Ratio fluorescence sensing system and preparation method and application thereof

Abstract

The invention provides a ratiometric fluorescence sensing system and a preparation method and application thereof, and the preparation method comprises the following steps: respectively preparing a silicon oxide-based aggregation-induced emission material and a copper-doped carbon dot material, and mixing the silicon oxide-based aggregation-induced emission material and the copper-doped carbon dot material with an ascorbic acid-2-phosphoric acid material to construct the ratiometric fluorescence sensing system. The fluorescent probe is applied to ferric ion detection, in-vitro detection of biomarkers or fluorescence imaging. The technical scheme provided by the invention has good stability, accuracy and practical application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of detection and sensing materials, and more particularly to a ratiometric fluorescence sensing system and its preparation and application. Background Art

[0002] A variety of analytical methods, including atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), surface-enhanced Raman scattering (SERS), and electrochemical determination, have been reported for the measurement of disease markers. These techniques generally involve complex operating procedures, professional technicians, and long detection times.

[0003] In recent years, fluorescence detection technology has attracted much attention due to its high sensitivity, cost-effectiveness, and simple operation. However, most detection probes only exhibit a single-color fluorescence signal, and the inherent background fluorescence and complex detection environment may lead to insufficient accuracy of results, poor reproducibility, and difficulty in visual discrimination by the naked eye. In contrast, ratiometric fluorescence sensing systems have dual-emission fluorescence signals and built-in self-calibration functions, which can improve the accuracy, reliability, and signal-to-noise ratio of quantitative detection results. In addition, their fluorescence colors are easily changed during the detection process, with a wide range of color gamut changes that can be distinguished by the naked eye, endowing the probes with the ability of visual detection.

[0004] Among them, the selection of a reference fluorescence probe is the key to constructing an ideal ratiometric fluorescence sensing system. Traditional fluorescent dyes have the phenomenon of aggregation-caused quenching, with single functions, fast photobleaching rates, and small Stokes shifts, which have great limitations in constructing dual fluorescence sensors. Aggregation-induced emission (AIE) molecules overcome the limitations of traditional dyes, with high emission efficiency, large Stokes shifts, and low background noise, and have significantly enhanced detection performance in the aggregated state. However, the water solubility and aggregation controllability of AIE molecules in complex physiological environments are insufficient at present. Summary of the Invention

[0005] Aiming at the problems in the prior art, the purpose of the present invention is to provide a novel ratiometric fluorescence sensing system and its preparation method and application.

[0006] On the one hand, the present invention provides a ratiometric fluorescence sensing system, including a reference probe and a response probe, wherein the reference probe is a silica-based aggregation-induced emission material, and the response probe is a copper-doped carbon dot material.

[0007] Preferably, the mass ratio of the silica-based aggregation-induced emission material to the copper-doped carbon dot material is 3.70 - 25.93:1.

[0008] Preferably, it further includes a detection medium, and the detection medium is ascorbic acid-2-phosphate material, which enhances the fluorescence performance of the response probe copper-doped carbon dots.

[0009] Preferably, the g / mol value of the copper-doped carbon dot material and the ascorbic acid-2-phosphate material is 16.2 g / mol, which is the ratio of the concentration of the copper-doped carbon dot material in mg / ml to the concentration of the ascorbic acid-2-phosphate material in mM.

[0010] Preferably, the silica-based aggregation-induced emission material is an aggregation-induced emission material loaded on silica, and the structural formula of the aggregation-induced emission material is:

[0011]

[0012] On the other hand, the present invention provides a method for preparing the above ratio fluorescence sensing system, including:

[0013] Step 1, respectively prepare a silica-based aggregation-induced emission material and a copper-doped carbon dot material;

[0014] Step 2, mix the silica-based aggregation-induced emission material, the copper-doped carbon dot material and the ascorbic acid-2-phosphate material to construct a ratio fluorescence sensing system.

[0015] Preferably, in Step 1, porous silica is synthesized by a one-pot method and dispersed in dimethyl sulfoxide, and then a molecular working solution of the aggregation-induced emission material is added and stirred at room temperature to obtain the silica-based aggregation-induced emission material.

