Preparation method and application of long-chain non-coding RNA response type urine PCA3 detection nano-motor probe

Abstract

The invention discloses a preparation method and application of a long-chain non-coding RNA response type nano motor probe for urine PCA3 detection, and belongs to the field of nano medicine. The technical problem to be solved by the invention is to provide a nucleic acid response type Janus nano motor probe for accurately and rapidly detecting PCA3 in urine. According to the method, a PCA3 sequence is used as a target lncRNA, and stem-loop hDNA molecules marked with NIR fluorescence and quenching molecules are innovatively and directionally assembled with Fe3O4 / Au nanoparticles and Urease, so that the Janus nano motor probe (Janus hDNA-Fe3O4 / Au-Urease) with a magnetic enrichment effect is formed. Urease catalyzes urea to generate gas, so that the Janus nano-motor probe prepared by the invention can autonomously move in a complex biological environment (urine and tumor cells), thereby realizing efficient recognition of PCA3. On the basis of a pairing effect with a characteristic sequence in a PCA3 long chain, the stem-loop hDNA molecule and the quenching molecule are disassembled, so that an NIR fluorescence signal of the nanoprobe is lightened.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedicine, and in particular relates to a preparation method of a long-chain non-coding RNA responsive nanomotor probe and an application thereof. Background Art

[0002] Prostate cancer (PCa) is currently the most common malignant tumor of the male reproductive system. Its incidence rate (14.1%) is second only to lung cancer and is the second most common cancer in men. The malignancy of prostate cancer depends on the invasion range and stage of the tumor. Even after systematic treatment, the 5-year survival rate of patients with stage IV prostate cancer is still very low (<30%). If PCa can be diagnosed early, the patient's 5-year survival rate after related treatment (surgery, chemotherapy, etc.) will be significantly improved. Therefore, early diagnosis is the key to reducing the mortality rate of prostate cancer.

[0003] At present, the clinical diagnosis methods of prostate cancer include biochemical testing, puncture biopsy and imaging examination. Since the 1980s, prostate-specific antigen (PSA) has gradually become a widely used early screening indicator for prostate cancer in clinical practice. PSA is mainly secreted by prostate epithelial cells. The rapid proliferation of prostate cancer cells will lead to an abnormal increase in serum PSA levels. Prostate tissue puncture biopsy combined with PSA levels ≥ 10 μg / L can greatly improve the sensitivity of prostate cancer diagnosis. However, PSA is only a prostate organ-specific marker and is easily affected by many factors such as prostate inflammation and hyperplasia, mechanical stimulation, and urinary retention. Especially when the PSA level is in the "gray zone" of 4~10 μg / mL, the positive rate of prostate cancer biopsy is less than 40%, and the examination specificity is poor, resulting in meaningless puncture and misdiagnosis. Transrectal ultrasound (TRUS) is used to guide puncture biopsy of specific parts of prostate cancer. It has high accuracy, but TRUS cannot detect early tumors. In addition, due to the limited sampling area and high heterogeneity of the tumor, it is also difficult to collect the lesion information of the entire tumor. Studies have shown that prostate cancer antigen 3 (PCA3) is a long non-coding RNA produced by androgen-induced prostate cancer cells. Compared with the correlation between other prostate cancer-related lncRNAs and various tumors, PCA3 is only specifically and highly expressed in prostate cancer tissues or their metastases. And when a small amount of prostate tissue undergoes early canceration, PCA3 will be overexpressed and can be excreted into the urine in large quantities through the prostate duct, showing extremely high specificity and sensitivity to prostate cancer. It can be seen that PCA3 can provide specific markers and targets for the early diagnosis of prostate cancer, and is expected to improve the diagnostic accuracy of prostate cancer. Therefore, designing a multifunctional nucleic acid probe, targeting PCA3, and realizing accurate urine detection and exfoliated tumor cell imaging labeling are of great significance for the early diagnosis and prognosis of prostate cancer.

