Bimodal nanoprobe for detecting uric acid and preparation method and application thereof

Abstract

The application relates to the technical field of nanobiosensors, and discloses a bimodal nanoprobe for uric acid detection and a preparation method and application thereof. The bimodal nanoprobe is a fluorescence-photothermal bimodal nanoprobe, and is a core-shell structure nanoparticle, which comprises, from the inner layer to the outer layer, a) a fluorescent inner core composed of carbon dots and a silica layer coated outside the carbon dots; b) a manganese dioxide layer coated outside the fluorescent inner core; and c) a molecularly imprinted polymer layer coated outside the manganese dioxide layer. The bimodal nanoprobe in the application integrates a fluorescence signal source (carbon dots), a signal modulation and photothermal unit and a specific recognition layer (a uric acid molecularly imprinted polymer) through ingenious structure design, realizes bimodal sensing of "specific recognition -> MnO2 etching -> fluorescence recovery / photothermal weakening" on UA, and effectively solves the problems of poor selectivity, complicated operation and high cost of traditional methods.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensor technology, and in particular to a dual-modal nanoprobe for uric acid detection, its preparation method, and its application. Background Technology

[0002] Uric acid (UA) is the end product of purine metabolism in the human body, and abnormal UA concentrations are key diagnostic indicators for various diseases such as gout, hyperuricemia, and kidney stones. Clinically commonly used UA detection methods mainly include the uricase method and the phosphotungstic acid method. The uricase method has good specificity, but the enzyme is expensive, has poor stability, and is cumbersome to operate (often requiring coupling with peroxidase for color development). While the phosphotungstic acid method is simple, it is easily interfered with by other reducing substances in the sample (such as vitamin C and glutathione), has poor specificity, and requires complex pretreatment.

[0003] In recent years, sensing platforms based on nanomaterials (such as manganese dioxide nanozymes) have attracted attention due to their high stability and low cost. Manganese dioxide (MnO2) can not only oxidize UA, but also possesses excellent fluorescence quenching ability and photothermal conversion performance, making it suitable for constructing sensing platforms. However, bare MnO2 nanozymes respond to a variety of reducing substances, exhibiting a severe lack of selectivity, and thus cannot achieve accurate quantification in complex real biological samples (such as serum). Therefore, developing a new method for UA detection that combines high selectivity, high sensitivity, ease of operation, and strong anti-interference ability is a pressing technical problem to be solved in the field of point-of-care testing (POCT). Summary of the Invention

[0004] The purpose of this invention is to provide a dual-modal nanoprobe for uric acid detection, its preparation method, and its application. Through ingenious structural design, the dual-modal nanoprobe integrates a fluorescence signal source (carbon dot), a signal modulation and photothermal unit, and a specific recognition layer (uric acid molecularly imprinted polymer), realizing dual-modal sensing of UA through "specific recognition → MnO2 etching → fluorescence recovery / photothermal attenuation". This effectively solves the problems of poor selectivity, complicated operation, and high cost of traditional methods.

[0005] To achieve the above objectives, this invention provides a dual-modal nanoprobe for uric acid detection. The dual-modal nanoprobe is a fluorescence-photothermal dual-modal nanoprobe, a core-shell structured nanoparticle comprising, from the inner layer to the outer layer:

[0006] a) The fluorescent core consists of carbon dots and a silicon dioxide layer covering the carbon dots;

[0007] b) A manganese dioxide layer covering the fluorescent core;

[0008] c) A molecularly imprinted polymer layer covering the manganese dioxide layer, the molecularly imprinted polymer layer being formed using uric acid as a template molecule.

[0009] Furthermore, the manganese dioxide layer can quench the fluorescence of the carbon dots and has photothermal conversion properties; the molecularly imprinted polymer layer enables the nanoprobe to specifically bind uric acid molecules; when uric acid is present, the manganese dioxide layer is reduced and decomposed, resulting in the recovery of the fluorescence of the carbon dots and a reduction in the photothermal effect of the nanoprobe.

[0010] Furthermore, the molecularly imprinted polymer layer is polymerized using a silane coupling agent containing a urea group as a functional monomer and tetraethyl orthosilicate as a crosslinking agent; the volume ratio of the functional monomer to the crosslinking agent is 1:1 to 1:4.

[0011] Furthermore, the silane coupling agent containing urea groups is ureapropyltriethoxysilane.