[0016] Preferably, in Step 1, the copper-doped carbon dot material is prepared by a hydrothermal method.

[0017] On the other hand, the present invention provides the application of the above ratio fluorescence sensing system in the detection of ferric ions.

[0018] On the other hand, the present invention provides the application of the above ratio fluorescence sensing system in the in vitro detection or fluorescence imaging of biomarkers.

[0019] The beneficial effects of the technical solution of the present invention are as follows:

[0020] 1. The present invention proposes a novel method for constructing a ratio fluorescence sensing system based on an aggregation-induced emission material. By adopting a proportional signal response mode, the accuracy of the detection result is significantly improved, with a fast response speed (2 min), a wide linear response range (2 - 400 μM), and a low detection limit for Fe 3+ (1.53 μM).

[0021] 2. The ratio fluorescence sensing system obtained by the present invention can produce rich changes in fluorescent colors recognizable by the human eye within a wider color gamut and has visual sensing ability.

[0022] 3. The ratio fluorescence sensing system obtained by the present invention has a good detection effect on Fe in actual human serum samples 3+The detection also has good linearity and acceptable sensitivity.

[0023] 4. The construction method of the ratiometric fluorescence sensing system of the present invention provides a valuable reference for the design of dual-signal detection systems and has good application potential in the field of biomarker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects, and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0025] Figure 1 It is the water contact angle diagram of the hydrophobic porous silica hMSN obtained in Example 1 of the present invention;

[0026] Figure 2 It is the TEM diagram (A) of the silicon-based AIE material hMSNA obtained in Example 1 of the present invention;

[0027] Figure 3 It is the normalized fluorescence intensity bar chart of the silicon-based AIE material hMSNA loaded with different volumes of AIE molecules obtained in Example 1 of the present invention;

[0028] Figure 4 It is the fluorescence spectrum diagram (excitation light is 545 nm) of the silicon-based AIE material hMSNA obtained in Example 1 of the present invention, and the inset is the digital photo of the sample under daylight and ultraviolet light;

[0029] Figure 5 It is the TEM diagram (A) and fluorescence spectrum diagram (B) (excitation light is 371 nm) of the copper-doped carbon dots Cu-CDs obtained in Example 2 of the present invention, and the inset is the digital photo of the sample under daylight and ultraviolet light;

[0030] Figure 6 It is the bar chart of the relationship between the amount of Cu-CDs adjusted during the construction of the ratiometric fluorescence sensing system in Example 3 of the present invention and the fluorescence intensity of the sample (excitation light is 371 nm, and the fluorescence intensity at 435 nm is collected) (A), the time-point line chart of the incubation time of Cu-CDs and AAP (excitation light is 371 nm, and the fluorescence intensity at 435 nm is collected) (B), and the point-line chart of the relationship between the AAP concentration and the fluorescence intensity of the Cu-CDs sample solution (excitation light is 371 nm, and the fluorescence intensity at 435 nm is collected) (C);

[0031] Figure 7 It is the fluorescence digital photo of the corresponding sample solution during the construction of the ratiometric fluorescence sensing system in Example 3 of the present invention as the volume ratio of Cu-CDs:hMSNA and the AAP concentration increase;

[0032] Figure 8It is a graph showing the change of fluorescence intensity of each sensing element in the ratio fluorescence sensing system obtained in Example 3 of the present invention with the illumination time under continuous light excitation;

[0033] Figure 9 It is the fluorescence emission spectra of Cu-CDs, Cu-CDs+AAP, Cu-CDs+hMSNA, Cu-CDs+hMSNA+AAP and hMSNA in the ratio fluorescence sensing system obtained in Example 4 of the present invention. (The excitation light is 371 nm). The inset is the corresponding fluorescence image under ultraviolet light;

[0034] Figure 10 It is an application example of using the ratio fluorescence sensing system to detect Fe 3+ in Example 5 of the present invention. Figure (A) is the fluorescence spectrum of Cu-CDs / hMSNA / AAP with the increase of Fe 3+ concentration (the excitation light is 371 nm). The inset is the corresponding fluorescence digital photo under ultraviolet light; Figure (B) is the fitting relationship diagram of Ln(I 435 / I 655 ) and Fe 3+ concentration (the excitation light is 371 nm);

