A fluorescent aptamer sensor and a preparation method and application thereof

Abstract

The application discloses a fluorescent aptamer sensor and a preparation method and application thereof, and the fluorescent aptamer sensor is prepared by coupling amino-modified UCNPs and PA nucleic acid aptamers, and then introducing MoS2 for incubation, wherein the sequence of the PA nucleic acid aptamer is shown as SEQ ID NO. 1. The application utilizes the unique up-conversion fluorescence emission of UCNPs, the strong quenching characteristics of MoS2 nanosheets and the significant binding force between the aptamer and PA, so that the PA in an infected wound can be detected with high sensitivity, high specificity and high selectivity.

Description

Technical Field

[0001] This invention belongs to the field of sensor application technology, and relates to a fluorescent aptamer sensor, its preparation method and application, and particularly to the detection of Pseudomonas aeruginosa in wound skin based on fluorescence resonance energy transfer generated between aptamer-modified upconversion nanoparticles (UCNP) (donor) and molybdenum disulfide (MoS2) nanosheets (acceptor). Background Technology

[0002] In recent years, extensive research has been conducted on the mechanisms and prevention of burns and trauma. Related studies have also shown that infection is a common complication of surgical diseases, particularly prominent in burn treatment. Currently, wound infection remains a leading cause of death in burn patients, and the analysis of bacterial species in local wounds has become a hot topic in clinical treatment. For many years, bacteriological surveys of burn wounds have consistently shown that *Pseudomonas aeruginosa* (PA) and *Staphylococcus aureus* (a group of bacteria in the genus *Staphylococcus*) are the most prevalent and life-threatening bacteria in burn patients. It is also a significant cause of infection in burn patients, wound infections, cystic fibrosis patients, and patients with respiratory-related pneumonia. Epidemiological data shows that PA accounts for approximately 8.3%-11.1% of skin and soft tissue infections. Recent studies have found that PA accounts for a considerable proportion (<50%) of hospital-acquired infections, and this proportion is increasing, especially in orthopedic trauma and amputation patients.

[0003] Currently, conventional methods for PA detection mainly include standard culture colony counting, polymerase chain reaction (PCR) molecular methods, and enzyme-linked immunosorbent assay (ELISA). However, these traditional methods have limitations, such as time-consuming culture processes, reliance on sophisticated instruments, professional personnel, and expensive reagents. This significantly hinders the practical application of PA detection. Therefore, developing a new method for rapid and accurate detection of PA in infected samples is of great significance for early clinical diagnosis. In recent years, fluorescent biosensors have attracted great interest in the pharmaceutical and food safety fields due to their high sensitivity, rapid response, low cost, and simple detection process, and are considered a promising alternative to commonly used pathogen detection methods.

[0004] Due to their unique anti-Stokes luminescence properties, rare-earth-doped upconversion nanoparticles (UCNPs), as a new generation of fluorescent dyes, can emit strong visible light through continuous multiphoton absorption or energy transfer under near-infrared (NIR) excitation, exhibiting advantages such as narrow emission peaks, high biocompatibility, and long fluorescence lifetimes. Compared to traditional fluorophores (Stokes luminescence) such as organic dyes, they effectively avoid interference from background autofluorescence. Given these special optical properties, UCNPs have become a new generation of fluorophores for analyzing food hazards such as foodborne pathogens, heavy metal ions, and pesticide residues. Furthermore, portable upconversion devices can be used for on-site detection, enabling point-of-care testing.

[0005] In addition, two-dimensional (2D) layered nanomaterials have attracted widespread interest due to the related physicochemical properties arising from the quantum size effect of their ultrathin structures. Layered molybdenum disulfide (MoS2), as an emerging 2D material, possesses a wide intrinsic band gap and the advantage of direct dispersibility in aqueous solutions, and has already garnered significant attention in the field of biosensors. Furthermore, MoS2 nanosheets exhibit broad absorption peaks and excellent absorption capabilities in the 200–700 nm range, overlapping with the emission spectra of many fluorescent materials, and can quench the fluorescence emitted by various materials through FRET (fluorescence resonance energy transfer). Due to these unique electronic and optical properties, MoS2 becomes an effective UCNP acceptor.

