WO2022027976A1 - Method for detecting prostate cancer exosome on basis of fe3o4@sio2@tio2 nanoparticle enrichment and psma sensor - Google Patents

Abstract

Provided is a method for detecting a prostate cancer exosome on the basis of Fe3O4@SiO2@TiO2 nanoparticle enrichment and a PSMA sensor. According to the method, Fe3O4@SiO2@TiO2 nanoparticles are used for enriching an exosome, then a hairpin-like PSMA aptamer sensor is constructed to quantify the exosome by means of measuring a change in fluorescence intensity. The method can be used to successfully distinguish a prostate cancer cell exosome from a normal prostate cell exosome. The method is used for clinical prostate cancer patient serum samples and normal human serum samples, can quickly and accurately detect prostate cancer in patients, and is verified by a traditional ELISA method. The method is simple and fast, exosome enrichment may be completed within 8 minutes, and the enrichment efficiency is 91.5%, thereby having a high enrichment efficiency. The detection limit is 9×103 exosomes/μL, the sensitivity is high, and the specificity is strong, and clinical prostate cancer patients and healthy people can be distinguished by means of same.

Description

Fe-based 3O 4@SiO 2@TiO 2 Nanoparticle enrichment and PSMA sensor for detection of prostate cancer exosomes technical field

The invention is applied to the technical field of detection and analysis of exosome markers, and relates to a rapid and accurate analysis and determination of prostate cancer exosomes based on _TiO2 and PSMA aptamers.

Background technique

Prostate cancer (PCa) is the most common solid malignancy in men worldwide. At an early stage, it has a high cure rate, so the treatment of prostate cancer relies on early diagnosis. Prostate-specific antigen (PSA) is a serum biomarker for prostate cancer diagnosis. However, although PSA has high sensitivity, its specificity is poor, especially in men with serum PSA levels of 2-10 ng/mL, therefore, other specific PCa biomarkers are needed in urology. Exosomes are biological vesicles ranging in size from 30 to 200 nanometers, encapsulated within a lipid bilayer membrane, secreted by normal and tumor cells. Exosomes play different roles in intercellular communication and are widely found in body fluids, including blood, urine, saliva, etc. Tumor exosomes contain tumor-specific proteins and RNAs, and are involved in tumorigenesis and pathogen spread. Many studies have shown that tumor exosomes are an ideal marker for diagnosing cancer, but there are still challenges in their isolation and detection methods. Ultracentrifugation is the most commonly used method for exosome isolation, but the steps are complicated and the isolation efficiency is low. Commercial ELISA kits use CD-63, CD-9 (exosome surface antibody) as targets to isolate and detect exosomes based on immunoaffinity methods, which have high sensitivity, but the process is complicated and costly.

Due to its reversible binding effect with phosphate groups, TiO ₂ is widely used in the separation and enrichment of phosphopeptides and proteins, while the surface of exosomes is rich in phosphate groups, so TiO ₂ can be used to separate and enrich exosomes. It has been reported that TiO ₂ can successfully enrich exosomes, but subsequent solid-liquid separation by centrifugation may lose a large amount of exosomes.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a rapid, highly sensitive and highly specific method for detecting prostate cancer exosomes.

The technical solutions of the present invention are specifically introduced as follows.

The present invention provides a method for detecting prostate cancer exosomes based on Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticle enrichment and PSMA sensor, comprising the following steps:

1) Synthesize Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles, co-incubate with samples containing prostate cell exosomes and magnetically separate them to achieve the separation of exosomes;

2) Co-incubate the isolated exosomes with a prostate-specific membrane antigen (PSMA) sensor, and obtain a fluorescence intensity signal, and quantify the exosomes by measuring the change in fluorescence intensity.

Further, in step 1), the preparation method of Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles is as follows:

Dissolve Fe ₃ O ₄ in a mixture of ethanol, deionized water and ammonia water, sonicate, then add TEOS, and continuously stir the reaction at room temperature to obtain Fe ₃ O ₄ @SiO ₂ ;

Fe ₃ O ₄ @SiO ₂ was dissolved in a mixture of ethanol and ammonia water, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. The reaction was continuously stirred, and the prepared product was washed with ultrapure water and ethanol, and dried to obtain Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles.

Further, the optimal conditions for exosome isolation in the present invention are: 1.2 mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ , incubated at room temperature for 8 minutes.

Further, in step 1), after magnetic separation, alkaline solution is eluted.

