A preparation method of an on-off-on fluorescent nanosensor based on NCl-CQDs

Abstract

The application belongs to the technical field of fluorescent nanosensor, and particularly relates to a preparation method of an 'on-off-on' fluorescent nanosensor based on NCl-CQDs. Nitrogen-chlorine co-doped carbon quantum dots NCl-CQDs are used as a fluorescent needle, 2,6-dichloroindophenol sodium DCIP is used as a fluorescence quencher, and ascorbic acid AA is used as a fluorescence restorer. The three are specifically combined in a specific sample adding sequence to obtain the 'on-off-on' fluorescent nanosensor. The preparation method specifically comprises the following steps: nitrogen-chlorine co-doped carbon quantum dots NCl-CQDs are prepared by using glucose Glu, ethylenediamine EDA and concentrated hydrochloric acid HCl as raw materials and adopting a dehydration heat carbonization method; when the excitation wavelength is 390 nm, the maximum fluorescence emission wavelength of the NCl-CQDs is 467 nm, and at this time, the fluorescence intensity is in an 'ON' state; when the DCIP is introduced into the NCl-CQDs solution, due to the photoinduced electron transfer effect, the fluorescence of the NCl-CQDs is quenched, the fluorescence signal intensity is turned off, and is in an 'OFF' state; the continuous introduction of the AA into the NCl-CQDs / DCIP system can significantly restore the fluorescence intensity of the NCl-CQDs, and at this time, the fluorescence intensity is in an 'ON' state.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent nanosensors, and particularly relates to a preparation method of an "on-off-on" fluorescent nanosensor based on NCl-CQDs. BACKGROUND

[0002] Carbon quantum dots (CQDs) are a new type of fluorescent carbon nanomaterial composed of dispersed spherical particles with extremely small sizes (≤10 nm below), and have the advantages of low cost, high stability, good biocompatibility, and no need for complex steps. As a new type of nanomaterial with low toxicity and superior optical properties, CQDs have different application prospects in various fields, such as biological sensing, biological imaging, photodynamic therapy, and drug delivery. Due to its unique properties and wide applications, CQDs have been synthesized in large quantities. However, all methods have the disadvantages of complex synthesis process, time-consuming, and complex reaction conditions, which limit their wide application. In addition, most CQDs have the defects of relatively low fluorescence quantum yield (QY), few active sites, and poor selectivity, which usually cannot be well used in practical applications. Therefore, in order to improve the optical and chemical properties of carbon dots, researchers have proposed the method of heteroatom doping. Among different heteroatoms, nitrogen-doped CQDs have higher QY, which is conducive to stabilizing the surface defects of CQDs, thereby improving the fluorescence emission ] However, pure nitrogen doping may not meet the specific performance requirements in some aspects, such as some special catalytic reactions or optical applications. Among the numerous reported modification methods of carbon dots, nitrogen atom co-doping with other heteroatoms is the most studied method, mainly including N / S, N / B, N / F, N / P, N / Mg, N / Cu, and other co-doping methods.

[0003] Therefore, based on these studies, more and more people are committed to choosing N and Cl as co-dopants. Compared with single-doped N or Cl carbon dots, co-doped N and Cl has several significant advantages, including enhanced fluorescence emission, higher sensitivity, and the ability to adjust the optical and electrical properties of quantum dots. In 2021, Yin synthesized new NCl co-doped carbon dots (NCl-CQDs) using choline chloride / urea DES and glycine as a C atom source. The prepared NCl-CQDs have strong fluorescence emission and good thermal stability. The NCl-CQDs fluorescence probe was used to determine the main alkaloid morphine in poppy husks for high-sensitivity analysis of trace amounts of poppy husks in food. In 2022, Cao et al. used 4-chlorobenzene-1,2-diamine, dilute hydrochloric acid, and deionized water as raw materials to prepare nitrogen-chlorine co-doped CQDs (R-CQDs) with bright red fluorescence using a simple one-step hydrothermal method. The R-CQDs have high PLQY red fluorescence. In 2023, R Tabaraki et al. used a microwave method to prepare NCl-doped carbon dots (NCl-CQDs) in a choline chloride-glycerol deep eutectic solvent (DES), and the surface was modified with vancomycin for the detection of S. aureus bacteria. Therefore, based on previous research, this study is committed to exploring the synthesis of NCl-CQDs to achieve the detection of AA.