[0004] In recent years, with the rapid development of nanotechnology, the functionalization of nucleic acid molecular probes using nanotechnology has provided new ideas and methods for PCA3 in vivo multimodal imaging. Compared with nucleic acid molecular probes, nano-sized nucleic acid probes can be multifunctionalized through reasonable nanomaterial design, thereby integrating multiple signal molecules into one for multimodal imaging analysis. For example, nanomaterials such as metal nanoclusters, inorganic quantum dots, and carbon quantum dots have unique fluorescent properties and can be used to assist in the construction of fluorescent nucleic acid probes for fluorescent imaging analysis. In addition, nanoparticles can also effectively protect nucleic acid probes from being degraded by nucleases and enhance their in vivo stability. In addition, nanomaterials can also control their pharmacokinetics through changes in their size, surface properties, and shape, and are easier to amplify imaging signals, providing a good material platform for the establishment of efficient multimodal nucleic acid nanoprobes.

[0005] Currently, many clinical imaging methods, such as computed tomography (CT), magnetic resonance imaging (MRI), and choline positron emission tomography (PET), have been used for early diagnosis and efficacy evaluation of prostate cancer. However, due to nonspecific recognition and insufficient resolution, early diagnosis and accurate staging through conventional imaging methods are still very challenging, especially for asymptomatic elderly patients. If contrast agents can achieve accurate detection at the molecular level, it will largely overcome the limitations of current clinical diagnostic techniques. With the rapid development of nanotechnology in the medical field, many scholars have found that composite multifunctional nanomaterials based on nanotechnology can effectively improve the sensitivity and specificity of prostate cancer diagnosis and treatment. Cleverly designed nanoparticles (through specific targeting, contrast-enhanced imaging, and multimodal imaging) can not only target tumor lesions, but also respond to physiological environments (such as overexpressed enzymes, changing pH) or external stimuli (such as magnetic fields, ultrasound, and near-infrared red light), showing great potential for early diagnosis of prostate cancer. In addition to conventional imaging methods, traditional optical imaging such as fluorescence imaging, Raman imaging, luminescence imaging, and diffuse optical tomography have become emerging imaging methods for guiding prostate cancer surgery due to real-time visual recognition and high-sensitivity detection of tumor lesions. In recent years, research on fluorescence imaging technology has mainly focused on the near-infrared window (NIR). Compared with ordinary fluorescence, NIR fluorescence (650~1700 nm) has a longer wavelength, which can greatly reduce tissue absorption, scattering and autofluorescence interference, and has strong tissue penetration, which can achieve high-sensitivity in vivo imaging of deep tissues.

[0006] Specific molecular probes are the basis of molecular imaging technology. They are an organic combination of imaging technology with disciplines such as biology, medicine, materials science and nanotechnology. Molecular probes are usually composed of recognition molecules and signal molecules bonded by chemical bonds, and they recognize and image specific targets respectively. The specific recognition of molecular probes and target molecules is generally based on specific interactions between biological molecules, such as nucleic acid base pairing or antigen-antibody reactions. Therefore, depending on the molecular type of the object to be identified, molecular probes need to be reasonably designed to ensure their specificity and targeting. In the study of lncRNA such as PCA3, nucleic acid molecular probes based on nucleic acid hybridization technology have unique advantages in their detection analysis and high-sensitivity imaging. Therefore, the development of NIR fluorescence imaging methods for PCA3 molecules is expected to achieve high-sensitivity, non-destructive, high-resolution, high-penetration real-time urine and exfoliated tumor cell detection of PCA3, providing more comprehensive and complementary imaging information for the diagnosis of prostate cancer. Summary of the invention

[0007] The purpose of the present invention is to provide a Janus nanomotor probe as a carrier-driven nucleic acid probe for highly sensitive urine detection of prostate cancer-related PCA3 and precise tumor cell capture, in order to achieve early non-destructive body fluid testing and in situ cell imaging diagnosis of prostate cancer.

