NSP-CDs / Ag + Fluorescent nanoprobes and their use in detecting alpha-lipovmycin

Abstract

The application discloses NSP-CDs / Ag + The application discloses a fluorescent nanoprobe and application thereof in detection of alpha-lipomycin, and belongs to the field of nanomaterials and medical detection. The application prepares nitrogen-sulfur-phosphorus co-doped quantum dots by taking o-phenylenediamine as a carbon source and a nitrogen source, taking thiourea as a sulfur source, and taking adenosine triphosphate as a phosphorus source. The quantum dots have strong yellow fluorescence, the optimal excitation wavelength is 415 nm, and the optimal emission wavelength is 570 nm. Then, the quantum dots are combined with Ag + to form NSP-CDs / Ag + The application further discloses a fluorescent probe, Ag + quenches the fluorescence of NSP-CDs, and NSP-CDs / Ag + is almost non-fluorescent. Finally, the NSP-CDs / Ag + fluorescent probe is used for detecting alpha-lipomycin. In the detection, alpha-lipomycin competes with NSP-CDs for the combination of Ag + . At this time, the fluorescence of NSP-CDs is recovered, the whole process is a direct reaction in an instant, and the detection time is shortened.

Description

Technical Field

[0001] This invention relates to an NSP-CDs / Ag + Fluorescent nanoprobes and their application in the detection of α-lipomycin belong to the fields of nanomaterials and medical detection. Background Technology

[0002] Carbon quantum dots (CDs) are quasi-zero-dimensional nanomaterials with sizes ranging from 1 to 10 nm. Compared to traditional semiconductor quantum dots and organic fluorophores, quantum dots are widely used in analytical detection, catalytic degradation, bioimaging, drug delivery, and biosensing due to their advantages such as simple preparation methods, strong photoluminescence properties, excellent stability, water solubility, and high selectivity. Furthermore, quantum dots possess excellent fluorescence properties, including a continuous and broad excitation spectrum, a symmetrical and narrow emission spectrum, rich colors, high photochemical stability, and long fluorescence lifetime, as well as the ability to easily modify their surface. These characteristics make them ideal fluorescent probes, showing great promise for applications in fluorescence imaging and analytical detection in complex environments.

[0003] Alpha-lipomycin is an acyclic polyene antibiotic isolated from the Gram-positive bacterium *Streptomyces aureus* Tü117. Previous studies have shown that alpha-lipomycin is highly expressed in hypertensive individuals. Conventional analytical methods for determining alpha-lipomycin include high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), electrochemical methods, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these methods offer good accuracy and selectivity, they suffer from drawbacks such as complex sample pretreatment, cumbersome procedures, expensive equipment, and long analysis times, limiting their application. Fluorescence spectrophotometry, due to its high precision, excellent selectivity and sensitivity, ease of operation, and cost-effectiveness, has gradually become a highly efficient detection method.

[0004] Quantum dot-based fluorescent probes have been widely used in metal ion detection. Antibiotics, as special commodities affecting physiological, biochemical, and pathological processes and used for the prevention, diagnosis, and treatment of diseases, are closely related to the health and lives of the people. Currently, research on the synthesis and application of luminescent quantum dots based on fluorescent probes for antibiotic detection is limited. Therefore, research on the preparation of novel luminescent quantum dots and their application in antibiotic fluorescence analysis based on single-peak response has significant theoretical and practical value. However, most conventionally prepared quantum dots exhibit blue or green fluorescence, which undoubtedly interferes with the determination of α-lipomycin due to the autofluorescence of biological samples. This also hinders the application of quantum dots in bioimaging, making accurate detection difficult in complex environments such as serum. Furthermore, there are few reports in the literature on the detection of α-lipomycin using fluorescence colorimetry.

[0005] Therefore, there is an urgent need to find a simple, rapid and highly sensitive detection method for α-lipomycin. Summary of the Invention

[0006] [Technical Issues]

[0007] Conventional quantum dots emit blue and green light, making them difficult to use for detecting α-lipomycin in complex environments; and currently, no literature mentions the use of fluorescence colorimetry to detect α-lipomycin.

[0008] [Technical Solution]

[0009] To address the aforementioned problems, this invention first prepared nitrogen-sulfur-phosphorus co-doped quantum dots (NSP-CDs) using 1,2-Phenylenediamine tablet (OPD) as the carbon and nitrogen source, thiourea (Tu) as the sulfur source, and adenosine triphosphate (ATP) as the phosphorus source. These quantum dots exhibit strong yellow fluorescence, with an optimal excitation wavelength of 415 nm and an optimal emission wavelength of 570 nm. Subsequently, they were combined with Ag... + Complex formation of NSP-CDs / Ag + fluorescent probe, Ag + The fluorescence of NSP-CDs was quenched, and the NSP-CDs / Ag ratio was also observed. + The fluorescent probe showed almost no fluorescence; finally, NSP-CDs / Ag + The fluorescent probe is used to detect α-lipomycin, during which α-lipomycin competitively binds to Ag with NSP-CDs. + At this point, the fluorescence of NSP-CDs is restored. The whole process is an instantaneous and direct reaction, which is simple, fast, low-cost, and easy to operate, shortening the detection time.