[0004] 2,6-dichloroindophenol sodium (DCIP) is a deep blue crystal powder that is soluble in ethanol and ether, slightly soluble in water, but insoluble in non-polar solvents. As a well-known analytical reagent, DCIP has redox properties and can be used for volumetric and photometric determination of ascorbic acid (AA) and other reducing agents. Because of its obvious color change, it is also commonly used in kits for enzyme activity determination, enzyme-linked immunosorbent assay, drug determination, etc. When the pH value is higher than 6.0, the oxidized form of DCIP aqueous solution is deep blue in solution, with an absorption spectrum of about 420-750 nm, while the reduced form of DCIP is colorless. Due to the presence of photo-induced electron transfer effect (PET) between NCl-CQDs and DCIP in the system, fluorescence quenching occurs.

[0005] It is well known that there is a specific reduction reaction between 2,6-dichloroindophenol sodium and ascorbic acid [They are often used as active molecule pairs in signal switch sensing systems. Ascorbic acid (AA, also known as vitamin C) is a highly water-soluble polyhydroxyl compound, and is also an important nutrient, which plays a leading role in human health in various biological processes by reducing oxidative damage to proteins, DNA and lipids. Secondly, it has strong reducing property and can participate in redox reactions and various hydroxylation reactions in the body. When the body lacks ascorbic acid, it may cause symptoms such as weakness of limbs and mental fatigue, and in severe cases, it may also cause bone pain and osteoporosis. Generally, ascorbic acid can also promote the synthesis of collagen and connective tissue in the body, help wound recovery and healing, and can also scavenge free radicals in the body to avoid oxidative damage to cells and improve the body's immune ability. According to the evaluation, the tolerable daily intake of AA is 2000mg / d. As an indispensable micronutrient, AA cannot be synthesized in the body and must be taken from exogenous sources such as fruits or the dining table to meet the body's needs. Therefore, it is necessary to detect a large amount of AA in the body. At present, the main methods for detecting AA include electrochemical analysis, chemiluminescence, fluorescence spectroscopy and liquid chromatography. Among them, fluorescence spectroscopy is of great concern due to its simple operation, high sensitivity and visual detection results. SUMMARY

[0006] The present application aims at the problems existing in the prior art, and provides a preparation method of an "on-off-on" fluorescent nanosensor based on NCl-CQDs.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0008] The preparation method of the "on-off-on" fluorescent nanosensor based on NCl-CQDs is characterized by: using nitrogen-chlorine co-doped carbon quantum dots NCl-CQDs as a fluorescent needle, 2,6-dichloroindophenol sodium DCIP as a fluorescent quencher, and ascorbic acid AA as a fluorescent restorer, and the three are specifically combined in a specific sample adding sequence to obtain the "on-off-on" fluorescent nanosensor; and the specific execution is as follows:

[0009] Step one, using glucose Glu, ethylenediamine EDA and concentrated hydrochloric acid HCl as raw materials, nitrogen-chlorine co-doped carbon quantum dots NCl-CQDs with a concentration of 44.4μM are prepared by a dehydration exothermic carbonization method; when the excitation wavelength is 390nm, the maximum fluorescence emission wavelength of NCl-CQDs is 467nm, at which time the fluorescence intensity is in the "ON" state;

[0010] Step two, when 1 mL of 30.77 μM DCIP is introduced into 1 mL of 44.4 μM NCl-CQDs solution, the fluorescence of NCl-CQDs is quenched due to the photoinduced electron transfer effect, and the fluorescence emission intensity decreases from 910 nm to about 50 nm when the excitation wavelength is 390 nm, at this time the fluorescence is in the "OFF" state;

[0011] Step three, the continuous introduction of 85.5 μM AA in the NCl-CQDs / DCIP system can significantly restore the fluorescence intensity of NCl-CQDs, and when the excitation wavelength is 390 nm, the signal intensity can rise from 148 nm to about 748 nm, at this time the fluorescence intensity is in an "ON" state; thus obtaining the "on-off-on" fluorescence nanosensor of NCl-CQDs / 2,6-dichloroindolinesodium+AA.

[0012] Preferably, the preparation steps of the nitrogen and chlorine co-doped carbon quantum dots NCl-CQDs are as follows:

[0013] Step (1) 0.8g of glucose and 12ml of EDA are completely dissolved in 20ml of ultrapure water by ultrasonic and vortex;

[0014] Step (2) 4ml of concentrated hydrochloric acid is added to the above mixture, a large amount of white smoke and heat are released, and after cooling to room temperature, a brown yellow substance is obtained;

[0015] Step (3) the above brown yellow substance is dissolved in 50ml of ultrapure water, then the solution is centrifuged at 4000rpm for 10min, then microfiltration is carried out using a 0.22 μM filter, a brown solution is obtained, and the solution is freeze-dried to obtain pure NCl-CQDs brown solid powder.

[0016] Preferably, in step one, the fluorescence quantum yield of the prepared NCl-CQDs is 48.5%

[0017] Preferably, in step two, the reaction time is 5min.

