CN110531061B - A circulating immunodetection method for tumor markers based on core-shell nanomaterials - Google Patents

Abstract

The invention discloses a preparation method of ferroferric oxide/titanium dioxide/silver core-shell nano material and direct recyclable immunodetection application thereof, which is characterized in that a triple core-shell nano material is activated to be combined with antigen to be detected, the direct Raman detection of the antigen to be detected is realized by utilizing the surface enhanced Raman scattering effect, after the Raman detection is finished, the antigen is degraded by ultraviolet illumination on the immune combined nano material, and Fe without the antigen existing on the surface is recovered by magnetic force₃O₄/TiO₂The Ag core-shell nano material is used for detecting an antigen to be detected next time, the Raman detection-catalytic degradation-Raman detection process is repeated, and the tumor marker can be directly subjected to circulating immunodetection.

Description

A circulating immunodetection method for tumor markers based on core-shell nanomaterials

technical field

The invention relates to the fields of material engineering and nanotechnology, in particular to a preparation method of a ferric tetroxide/titanium dioxide/silver (Fe ₃ O ₄ /TiO ₂ /Ag) core-shell nanomaterial and its recyclable immunodetection application.

Background technique

In recent years, with the intensification of environmental pollution, the continuous change of the global climate, the occurrence of food crisis and the increase of people's living pressure, cancer frequently occurs, and its high mortality has become a serious threat to human health. Therefore, a series of advanced immune technologies, such as chemiluminescence and enzyme-linked immunoassay, have been developed to detect and treat cancer patients. However, for patients in the early stages of cancer, their blood levels of cancer markers are low, making it difficult to detect and treat early. The surface-enhanced Raman scattering immune technology developed in recent years takes full advantage of the high electromagnetic field enhancement and single-molecule level of surface-enhanced Raman scattering to achieve trace detection of tumor markers. However, the materials based on surface-enhanced Raman scattering technology are mostly noble metal nanomaterials with high preparation cost, which is not conducive to its practical application in clinical immunodetection. Therefore, it is necessary to develop a circulating immunoassay technique. Titanium dioxide nanomaterials have excellent photocatalytic activity and are a potential key material to realize this recyclable immunodetection technology. At the same time, how to use diversified nanomaterials with excellent opto-electromagnetic properties to further improve the photocatalytic activity of composite nanomaterials and promote the application of recyclable immunoassay technology in in-situ rapid immunoassay is still the direction of researchers' efforts.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a preparation method of ferric tetroxide/titanium dioxide/silver core-shell nanomaterials that not only has the functions of magnetic separation and Raman enhancement, but also has low detection limit for catalytic degradation of cancer markers. It can be used for circulating immunoassay applications.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of ferric tetroxide/titanium dioxide/silver core-shell nanomaterial, comprising the following steps:

(1) Dissolve 1.35-6.75 g of ferric chloride hexahydrate, 3.6-18.0 g of sodium acetate and 1.0-5.0 g of polyethylene glycol in 40-200 ml of ethylene glycol, sonicate until completely dissolved, then transfer to water In a thermal reaction kettle, react at 200°C for 10 hours, centrifuge and wash with ethanol and collect the precipitate to obtain Fe ₃ O ₄ nanoparticles;

(2) Weigh 50 milligrams of Fe ₃ O ₄ nanoparticles obtained in step (1), dissolve them in an aqueous hydrochloric acid solution with a concentration of 0.1 mmol per milliliter, and ultrasonically for 15 minutes, magnetically separate and wash three times with deionized water; after magnetic separation again, the The Fe ₃ O ₄ nanoparticles were dissolved in 120 ml of a mixed solution of ethanol and acetonitrile in a volume ratio of 3:1. After ultrasonically dissolving it completely, 500 microliters of ammonia water was added to it, and the mixture was uniformly mixed by ultrasonication for 5 minutes. Then, 1-6 ml of tetrabutyl titanate was added dropwise under stirring conditions, and after 1.5 hours of reaction, magnetic separation and three washings with ethanol, Fe ₃ O ₄ /TiO ₂ nanoparticles were obtained;

