A fluorescent silicon dot, a fluorescent silicon dot preparation method, a pH detection method and a biosensor

Abstract

The fluorescent silicon dots and the preparation method thereof are provided, wherein DAMO is used as a silicon source, amoxicillin is used as a reactant, and a microwave-assisted synthesis method is used to synthesize the fluorescent silicon dots with double emission centers; the fluorescent silicon dots have rich surface functional groups such as hydroxyl groups, carboxyl groups and amino groups, have good stability and biocompatibility, can realize pH detection, and have high detection precision and a wide precision range; the application further provides a biosensor comprising the fluorescent silicon dots; the fluorescent silicon dots are combined with urea hydrolysis-mediated pH change to perform urease detection, and the detection is more sensitive; in addition, the double emission centers of the fluorescent silicon dots are successfully used to perform pH monitoring on living HeLa cells, and the detection precision is high.

Description

Technical Field

[0001] This application relates to the field of biosensing technology, and in particular to a fluorescent silicon dot, a method for preparing the fluorescent silicon dot, a pH detection method, and a biosensor. Background Technology

[0002] pH is crucial for the normal morphology and function of cells and organisms. Abnormal intracellular pH can affect signal transduction, enzyme activity, endocytosis, cell proliferation, and apoptosis, leading to a series of adverse reactions. Furthermore, abnormal pH can influence the pathological processes of certain diseases. For example, even small changes in cellular pH can affect the proliferation, metabolism, and metastasis of cancer cells. Additionally, after infection, the pH of the wound environment shifts from the slightly acidic pH (~5.5) of healthy skin to an alkaline pH (~7-9). Therefore, high-precision and wide-range pH detection methods are essential for medical diagnosis and treatment; however, meeting both requirements remains a significant challenge.

[0003] Ratio-fluorescence methods have been developed for pH detection due to their advantages such as simplicity, fast real-time response, high sensitivity, and good accuracy. Most previously developed ratio-fluorescent probes are constructed by covalently coupling two different fluorescent emitting materials, such as organic molecules, carbon dots, and semiconductor quantum dots (QDs), requiring multi-step synthesis, which reduces fluorescence efficiency and stability. Previously, internally dual-emitting CdZnSe / Mn:ZnS quantum dots were reported to have the potential for intracellular pH detection. However, the presence of heavy metals in semiconductor quantum dots severely limits their biological applications. Summary of the Invention

[0004] Therefore, it is necessary to provide a fluorescent silicon dot, a method for preparing the fluorescent silicon dot, a pH detection method, and a biosensor that can achieve high-precision, wide-range pH detection, addressing the current technical problem of low pH detection accuracy.

[0005] To solve the above problems, this application adopts the following technical solution:

[0006] One objective of this application is to provide a method for preparing fluorescent silicon dots, comprising the following steps:

[0007] Fluorescent silicon dots with dual emission centers were synthesized using DAMO as the silicon source and amoxicillin as the reactant via microwave-assisted synthesis. The fluorescent silicon dots possess surface functional groups, including at least one of hydroxyl, carboxyl, or amino groups. Specifically, the volume-to-mass ratio of DAMO to amoxicillin was 10:7–10:100 (L / g).

[0008] In some embodiments, the fluorescent silicon dot has two emission centers at 427 nm and 500 nm, respectively.

[0009] In some embodiments, the color of the fluorescent silicon dots changes from light green to blue when the ambient pH increases from 7.0 to 10.0, and changes from blue to dark green when the ambient pH increases from 10.0 to 12.0.

[0010] The second objective of this application is to provide a fluorescent silicon dot, which is prepared by any of the preparation methods described herein.

[0011] The third objective of this application is to provide a pH detection method, comprising the following steps:

[0012] Fluorescent silicon dots with dual emission centers were synthesized using DAMO as the silicon source and amoxicillin as the reactant via microwave-assisted synthesis. The fluorescent silicon dots have surface functional groups, including at least one of hydroxyl, carboxyl, or amino groups. The fluorescent silicon dots can be used for pH detection.