[0018] Preferably, in step three, the reaction time is 1.5min.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1. The present application uses a dehydration carbonization method to synthesize NCl-CQDs, and the synthesized NCl-CQDs have uniform particle distribution, good dispersibility, an average particle size of 2.96nm, and obvious luminescent characteristics and excitation wavelength dependence.

[0021] 2、The application establishes a novel "on-off-on" type detection method of AA based on NC1-CQDs fluorescent probe.In the experiment, NC1-CQDs is used as a fluorescent probe, and based on the principle that 2,6-dichloroindophenol sodium and NC1-CQDs photoinduced electron transfer makes the fluorescence spectrum of NC1-CQDs change, a "on-off" type fluorescent sensor of NC1-CQDs and 2,6-dichloroindophenol sodium is formed, then 2,6-dichloroindophenol sodium and AA are introduced into the system to form a "off-on" type fluorescent sensor.Based on the specific reaction between AA and 2,6-dichloroindophenol sodium, AA competes with 2,6-dichloroindophenol sodium on the surface of NC1-CQDs, so that the UV-visible absorption spectrum and fluorescence signal of NC1-CQDs are restored, and then an "on-off-on" type fluorescent sensor of NC1-CQDs / 2,6-dichloroindophenol sodium+AA is established.

[0022] 3、The fluorescence analysis method established by the application can be used for the detection of AA in actual serum samples by standard addition recovery, which shows that the fluorescence sensing system has better utilization value for AA in actual complex samples, and compared with the traditional detection method, the method has good application prospect for monitoring the health level of human body. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a schematic diagram of NC1-CQDs for DCIP and AA detection in the application;

[0024] Figure 2 In the figure, A and B are TEM images of NC1-CQDs at 10nm and 20nm scales respectively; C is the lattice spacing of the HRTEM image of NC1-CQDs; D is the particle size distribution histogram of NC1-CQDs; E is the XRD spectrum of NC1-CQDs; F is the Raman scattering diagram of NC1-CQDs;

[0025] Figure 3 In the figure, A is the ultraviolet-visible absorption, excitation spectrum and emission spectrum of NC1-CQDs (insert: image of NC1-CQDs under visible light and 365nm ultraviolet light); B is the fluorescence emission spectrum of NC1-CQDs under different excitation wavelengths (350-450nm); C is the total luminescence spectrum (220-800nm) of NC1-CQDs; D is the CIE color coordinate of NC1-CQDs;

[0026] Figure 4 In the figure, A is the XPS spectrum of NC1-CQDs; (B) high-resolution XPS spectrum of C-1s, (C) O-1s, (D) N-1s and (E) Cl-2p; (F) FT-IR spectrum of NC1-CQDs.

[0027] Figure 5 The fluorescence intensity of NCl-CQDs corresponding to different xenon lamp irradiation time (A), different storage time (B) and different NaCl solution concentration (C);

[0028] Figure 6 The "ON-OFF-ON" fluorescence sensing system is constructed.

[0029] Figure 7 A is the reaction time optimization of NCl-CQDs and DCIP; B is the reaction time optimization of NCl-CQDs-DCIP system and AA.

[0030] Figure 8 The fluorescence spectrum of NCl-CQDs under the condition of 0-30.77 μM DCIP;

[0031] Figure 9 The influence of different substances on the fluorescence emission intensity of NCl-CQDs / DCIP system;

[0032] Figure 10 A is the influence of different concentrations of AA (9.09-85.5 μM) on the fluorescence spectrum of NCl-CQDs / DCIP system; B is the curve relationship obtained by the influence of different concentrations of AA on the fluorescence intensity of NCl-CQDs / DCIP system; C and D are the piecewise linear relationship between the fluorescence intensity of NCl-CQDs / DCIP system and different concentrations of AA.

[0033] Figure 11 A is the "ON-OFF-ON" fluorescence sensing system; B and C are the ultraviolet-visible absorption spectra of various substances. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0035] Example 1 experiment

[0036] 1. Materials and instruments

[0037] All reagents were of analytical reagent grade and used without further purification. Glucose (Glu) was purchased from Shanghai Wakai Biotechnology Co., Ltd., ascorbic acid was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., 11 kinds of amino acids (L-alanine, L-cysteine, L-threonine, L-arginine, L-histidine, L-methionine, L-leucine, L-serine, L-aspartic acid, L-valine, L-isoleucine) were purchased from Shanghai Aladdin Pharmaceutical Co., Ltd., 2,6-dichloroindophenol sodium was purchased from Shanghai Zhongqin Chemical Reagent Co., Ltd. (Shanghai, China). Ethylenediamine was purchased from Tianjin Damao Chemical Reagent Factory, hydrochloric acid was purchased from Hedyang Reagent Factory (Tianjin, China). Metal ion solution preparation reagent was purchased from National Pharmaceutical Chemical Reagent Co., Ltd. (Shenyang, China).

