Preparation method and application of receptaculum nelumbinis derived fluorescent carbon dots

Abstract

The invention discloses a preparation method and application of receptaculum nelumbinis-derived fluorescent carbon dots, the receptaculum nelumbinis is used as a natural precursor, and the fluorescent carbon dots are synthesized through a one-step hydrothermal method.The receptaculum nelumbinis is used as the natural precursor to prepare the fluorescent carbon dots, and the preparation method has the advantages of being environmentally friendly, low in cost, simple in synthesis, high in detection method sensitivity and good in selectivity; the method can rapidly and accurately detect the activity of alpha-glucosidase, is simple and convenient to operate, and is suitable for practical application.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent carbon dots, and in particular relates to a preparation method and application of lotus pod-derived fluorescent carbon dots. Background Art

[0002] Neutral α-glucosidase (NAG) is an enzyme that has the function of hydrolyzing α-1,4-glycosidic bonds. It is only produced in the epididymis and is considered to be a reliable biomarker for evaluating epididymal function. Therefore, seminal plasma NAG activity is a key parameter for evaluating sperm quality and has clear clinical value in assisting the diagnosis of male infertility. Therefore, people have been working hard to develop methods for detecting seminal plasma NAG activity. Some studies have proposed to detect the activity of NAG by measuring the ultraviolet absorption of p-nitrophenol produced by NAG hydrolysis of p-nitrophenyl-α-D-glucoside (PNPG) at around 405nm. However, this method has problems with matrix interference, high cost, poor selectivity and sensitivity. Therefore, it is of great significance to develop a simple, sensitive and reliable method to determine NAG activity to assist in the assessment of sperm quality.

[0003] In recent years, fluorescent carbon dots (CDs) have been used as sensors for biosensing applications due to their excellent optical properties, good biocompatibility, and high resistance to photobleaching. However, most of these methods use expensive and toxic chemical reagents, which will cause environmental pollution and increase production costs. It is worth noting that natural biomass contains complex chemical compositions and abundant heteroatoms, which is conducive to the generation of self-doping during the preparation of CDs. Studies have shown that lotus pods contain a variety of chemical substances, such as flavonoids, alkaloids, and amino acid compounds, which can provide rich carbon sources and heteroatom doping for the synthesis of CDs. Therefore, using lotus pods as precursors to prepare fluorescent CDs and using them to construct a highly sensitive NAG activity detection method can not only promote the sustainable utilization of waste resources, but also facilitate the clinical diagnosis of NAG-related diseases. Therefore, a preparation method and application of lotus pod-derived fluorescent carbon dots are needed. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of lotus pod-derived fluorescent carbon dots.

[0005] A method for preparing lotus pod-derived fluorescent carbon dots, using lotus pods as natural precursors and synthesizing fluorescent carbon dots by a one-step hydrothermal method, specifically comprising:

[0006] The lotus seed pod powder was dissolved in water at a solid-liquid ratio of 0.03 g / mL, and ultrasonicated for 10 minutes to obtain a mixture;

[0007] heating the mixture in a high pressure reactor at 200° C. for 6 hours to obtain a reaction solution;

[0008] After cooling naturally to room temperature, the reaction solution was centrifuged at 10000 rpm for 10 minutes and passed through a 0.22 μm filter membrane to obtain a supernatant;

[0009] The supernatant was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried under vacuum to obtain fluorescent carbon dots.

[0010] The fluorescent carbon dots obtained by the method for preparing lotus pod-derived fluorescent carbon dots are used in the activity detection of NAG, and the fluorescence of the fluorescent carbon dots can be detected by Fe 3+ quenched and subsequently restored by ascorbic acid.

[0011] Furthermore, the fluorescence intensity of the fluorescent carbon dots changes with the activity of α-glucosidase in the range of 2 to 1200 UL -1 There is a good linear relationship within the range.

[0012] Furthermore, the ascorbic acid recovery time is selected to be 15 minutes, and the incubation temperature is 37°C.