[0013] In some embodiments, the fluorescent silicon dot has two emission centers at 427 nm and 500 nm, respectively.

[0014] In some embodiments, the color of the fluorescent silicon dots changes from light green to blue when the ambient pH increases from 7.0 to 10.0, and changes from blue to dark green when the ambient pH increases from 10.0 to 12.0.

[0015] The fourth objective of this application is to provide a biosensor, which includes the aforementioned fluorescent silicon dots.

[0016] Fifthly, this application provides an application of the aforementioned biosensor in detecting urease and / or detecting the pH of living cells.

[0017] The present application adopts the above technical solution, and its beneficial effects are as follows:

[0018] The fluorescent silicon dots and their preparation method provided in this application use DAMO as the silicon source and amoxicillin as the reactant to synthesize fluorescent silicon dots with dual emission centers. The fluorescent silicon dots have abundant surface functional groups, such as hydroxyl, carboxyl, and amino groups, and exhibit good stability and biocompatibility, enabling high-precision and wide-range pH detection. This application also provides a biosensor including fluorescent silicon dots, which combines with urea hydrolysis-mediated pH changes for urease detection, resulting in more sensitive detection. Furthermore, the dual emission centers of the fluorescent silicon dots have been used to successfully monitor the pH of live HeLa cells with high detection accuracy. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The diagram illustrates the method for preparing fluorescent silicon dots and their application in the sensitive detection of urease and pH detection of live cells, as provided in the embodiments of the present invention.

[0021] Figure 2 This is a schematic diagram illustrating the preparation and characterization of fluorescent silicon dots with dual emission centers, provided in an embodiment of the present invention.

[0022] Figure 3 This is a schematic diagram of two emission centers of a silicon dot being compared, provided as an embodiment of the present invention.

[0023] Figure 4 This is a high-resolution XPS spectrum diagram of a silicon dot provided in an embodiment of the present invention.

[0024] Figure 5 Synchronous fluorescence spectra of fluorescent silicon dots at different Stokes shifts provided in embodiments of the present invention.

[0025] Figure 6 A schematic diagram illustrating the stability of silicon dots provided in an embodiment of the present invention.

[0026] Figure 7 This is a schematic diagram of the fluorescence response of silicon dots to pH provided in an embodiment of the present invention.

[0027] Figure 8 The schematic diagram of the fluorescence response mechanism of silicon dots to pH is provided for the embodiment of the present invention, which uses (A) UV-Vis absorption spectra and (B) surface potential of silicon dots.

[0028] Figure 9 For embodiments of the present invention, at different pH values ​​(5, 9, 12), (A) 427 nm and (B)

[0029] A schematic diagram of fluorescence lifetime decay curve at 500nm.

[0030] Figure 10 This is a schematic diagram of detecting urease using pH-responsive silicon dots, provided in an embodiment of the present invention.

[0031] Figure 11 This is a schematic diagram illustrating pH monitoring of HeLa cells using dual-emission silicon dots, as provided in an embodiment of the present invention.

[0032] Figure 12This is a schematic diagram illustrating the activity study of cells treated with different concentrations of silicon dots for 2 hours, as provided in an embodiment of the present invention.

[0033] Figure 13 This is a schematic diagram illustrating the concentration optimization of silicon dots for pH imaging, provided in an embodiment of the present invention. Detailed Implementation

[0034] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0035] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0036] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0037] This application employs a microwave-assisted synthesis method, using DAMO as the silicon source and amoxicillin as the reactant, to synthesize fluorescent silicon dots with dual emission centers. These fluorescent silicon dots also possess abundant surface functional groups, such as hydroxyl, carboxyl, and amino groups, which can be easily modulated for pH detection.

[0038] Please see Figure 1 The diagrams below illustrate the preparation method of fluorescent silicon dots provided in this application and their application in sensitive detection of urease and pH detection in live cells. (A) shows the preparation of pH-responsive dual-emission silicon dots using amoxicillin and DAMO. (B) shows the detection of urease using pH-responsive silicon dots via urease-mediated urea hydrolysis. (C) shows the use of dual-emission silicon dots for pH monitoring in live cells.