[0010] The first objective of this invention is to provide a method for preparing nitrogen-thiol-phosphorus co-doped quantum dots for detecting α-lipomycin. The method employs a one-step hydrothermal synthesis and specifically includes the following steps:

[0011] o-phenylenediamine, thiourea, and adenosine triphosphate were added to water and mixed thoroughly to obtain a mixed solution. The mixed solution was then sealed and reacted at 200°C for 6 hours. After the reaction was completed, the solution was cooled, centrifuged, and dialyzed to obtain nitrogen-thiophosphorus co-doped quantum dots (NSP-CDs) for the detection of α-lipomycin.

[0012] The mass ratio of o-phenylenediamine, thiourea, adenosine triphosphate, and water is 1:2:1:30.

[0013] In one embodiment of the present invention, the o-phenylenediamine, thiourea, and adenosine triphosphate are all solids, and their final concentrations after mixing with water are 30.82 mM, 87.58 mM, and 6.57 mM, respectively.

[0014] In one embodiment of the present invention, the water is ultrapure water.

[0015] In one embodiment of the present invention, the uniform mixing is achieved by ultrasonic mixing for a duration of 20 minutes.

[0016] In one embodiment of the present invention, the reaction is carried out in a high-pressure reactor lined with stainless steel and containing polytetrafluoroethylene.

[0017] In one embodiment of the invention, the cooling is natural cooling to room temperature.

[0018] In one embodiment of the present invention, the centrifugation is performed by centrifuging the obtained brownish-yellow product to separate the insoluble particles.

[0019] In one embodiment of the present invention, the dialysis is performed by pouring the supernatant obtained by centrifugation into a dialysis bag with a molecular weight of 1000 and dialyzing with ultrapure water for 4 hours.

[0020] In one embodiment of the present invention, the nitrogen-sulfur-phosphorus co-doped quantum dots are prepared and then stored in a refrigerator at 4°C in the dark.

[0021] The second objective of this invention is to prepare nitrogen-thiol-phosphorus co-doped quantum dots (NSP-CDs) for detecting α-lipomycin using the method described herein.

[0022] The third objective of this invention is to provide a method for preparing NSP-CDs / Ag. + The method for using fluorescent probes includes the following steps:

[0023] Nitrogen-thiophosphorus co-doped quantum dots for detecting α-lipomycin were mixed with Tris-HCl buffer, and then Ag was added. + The solution was incubated to obtain NSP-CDs / Ag + Fluorescent probe.

[0024] In one embodiment of the present invention, the nitrogen-thiol-phosphorus co-doped quantum dots, Tris-HCl buffer solution, and Ag used for detecting α-lipomycin are described. + The volume ratio of the solutions is 1:9:5.

[0025] In one embodiment of the present invention, the concentration of the Tris-HCl buffer is 20 mM and the pH is 7.

[0026] In one embodiment of the present invention, the Ag + The solution is a silver nitrate solution with a concentration of 800 μM.

[0027] In one embodiment of the present invention, the incubation is performed at 20-30°C (room temperature) for 2-3 minutes.

[0028] The fourth objective of this invention is to prepare NSP-CDs / Ag using the method described herein. + Fluorescent probe.

[0029] The fifth objective of this invention is to provide a NSP-CDs / Ag-based... + The method for detecting α-lipomycin using fluorescent probes includes the following steps:

[0030] NSP-CDs / Ag + The fluorescent probe was placed in the test solution containing α-lipomycin, and then the NSP-CDs / Ag ratio was recorded at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + The fluorescence intensity difference was used to calculate the concentration of α-lipomycin in the test solution containing α-lipomycin.

[0031] In one embodiment of the present invention, NSP-CDs / Ag + The volume ratio of the fluorescent probe to the test solution containing α-lipomycin is 3:1.

[0032] In one embodiment of the present invention, the test solution containing α-lipomycin includes an aqueous solution containing α-lipomycin and a serum solution containing α-lipomycin.

[0033] [Beneficial Effects]

[0034] (1) The nitrogen-sulfur-phosphorus co-doped quantum dots for detecting α-lipomycin described in this invention have the advantages of simple preparation method, high stability, good water solubility and high selectivity.

[0035] (2) This invention is based on NSP-CDs / Ag + The fluorescent probe method for detecting α-lipomycin does not require sample pretreatment and can directly and rapidly detect α-lipomycin.