[0013] Compared with the prior art, the present invention has the following effects:

[0014] The present invention uses lotus pods as natural precursors to prepare fluorescent carbon dots, which are environmentally friendly, low-cost, and simple to synthesize. The detection method has high sensitivity and good selectivity, can quickly and accurately detect α-glucosidase activity, and is easy to operate and suitable for practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic diagram of the optimization of the preparation conditions of the fluorescent carbon dots of the present invention; Figure 1 A is the fluorescence spectra of carbon dots prepared with different raw material addition amounts. Figure 1 B is the fluorescence intensity of carbon dots prepared with different raw material addition amounts. Figure 1 C is the yield of carbon dots prepared with different raw material addition amounts. Figure 1 D is the fluorescence spectra of carbon dots prepared at different temperatures. Figure 1 E is the fluorescence intensity of carbon dots prepared at different temperatures. Figure 1 F is the yield of carbon dots prepared at different temperatures, Figure 1 G is the fluorescence spectra of carbon dots prepared at different reaction times. Figure 1 H is the fluorescence intensity of carbon dots prepared at different reaction times, Figure 1 I is the yield of carbon dots prepared at different reaction times.

[0016] Figure 2 Schematic diagram of the characterization of the fluorescent carbon dots of the present invention; Figure 2 A is the TEM image, Figure 2 B is the HRTEM image, Figure 2 C is the particle size distribution statistics, Figure 2 D is the XRD spectrum, Figure 2 E is the FT-IR spectrum, Figure 2 F is the full scan XPS spectrum, Figure 2 G is the C1s high-resolution XPS spectrum, Figure 2 H is N1s high-resolution XPS spectrum, Figure 2 I is the O1s high-resolution XPS spectrum.

[0017] Figure 3 Schematic diagram of the optical properties and metal ion selectivity of the fluorescent carbon dots of the present invention; Figure 3 A is the UV-visible absorption spectrum, fluorescence excitation spectrum and emission spectrum, Figure 3 B is the CIE coordinate diagram, Figure 3 C is the fluorescence emission spectrum, Figure 3 D is the stability of fluorescent carbon dots under ultraviolet light. Figure 3 E is the storage stability of fluorescent carbon dots under sunlight, Figure 3 F is the effect of different metal ions on the fluorescence of carbon dots. Figure 3 G is the selectivity of carbon dots to metal ions, Figure 3 H is different Fe 3+ Fluorescence spectra at different concentrations, Figure 3 I is the fluorescence of carbon dots and the addition of Fe 3+ The relationship between concentration.

[0018] Figure 4 A schematic diagram of the research on the fluorescence quenching mechanism of carbon dots and the optimization of detection parameters of the present invention; Figure 4 A is the fluorescence spectrum of carbon dots and Fe 3+ The UV absorption spectrum of Figure 4 B is the change of Zeta potential value, Figure 4 C is the fluorescence lifetime decay curve, Figure 4 D is CDs@Fe 3+ Fluorescence spectra under different concentrations of AA, Figure 4 E is the relationship between carbon dots fluorescence and AA concentration, where F0 and F represent CDs@Fe 3+ Fluorescence intensity in the presence and absence of AA, Figure 4 F is the incubation temperature optimization, Figure 4 G is the AA2G concentration optimization, Figure 4 H is the incubation time optimization, Figure 4 I is recovery time optimization.

[0019] Figure 5 This is a schematic diagram of the human seminal plasma NAG activity detection system constructed by using carbon dots in the present invention and its application; Figure 5 A is the fluorescence spectrum of carbon dots in the presence of different substances, Figure 5B is a selective test for α-glucosidase. The concentration of interfering ions is 1mM, and the concentrations of BSA, GSH, Glucose, Trp, Cys, Gly, Leu, TRY, AMS, β-Glu, α-glucosidase, Pepsin, and Lipase are 1000μL -1 , Figure 5 C is the fluorescence spectrum change, Figure 5 D is the relationship between the concentration of α-glucosidase and the degree of fluorescence recovery. F0 and F represent the fluorescence intensity in the presence and absence of α-glucosidase, respectively. Figure 5 E is the NAG activity of different samples, Figure 5 F is the comparison of methods for determining NAG levels in samples. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0021] In the present invention, lotus pod-derived fluorescent carbon dots (LS-CDs) and a green preparation method thereof are proposed, and applied to the highly sensitive and selective detection of seminal plasma NAG activity.