[0035] Figure 11 It is the fitting relationship diagram of Ln(I 3+ / I 435 / I 655 ) and Fe 3+ concentration when using the ratio fluorescence sensing system to detect Fe

[0036] Figure 12 in real human serum samples in Example 6 of the present invention (the excitation light is 371 nm); 3+ It is when using a single-color fluorescence probe to detect Fe 3+ in Comparative Example 1. Figure (A) is the fluorescence spectrum of Cu-CDs / AAP with the increase of Fe 435 concentration (the excitation light is 371 nm). The inset is the corresponding fluorescence digital photo under ultraviolet light; Figure (B) is the fitting relationship diagram of Ln(I 3+ ) and Fe concentration (the excitation light is 371 nm).

[0037] Now, the exemplary embodiments will be described more fully with reference to the accompanying drawings. However, the exemplary embodiments can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. Identical reference numerals in the figures denote identical or similar structures, and thus their repetitive description will be omitted.

[0038] In the embodiments of the present invention, it is found through research that the porous silica material has excellent colloidal stability, rich pore structures, and active sites that are easy to modify. By integrating the silica matrix with AIE molecules, the prepared silica-based AIE material not only combines the advantages of inorganic materials such as controllable morphology and excellent water dispersibility, but also inherits the unique fluorescence properties of AIE molecules, having a wide excitation region, enabling it to balance the excitation characteristics of multiple other fluorophores while maintaining its own fluorescence intensity. Therefore, the silica-based AIE material becomes an ideal choice for constructing a dual-emission ratio fluorescence sensing system.

[0039] For this reason, the embodiments of the present invention provide a ratio fluorescence sensing system, its preparation method and application. A ratio fluorescence sensing system is designed and constructed by using fluorescent carbon dots and silica-based AIE materials for the detection of biochemical indicators such as Fe 3+ , realizing early screening.

[0040] A ratio fluorescence sensing system according to an embodiment of the present invention includes a reference probe, a response probe, and a detection medium. The reference probe is a silica-based aggregation-induced emission material, the response probe is a copper-doped carbon dot material, and the detection medium is an ascorbic acid-2-phosphate material.

[0041] The silica-based AIE material of the reference probe, the copper-doped carbon dots of the response probe, and the ascorbic acid-2-phosphate of the detection medium coexist in the sensing system in a physically mixed manner.

[0042] The mass ratio of the silica-based aggregation-induced emission material to the copper-doped carbon dot material is 3.70 - 25.93:1.

[0043] The g / mol value of the copper-doped carbon dot material to the ascorbic acid-2-phosphate material is 16.2.

[0044] The silica-based aggregation-induced emission material is an aggregation-induced emission material loaded on silica. The hydrophobic porous silica loads AIE molecules through hydrophobic and electrostatic interactions.

[0045] The AIE molecule has a positive charge property and a hydrophobic group, emits red fluorescence, and its structural formula is:

[0046]

[0047] The preparation method of the ratio fluorescence sensing system according to an embodiment of the present invention includes:

[0048] Step 1, respectively prepare the silica-based aggregation-induced emission material and the copper-doped carbon dot material;

[0049] Step 2: Mix the silica-based aggregation-induced emission (AIE) material, copper-doped carbon dots (Cu-CDs) material, and ascorbic acid-2-phosphate (AAP) material, and precisely adjust the dosage of Cu-CDs, the blending ratio of the three sensing elements of hMSNA, Cu-CDs, and AAP, and the incubation time, etc. Under the optimal conditions, a ratio fluorescence sensing system is constructed.

[0050] Among them, in the embodiment of the present invention, in Step 1, porous silica is synthesized by a one-pot method and dispersed in dimethyl sulfoxide, and then the AIE material molecular working solution is added and stirred at room temperature to obtain the silica-based AIE material.

[0051] The one-pot method is preferably using tetraethyl orthosilicate (TEOS) as the silicon source, octadecyltrimethoxysilane (ODMS) as the hydrophobic modifier and pore-forming agent, and the volume ratio of the two is 2.5:1, and ammonia water is used to adjust the alkaline environment to prepare hydrophobic porous silica.