[0012] Furthermore, the present invention also provides a method for preparing the above-mentioned dual-modal nanoprobe for uric acid detection, comprising the following steps:

[0013] Step S1: Using carbon dots as the core, a silica layer is coated by the sol-gel method to obtain fluorescent silicon spheres;

[0014] Step S2: In situ reduction of permanganate on the surface of the fluorescent silicon sphere to deposit a manganese dioxide layer, thereby obtaining the precursor;

[0015] Step S3: On the surface of the precursor, using uric acid as a template molecule, a molecularly imprinted polymer layer is formed by sol-gel polymerization. Subsequently, the template molecules are eluted to obtain a dual-modal nanoprobe.

[0016] Furthermore, the present invention also provides the application of the above-mentioned dual-modal nanoprobe in the preparation of uric acid detection products.

[0017] Furthermore, the detection is based on the enhancement of the fluorescence signal and / or the reduction of the photothermal signal of the dual-modal nanoprobe.

[0018] Furthermore, the uric acid detection product is used to detect the concentration of uric acid in serum, urine, or synovial fluid.

[0019] Furthermore, the present invention also provides a uric acid detection kit, the uric acid detection kit comprising the above-mentioned dual-modal nanoprobe.

[0020] Furthermore, the present invention also provides a uric acid test strip, wherein the detection area of ​​the uric acid test strip is fixed with the above-mentioned dual-modal nanoprobe.

[0021] The advantages and positive effects of the dual-modal nanoprobe for uric acid detection, its preparation method, and its application described in this invention are as follows:

[0022] 1. The innovative molecular imprinted layer acts like an "artificial key," giving the probe the ability to specifically recognize UA, effectively shielding it from the influence of dozens of common biological interfering substances such as vitamin C, glutathione, dopamine, and glucose, thus solving the fundamental problem of poor selectivity in MnO2-type sensors.

[0023] 2. Dual signal output, reliable results: This invention innovatively integrates two independent detection modes, "fluorescence enhancement" and "photothermal attenuation," onto the same probe. Both signals are correlated with UA concentration and can be mutually calibrated and verified, greatly improving the accuracy and reliability of the detection results, making it particularly suitable for the precise analysis of complex samples.

[0024] 3. Fast, simple, and no pretreatment required: The detection process is a "one-step mixing" reaction that can be completed in 5-10 minutes. It does not require complicated protein removal or separation steps for samples, nor does it require expensive enzyme reagents, making it very suitable for developing point-of-care testing (POCT) products.

[0025] 4. Sensitivity and linear range meet clinical needs: The detection limit in fluorescence mode is as low as 1.39 μM, and the detection limit in photothermal mode is 5.2 μM. The linear range covers the routine dilution concentrations of clinical samples and has good correlation with the gold standard method.

[0026] 5. Good stability and low cost: The probe is composed of inorganic materials and silicon-based polymers, which are much more stable than enzymes. It retains more than 95% of its activity after being stored at room temperature for one month. Moreover, the raw materials are cheap and readily available, and it has the potential for large-scale production and application.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation process of the nanoprobe MMSC in this embodiment of the invention and a schematic diagram of the principle of "fluorescence-photothermal" dual-modal detection of UA;

[0029] Figure 2 These are transmission electron microscope (TEM) images and Mn and Si elemental distribution maps of MMSC in embodiments of the present invention, wherein A is a TEM image, B is an Mn elemental distribution map, C is a Si elemental distribution map, and D is a Mn+Si elemental distribution map.

[0030] Figure 3 The following are fluorescence response spectra and calibration curves of MMSCs to different concentrations of UA in this embodiment of the invention, where A is the fluorescence response spectrum and B is the calibration curve;

[0031] Figure 4The images show the photothermal response curves (temperature-time curves) and calibration curves of MMSC to different concentrations of UA in this embodiment of the invention, where A is the photothermal response curve and B is the calibration curve.

[0032] Figure 5 This is a selective test diagram (fluorescence mode and photothermal mode) of MMSC for various potential interfering substances in an embodiment of the present invention, where A is the fluorescence mode and B is the photothermal mode;

[0033] Figure 6 This invention illustrates the effect of different UPTEOS+TEOS dosages on the uric acid / glutathione responsiveness of MMSCs in various embodiments. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0036] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0037] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0038] This invention discloses a fluorescent-photothermal dual-modal nanoprobe for the specific detection of uric acid. The fluorescent-photothermal dual-modal nanoprobe is a core-shell structured nanoparticle, comprising, from the inner layer to the outer layer:

[0039] Fluorescent core: Core-shell nanospheres (SC) consisting of fluorescent carbon dots (CDs) and an outer layer of silica (SiO2). The SiO2 layer protects the CDs from damage during subsequent synthesis.