[0022] Specifically, LS-CDs with excellent optical properties were synthesized by a one-step hydrothermal method using lotus pods as natural precursors. 3+ The fluorescence of LS-CDs is quenched and then recovered by ascorbic acid (AA), which can be produced by the hydrolysis of ascorbic acid 2-glucoside (AA2G) catalyzed by NAG. Therefore, the activity of NAG involved in the hydrolysis process can be indicated by the degree of fluorescence recovery of LS-CDs. Since the preparation of LS-CDs does not involve toxic reagents and extremely harsh conditions, LS-CDs have the advantages of environmental protection, simple synthesis and low cost. In addition, the proposed detection method has the characteristics of fast response, high sensitivity and good selectivity, and can accurately measure the NAG activity in human seminal plasma samples.

[0023] Example 1 Preparation of Lotus Pod-derived Fluorescent Carbon Dots

[0024] The synthesis parameters of lotus pod derived fluorescent carbon dots (LS-CDs) are as follows: 0.600 g of lotus pod powder was dissolved in 20 mL of water and ultrasonicated for 10 min. Then, the mixture was transferred to a polytetrafluoroethylene-lined autoclave and heated at 200 °C for 6 h. Subsequently, it was naturally cooled to room temperature, and the black solution was centrifuged at 10,000 rpm for 10 min and passed through a 0.22 μm filter membrane to obtain the supernatant. Finally, the lotus pod derived fluorescent carbon dots solution was further purified in a dialysis bag (1000 Da) for 24 h to obtain lotus pod derived fluorescent carbon dots, which were then freeze-dried under vacuum for subsequent use.

[0025] Characterization of LS-CDs

[0026] Transmission electron microscopy (TEM) images were taken on a Talos F200X microscope. UV-visible absorption and fluorescence spectra were tested on a Cary60 UV-visible spectrophotometer and an F-7000 fluorophotometer, respectively. Fourier transform infrared (FT-IR) spectra were obtained using a Nicolet 6700 FT-IR spectrometer. X-ray photoelectron spectroscopy (XPS) was measured by a Thermoescalab 250XI. X-ray diffraction (XRD) data were measured on a D8Advance (Brucker, Germany). Quantum yield (QY) was determined using a Zetasizer Nano.

[0027] Quinine sulfate solution (0.1M H2SO4 as solvent) was used as the standard. Its QY and refractive index are 0.577 and 1.33, respectively. The QY calculation formula of LS-CDs is as follows:

[0028] QYS(%)=QYR×(F / FR)×(AR / A)×(η / ηR)2

[0029] The subscripts "S" and "R" represent the sample and reference substance, respectively. "F" represents the fluorescence value, "A" represents the absorbance, and "η" represents the refractive index of the solvent.

[0030] Determination of α-glucosidase activity

[0031] First, LS-CDs (0.6 mg / mL) and Fe 3+ Solution (120 μM) preparation of CDs@Fe 3+ Mix the solution. 25 μL PBS, 25 μL 10 mM AA2G solution, and 50 μL α-glucosidase solution of different concentrations were added to the 96-well plate in sequence, shaken for 2 minutes, and then placed in a 37°C incubator for 35 minutes. Subsequently, 100 μL CDs@Fe 3+ The solution was shaken for 15 minutes and then the fluorescence spectrum at the optimal excitation wavelength was recorded using an ELISA reader.

[0032] Determination of NAG activity in human seminal plasma

[0033] 12 seminal plasma samples were provided by volunteers, and all participants provided informed consent. NAG activity analysis was performed according to the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (World Health Organization 2021). First, semen samples were collected by masturbation after 3-7 days of abstinence and centrifuged after complete liquefaction at 37°C. Seminal plasma was obtained by transferring the supernatant and then sealed and stored in a -20°C refrigerator. To determine the activity of NAG, 30μL of 1% SDS solution and 20μL of seminal plasma were added to a 96-well plate in sequence and incubated for 5 minutes to eliminate the influence of acid α-glucosidase. Then, 25μL PBS and 25μL 10mM A2G solution were added, and the mixture was placed in an incubator at 37°C for 35 minutes. Next, 100μL CDs@Fe was added. 3+ The solution was shaken for 15 minutes and then the fluorescence spectrum at the optimal excitation wavelength was recorded using an ELISA reader.