(3) Dissolve 81 mg of Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (2) and 75.4 mg of ammonium fluoride in 20.45 ml of a mixed solution of ethanol and water in a volume ratio of 13.5:6.95, ultrasonically After it was fully dissolved, the reaction solution was placed at room temperature and continued to stir for 1 hour at a stirring speed of 180-200 rpm. After the stirring was completed, the reaction solution was transferred to the reaction kettle and reacted at 180 ° C for 24 hours. After the reaction kettle was naturally cooled to room temperature, magnetically separated and washed three times with deionized water to obtain crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles;

(4) Dissolve 81 mg of the crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (3) in 33.4 ml of ethanol, add 25 μl of 3-aminopropyltrimethoxysiloxane dropwise with continuous stirring alkane, the reaction solution was condensed and refluxed at 80°C for 4 hours, after it was naturally cooled to room temperature, magnetically separated and washed with ethanol three times to obtain aminated Fe ₃ O ₄ /TiO ₂ nanoparticles;

(5) 1 g of polyvinyl pyrrolidone (molecular weight 5800 g per mole) and 28 mg of the aminated Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (4) were dissolved in 10 ml of deionized water and mixed uniformly, and then placed in a mechanical The silver ammonia solution was added dropwise under stirring and transferred to a hydrothermal reactor, sealed and then placed at 120°C for 11 hours of reaction. After the reactor was naturally cooled to room temperature, magnetically separated and washed sequentially with tetrahydrofuran, ethanol and deionized water. , namely obtain Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, and store them in deionized water for later use, wherein the preparation method of silver ammonia solution is: take 2 ml of silver nitrate aqueous solution with a concentration of 0.01-0.08 mg per ml , adding excess ammonia water until the reaction solution becomes clear to obtain silver ammonia solution.

The circulating immunodetection method for tumor markers based on the ferric oxide/titanium dioxide/silver core-shell nanomaterial obtained by the above preparation method includes the following steps:

(1) Dissolve 15 mg of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials in 2 ml of dimethylformamide (DMF) solution, then add 1 ml of succinic anhydride in DMF solution, slowly at 70°C After shaking and incubating for 24 hours, magnetic separation and washing twice with deionized water yielded carboxylated Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, which were dissolved in 1 mL of water, and then 1 mL of EDC/NHS was added. PBS solution, incubate with slow shaking at 30 °C for 1 hour, wash with deionized water once, and then dissolve the washed Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials in 1 ml of PBS buffer and water Add 100 μg/ml of tumor marker solution to the mixed solution mixed in equal volume ratio, incubate with slow shaking at 30°C for 3.5 hours, wash once with deionized water, and magnetically separate the tumor marker Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, and the content of tumor markers to be detected can be detected by spectral measurement of Raman spectrometer;

(2) After the detection, the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials labeled with tumor markers were dissolved in deionized water, transferred to a transparent glass bottle, and gathered by small magnet attraction Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanoparticles were irradiated with a UV lamp with a wavelength of 265 nm, under the action of photocatalysis until the tumor markers were completely degraded, and then the catalyzed Fe ₃ O ₄ /TiO ₂ / The Ag core-shell nanomaterial repeats the method of step (1) to link the new tumor marker, so that the direct circulating immune detection of the tumor marker can be realized.

The concentration of succinic anhydride in the DMF solution of succinic anhydride described in step (1) is 10.3 milligrams per milliliter; the concentration of EDC in the PBS solution of described EDC/NHS is 10 milligrams per milliliter, and the concentration of NHS is 10 milligrams per milliliter. milligrams per milliliter.

The tumor markers include prostate specific antigen PSA, alpha-fetoprotein AFP, immunoglobulin IGg, carbohydrate antigen CA199 and carcinoembryonic antigen CEA.