[0038] An electronic balance of FA2104 model was used to weigh the sample (Shanghai Yuheng Scientific Instrument Co., Ltd., China). Transmission electron microscope images were obtained with a JEM-2100F microscope (Japan JEOL). X-ray powder diffraction patterns of the samples were collected with a Bruker D2 phase shift diffractometer. Fluorescence spectra were measured with an RF-5301-PC fluorescence spectrometer (Shimadzu Corporation, Tokyo, Japan). Ultraviolet-visible absorption spectra were recorded on a T6 new century type ultraviolet-visible spectrophotometer produced by Beijing Purui General Instrument Co., Ltd. (Beijing Purui General Instrument Co., Ltd., China). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB250Xi instrument. Fourier transform infrared spectroscopy (FT-IR) was measured by an IR Prestige 21 spectrometer from Shimadzu Corporation, Tokyo, Japan. The numerical control ultrasonic cleaning machine used KH-300DE model (Kunshan Hechuang Ultrasonic Instrument Co., Ltd., China).

[0039] 2. Synthesis of NCI-CQDs

[0040] NCl-CQDs were synthesized by dehydration exothermic carbonization method using glucose (Glu), ethylenediamine (EDA) and concentrated hydrochloric acid as raw materials. First, 0.8 g of glucose and 12 ml of EDA were completely dissolved in 20 ml of ultrapure water by ultrasonic and vortex. 4 ml of concentrated hydrochloric acid was added to the above mixture, releasing a large amount of white smoke and heat. After cooling to room temperature, a brownish yellow substance was obtained, which was dissolved in 50 ml of ultrapure water. Then the solution was centrifuged at 4000 rpm for 10 min, and then microfiltered using a 0.22 μM filter to obtain a brown solution. The solution was freeze-dried to obtain pure NCl-CQDs brown solid powder. Finally, the black brown solid powder was stored in a refrigerator at 4°C for further characterization and application.

[0041] 3. Calculation of fluorescence quantum yield

[0042] Fluorescence quantum yield (QY) is an important luminescence parameter of fluorescent substance, which is defined as the ratio of the number of emitted fluorescent photons to the number of absorbed excitation photons. The greater the value of QY, the stronger the fluorescence of the fluorescent substance, but its value is generally less than 1 according to the definition. The calculation of QY generally adopts the reference method by comparing the integrated emission spectrum and absorbance of the fluorescent substance to be tested and the known fluorescent quantum yield substance under the same determination conditions. A known fluorescent quantum yield of quinine sulfate (QY = 54%) is used as a standard substance to calculate the fluorescence quantum yield of NCl-CQDs. 0.04 g of quinine sulfate is diluted with 0.05 mol / l sulfuric acid solution to constant volume, 0.05 mol / l sulfuric acid solution is used as a reference, the maximum absorption wavelength of the tested fluorescence is used as the excitation wavelength, and the fluorescence emission intensity under the excitation wavelength is measured by a fluorescence spectrophotometer, and the absorbance of the quinine sulfate solution under the wavelength is measured by a UV-visible spectrophotometer at the same time.

[0043]

[0044] wherein I is the fluorescence integral area of the emission spectrum, A is the UV-visible absorbance under the fluorescence excitation wavelength, and η is the refractive index of the solvent (the refractive index of the solvent water is 1.33, and the refractive index of the concentrated sulfuric acid solution is 1.418).

[0045] 4. Construction of sensing system

[0046] AA is detected by using NCl-CQDs and DCIP as fluorescent probes. In order to construct the NCl-CQDs / DCIP fluorescence sensing platform, 30 μl of NCl-CQDs is mixed with different concentrations of DCIP, and the fluorescence emission intensity is measured at an excitation wavelength of 390 nm. The concentration ratio of the fluorescence emission intensity with the best quenching effect is the optimal system. Subsequently, different concentrations of AA are added to the NCl-CQDs / DCIP sensing system, and the fluorescence spectrum generated is recorded under an excitation wavelength of 390 nm. The concentration ratio of the fluorescence emission intensity with the best recovery effect is the optimal system.

[0047] 5. Detection of DCIP and AA in actual samples

[0048] The human blood sample for actual sample analysis is from the Fourth People's Hospital of Qinghai Province. The collected fresh blood sample is left to stand at room temperature for 30 min, centrifuged at 10000 r / min for 25 min, and then the supernatant is filtered by a 0.22 nm microporous filter to remove large particle impurities. Subsequently, 1 ml of the supernatant is diluted 200 times to obtain a serum sample. Different time periods of serum samples are taken and different concentrations of AA are added, and the fluorescence intensity is detected. The reliability and accuracy of the fluorescence sensing system for AA detection are proved by the standard addition recovery rate.