[0034] Example 2 Preparation conditions of carbon dots

[0035] Under the same conditions of reaction temperature of 180℃ and reaction time of 4h, the amount of lotus pod raw material added was changed to investigate the effect of different amounts of lotus pod added (0.2, 0.4, 0.6, 0.8 and 1.0g) on ​​the fluorescence intensity and yield of lotus pod carbon dots. Figure 1 As shown in A, B, and C, when the amount of lotus pod added increased from 0.2 g to 0.6 g, the carbon dot yield and fluorescence intensity continued to increase, because the raw materials were fully carbonized under appropriate conditions. However, when the amount of raw materials continued to increase, the yield and fluorescence intensity of lotus pod carbon dots began to show a downward trend, which may be due to the accumulation of too much raw material in the reactor, resulting in the inability to fully carbonize the raw materials, or even only a small part of the raw materials can undergo carbonization reaction. Therefore, the optimal raw material addition amount is 0.6 g.

[0036] Under the same conditions of 0.6 g lotus pod addition and 4 h reaction time, the effects of different reaction temperatures (140, 160, 180, 200, 220°C) on the fluorescence intensity and yield of lotus pod carbon dots were investigated. Figure 1 As shown in D, E, and F, with the increase of reaction temperature, the yield and fluorescence intensity of carbon dots are significantly enhanced. When the reaction temperature reaches 200°C, the yield and fluorescence intensity reach the highest point. When the temperature rises to 220°C, the yield and fluorescence intensity of carbon dots begin to decrease. Studies have shown that too high a temperature may promote the aggregation of carbon dots, resulting in a decrease in yield and fluorescence intensity. Therefore, 200°C is selected as the optimal reaction temperature.

[0037] The experiment explored the effect of different reaction times (2, 4, 6, 8, 10 h) on the yield and fluorescence intensity of carbon dots. Figure 1 G, Figure 1 H, Figure 1As shown in Figure 1, when the reaction time increases from 2h to 6h, the yield and fluorescence intensity of LS-CDs gradually increase and reach the maximum at 6h. This is because the reaction time is too short, resulting in incomplete carbonization of lotus pods, so the carbon point yield and fluorescence intensity continue to increase with the increase of carbonization time. However, when the reaction time is increased, the fluorescence intensity begins to decline. This may be due to the long reaction time, the surface structure of the carbon points is destroyed, and the fluorescence quenching phenomenon occurs. Therefore, 6h is selected as the optimal reaction time.

[0038] The single factor experiment showed that the best conditions for preparing lotus pod carbon dots were 0.6 g lotus pod addition, 200 °C reaction temperature and 6 h reaction time. Under these conditions, the yield and quantum yield of LS-CDs were 2.11% and 9.25%, respectively.

[0039] Characterization of carbon dots

[0040] Transmission electron microscopy (TEM) was further used to characterize the morphology, size, and microstructure of LS-CDs. Figure 2 As shown in A and B, LS-CDs are spherical and have good solution dispersibility. The particle size statistical analysis is shown in Figure 2 C, the particle size of LS-CDs ranges from 2-4nm, with an average particle size of 2.63±0.37nm. At the same time, the lattice structure can be clearly observed from the HRTEM image, and its lattice spacing is 0.21nm, which matches the graphene (100) crystal plane. The XRD pattern of LS-CDs ( Figure 2 D) shows sharp peaks at 28.33°, 40.47°, and 50.12°, corresponding to the (002), (100), and (102) planes of sp2 hybridized graphitic carbon. The peaks at 58.55° and 66.26° correspond to the (600) and (220) planes of sp3 hybridized diamond-carbon structure. This indicates that LS-CDs have a similar structure to other biomass-based CDs.