Compared with the prior art, the present invention has the advantages that: the present invention discloses for the first time a preparation method of ferric oxide/titanium dioxide/silver core-shell nanomaterials and a method for circulating immune detection of tumor markers, and a three-layer core-shell structure. The Fe ₃ O ₄ core in the core makes the triple core-shell structure have the effect of magnetic separation, which can make the sample cleaning and separation easier and faster. At the same time, due to the existence of Fe ₃ O ₄ core, when the tumor markers bound on the surface of core-shell nanomaterials are photocatalytically degraded, the core-shell nanomaterials can be agglomerated and enriched to a certain extent by applying an external magnetic field to enhance the catalytic performance. efficiency. The _TiO2 intermediate shell has good photocatalytic activity, the mechanism is that under ultraviolet light, since the energy of the photon is greater than the band gap of _TiO2 , the electrons (e ^- ) in the valence band absorb the photon energy and are excited to the conduction band , while leaving a hole (h ⁺ ) in the valence band. When TiO ₂ has surface defects or suitable capture agents, the recombination of electrons and holes is inhibited, and an oxidation-reduction reaction occurs on the surface of TiO ₂ . The valence band holes are good oxidants, which can combine with H ₂ O molecules adsorbed on the surface of TiO ₂ or OH ^- dissolved in the reaction solution to form hydroxyl radicals (·OH) with very active oxidative waves. The conduction band electron is a good reducing agent. Through a series of intermediate reactions with _O2 adsorbed on the surface of _TiO2 or dissolved in the reaction solution, it can also form hydroxyl radicals ( OH) and superoxide radicals with very active oxidative waves finally. Oxygen ion radical (·O ₂ ^- ). Hydroxyl radicals (·OH) and superoxide ion radicals (·O ₂ ^- ) can oxidize various organic substances (including single-chain glycoproteins such as prostate specific antigen PSA) into inorganic small molecules such as CO ₂ and H ₂ O , and because of their strong oxidizing ability, it can ensure that the oxidation reaction generally does not stay in the intermediate steps and does not produce intermediate products. The Ag shell is composed of a large number of dense Ag nanoparticles. When there is external light, the surface electrons will be excited to form localized surface plasmon resonance. When there is _TiO2 around the Ag nanoparticles, some of these excited electrons will be excited. It can reach the conduction band of TiO ₂ through the process of charge transfer, and the increased electrons in the conduction band will significantly promote the catalytic degradation ability of TiO ₂ to tumor markers. After catalytic degradation, through proper cleaning, the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials can be recycled and combined with new antigens to be tested for detection, thereby realizing a circular immunodetection. Meanwhile, the Ag shell layer coated by one-step hydrothermal method is more tightly and uniformly adsorbed on the surface of aminated _TiO2 than Ag particles synthesized by other methods such as electrostatic adsorption. It is beneficial to the promotion effect of the charge transfer process between Ag and _TiO2 , thereby significantly enhancing the photocatalytic activity of _TiO2 , and realizing the catalytic degradation of macromolecules such as tumor markers. Moreover, the localized electromagnetic field induced by the resonance of its surface electrons under the action of excitation light can significantly enhance the specific Raman signal of tumor markers. The Ag Raman scattering cross section is larger, and the enhanced Raman scattering efficiency is higher, which is conducive to the realization of high-sensitivity and high-specificity surface-enhanced Raman scattering-based direct immunodetection of tumor markers, avoiding the need to introduce additional Raman molecules for indirect detection. , the disadvantage of more complicated operation steps. In conclusion, Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials not only have the function of magnetic separation, catalytic degradation of tumor markers, but also enhance the Raman signal of tumor markers, which is a novel multifunctional nanomaterials. By designing and synthesizing this novel triple core _- shell structure, especially by systematically adjusting the reaction conditions during the reaction, controlling the diameter of _Fe3O4 , the interlayer thickness of _TiO2 , and the density of Ag shell layers, we were able to synthesize the Utilizing the respective advantages of the above three materials, high-sensitivity and high-specificity direct circulating immunodetection of tumor markers can be achieved.