Further, the elution step used 10% ammonia water to co-incubate with magnetically separated Fe ₃ O ₄ @SiO ₂ @TiO ₂ /exosome complexes with shaking.

Preferably, in the elution step, 10% ammonia water and the magnetically separated Fe ₃ O ₄ @SiO ₂ @TiO ₂ /exosome complexes are used for shaking and co-incubating at 4 degrees Celsius for 10 minutes to achieve sufficient elution.

Further, in step 2), the hairpin-shaped PSMA sensor is constructed as follows: the PSMA antibody sequence is designed to form a hairpin-shaped structure, and a carboxytetramethylrhodamine fluorescent group ( TAMRA) and azobenzoic acid (Dabcyl) quenching groups.

Further, in step 2), the PSMA antibody nucleotide sequence is shown in SEQ No.3.

Further, the PSMA sensor concentration was 400 nM, and the cells were incubated at 25 degrees for 30 minutes.

Further, in step 2), the conditions for obtaining the fluorescence intensity: the excitation light is 557 nanometers, and the emission light is 580 nanometers.

The present invention also provides a Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticle, and the preparation method of the nanoparticle is as follows:

Dissolve Fe ₃ O ₄ in a mixture of ethanol, deionized water and ammonia water, sonicate, then add TEOS, and continuously stir the reaction at room temperature to obtain Fe ₃ O ₄ @SiO ₂ ;

Fe ₃ O ₄ @SiO ₂ was dissolved in a mixture of ethanol and ammonia water, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. The reaction was continuously stirred, and the prepared product was washed with ultrapure water and ethanol, and dried to obtain Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles.

The invention also provides the use of Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles in the detection of prostate cancer exosomes. Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles are used to co-incubate with exosomes and Magnetic separation to achieve separation and enrichment of exosomes.

The invention provides a prostate cancer exosome detection device, in which Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles are used to separate and enrich exosomes, and are incubated with a PSMA sensor.

Specifically, in an embodiment of the present invention,

The synthesis of Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles in the present invention for the enrichment of exosomes is mainly based on the reversible combination of TiO 2 and phosphate groups. A prostate-specific membrane antigen (PSMA) sensor was then designed.

PSMA is a specific protein on the surface of prostate cancer exosomes. It is highly expressed on the surface of prostate disease exosomes and lowly expressed on normal exosomes. The specific detection of prostate exosomes can be achieved by detecting PSMA. The PSMA sensor is a hairpin structure composed of PSMA protein aptamer, TAMRA fluorophore and Dabcyl quenching group. After the construction is completed, the fluorescence of PSMA sensor TAMRA is quenched due to the fluorescence resonance energy transfer effect. When it encounters exosomes When the PSMA aptamer specifically binds to the PSMA protein on the surface of exosomes, the fluorescence resonance energy transfer effect disappears, and TAMRA re-emits fluorescence.

Compared with the prior art, the present invention has the following beneficial effects:

Fe ₃ O ₄ @SiO ₂ @TiO 2 is synthesized in the present invention, and Fe ₃ O ₄ is used as the inner core, and SiO ₂ is beneficial to the connection of the two materials, and after the enrichment is completed, it can be rapidly magnetically separated to reduce the loss of exosomes. The present invention combines the advantages of rapid magnetic separation of Fe ₃ O ₄ and the wide compatibility of SiO ₂ to synthesize Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles for rapid and efficient separation of exosomes. At the same time, a prostate cancer-specific protein PSMA sensor was designed to specifically recognize prostate cancer exosomes. The invention integrates enrichment and detection, and realizes rapid and accurate analysis and identification of prostate cancer exosomes

Specifically, the method for enriching exosomes of the present invention is based on Fe3O4@SiO2@TiO2 nanoparticles. The enrichment of exosomes is based on the specific binding of TiO ₂ and phosphate groups. The size of TiO ₂ is about 600 nanometers. Compared with the size of exosomes, it has a large specific surface area and can bind more exosomes. Therefore, the enrichment efficiency is high, and the enrichment efficiency is 91.5%. The Fe3O4@SiO2@TiO2 nanoparticles synthesized by the present invention can also enrich other polypeptides, proteins, etc. containing phosphate groups. With Fe ₃ O ₄ as the core, it can be quickly magnetically separated after enrichment, and the enrichment can be completed within 8 minutes, which is simple and fast. The method for detecting exosomes of the invention has high sensitivity, strong specificity, and a detection limit of 9×10 ³ /μL, can significantly distinguish clinical prostate cancer patients and healthy people (P<0.01), and is verified by traditional ELISA method . The invention includes two parts of enrichment and detection of exosomes, the separation method is simple and rapid, the efficiency is high, the detection method has high sensitivity and good specificity, and is suitable for early diagnosis of clinical prostate cancer.