[0041] The functional groups contained in carbon dots play a very important role in their optical properties and their interactions with other substances. In order to explore the functional group structure on the surface of carbon dots, FT-IR was used to characterize the carbon dots. The results are shown in Figure 2. Figure 2 As shown in E, the broad absorption peak at 3233cm-1 is the stretching vibration peak of OH and NH bonds; the absorption peak at 2930cm-1 can be attributed to the stretching vibration peak of CH on saturated carbon; a strong absorption peak was observed at 1602cm-1, which is attributed to the stretching vibration of C=C / C=O; the absorption peak at 1405cm-1 can be attributed to the stretching vibration peak of CN; the absorption peak at 1063cm-1 can be attributed to the stretching vibration absorption peak of CO; the absorption peak at 881cm-1 can be attributed to the stretching vibration absorption peak of NH.

[0042] XPS analysis was used to further investigate the elemental composition and related chemical bonds. The XPS full spectrum showed ( Figure 2 F), there are three obvious peaks at 284.8, 399.8 and 532.2 eV, corresponding to the characteristic binding energies of C1s, N1s and O1s, respectively, proving that the prepared LS-CDs have C, N and O elements. Their proportions are C: 60.34%, N: 4.94%, O: 34.72%. In detail, from the peak fitting diagram of C1s ( Figure 2 G) shows that there are three obvious peaks at 284.8, 286.2 and 288.1 eV, which are respectively attributed to CC / C=C, CO / CN and C=O groups. Figure 2 H) showed two peaks at 399.92eV and 401.56eV, corresponding to the CNC and NH functional groups. Figure 2 I), the two peaks of 531.31eV and 532.66eV come from C=O and CO groups, respectively. Based on the results of FTIR and XPS, the surface of LS-CDs has abundant hydrophilic groups, such as hydroxyl (-OH), carboxyl (-COOH), amine (-NH2), etc., which help to improve the fluorescence properties of LS-CDs and increase their solubility in aqueous solution.

[0043] Optical properties of LS-CDs

[0044] In order to explore the optical properties of LS-CDs, their UV-visible absorption spectra and fluorescence spectra were studied. Figure 3 As shown in A, the UV-visible absorption spectrum of LS-CDs shows that CDs has an obvious characteristic absorption peak at 270nm, which is a typical aromatic hydrocarbon absorption peak, which comes from the π-π* transition of the aromatic sp2 structure. The maximum excitation wavelength of the LS-CDs solution is 310nm, and the emission wavelength is at 415nm, showing that the LS-CDs aqueous solution is transparent brown-yellow under visible light and emits bright blue fluorescence under 365nm ultraviolet light irradiation. The CIE chromaticity coordinates are (0.1595, 0.1231), which is located in the blue light region, further proving the fluorescence characteristics of LS-CDs ( Figure 3 B). It is worth noting that LS-CDs exhibit certain excitation wavelength dependence. Figure 3C shows the fluorescence intensity of LS-CDs obtained at different excitation wavelengths (ranging from 300 to 400 nm, with an increment of 10 nm). In the 300-400 nm wavelength detection range, as the excitation wavelength gradually increases, the fluorescence intensity of LS-CDs shows a trend of first increasing and then decreasing. When the excitation wavelength increases from 300 nm to 310 nm, the fluorescence intensity of CDs reaches its maximum value. However, when the excitation wavelength continues to increase, the fluorescence intensity decreases significantly and the peak position shows an obvious red shift. It is speculated that this may be caused by the uneven size of carbon dots and different surface defect states. In addition, the optical stability of LS-CDs was investigated. Figure 3 As shown in Figure D, the fluorescence intensity of LS-CDs did not change significantly within 3 hours after the LS-CDs solution was irradiated with UV light, indicating that it has good anti-photobleaching ability. In addition, LS-CDs themselves have high stability. After three weeks of storage, the fluorescence intensity of LS-CDs remained basically stable ( Figure 3 E), reflecting the long-lasting luminescence properties of LS-CDs.