[0006] Current biosensors still need improvement in areas such as the selection of recognition elements, system construction, and photoelectric signal conversion. Therefore, there is still great potential for the research and application of fluorescent aptamer biosensors. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a UCNP-MoS2 fluorescent aptamer sensor that can achieve highly sensitive detection of PA in burn skin wounds, with a detection limit as low as 15.5 cfu / mL.

[0008] Another technical problem that this invention aims to solve is to provide a method for preparing a fluorescent aptamer sensor.

[0009] The final technical problem to be solved by this invention is to provide the use of a fluorescent aptamer sensor in the detection of PA.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a fluorescent aptamer sensor, which is prepared by coupling amino-modified UCNPs with PA nucleic acid aptamers and then incubating them with MoS2. The sequence of the PA nucleic acid aptamer is shown in SEQ ID NO.1.

[0011] The PA nucleic acid aptamer sequence is 5'-COOH-CCC CCG TTG CTT TCG CTT TTC CTT TCGCTT TTG TTC GTT TCG TCG TCC CTG TTG CTT CCT TTC-3'.

[0012] The concentration of the amination-modified UCNPs is 0.01–0.1 mg / mL, and the concentration of the PA nucleic acid aptamer is 25–100 μM.

[0013] Preferably, the concentration of the amination-modified UCNPs is 0.1 mg / mL, and the concentration of the PA nucleic acid aptamer is 100 μM.

[0014] The concentration of MoS2 is 0.09–0.1 mg / mL.

[0015] The present invention also includes a method for preparing the aforementioned fluorescent aptamer sensor, comprising the following steps:

[0016] 1) Aminated UCNPs:

[0017] UCNPs powder was added to cyclohexane and sonicated until it was completely dispersed to obtain a cyclohexane solution of UCNPs. Then, lgepal CO-520 and cyclohexane were added and mixed and stirred to obtain a solution. lgepal CO-520 and ammonia were added to the above solution, the container was sealed and sonicated to form a transparent emulsion. Then, TEOS was added to the emulsion and rotated. Then, APTEs were added and stirring was continued to form aminated upconversion particles UCNPs-SiO2-NH2. Acetone was added to precipitate the particles and the mixture was washed twice with ethanol / water. The precipitate was then stored in water.

[0018] 2) Bioconjugation with PA nucleic acid aptamers: UCNP-apt was prepared by the 25% glutaraldehyde crosslinking method: The prepared UCNP-SiO2-NH2 was added to PBS buffer and ultrasonically dispersed. Then, 25% glutaraldehyde aqueous solution was added under light-protected conditions and shaken. The solution was then centrifuged and the precipitate was washed with PBS buffer. The activated UCNPs were redispersed in PBS buffer, PA aptamer solution was added and incubated with shaking. Unbound aptamers were removed by centrifugation, and finally UCNPs-apt were obtained.

[0019] 3) MoS2 introduction: MoS2 was added to UCNPs-apt and incubated to complete the construction of the fluorescent aptamer sensor.

[0020] In step 1), after adding TEOS, the rotation is carried out for 11 to 12 hours, and then APTES is added dropwise and rotated for 1 to 2 hours. The rotation speed is 600 to 700 rpm / min.

[0021] Preferably, in step 1), after adding TEOS, the rotor is rotated for 11 hours, and then APTES is added dropwise and rotated for 1 hour, with the rotation speed being 600 rpm / min.

[0022] In step 2), after adding 25% glutaraldehyde, shake for 1-2 hours. The temperature should not be too high during the shaking process.

[0023] In step 3), the concentration of MoS2 is 0.09–0.1 mg / mL.

[0024] In step 3), the pH for incubation is 5–10, and the incubation time is 5–35 min.

[0025] The present invention also includes the application of the fluorescent aptamer sensor in the detection of Pseudomonas aeruginosa.

[0026] The concentration of *Pseudomonas aeruginosa* was 8.7 × 10⁻⁶. 1 -8.7×10 7 cfu / mL.

[0027] Furthermore, based on the FRET that occurs between UCNP and MoS2, this invention enables sensitive detection of wound PA by observing the "on-off" of fluorescence.