[0036] (3) The method of the present invention is applicable to Ag + It is specific for the detection of α-lipomycin and has good anti-interference ability.

[0037] (4) The method of the present invention can detect low concentrations of α-lipomycin with high sensitivity. Attached Figure Description

[0038] Figure 1 For NSP-CDs / Ag + Flowchart for the detection of α-lipomycin using a fluorescent probe.

[0039] Figure 2 TEM images (scale bar of A is 100 nm, scale bar of B is 50 nm) of nitrogen-sulfur-phosphorus co-doped quantum dots (NSP-CDs) at different magnifications, AFM images (C, (a) and (b) represent different thicknesses), EDS images (D), 3D images of AFM from different perspectives (E, F), particle size distribution (G), thickness distribution curves (H) and (I) corresponding to line segments (a) and (b) in AFM images.

[0040] Figure 3 XPS full spectrum (A), C1s spectrum (B), Ols spectrum (C), N1s spectrum (D), S2s spectrum (E), P2p spectrum (F), XRD (G) and infrared spectrum (H) of nitrogen, sulfur and phosphorus co-doped quantum dots (NSP-CDs).

[0041] Figure 4 Comparison of UV-Vis absorption spectrum, fluorescence excitation spectrum, and emission spectrum of NSP-CDs (A); NSP-CDs, NSP-CDs / Ag + and NSP-CDs / Ag + -α-lipomycin fluorescence spectrum (B).

[0042] Figure 5 For NSP-CDs detection of different concentrations of Ag + The response fluorescence spectrum (A) and the fitted standard curve (B).

[0043] Figure 6 The fluorescence spectra of NSP-CDs in response to various interfering substances are shown in (A) and the corresponding net response histograms are shown in (B).

[0044] Figure 7 For NSP-CDs / Ag + Fluorescent nanoprobes were used to detect the response fluorescence spectra (A) and fitted standard curves (B) to different concentrations of α-lipomycin.

[0045] Figure 8 For NSP-CDs / Ag + Fluorescence spectra of fluorescent nanoprobes in response to various interfering substances (A) and corresponding net response histograms (B).

[0046] Figure 9 For NSP-CDs / Ag + Detection of α-lipomycin (A) in the serum of normal mice (WT), high-salt induced hypertensive mice (HS), and high-fat induced hypertensive mice (HFD), and α-lipomycin (B) in the serum of normal humans (Nor) and hypertensive individuals (HTN).

[0047] Figure 10 For NSP-CDs, NSP-CDs / Ag + NSP-CDs / Ag + Fluorescence decay curve of α-lipasein.

[0048] Figure 11 For NSP-CDs, NSP-CDs / Ag + NSP-CDs / Ag + Results of tests on the imaging capabilities of α-lipase within cells.

[0049] Figure 12 The results are for the cytotoxicity test of NSP-CDs nanoprobes; where A is MDA-MB-231 and B is HEK-293.

[0050] Figure 13 This describes the ion screening process; where A is the excitation and emission fluorescence spectrum of lipase itself; B is the fluorescence spectrum after the reaction of α-lipase with ions; C is an image of B under a UV lamp; D is the UV absorption spectrum of lipase itself; E is the UV absorption spectrum after the reaction of α-lipase with ions; and F is an image of E under a fluorescent lamp. Detailed Implementation

[0051] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0052] Unless otherwise specified, all solutions mentioned in the examples use water as the solvent.

[0053] Example 1 NSP-CDs

[0054] A method for preparing nitrogen-sulfur-phosphorus co-doped quantum dots for detecting α-lipomycin, the method comprising a one-step hydrothermal synthesis, specifically including the following steps:

[0055] Add 0.1g of o-phenylenediamine, 0.2g of thiourea and 0.1g of ATP to 30mL of ultrapure water, and then sonicate for 20min to mix them evenly to obtain a mixed solution;

[0056] The mixed solution was then placed in a 50 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor, sealed, and reacted at 200 °C for 6 h.

[0057] After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The resulting brownish-yellow product was centrifuged to separate the insoluble particles. The collected supernatant was poured into a dialysis bag with a molecular weight of 1000 and dialyzed with ultrapure water for 4 hours to obtain a nitrogen-sulfur-phosphorus co-doped quantum dot solution (NSP-CDs) for the detection of α-lipomycin. The solution was then stored in a refrigerator at 4°C in the dark.