[0039] from Figure 1 As shown in A, when the pH increases from 7.0 to 10.0, the color of the prepared silicon dots changes from light green to blue, and when the pH increases from 10.0 to 12.0, the color of the silicon dots changes from blue to dark green.

[0040] Based on this, this application also provides a pH detection method based on fluorescent silicon dots. For example... Figure 1 As shown in Figure B, the pH-responsive dual-emission silicon dots identify and detect the urease-catalyzed hydrolysis of urea to produce NH4. + The method mediates an increase in pH to indirectly detect urease, with a detection limit of 0.28 U / L.

[0041] Furthermore, silicon dots exhibit high stability and biocompatibility, and pH changes in live HeLa cells were successfully monitored using confocal imaging, such as... Figure 1 As shown in Figure C, when the pH of HeLa cells increased from 7.0 to 10.0, the blue fluorescence of the silicon dots was significantly enhanced, and the color of the silicon dots changed from green to blue.

[0042] Therefore, internal dual-emission silicon dots can provide a simple and versatile platform for developing pH-related biosensors and bioimaging applications.

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0044] Example

[0045] 1. Experimental Section

[0046] 1.1 Preparation of silicon dots

[0047] In one synthesis system, 125 μL of amoxicillin (100 mM) and 200 μL of DAMO were mixed with ddH2O to a final 1000 μL solution, which was then transferred to a special glass vial in a microwave synthesis system. The microwave synthesizer program was set as follows: pre-stirring for 2 min, reaction temperature 175 °C, reaction time 20 min, to prepare silicon dots. Subsequently, the prepared silicon dots were mixed with acetonitrile at a volume ratio of 1:4, the precipitate was extracted, and then centrifuged at 8000 rpm for 15 min. The supernatant was discarded, and the mixture was washed twice in the same manner. The purified silicon dot crystals were dried in an oven at 70 °C for approximately 48 hours and stored at 4 °C for further experiments.

[0048] 1.2 pH detection

[0049] First, silicon dots (0.1 mg / mL) were added to a pH solution with a range of 6-12, defined by an electronic pH meter, with intervals of 0.5. Synchronous fluorescence spectra of the silicon dots were acquired at excitation wavelengths of 310–450 nm. Simultaneously, the response stability of the pH sensor was investigated by collecting the synchronous fluorescence spectra of the silicon dots.

[0050] 1.3 Cytotoxicity Studies

[0051] The cytotoxicity of silicon dots was assessed using the Super-Enhanced Cell Counting Kit-8 (CCK-8). HeLa cells were first counted in 96-well plates at 5 × 10⁶ cells / well. 3 Cells were incubated at a density of cells / well for 24 hours. Subsequently, they were incubated with 100 μL of culture medium containing different silicon dot concentrations (0, 10, 25, 50, 100, 150, 100, and 200 μg / mL). After 24 hours, the supernatant was collected and incubated with 10 μL of Super-Enhanced Cell Counting Kit-8 solution for 30 minutes. The absorbance at 450 nm was measured using a BioTek microplate reader.

[0052] 1.4 pH monitoring and imaging

[0053] HeLa cells were incubated with different concentrations of silica dots (0, 50, 100, and 250 μg / mL) for 24 hours (37°C, 5% CO2), then the supernatant was discarded, and the cells were washed three times with PBS. For optimal silica dot utilization, fluorescence imaging of the cells was performed using a confocal microscope equipped with 405 nm and 488 nm lasers. In pH imaging, HeLa cells were treated with 100 μg / mL silica dots for 24 hours (37°C, 5% CO2). The cells were then washed three times with PBS and incubated with PBS buffer containing 100 μM nigericin at different pH values ​​(6.0, 7.0, 8.0, 9.0, and 10.0) for 30 min. Fluorescence imaging of the cells was performed using a confocal microscope equipped with 405 nm and 488 nm lasers. The location of the silica dots in the cells was then determined after HeLa cells were initially incubated with 100 μg / mL silica dots for 24 hours (37°C, 5% CO2). Cells were then washed three times with PBS; for cell membrane staining, cells were incubated with Vybrant DiD cell labeling solution (1:200 dilution) at 37°C for 8 min; for nuclear staining, cells were incubated with SYTO 82 staining solution (5 μM) at a 1:1000 dilution at 37°C for 10 min; for lysosomal staining, cells were incubated with Lyso-Tracker Red staining solution (1 mM) at a 1:2000 dilution at 37°C for 10 min; for endoplasmic reticulum (ER) staining, cells were incubated with ER-Tracker Red at a 1:1000 dilution at 37°C for 15 min. Fluorescence images of the cells were captured using a confocal microscope equipped with 405 nm, 488 nm, and 561 nm lasers.