[0008] The preparation method and application of the long-chain non-coding RNA responsive urine PCA3 detection nanomotor probe of the present invention firstly uses the PCA3 sequence as the target lncRNA, prepares a functionalized stem-loop DNA (hDNA) molecular probe and labels it with NIR fluorescence and quenching molecules, and then directionally assembles it with Fe3O4 / Au nanoparticles and Urease to form a Janus nanomotor probe (Janus hDNA-Fe3O4 / Au-Urease, JNMPs).

[0009] The Janus nanomotor probe of the present invention utilizes urease to catalyze urea to realize autonomous movement of the JNMPs probe in a complex biological environment (urine, tumor cells), thereby enhancing the recognition and separation efficiency of PCA3. In the JNMPs probe, the stem-loop hDNA molecule is paired with the characteristic sequence in the PCA3 long chain, thereby disassembling with the quenching molecule, thereby causing the lighting of the NIR fluorescence signal. Fe3O4 is utilized to obtain an effective magnetic enrichment effect, thereby realizing highly sensitive detection of urine PCA3 and highly sensitive fluorescence images of tumor cells. Through the implementation of the present invention, new ideas and means are provided for the early clinical non-destructive diagnosis of prostate cancer.

[0010] A method for preparing a nucleic acid-responsive driven nano prostate cancer detection agent comprises the following steps: (1) 0.1-1.0 g of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSAM) and 0.2-1.0 g of ferric chloride hexahydrate (FeCls·6H2O) were added to 10-50 mL of ethylene glycol, and an aqueous solution of ferrosoferric oxide nanoparticles (Fe3O4NPs) was prepared by a hydrothermal method.

[0011] (2) 5-20 μg of Fe3O4NPs were dispersed in 5-30 mL of ethanol and vacuum dried to form a Fe3O4NPs monolayer structure on a coverslip. Next, 1-100 μg of gold (Au) was deposited on the Fe3O4NPs monolayer structure by sputtering, and the coverslip was gently ultrasonicated in deionized water to obtain Fe3O4 / AuJNMs nanoparticles with half of the gold coating.

[0012] (3) Add 0.05-0.1 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.02-0.1 g N-hydroxysuccinimide (NHS) to 5-50 mL of 0.5-5 mg / mL Fe3O4 / Au JNMs. Then, 20-100 mg of urease was added to the solution and the grafting reaction was carried out at room temperature for 24-72 h to obtain the Janus Fe3O4 / Au-Urease nano-aqueous solution.

[0013] (4) Dissolve 1-50 μg of stem-loop DNA and 1-50 μg of tri(2-carboxyethyl)phosphine (TCEP) in 1-10 mL of 0.1-1 mol / L Fe3O4 / Au-Urease nanoaqueous solution, mix and stir the solution for 6-24 h, and obtain the final product JNMPs hDNA-Fe3O4 / Au-Urease (JNMPs) nanoparticles through Au-S bond coupling.

[0014] Furthermore, the particle size of the ferrosoferric oxide nanoparticles prepared in step 1 is 220 to 250 nm, and the surface charge is -35 to -40 mV.

[0015] Furthermore, the particle size of the Fe3O4 / Au JNMs prepared in step 2 is 230 to 260 nm, and the surface charge is -35 to -40 mV.

[0016] Furthermore, the surface structure of the Fe3O4 / Au JNMs prepared in step 2 is that the Au nanolayer covers the hemispherical surface of Fe3O4, presenting a Janus structure.

[0017] Furthermore, the particle size of Janus Fe3O4 / Au-Urease prepared in step 3 is 250-280 nm, and the surface charge is -25--29 mV.

[0018] Furthermore, the surface structural feature of Janus Fe3O4 / Au-Urease prepared in step 3 is that urease is coupled to the side of Fe3O4 without Au nanolayer through amidation reaction.