[0049] Example 2

[0050] 1. TEM and XRD characteristics of NCl-CQDs

[0051] To demonstrate the successful synthesis of NCl-CQDs, they were characterized using transmission electron microscopy and XRD. Figure 2 As shown, the morphology, particle size, and dispersibility of the synthesized NCl-CQDs were characterized using transmission electron microscopy. (The image shows the morphology, particle size, and dispersibility of the NCl-CQDs at a 10 nm diameter.) Figure 2 A) and 20nm ( Figure 2 TEM at position B) indicates that the prepared NCl-CQD consists of dispersed spherical carbon particles with extremely small size, spherical morphology, uniform particle size, and good dispersibility. HR-TEM ( Figure 2 C) It can be clearly observed that the prepared NCl-CQDs have obvious crystal characteristics, and their lattice spacing was measured to be 0.23 nm. For example... Figure 2 As shown in Figure D, the particle size distribution of the synthesized NCl-CQD was investigated, and the results showed that its particle size ranged from 1.5 to 5.5 nm, with an average particle size of 2.96 nm. Furthermore, the X-ray diffraction pattern (...) Figure 2 E) A strong diffraction peak appears at 2θ = 22.71°, corresponding to the (002) crystal plane of the graphite structure, indicating the presence of highly disordered carbon atoms. Based on the above results, the synthesized NCl-CQDs possess a highly crystalline and graphitized structure. Figure 2 In the Raman scattering spectrum of F, the D peak represents amorphous carbon in the material, and the G peak represents crystalline carbon. The G peak is significantly higher than the D peak, and the peak shape of the G peak is sharper, which indicates that the synthesized carbon quantum dots have a higher degree of crystallinity.

[0052] 2. Optical properties of NCl-CQDs

[0053] The optical properties of the synthesized NCl-CQDs were analyzed using UV-Vis absorption spectroscopy and fluorescence spectroscopy. For example... Figure 3 As shown, the UV-Vis absorption spectrum exhibits an absorption band of 220–650 nm, with a maximum characteristic absorption wavelength at 336 nm, which may be attributed to the π-π* or n-π* transitions in carbon nanomaterials. Further fluorescence property studies indicate that the optimal excitation wavelength (Ex) and maximum emission wavelength (Em) are located at 390 nm and 467 nm, respectively. Notably, the excitation and emission spectra exhibit mirror symmetry, determined by the symmetry of the energy level structure within the atoms or molecules, consistent with fluorescent carbon nanomaterials. The NCl-CQDs solution appears brown under visible light but exhibits blue fluorescence under 365 nm UV irradiation. Figure 3a inset). This fluorescence emission behavior is due to the photoinduced charge separation and the surface trapping, which leads to the radiative recombination between the hole and electron pairs. In addition, the excitation wavelength dependence of the prepared NCl-CQDs was also investigated. As shown in Figure 3 B, the fluorescence spectra of NCl-CQDs with different excitation wavelengths (350-450 nm) show typical excitation-dependent behavior. With the increase of excitation wavelength, the emission wavelength red-shifts, showing a slight excitation wavelength dependence. The fluorescence emission intensity gradually increases with the excitation wavelength increasing from 350 nm to 390 nm, and then gradually decreases with the excitation wavelength increasing from 390 nm to 450 nm, which is considered to be caused by the uneven size of the clusters. In order to further study its optical properties, the total luminescence spectrum (220-800 nm) was obtained, and the excitation-emission matrix spectrum shows that the excitation wavelength between 350-400 nm shows certain excitation wavelength independent characteristics, as shown in Figure 2 C. Figure 3 In D, the color coordinates (0.1395, 0.1839) were also simulated using CIE chart simulation software, which can be seen at a glance that the position of NCl-CQDs blue light. In addition, according to the quantum yield calculation formula, the fluorescence quantum yield of the prepared NCl-CQDs was calculated to be 48.5%, which is a favorable choice for constructing a fluorescent sensing system with potential application prospects.

