An ultra-fast fungal trapping agent, its preparation method and uses

Abstract

This invention relates to an ultrafast intelligent fungal capture agent, which is a core-shell nanostructure. The core is a magnetic nanoparticle, and the shell is a thermosensitive polymer. The surface of the thermosensitive polymer is coupled with substances that specifically bind to fungi. This ultrafast fungal capture agent exhibits rapid capture speed, successfully isolating approximately 75% or more of the fungi, which can then be immediately detected using SERS. The preparation method of this fungal capture agent is simple and mild, allowing for repeated recycling using an external magnetic field, and the fungal capture capacity remains largely unchanged. It is significantly economical for industrial applications and suitable for large-scale use. This fungal capture agent possesses intelligent capture and release behavior. By controlling the temperature, an intelligent and efficient fungal capture-detection process can be completed, reducing the entire process time to within 10 minutes, thus improving diagnostic speed and reducing patient mortality.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial synthesis technology, specifically relating to an ultra-fast fungal capture agent, its preparation method, and its uses. Background Technology

[0002] Over the past two decades, with the use of immunosuppressants and broad-spectrum antibiotics, the increase in invasive treatments such as transplantation and intubation, and the rise in immunocompromised patients and sub-healthy individuals, the global rate of invasive fungal infections has increased significantly. The situation of invasive fungal infections in my country is severe, making effective control of infections and reducing patient mortality an urgent priority. Invasive fungal infections (IFIs) seriously threaten patient health due to their long diagnostic time (more than 7 days) and frequent misdiagnosis. Therefore, the mortality rate of IFIs is as high as 39%-60%. Traditional fungal detection methods are very time-consuming and often miss pathogens. Polymerase chain reaction (PCR) is a commonly used clinical technique for detecting invasive fungi, but its DNA extraction efficiency is poor, limiting its clinical application. The main diagnostic methods for fungal infections rely on imaging examinations such as X-rays and CT scans, but existing methods cannot meet the requirements for rapid diagnosis. Therefore, early, accurate, and efficient detection of yeast-like pathogens is of great significance for improving patients' quality of life, reducing medical costs, and alleviating drug resistance pressure.

[0003] Isolation and identification of pathogenic fungi are challenging due to the need for lengthy in vitro cultures, which can lead to high mortality rates. Direct detection of invasive fungi from clinical specimens is therefore of great importance. Surface-enhanced Raman scattering (SERS) is a promising non-destructive tool for the direct detection of fungi from clinical samples; however, capturing fungi directly from clinical samples remains time-consuming, and SERS substrates are typically non-selective and cannot yet be used for pathogen detection in clinical samples.

[0004] Existing technologies have reported some alternative strategies. For example, reference 1 (S.Hu, H.Kang, F.Gu, C.Wang, S.Cheng, W.Gong, L.Wang, B.Gu, Y.Yang, Int J Nanomedicine. 2021, 16, 941-950) reports a method involving pre-modified magnetic nanoparticles and a positively charged substrate to directly isolate fungi from samples and detect SERS signals. However, fungal capture takes more than 20 minutes, and the entire operation takes several hours, which is insufficient for practical needs. Therefore, we continue to develop a method for ultra-fast fungal capture and detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an ultrafast fungal capture agent, its preparation method, and its applications. This invention utilizes ultrasound-initiated polymerization to coat the surface of magnetic nanoparticles with a temperature-sensitive block polymer, endowing the fungal capture agent with intelligence. Furthermore, it leverages coupling sites provided by the polymer to connect to molecular structures targeting fungal cell walls, giving it specificity for fungal capture. The fungal capture agent of this invention exhibits stable size and morphology, ultrafast fungal capture capability, and good potential for clinical application. The preparation method of this ultrafast fungal capture agent is simple and reproducible, enabling ultrafast capture of fungi from complex samples using an intelligent strategy. The capture time is only 3 seconds above the lower critical solution temperature (LCST), after which the temperature decreases below the LCST, and the capture agent releases fungi onto a SERS substrate for detection. The entire operation takes less than 10 minutes.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] An ultra-fast intelligent fungal trapping agent has a core-shell nanostructure, with the core being a magnetic nanoparticle and the shell being a thermosensitive polymer. The surface of the thermosensitive polymer is coupled with a substance that can specifically bind to fungi.