[0040] Signal modulation and conversion intermediate layer: a manganese dioxide (MnO2) layer (collectively MSC) covering the SC. This MnO2 layer can efficiently quench the fluorescence of the core CDs through the internal filtering effect (IFE) (forming an "OFF" state), while giving the material near-infrared photothermal conversion properties.

[0041] Specific recognition shell: The outermost layer is a molecularly imprinted polymer (MIP) layer (collectively denoted as MMSC). This MIP layer is polymerized using uric acid (UA) as a template molecule and a urea-containing silane coupling agent (preferably ureapropyltriethoxysilane, UPTEOS) as a functional monomer. This layer has imprinted cavities that are complementary to the UA molecule in shape, size, and hydrogen bonding sites, enabling it to specifically capture and enrich UA molecules in the sample.

[0042] The detection mechanism of the nanoprobe is ( Figure 1 As shown): When the target molecule UA is present, it specifically binds to the MIP imprint cavity and rapidly reduces the MnO2 layer in the middle of the etching, causing it to decompose into Mn. 2+ This process causes two synchronized signals to change:

[0043] Fluorescence signal "ON": The MnO2 quenching layer is destroyed, the fluorescence of the core CDs is restored, and the fluorescence intensity is enhanced.

[0044] Photothermal signal "OFF": With the reduction of MnO2 photothermal agent, the temperature rise effect of the material under near-infrared light irradiation is weakened.

[0045] The concentration of UA can be quantitatively detected by monitoring the fluorescence recovery degree (F / F0) or the photothermal temperature rise decay degree (ΔT), and the results of the two modes can be mutually verified.

[0046] The carbon dots (CDs) are preferably synthesized via a solvothermal method using polyethylenepolyamine as the carbon source, exhibiting blue fluorescence with a maximum excitation / emission wavelength of approximately 350 / 450 nm. Further, the manganese dioxide (MnO2) is an amorphous nanolayer generated through in-situ reduction of potassium permanganate on the SC surface. In the molecularly imprinted polymer (MIP) layer, the volume ratio of the functional monomer UPTEOS to the crosslinking agent tetraethyl orthosilicate (TEOS) is preferably 1:1 to 1:4, more preferably 1:2. The nanoprobe MMSC has a particle size of 60-100 nm and exhibits optimal response to UA under pH 4-7 and room temperature conditions.

[0047] This invention also provides a method for preparing the above-mentioned nanoprobe (MMSC). Figure 1 (As shown), including the following steps:

[0048] S1. Synthesis of fluorescent silicon spheres (SC): Carbon dots (CDs) are dispersed in an alcohol-water mixed solvent. Under the catalysis of ammonia, tetraethyl orthosilicate (TEOS) is added to carry out hydrolysis and condensation, and a silica layer is coated on the surface of CDs to obtain SC.

[0049] S2. Constructing the MnO2 signal layer (obtaining MSCs): Disperse SCs in water, add potassium permanganate (KMnO4), and utilize the reducing properties of SCs or their surface groups to reduce KMnO4 in situ and deposit a thin layer of MnO2 on the SC surface to obtain MSCs with quenched fluorescence.

[0050] S3. Constructing a molecularly imprinted recognition layer (obtaining MMSCs): MSCs are dispersed in an alcohol solvent, and template molecules uric acid (UA), functional monomers UPTEOS and crosslinking agent TEOS are added. A sol-gel reaction is carried out under alkaline conditions to polymerize on the surface of MSCs to form a SiO2-based molecularly imprinted polymer layer. Finally, the template molecule UA is eluted to remove it, and MMSCs with UA-specific recognition cavities are obtained.

[0051] This invention also provides the application of the above-mentioned nanoprobes in the preparation of uric acid detection reagents or devices. Detection can be performed on a solid-phase carrier such as a solution phase or test strip, and can be used for: ① quantitative detection of uric acid concentration in biological samples such as human serum, urine, and synovial fluid; ② auxiliary diagnosis or efficacy monitoring of diseases such as gout and hyperuricemia.