[0028] Mechanism of the invention: The fluorescent aptamer biosensor of the present invention mainly consists of a recognition element, a signal transduction element, and a signal amplification element. Its principle is that a biologically active receptor (aptamer) reacts with the target analyte and generates a signal. This signal is then converted into detectable and analyzable light, electrical, or other signals by a transduction element combined with optical technology. Finally, the amplified signal is output to achieve qualitative or quantitative detection of the analyte. Specifically, the present invention provides a fluorescent aptamer sensor based on UCNP-MoS2. This sensor uses UCNP as its fluorescent donor, MoS2 as its fluorescent acceptor, and PA nucleic acid aptamer as a specific capture element. PA is detected via FRET. The working principle of the sensor is described in [reference needed]. Figure 1MoS2 nanosheets are primarily modified to induce a quenching effect through electrostatic adsorption on the UCNP surface. Furthermore, MoS2 nanosheets spontaneously adsorb single-stranded DNA via van der Waals forces and base interactions, thus exhibiting strong absorption and fluorescence quenching efficiency in the near-infrared region. Figure 4 The UV-Vis absorption spectrum of MoS2 nanosheets and the 980 nm fluorescence emission spectrum of UCNPs are shown. Aptamer-functionalized upconversion nanoparticles (UCNP-apt) serve as fluorescence donors, MoS2 nanosheets as fluorescence acceptors, and the aptamers are selected as specific recognition elements for PAs. The aptamers adsorb onto the MoS2 nanosheets via van der Waals forces, effectively quenching UCNP fluorescence. However, when PAs are introduced into the system, they bind to the specific aptamers and change their conformation, causing the UCNP-apt to dissociate from the MoS2 nanosheets, thus partially restoring the quenched fluorescence. This invention is based on the FRET that occurs between UCNPs and MoS2, and according to the "on-off" nature of fluorescence, it enables sensitive detection of PAs in wounds.

[0029] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention is the first to obtain a UCNP-MoS2 sensor with high sensitivity, high specificity, and high selectivity. Based on the unique upconversion fluorescence emission of UCNP, the strong quenching properties of MoS2 nanosheets, and the significant binding force between the aptamer and PA, this invention can detect PA in infected wounds with high sensitivity, high specificity, and high selectivity, with a detection limit as low as 15.5 cfu / mL. Fluorescent aptamer biosensors, compared with other conventional methods for detecting pathogenic bacteria, have advantages such as high sensitivity and fast detection speed, and are widely used in the detection of microorganisms in biomedicine. This invention eliminates the disadvantage of long detection time in the original method (bacterial culture), reducing the detection time from 2.5 days to 2 hours. Attached Figure Description

[0030] Figure 1 Schematic diagram of the UCNP-MoS2 fluorescent aptamer sensor for PA detection.

[0031] Figure 2 (A) Synthesized UCNPs; (B) UCNPs after cleaning under 980nm laser irradiation.

[0032] Figure 3 Characterization of UCNPs: (A) Electron micrograph of UCNP before silicon coating; (B) XRD pattern of UCNP; (C) Electron micrograph of UCNP after silicon coating; (D) Electron micrograph of MoS2.

[0033] Figure 4 UCNP-MoS2 fluorescence superposition image.

[0034] Figure 5FTIR spectra of UCNP.

[0035] Figure 6 Characterization of UCNP-apt-PA.

[0036] Figure 7 1. Optimization of conditions for the UCNP-MoS2 fluorescence sensor: (A) concentration of MoS2 nanosheets; (B) UCNP / MoS2 incubation time; (C) system pH; (D) response time of PA.

[0037] Figure 8 Detection of PA by UCNP-MoS2 fluorescence sensor.

[0038] Figure 9 Selectivity analysis of the UCNP-MoS2 fluorescence sensor. Detailed Implementation

[0039] Example 1: Preparation of UCNP

[0040] YCl3·6H2O (0.3124 g), YbCl3·6H2O (0.1006 g), and ErCl3·6H2O (0.011 g) (Y:Yb:Er = 80:18:2) were added to a 250 mL three-necked flask. Then, 12 mL of oleic acid (OA) and 30 mL of octadecene (ODE) were added to the flask with magnetic stirring, and nitrogen gas was purged for 10 minutes to remove any remaining gases. The solution was heated to 160 °C to form a homogeneous solution, and then the temperature was lowered to 60 °C. 20 mL of a methanol solution containing sodium hydroxide (0.2 g) and NH4F (0.296 g) was slowly added to the flask and maintained for 30 minutes to remove the methanol. The solution was then heated to 285 °C and maintained under nitrogen for 1 hour. After the solution was cooled to room temperature, nanocrystals were precipitated from the solution with ethanol. The UCNPs were collected by centrifugation at 8000 rpm for 10 minutes and then washed three times with ethanol / cyclohexane (1:1 v / v). Finally, they were dried in an oven to obtain UCNPs.