[0008] Furthermore, the magnetic nanoparticles are modified with amino groups on their surface, preferably Fe3O4 nanoparticles coated with chitosan containing amino groups, with a particle size of 300-600 nm, more preferably 400-500 nm.

[0009] Furthermore, the carboxyl-containing thermosensitive polymer is a thermosensitive unsaturated monomer obtained by copolymerizing the carboxyl-containing unsaturated monomer with a crosslinking agent.

[0010] Furthermore, the thermosensitive unsaturated monomer is selected from at least one of N-isopropylacrylamide, N-vinylcaprolactam, N-vinylisopropionamide, N,N-diethyl-2-acrylamide, and N-vinylcaprolactam; the carboxyl-containing unsaturated monomer is selected from at least one of (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; and the crosslinking agent is selected from at least one of polyethylene glycol diacrylate, methylenebisacrylamide, di(methacryloyloxyethyl) hydrogen phosphate, and bis(2-methylpropene)ethoxydisulfide.

[0011] Furthermore, the molar ratio of the thermosensitive unsaturated monomer, the carboxyl-containing unsaturated monomer, and the crosslinking agent is 10-15:1-2:1-1.3. 30-50% of the carboxyl-containing unsaturated monomer is first coupled with the amino-modified magnetic nanoparticles before participating in the polymerization reaction.

[0012] Furthermore, the molar amount of the carboxyl-containing unsaturated monomer and the mass ratio of the magnetic nanoparticles are 0.2-0.5 mmol:1g; preferably 0.34-0.4 mmol:1g.

[0013] Furthermore, the substance capable of specifically binding to fungi is selected from at least one of caspofungin, amphotericin B, and anidulafungin; the mass ratio of the substance capable of specifically binding to fungi to the magnetic nanoparticles is 0.5 × 10⁻⁶. -5 -5×10 -5 :1.

[0014] Caspofungin, rich in amino groups, can couple with the carboxyl groups on the surface of thermosensitive polymer-coated magnetic nanoparticles. Furthermore, with the aid of a crosslinking agent, caspofungin can uniformly and stably modify the surface of the magnetic nanoparticles through chemical bonds, thereby specifically binding to fungi in the test system. Because the polymer on the surface of the magnetic nanoparticles is thermosensitive, below the LCST (lowest temperature range), the polymer is in a stretched state, facilitating adsorption and affinity for more fungi; above the LCST, the polymer chains begin to contract, capturing the fungi. This invention intelligently controls the capture and release of fungi through changes in external temperature, achieving intelligent capture and testing, avoiding matrix interference, accelerating the capture speed, and improving fungal detection efficiency.

[0015] The fungal capture agent of the present invention is prepared by dispersing in water to form a dispersion for storage and use. The concentration of the dispersion is 10-100 μg / mL, preferably 20-50 μg / mL.

[0016] This invention also provides a method for preparing an ultra-fast fungal scavenger, comprising the following steps:

[0017] (1) Magnetic nanoparticles with amino groups on their surface are coupled with 30-50% of unsaturated monomers containing carboxyl groups.

[0018] (2) Using dispersion polymerization, the remaining carboxyl-containing unsaturated monomer, thermosensitive unsaturated monomer, crosslinking agent, emulsifier, and initiator are added to the dispersion of the product obtained in step (1), and copolymerized under ultrasonic conditions to obtain polymer-coated magnetic nanoparticles.

[0019] (3) The polymer-coated magnetic nanoparticles prepared in step (2) are coupled with the carboxyl groups on their surface and the amino groups on substances that can specifically bind to fungi to obtain the fungal scavenger.

[0020] Further, in step (1), the coupling reaction is carried out under activation conditions, and the activating agent is EDC / NHS. After reacting for 10-15 hours, it is stored at a low temperature of 2-8°C for later use.