[0054] 1.5 Determination of urease activity

[0055] To complete the urea hydrolysis reaction, different concentrations of urease were added to a 500 μL mixed solution containing 50 μL of urea (500 mM) and incubated at 37 °C for 60 min. Then, 1 μL of silica dot solution (50 mg / mL) at pH 7 was added to the mixture, and synchronous fluorescence spectra were measured at excitation wavelengths of 310–450 nm.

[0056] Please see Figure 2 This embodiment describes the preparation and characterization of fluorescent silicon dots with dual emission centers. (A) Fluorescence emission spectrum of silicon dots at excitation wavelengths of 320-460 nm. (B) UV-Vis absorption spectrum (green line) and fluorescence excitation spectrum of silicon dots at 427 nm (red line) and 500 nm (blue line). (C) TEM image of silicon dots. Scale bar: 10 nm. Insert: Size distribution of silicon dots. (D) XRD analysis of silicon dots. (E) Infrared spectra of DAMO, amoxicillin, and the prepared silicon dots. (F) XPS analysis of silicon dots.

[0057] 2. Experimental Results

[0058] 2.1 Preparation and Characterization of Silicon Dots

[0059] Please see Figure 3 , which means that the preparation conditions of silicon dots were optimized by comparing the two emission centers of silicon dots, where (A) is the concentration of amoxicillin, (B) is the preparation time and (C) is the preparation temperature.

[0060] Please see Figure 4 , representing the high-resolution XPS spectra of silicon points, including (A) C1s, (B) N1s, (C) O1s, and (D) Si2p. Peak sums are the sums of peak values ​​obtained by fitting the original data.

[0061] Please see Figure 5 The denoted is the synchronous fluorescence spectrum of the fluorescent silicon dots at different Stokes shifts. Silicon dots were prepared using a microwave-assisted method with DAMO and amoxicillin. The final optimized conditions for silicon dot preparation were: 12.5 mM amoxicillin concentration (…). Figure 3 (A), the reaction temperature is 200°C. Figure 3 (B), reaction time is 20 min ( Figure 3 (C)

[0062] To investigate the optical properties of silicon dots, their fluorescence and UV-Vis absorption spectra were measured. The fluorescence emission of the silicon dots was related to the excitation wavelength, with two main emission centers observed at 427 nm and 500 nm. Figure 2 (A) Figure 2The fluorescence excitation spectrum in B shows that the optimal emission at 427 nm is achieved by excitation at 347 nm, while the optimal emission at 500 nm is achieved by excitation at both 347 nm and 420 nm.