[0058] The obtained nitrogen-sulfur-phosphorus co-doped quantum dots (NSP-CDs) were subjected to performance testing, and the test results are as follows:

[0059] Figure 2 TEM images (scale bar 100 nm for A, 50 nm for B) of nitrogen-sulfur-phosphorus co-doped quantum dots (NSP-CDs) at different magnifications, AFM images (C, (a) and (b) represent different thicknesses), EDS images (D), 3D AFM images from different viewpoints (E, F), particle size distribution plot (G), thickness distribution curves corresponding to line segment (a) and (b) in AFM images (H and I). Figure 2 As can be seen, NSP-CDs have good dispersibility and are monodisperse layered structures. Figure 2 The particles are classified as A, B, C, E, and F, with a particle size range of 6-17 nm. Figure 2 (G), thickness approximately 2-4 nm ( Figure 2 In NSP-CDs, the main component is carbon (C), with nitrogen (N), oxygen (O), sulfur (S), and phosphorus (P) distributed on the surface. Figure 2 The presence of D indicates that N, S, and P have been successfully doped.

[0060] Figure 3 XPS full spectrum (A), C1s spectrum (B), Ols spectrum (C), N1s spectrum (D), S2s spectrum (E), P2p spectrum (F), XRD (G), and infrared spectrum (H) of nitrogen-sulfur-phosphorus co-doped quantum dots (NSP-CDs). Figure 3It can be seen that NSP-CDs have five unique peaks. The sharp peaks at 161.96 eV and 132.80 eV correspond to the binding energy peaks of S2p and P2p. The other three sharp peaks appear at 284.22 eV, 398.76 eV, and 531.07 eV, corresponding to the binding energy peaks of C1s, N1s, and O1s, indicating that the chemical elements of NSP-CDs mainly include C, N, O, S, and P. Figure 3 (A). High-resolution energy spectrum of C1s ( Figure 3 From (B), we can see that the absorption peaks at 284.8, 286.0, 286.7, and 287.8 eV correspond to the chemical bonds CC, CO, CN, and COC, respectively. This is based on the high-resolution energy spectrum of O1s. Figure 3 From the C) diagram, we can see that the absorption peaks appear at 531.6, 532.6, and 533.0 eV, which are related to the chemical bonds OH, CO, and COC, respectively. From the N1s high-resolution energy spectrum ( Figure 3 The three absorption peaks at 397.6, 398.9, and 400.2 eV on the S2s spectrum correspond to the chemical bonds NH, NCN, and CN. (This is from the S2s high-resolution energy spectrum.) Figure 3 The three absorption peaks on the (E) are located at 162.1, 163.3, and 168.5 eV, respectively, and can be attributed to the presence of S. 2- S2 2- and SO4 2- From the P2p high-resolution energy spectrum ( Figure 3 Two absorption peaks were observed on the graphene surface (F), located at 133.2 and 134.0 eV, which can be attributed to the presence of CP and PO. The NSP-CDs exhibited distinct diffraction peaks at 2θ = 10.6°, 16.7°, 23.8°, 28.8°, and 44.9°. Comparison with standard cards revealed that these peaks correspond to the (001), (010), (002), (011), and (100) planes of graphene. Figure 3 (G); Fourier transform infrared spectroscopy was used to further confirm the chemical functional groups present on the surface of NSP-CDs, such as... Figure 3 As shown in Figure H, a very obvious absorption peak appears at 3446.99 cm⁻¹. -1 This absorption peak is attributed to the stretching vibrations of the OH and NH bonds, indicating that the synthesized NSP-CDs contain abundant functional groups such as amino and carboxyl groups. The presence of these functional groups significantly improves the water solubility of NSP-CDs; 2360.41 cm⁻¹ -1 The absorption peak at -C=N + -Related to sensual groups; 2065.98cm -1 The absorption peak at 1558.75 cm⁻¹ is related to the C≡C functional group. -1The absorption peak is due to the unsaturated stretching and bending vibrations of CH; it appears at 1635.98 cm⁻¹. -1 The strong absorption peak originates from the bending vibration of NH, and 1457.30 cm⁻¹ -1 The peak value at point P=O originates from the stretching vibration peak of CN at 1399.25 cm⁻¹. -1 At this point, the absorption peak of the PO bond stretching vibration is located at 1081.09 cm⁻¹. -1 937.95cm -1 The peak value represents the stretching vibration of PN; while 537.01 cm -1 The presence of these functional groups is primarily associated with the CH bending vibrations on the benzene ring; the presence of these functional groups indicates the successful synthesis of NSP-CDs.

[0061] The optical properties of NSP-CDs were investigated using ultraviolet absorption and fluorescence spectra. Figure 4 As can be seen from A, NSP-CDs emit a wavelength of 570nm under 415nm excitation, indicating that NSP-CDs emit yellow fluorescence. Figure 4 From B, we can see that: Ag + It can effectively quench the fluorescence of NSP-CDs, effectively "turning off" the fluorescence signal of NSP-CDs. When α-lipomycin is added to the quenched NSP-CDs, the fluorescence of the NSP-CDs is restored, and the fluorescence signal of the system is put into an "on" state. Therefore, NSP-CDs / Ag + It is feasible to use fluorescent probes to detect α-lipomycin.