Description of drawings

1 is a transmission electron microscope photograph of Fe ₃ O ₄ nanomaterials prepared in Example 1 of the present invention;

Fig. 2 is the transmission electron microscope photograph of Fe ₃ O ₄ /TiO ₂ core-shell nanomaterial prepared in Example 1 of the present invention;

3 is a scanning electron microscope photograph of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention;

Fig. 4 is the scanning electron of Fe ₃ O ₄ /TiO ₂ /Ag triple core-shell nanomaterial formed by encapsulating Ag particles on the outer surface of Fe ₃ O ₄ /TiO ₂ core-shell nanomaterial prepared in Example 1 by electrostatic adsorption microscope photos;

Fig. 5 shows the effect of Fe _{3 O 4 /TiO 2 /Ag core-shell nanomaterial prepared in Example 1 of the present invention and Fe 3 O 4 / TiO 2 /} Ag triple core-shell nanomaterial prepared by electrostatic adsorption on small molecules 4 - the result of catalysis by mercaptobenzoic acid (0.01 mmol per ml);

Figure 6 is the result of immunodetection and catalysis of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention to prostate specific antigen PSA (10 micrograms per milliliter);

Figure 7 shows the results of immunodetection and catalysis of prostate specific antigen PSA (10 micrograms per milliliter) by Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption;

Fig. 8 is the direct circulating immunodetection result of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention on prostate specific antigen PSA;

Fig. 9 is the characteristic peak intensity with a frequency shift of 1264 cm ^-1 in the Raman spectrum of the direct circulating immunity of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention to prostate specific antigen PSA Change graph of the concentration of the antigen to be tested;

Fig. 10 is the characteristic peak intensity with frequency shift of 1264 cm ^-1 in the Raman spectrum of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 2 of the present invention in direct circulating immunity to prostate specific antigen PSA Change graph of the concentration of the antigen to be tested;

Figure 11 shows the characteristic peak intensity with a frequency shift of 1264 cm ^-1 in the Raman spectrum of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 3 of the present invention in direct circulating immunity to prostate specific antigen PSA Graph of changes in the concentration of the antigen to be tested.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities. The Raman spectrometer BWS415 used in the examples was purchased from B&W Tek Inc., USA.

Example 1

A Fe ₃ O ₄ /TiO ₂ /Ag triple core-shell nanomaterial and its direct recyclable immunodetection method for prostate specific antigen PSA, comprising the following steps:

1. Preparation of Fe ₃ O ₄ /TiO ₂ /Ag triple core-shell nanomaterials

(1) Dissolve 3.5 grams of ferric chloride hexahydrate, 10.8 grams of sodium acetate and 3 grams of polyethylene glycol in 120 milliliters of ethylene glycol, ultrasonically until completely dissolved, transfer to a hydrothermal reactor, and heat at 200° C. The reaction was carried out for 10 hours, and the precipitate was collected after centrifugation and washing with ethanol to obtain Fe ₃ O ₄ nanoparticles;

(2) Weigh 50 milligrams of Fe ₃ O ₄ nanoparticles obtained in step (1), dissolve them in an aqueous hydrochloric acid solution with a concentration of 0.1 mmol per milliliter, and ultrasonically for 15 minutes, magnetically separate and wash three times with deionized water; after magnetic separation again, the The Fe ₃ O ₄ nanoparticles were dissolved in 120 ml of a mixed solution of ethanol and acetonitrile in a volume ratio of 3:1. After ultrasonically dissolving it completely, 500 microliters of ammonia water was added to it, and the mixture was uniformly mixed by ultrasonication for 5 minutes. Then, 3 ml of tetrabutyl titanate was added dropwise under stirring conditions and reacted for 1.5 hours. After the reaction, magnetic separation was performed and washed with ethanol three times to obtain Fe ₃ O ₄ /TiO ₂ nanoparticles;