[0063] It is understandable that, since the Stokes shifts of the two emission centers are similar, this embodiment performed synchronous fluorescence spectroscopy analysis on the silicon dot, obtaining two emission peaks with good resolution. The fluorescence intensity was highest when the difference was 80 nm, corresponding to the Stokes shift. Figure 4 The ultraviolet-visible spectrum of silicon dots ( Figure 2 In sample B), an absorption peak is observed at approximately 350 nm, belonging to the n-π* transition of amide and carboxyl groups. This peak also overlaps well with the excitation spectrum emitted by the silicon dot at 427 nm. Therefore, the emission at 427 nm originates from the surface of the silicon dot. Subsequently, the morphology and size of the silicon dot were characterized using transmission electron microscopy. Figure 2 As shown in Figure C, the silicon dots are nearly spherical, uniformly distributed, and have an average diameter of 2.35 nm. The XRD pattern of the silicon dots shows only one broad peak at 21.8°, indicating the amorphous structure of the silicon dots. Figure 2 (D). Infrared spectroscopy and XPS measurements were used to characterize the chemical bonds and elemental composition of the silicon dot surface. At 1130 cm⁻¹ -1 and 1642cm -1 The absorption peaks on the left and right are due to the Si-O stretching vibration and the NH bending vibration, respectively. Figure 2 (E). At 2934cm -1 The characteristic peak at 3439 cm⁻¹ is related to the CH stretching vibrations of -CH₂ and -CH₃. Additionally, the peak at 3439 cm⁻¹... -1 The strong characteristic peaks at the silicon dot are attributed to the stretching vibrations of NH and OH. These results preliminarily indicate the presence of amino and hydroxyl groups on the silicon dot surface. Furthermore, XPS was used to further characterize the elemental composition and chemical bonds in the silicon dot structure. Figure 2 China F and Figure 5 The results show that the silicon dots contain C, N, O, S, and Si elements. Figure 2 Further investigation was conducted using XPS high-resolution spectra of C, N, O, and Si elements, revealing the presence of amino and hydroxyl groups on the surface of silicon dots.

[0064] 2.2 Stability Study of Silicon Points

[0065] Please see Figure 6 This study investigates the stability of the silicon dots prepared in this embodiment. (A) Photostability of the silicon dots at emission wavelengths of 427 nm (Ex = 347 nm) and 500 nm (Ex = 420 nm), and photostability of FITC at an emission wavelength of 520 nm (Ex = 490 nm). (B) Fluorescence intensity ratio of different ions (100 μM) on the silicon dots (F... 347 / F420 (C) The ratio of fluorescence intensity of silicon dots to different amino acids and small biomolecules (100 μM) (F) 347 / F 420 (D) The fluorescence intensity of silicon dots stored at 4℃ for different times was compared with (F) 347 / F 420 ) changes.

[0066] Prior to the biomedical application of silicon dots, their photostability, storage stability, and chemical stability were tested. After 2 hours of continuous light irradiation, the fluorescence intensity of both emission centers of the silicon dots remained near 100%. Figure 6 The fluorescence concentration of silicon dots was higher than that of FITC (35%), indicating that the silicon dots possess good photostability. Subsequently, the chemical stability of the silicon dots was also tested; most metal ions and small molecules had little effect on the fluorescence of the silicon dots, demonstrating their good chemical stability. Figure 6 (Central BC). Furthermore, the fluorescence intensity of the silicon dots showed almost no decrease after being stored at 4°C for one month. Figure 6 The presence of D indicates good storage stability. These findings collectively confirm the excellent stability of the prepared silicon dots, meaning they can be used for complex matrix analyses.

[0067] 2.3 pH response of silicon points

[0068] Figure 7 The fluorescence response of silicon dots to pH is shown. (A) Fluorescence emission spectra of silicon dots at different pH values ​​under excitation at 347 nm and 420 nm. (B) Synchronous fluorescence spectra of silicon dots at pH 6.0–9.5. (C) Fluorescence intensity ratio (F) 347 / F 420 (A) Linear relationship between pH and pH range 7.0–9.5. (D) Synchronous fluorescence spectra of silicon points at pH 10.0–12.5. (E) Fluorescence intensity ratio (F) 347 / F 420 (F) Linear relationship between pH value and pH range of 10.0–12.0. Reversible pH response of silicon points in three cycles from pH 5 to 9 to 12.