[0052] And preferably when preparing the reference probe silica-based AIE material, the feeding mass ratio of hydrophobic porous silica to AIE molecules is 800:1 to 133:1, and stir at room temperature for 4 h.

[0053] In addition, in the embodiment of the present invention, it is preferred to prepare the copper-doped carbon dots material by a hydrothermal method in Step 1.

[0054] And preferably the hydrothermal method uses urea, resorcinol, and CuSO4·5H2O as raw materials, which are dissolved in deionized water at a molar concentration ratio of 11:12:1, the reaction temperature is 180 °C, and the reaction time is 7 h.

[0055] In addition, in the embodiment of the present invention, it is preferred that the incubation time of ascorbic acid-2-phosphate with the two probes in Step 2 is 1 to 10 min.

[0056] The preparation process, construction method, and application examples of a novel ratio fluorescence sensing system based on aggregation-induced emission materials in the embodiments of the present invention break the limitations of traditional fluorescent dyes in constructing dual-emission fluorescence sensors, solve the technical problems such as high inherent background, insufficient accuracy, difficult visual discrimination, and insufficient practicality in complex detection environments of single fluorescence detection technology, and provide a fluorescence sensor construction method with practical application potential.

[0057] The following describes the present invention with specific examples and comparative examples:

[0058] Example 1

[0059] Prepare the reference probe silica-based AIE material, and the process is as follows:

[0060] The reaction was carried out in a single-neck round-bottom flask. 5 mL of TEOS and 2 mL of C18TMS (ODMS) were added to a mixture of 142.8 mL of ethanol, 20 mL of deionized water, and 3.14 mL of ammonia water. The mixture was stirred at 30 °C for 1 h, and the product was collected by centrifugation (13500 rpm, 10 min). The mixture was washed three times with ethanol and water respectively, and then freeze-dried to obtain hydrophobic porous silica nanoparticles (hMSN). It can be seen from the BET test that the specific surface area of hMSN is 57.03 m 2 / g, and the pore diameter is 1.5 nm, confirming that hMSN has a porous structure. As can be seen from the attached Figure 1 figure, the water contact angle of hMSN is 112.4°, showing hydrophobic properties.

[0061] 20 mg of hMSN was resuspended in 1.7 mL of dimethyl sulfoxide (DMSO), ultrasonically dispersed evenly, and DMSO solutions (1 mg / mL) of AIE molecules with different volumes (25 μL, 50 μL, 75 μL, 100 μL, 150 μL) were added. The mixture was stirred at room temperature for 4 h, and the product was collected by centrifugation (14500 rpm, 6 min). After washing 3 times with ethanol and deionized water, it was dispersed in 2 mL of deionized water, and finally the reference fluorescence probe hMSNA was obtained. As can be seen from the attached Figure 2 figure, hMSNA presents a regular spherical morphology. As can be seen from the attached Figure 3 figure, the fluorescence intensity of the reference probe hMSNA gradually increases with the increase of the feeding amount of AIE molecules, indicating that hMSNA has successfully loaded AIE molecules through hydrophobic interaction, and its rich pore structure and limited pore volume can effectively limit the non-radiative transition channels of AIE molecules to achieve aggregation-induced emission. As can be seen from the attached Figure 4 figure, the regulated hMSNA emits bright red fluorescence, with high fluorescence intensity and large Stokes shift, and the best emission peak is located at 655 nm.

[0062] Example 2

[0063] The preparation of the responsive probe copper-doped carbon dots is as follows:

[0064] An aqueous deionized solution (40 mL) of resorcinol (0.12 M), urea (0.11 M), and CuSO4·5H2O (0.01 M) was ultrasonically dispersed evenly and then transferred to a stainless-steel sealed Teflon reactor and reacted at 180 °C for 420 min. The product solution was centrifuged at high speed 3 times (13500 rpm, 15 min) to remove millimeter-sized precipitates, and then a 0.22 μm needle filter was used to further remove micron-sized impurity particles in the reaction solution. It was further purified using a dialysis bag (200 Da) for 10 h. Finally, the sample in the bag was collected and quantified to obtain the responsive probe Cu-CDs. As shown in attached Figure 5 (A) and attachedFigure 5 From the characterization results of (B), it can be seen that Cu-CDs exhibit a regular spherical morphology, have good dispersibility, a size of approximately 5 nm, and Cu-CDs emit bright blue fluorescence under photoexcitation, with the optimal emission peak located at 435 nm.