(3) Dissolve 81 mg of Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (2) and 75.4 mg of ammonium fluoride in 20.45 ml of a mixed solution of ethanol and water in a volume ratio of 13.5:6.95, ultrasonically After it was fully dissolved, the reaction solution was placed at room temperature and continued to stir for 1 hour at a stirring speed of 180-200 rpm. After the stirring was completed, the reaction solution was transferred to the reaction kettle and reacted at 180 ° C for 24 hours. After the reaction kettle was naturally cooled to room temperature, magnetically separated and washed three times with deionized water to obtain crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles;

(4) Dissolve 81 mg of the crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (3) in 33.4 ml of ethanol, add 25 μl of 3-aminopropyltrimethoxysiloxane dropwise with continuous stirring alkane, the reaction solution was condensed and refluxed at 80°C for 4 hours, after it was naturally cooled to room temperature, magnetically separated and washed with ethanol three times to obtain aminated Fe ₃ O ₄ /TiO ₂ nanoparticles;

(5) 1 g of polyvinyl pyrrolidone (molecular weight 5800 g per mole) and 28 mg of the aminated Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step (4) were dissolved in 10 ml of deionized water and mixed uniformly, and then placed in a mechanical The silver ammonia solution was added dropwise under stirring and transferred to a hydrothermal reactor, sealed and then placed at 120°C for 11 hours of reaction. After the reactor was naturally cooled to room temperature, magnetically separated and washed sequentially with tetrahydrofuran, ethanol and deionized water. , namely obtain Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, and store them in deionized water for later use, wherein the preparation method of silver ammonia solution is: take 2 ml of silver nitrate aqueous solution with a concentration of 0.01-0.08 mg per ml , adding excess ammonia water until the reaction solution becomes clear to obtain silver ammonia solution.

2. Direct circulating immunodetection of prostate characteristic antigen PSA based on Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials

(1) 15 mg of Fe ₃ O ₄ /TiO ₂ /Ag triple core-shell nanomaterials were dissolved in 2 ml of dimethylformamide (DMF) solution, and then 1 ml of succinic anhydride with a concentration of 10.3 mg per ml was added DMF solution was incubated at 70°C with slow shaking for 24 hours, magnetically separated and washed twice with deionized water to obtain carboxylated Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, which were dissolved in 1 ml of water. Then add 1 ml of EDC/NHS in PBS, incubate with slow shaking at 30°C for 1 hour, wash once with deionized water, and dissolve the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials after washing. Add 100 micrograms per milliliter of prostate-specific antigen PSA solution to 1 ml of a mixed solution consisting of PBS buffer and water in an equal volume ratio. Washing once, magnetic separation to obtain Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials labeled with PSA, and the content of the antigen PSA to be detected can be detected by spectral measurement of Raman spectrometer;

(2) After the detection, the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials labeled with PSA were dissolved in deionized water, transferred to a transparent glass bottle, and the nanoparticles were aggregated by attracting small magnets , irradiated with a UV lamp with a wavelength of 265 nm, and detected the Raman signal every ₂₀ minutes until the Raman characteristic peak of PSA disappeared, which proved that the PSA had been degraded completely _; /TiO ₂ /Ag core-shell nanomaterials, and then link to a new PSA, conduct Raman detection and then UV light for catalysis, and repeat this process of immune linking, Raman detection and photocatalytic degradation cyclically to achieve direct recyclable immunity detection.

Figure 1 shows a transmission electron microscope photograph of the Fe ₃ O ₄ nanomaterials prepared in this example. It can be seen from Fig. 1 that the diameter of the as _- prepared _Fe3O4 nanomaterials is 240-280 nm.

Figure 2 shows the transmission electron microscope photo of the Fe ₃ O ₄ /TiO ₂ core-shell nanomaterial prepared in this example. It can be seen from Figure 2 that the prepared material has a core-shell structure, and the thickness of the TiO ₂ shell is 20 nano.