[0054] 3. XPS and FT-IR characterization of NCl-CQDs

[0055] Further study the chemical element composition and the chemical state of the elements of NCl-CQDs by X-ray photoelectron spectroscopy (XPS). As shown in Figure 4 A, four different element peaks can be seen: O1s, N1s, C1s and Cl2p. Subsequently, the four elements were further characterized by high-resolution XPS spectra. In Figure 4 B, four significant related peaks of C1s were shown in high-resolution XPS, which were 284.4 eV (C-C / C=C), 285.5 eV (C-N), 286.2 eV (C-O) and 287.9 eV (C=O). Figure 4Two different states of Ols were shown in C: 531.4 eV (C=O), 532.6 eV (C-O). In addition, high resolution XPS spectra were performed for Nls, and three different peaks of N element were obtained, respectively, which were amino state nitrogen (398.8 eV), pyrrole type nitrogen (400.1 eV), pyridine type nitrogen (401.2 eV). Finally, Cl2p was also split into two different states of peaks: the peak value of Cl2p (3 / 2) was 198.2 eV, and the peak value of Cl2p (1 / 2) was 199.8 eV. Through XPS characterization, it can be better proved that NCl-CQDs are composed of O, N, C and Cl four elements, which has important research significance for identifying the surface chemical properties and composition analysis of the sample.

[0056] In the study of NCl-CQDs, FT-IR spectrum provides valuable information about the existence of different functional groups. In order to better understand the types of functional groups of NCl-CQDs, infrared spectrum scanning was carried out. As shown in Figure 4 F, 3454 cm-1 is N / O-H stretching vibration, the weak peak at 2079 cm-1 may be C≡C, which may be due to the weakening of absorption in the carbon structure inside the alkyne, 1638 cm-1 is the stretching vibration peak of C=O / N, 1328 cm-1 is the stretching vibration of C-N, and 574 cm-1 is the stretching vibration of C-Cl. The infrared spectrum can show that N and Cl are successfully doped in carbon quantum dots.

[0057] 4、Stability study of NCl-CQDs

[0058] In order to explore the stability of the carbon dots, the stability test was carried out under xenon lamp simulated sunlight irradiation time, placement days and different concentrations of NaCl salt solution, respectively. The results showed that under the irradiation of xenon lamp for different time (0-70 min), the fluorescence intensity of carbon dots did not change obviously with the extension of irradiation time, as shown in Figure 5 A; the carbon dots were placed in the refrigerator at 4℃ for about 8 days, and the fluorescence intensity was measured every day, from Figure 5 B, it can be seen that the fluorescence intensity did not change obviously; in addition, the salt resistance of carbon dots was detected in different concentrations (0-1.0 M) of NaCl salt solution, and it can be seen that the fluorescence intensity also remained almost unchanged, as shown in Figure 5 C. Therefore, from the above results, it can be shown that the prepared NCl-CQDs have good stability, which can be better applied in practical life detection.

[0059] Example 3

[0060] 1、Construction of "ON-OFF-ON" sensing system

[0061] The maximum fluorescence emission wavelength of the synthesized NCI-CQDs fluorescent probe under 390 nm excitation is 467 nm. At this time, the fluorescence signal of NCI-CQDs is in the ON state. However, when 2,6-dichloroindophenol sodium is added to the sensing system, the fluorescence intensity of NCI-CQDs is significantly quenched, resulting in the fluorescence signal being in the OFF state, and then the addition of ascorbic acid (AA) in the NCI-CQDs / 2,6-dichloroindophenol sodium solution restores the fluorescence signal to the "ON" state, as shown in Figure 6 Thus, a "turn-on-turn-off-turn-on" fluorescent sensing system based on NCI-CQDs / 2,6-dichloroindophenol sodium-AA is successfully constructed.

[0062] 2. Condition optimization of the sensing system

[0063] In order to study whether the fluorescence intensity is affected by the reaction time, it is necessary to study the reaction time between the fluorophore and the specific analyte. For the NCI-CQDs / DCIP sensing system, the fluorescence emission intensity was determined at an excitation wavelength of 390 nm, and the determination was performed every 60 s, Figure 7 As can be seen from A, with the increase of the reaction time, the fluorescence intensity gradually decreases, and after 5 min the fluorescence intensity tends to be stable, indicating that the optimal reaction time of the system is 5 min. At the same time, the reaction time of NCI-CQDs / DCIP and AA was studied, and the determination was performed every 30 s, and it was found that after 1.5 min, the fluorescence intensity no longer increased but began to decrease, and the fluorescence intensity reached the highest at 1.5 min, as shown in Figure 7 B.

[0064] Example 3 Test optimization design

[0065] 1. DCIP concentration optimization

[0066] As shown in Figure 8 , the fluorescence was determined, and 2,6-dichloroindophenol sodium was added from low concentration to high concentration (0 μM, 2.26 μM, 4.6 μM, 13.8 μM, 15.6 μM, 20.7 μM, 23.6 μM, 24.8 μM, 26.3 μM, 27.7 μM, 29.4 μM, 30.77 μM) in the optimized NCI-CQDs solution. With the continuous increase of 2,6-dichloroindophenol sodium, the fluorescence intensity of NCI-CQDs decreased, and NCI-CQDs and 2,6-dichloroindophenol sodium had a good rising and falling relationship.