[0021] Further, in step (2), the emulsifier is selected from sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, and the amount of emulsifier is 0.5-2% of the total molar amount of monomers (the sum of unsaturated monomers containing carboxyl groups, thermosensitive unsaturated monomers, and crosslinking agents); the initiator is a water-soluble initiator, specifically selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate, and the amount of initiator is 0.5-2% of the total molar amount of monomers. The reaction is carried out at 30-40℃ and 60-100MHz ultrasonic environment for 3-5 hours.

[0022] Traditional polymerization methods often lead to the aggregation of magnetic nanoparticles. This invention utilizes ultrasonic-initiated polymerization, significantly improving the thermodynamic stability of the resulting temperature-sensitive polymer-coated magnetic nanoparticles. Carboxyl-containing monomers and temperature-sensitive monomers are polymerized into the shell and tightly cross-linked with the magnetic nanoparticles through a cross-linking agent, rather than adsorbing onto the surface of the magnetic nanoparticles via electrostatic interactions. This results in greater stability, more accurate detection results, and the ability to be recycled.

[0023] Further, in step (3), the product obtained in step (2) is first activated, specifically by activating it with EDC / NHS for 8-15 hours, and then an aqueous solution of a substance that specifically binds to fungi is added, and the reaction is carried out for 10-20 hours. The obtained product is centrifuged and washed to obtain the fungal scavenging agent. The concentration of the aqueous solution of the substance that specifically binds to fungi is 10-50 μg / mL.

[0024] The magnetic nanoparticles coated with the thermosensitive polymer obtained in this invention have carboxyl groups on their surface, which can easily react with some groups, such as the amino groups in caspofungin (CAS), to smoothly couple with substances that specifically bind to fungi, thereby capturing fungi in the sample.

[0025] The fungal capture agent obtained by this invention changes the dispersion state of the polymer chain according to the ambient temperature, thereby enabling the capture or release of fungi through temperature control. The capture and collection process is very fast, requiring only about 3 seconds.

[0026] This invention also provides an ultra-rapid method for detecting fungi, comprising the following steps:

[0027] (S1) At a temperature below the low critical transition temperature (LCST) of the thermosensitive polymer, the above-mentioned ultra-fast fungal capture agent and the sample containing fungi are mixed, and then the temperature is raised to above the low critical transition temperature to complete the fungal capture and obtain a complex of fungal capture agent and fungi.

[0028] (S2) The fungal capture agent-fungal complex was enriched using an external magnetic field. The supernatant was discarded, the precipitate was mixed with the SERS substrate, dried, and then subjected to SERS detection.

[0029] The SERS substrate is not particularly limited, such as silver nanoparticles (AgNPs) or gold nanoparticles (AuNPs); its preparation method is well known in the art. In one specific embodiment of the present invention, a mixed solution of silver salt, surfactant and ammonia water and a mixed solution containing surfactant and reducing agent are stirred in an ice bath for 4-6 hours, then the solution is heated for 5-10 minutes, cooled, and stored at 2-5°C for later use.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The ultra-fast fungal capture agent of this invention has a fast capture speed, which can complete the capture of fungi within 3 seconds. It can successfully isolate more than 75% of fungi and immediately use SERS for detection. The whole process only takes 10 minutes, which is crucial for direct SERS detection of clinical samples, can improve the speed of diagnosis and reduce patient mortality.

[0032] 2. The preparation method of the fungal capture agent described in this invention is simple and mild. It can be recycled and reused repeatedly by applying an external magnetic field, and the fungal capture capacity will not decrease significantly. It has significant economic advantages suitable for industrial use and is suitable for large-scale application.

[0033] 3. The fungal capture agent of this invention exhibits intelligent capture and release behavior. When the temperature is below the LCST, the thermosensitive polymer is in a stretched state, facilitating adsorption and fungal capture. Raising the ambient temperature above the LCST causes the thermosensitive polymer to contract, completing the fungal capture. Furthermore, by controlling the temperature, when the ambient temperature is above the LCST, external magnetic force can bind the fungal-captured agent to the bottle wall in just 3 seconds, completing an ultra-fast capture process; while when the temperature is below the LCST, it takes more than 30 minutes. Based on the aforementioned fungal capture and magnetic behavior of the thermosensitive polymer at different temperatures, an intelligent and efficient fungal capture-detection process can be completed by controlling the temperature, increasing the entire process speed to 10 minutes.