[0062] Example 2: NSP-CDs and Ag + bonding performance

[0063] (1) Detection of silver ions

[0064] 100 μL of NSP-CDs from Example 1 was added to 900 μL of Tris-HCl buffer (20 mM, pH 7), followed by the addition of 1 mL of silver nitrate solutions of different concentrations (50, 100, 150, 200, 250, 300, 350, 400, 600, 800, 1000, 1200, 1400 μM) to obtain a mixed solution.

[0065] Then place the mixed solution on a vortex mixer to mix it rapidly, and incubate at room temperature for 2 minutes;

[0066] The fluorescence intensity of NSP-CDs was recorded using a Bio-Tek Synergy H4 microplate reader at the optimal excitation wavelength of 415 nm and the optimal emission wavelength of 570 nm.

[0067] Let ΔF1 represent the addition of Ag. + The difference in fluorescence intensity before and after NSP-CDs. Calculate the fluorescence response value and Ag. + The linear relationship between concentrations was determined, a standard curve was fitted, the fitting equation was determined, and the detection limit was calculated.

[0068] The results are as follows Figure 5 .from Figure 5 As can be seen from A, increasing the concentration of silver ions leads to a continuous decrease in the fluorescence value of NSP-CDs at 570 nm. From... Figure 5 As can be seen from Figure B, a linear curve showing the change in fluorescence intensity ΔF1 corresponds to the change in silver ion concentration. When Ag... + When the concentration varied from 25.00 μM to 150.00 μM, the linear curve showed a good linear relationship, with the linear equation being Y = 85.25X + 192.1, where Y is ΔF1, the concentration of Ag added. + The difference in fluorescence intensity of NSP-CDs before and after; X is the concentration of silver ions, and the correlation coefficient (R) is... 2 (It is 0.9769). Ag was calculated using the 3σ / K formula. + The detection limit was 0.200 μM. Specifically, when Ag... + At a concentration of 200 μM, the fluorescence quenching of NSP-CDs almost reached saturation, and subsequent NSP-CDs / Ag ratio was established. + Ag in fluorescent nanoprobes + Set this as the standard.

[0069] (2) Selectivity for silver ions

[0070] Add 100 μL of NSP-CDs to 900 μL of Tris-HCl buffer (20 mM, pH 7), followed by 1 mL of other interfering substances such as tryptophan (Trp) and sodium chloride (Na). + ), potassium chloride (K) + Sodium bicarbonate (HCO3) - Anhydrous calcium chloride (Ca) 2+ ), cobalt chloride (Co) 2+ ), zinc chloride (Zn 2+ Nickel chloride (Ni) 2+ Anhydrous manganese chloride (Mn) 2+ Sodium thiocyanate (SCN) - ), potassium iodide (I - ), potassium bromide (Br) - ), ammonium chloride (NH4) + Sodium nitrite (NO2) - ), chromium chloride (Cd) 2+ Sodium hypochlorite (ClO)- The final concentration of the interfering substance was set to 500 μM, Ag. + The final concentration was set to 200 μM; the mixture was thoroughly mixed and incubated at room temperature for 2 min, and the fluorescence intensity was recorded.

[0071] The results are as follows Figure 6 ,from Figure 6 It can be seen that none of the interfering ions can cause a change in the fluorescence intensity of NSP-CDs if and only if Ag + The fluorescence intensity of NSP-CDs only changed significantly when silver ions were present, indicating that NSP-CDs are specific for detecting silver ions, and the fluorescence quenching of NSP-CDs can only be caused by the presence of silver ions.

[0072] Example 3

[0073] A method for preparing NSP-CDs / Ag + The method for using fluorescent probes includes the following steps:

[0074] Mix 100 μL of nitrogen-thiophosphorus co-doped quantum dots for the detection of α-lipomycin with 900 μL of Tris-HCl buffer (20 mM, pH 7), then add 0.5 mL of Ag + Incubate the solution (silver nitrate solution, 800 μM) at room temperature for 2 min to obtain NSP-CDs / Ag. + Fluorescent probe.

[0075] Example 4

[0076] A method based on NSP-CDs / Ag + The method for detecting α-lipomycin using fluorescent probes includes the following steps:

[0077] 1500 μL NSP-CDs / Ag + The fluorescent probe was placed in 500 μL of a series of α-lipomycin standard methanol solutions of various concentrations (0.2, 0.3, 0.4, 0.8, 1, 1.2, 1.6, 2, 3, 4, 10, 20, 30, 40 μM) to make a final volume of 2 mL. The NSP-CDs / Ag ratio was then recorded at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + Fluorescence intensity difference; ΔF2 represents the NSP-CDs / Ag ratio before and after the addition of α-lipomycin. + The difference in fluorescence emission intensity.