[0052] The following examples provide a detailed explanation.

[0053] Example 1: Preparation and Characterization of Nanoprobe MMSCs

[0054] S1. Synthesis of fluorescent carbon dots (CDs):

[0055] 10 mL of polyethylene polyamine was placed in a 50 mL polytetrafluoroethylene-lined reactor, sealed, and placed in an oven at 200 °C for 12 hours. After natural cooling to room temperature, a brownish-yellow viscous liquid was obtained. The product was dissolved in ultrapure water and transferred to a dialysis bag with a molecular weight cutoff of 500 Da. Dialysis was performed with ultrapure water for 48 hours to remove small molecule impurities, with the water changed every 12 hours. The liquid after dialysis was freeze-dried to obtain solid CDs powder. Transmission electron microscopy (TEM) showed that the CDs particle size was approximately 3 nm. Fluorescence spectroscopy showed that its maximum excitation / emission wavelengths were 350 / 450 nm.

[0056] Synthesis of S2, carbon dot-silica core-shell nanospheres (SC):

[0057] 10 mg of the above-mentioned CDs solid was dissolved in 20 mL of anhydrous ethanol and ultrasonically dispersed. 2 mL of 25% ammonia solution was added to the solution, followed by slow dropwise addition of 0.3 mL of tetraethyl orthosilicate (TEOS) under vigorous stirring. The reaction was carried out at room temperature with continuous stirring for 12 hours. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the white precipitate was collected. The precipitate was washed three times alternately with ultrapure water and ethanol to obtain carbon dot-silica core-shell nanospheres (SC). TEM showed that SC were uniformly spherical with a particle size of approximately 50 nm, and their fluorescence spectrum was consistent with that of CDs.

[0058] Synthesis of S3 and manganese dioxide-coated SC (MSC):

[0059] The above-mentioned SCs were dispersed in ultrapure water to prepare a dispersion of 0.5 mg / mL. 10 mL of this dispersion was taken, and potassium permanganate (KMnO4) solution was added with stirring to bring the final concentration of KMnO4 in the system to 0.3 mM (based on manganese element). The reaction was carried out at room temperature in the dark with stirring for 24 hours. After the reaction, the mixture changed from colorless to brownish-red. The reaction solution was centrifuged at 10,000 rpm for 10 minutes, and the brownish-red precipitate was collected and washed three times with ultrapure water to obtain MnO2-coated SCs, designated as MSCs. TEM showed that the MSCs had a uniform thin layer of coating on their surface, and their fluorescence was almost completely quenched.

[0060] S4. Molecularly imprinted layer modification yields MMSCs:

[0061] 50 mg of MSCs were dispersed in 50 mL of anhydrous ethanol. In a separate container, 0.1 mL of ureapropyltriethoxysilane (UPTEOS, 50% methanol solution) and 2 mg of uric acid (UA) were dissolved in 10 mL of methanol and sonicated for 30 minutes to form a pre-assembled solution. This pre-assembled solution was added to the ethanol dispersion of MSCs, followed by 0.1 mL of TEOS and 0.25 mL of concentrated ammonia (25%). The mixture was stirred and polymerized at room temperature for 24 hours. After the reaction was complete, the solid was collected by centrifugation and repeatedly washed with an ethanol solution containing 0.1% ammonia until the UV absorbance of the washing solution at 290 nm was below 0.01, ensuring complete elution of the template molecule UA. Finally, the mixture was washed twice with ultrapure water and freeze-dried to obtain the final product, MMSCs.

[0062] Characterization results:

[0063] Transmission electron microscopy (TEM) observation Figure 2 As shown in the figure, MMSCs are regularly spherical with an average particle size of approximately 80 nm and exhibit a clear core-shell structure. The elemental distribution map shows that the Si element signal is uniformly distributed, while the Mn element signal is concentrated inside the particles, confirming that the "SiO2 / MnO2 core-shell" structure was successfully encapsulated within the molecularly imprinted polymer layer.

[0064] Example 2: Evaluation of MMSC Detection Performance

[0065] S1. Screening for optimal reaction conditions:

[0066] A series of acetate-sodium acetate buffer or phosphate buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.0, and 8.0 were prepared. Equal volumes of MMSCs (final concentration 0.5 mg / mL) and uric acid (final concentration 50 μM) were added to each buffer solution. After reacting at room temperature for 5 minutes, the change in fluorescence intensity (F / F0, where F is the fluorescence intensity after the reaction and F0 is the fluorescence intensity before the reaction) was measured. The results showed that the fluorescence response (F / F0) was strongest at pH 5.0; therefore, pH 5.0 was selected as the optimal detection pH.