[0069] Fluorescence pH response analysis was performed using the prepared dual-emission fluorescent silicon dots. When the pH increased from 6.0 to 12.0, the fluorescence intensity at 500 nm of the silicon dots only increased slightly. Figure 7 (A); however, the fluorescence intensity at 427 nm of the silicon dot increased with increasing pH from 6.0 to 10.0, but decreased rapidly with increasing pH from 10.0 to 12.0. Figure 7(A). In synchronous fluorescence mode, as the pH increased from 6.0 to 9.5, the fluorescence intensity of the silicon spot at excitation at 347 nm increased, while it remained almost unchanged at 420 nm. Figure 7 (B). Therefore, the ratio of pH to synchronous fluorescence intensity (F) 347 / F 420 A linear relationship was established () Figure 7 In the case of C), the linear equation is: F 347 / F 420 = 1.257 * pH - 8.499. Additionally, as the pH increases from 10.0 to 12.0, the fluorescence enhancement at 347 nm of the silicon dot decreases, while the fluorescence enhancement at 420 nm increases. Figure 7 (middle D), synchronous fluorescence intensity ratio (F) 347 / F 420 The relationship between pH and the increase of synchrotron fluorescence intensity (F) was negatively correlated, and the relationship between pH and the ratio of synchrotron fluorescence intensity (F) was also negatively correlated. 347 / F 420 There is a good linear relationship between them, and the equation is: F 347 / F 420 = -1.560 * pH + 18.85 Figure 7 (E). We then investigated the reversibility of using silicon dots for pH detection. The results showed that the fluorescence intensity ratio of the silicon dots remained stable after three cycles of acidic and alkaline pH. Figure 7 The results (F) indicate that the silicon dots exhibit good reversibility in pH detection. All the above findings confirm that the prepared silicon dots can serve as an ideal optical material for rapid, sensitive, and stable pH detection.

[0070] 2.4 Fluorescence response mechanism of silicon dots to pH

[0071] Please see Figure 8 The fluorescence response mechanism of silicon dots to pH was studied using (A) UV-Vis absorption spectra and (B) surface potential of silicon dots.

[0072] Please see Figure 9 The figures represent the fluorescence lifetime decay curves at different pH values ​​(5, 9, 12) for (A) 427 nm and (B) 500 nm.

[0073] Consistent with the change in fluorescence intensity at 427 nm, the UV-Vis absorption spectra of silicon dots at different pH values ​​show that the absorption at 350 nm gradually increases from pH 5.0 to 9.0, and gradually decreases from pH 9.0 to 12.0. Figure 8 Therefore, the fluorescence quenching and recovery of silicon dots at 427 nm can be attributed to changes in absorption. We investigated the time-resolved fluorescence decay behavior of silicon dots at different pH values ​​and found that the average fluorescence lifetime of silicon dots at 427 nm was almost independent of pH. Figure 8Based on the absorption spectrum and time-resolved fluorescence decay (see Table A and Table 1), we hypothesize that the fluorescence response of the silicon spot at 427 nm to pH is due to absorption changes caused by protonation and deprotonation. Furthermore, the zeta potential of the silicon spot gradually decreases from 33.03 mV to -3.21 mV as pH increases from 5.0 to 12.0. Figure 8 (B) further illustrates the protonation and deprotonation of hydroxyl and amino groups. Furthermore, the average fluorescence lifetime of the silicon dot at 500 nm increases with increasing pH. Figure 9 (See Table B and Table 2) to show that a decrease in pH will lead to dynamic quenching. At 500 nm, the decrease in fluorescence intensity caused by a decrease in pH may be due to the increased intramolecular electron transfer ability between silicon dots and amino groups.

[0074] Table 1 shows the fluorescence lifetime emitted by silicon dots at 427 nm at different pH values.

[0075] pH 5 2.4952 86.16 11.8606 13.84 3.7914 pH 9 2.5558 87.81 12.8974 12.19 3.8164 pH 12 2.7675 82.29 10.7494 17.71 4.1811

[0076] Table 2 shows the fluorescence lifetime of silicon dots at 500 nm under different pH values.

[0077] pH 5 2.6667 78.31 8.9749 21.69 4.0349 pH 9 3.5884 89.76 12.5937 10.24 4.5105 pH 12 4.3145 91.44 13.3013 9.56 5.1736

[0078] in: [a] Fluorescence lifetime. [b] Fraction contribution. [c] Weighted mean fluorescence lifetime

[0079] 2.5 Urease Activity Detection

[0080] Urease is an enzyme that specifically catalyzes the hydrolysis of urea and is distributed in various biological systems, such as animal blood and urine, microorganisms, and plants. Bacterial urease is associated with a variety of human diseases, including infectious stones and peptic ulcers. This embodiment establishes a simple and sensitive sensing platform to detect urease activity. The catalytic decomposition of urea into ammonium carbonate by urease increases the pH of the system; this phenomenon can be monitored using the pH-responsive silicon dots prepared in this application.