[0041] Example 2: Fabrication of UCNP-MoS2 fluorescent aptamer sensor

[0042] After synthesizing UCNPs in the first step, this invention modifies them to transform them from oil-soluble particles into water-soluble particles. To obtain hydrophilic UCNPs, 10 mg of UCNPs powder is added to 4.0 mL of cyclohexane and sonicated until completely dispersed to obtain a cyclohexane solution of UCNPs. Then, 0.1 mL of Lgepal CO-520 and 6.0 mL of cyclohexane are added and mixed, and stirred for 10 minutes to obtain a solution. Next, 0.4 mL of Lgepal CO-520 and 0.08 mL of ammonia (wt. / v = 30%) are added to the above solution, the container is sealed and sonicated for 20 minutes to form a transparent emulsion. Then, 0.04 mL of LTEOS is added to the emulsion and the mixture is rotated at 600 rpm / min for 11 hours. Then, 15 μL of LAPTEs is added and stirring is continued for 1 hour to form aminated upconversion particles UCNPs-SiO2-NH2. UCNPs-SiO2-NH2 was precipitated by adding acetone, washed twice with ethanol / water (v:v = 1:1), and then stored in water.

[0043] Next, UCNP was bioconjugated with aptamers. UCNP-apt was prepared via glutaraldehyde cross-linking. 20 mg of pre-prepared UCNP-SiO2-NH2 was added to 10 mL of PBS buffer (pH 7.4, 20 mM) and sonicated for 10 min. Subsequently, 2.5 mL of 25% glutaraldehyde aqueous solution was added under light-protected conditions and shaken for 2 h. The solution was then centrifuged at 9000 rpm for 10 min, and the precipitate was washed three times with PBS buffer. The activated UCNP was redispersed in 10 mL of PBS buffer, 50 μL of 100 μM PA aptamer solution was added, and the mixture was incubated at 37 °C with shaking for 10 h. Unbound aptamers were removed by centrifugation at 8000 rpm, finally obtaining aptamer-modified UCNP-apt. Figure 6 Electron microscopy revealed the specific recognition of PA by the aptamer, with UCNP-apt tightly adsorbed around the PA. Finally, 300 μL of 0.1 mg / mL MoS2 was introduced into the system to induce FRET between UCNPs and MoS2, thus completing the construction of the fluorescent aptamer sensor.

[0044] Example 3 Characterization of UCNP and UCNP-MoS2 fluorescent aptamer sensors

[0045] This invention characterizes UCNPs using a series of methods, including TEM, FTIR, and XRD, to evaluate their morphology, size, and crystal form. For example... Figure 3 A. TEM analysis shows that the synthesized UCNPs have a regular hexagonal morphology and uniform size, with a particle size of approximately 88 nm. The XRD pattern of the UCNPs (...) Figure 3B) It can be clearly seen that the diffraction peaks of the prepared UCNPs almost perfectly match those of the standard card JCPDS No. 28-1192, indicating that the prepared material has a pure crystalline phase structure from a crystal structure analysis perspective. After coating its surface with SiO2, as... Figure 3 C shows that these UCNPs are encased in a uniformly thick silicon layer, and their size has increased to 99.8 nm. In addition, Figure 5 FT-IR characterization was performed on UCNPs before and after silicon coating, as shown by the black line in the figure (UCNP-OA). Due to the presence of oleic acid molecules on the UCNP surface, at 2928 cm⁻¹... -1 and 2855cm -1 At 1562 cm⁻¹, peaks corresponding to the asymmetric and symmetric stretching vibrations of the methylene group (-CH₂-) in oleic acid appear. -1 and 1468cm -1 Symmetric and asymmetric stretching vibration peaks of the oleic acid carboxyl group (COO-) appear at 1049 cm⁻¹. After amino modification, the peak at 1049 cm⁻¹ is observed in UCNP-SiO₂-NH₂ (red line). -1 A symmetrical stretching vibration peak belonging to the silicon-oxygen bond (Si-O) appears at 3440 cm⁻¹. -1 A stretching vibration peak of amino group (-NH2-) appears at 1637 cm⁻¹. -1 The appearance of a bending vibration peak of amino (-NH2-) indicates that SiO2 has been coated on the outer layer of UCNPs and that amination has been successful.