[0045] Example 3 Construction of fluorescence sensor

[0046] Fe 3+ The selectivity of LS-CDs to various ions was evaluated by fluorescence spectroscopy. 3+ 、Na + , K + 、Zn 2+ , Cu 2+ , Pb 2+ , Fe 3+ , Fe 2+ , Ca 2+ Mg 2+ , Hg 2+ , Mn 2+ 、Ag + 、Co 2+ 、Cd 2+ ) selection recognition performance. Figure 3 It can be seen from F that adding Fe 3+ After that, the fluorescence of LS-CDs almost disappeared under 365nm irradiation. Figure 3 G) Fe can be clearly seen 3+ It exhibits the strongest fluorescence quenching effect on LS-CDs, while other metal ions have no significant effect on the fluorescence of carbon dots. Figure 3 H is the fluorescence intensity of carbon dots with Fe 3+ concentration (0-160μM), with the Fe 3+ With the increase of concentration, the fluorescence intensity of LS-CDs gradually decreased. Figure 3I shows that the fluorescence intensity of LS-CDs at the optimal excitation wavelength and emission wavelength varies with Fe 3+ concentration changes. It can be seen that when Fe 3+ When the concentration increased from 0 to 120 μM, the fluorescence intensity of LS-CDs at 415 nm decreased the most. 3+ When the concentration was higher than 120 μM, the fluorescence intensity was slightly quenched. 3+ The optimal concentration was 120 μM.

[0047] In order to explore the relationship between LS-CDs and Fe 3+ The quenching mechanism between Fe 3+ 、CDs@Fe 3+ UV-visible absorption spectra and fluorescence spectra of LS-CDs. Figure 4 A shows the excitation spectrum of LS-CDs and Fe 3+ There is an obvious overlap between the UV absorption spectra of Fe 3+ The addition of may absorb the excitation light energy of LS-CDs first, thereby causing the quenching of the LS-CDs' own fluorescence signal. Figure 4 B shows that as Fe 3+ The Zeta potential of LS-CDs aqueous solution changed from -16.02 to -4.76, which may be due to the addition of Fe 3+ The binding to the hydroxyl and carboxyl groups on the surface of LS-CDs led to the consumption of negative charges. 3+ The effect of the fluorescence lifetime of LS-CDs before and after addition was further analyzed to analyze its detection mechanism. If the fluorescence lifetime values ​​before and after addition are almost the same, it is a static quenching process. If not, it is a dynamic quenching process. The fluorescence decay curve after fitting with the double exponential equation is shown in Figure 4 As shown in C, the two fluorescence lifetimes of LS-CDs are τ1 = 6.9962 (88.18%), τ2 = 12.8578 (11.82%), and χ 2 =1.065, the average fluorescence lifetime is 8.1547ns, and Fe is added 3+ After that, the two fluorescence lifetimes are τ1=6.9450(86.93%), τ2=12.5230(13.07%), χ 2 =1.065, and the average fluorescence lifetime is 8.1347ns. 3+ The CDs fluorescence lifetime value only changed slightly before and after the addition of Fe 3+ It is through the formation of non-fluorescent complexes by combining with the ground-state molecules on the surface of LS-CDs, i.e., static quenching process. 3+The interaction mechanism between LS-CDs and the LS-CDs is through the combined action of inner filter effect and static quenching.

[0048] Restoration effect of AA on fluorescence of LS-CDs

[0049] As a reducing substance, ascorbic acid (AA) and Fe 3+ It has strong binding force and can convert Fe 3+ Reduction to Fe 2+ Therefore, adding AA to the detection system can provide a reducing environment and partially restore the fluorescence of LS-CDs. Considering that the established NAG detection system is related to the production of AA, the CDs@Fe 3+ Sensitivity to AA. Figure 4 As shown in D, with the addition of AA, the fluorescence of the mixture gradually increased. In the range of 1 to 140 μM, there is a good linear relationship between F0 / F and AA concentration ( Figure 4 E), the linear equation is F / F0=0.0113[C AA ]+1.0298, and the correlation coefficient is 0.9964. In theory, NAG specifically hydrolyzes AA2G to produce AA, so this method can be used to detect the activity of NAG.

[0050] In addition, the parameters affecting the fluorescence recovery process in the established detection method were further optimized. Figure 4 As shown in F, G, H, and I, the optimal incubation temperature is 37°C. When the concentration of AA2G is 10.0 mM, the fluorescence recovery effect reaches the maximum value and remains unchanged. The incubation time of the enzyme reaction directly affects the production of AA. The fluorescence intensity gradually increases with the incubation time from 10 minutes to 45 minutes and reaches a stable state at 35 minutes. The optimization results of the fluorescence recovery time show that the optimal recovery time is 15 minutes.