FIG. 3 shows the scanning electron microscope photo of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in this example. It can be seen from FIG. 3 that the prepared Fe ₃ O ₄ /TiO ₂ /Ag core-shell The surface of the nanomaterial is a large number of silver nanoparticles, which are closely arranged.

Fig. 4 shows the scanning electron microscope of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial formed by encapsulating Ag particles on the outer surface of Fe ₃ O ₄ /TiO ₂ core-shell nanomaterial prepared in this example by electrostatic adsorption As can be seen from Figure 4, the surface of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared is a small amount of sparse silver nanoparticles, and the silver nanoparticles and the outer surface of Fe ₃ O ₄ /TiO ₂ are relatively combined Loose and uneven.

Fig. 5 shows the effects of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared in Example 1 of the present invention and Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption on small molecules 4- The result of catalysis by mercaptobenzoic acid (0.01 mmol per milliliter), it can be seen from Figure 5 that when the catalysis time is extended to 70 minutes, 4-mercaptobenzoic acid has been prepared by Fe ₃ O prepared in Example 1 of the present invention. The catalytic degradation of ₄ /TiO ₂ /Ag core-shell nanomaterials was complete, while the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption did not catalyze the degradation of 4-mercaptobenzoic acid molecules completely. Therefore, the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention has higher catalytic activity.

Figure 6 shows the immunodetection and catalysis results of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared in Example 1 of the present invention for prostate specific antigen PSA (10 micrograms per milliliter), as can be seen from Figure 6 It was found that a relatively obvious Raman spectrum appeared after immunocombination, which proved that the antigen PSA had been detected in combination with the core-shell nanomaterials. After UV light catalysis, the characteristic Raman spectrum intensity is almost reduced to zero, which proves that the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention has completely catalyzed and decomposed the test antigen PSA. This is mainly due to the fact that the Ag particles coated by the hydrothermal method are more closely and uniformly distributed on the outer surface of the Fe ₃ O ₄ /TiO ₂ core-shell nanomaterial, which is beneficial to the charge transfer process between Ag and TiO ₂ . The promotion effect, which in turn significantly enhances the photocatalytic activity of _TiO2 , realizes the catalytic degradation of macromolecules such as tumor markers.

Figure 7 shows the immunodetection and catalysis results of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption on prostate specific antigen PSA (10 micrograms per milliliter). After immunocombination, a relatively obvious Raman spectrum also appeared, which proves that Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption also have a relatively good SERS enhancement effect, which can realize the immunodetection of antigen PSA. However, after catalysis by ultraviolet light, it can be found that the above characteristic Raman spectrum intensity is only slightly reduced, which proves that Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials prepared by electrostatic adsorption method cannot catalyze the antigen PSA to be tested. break down. This is mainly because the Ag particles coated by electrostatic adsorption method are relatively loose and unevenly distributed on the outer surface of Fe ₃ O ₄ /TiO ₂ core-shell nanomaterials, which is not conducive to the realization of charge transfer between Ag and TiO ₂ . The promotion of the process cannot enhance the photocatalytic activity of TiO ₂ , and thus cannot achieve the catalytic degradation of macromolecules such as tumor markers. Therefore, by using the hydrothermal synthesis method to coat silver nanoparticles, a triple core-shell nanomaterial with good structure can be obtained, and a relatively good catalytic activity and cyclic immunocatalytic effect can be obtained.

FIG. 8 is the direct circulating immunodetection result of the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial prepared in Example 1 of the present invention for prostate specific antigen PSA. It can be seen from Figure 8 that the Raman spectrum of the Raman-labeled molecule disappears after each photocatalytic degradation process, indicating that the antigen-antibody has been completely catalyzed. After eight cycles of immunolinking, Raman detection, and photocatalytic degradation, the detection limit of prostate-specific antigen (PSA) can reach 100 pg/mL.