[0034] 4. The fungal capture agent described in this invention is specific to fungi, provides accurate detection results, can be used repeatedly, and the fungal capture efficiency does not decrease significantly.

[0035] 5. The fungal capture agent of the present invention has good biocompatibility and has great potential in clinical fungal detection applications. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the preparation of the ultra-fast fungal scavenger obtained in Example 1;

[0037] Figure 2 This is a TEM image of the silver nanoparticles obtained in the preparation example;

[0038] Figure 3 The FTIR spectrum of the ultra-fast fungal scavenger prepared in Example 1 of this invention;

[0039] Figure 4 TEM image of the ultra-fast fungal scavenger prepared in Example 1 of this invention;

[0040] Figure 5 This is a SEM elemental distribution diagram of the ultra-fast fungal scavenger prepared in Example 1 of the present invention;

[0041] Figure 6 This is a performance test of the ultra-fast fungal capture agent in Example 1 of the present invention;

[0042] Figure 7 This is a schematic diagram of the process for detecting fungi using an ultra-fast fungal capture agent according to the present invention;

[0043] Figure 8 This invention utilizes the magnetic force test of ultra-fast fungi at different temperatures;

[0044] Figure 9 These are the SERS spectra of the three captured fungi;

[0045] Figure 10 These are the OPLS-DA analysis results of the SERS spectra of the three captured fungi. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as a limitation of the present invention.

[0047] MNP was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Caspofungin (CAS), acrylic acid (AA), N-isopropylacrylamide (NIPAM), ammonium persulfate (APS), bis(2-methylpropene)ethoxydisulfide (BMOD), sodium dodecyl sulfate (SDS), silver nitrate (AgNO3) with a purity of 99%, and sodium borohydride (NaBH4) were all purchased from Sigma-Aldrich. All chemicals used in cell experiments were purchased from Keygen Biotech Co., Ltd. (Nanjing, China). Monocrystalline silicon wafers were provided by Xuzhou Tongxin Optoelectronic Technology Co., Ltd. (Xuzhou, China). Clinical samples were provided by Guangdong Provincial People's Hospital.

[0048] Preparation Example

[0049] Preparation of positively charged silver nanoparticles (AgNPs) +As the SERS substrate: Solution A, containing AgNO3 (21.92 mg), CTAB (7.3 mg), and 25% NH4OH (1 mL), was added to 39 mL of ultrapure water. Solution B, containing CTAB (7.3 mg) and NaBH4 (12.1 mg) dissolved in 40 mL of ultrapure water, was also prepared. Solutions A and B were placed in an ice bath for 10 minutes. Solution A was then stirred in an Erlenmeyer flask using a magnetic stirrer under ice bath conditions. Solution B was added dropwise to solution A during stirring until the solution turned dark yellow. Stirring was then stopped after 4 hours, and the solution was heated for 8 minutes. Stirring continued until the sample cooled to room temperature. The volume was brought to 80 mL, and the sample was stored at 4 °C. The resulting AgNPs... + It is nanosphere-shaped, and its TEM image is as follows. Figure 2 As shown.

[0050] Example 1

[0051] (1) The acrylic acid (AA, 100mM, 0.34mL) solution was activated with EDC / NHS and reacted with chitosan-coated magnetic nanoparticles (NMP, 0.1mg / L, 2mL) at room temperature for 12h and stored at 4℃ for later use.

