessary to develop a kind of circulation immunity detection technique.Titanium dioxide is received Rice material has excellent photocatalytic activity, is a kind of potential critical material for realizing this recyclable immunoassay technology.Together When, how to be urged using the light that the diversified nano material with performances such as excellent photoelectromagnetics further increases composite nano materials Change activity, promoting application of the recyclable immunoassay technology in former field tachysynthesis detection is still the side that researcher is made great efforts To.
Summary of the invention
Technical problem to be solved by the invention is to provide one kind not only to have had the function of Magnetic Isolation and Raman enhancing, but also tool There is the detection of catalytic degradation cancer markers to limit low ferroso-ferric oxide/titanium dioxide/silver core-shell nano material preparation method And its recyclable immune detection application.
The technical scheme of the invention to solve the technical problem is: a kind of ferroso-ferric oxide/titanium dioxide/silver core The preparation method of shell nanometer material, comprising the following steps:
(1) 1.35-6.75 grams of Iron(III) chloride hexahydrate, 3.6-18.0 grams of sodium acetate and 1.0-5.0 grams of polyethylene glycol are dissolved in 40- In 200 milliliters of ethylene glycol, after ultrasound is until be completely dissolved, it is transferred in hydrothermal reaction kettle, is reacted 10 hours at 200 DEG C, from The heart simultaneously collects precipitating after being cleaned with ethyl alcohol to get to Fe3O4Nano particle；
(2) Fe that 50 milligrams of steps (1) obtain is weighed3O4It is that 0.1 mM of every milliliter of hydrochloric acid is water-soluble that nano particle, which is dissolved in concentration, Ultrasound 15 minutes in liquid, Magnetic Isolation is simultaneously cleaned three times with deionized water；Again after Magnetic Isolation, by Fe3O4Nano particle is dissolved in 120 milliliters by ethyl alcohol and the acetonitrile mixed solution that 3:1 is mixed by volume, after ultrasound makes it completely dissolved, Xiang Qijia Enter 500 microlitres of ammonium hydroxide, ultrasound is uniformly mixed it；Then 1-6 milliliters of four fourths of metatitanic acid are added dropwise under agitation Ester, reaction 1.5 hours after, Magnetic Isolation and with ethanol washing three times after to get arrive Fe3O4/TiO2Nano particle；
(3) Fe for taking 81 milligrams of steps (2) to obtain3O4/TiO2Nano particle and 75.4 milligrams of ammonium fluorides be dissolved in 20.45 milliliters by In the second alcohol and water mixed solution that 13.5:6.95 is mixed by volume, ultrasound puts it by reaction solution after completely dissolution It is placed in and persistently stirs 1 hour at room temperature, mixing speed is 180-200 rpms, and after the completion of stirring, reaction solution is transferred to It in reaction kettle, is reacted 24 hours at 180 DEG C, after reaction kettle cooled to room temperature, Magnetic Isolation is simultaneously washed with deionized water 3 times are washed to get the Fe for arriving crystallization3O4/TiO2Nano particle；
(4) Fe for the crystallization for taking 81 milligrams of steps (3) to obtain3O4/TiO2Nano particle is dissolved in 33.4 milliliters of ethyl alcohol, is continued 25 microlitres of 3- aminopropyl trimethoxy siloxanes are added dropwise under stirring, reaction solution is placed at 80 DEG C and is condensed back 4 hours, After its cooled to room temperature, Magnetic Isolation and with ethanol washing three times to get arrive amidized Fe3O4/TiO2Nanometer Grain；
(5) 1 gram of polyvinylpyrrolidone (5800 grams every mole of molecular weight) and 28 milligrams of steps (4) are obtained amidized Fe3O4/TiO2Nano particle is dissolved in 10 ml deionized waters after mixing, and silver ammino solution is added dropwise under mechanical stirring And be transferred in hydrothermal reaction kettle, sealing, which is placed at 120 DEG C, reacts 11 hours, after reaction kettle cooled to room temperature, magnetic Property separation and successively with tetrahydrofuran, ethyl alcohol and deionized water wash to get arrive Fe3O4/TiO2/ Ag core-shell nano material, juxtaposition It is stored in deionized water for use, wherein silver ammino solution is the preparation method comprises the following steps: taking 2 milliliters of concentration is 0.01-0.08 milligrams every milliliter Silver nitrate aqueous solution, be added excessive ammonium hydroxide until reaction solution become clarification obtain silver ammino solution.