[0019] Furthermore, the particle size of JNMPs hDNA-Fe3O4 / Au-Urease prepared in step 4 is 255 to 290 nm, and the surface charge is -33 to -36 mV.

[0020] Furthermore, the characteristic of preparing JNMPs hDNA-Fe3O4 / Au-Urease in step 4 is that hDNA is coupled to the Au nanolayer covered side through Au-S bonds, and the stem-loop DNA can light up the fluorescence by specifically binding to PCA3.

[0021] Furthermore, the Janus nanomotor probe can not only perform specific and accurate detection of free PCA3 in urine, but also capture detached prostate cancer cells in urine and perform fluorescence imaging of in situ targeted PCA3, providing new ideas for the early diagnosis of prostate cancer.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Driving the JNMPs probe to rapidly detect PCA3 in urine, a highly sensitive and specific screening for prostate cancer can be achieved in vitro, with a detection threshold of PCA3 concentration of 1×10 -12 mol / L.

[0023] (2) Use the driving strategy to assist the JNMPs probe to achieve real-time urine and exfoliated tumor cell detection and labeling, provide more accurate and complementary in situ cell imaging information for early screening and diagnosis of prostate cancer, achieve early detection efficiency of prostate cancer, and improve patient prognosis.

[0024] (3) The preparation method is stable and reliable with good reproducibility. The prepared Janus nanomotor probe not only has good dispersibility, stability and targeting, but can also accurately identify the prostate cancer-specific nucleic acid marker PCA3 through base complementary pairing, and can light up the fluorescence by opening the hDNA stem-loop, effectively realizing the detection of free PCA3 in urine and intracellular labeling, which is expected to provide new ideas for the clinical early diagnosis and precision treatment of prostate cancer.

[0025] With reference to the following description and the accompanying drawings, the specific embodiments of the present invention are disclosed in detail, indicating the manner in which the principles of the present invention can be adopted. It should be understood that the embodiments of the present invention are not limited in scope thereby.

[0026] Features described and / or illustrated with respect to one embodiment may be used in the same or similar manner in one or more other embodiments, combined with features in other embodiments, or substituted for features in other embodiments.

[0027] It should be emphasized that the term “include / comprises” when used herein refers to the presence of features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps or components. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 The transmission electron microscope and mapping images of Fe3O4 and Fe3O4 / Au nanoparticles of the present invention; Figure 2 The design diagram, fluorescence recovery mechanism, gel electrophoresis results and fluorescence spectrum diagram of the stem-loop hDNA of the present invention are shown; Figure 3 A is the hydration particle size diagram of Fe3O4, Fe3O4 / Au, Fe3O4 / Au@Urease and Janus hDNA-Fe3O4 / Au-Urease of the present invention; Figure 3 B is a surface charge comparison diagram of Fe3O4, Fe3O4 / Au, Fe3O4 / Au@Urease and Janus hDNA-Fe3O4 / Au-Urease of the present invention; Figure 4 The ultraviolet absorption spectra of Fe3O4, Fe3O4 / Au, Fe3O4 / Au@Urease and Janus hDNA-Fe3O4 / Au-Urease of the present invention are shown; Figure 5 The magnetic force curves of Fe3O4 and Janus hDNA-Fe3O4 / Au-Urease of the present invention; Figure 6 is the decomposition rate of urea of ​​the present invention under the catalysis of Janus hDNA-Fe3O4 / Au-Urease; Figure 7 A is a graph showing the fluorescence spectrum changes of Janus hDNA-Fe3O4 / Au-Urease after co-incubation with PCA3 of different concentrations; Figure 7 B is a graph showing the fluorescence intensity changes of Janus hDNA-Fe3O4 / Au-Urease of the present invention after co-incubation with PCA3 of different concentrations; Figure 7 C and 7D are the standard curves of Janus hDNA-Fe3O4 / Au-Urease for detecting PCA3 of the present invention; Figure 8 The Janus hDNA-Fe3O4 / Au-Urease of the present invention catalyzes urea solutions of different concentrations to detect the change of PCA3 fluorescence signal intensity over time; Fig. 9 The comparison of PCA3 fluorescence signal intensity detected by Janus hDNA-Fe3O4 / Au-Urease under magnetic enrichment / non-magnetic enrichment conditions; Fig.10 The Janus hDNA-Fe3O4 / Au-Urease of the present invention detects the fluorescence signal intensity of other RNA in urine; Fig.11 The accuracy of the urine spiked PCA3 detected by Janus hDNA-Fe3O4 / Au-Urease of the present invention; Fig.12 The fluorescence images of Janus hDNA-Fe3O4 / Au-Urease co-incubated with prostate cancer cells for different time periods according to the present invention. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items. Example