[0052] (2) Add N-isopropylacrylamide (PNIPAM, 100mM, 6mL), acrylic acid (AA, 100mM, 0.34mL), bis(2-methylpropene)ethoxydisulfide (CAS: 36837-97-5, 50mM, 1.2mL), sodium dodecyl sulfate (SDS) (100mM, 100μL) aqueous solution to the reaction vessel, mix well, add the magnetic nanoparticles coated with acrylic acid and chitosan obtained in step (1), blow nitrogen to remove air for 20min, add ammonium persulfate (10mM, 1mL), react at 35℃ and 100MHz ultrasonic environment for 4h. After polymerization, centrifuge (14000rpm, 10min) to collect the magnetic nanoparticles MNP@PNIPAMAA coated with temperature-sensitive polymer, wash three times with ultrapure water to remove small molecules and homopolymers. That is, the monomer molar ratio of the thermosensitive polymer is 10:1.13:1, and 50% of the acrylic acid is first coupled with the chitosan-coated magnetic nanoparticles in step (1).

[0053] (3) The magnetic nanoparticles coated with the thermosensitive polymer prepared in step (2) were activated with EDC / NHS for 12 hours, caspofungin (CAS) aqueous solution (10 μg / mL, 2 mL) was added, and the reaction was carried out for 12 hours. The product was washed three times and purified by centrifugation (14000 rpm, 30 min). The final product MNP@PNIPAMAA-CAS was dispersed in water and prepared as a 20 μg / mL dispersion, which was stored at 4℃ for later use.

[0054] Figure 3 The FTIR spectrum of the obtained ultra-fast fungal scavenger MNP@PNIPAMAA-CAS confirms the accuracy of the structure. The main peak is assigned as follows: 3285 cm⁻¹ -1 The absorbance at 1642 and 1554 cm⁻¹ is attributed to the stretching of secondary amide NH₄⁺. -1 The absorption peak of -Co-NH- is attributed to this, and the characteristic absorption peak of -CH3 is located at 2794 cm3. These results indicate successful coupling of NIPAM. Comparing the FTIR spectra of MnP@PNIPAMAA and MnP@PNIPAMAA-CAS, the absorption of NH and -Co-NH- is significantly increased due to the coupling of caspofungin.

[0055] The size and morphology of the ultra-rapid fungal scavenger MNP@PNIPAMAA-CAS were tested using transmission electron microscopy (TEM). Figure 4 (As shown). The surface of MnP@PNIPAMAA-CAS is mainly composed of NIPAM with an outer coating layer. This fungal scavenger is regularly spherical with a good morphology and a particle size of about 600 nm. Dynamic light scattering (DLS) data show that the hydrated particle size is 1316 nm due to swelling of MNP@PNIPAMAA-CAS in room temperature aqueous solution.

[0056] The elemental distribution of the ultra-rapid fungal scavenger was tested using scanning electron microscopy (SEM). The prepared ultra-rapid fungal scavenger was diluted 50-fold, and 10 μL of the aqueous solution was dropped onto a copper mesh for electron microscopy. After drying, the elemental distribution of the scavenger was tested using SEM. The figure mainly shows the abundant surface distribution of C, N, O, and Si elements (e.g., ...). Figure 5 (As shown). C, N, and other elements were observed in the CAS-targeted structure. This confirms the successful encapsulation of NIPAM and the successful coupling of CAS.

[0057] To further verify that the fungal trapping agent of this invention is non-toxic and has good biocompatibility, its cytotoxicity against 4T1 cells was studied using the MTT assay. The results are as follows: Figure 6 As shown, the fungal scavenging agent of the present invention is not toxic to 4T1 cells.

[0058] To verify the thermosensitivity of the obtained MNP@PNIPAMAA-CAS, the nanoparticle size of the smart nanotrapper above and below LCST was measured by DLS, such as... Figure 6As shown, the size of the fungal scavenger MNP@PNIPAMAA-CAS prepared in this invention is approximately 1.3 μm at 15 °C, but only 480 nm above 32 °C. This is because the thermosensitive polymer is in a stretched state in solution at temperatures below the LCST, resulting in larger block spacing. At temperatures above the LCST, the polymer shrinks, causing rapid fungal aggregation. Therefore, the fungal scavenger provided by this invention offers convenient intelligence for fungal capture and detection, enabling rapid fungal capture in the system.