[0067] 2. Selectivity of NCI-CQDs sensing system

[0068] To verify whether the fluorescent sensor can be well applied to the detection of actual samples, its selectivity was investigated. In the NCl-CQDs / 2,6-dichloroindophenol sodium system, 10 μL of AA, Ba 2+ , Ca 2+ , Co 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Hg 2+ , K + , Ni 2+ , Sr 2+ , L-alanine, L-cysteine, L-threonine, L-arginine, L-histidine, L-methionine, L-leucine, L-serine, L-aspartic acid, L-valine, and L-isoleucine were added, respectively, to obtain an interesting result. As can be seen from Figure 9 , when the concentration of the interfering substance was 100 times that of AA, only ascorbic acid (AA) could make the fluorescence emission intensity of NCl-CQDs / DCIP increase again, and the effects of other substances on the fluorescence intensity were negligible. The above results show that the NCl-CQDs / DCIP system has good selectivity for AA and can be further applied to the detection of actual samples.

[0069] 3. Linear relationship construction of NCl-CQDs-DCIP and AA system

[0070] In the NCl-CQDs / DCIP system, 1×10 -5 mol / L to 1×10 -4 mol / L of AA was added, the excitation light was set to 390 nm, the slit width was 3×5, and the fluorescence intensity of the NCl-CQDs-2,6-dichloroindophenol sodium-AA system was determined in the range of 400-600 nm. The concentrations of AA from bottom to top were 9.09 μM, 23.07 μM, 33.3 μM, 44.4 μM, 52.38 μM, 58.33 μM, 65.51 μM, 70.58 μM, 74.35 μM, 77.27 μM, 79.59 μM, 81.48 μM, 83.05 μM, 84.37 μM, 85.5 μM. With the continuous increase of the concentration of AA, the fluorescence emission peak was restored by AA. When the concentration of AA added was 85.5 μM, the fluorescence intensity was restored to the highest, as shown in Figure 10 A.

[0071] The fluorescence intensity of NCl-CQDs-2,6-dichloroindophenol sodium-AA was used as the ordinate, and the concentration of AA was used as the abscissa to draw the working curve, as shown in Figure 10As shown in Figure B. The results indicate that when the concentration is between 9.09 μM and 65.51 μM, the linear equation is Y = 4.606X + 102.438, R0. 2 =0.991, linear range is 144.31μM~404.18μM, detection limit is 13.137μM, such as Figure 10 As shown in Figure C; when the concentration is between 70.58 μM and 85.5 μM, its linear equation is Y = 19.429X - 917.395, R 2 =0.990, linear range is 453.90μM~743.78μM, detection limit is 0.80μM, such as Figure 10 As shown in D, this indicates that there is a good linear relationship between AA and the NCl-CQDs-2,6-dichlorophenolindophenol sodium system, which enables the detection of AA in actual samples.

[0072] Example 4: Practical Application of the "ON-OFF-ON" Sensing System

[0073] Therefore, it is necessary to evaluate the feasibility of using a fluorescence sensor to detect analytes in actual samples. The proposed NCl-CQDs / DCIP-AA sensing platform was used to detect analytes in serum samples, and the average recovery rate of AA was 97.20%–103.90%, with a relative standard deviation of no more than 0.65%, which is within an acceptable range, as shown in Table 1. In conclusion, the NCl-CQDS / DCIP-AA fluorescence sensing platform can effectively monitor the content of AA in serum and has certain practical application potential.

[0074] Table 1. Detection of AA in actual samples

[0075]

[0076]

[0077] Discussion on the Mechanism of "ON-OFF-ON" Sensing System

[0078] like Figure 11 As shown in Figure A, NCl-CQDs exhibit significant fluorescence absorption, but fluorescence quenching can be observed after the addition of DCIP. To demonstrate the quenching mechanism of its fluorescence intensity, we investigated its ultraviolet-visible absorption spectrum (UV-vis). Figure 11It can be seen that NCl-CQDs has a distinct UV absorption peak at about 287 nm, and DCIP has a sharp absorption peak at 270 nm. When DCIP is introduced into NCl-CQDs, the absorption peak shows red shift and absorption peak enhancement. It is preliminarily inferred that there is potential n→π* and π→π* transition between the two, which causes the energy of the electrons on the bonding orbital in the molecule to be excited to the antibonding orbital, thereby forming a non-luminescent ground-state complex (NCl-CQDs / DCIP) through photoelectron-induced transfer, so that fluorescence quenching phenomenon occurs.