Figure 9 is a graph showing the variation of the characteristic peak intensity with a frequency shift of 1264 cm ^-1 in the Raman spectrum with the concentration of the antigen to be tested. It can be seen from fitting that when the concentration of the antigen to be tested changes from 100 pg/ml to 0.1 mg/ml, The intensity of Raman characteristic peaks varies linearly with concentration. The fitting results show that this trend is in line with the linear equation of Y=8750+792X, and the fitting degree is 0.985.

Example 2

With the above-mentioned embodiment 1, the difference is: in the preparation of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials: in step (1), 1.35 grams of ferric trichloride hexahydrate, 3.6 grams of sodium acetate and 1.0 grams of poly Ethylene glycol was dissolved in 40 ml of ethylene glycol, sonicated until completely dissolved, transferred to a hydrothermal reactor, reacted at 200 ° C for 10 hours, centrifuged and washed with ethanol to collect the precipitate to obtain Fe ₃ O ₄ nanoparticles ; The volume of tetrabutyl titanate added in step (2) is 1 ml.

Figure 10 is a graph showing the variation of the characteristic peak intensity with a frequency shift of 1264 cm ^-1 in the Raman spectrum with the concentration of the antigen to be tested. It can be seen from the fitting that when the concentration of the antigen to be tested changes from 100 pg/ml to 0.1 mg/ml, The intensity of Raman characteristic peaks varies linearly with concentration. The fitting result shows that this change trend is in line with the linear equation of Y=8734+806X, and the fitting degree is 0.972.

Example 3

With the above embodiment 1, the difference is: in the preparation of Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials: in step (1), 6.75 grams of ferric chloride hexahydrate, 18.0 grams of sodium acetate and 5.0 grams of poly Ethylene glycol was dissolved in 200 ml of ethylene glycol, sonicated until completely dissolved, transferred to a hydrothermal reactor, reacted at 200°C for 10 hours, centrifuged and washed with ethanol to collect the precipitate to obtain Fe ₃ O ₄ nanoparticles ; The volume of tetrabutyl titanate added in step (2) is 6 ml.

Figure 11 is a graph showing the variation of the intensity of the characteristic peak with a frequency shift of 1264 cm ^-1 in the Raman spectrum with the concentration of the antigen to be tested. It can be seen from fitting that when the concentration of the antigen to be tested changes from 100 pg/ml to 0.1 mg/ml, The intensity of Raman characteristic peaks varies linearly with concentration. The fitting result shows that this change trend conforms to the linear equation of Y=8930+840X, and the fitting degree is 0.981.

In addition to the above examples, the tumor markers can also be alpha-fetoprotein AFP, immunoglobulin IGg, carbohydrate antigen CA199, carcinoembryonic antigen CEA, and the like.

The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention should also belong to the protection scope of the present invention.

Claims (2)