[0059] The magnetic force of the fungal trap was tested using an external magnet at 35°C and 10°C. At 10°C, the fungal trap exhibited a slower enrichment phenomenon, while at 35°C, the enrichment time of the fungal trap was 3 seconds.

[0060] Example 2

[0061] Other conditions and operations are the same as in Example 1, except that (1) the acrylic acid (AA, 100mM, 0.24mL) solution is activated with EDC / NHS and reacted with chitosan-coated magnetic nanoparticles (NMP, 0.1mg / L, 2g) at room temperature for 12h and stored at 4℃ for later use.

[0062] (2) Add N-isopropylacrylamide (PNIPAM, 100mM, 6mL), acrylic acid (AA, 100mM, 0.56mL), bis(2-methylpropene)ethoxydisulfide (CAS: 36837-97-5, 52mM, 1.2mL), sodium dodecyl sulfate (SDS) (100mM, 100μL) aqueous solution to the reaction vessel, mix well, add the magnetic nanoparticles coated with acrylic acid and chitosan obtained in step (1), blow nitrogen to remove air for 20min, add ammonium persulfate (10mM, 1mL), react at 35℃ and 100MHz ultrasonic environment for 4h. After polymerization, centrifuge (14000rpm, 10min) to collect the magnetic nanoparticles MNP@PNIPAMAA coated with temperature-sensitive polymer, wash three times with ultrapure water to remove small molecules and homopolymers. That is, the monomer molar ratio of the thermosensitive polymer is 15:2:1.3, and 30% of the acrylic acid is first coupled with the chitosan-coated magnetic nanoparticles in step (1).

[0063] Example 3

[0064] The other conditions and operations are the same as in Example 1, except that the amount of the crosslinking agent bis(2-methylpropene)ethoxydisulfide is changed to 1 mL.

[0065] Example 4

[0066] The other conditions and operations are the same as in Example 1, except that the amount of crosslinking agent bis(2-methylpropene)ethoxydisulfide is changed to 2 mL.

[0067] Example 5

[0068] The other conditions and operations are the same as in Example 1, except that the crosslinking agent used is polyethylene glycol diacrylate.

[0069] Example 6

[0070] The other conditions and operations are the same as in Example 1, except that the crosslinking agent used is methylene bisacrylamide.

[0071] Example 7

[0072] The other conditions and operations are the same as in Example 1, except that the crosslinking agent is di(methacryloyloxyethyl) phosphate.

[0073] Application examples

[0074] Figure 7 This is a schematic diagram of the process for detecting fungi using an ultra-fast fungal capture agent according to the present invention. Figure 8 This invention utilizes ultra-fast magnetic force testing of fungi at different temperatures. When the ambient temperature is above the LCST (35°C), an external magnet can completely bond MNP@PNIPAMAA-CAS to the bottle wall in just 3 seconds. However, when the temperature is below the LCST, such as 10°C, it takes 30 minutes to completely bond to the bottle wall, and there is no visible change in the solution after 3 seconds. This is because the MNP@PNIPAMAA-CAS shell is in a stretched state below the LCST, resulting in a larger size and reduced magnetic force from the Fe3O4 core. Conversely, when the material is above the LCST, the MnP@PNIPAMAA-CAS coating shell shrinks, and the magnetic force is enhanced.

[0075] This invention investigates the SERS detection performance of MNP@PNIPAMAA-CAS, using Candida albicans, Candida tropicalis, and Candida krusei as representatives.