[0078] The results are as follows Figure 7 .

[0079] from Figure 7 It can be seen that: with the increase of α-lipomycin concentration, NSP-CDs / Ag +The fluorescence emission peak intensity at 570 nm gradually increased, and when the concentration of α-lipomycin was 10.00 μM, its fluorescence intensity recovery almost reached saturation. When the concentration of α-lipomycin ranged from 0.05 to 10 μM, a good linear relationship was observed between the fluorescence intensity difference ΔF2 and the logarithm of the α-lipomycin concentration, with a linear regression coefficient R0. 2 =0.9742. The linear equation for the detection of α-lipomycin by this fluorescent probe is ΔF2 = 3701 × logC + 5091 (where C is the concentration of α-lipomycin). According to the 3σ / K formula, the detection limit of this nanoprobe can be calculated to be 5.80 nM, providing a new detection method for trace amounts of α-lipomycin.

[0080] Example 5 NSP-CDs / Ag + Specificity of fluorescent probes

[0081] Add 100 μL of NSP-CDs to 900 μL of Tris-HCl buffer (20 mM, pH 7), then add 0.50 mL of Glucoside. + (silver nitrate solution, 800 μM) solution, to obtain a mixed solution;

[0082] Place the mixed solution in a centrifuge tube, and then add 0.50 mL of interfering antibiotic solution (asparagine Asp, amoxicillin AMX, chloramphenicol CAP, oxytetracycline OTC, ciprofloxacin CIP, ampicillin AMP, penicillin G sodium PG, tetracycline TCY, tryptophan Try, glutathione GSH, adenosine triphosphate ATP, bovine serum albumin BSA, heme chloride Hemin, α-lipomycin; wherein the final concentration of α-lipomycin was set to 10 μM, and the final concentration of the other interfering antibiotics was set to 5 times that amount;

[0083] Place each centrifuge tube in a vortex mixer to mix the solution thoroughly, and incubate at room temperature for 2 minutes;

[0084] Finally, NSP-CDs / Ag were recorded using a full-function microporous detection plate at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + Fluorescence intensity difference.

[0085] The results are as follows Figure 8 .

[0086] from Figure 8 It can be seen that when other potentially interfering antibiotics or other substances are added, their presence does not affect NSP-CDs / Ag. + A significant change in fluorescence was observed at 570 nm; only in the presence of α-lipomycin did NSP-CDs / Ag show a change. +The fluorescence intensity only showed a significant enhancement at 570 nm, indicating that the constructed NSP-CDs / Ag + Fluorescent nanoprobes exhibit strong specificity for the detection of α-lipomycin and can be used for trace monitoring of α-lipomycin.

[0087] Example 6: Spike Recycling in Tap Water

[0088] A method based on NSP-CDs / Ag + A method for detecting α-lipomycin in tap water using a fluorescent probe includes the following steps:

[0089] The water sample was centrifuged at 5000 rpm for 5 minutes, and then filtered through a 0.22 μM filter to obtain the sample solution.

[0090] Add α-lipomycin to the sample solution, 1.5 mL NSP-CDs / Ag + The fluorescent probe was mixed with 0.5 mL of the sample to achieve final concentrations of α-lipomycin of 0.10, 0.50, and 5.00 μM, respectively. The NSP-CDs / Ag ratio was then recorded at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + The concentration of α-lipomycin in the sample was obtained by calculating the fluorescence intensity difference using the standard curve from Example 4.

[0091] The results are shown in Table 1.

[0092] Table 1 NSP-CDs / Ag + Determination of α-lipomycin in real samples by fluorescent nanoprobes (n=3)

[0093]

[0094] As shown in Table 1, the recovery rate in tap water was 97.36-99.04%, and the relative standard deviation (RSD) was between 2.28-2.95%.

[0095] Example 7: α-Lipomycin in serum

[0096] A method based on NSP-CDs / Ag + A method for detecting α-lipomycin in serum using a fluorescent probe includes the following steps:

[0097] Serum from normal mice (WT), high-salt induced hypertensive mice (HS), high-fat induced hypertensive mice (HFD), normal humans (Nor), and hypertensive individuals (HTN) was collected as sample solutions.

[0098] Prepare NSP-CDs / Ag at the same concentration as in Example 4 +Fluorescent probe, then 75 μL NSP-CDs / Ag + The fluorescent probe was incubated in 25 μL of sample solution for 2 minutes, and then the NSP-CDs / Ag ratio was recorded at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + The concentration of α-lipomycin in the sample solution was obtained by calculating the fluorescence intensity difference using the standard curve from Example 4.

[0099] The results are as follows Figure 9 .