[0032] This embodiment provides a method for preparing a nucleic acid-responsive nano-prostate cancer detection agent, comprising the following steps: (1) Preparation of Fe3O4 nanoparticles: 0.5 g PSSAM and 0.54 g FeCls-6H2O were added to 20 mL ethylene glycol and stirred until transparent. Then 1.5 g sodium acetate was added to the solution and the mixed solution was sealed in a stainless steel autoclave and heated at 200°C for 10 hours to obtain Fe3O4 NPs. Figure 1 As shown in A, Fe3O4 nanoparticles are spherical nanoparticles with a particle size of about 220 nm.

[0033] (2) Preparation of Janus structure Fe3O4 / Au: 10 μg of Fe3O4NPs were dispersed in 100 μL of ethanol and vacuum dried to form a Fe3O4NPs monolayer structure on a coverslip. Next, 10 μg of Au was deposited on the Fe3O4NPs monolayer structure by sputtering, and the coverslip was gently ultrasonicated in deionized water to obtain Fe3O4 / Au JNMs nanoparticles with half of the gold coating. Figure 1 As shown in B, Fe3O4 / Au NPs present a Janus structure and the particle size is about 230 nm.

[0034] (3) Fe3O4 / Au immobilized Urease: 0.076g EDC and 0.048g NHS were added to 10 mL of 1mg / mL Fe3O4 / Au JNMs nano-aqueous solution to activate the carboxyl groups on its surface. Then, 50 mg urease was added to the solution and the grafting reaction was carried out at room temperature for 48 h to obtain Janus Fe3O4 / Au-Urease nano-aqueous solution. (4) Preparation of hDNA-Fe3O4 / Au-Urease: 10ul of 100mM stem-loop DNA and 10 μL TCEP were dissolved in 0.48 mL of Fe3O4 / Au-Urease (1mg) nano-water solution, and the solution was mixed and stirred for 12 h. The final product, JNMPs hDNA-Fe3O4 / Au-Urease nanoparticles, was obtained through Au-S bond coupling. Finally, JNMPs hDNA-Fe3O4 / Au-Urease was dispersed in DEPC-treated PBS buffer (200 μL, 0.01 mol / L, pH =7.2~7.4, RNase-free and DNase-free) and stored at 4°C.