[0076] Serum samples from patients with candidiasis were used: 58 cases of Candida albicans, 24 cases of Candida tropicalis, and 15 cases of Candida krusei. Samples were placed in 5 mL Eppendorf pipettes. The serum was heated to 35°C, and 2 mL of 20 μg / mL MNP@PNIPAMAA-CAS prepared in Example 1 was added to 5 mL of serum to form an MNP@PNIPAMAA-CAS@fungal complex. The MNP@PNIPAMAA-CAS@fungal complex was captured at the bottom of the tube using a strong magnet, and the supernatant was discarded. AgNPs obtained in the preparation example were added.+ This forms MNP@PNIPAMAA-CAS@fungi@AgNPs + For the composite, 2 μL of sample was placed on a silicon wafer, dried, and then subjected to SERS detection. Detection conditions: the excitation wavelength of the Raman spectrometer was set to 785 nm, the spot diameter to 105 μm, and the maximum laser power to 275 mW. The excitation power was 5% of the maximum power, and the integration time was 20 s. The average value of five sites was taken from each sample used for testing. The Raman spectra of the samples may contain varying levels of fluorescence or thermal background, which was then subtracted from the baseline. After baseline subtraction, spectral smoothing was performed to improve the signal-to-noise ratio. Figure 9 This is the SERS spectrum of the three captured fungi, among which... Figure 9 (A) is the SERS spectrum of Candida albicans. Figure 9 (B) is the SERS spectrum of Candida tropicalis. Figure 9 (C) is the SERS spectrum of Candida krusei. The captured spectrum is consistent with the standard spectrum. By analyzing the micrographs of clinical samples and the supernatants after capture of Candida albicans, Candida tropicalis, and Candida krusei, the capture efficiency was calculated to be 75.2% for Candida albicans, 76.7% for Candida tropicalis, and 70.5% for Candida krusei. The accurate detection results obtained in such a short time are attributed to the rational structural design and specificity of the fungal capture agent of this invention.

[0077] To further differentiate the detected fungi, OPLS-DA ( Figure 10 The SERS spectra of 58 clinical samples of *Candida albicans*, 24 *Candida tropicalis*, and 15 *Candida krusei* were compared. Each point represents one sample in the figure. All three fungi detected in this experiment were well isolated. 2 X(Cum) = 0.884, R 2 Y(Cum) = 0.954, Q 2 (Cum) = 0.937. R 2 X, R 2 Y and Q 2 All values ​​were greater than 0.5 and were close in magnitude, indicating that the experimental model was well-established and had good predictive ability. The experimental spectral data were analyzed using SIMCA 14.1 software (Umetrics, Sweden). All SERS fingerprint data were randomly divided into 10 parts: 9 parts were used as training data and 1 part as test data for 10-fold cross-validation to evaluate the model's classification ability. The average accuracy of the training and test data was obtained. The results are shown in Table 1 below. The average accuracy of the training and test data were 100% and 100%, respectively, indicating that the model has good classification ability.

[0078] Table 1

[0079] frequency Accuracy of training data Accuracy of test data 1 100％ 99.34％ 2 100％ 98.67％ 3 100％ 100％ 4 100％ 100％ 5 100％ 100％ 6 100％ 100％ 7 100％ 100％ 8 100％ 100％ 9 100％ 100％ 10 100％ 100％

[0080] As can be seen, using the rapid fungal capture agent of this invention, fungi can be detected directly from clinical samples in a non-destructive manner within 10 minutes via SERS. In contrast, previous clinical trials required several days for the entire detection process due to the need to culture the fungi. The method of this invention uses an ultra-rapid fungal capture agent to capture the fungi, followed by SERS detection, eliminating the need for culture and making it faster and more accurate. Therefore, it has significant value in the field of early fungal diagnosis in clinical laboratories.

[0081] Application Example 2

[0082] To verify that the fungal scavenger obtained in this invention can be recycled and reused, the fungal scavenger in the example was recycled and reused 5 times. Specifically, the fungal scavenger was dispersed in ultrapure water at 10°C at a concentration of 100 μg / mL, and the ultrapure water was replaced every 8 hours. After that, the fungal scavenger was recovered by magnetic adsorption, and the capture efficiency against Candida albicans was tested again. The results are shown in Table 2 below:

[0083] Table 2

[0084]