[0100] from Figure 9 It can be seen that the serum α-lipomycin level in the high-salt-induced hypertensive mouse model was significantly higher than that in the other two groups. Furthermore, the serum α-lipomycin level in hypertensive individuals was also significantly higher than that in the normal control group. This is because the circulating level of α-lipomycin is regulated by dietary NaCl intake. These results indicate that the constructed fluorescent nanoprobe has certain practical application potential.

[0101] Example 8: Mechanism Discussion

[0102] For NSP-CDs and NSP-CDs / Ag + Fluorescence lifetime was measured, and the results are shown in Table 2 and... Figure 10 As shown.

[0103] from Figure 10 As shown in Table 2, their weighted average fluorescence lifetimes were 2.80 ns and 2.74 ns, respectively. The fluorescence lifetimes of NSP-CDs and NSP-CDs / Ag... + Compared to no significant change, this confirms that Ag + The fluorescence quenching of NSP-CDs is static quenching, i.e., Ag... + The formation of non-fluorescent or weakly fluorescent ground-state complexes with excited-state NSP-CDs leads to fluorescence quenching of NSP-CDs.

[0104] Table 2 NSP-CDs, NSP-CDs / Ag + NSP-CDs / Ag + +α-lipase fluorescence lifetime value

[0105] NSP-CDs 1.16 3.36 2.80 0.99969 <![CDATA[NSP-CDs / Ag + ]]> 1.24 3.62 2.74 0.99963 <![CDATA[NSP-CDs / Ag + +α-lipomycin]]> 1.25 3.61 2.76 0.99961

[0106] The hypothesized fluorescence quenching-recovery reaction mechanism of the system is as follows: Ag + It has a strong complexing effect; when added to NSP-CDs solution, Ag... + A certain interaction occurs between the NSP-CDs and the surface groups of NSP-CDs to form NSP-CDs / Ag +The complex effectively quenches the fluorescence of NSP-CDs, reducing their fluorescence intensity; after quenching, the NSP-CDs / Ag... + After the addition of α-lipase to the complex, α-lipase will remove NSP-CDs from NSP-CDs / Ag + The fluorescence of NSP-CDs is restored after the complex is released, and the fluorescence signal is "reactivated." The specific process is as follows: Figure 1 As shown.

[0107] Example 9 Cell Imaging

[0108] To evaluate the intracellular imaging capability of NSP-CD nanoprobes, 50 μL of NSP-CD nanoprobes were added to 950 μL of DMEM medium containing 10% serum, and MDA-MB-231 cells were cultured at 37°C and 5% CO2 for 12 h. NSP-CDs + Ag + The group was based on the NSP-CDs group, with a new culture medium and Ag added. + The final concentration was adjusted to 200 μM, and the mixture was further incubated at 37 °C and 5% CO2 for 12 hours. NSP-CDs+Ag + +α-lipomycin group in NSP-CDs+Ag + The culture medium was replaced with fresh DMEM and α-lipomycin was added to bring the final concentration to 10 μM. The culture was then incubated at 37 °C and 5% CO2 for 12 hours.

[0109] like Figure 11 As shown; from Figure 11 It can be seen that: no external Ag was added. + At that time, strong yellow fluorescence was observed in the cells after incubation with NSP-CDs nanoprobes, indicating that the NSP-CDs nanoprobes successfully took up into MDA-MB-231 cells and accumulated in the cytoplasm. Conversely, after incubation with NSP-CDs nanoprobes followed by the addition of Ag... + No fluorescence signal was observed during incubation. However, upon adding α-lipase to the culture system, a clear recovery of yellow fluorescence was observed. It can be inferred that α-lipase removes NSP-CDs from the NSP-CDs / Ag... + The NSP-CDs were released from the complex, restoring their fluorescence and consistent with the results obtained in vitro, thus validating their potential as fluorescent probes for bioimaging and other basic biological research.

[0110] Example 10 Cytotoxicity

[0111] MTT assay was used to detect the cytotoxicity of NSP-CDs nanoprobes on MDA-MB-231 and HEK-293 cells.

[0112] Specifically, cells are seeded at 1×10⁻⁶. 4 Cells were cultured in 96-well plates at 37°C and 5% CO2 for 24 hours in DMEM medium containing 10% serum. Then, the DMEM was aspirated, and fresh DMEM containing different quantum dot concentrations (0, 50, 100, 150, 200, 250, and 300 μg / mL) was added, followed by another 24 hours of culture. Subsequently, the medium was aspirated, and cells were treated with 5 mg / mL MTT (10 μL per well) and cultured for another 4 hours (37°C, 5% CO2). Next, dimethyl sulfoxide (100 μL per well) was added to dissolve the purple methoxynitrite. Finally, the absorbance of MTT was recorded at 570 nm using a microplate reader.