[0067] The reaction time was investigated at pH 5.0 and room temperature (25°C). After mixing MMSCs and UA, the fluorescence intensity was measured every 30 seconds. The results showed that the reaction reached a plateau within 5 minutes, so 5 minutes was selected as the incubation time for fluorescence detection.

[0068] S2. Establishment of calibration curve for fluorescence detection mode:

[0069] In an acetate-sodium acetate buffer solution at pH 5.0, MMSCs were added to a final concentration of 0.5 mg / mL, followed by different concentrations of uric acid standards (0, 10, 20, 50, 80, 100, 150, 200 μM). The mixture was reacted at room temperature for 5 minutes. The fluorescence intensity F of each tube was measured using a fluorescence spectrophotometer (excitation wavelength 350 nm, emission wavelength 450 nm). The fluorescence intensity of the tube without UA was taken as F0, and F / F0 was calculated.

[0070] like Figure 3 As shown, the fluorescence recovery rate (F / F0) exhibits a good linear relationship with UA concentration in both the 10-100 μM and 100-200 μM ranges. The linear equations are as follows: 10-100 μM: F / F0 = 0.00502[UA] + 1.04255 (R0) 2 =0.996); 100-200μM: F / F0=0.01265[UA]+0.31507 (R 2 =0.997). Based on a signal-to-noise ratio of 3, the detection limit for fluorescence mode is 1.39 μM.

[0071] S3. Establishment of calibration curve for photothermal detection mode:

[0072] The solution obtained after the above reaction was transferred to a 96-well plate. An 808 nm near-infrared laser (power 1.5 W / cm²) was used. 2The solution surface was vertically irradiated for 10 minutes, and the temperature (T) at the 9th minute was recorded using a handheld infrared thermal imager. The temperature rise decay was calculated with ΔT0 for the tube without UA and ΔT for the tube containing UA.

[0073] like Figure 4 As shown, the temperature rise decay (ΔT) is linearly negatively correlated with the UA concentration in the range of 10-100 μM, and the linear equation is: ΔT = 23.58501 - 0.08066 [UA] (R 2 =0.993). The detection limit for photothermal mode is 5.2 μM.

[0074] Example 3: Selectivity and anti-interference capability test of MMSC

[0075] S1. Interference Test:

[0076] Under optimal detection conditions, uric acid was replaced with potential interfering substances and reacted with MMSCs. The interfering substances tested included: Fe... 3+ The following reagents were used: (10 μM) urea (urea, 1 mM), glucose (Glc, 5 mM), bovine serum albumin (BSA, 1 mg / mL), histidine (His, 50 μM), glutathione (GSH, 50 μM), xanthine (Xat, 50 μM), cysteine ​​(Cys, 50 μM), tyrosine (Tyr, 50 μM), citric acid (CA, 50 μM), dopamine (DA, 50 μM), and ascorbic acid (VC, 50 μM). The fluorescence response (F / F0) and photothermal response (ΔT) were measured after the reaction.

[0077] The results are as follows Figure 5 As shown, in fluorescence mode, only UA caused significant fluorescence enhancement (F / F0 > 1.5), while even the strong reducing agents VC, GSH, and DA had F / F0 values ​​of only 1.23, 1.18, and 1.21, respectively. In photothermal mode, only UA caused significant temperature rise decay (ΔT decrease of approximately 15.7℃), while the ΔT changes caused by other interfering agents were all less than 3℃. This indicates that MMSCs have high specificity for UA.

[0078] S2. Stability Test:

[0079] The MMSC dispersion (1 mg / mL in PBS) was stored at 4°C in the dark for 30 days. A sample was taken every 3 days, and its fluorescence response to 50 μM UA was measured using standard methods. The results showed that after 30 days of storage, the fluorescence response value (F / F0) was 94.3% of the initial value, indicating good storage stability. Eight consecutive laser irradiation-cooling cycles were performed on the same MMSC dispersion, and the photothermal heating curves highly overlapped, with ΔT fluctuations less than 1.5°C, indicating stable photothermal properties.