[0081] Figure 10This is a schematic diagram of the pH-responsive silicon dot detection of urease in this embodiment. (A) Fluorescence intensity ratio of silicon dots after reaction with urease at different concentrations of urea. Reaction time: 60 min. (B) Fluorescence kinetics of silicon dots at 427 nm emission for different concentrations of urease (15, 50, and 100 U / L). Urea = 50 mM, excitation wavelength = 340 nm. F0 and F represent the fluorescence intensity at 427 nm before and after the addition of urease, respectively. (C) Synchronous fluorescence spectra of silicon dots responding to different concentrations of urease. Urea: 50 mM, reaction time: 60 min. (D) Calibration curve for urease detection. (E) Selectivity of urease detection. Urea: 20 U / L. The concentration of interfering protein is 10 times that of urease. (F) Comparison of urease detection results in ddH2O and 1% human oral extract.

[0082] To obtain the maximum fluorescence change in urease detection, the amount of urea used and the reaction time were first optimized. The optimal conditions were selected as 50 mM urea and 60 min reaction time for the next experiment. Figure 10 (AB). As the urease concentration increased from 0 to 100 U / L, the synchronous fluorescence of the silicon dots gradually recovered at 347 nm (F). 347 ), at 420nm (F 420 ) remains unchanged, fluorescence intensity ratio (F 347 / F 420 ) gradually increase ( Figure 10 (C) F 347 / F 420 There is a good linear relationship between the concentration of urease and the concentration of urease in the range of 1-80 U / L. Figure 10 In the middle (D), the linear equation is: F 347 / F 420 =0.03403*C Urease +0.7028, with a detection limit of 0.28 U / L, lower than most previous reports (Table S3). Selectivity experiments showed that interfering proteins at concentrations 10 times higher than urease, including horseradish peroxidase (HRP), acetylcholinesterase (Ache), lysozyme (Lys), and BSA, did not affect the fluorescence signal of the silicon dots. Figure 10 E). The anti-interference ability of silicon dots was further evaluated by detecting urease in oral extracts. The linear relationship between the urease detection of oral extracts and ddH2O was similar ( Figure 10 The result (F) indicates that our method is highly feasible in the detection of urease in real samples.

[0083] Table 3 compares the previously reported methods for detecting urease with the method used in this study.

[0084]

[0085] 2.6 Detection of Intracellular pH Changes Using Silicon Dots

[0086] Please see Figure 11 To monitor the pH of HeLa cells using dual-emission silicon dots. (A) Confocal microscopy images of HeLa cells treated with 100 μg / mL silicon dots for 30 min at pH 6-10. Scale bar: 20 μm. (B) Linear relationship between the fluorescence intensity ratio (B / G) of silicon dots and pH. (C) Colocalization coefficients of different organelles in HeLa cells stained with conventional dyes and the newly developed silicon dots, including (1) nucleus, (2) endoplasmic reticulum (ER), (3) cell membrane, and (4) lysosome. (D) Confocal microscopy colocalization images of HeLa cells stained with conventional dyes and the newly developed silicon dots. Scale bar: 20 μm. Cells were incubated with silicon dots and stained with (a) SYTO 82 for the nucleus, (b) ER-Tracker Red for the ER, (c) DiD for the cell membrane, and (d) Lyso-Tracker Red for the lysosome.

[0087] Please see Figure 12 This is a schematic diagram illustrating the activity study of cells after treatment with different concentrations of silicon dots for 2 hours.

[0088] Please see Figure 13 The diagram illustrates the concentration optimization of silicon dots for pH imaging. Confocal microscopy images of HeLa cells treated with different concentrations of silicon dots (0, 50, 100, 250 μg / mL) for 30 min are shown. Scale bar: 20 μm.