Description of drawings

Figure 1 is the characterization of Fe ₃ O ₄ @SiO ₂ @TiO ₂ . (a ₎ Transmission electron microscope (TEM) image of _Fe3O4 . Ruler, 100 nm. (b ₎ TEM of _Fe3O4 @ _SiO2 . Scale bar 0.2 μm. (c) TEM of _{Fe3O4 @ SiO2} @ _TiO2 . Scale bar 500 nm. (d) Fe ₃ O ₄ @SiO ₂ @TiO ₂ TEM magnified image. Scale bar 200 nm. (e) Scanning electron microscope (SEM) image of _Fe3O4 @ _{SiO2 @ TiO2} . Scale bar 200 nm. (f) Particle size distribution of _{Fe3O4 @ SiO2} @ _TiO2 .

Figure 2 shows the optimal conditions for fluorescence images and exosome isolation. (ac) Bright _- field and fluorescence images of exosomes, _Fe3O4 @ _SiO2 @ _TiO2 /exosomes, magnetically separated washings after repeated washing. Scale bar 50 μm. (d) Optimizing the amount of Fe ₃ O ₄ @SiO ₂ @TiO ₂ for exosome isolation. (e) Optimized incubation time for exosome isolation. (f) Comparison of the efficiency of _{Fe3O4 @ SiO2} @ _TiO2 nanoparticles and ultracentrifugation to separate exosomes in serum and PBS. All experiments were performed independently at least three times. Error bars represent the standard deviation of three replicate measurements for each sample.

Figure 3 shows the optimal detection conditions for exosomes. (a) Fluorescence spectra of single detection sensor 1, sensor 2, and sensor 3. (b) Scatter plots of the same exosome content in the presence of different concentrations of sensors. (c) Fluorescence intensity time and temperature plots. (d) Fluorescence spectrogram. Exosomes at different concentrations (ah: 0.09, 0.69, 6.93, 2.08, 3.12, 6.25, 8.3, ¹⁰ , 12.5 x 105/μL, respectively). (e) Linear relationship between different concentrations of exosomes and sensors. (f) Specificity histogram. Error bars represent standard errors based on three replicate experiments.

Figure 4 Quantitative detection of exosomes in clinical serum samples. (a) Detection results of the PSMA sensor. (b) Dot plot for analysis of clinical samples for PSMA sensors. (c) ELISA test results. (d) Dot plot of clinical sample analysis by ELISA.

detailed description

The present invention will be further described in detail below with reference to the embodiments and the accompanying drawings, but the implementation of the method of the present invention is not limited thereto.

The reagents and instruments employed in the examples are as follows:

1. Reagents

Fe ₃ O ₄ was purchased from Suzhou Beaver Company

Tetraethoxysilane (TEOS) and tetrabutyl titanate (TBOT) are both produced by Shanghai Hushi Co., Ltd.

PBS buffer (pH 7.4), ammonia water, 0.22 μm ultrafiltration membrane, PKH26 were purchased from sigma company in the United States

ELISA was purchased from SBI, USA

PSMA aptamers were purchased from Shanghai Sangon Co., Ltd.

2. Instruments

Transmission electron microscope 200KV (model: JEM 2011, manufacturer: JEOL, Japan), F7000f fluorescence spectrometer (Japan), Rili S-4800 field emission scanning electron microscope, ZS90 nanopotentiometer (Malvern, UK).

Example 1

Synthesis of _{Fe3O4 @ SiO2} @ _TiO2 nanoparticles

1. Dissolve 0.15 g Fe ₃ O ₄ in a mixture of ethanol (280 mL), deionized water (70 mL) and ammonia (5 mL, 28 wt %), and sonicate for 15 min. Then, 4 mL of TEOS was added, and the reaction was continuously stirred for 10 h at room temperature to obtain Fe ₃ O ₄ @SiO ₂ . Figure 1a is the TEM image of _Fe3O4 , and Figure _1b is the TEM image of _{Fe3O4 @ SiO2} . These two figures illustrate that SiO ₂ is successfully wrapped around Fe ₃ O ₄ .