[0065] Example 3

[0066] Construct a ratio fluorescence sensing system, and the process is as follows:

[0067] The ratio fluorescence sensing system in Example 3 of the present invention is composed of three sensing elements, namely hMSNA, Cu-CDs, and AAP, which are mixed in an appropriate ratio.

[0068] First, mix and incubate Cu-CDs and AAP in a volume ratio of 1:1, and dilute with deionized water. The total reaction volume is 2 mL.

[0069] Record the fluorescence spectra and the fluorescence intensity at 435 nm under different conditions, including the relationship between the dosage of Cu-CDs (10, 15, 20, 30, 40 μL, 2.7 mg / mL) and the fluorescence intensity at the same AAP concentration (1.5 mM) (attached Figure 6 (A)), the relationship between the reaction time (0 - 10 min) of the two and the fluorescence intensity (attached Figure 6 (B), the reaction time of 15 μL of Cu-CDs and 1.25 mM of AAP, with a total system volume of 2 mL), and the relationship between the dosage of AAP (0 - 1.5 mM) and the fluorescence intensity at the same Cu-CDs dosage (working concentration of 0.02 mg / mL) (attached Figure 6 (C)). From the results, it can be seen that when the dosage of Cu-CDs is 15 μL (working concentration of 0.02 mg / mL), the reaction time is 4 min, and the working concentration of AAP is 1.25 mM, the fluorescence intensity of the response probe is the strongest, which is suitable for quantitative detection.

[0070] Secondly, the chromaticity difference between the response probe and the reference probe is large. Therefore, it is crucial to further adjust the volume ratio of Cu-CDs (2.7 mg / mL) to hMSNA (10 mg / mL) in the sensing system.

[0071] When the volume ratio of Cu-CDs (2.7 mg / mL) to hMSNA (10 mg / mL) is 1:1, 1:3, 1:5, and 1:7, record the changes in the fluorescence color of the sensing system under ultraviolet light excitation in the form of digital photos, as shown in the attachment Figure 7 As shown, when the volume ratio of the two is 1:7, with the change of the concentration of the detection medium AAP, the sensing system shows the richest color changes, endowing the sensing system with the ability of visual detection.

[0072] In addition, each sensing element maintains good fluorescence stability under continuous light excitation (see Attachment Figure 8 , 1.25 mM AAP, 0.525 mg / mL hMSNA, 0.02 mg / mL Cu-CDs, 2 mL of the system, continuously excited and detected with a fluorescence spectrophotometer), ensuring the accuracy of the quantitative detection results.

[0073] Example 4

[0074] Construct a ratio fluorescence sensing system as follows:

[0075] Use the optimal detection conditions obtained in Example 3 of the present invention to construct a ratio fluorescence sensing system to achieve the analytical detection of Fe 3+ .

[0076] First, dilute Cu-CDs (2.7 mg / mL) and hMSNA (10 mg / mL) with deionized water according to a volume ratio of 1:7. For example, mix 15 μL of the Cu-CDs solution with 105 μL of the hMSNA solution and dilute to a total volume of 800 μL with deionized water to prepare a detection probe stock solution.

[0077] Then, calculate the required amount of AAP according to the g / mol value of Cu-CDs to AAP of 16.2, and control the volume ratio of the detection probe stock solution to the detection medium AAP solution to be 1:1, that is, mix 800 μL of the probe stock solution with 800 μL of the detection medium AAP solution and incubate for 4 min.

[0078] Finally, an ideal ratio fluorescence sensing system (Cu-CDs / hMSNA / AAP) is constructed.