[0080] Example 4: Application and Recovery of MMSCs in Real Samples (Human Serum, Urine)

[0081] S1. Human urine sample testing:

[0082] First morning urine was collected from healthy volunteers, centrifuged at 10,000 rpm for 10 minutes to remove precipitate, and the supernatant was diluted 10-fold with ultrapure water. Using the standard addition method: 0.2 mL of diluted urine was taken, and 1.6 mL of pH 5.0 buffer and 0.2 mL of MMSC (5 mg / mL) were added. Detection was performed using fluorescence and photothermal methods, respectively. Simultaneously, known amounts of UA standards (spiking concentrations of 100, 200, and 400 μM) were added to the samples for recovery experiments.

[0083] The results are shown in Table 1. The recovery rate of the fluorescence method was between 96.81% and 98.91%, with a relative standard deviation (RSD) of 1.2% to 2.3%; the recovery rate of the photothermal method was between 99.15% and 104.6%, with an RSD of 0.6% to 1.9%. This indicates that MMSCs can still accurately quantify UA in complex urine matrices.

[0084] Table 1. Detection results of MMSC in urine and serum samples.

[0085]

[0086] S2. Human serum sample detection and comparison:

[0087] Clinical serum samples (including those from healthy individuals and patients with hyperuricemia) were obtained from the hospital and diluted 5-fold with PBS. MMSCs were then analyzed using the method described above. Simultaneously, the same samples were analyzed in parallel using both a commercial uricase kit (enzymatic method) and the phosphotungstic acid method (PTA method).

[0088] As shown in Table 2, the detection results of MMSC fluorescence method, photothermal method, enzymatic method, and PTA method were highly consistent (relative errors were all <5%). Linear regression analysis was performed on 10 samples with different concentrations, and the correlation coefficient R between the MMSC fluorescence method and the enzymatic method was [value missing]. 2 =0.994, indicating that the method of the present invention has a good correlation with the clinical gold standard method.

[0089] Table 2. Results of MMSC assay and clinical standard methods (urate oxidase method, phosphotungstic acid method) for detecting actual serum samples.

[0090]

[0091] Performance testing of the control probe (MSC) without molecular imprinting layer in Comparative Example 1

[0092] MSCs were synthesized according to the method of Example 1 (i.e., step S4 was omitted). The fluorescence response of MSCs to UA and various interfering substances (VC, GSH, DA) was tested under the same conditions as in Example 3.

[0093] Figure 5 The results showed that MSCs exhibited a 2.1-fold fluorescence enhancement to 50 μM UA, but also significant enhancements to 50 μM VC, GSH, and DA (1.9-fold, 1.8-fold, and 2.5-fold, respectively, with DA showing a stronger signal due to fluorescence generated by oxidative polymerization). This indicates that while MSCs without molecular imprinting respond to UA, they lack selectivity and cannot specifically detect UA in complex samples. This comparative example highlights the crucial and indispensable role of the molecular imprinting layer in achieving highly selective detection in this invention.

[0094] Comparative Example 2: Probe performance with different MIP layer thicknesses

[0095] In step S4 of Example 1, a series of MMSCs with different MIP layer thicknesses were prepared by varying the total dosage of TEOS and UPTEOS ([Si]). With a fixed UA concentration (50 μM), their fluorescence response ratio (i.e., selectivity) to UA and the interfering agent GSH (50 μM) was tested.

[0096] The results are as follows Figure 6 As shown, when the [Si] dosage is too low, the probe exhibits strong responses to both UA and GSH, but poor selectivity. With the [Si] dosage increasing to 30 μL / mg MSC, the response retention to UA reaches 61%, while the response to GSH is suppressed to <5%, achieving optimal selectivity. Further increasing the [Si] dosage leads to excessive suppression of the response to UA. This comparative example illustrates that optimizing the MIP layer thickness is crucial for balancing detection sensitivity and selectivity; this invention determines the optimal process parameters through optimization.

[0097] The present invention has the following beneficial effects:

[0098] The innovative molecularly imprinted layer acts like an "artificial key," endowing the probe with the ability to specifically recognize UA, effectively shielding it from the influence of dozens of common biological interfering substances such as vitamin C, glutathione, dopamine, and glucose, thus solving the fundamental problem of poor selectivity in MnO2-type sensors.