[0046] Example 4: PA strain culture and plate counting

[0047] 100 μL of PA (CMCC10104) was inoculated and grown in Luria Bertani broth (10 mg·mL⁻¹). -1 Medium tryptone, 5 mg / mL -1 One part yeast extract, 10 mg / mL -1 NaCl (pH 7.0) was added and the mixture was shaken at 200 rpm for 18-24 hours at 37°C. Before use, bacterial cells were centrifuged at 10,000 rpm for 1 minute to separate them from the broth. The separated bacterial cells were then washed three times with PBS buffer (20 mM, pH 7.4), and the optical density (OD) was measured on a UV spectrophotometer. 600 Then, the desired bacterial concentration (8.7 × 10⁻⁶) was adjusted by measuring the optical density. 0 -8.7×10 7 (cfu / mL). Subsequently, the bacterial suspension was serially diluted 10-fold. The diluted bacteria were inoculated onto sterile culture medium and incubated at 37°C for 48 hours, during which the bacterial cells were counted. Bacterial culture and sample processing were performed in a sterile cleanroom.

[0048] Example 5: Optimization of Fluorescent Aptamer Sensor Fabrication Conditions

[0049] First, the effect of MoS2 concentrations (0.01, 0.03, 0.05, 0.07, 0.09, and 0.10 mg / mL) on the quenching of UCNP fluorescence was investigated at 0.1 mg / mL UCNP-apt and pH 7.4. Following a typical carbodiimide coupling procedure, specific *Pseudomonas aeruginosa* aptamers were coupled to the surface of UCNPs. These UCNP-apts exhibited strong upconversion fluorescence at an excitation wavelength of 980 nm. Since MoS2 has a broad absorption range of 200–800 nm, it can overlap well with the fluorescence emission spectrum of UCNPs-apt. Therefore, the strong upconversion fluorescence of UCNP-apts was maximally quenched upon the addition of MoS2. Figure 7 As can be seen from A, the fluorescence intensity decreases continuously with the increase of MoS2 concentration until the concentration of MoS2 is 0.09-0.1 mg / mL, at which point the fluorescence intensity stops decreasing. Therefore, the optimal concentration of MoS2 is 0.1 mg / mL.

[0050] Secondly, the effect of UCNP / MoS2 incubation time on fluorescence intensity was investigated under the conditions of 0.1 mg / mL UCNP-apt, 0.1 mg / mL MoS2, and pH 7.4. Figure 7 As can be seen from B, the present invention selects (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min as the optimal time). As the incubation time is continuously extended, the fluorescence intensity continuously decreases until after 30 min, the fluorescence intensity no longer changes. Therefore, 30 min is selected as the optimal incubation time for UCNP / MoS2.

[0051] Furthermore, under the conditions of 0.1 mg / mL UCNP-apt, 0.1 mg / mL MoS2, and UCNP / MoS2 incubation time of 30 min, pH values ​​of 5, 6, 7.4, 8, 9, and 10 were selected as the optimal pH conditions, as shown in Figure 7C. Under the above optimal conditions, the fluorescence intensity reached its maximum when the system pH was 7.4. Therefore, pH 7.4 was selected as the optimal condition for subsequent experiments in this invention.