[0035] In order to explore the Janus microstructure of Fe3O4 / Au, we performed TEM imaging and mapping element analysis. Figure 1 C shows that Au elements are evenly distributed on one half of Fe3O4, proving the successful assembly of the Janus structure of Fe3O4 / Au. In order to synthesize capture probes targeting PCA3, we screened three PCA3 base sequences from the NCBI library ( Figure 2 A), the schematic diagram of the synthesis process is shown in Figure 2 B. Gel electrophoresis results ( Figure 2 C) and fluorescence images ( Figure 2 D) Consistently shows that the first hDNA sequence designed specifically binds to PCA3 with the best effect. Malvern particle size tester test results show that the hydrated particle sizes of Fe3O4, Fe3O4 / Au, Fe3O4 / Au-Urease and hDNA-Fe3O4 / Au-Urease are approximately 228nm, 255nm, 287nm and 295nm respectively ( Figure 3 A). After comparing their surface charges, we found that ( Figure 3 B), Fe3O4 / Au has a negative charge on the surface. After activation by the amide method, its surface has a positive charge. The negatively charged Urease makes the surface of Fe3O4 / Au-Urease negatively charged. The Fe3O4 / Au bonded to hDNA through the thiol group makes the final prepared hDNA-Fe3O4 / Au-Urease have a negative charge on the surface potential, which to a certain extent proves the successful preparation of hDNA-Fe3O4 / Au-Urease. In addition, we conducted UV spectroscopy tests on different probes, and the results are as follows Figure 4 As shown. Compared with the spectrum of Fe3O4 / Au, the spectrum of hDNA-Fe3O4 / Au-Urease shows the characteristic absorption peaks of protein and nucleic acid of Urease at 280 nm and 260 nm, respectively, which further proves the successful preparation of Janus nanomotor probe hDNA-Fe3O4 / Au-Urease. By comparing the saturation magnetization intensity (MS) of Fe3O4 and hDNA-Fe3O4 / Au-Urease NPs with a Lake Shore 8604 vibrating sample magnetometer, it was found that ( Figure 5 ), the MS of Fe3O4 and hDNA-Fe3O4 / Au-Urease NPs were 60.1 and 57.6 emu g -1 , both curves intersect with the origin, indicating that hDNA-Fe3O4 / Au-Urease NPs are still in the superparamagnetic state at room temperature.

[0036] Urease can catalyze urea to produce NH3 and CO2. Therefore, the catalytic activity of urease-mediated Janus nanomotor probe can be explored by detecting the concentration of NH3. When the urea concentration is constant (100mM), the rate at which the Janus nanomotor probe catalyzes urea to produce NH3 increases with the increase of the probe concentration ( Figure 6 A). When the concentration of Janus nanomotor probe is constant (100ug / mL), the ammonia production rate increases with the increase of urea concentration ( Figure 6 B). When the urea concentration ranges from 100 to 500 mM, the Janus nanomotor probe hDNA-Fe3O4 / Au-Urease NPs (100 ug / mL) prepared by the present invention can continuously provide efficient urease catalytic activity within 60 min. As time goes by, although the consumption of the probe will reduce the rate of the enzymatic reaction, it can still generate enough NH3 and CO2 to provide kinetic energy for the Janus nanomotor probe.

[0037] like Figure 7 As shown in A, the fluorescence of the Janus nanomotor probe (100ug / mL) increases with the addition of PCA3. This is because the complementary base pairing between DNA and PCA3 opens the hDNA stem loop, causing the fluorescent spot Cy5 labeled on one side to separate from the quenching factor BHQ-3, thereby lighting up the fluorescence. Moreover, the fluorescence intensity of the Janus nanomotor probe at 665nm gradually increases with the increase in PCA3 concentration ( Figure 7 B), which may be related to the opening of more hDNA. In addition, we also found that the minimum detection threshold of the Janus nanomotor probe of the present invention is 1pM. When the PCA3 concentration ranges from 1-100 pM and 100-200000 pM, the PCA3 concentration is linearly correlated with the fluorescence intensity. The standard curves are as follows: Figure 7 C, 7D. This result shows that within a certain concentration range, the PCA3 content can be relatively accurately estimated based on the fluorescence intensity by using the Janus nanomotor probe of the present invention to detect the target sample. In order to verify the performance of urease in promoting the detection of PCA3 by the Janus nanomotor probe, the appropriate time and fluorescence intensity of the probe to detect PCA3 in the presence or absence of urea were investigated. The results are shown in Figure 8As shown. The fluorescence intensity of the urea group was significantly higher than that of the non-urea group, and the fluorescence intensity of the urea group almost reached saturation only after incubation for 10 minutes. In the non-urea group, since there was no urease catalysis to provide motor power, the saturation time of incubation time was as long as 30 minutes, which was still lower than the fluorescence intensity of the urea group. Compared with the 100mM urea group, the fluorescence intensity of the 300mM urea group was slightly higher, but it almost reached saturation within 10 minutes, which shows that the Janus nanomotor probe can complete PCA3 detection in urine containing different concentrations of urea in only 10 minutes. In addition to the gas drive provided by urease, the magnetism of the Janus nanomotor probe can also improve its detection efficiency. On the one hand, the magnetic adsorption function of the Janus nanomotor probe can assist in the rapid collection of samples and improve detection efficiency. On the other hand, the magnetic enrichment property of the Janus nanomotor probe enables it to accelerate sample concentration, thereby increasing the fluorescence intensity ( Fig. 9 ), indicating that magnetic enrichment can effectively reduce the detection threshold of PCA3 and improve the detection accuracy of PCA3.