2. Dissolve 0.05 g Fe ₃ O ₄ @SiO ₂ in a mixture of ethanol (100 mL) and ammonia (0.3 mL, 28 wt%), and after sonicating for 15 min, add 0.75 mL of TBOT in ethanol dropwise with continuous stirring. The reaction was continuously stirred at 45 °C for 24 h, the prepared product was washed three times with ultrapure water and ethanol, and dried at 60 °C overnight. Figure 1c is a TEM image of _{Fe3O4 @ SiO2} @ _TiO2 , and Figure 1d is a partial enlarged image of _{Fe3O4 @ SiO2} @ _TiO2 , these two images illustrate _{Fe3O4 @ SiO2} @ _TiO2 was successfully synthesized, and the particle size was uniform. Figure 1e is the SEM image of Fe ₃ O ₄ @SiO ₂ @TiO ₂ , indicating that the synthesis was successful and the particle size was uniform. Figure 1f is a particle size distribution diagram of Fe ₃ O ₄ @SiO ₂ @TiO ₂ , indicating that the particle size is about 600 nm.

Example 2

Exosome isolation.

1. Prepare two parallel samples: LNCaP exosome solution ( ¹⁰⁷ /μL). Exosomes from one sample were stained with PKH26 dye. The exosomes of another sample were stained and incubated with 1.2 mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ at room temperature for 8 minutes, then rapidly magnetically separated with a magnetic stand, and washed with PBS for 3 times. Figure 2a is the bright field and fluorescence images of exosomes after staining, Figure 2b is the bright field and fluorescence images of Fe ₃ O ₄ @SiO ₂ @TiO ₂ /exosomes, and Figure 2c is the bright field of washings after repeated washing and fluorescence maps. These three figures illustrate that Fe ₃ O ₄ @SiO ₂ @TiO ₂ can successfully isolate exosomes, and the number of enriched exosomes is large.

2. Prepare exosomes at a concentration of 10 ⁷ /μL, stain with PKH26, and record the fluorescence intensity as F ₀ . Prepare the same concentration of exosomes and incubate with 0.2, 0.4, 0.6, 0.8, 1.2, 1.4, 1.6 mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles for 8 minutes, after magnetic separation, wash with 150 μL PBS for 3 times. , the fluorescence intensity of the supernatant was measured as F ₁ , each experiment was performed in parallel three times, and the separation efficiency was obtained by calculating F ₀ -F ₁ /F ₀ . Figure 2d is the effect of different quality Fe ₃ O ₄ @SiO ₂ @TiO ₂ on the separation efficiency of exosomes. With the increase of the amount of Fe ₃ O ₄ @SiO ₂ @TiO ₂ (0.2-1.2 mg), the separation efficiency of exosomes gradually increased, and then with the increase of the amount of Fe ₃ O ₄ @SiO ₂ @TiO ₂ ( 1.2-1.6mg), the exosome isolation efficiency decreased slowly, so 1.2mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ was selected as the optimal condition.

3. The fluorescence intensity of exosome staining was recorded as F ₀ (10 ⁷ cells/μL), and the same concentration of exosomes was incubated with the optimal Fe ₃ O ₄ @SiO ₂ @TiO ₂ mass obtained in step 2 for 1- 10 minutes, rapid magnetic separation, and repeated washing with PBS for 3 times, the fluorescence intensity was recorded as F ₁ , each group of experiments was performed in parallel three times, and the separation efficiency was obtained by calculating F ₀ -F ₁ /F ₀ . Figure 2e shows the effect of different incubation times on the isolation efficiency of exosomes. With the increase of incubation time (1-10 minutes), the separation efficiency gradually increased and then almost unchanged, so the incubation time of 8 minutes was chosen.