[0052] Finally, the effect of UCNP-MoS2 / PA incubation time on the system fluorescence recovery value was investigated. Keeping other conditions optimal (0.1 mg / mL UCNP-apt, 0.1 mg / mL MoS2, pH 7.4), the optimal incubation times were selected as (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min). Figure 7 As shown in D, in the presence of PA in the system, the specific aptamer preferentially binds to PA, leading to a conformational change in the aptamer. This phenomenon causes UCNP-apt to dissociate from the MoS2 surface. The process of MoS2 quenching UCNP fluorescence is inhibited in the system. Fluorescence recovery is rapid within 15 minutes after the addition of PA, and the fluorescence intensity stabilizes after 20 minutes. Therefore, 20 minutes is chosen as the optimal incubation time for UCNP-MoS2 / PA.

[0053] Example 6: Detection of PA using the UCNP-MoS2 fluorescent aptamer sensor

[0054] A fluorescence detection procedure for PA was developed. PA was detected using optimized conditions (0.1 mg / mL UCNP-apt, 0.1 mg / mL MoS2, pH 7.4, and UCNP / MoS2 incubation time of 30 min). Quantitative analysis of PA was performed as follows: First, UCNP-apt was diluted to 0.1 mg / mL with PBS (20 mM, pH 7.4) buffer. 300 μL of this solution was added to 300 μL of 0.1 mg / mL MoS2 dispersion, mixed, and incubated at 37°C for 15 min to obtain the UCNP / MoS2 fluorescent aptamer sensor. Then, 300 μL of different concentrations of target bacterial solutions (8.7 × 10⁻⁶) were added. 1 8.7×10 2 8.7×10 3 8.7×10 4 8.7×10 5 8.7×10 6 8.7×10 7 cfu·mL -1 CFU / mL was mixed and incubated at 37°C for 30 minutes. Upconversion fluorescence emission spectra in the 400-750 nm range were collected under 980 nm laser excitation. To verify the sensing performance of the constructed fluorescence sensor, different concentrations (8.7 × 10⁻⁶ CFU / mL) were added to the system. 1 8.7×10 2 8.7×10 3 8.7×10 4 8.7×10 5 8.7×10 6 8.7×10 7 cfu·mL -1 The performance of the fluorescence sensor was assessed by observing the change in fluorescence value at 654 nm, and the results are as follows: Figure 8(A). After PA is added to the system, the aptamer preferentially binds to the bacteria, which will cause the fluorescence of the sensor to recover. The recovery value of the system fluorescence intensity (ΔF = F - F0) is linearly related to the bacterial concentration (Log CFU / mL), such as... Figure 8 As shown in (B), ΔF represents the difference in upconversion fluorescence intensity recovery value under 980nm laser excitation with and without PA. (where R...) 2 =0.9941, limit of detection (LOD) = 5.5 CFU / mL.

[0055] Example 7: Selectivity Analysis of Fluorescent Aptamer Sensors

[0056] To evaluate the specificity of the fluorescent sensor prepared in this invention, Staphylococcus aureus (S. aureus A TCC 25923), Escherichia coli (E. coli BL21), and lactic acid bacteria (LAB (sourdough fermentation)) were used for specificity detection, with a uniform concentration of 1×10⁻⁶ for each bacterium. 6 cfu / mL. For example... Figure 9 As shown, compared with other bacteria, the entire system showed significant fluorescence recovery after the addition of PA. Furthermore, it can be observed that the fluorescence intensity in the presence of the target bacteria was significantly stronger than that of the other control groups, clearly demonstrating that the fluorescent aptamer sensor is suitable for the selective detection of PA.

[0057] Example 8: PA Detection Based on UCNP-MoS2 Fluorescent Aptamer Sensor

[0058] Detection of PA in real samples. To evaluate the accuracy of the prepared fluorescence sensor in the detection of real samples, this invention selected the burn wound skin of laboratory mice for measurement. Concentrations of 1.0 × 10⁻⁶ were added to the wound skin of three mice, respectively. 4 1.0×10 5 1.0×10 6 cfu.mL -1PA was cultured for 24h, 48h, and 72h. Then, 10μL of PA-containing tissue fluid was taken from the wounded skin of different mice and diluted with 1mL of PBS (20mM, pH 7.4) buffer to obtain the test solution. Following the method in Example 6, PA in the wounded skin of three mice was quantitatively detected using optimized conditions (0.1mg / mL UCNP-apt, 0.1mg / mL MoS2, pH 7.4, UCNP / MoS2 incubation time 30min). First, UCNP-apt was diluted to 0.1mg / mL with PBS (20mM, pH 7.4) buffer. 300μL of this solution was added to 300μL of 0.1mg / mL MoS2 dispersion, mixed, and incubated at 37℃ for 15 minutes to obtain the UCNP / MoS2 fluorescent aptamer sensor. Then, 300μL of the test solution was added, mixed, and incubated at 37℃ for 30 minutes. Upconversion fluorescence emission spectra in the 400-750nm range were collected under the excitation of a 980nm laser, and three parallel experiments were conducted to verify the performance of the prepared fluorescence sensor. The entire detection process took about two hours.