[0079] Subsequently, in order to prove the phenomenon that the fluorescence intensity of NCl-CQDs / DCIP system gradually recovers after AA is introduced, the UV-vis mechanism is also discussed. As shown in FIG. 4B, AA has no UV absorption peak. When AA is introduced into NCl-CQDs / DCIP system, the UV absorption peak disappears and obvious red shift occurs, which is due to the redox electron transfer between DCIP and AA, which causes chemical reaction in the ground state. At this time, the reduced ascorbic acid reduces 2,6-dichloro indophenol sodium, and at the same time is oxidized to dehydroascorbic acid, thereby recovering the fluorescence of NCl-CQDs. Therefore, by using photoelectron-induced transfer and redox reaction, we successfully constructed an NCl-CQDs / DCIP / AA "ON-OFF-ON" fluorescence sensing system, which realizes rapid and efficient detection of AA. Figure 11

[0080] ​In summary, the application synthesizes NCl-CQDs by using the dehydration carbonization method, the synthesized NCl-CQDs has uniform particle distribution, good dispersibility, an average particle size of 2.96 nm, obvious luminescence characteristics and excitation wavelength dependence. A novel "on-off-on" type AA detection method is established based on the NCl-CQDs fluorescent probe. In the experiment, NCl-CQDs is used as a fluorescent probe, based on the principle that 2,6-dichloroindophenol sodium and NCl-CQDs photoinduced electron transfer makes the fluorescence spectrum of NCl-CQDs change, and a "on-off" type fluorescent sensor of NCl-CQDs and 2,6-dichloroindophenol sodium is constructed. Then, 2,6-dichloroindophenol sodium and AA are introduced into the system to construct a "off-on" type fluorescent sensor. Based on the specific reaction between AA and 2,6-dichloroindophenol sodium, AA competes with 2,6-dichloroindophenol sodium on the surface of NCl-CQDs, so that the ultraviolet-visible absorption spectrum and fluorescence signal of NCl-CQDs are restored, and an "on-off-on" type fluorescent sensor of NCl-CQDs / 2,6-dichloroindophenol sodium+AA is established. The established fluorescent analysis method can be used for the detection of AA in actual serum samples by standard addition recovery, which shows that the fluorescent sensing system has better utilization value for AA in actual complex samples. Compared with the traditional detection method, the method has a good application prospect for monitoring the health level of human body.

[0081] The above is only a preferred embodiment of the application, and is not intended to limit the application. Any modification, equivalent replacement and improvement within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A method for fabricating an on-off-on fluorescent nanosensor based on NCl-CQDs, characterized in that, Includes the following steps: Step 1: Using glucose (Glu), ethylenediamine (EDA), and concentrated hydrochloric acid (HCl) as raw materials, nitrogen-chlorine co-doped carbon quantum dots (NCl-CQDs) are prepared by dehydration exothermic carbonization method, specifically including: (1) Dissolve 0.8g glucose and 12ml EDA completely in 20mL ultrapure water by sonication and vortexing; (2) Add 4 ml of concentrated hydrochloric acid to the above mixture. A large amount of white smoke and heat are released. After cooling to room temperature, a brownish-yellow substance is obtained. (3) Dissolve the above brownish-yellow substance in 50 ml of ultrapure water, then centrifuge the solution at 4000 rpm for 10 min, and then microfilter it using a 0.22 μM filter to obtain a brown solution. Freeze-dry the solution to obtain pure NCl-CQDs brown solid powder. The prepared NCl-CQDs had a concentration of 44.4 μM and a fluorescence quantum yield of 48.5%. When the excitation wavelength was 390 nm, the maximum fluorescence emission wavelength was 467 nm, at which point the fluorescence intensity was in the "ON" state. Step 2: Introduce 1 mL of 30.77 μM sodium 2,6-dichlorophenolindophenol (DCIP) into 1 mL of 44.4 μM NCl-CQDs solution and react for 5 min. Due to the photoinduced electron transfer effect, the fluorescence of NCl-CQDs is quenched and the fluorescence intensity is in the "OFF" state. Step 3: 85.5 μM ascorbic acid AA was continuously introduced into the NCl-CQDs / DCIP system. After reacting for 1.5 min, the fluorescence intensity of NCl-CQDs was significantly restored, and the fluorescence intensity was in the "ON" state. This yields an on-off-on fluorescent nanosensor based on NCl-CQDs / DCIP / AA for the specific detection of ascorbic acid.

2. The method according to claim 1, characterized in that, In step two, the fluorescence quenching is due to the photoinduced electron transfer effect between DCIP and NCl-CQDs.

3. The method according to claim 1, characterized in that, The sensor is used for the quantitative detection of ascorbic acid in serum samples, with a spiked recovery rate of 97.20%–103.90%.

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