4. Prepare model exosomes (exosomes secreted by standard cells), dissolve in PBS (10 ⁷ /μL), stain and record the fluorescence intensity as F ₀ . The same concentration of exosomes are stained and obtained by traditional ultracentrifugation. The fluorescence intensity of the supernatant was denoted as F ₁ , the same concentration of exosomes was stained with 1.2 mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ and incubated for 8 minutes, rapidly magnetically separated, washed three times with PBS, and the supernatant fluorescence intensity Denoted as F ₁ , each group of experiments was performed in parallel three times. The separation efficiency was obtained by calculating F ₀ -F ₁ /F ₀ . Serum exosomes (10 ⁷ /μL) were prepared, stained and recorded as F ₀ . The fluorescence intensity of the supernatant obtained by the traditional ultracentrifugation method after the same concentration of exosomes was stained was recorded as F ₁ , and the same concentration of exosomes was recorded as F 1 . After exosomes were stained, they were incubated with 1.2 mg Fe ₃ O ₄ @SiO ₂ @TiO ₂ for 8 minutes, rapidly magnetically separated, and washed three times with PBS. The fluorescence intensity of the supernatant was recorded as F ₁ . The separation efficiency was obtained by calculating F ₀ -F ₁ /F ₀ . Figure 2f is a comparison diagram of the traditional ultracentrifugation method and the Fe ₃ O ₄ @SiO ₂ @TiO ₂ method for isolating exosomes from model exosomes and serum exosomes, Fe ₃ O ₄ @SiO ₂ @TiO ₂ for isolating exosomes The separation efficiency of both model exosomes and serum exosomes is much higher than that of traditional ultracentrifugation methods.

Embodiment 3 Exploration of optimal conditions for exosome detection, including the following steps:

1. Optimization of PSMA sensor. First design and build three PSMA sensors, the PSMA1 sequence is shown in the following table:

After synthesis, chain unwinding at a high temperature of 100 degrees, followed by annealing experiments, and then the three sensors were diluted to a concentration of 200 nM and their fluorescence intensities were measured (excitation: 557 nm, emission 580 nm). Figure 3a is a graph of the fluorescence intensity of three sensors with the same concentration, in which sensor 3 has the smallest fluorescence intensity and the smallest background, so sensor 3 was selected for exosome detection in the experiment.

2. Prepare exosomes with a concentration of 106/μL, incubate with sensor 3 at different concentrations (0, 20, 50, 100, 200, 300, 400, 500, 600nM) at room temperature for 30 minutes, and measure the fluorescence intensity (excitation: 557 nm, emission 580 nm), each experiment was performed in parallel three times. Figure 3b shows the fluorescence intensities of different concentrations of sensor 3 incubated with the same concentration of exosomes. With the increase of the concentration of sensor 3, the fluorescence intensity continued to increase until almost unchanged, so 400 nM was selected as the detection concentration.

3. Prepare exosomes with a concentration of 10 ⁶ /μL, incubate with 200nM sensor 3 at different temperatures (4 degrees, 25 degrees, 37 degrees) for different times and measure the fluorescence intensity (excitation: 557nm, emission 580nm), each Group experiments were performed in parallel three times. Figure 3c is a graph showing the change of fluorescence intensity with time at different temperatures. As time increases, the fluorescence intensity increases. When the temperature is 25 degrees, the fluorescence increases faster and the fluorescence is stronger. Therefore, 25 degrees and 30 minutes are selected as the detection temperature and time.

4. The PSMA sensor concentration was 400nM, and were incubated with different concentrations (0.09, 0.69, 0.93, 2.08, 3.12, 6.25, 8.3, ¹⁰ , 12.5 × 105/μL LNCaP exosomes for 30 minutes at 25°C, respectively) The fluorescence intensity was measured (excitation: 557 nm, emission 580 nm). Figure 3d is a graph of the fluorescence response signal of the PSMA sensor to different concentrations of exosomes. With the increase of exosome concentration, the fluorescence intensity gradually increased.

5. In step 4, each group of experiments was performed in parallel three times, and the obtained average fluorescence intensity was fitted to the exosome concentration by curve fitting. As shown in Figure 3e, the fluorescence intensity and exosome concentration showed a good linear relationship y=39.62X+131.14, R ² =0.9942, indicating the feasibility of the PSMA sensor to detect exosomes, and the actual detection limit is 9×10 ³ /μL.

6. Specificity of the PSMA sensor. Prepare LNCaP exosomes with a concentration of 10 ⁶ /μL and the same concentration of PrEC exosomes and breast cancer MB-231 exosomes, respectively, incubate with 400nM PSMA sensor for 30 minutes at 25 degrees, and measure the fluorescence intensity (excitation: 557 nm, emission 580 nm), each experiment was performed in parallel three times. Figure 3f shows the fluorescence response signals of different types of exosomes at the same concentration to the PSMA sensor. LNCaP exosomes had the largest fluorescence response signal, followed by MB-231 exosomes, and PrEC exosomes had the smallest response, indicating that the PSMA sensor is specific to LNCaP exosomes and can successfully distinguish prostate cancer from normal people.