[0089] This embodiment uses the standard CCK-8 assay to evaluate the cytotoxicity of silicon dots in HeLa cells. Within the concentration range of 10–200 μg / mL, the cytotoxicity of silicon dots to HeLa cells is negligible. Figure 12 These concentrations of silicon dots can be used for further fluorescence imaging. We then observed the pH fluctuations in cells monitored by the silicon dots using confocal microscopy. First, the concentration of silicon dots was optimized to obtain the best fluorescence imaging results; when the concentration of silicon dots increased from 0 to 200 μg / mL, the intensities of both the blue and green channels increased. Figure 13 Considering the results of the cytotoxicity assay and for optimization purposes, a silicon dot concentration of 100 μg / mL was subsequently selected for cell pH monitoring. HeLa cells were incubated with silicon dots for 2 hours, followed by treatment with PBS containing 100 μM nigericin. As the pH increased (from 6.0 to 10.0), the fluorescence of the blue channel increased, while the fluorescence of the green channel remained almost unchanged. Figure 11 In the merged channel (A), the cell color changes from green to blue. Figure 11Figure B shows the linear relationship between the intensity ratio of blue-green fluorescence (B / G) in HeLa cells and pH. The intensity ratio of blue-green fluorescence (B / G) gradually increases with increasing intracellular pH, and the linear equation is: B / G = 0.4976 * pH - 2.424, with a ΔpH of 0.1. These results indicate that dual-emission silicon dots can be applied to a wide range and high precision of intracellular pH measurements.

[0090] The localization of silicon dots in cells was determined by staining the cell membrane, nucleus, lysosomes, and endoplasmic reticulum (ER) with commercial reagents, including Vybrant DiD cell-labeling solution, the SYTO 82 staining solution, Lyso-Tracker Red, and ER-Tracker Red. The cell membrane and lysosomes showed the highest Pearson coefficients, at 0.385 and 0.605, respectively, while the nucleus and ER showed Pearson coefficients close to 0. Figure 11 C and Figure 11 The data (D) indicates that most silicon dots are distributed in the cell membrane and lysosomes.

[0091] In summary, this application provides a novel method for pH detection using fluorescent silicon dots with internal dual emission centers. The prepared silicon dots possess abundant surface groups, including -OH, -COOH, and -NH2, exhibiting good stability and biocompatibility, enabling high-precision and wide-range pH detection. The high-precision pH-responsive silicon dots were combined with urea hydrolysis-mediated pH changes for urease detection, achieving a linear concentration range of 1–80 U / L and a LOD of 0.28 U / L, which is more sensitive than most previously reported methods. Furthermore, high-precision pH monitoring of live HeLa cells was successfully performed using the two bright fluorescence emissions of the silicon dots. Besides providing a new strategy for high-precision and wide-range pH detection, this application also opens new horizons for constructing more sensitive biosensors and ratio determination techniques for bioimaging applications.

[0092] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A method for preparing fluorescent silicon dots, characterized in that, Includes the following steps: 125 μL of 100 mM amoxicillin and 200 μL of DAMO were mixed with ddH2O to a final 1000 μL solution, which was then transferred to a special glass bottle in a microwave synthesis system. The microwave synthesizer program was set as follows: pre-stirring for 2 min, reaction temperature 175 °C, and reaction time 20 min, to obtain fluorescent silicon dots with dual emission centers. The fluorescent silicon dots have surface functional groups, which include at least one of hydroxyl, carboxyl, or amino groups. The fluorescent silicon dots have two emission centers at 427 nm and 500 nm, respectively; When the ambient pH increases from 7.0 to 10.0, the color of the fluorescent silicon dots changes from light green to blue; when the ambient pH increases from 10.0 to 12.0, the color of the fluorescent silicon dots changes from blue to dark green.

2. A fluorescent silicon dot, characterized in that, It is prepared by the preparation method described in claim 1.

3. The application of the fluorescent silicon dot as described in claim 2 in a pH detection method.

4. A biosensor, characterized in that, The biosensor includes the fluorescent silicon dot as described in claim 2.

5. The use of the biosensor of claim 4 in detecting urease and / or detecting pH in living cells.

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