[0099] Dual signal output for reliable results: This invention innovatively integrates two independent detection modes, "fluorescence enhancement" and "photothermal attenuation," onto the same probe. Both signals are correlated with UA concentration and can be mutually calibrated and verified, greatly improving the accuracy and reliability of the detection results, making it particularly suitable for the precise analysis of complex samples.

[0100] Fast, simple, and requires no pretreatment: The detection process is a "one-step mixing" reaction that can be completed in 5-10 minutes. It does not require complicated protein removal or separation steps for samples, nor does it require expensive enzyme reagents, making it ideal for developing point-of-care testing (POCT) products.

[0101] Sensitivity and linear range meet clinical needs: the detection limit in fluorescence mode is as low as 1.39 μM, the detection limit in photothermal mode is 5.2 μM, the linear range covers the routine dilution concentrations of clinical samples, and it has good correlation with the gold standard method.

[0102] High stability and low cost: The probe is composed of inorganic materials and silicon-based polymers, which are far more stable than enzymes. It retains more than 95% of its activity after being stored at room temperature for one month. Moreover, the raw materials are cheap and readily available, making it a potential candidate for large-scale production and application.

[0103] Therefore, this invention employs the aforementioned dual-modal nanoprobe for uric acid detection, its preparation method, and its application. Through ingenious structural design, the dual-modal nanoprobe integrates a fluorescence signal source (carbon dot), a signal modulation and photothermal unit, and a specific recognition layer (uric acid molecularly imprinted polymer), achieving dual-modal sensing of UA through "specific recognition → MnO2 etching → fluorescence recovery / photothermal attenuation," effectively solving the problems of poor selectivity, cumbersome operation, and high cost of traditional methods.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A dual-modal nanoprobe for uric acid detection, characterized in that: The dual-modal nanoprobe is a fluorescence-photothermal dual-modal nanoprobe, consisting of core-shell structured nanoparticles, with the following layers from the inner to the outermost: a) The fluorescent core consists of carbon dots and a silicon dioxide layer covering the carbon dots; b) A manganese dioxide layer covering the fluorescent core; c) A molecularly imprinted polymer layer covering the manganese dioxide layer, the molecularly imprinted polymer layer being formed using uric acid as a template molecule.

2. The dual-modal nanoprobe for uric acid detection according to claim 1, characterized in that: The manganese dioxide layer can quench the fluorescence of the carbon dots and has photothermal conversion properties; the molecularly imprinted polymer layer enables the nanoprobe to specifically bind uric acid molecules; when uric acid is present, the manganese dioxide layer is reduced and decomposed, resulting in the recovery of the fluorescence of the carbon dots and a reduction in the photothermal effect of the nanoprobe.

3. The dual-modal nanoprobe for uric acid detection according to claim 1, characterized in that: The molecularly imprinted polymer layer is polymerized using a urea-containing silane coupling agent as a functional monomer and tetraethyl orthosilicate as a crosslinking agent; the volume ratio of the functional monomer to the crosslinking agent is 1:1 to 1:

4.

4. The dual-modal nanoprobe for uric acid detection according to claim 3, characterized in that: The urea-containing silane coupling agent is ureapropyltriethoxysilane.

5. The method for preparing the dual-modal nanoprobe for uric acid detection according to any one of claims 1-4, characterized in that, Includes the following steps: Step S1: Using carbon dots as the core, a silica layer is coated by the sol-gel method to obtain fluorescent silicon spheres; Step S2: In situ reduction of permanganate on the surface of the fluorescent silicon sphere to deposit a manganese dioxide layer, thereby obtaining the precursor; Step S3: On the surface of the precursor, using uric acid as a template molecule, a molecularly imprinted polymer layer is formed by sol-gel polymerization. Subsequently, the template molecules are eluted to obtain a dual-modal nanoprobe.

6. The application of the dual-modal nanoprobe according to any one of claims 1-4 in the preparation of uric acid detection products.

7. The application according to claim 6, characterized in that: The detection is based on the enhancement of fluorescence signal and / or the reduction of photothermal signal of the dual-modal nanoprobe.

8. The application according to claim 6, characterized in that: The uric acid detection product is used to detect the concentration of uric acid in serum, urine, or synovial fluid.

9. A uric acid detection kit, characterized in that: It includes the dual-modal nanoprobe as described in any one of claims 1-4.

10. A uric acid test strip, characterized in that: The detection area of ​​the uric acid test strip is fixed with the dual-modal nanoprobe as described in any one of claims 1-4.

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