[0059] Table 1

[0060]

[0061] a RSD: (standard deviation) / mean×100%, n=3

[0062] b p>0.05, indicate: no significant difference

[0063] As shown in Table 1, the detection results of this method are similar to those of traditional methods, and no significant difference was found after statistical t-test, indicating that the performance of this method is better and can be used for practical application detection of PA. The recovery rate of this method is high (82.3%-92.9%). Therefore, it is proven that the sensor constructed in this invention has good accuracy, is easy to operate, saves time, and is less affected by external factors, and can be used in actual experiments to detect whether a wound is infected with PA.

Claims

1. A fluorescent aptamer sensor, characterized in that, The fluorescent aptamer sensor was prepared by coupling aminated UCNPs with Pseudomonas aeruginosa PA nucleic acid aptamers and then incubating them with MoS2. The sequence of the PA nucleic acid aptamer is shown in SEQ ID NO.

1. The concentration of the aminated UCNPs is 0.1 mg / mL, the concentration of the PA nucleic acid aptamer is 100 μM, and the concentration of MoS2 is 0.1 mg / mL.

2. The method for preparing the fluorescent aptamer sensor according to claim 1, characterized in that, Includes the following steps: 1) Aminated UCNPs: UCNPs powder was added to cyclohexane and sonicated until it was completely dispersed to obtain a cyclohexane solution of UCNPs. Then, lgepal CO-520 and cyclohexane were added and mixed and stirred to obtain a solution. lgepal CO-520 and ammonia were added to the above solution, the container was sealed and sonicated to form a transparent emulsion. Then, TEOS was added to the emulsion and rotated. Then, APTEs were added and stirring was continued to form aminated upconversion particles UCNPs-SiO2-NH2. Acetone was added to precipitate the particles and the mixture was washed twice with ethanol / water. The precipitate was then stored in water. 2) Coupling with PA aptamers: The prepared UCNPs-SiO2-NH2 was added to PBS buffer and ultrasonically dispersed. Then, glutaraldehyde aqueous solution was added and shaken under light-protected conditions. The solution was then centrifuged, and the precipitate was washed with PBS buffer. The activated UCNPs were redispersed in PBS buffer, and PA aptamer solution was added and incubated with shaking. Unbound aptamers were removed by centrifugation, and finally UCNPs-apt was obtained. The concentration of the amino-modified UCNPs was 0.1 mg / mL, and the concentration of PA aptamers was 100 μM. 3) MoS2 introduction: MoS2 was added to UCNPs-apt and incubated to complete the construction of the fluorescent aptamer sensor. The concentration of MoS2 was 0.1 mg / mL.

3. The method for preparing the fluorescent aptamer sensor according to claim 2, characterized in that, Step 1) After adding TEOS, rotate for 11 to 12 hours, then add APTES dropwise and rotate for 1 to 2 hours. The rotation speed is 600 to 700 rpm.

4. The method for preparing the fluorescent aptamer sensor according to claim 2, characterized in that, In step 2), add a 25% glutaraldehyde aqueous solution and shake for 1-2 hours.

5. The method for preparing the fluorescent aptamer sensor according to claim 2, characterized in that, Step 3) The incubation pH is 5-10, and the incubation time is 5-35 min.

6. The fluorescent aptamer sensor according to claim 1 in Pseudomonas aeruginosa (… Pseudomonas aeruginosa Applications of ) in non-diagnostic testing.

7. The application according to claim 6, characterized in that, The Pseudomonas aeruginosa ( Pseudomonas aeruginosa The concentration of ) was 8.7 × 10 1 -8.7×10 7 cfu / mL.

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