[0038] In order to explore the specificity of Janus nanomotor probe hDNA-Fe3O4 / Au-Urease NPs for PCA3 detection, we spiked urine with multiple groups of different types of RNA and PCA3 fragments with single and three base mismatches RNA fragments M1 and M3, respectively. The detection results are shown in Figure 2. Fig.10 As shown. Different from the obvious high fluorescence produced after co-incubation of PCA3 and the Janus nanomotor probe of the present invention, although the PCA3 mismatch base fragment M3 also expresses a small amount of fluorescence, it is not enough to interfere with the detection results, and the weak fluorescence expressed by the remaining RNA fragments in the urine can be ignored. This result proves that the Janus nanomotor probe hDNA-Fe3O4 / Au-Urease NPs of the present invention is highly specific for the detection of PCA3 in urine. In addition, we also added PCA3 to explore the accuracy of the Janus nanomotor probe of the present invention in detecting PCA3. The results are shown in Fig.11 As shown. -10 mol / L PCA3 was added to 5 groups of urine samples. The PCA3 concentration of the spiked samples detected by the Janus nanomotor probe hDNA-Fe3O4 / Au-Urease NPs of the present invention ranged from 0.79 to 1.2 mol / L, which was consistent with the spiked concentration (10 × 10 -10mol / L). However, the complex matrix effect of urine prevented all urine samples from directly detecting PCA3 by qRT-PCR, indicating that the designed probe has significant advantages in directly analyzing PCA3. To further explore the performance of the Janus nanomotor probe in capturing prostate cancer cells for in situ fluorescence imaging, LnCap cells were spiked into urine and hDNA-Fe3O4 / Au-Urease NPs (100ug / mL) probe was added for co-incubation for 0, 0.5, 1, and 2h, and then confocal fluorescence imaging was performed. Fig.12 As shown, a small amount of fluorescence can be observed in the cells after 0.5 hours of co-incubation. As the incubation time increases, the fluorescence gradually increases, indicating that the Janus nanomotor probe prepared by the present invention can effectively capture prostate cells, providing a possibility for the screening of prostate cancer cells in the urine of clinical prostate cancer patients.

[0039] Any numerical value cited herein includes all values ​​of lower and upper values ​​that increase by one unit from the lower limit to the upper limit, and there is at least a two-unit interval between any lower value and any higher value. For example, if the value of the quantity of a component or a process variable (such as temperature, pressure, time, etc.) is set forth to be from 1 to 90, preferably from 20 to 80, and more preferably from 30 to 70, the purpose is to illustrate that values ​​such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 are also explicitly listed in the specification. For values ​​less than 1, one unit is appropriately considered to be 0.0001, 0.001, 0.01, 0.1. These are merely examples that are intended to be clearly expressed, and it can be considered that all possible combinations of numerical values ​​listed between the lowest value and the highest value are clearly set forth in the specification in a similar manner.

[0040] Unless otherwise indicated, all ranges include the endpoints and all numbers between the endpoints. "About" or "approximately" used with a range applies to both endpoints of the range. Thus, "about 20 to 30" is intended to cover "about 20 to about 30", including at least the specified endpoints.