Based on the resulting ferroso-ferric oxide of above-mentioned preparation method/titanium dioxide/silver core-shell nano material tumor markers Recyclable immunologic detection method, comprising the following steps:
(1) by 15 milligrams of Fe3O4/TiO2/ Ag core-shell nano material is dissolved in 2 milliliters of dimethylformamide (DMF) solutions, then The DMF solution of 1 milliliter of succinic anhydride is added, after hatching being slowly shaken at 70 DEG C 24 hours, Magnetic Isolation simultaneously uses deionized water Washing 2 times to get the Fe for arriving carboxylated3O4/TiO2/ Ag core-shell nano material, and be dissolved in 1 milliliter of water, then it is added 1 milliliter The PBS solution of EDC/NHS is washed with deionized 1 time, then will be after washing after slowly shaking hatching at 30 DEG C 1 hour Fe3O4/TiO2/ Ag core-shell nano material is dissolved in 1 milliliter by PBS buffer solution and water by isometric mixed solution than mixing In, the tumor markers solution of 100 micrograms per millilitres is added thereto, in 30 DEG C slowly shake hatching 3.5 hours after, spend from Sub- water washing is primary, and Magnetic Isolation obtains the Fe for being marked with tumor markers3O4/TiO2/ Ag core-shell nano material, utilizes Raman Spectrometer, which carries out spectral measurement to it, can detect the content of tumor markers to be measured；
(2) after detecting, the Fe of tumor markers will be marked with3O4/TiO2/ Ag core-shell nano material is dissolved in deionized water, And be transferred into Clear glass bottles and jars, assemble Fe by the way of small magnet attraction3O4/TiO2/ Ag core-shell nano, is adopted It is irradiated with the ultraviolet lamp that wavelength is 265 nanometers, until tumor markers degradation completely, then will urge under photocatalysis Fe after change3O4/TiO2/ Ag core-shell nano material repeats step (1) method and links new tumor markers, can be realized to swollen The directly recyclable immune detection of tumor markers.
The concentration of succinic anhydride is 10.3 milligrams every milliliter in the DMF solution of succinic anhydride as described in step (1)；It is described EDC/NHS PBS solution in the concentration of EDC be 10 milligrams every milliliter, the concentration of NHS is 10 milligrams every milliliter.
The tumor markers include prostate-specific antigen PSA, AFP AFP, immune globulin IGg, carbohydrate Antigens c A199 and Carcinoembryonic Antigen CEA.
Compared with the prior art, the advantages of the present invention are as follows: present invention firstly discloses ferroso-ferric oxide/titanium dioxide/ The preparation method of galactic nucleus shell nanometer material and recyclable immunologic detection method to tumor markers, in three-layer nuclear shell structure Fe3O4Kernel makes triple core-shell structure have the function of Magnetic Isolation, and sample clean separation can be made more simple and quick. Simultaneously as Fe3O4The presence of kernel, when the tumor markers combined to core-shell nano material surface carry out photocatalytic degradation When, it can make core-shell nano material that enrichment of reuniting occur to a certain extent by externally-applied magnetic field, enhance the efficiency of catalysis. TiO2Subshell has good photocatalytic activity, and mechanism is under ultraviolet lighting, since the energy of photon is greater than TiO2Prohibit Bandwidth, the electronics (e in valence band-) absorption photon energy is excited on conduction band, while hole (h is left in valence band+). Work as TiO2There are when surface defect or suitable trapping agent, the compound of electrons and holes is inhibited, will be in TiO2Surface hair Raw oxidation-reduction reaction.Valence band hole is good oxidant, can be with TiO2The H of adsorption2In O molecule or reaction solution The OH of dissolution-In conjunction with the hydroxyl radical free radical (OH) for forming oxidisability wave very living.And conduction band electron is good reducing agent, is passed through With TiO2O that is adsorption or being dissolved in reaction solution2A series of intermediate reactions are carried out, finally can also form oxidisability very The hydroxyl radical free radical (OH) and Superoxide anion free radical (O of wave living2 -).Hydroxyl radical free radical (OH) and Superoxide anion free radical ( O2 -) various organic matters (including this single chain glycoprotein of prostate-specific antigen PSA) can be oxidized to CO2、H2O etc. is inorganic Small molecule, and because their oxidability is strong, it can be ensured that oxidation reaction does not stay in intermediate steps generally, does not generate Between product.Ag shell is made of the Ag nano particle largely to gather, and when there is ambient light photograph, surface electronic can be excited to be formed Local surface plasmon resonance has TiO around Ag nano particle2In the presence