[0079] As shown in the attachment Figure 9 , under light excitation, Cu-CDs show blue fluorescence at 435 nm, and hMSNA shows red fluorescence at 655 nm. At the same time, AAP can significantly enhance the fluorescence intensity of Cu-CDs. By adding hMSNA, the constructed system can emit two different and clearly distinguishable fluorescence signals simultaneously, and the positions of the emission peaks are exactly the same as those of Cu-CDs and hMSNA. In addition, adding AAP to the dual-emission system has a similar enhancing effect on the blue fluorescence part without affecting the fluorescence emission of hMSNA, and causes a change in fluorescence color, providing the possibility for visual semi-quantitative detection of Fe 3+ . The above results prove the successful construction of the ratio fluorescence sensing system (Cu-CDs / hMSNA / AAP).

[0080] Next, add 400 μL of the analyte solution (such as Fe 3+) Detection was carried out. In the final detection system, the working concentrations of the reference probe hMSNA, the response probe Cu-CDs, and the detection medium AAP were 0.525 mg / mL, 0.02 mg / mL, and 1.25 mM, respectively.

[0081] Example 5

[0082] Using the ratio fluorescence sensing system prepared in Example 4 of the present invention for the detection of Fe 3+ The application process is as follows:

[0083] According to the total volume of the ratio fluorescence sensing system (Cu-CDs / hMSNA / AAP) in Example 4 of the present invention being 1.6 mL, after ultrasonic treatment of the sensing stock solution until the components were evenly distributed, different concentrations of Fe 3+ solution (FeCl3·6H2O, 400 μL) was added. After thorough mixing and incubation for 2 min, the fluorescence spectrum (excitation light at 371 nm), the fluorescence intensities at 435 nm and 655 nm, and the fluorescence photograph of the corresponding sample solution under ultraviolet light were recorded using a fluorescence spectrophotometer.

[0084] From FIGS. Figure 10 (A) and 10(B), it can be seen that we found that after adding hMSNA, the sensing system showed a typical proportional response with the increase of Fe 3+ concentration, and the fluorescence color showed a significant change from purple to pink, and finally to dark red. In the range of 2 - 160 μM, the linear equation was y = -0.00657x + 0.83742, and the linear correlation coefficient R 2 was 0.9991 (the detection limit was 1.53 μM). In the range of 160 - 400 μM, the equation was y = -0.01300x + 1.92930, and R 2 was 0.9981.

[0085] In addition, combined with the digital photographs in FIGS. Figure 7 , further analysis showed that the ratio fluorescence sensing system presented rich color evolution within a wide color gamut, enabling it to perform visual semi-quantitative analysis on Fe 3+ .

[0086] Example 6

[0087] The application of the ratio fluorescence sensing system in Example 4 of the present invention for the detection of Fe 3+ in real human serum samples is as follows:

[0088] First, fetal bovine serum and acetonitrile were mixed at a volume ratio of 1:1, allowed to stand for 5 min, centrifuged, and the supernatant was taken and diluted 10 times with deionized water to obtain diluted serum. Different concentrations of Fe 3+Solution, as the actual required serum sample.

[0089] Then, repeat the steps in Example 5 to detect Fe using the ratio fluorescence sensing system 3+ to investigate the performance of the ratio fluorescence sensing system in actual samples.

[0090] By plotting and fitting the analysis of Ln(I 435 / I 655 ) versus the concentration of Fe in serum 3+ , it can be seen from the appendix Figure 11 that in actual human serum samples, the ratio fluorescence sensing system also maintains a high linear correlation coefficient, R 2 is 0.9900, indicating that Cu-CDs / hMSNA / AAP also has good accuracy in the detection of real samples. The good linear relationship provides sufficient basis and strong evidence for the clinical application of the ratio fluorescence sensing system.

[0091] Comparative Example 1

[0092] Use the single-signal probe copper-doped carbon dots (Cu-CDs / AAP) to detect Fe 3+ , and the process is as follows:

[0093] Prepare a mixed solution of Cu-CDs and AAP, dilute it to 1.6 mL with deionized water according to a volume ratio of 1:1, and incubate for 4 min.

[0094] Add solutions of different concentrations of Fe 3+ (400 μL) to ensure that the final concentrations of Cu-CDs and AAP in the mixed system are 0.02 mg / mL and 1.25 mM respectively. Mix well and incubate for 2 min, and record the fluorescence spectrum (excitation light is 371 nm), the fluorescence intensity at 435 nm, and the fluorescence photograph of the sample solution under ultraviolet light.