[0113] Based on the OD values ​​of the experimental group (containing cells, culture medium, MTT, and quantum dots), the control group (containing cells, culture medium, and MTT, but without quantum dots), and the blank group (containing no cells and quantum dots, but containing culture medium and MTT), the survival rate of quantum dots for the two cell types was calculated. The results are as follows: Figure 12 As shown.

[0114] from Figure 12 It can be seen that when the working concentration of the prepared quantum dots is as high as 300 ug / mL, the cell survival rate is still above 79%, indicating that the quantum dots have low cytotoxicity.

[0115] Comparative Example 1: Ion Screening Process

[0116] Mix 10 μL of estermycin (85.1 μM) with 10 μL of methanol at a volume ratio of 1:1, then add 10 μL of a 1 mM metal ion solution, such as silver nitrate (Ag). + Magnesium chloride (Mg) 2+ Cobalt chloride (Co) 2+ Copper sulfate (Cu) 2+ Aluminum nitrate Al 3+ Cerium nitrate (Ce) 3+ Lead nitrate (Pb) 2+ Manganese chloride (Mn) 2+ Potassium dichromate Cr2O7 2- Zinc chloride (Zn) 2+ ferric chloride (Fe) 3+ .

[0117] The results showed that, Figure 13 .

[0118] from Figure 13It can be seen that lipase itself exhibits orange fluorescence, with optimal excitation and emission wavelengths of 430 nm and 590 nm, respectively. Figure 13 (A) and the lipase solution is yellow, with the maximum absorption peak located at 405 nm. Figure 13 (D). Only when Ag + In its presence, both the fluorescence spectrum and ultraviolet absorption spectrum of lipase are significantly reduced. Figure 13 (B and E), accompanied by fluorescence quenching, the solution changes from yellow to colorless ( Figure 13 (C and F in the middle). This shows that the active groups of the estermycin molecule can chelate Ag. +

[0119] Comparative Example 2

[0120] Quantum dots, as the most promising alternative to traditional fluorescent dyes, have attracted increasing attention from researchers and sparked a research boom in recent decades. Although the synthesis of quantum dots is relatively easy and simple, and the precursor raw materials are also abundant, the fluorescence emission wavelength of most prepared quantum dots is in the blue or green region. This will undoubtedly interfere with the determination of α-lipomycin due to the autofluorescence of biological samples, and also hinder the application of quantum dots in the field of bioimaging.

[0121] Therefore, this invention achieves highly sensitive detection of α-lipomycin by synthesizing yellow quantum dots and ultimately applying them to construct a fluorescent sensor.

[0122] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing NSP-CDs / Ag + The method using fluorescent probes is characterized by, Includes the following steps: o-phenylenediamine, thiourea, and adenosine triphosphate were added to water and mixed thoroughly to obtain a mixed solution. The mixed solution was then sealed and reacted at 200°C for 6 hours. After the reaction was completed, the solution was cooled, centrifuged, and dialyzed to obtain nitrogen-thiophosphorus co-doped quantum dots for the detection of α-lipomycin. Nitrogen-thiophosphorus co-doped quantum dots for detecting α-lipomycin were mixed with Tris-HCl buffer, and then Ag was added. + The solution was incubated to obtain NSP-CDs / Ag + Fluorescent probe.

2. The method according to claim 1, characterized in that, The mass ratio of o-phenylenediamine, thiourea, adenosine triphosphate and water is 1:2:1:

30.

3. The method according to claim 1, characterized in that, The nitrogen-sulfur-phosphorus co-doped quantum dots, Tris-HCl buffer, and Ag used for the detection of α-lipomycin + The volume ratio of the solutions is 1:9:

5.

4. The method according to claim 1, characterized in that, The incubation is carried out at 20-30℃ for 2-3 minutes.

5. The NSP-CDs / Ag prepared by the method according to any one of claims 1-4 + Fluorescent probe.

6. A method based on NSP-CDs / Ag + A method for detecting α-lipomycin using a fluorescent probe, characterized in that, Includes the following steps: The NSP-CDs / Ag described in claim 5 + The fluorescent probe was placed in the test solution containing α-lipomycin, and then the NSP-CDs / Ag ratio was recorded at an excitation wavelength of 415 nm and an emission wavelength of 570 nm. + The fluorescence intensity difference was used to calculate the concentration of α-lipomycin in the test solution containing α-lipomycin.

7. The method according to claim 6, characterized in that, The NSP-CDs / Ag + The volume ratio of the fluorescent probe to the test solution containing α-lipomycin is 3:

1.

8. The method according to claim 6, characterized in that, Test solutions containing α-lipomycin include aqueous solutions containing α-lipomycin and serum solutions containing α-lipomycin.

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