Claims

1. An ultra-fast intelligent fungal trap, characterized in that, It has a core-shell nanostructure, with a magnetic nanoparticle core and a thermosensitive polymer shell. The surface of the thermosensitive polymer is coupled with a substance that specifically binds to fungi. The magnetic nanoparticles are modified with amino groups and have a particle size of 300-600 nm. The thermosensitive polymer containing carboxyl groups is a thermosensitive unsaturated monomer, copolymerized with a carboxyl-containing unsaturated monomer and a crosslinking agent. The thermosensitive unsaturated monomer is selected from at least one of N-isopropylacrylamide, N-vinylcaprolactam, N-vinylisopropylamide, N,N-diethyl-2-acrylamide, and N-vinylcaprolactam. The carboxyl-containing unsaturated monomer is selected from at least one of (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; the crosslinking agent is bis(2-methylpropene)ethoxydisulfide; the molar ratio of the thermosensitive unsaturated monomer, the carboxyl-containing unsaturated monomer, and the crosslinking agent is 10-15:1-2:1-1.3; the molar amount of the carboxyl-containing unsaturated monomer and the mass ratio of the magnetic nanoparticles are 0.2-0.5 mmol:1 g; the substance that specifically binds to fungi is caspofungin; the mass ratio of the substance that specifically binds to fungi to the magnetic nanoparticles is 0.5 × 10⁻⁶. -5 -5×10 -5 :

1.

2. The ultra-fast intelligent fungal trap according to claim 1, characterized in that, The magnetic nanoparticles are Fe3O4 nanoparticles coated with chitosan and containing amino groups on their surface, with a particle size of 400-500 nm.

3. The ultra-fast intelligent fungal trap according to claim 1, characterized in that, 30-50% of the unsaturated monomers containing carboxyl groups are first coupled with magnetic nanoparticles with amino groups on the surface, and then participate in the polymerization reaction.

4. The ultra-fast intelligent fungal trap according to claim 1, characterized in that, The molar amount of the unsaturated monomer containing carboxyl groups and the mass ratio of the magnetic nanoparticles are 0.34-0.4 mmol: 1 g.

5. A method for preparing the ultra-fast intelligent fungal trap according to any one of claims 1-4, comprising the following steps: (1) Magnetic nanoparticles with amino groups on their surface are coupled with 30-50% unsaturated monomers containing carboxyl groups. (2) Using dispersion polymerization, the remaining carboxyl-containing unsaturated monomer, thermosensitive unsaturated monomer, crosslinking agent, emulsifier, and initiator are added to the dispersion of the product obtained in step (1), and copolymerized under ultrasonic conditions to obtain polymer-coated magnetic nanoparticles. (3) The polymer-coated magnetic nanoparticles prepared in step (2) are coupled with the carboxyl groups on their surface and the amino groups on substances that can specifically bind to fungi to obtain the fungal capture agent.

6. The preparation method according to claim 5, characterized in that, In step (1), the coupling reaction is carried out under activation conditions, and the activating reagent is EDC / NHS; after the reaction is carried out for 10-15 hours, it is stored at a low temperature of 2-8℃ for later use. In step (2), the emulsifier is selected from sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, and the amount of emulsifier is 0.5-2% of the total molar amount of the monomer; the initiator is a water-soluble initiator, and the amount of initiator is 0.5-2% of the total molar amount of the monomer; the reaction is carried out at 30-40℃ and 60-100MHz ultrasonic environment for 3-5 hours. In step (3), the product obtained in step (2) is first activated, and then an aqueous solution of a substance that can specifically bind to fungi is added. The reaction is carried out for 10-20 hours. The product is centrifuged and washed to obtain the fungal capture agent. Preferably, the concentration of the aqueous solution of the substance that can specifically bind to fungi is 10-50 μg / mL.

7. The preparation method according to claim 6, characterized in that, The water-soluble initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate.

8. A method for ultra-rapid detection of fungi, comprising the following steps: (S1) At a temperature below the low critical transition temperature of the thermosensitive polymer, the ultra-fast intelligent fungal capture agent according to any one of claims 1-4 is mixed with a sample containing fungi, and then the temperature is raised to above the low critical transition temperature to complete the fungal capture and obtain a complex of fungal capture agent and fungi. (S2) The fungal capture agent-fungal complex was enriched using an external magnetic field. The supernatant was discarded, the precipitate was mixed with the SERS substrate, dried, and then subjected to SERS detection.

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