[0095] It can be seen from the appendix Figure 12 (A) and Figure 12 (B) that as the concentration of Fe 3+ increases, the fluorescence intensity of the single-signal probe gradually decreases, but the linear correlation coefficient R 2 is calculated to be 0.9786 in the low-concentration region. In the high-concentration region, R 2 is calculated to be 0.9689. The linear correlation coefficient of the single-signal probe's response to Fe 3+ is far lower than that of the ratio fluorescence sensing system in the full concentration range (R 2 =0.9991, R 2= 0.9981), which means that within the same detection range, the ratio fluorescence sensing system has higher detection accuracy and precision, increases the credibility of the detection data, and enhances the practicality of the detection system. This is mainly due to the inherent reliability of the dual-signal analysis.

[0096] In addition, Figure 12 (A) The digital photos show that the fluorescence change of the single-color fluorescent probe is hardly perceptible to the naked eye, only showing a slight chromaticity shift. In terms of visualization, the ratio fluorescence system has a more obvious advantage because it can produce a change in the fluorescence color that is easily recognizable to the naked eye as the concentration of the analyte changes, thus endowing the sensing system with the ability of visual detection and further increasing the practical usability of the ratio fluorescence sensing system.

[0097] In summary, through the design and regulation of key factors such as the loading amount of AIE molecules in hydrophobic porous silica, the working concentration of the response probe (Cu-CDs) in the system, the working volume ratio of the response probe (Cu-CDs) to the reference probe (hMSNA), the working concentration of the detection medium (AAP), and the incubation time in the embodiments of the present invention, a new and ideal ratio fluorescence sensing system is obtained. Using the sensing system of the present invention, the fluorescence quantitative detection and visual semi-quantitative sensing of Fe 3+ are further realized. Compared with the single-color fluorescent probe, this system has high accuracy, a wide detection range, strong visualization ability, good stability, and potential for practical clinical applications. The construction method of the ratio fluorescence sensing system in the embodiments of the present invention provides a valuable reference for the design of dual-signal detection systems and has good application prospects in the field of biomarker detection.

[0098] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A ratiometric fluorescence sensing system, characterized in that, It includes a reference probe and a response probe. The reference probe is a silica-based aggregation-induced emission material, and the response probe is a copper-doped carbon dot material.

2. The ratio fluorescence sensing system according to claim 1, characterized in that: The mass ratio of the silica-based aggregation-induced emission material to the copper-doped carbon dot material is 3.70 - 25.93:

1.

3. The ratio fluorescence sensing system according to claim 1, characterized in that: It further includes a detection medium, and the detection medium is ascorbic acid-2-phosphate material.

4. The ratiometric fluorescence sensing system according to claim 3, wherein: The g / mol value of the copper-doped carbon dot material to the ascorbic acid-2-phosphate material is 16.

2.

5. The ratio fluorescence sensing system according to claim 1, characterized in that: The silica-based aggregation-induced emission material is an aggregation-induced emission material loaded on silica, and the structural formula of the aggregation-induced emission material is:

6. The preparation method of the ratiometric fluorescence sensing system according to claim 3, characterized in that: It includes: Step 1: Prepare the silica-based aggregation-induced emission material and the copper-doped carbon dot material respectively. Step 2: Mix the silica-based aggregation-induced emission material, the copper-doped carbon dot material and the ascorbic acid-2-phosphate material to construct a ratio fluorescence sensing system.

7. The preparation method of the ratiometric fluorescence sensing system according to claim 6, characterized in that: In Step 1, porous silica is synthesized by a one-pot method and dispersed in dimethyl sulfoxide, and then a molecular working solution of the aggregation-induced emission material is added and stirred at room temperature to obtain the silica-based aggregation-induced emission material.

8. The preparation method of the ratio fluorescence sensing system according to claim 6, characterized in that: In Step 1, the copper-doped carbon dot material is prepared by a hydrothermal method.

9. Application of the ratio fluorescence sensing system according to any one of claims 1 - 5 in the detection of ferric ions.

10. Application of the ratio fluorescence sensing system according to any one of claims 1 - 5 in the in vitro detection or fluorescence imaging of biomarkers.

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