[0041] It should be understood that the above description is for illustration and not for limitation. Many embodiments and many applications beyond the examples provided will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present teachings should not be determined with reference to the above description, but should be determined with reference to the appended claims and the full scope of equivalents possessed by such claims. For the purpose of comprehensiveness, all articles and references, including disclosures of patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the preceding claims is not intended to be a waiver of the subject matter, nor should it be considered that the inventors did not consider the subject matter to be part of the disclosed inventive subject matter.

Claims

1. A method for preparing a long non-coding RNA responsive nanomotor probe for urine PCA3 detection, characterized in that: The method comprises the following steps: 0.1-1.0 g of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSAM) and 0.2-1.0 g of ferric chloride hexahydrate (FeCls·6H2O) were added to 10-50 mL of ethylene glycol, and an aqueous solution of ferrosoferric oxide nanoparticles (Fe3O4NPs) was prepared by a hydrothermal method; 5-20 μg of Fe3O4NPs were dispersed in 5-30 mL of ethanol and vacuum dried to form a Fe3O4NPs monolayer structure on a cover glass. Then, 1-100 μg of gold (Au) was deposited on the Fe3O4NPs monolayer structure by sputtering, and the cover glass was ultrasonically treated in deionized water to obtain Fe3O4 / AuJNMs nanoparticles with a gold coating. 0.05-0.1g 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.02-0.1g N-hydroxysuccinimide (NHS) are added to 5-20mg Fe3O4 / AuJNMs nano-aqueous solution to activate the surface carboxyl groups; then 20-100mg urease (Urease) is added to the solution, and the grafting reaction is carried out at room temperature for 24-72h to obtain JanusFe3O4 / Au-Urease nano-aqueous solution; 1-50 μg of stem-loop DNA and 1-50 μg of tri(2-carboxyethyl)phosphine (TCEP) were dissolved in 1-10 mL of 0.1-1 mol / L Fe3O4 / Au-Urease nanoaqueous solution, the solution was mixed and stirred for 6-24 h, and the final product, Janus nanomotor probe (JanushDNA-Fe3O4 / Au-Urease), was obtained through coupling of Au-S bonds.

2. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The particle size of the prepared ferroferric oxide nanoparticles is 220-250nm, and the surface charge is -35--40mV.

3. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The particle size of the prepared Fe3O4 / AuJNMs is 230-260nm, and the surface charge is -35--40mV.

4. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The surface structure of the prepared Fe3O4 / AuJNMs is that the Au nanolayer covers the hemispherical surface of Fe3O4, presenting a Janus structure.

5. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The particle size of the prepared JanusFe3O4 / Au-Urease is 250-280nm, and the surface charge is -25--29mV.

6. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The surface structural feature of the prepared JanusFe3O4 / Au-Urease is that urease is coupled to the side of Fe3O4 without Au nanolayer through amidation reaction.

7. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The particle size of the prepared JanushDNA-Fe3O4 / Au-Urease is 255 to 290 nm, and the surface charge is -33 to -36 mV.

8. The method for preparing a long non-coding RNA-responsive driven nanomotor probe according to claim 1, characterized in that: The prepared JanushDNA-Fe3O4 / Au-Urease is characterized in that hDNA is coupled to the Au nanolayer-covered side through an Au-S bond, and the stem-loop DNA can light up the fluorescence by specifically binding to PCA3.

9. The method for preparing a long non-coding RNA-responsive driven nanomotor probe as claimed in claim 2, characterized in that: The Janus nanomotor probe is used to specifically and accurately detect free PCA3 in urine, and can also be used to capture detached prostate cancer cells in urine and perform fluorescence imaging of in situ targeted PCA3.

10. A long non-coding RNA responsive nanomotor probe for urine PCA3 detection, characterized in that: A urease-driven Janus nanomotor probe with magnetic enrichment effect was constructed for specific fluorescence-triggered detection of PCA3 in urine.

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