Example 4

Detection of serum exosomes

Serum samples from clinical prostate cancer patients and normal people were passed through a 0.22 μm filter to remove macromolecules for later use. After the serum was separated by the above Fe ₃ O ₄ @SiO ₂ @TiO ₂ and detected by the PSMA sensor, the fluorescence response signal (excitation: 557nm, emission 580nm) was obtained, which was converted into exosome concentration according to the linear relationship. The same sample was experimentally verified by ELISA to obtain the corresponding exosome concentration. Each group of experiments was performed in parallel three times. Fig. 4a is the detection result of the prostate cancer patient serum and normal human serum by the PSMA sensor. The serum of prostate cancer patients contains a higher number of exosomes than normal people. Figure 4b is a dot plot representation of the data in Figure 4a, there is a significant difference between prostate cancer serum and normal people, P<0.01. Figure 4c shows the results of a commercial ELISA on the same sample. Compared with our method, the prostate cancer serum exosomes are much larger than normal human serum exosomes, indicating the feasibility of our method for detecting exosomes. Figure 4d is a dot plot of ELISA4c data, there is a significant difference between prostate cancer serum and normal people, P<0.01. ELISA is a detection method for all exosomes, including normal exosomes. Our method is based on the specificity of PSMA and is highly specific for detecting prostate cancer exosomes. At the same time, compared with the traditional method, our detection method is simple and convenient to operate, and the detection can be completed within 30 minutes.

Claims (10)

A method for detecting prostate cancer exosomes based on Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticle enrichment and PSMA sensor, characterized in that it includes the following steps: ₁ ) _Fe3O4 @ _SiO2 @ _TiO2 nanoparticles were synthesized, co-incubated with samples containing prostate cell exosomes and magnetically separated, To achieve the isolation of exosomes; 2) Co-incubate the isolated exosomes with the PSMA sensor to obtain a fluorescence intensity signal, and quantify the exosomes by measuring the change in the fluorescence intensity. The method according to claim 1, characterized in that, in step 1), the preparation method of Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles is as follows: Dissolve Fe ₃ O ₄ in a mixture of ethanol, deionized water and ammonia water, sonicate, then add TEOS, and continuously stir the reaction at room temperature to obtain Fe ₃ O ₄ @SiO ₂ ; Fe ₃ O ₄ @SiO ₂ was dissolved in a mixture of ethanol and ammonia water, and after sonication, tetrabutyl titanate dissolved in ethanol was added dropwise with continuous stirring. The reaction was continuously stirred, and the prepared product was washed with ultrapure water and ethanol, and dried to obtain Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles. The method according to claim 1, characterized in that, in step 1), after the magnetic separation, the alkaline solution is eluted. The method according to claim 3, characterized in that, in the elution step, 10% ammonia water is used to co-incubate the Fe ₃ O ₄ @SiO ₂ @TiO ₂ /exosome complex obtained by magnetic separation with shaking. The method according to claim 1, wherein, in step 2), the hairpin-shaped PSMA sensor is constructed as follows: by designing the PSMA antibody sequence, it forms a hairpin-shaped structure, and modifying the corresponding 3' end and 5' The upper carboxytetramethylrhodamine fluorescent group and the azobenzoic acid quenching group. The method according to claim 5, wherein in step 2), the PSMA antibody nucleotide sequence is shown in SEQ No.3. The method according to claim 1, wherein, in step 2), the conditions for obtaining the fluorescence intensity are: excitation light of 557 nanometers and emission light of 580 nanometers. Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles, characterized in that, the preparation method of the nanoparticles is as follows: 1) Dissolve Fe ₃ O ₄ in a mixture of ethanol, deionized water and ammonia water, sonicate, then add TEOS, and continuously stir the reaction at room temperature to obtain Fe ₃ O ₄ @SiO ₂ ; 2) Dissolve Fe ₃ O ₄ @SiO ₂ in a mixture of ethanol and ammonia water, after ultrasonication, add tetrabutyl titanate dissolved in ethanol dropwise with continuous stirring. The reaction was continuously stirred, and the prepared product was washed with ultrapure water and ethanol, and dried to obtain Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles. The application of Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles in the detection of prostate cancer exosomes is characterized in that, Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles are incubated with exosomes and magnetically separated, To achieve the isolation and enrichment of exosomes. The prostate cancer exosome detection device is characterized in that, Fe ₃ O ₄ @SiO ₂ @TiO ₂ nanoparticles are used in the detection device to separate and enrich the exosomes, and co-incubate with the PSMA sensor.

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