of, electronics a part of these excitations can lead to It crosses charge transfer process and reaches TiO2Among conduction band, the electrons increased among conduction band remarkably promote TiO2To tumor markers Catalytic degradation ability.After catalytic degradation, by suitably cleaning, Fe3O4/TiO2/ Ag core-shell nano material can also be recovered benefit With being detected in conjunction with new determined antigen again, realize a kind of detection of circulation immunity.Meanwhile passing through one step packet of hydro-thermal method The Ag shell covered is compared to the Ag particle that other methods such as Electrostatic Absorption method synthesizes, in amidized TiO2Adsorption is more tight It is close and uniform.Be conducive to Ag and TiO2Between charge transfer process facilitation effect, and then significantly increase TiO2Photocatalysis Activity realizes the catalytic degradation of this kind of macromolecular of tumor markers.Also, its surface electronic causes in excitation light action low-resonance Local electromagnetic field can significantly increase the specific Raman signals of tumor markers.Ag raman scattering cross section is larger, and enhancing is drawn Graceful scattering efficiency is higher, and the Surface enhanced Raman scattering base for being advantageously implemented the highly sensitive high specific of tumor markers is direct Immune detection avoids indirect detection and needs to introduce additional Raman molecular, the cumbersome disadvantage of operating procedure.In short, Fe3O4/ TiO2/ Ag core-shell nano material, not only had the function of Magnetic Isolation, catalytic degradation tumor markers effect but also have enhancing The effect of tumor markers Raman signal is a kind of novel multi-functional nanometer material.Pass through design synthesis this novel three Heavy nucleus shell structure especially controls Fe by systematically adjusting reaction condition during the reaction3O4Diameter, TiO2Centre The closeness of thickness degree and Ag shell, we can comprehensively utilize the respective advantage of above-mentioned three kinds of materials, realize tumor markers The directly recyclable immune detection of highly sensitive high specific.
Detailed description of the invention
Fig. 1 is the Fe prepared in the embodiment of the present invention 13O4The transmission electron microscope photo of nano material；
Fig. 2 is the Fe prepared in the embodiment of the present invention 13O4/TiO2The transmission electron microscope photo of core-shell nano material；
Fig. 3 is the Fe prepared in the embodiment of the present invention 13O4/TiO2The electron scanning micrograph of/Ag core-shell nano material；
Fig. 4 is that Ag particle is wrapped in Fe prepared by the present embodiment 1 using Electrostatic Absorption method3O4/TiO2Core-shell nano outer surface The Fe of formation3O4/TiO2The electron scanning micrograph of/Ag triple core-shell nano material；
Fig. 5 is that the Fe prepared in the embodiment of the present invention 1 is respectively adopted3O4/TiO2/ Ag core-shell nano material and Electrostatic Absorption legal system Standby Fe3O4/TiO2/ Ag triple core-shell nano material urges small molecule 4- mercaptobenzoic acid (0.01 mM every milliliter) The result of change；
Fig. 6 is the Fe prepared in the embodiment of the present invention 13O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA The carry out immune detection of (10 micrograms per millilitre) and the result being catalyzed；
Fig. 7 is the Fe prepared using Electrostatic Absorption method3O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA(10 Micrograms per millilitre) carry out immune detection and be catalyzed result；
Fig. 8 is the Fe prepared in the embodiment of the present invention 13O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA's Directly recyclable immune detection result；
Fig. 9 is the Fe prepared in the embodiment of the present invention 13O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA's Frequency displacement is 1264 cm in directly recyclable immune middle Raman spectrum-1Feature peak intensity with determined antigen concentration variation diagram；
Figure 10 is the Fe prepared in the embodiment of the present invention 23O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA It is directly recyclable immune in Raman spectrum frequency displacement be 1264 cm-1Feature peak intensity with determined antigen concentration variation diagram；
Figure 11 is the Fe prepared in the embodiment of the present invention 33O4/TiO2/ Ag core-shell nano material is to prostate-specific antigen PSA It is directly recyclable immune in Raman spectrum frequency displacement be 1264 cm-1Feature peak intensity with determined antigen concentration variation diagram.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.Embodiment Used in Raman spectrum detector BWS415 be purchased from U.S. Bi Da Imtech (B&W Tek Inc.).