[0051] Fluorescence detection of α-glucosidase activity

[0052] By studying the effects of different substances on the fluorescence of LS-CDs, the feasibility of this method for NAG determination was demonstrated. Figure 5 As shown in A, only when CDs@Fe 3+ When AA2G and α-glucosidase were co-incubated, the fluorescence of the mixture was restored. This can be explained by the fact that α-glucosidase catalyzes the hydrolysis of substrate AA2G to produce AA, which converts the Fe 3+ Reduction to Fe 2+ , thereby restoring the fluorescence of quenched LS-CDs. Based on this principle, the activity of α-glucosidase can be quantitatively analyzed by measuring the fluorescence intensity of LS-CDs. In addition, some metal ions, various common biomolecules and enzymes have a significant effect on CDs@Fe3+ The interference effects of fluorescence were investigated to evaluate the selectivity of the method. The effect of these potential interferences on the fluorescence recovery of quenched LS-CDs was negligible ( Figure 5 B), which indicates that this method has excellent selectivity for NAG detection.

[0053] Under the optimal sensing conditions, the fluorescence spectra of LS-CDs were recorded after adding α-glucosidase with different activities. Figure 5 As shown in C, as the α-glucosidase activity changes from 0 to 2000 U L -1 The fluorescence intensity of LS-CDs increased gradually. The fluorescence intensity of LS-CDs increased with the activity of α-glucosidase in the range of 2 to 1200 U L -1 There is a good linear relationship in the range ( Figure 5 D). The linear regression equation is expressed as F0 / F=0.0010[C alpha-glucosidase ]+1.0183(R 2 =0.9988), the detection limit is 0.8UL -1 (S / N=3). The relative standard deviation (RSD) obtained by repeatability evaluation (n=6) was 3.45%, indicating that the method has good reproducibility. In addition, compared with the previously reported method, the detection strategy proposed in the present invention has a wider detection range and less detection time.

[0054] Detection of NAG activity in human seminal plasma

[0055] Since seminal plasma also contains acid α-glucosidase produced by the prostate, sodium dodecyl sulfate was added to eliminate possible interference. In order to study the reliability of this method, the sample recovery rate of this method was determined. As shown in Table 1, when different concentrations of α-glucosidase were added, the recovery rate ranged from 97.3% to 101%, and the RSD did not exceed 2.69%, indicating that this method has high reliability and accuracy in the determination of NAG. Subsequently, the NAG activity in human seminal plasma samples was determined by the established detection system, and it was multiplied by the total volume of semen to calculate the total NAG activity of each ejaculation. Figure 5 As shown in Figure E, the total NAG activity varies greatly between different samples and is lowest in sample 9. In addition, the activity of NAG in seminal plasma samples was determined using a traditional method, and the results obtained were basically consistent with the experimental data determined in the present invention ( Figure 5 F) These results indicate that the method developed in the present invention has high reliability in detecting NAG activity.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A method for preparing lotus pod-derived fluorescent carbon dots, characterized in that: Fluorescent carbon dots were synthesized using lotus pods as natural precursors via a one-step hydrothermal method, including: The lotus seed pod powder was dissolved in water at a solid-liquid ratio of 0.03 g / mL, and ultrasonicated for 10 minutes to obtain a mixture; heating the mixture in a high pressure reactor at 200° C. for 6 hours to obtain a reaction solution; After cooling naturally to room temperature, the reaction solution was centrifuged at 10000 rpm for 10 minutes and passed through a 0.22 μm filter membrane to obtain a supernatant; The supernatant was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried under vacuum to obtain the fluorescent carbon dots.

2. Application of the fluorescent carbon dots obtained by the method for preparing lotus pod-derived fluorescent carbon dots as claimed in claim 1 in the activity detection of NAG, characterized in that: The fluorescence of the fluorescent carbon dots can be 3+ quenched and subsequently restored by ascorbic acid.

3. The use according to claim 2, characterized in that: The fluorescence intensity of the fluorescent carbon dots changes with the activity of α-glucosidase in the range of 2 to 1200 UL. -1 There is a good linear relationship within the range.

4. The use according to claim 2, characterized in that: The ascorbic acid recovery time was selected to be 15 minutes, and the incubation temperature was 37°C.

Images