1. a non-therapeutic and/or diagnostic purpose-based tumor marker circulatory immune detection method based on ferric oxide/titanium dioxide/silver core-shell nanomaterials, is characterized in that comprising the following steps: (1) the preparation method of described ferric oxide/titanium dioxide/silver core-shell nanomaterial 1-1. Dissolve 1.35-6.75 g of ferric chloride hexahydrate, 3.6-18.0 g of sodium acetate and 1.0-5.0 g of polyethylene glycol in 40-200 ml of ethylene glycol, ultrasonicate until completely dissolved, then transfer to In a hydrothermal reactor, react at 200° C. for 10 hours, centrifuge and wash with ethanol and collect the precipitate to obtain Fe ₃ O ₄ nanoparticles; 1-2. Weigh 50 mg of Fe ₃ O ₄ nanoparticles obtained in step 1-1, dissolve them in an aqueous hydrochloric acid solution with a concentration of 0.1 mmol per milliliter, and ultrasonicate for 15 minutes, separate magnetically and wash three times with deionized water; after magnetic separation again, Dissolve Fe ₃ O ₄ nanoparticles in 120 ml of a mixed solution of ethanol and acetonitrile in a volume ratio of 3:1, and after ultrasonication makes it completely dissolved, add 500 microliters of ammonia water to it, and ultrasonicate for 5 minutes to mix uniform; then add 1-6 ml of tetrabutyl titanate dropwise under stirring conditions, react for 1.5 hours, magnetically separate and wash with ethanol three times to obtain Fe ₃ O ₄ /TiO ₂ nanoparticles; 1-3. Dissolve 81 mg of Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step 1-2 and 75.4 mg of ammonium fluoride in 20.45 ml of a mixed solution of ethanol and water in a volume ratio of 13.5:6.95, After ultrasonically dissolving it fully, the reaction solution was placed at room temperature and stirred continuously for 1 hour, and the stirring speed was 180-200 rpm. After the stirring was completed, the reaction solution was transferred to the reaction kettle, and the reaction was carried out at 180 ° C for 24 hours. After the reaction kettle was naturally cooled to room temperature, magnetic separation was performed and washed three times with deionized water to obtain crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles; 1-4. Dissolve 81 mg of the crystallized Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step 1-3 in 33.4 ml of ethanol, and add 25 μl of 3-aminopropyltrimethoxysilicon dropwise with constant stirring Oxane, the reaction solution was condensed and refluxed at 80°C for 4 hours, after it was naturally cooled to room temperature, magnetically separated and washed with ethanol three times to obtain aminated Fe ₃ O ₄ /TiO ₂ nanoparticles; 1-5. Dissolve 1 g of polyvinylpyrrolidone and 28 mg of the aminated Fe ₃ O ₄ /TiO ₂ nanoparticles obtained in step 1-4 in 10 ml of deionized water and mix well, then add silver dropwise under mechanical stirring The ammonia solution was transferred to a hydrothermal reactor, sealed and then placed at 120°C for reaction for 11 hours. After the reactor was naturally cooled to room temperature, it was magnetically separated and washed with tetrahydrofuran, ethanol and deionized water in turn to obtain Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, and placed in deionized water for storage for later use, wherein the preparation method of silver ammonia solution is: take 2 ml of silver nitrate aqueous solution with a concentration of 0.01-0.08 mg per ml, add excess The ammonia water until the reaction solution becomes clear to obtain silver ammonia solution; (2) The tumor marker circulating immune detection method 2-1. Dissolve 15 mg Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials in 2 ml of dimethylformamide solution, then add 1 ml of succinic anhydride in DMF solution, and incubate with slow shaking at 70°C After 24 hours, magnetic separation was performed and washed twice with deionized water to obtain carboxylated Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, which were dissolved in 1 mL of water, and then 1 mL of EDC/NHS in PBS was added. , incubate at 30℃ for 1 hour, wash with deionized water once, and then dissolve the washed Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterial in 1 ml by PBS buffer and water, etc. Add 100 μg/ml of tumor marker solution to the mixed solution by volume ratio, incubate with slow shaking at 30°C for 3.5 hours, wash once with deionized water, and magnetically separate Fe labeled with tumor markers. ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials, the content of tumor markers to be tested can be detected by spectral measurement of Raman spectrometer; 2-2. After the detection, dissolve the Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials marked with tumor markers in deionized water, and transfer them to a transparent glass bottle, using a small magnet to attract Aggregate Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanoparticles, irradiated with a UV lamp with a wavelength of 265 nm, under photocatalysis until the tumor markers are completely degraded, and then catalyzed Fe ₃ O ₄ /TiO ₂ /Ag core-shell nanomaterials repeat step 2-1 to link new tumor markers, so that the direct circulating immune detection of tumor markers can be realized. 2. a kind of non-treatment and/or diagnostic purpose-based tumor marker circulatory immunodetection method based on ferric oxide/titanium dioxide/silver core-shell nanomaterials according to claim 1, is characterized in that: step 2- The concentration of succinic anhydride in the DMF solution of succinic anhydride described in 1 is 10.3 mg per ml; the concentration of EDC in the PBS solution of EDC/NHS is 10 mg per ml, and the concentration of NHS is 10 mg per ml .

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