CN116855247A - Preparation, processing method and application of carbon nanodots - Google Patents

Abstract

The invention provides a preparation and treatment method of carbon nano dots, which comprises the following steps: (1) cutting mango pulp, reacting at 170 ℃ and filtering to obtain carbon nanodots; (2) dispersing the carbon nano-dots in NaOH solution, heating and introducing ozone for treatment, and obtaining the carbon nano-dots with the molecular weight of 3 kDa-50 kDa through ultrafiltration. The prepared carbon nano dot has high fluorescence quantum efficiency, and can improve the stability of the colorimetric probe 3,3', 5' -tetramethyl benzidine oxidation product. The invention also provides a ratio fluorescence-colorimetric dual-mode detection method based on the carbon nano dots and 3,3', 5' -tetramethyl benzidine.

Description

Preparation, processing method and application of carbon nanodots

Technical field

The invention relates to a preparation, processing method and application of carbon nanodots, and belongs to the technical field of nanomaterials.

Background technique

Dual-mode detection uses two different detection methods to detect samples at the same time, providing results that are mutually authenticated, improving the reliability and accuracy of measurement. 3,3',5,5'-Tetramethylbenzidine (TMB) is a commonly used chromogenic substrate in dual-mode detection. It is oxidized during the detection process, and the generated product (oxTMB or diimineoxTMB) displays in the visible light region. Strong absorbance. However, researchers found that the oxidation product is unstable under many working conditions, and the color of the product becomes lighter or even disappears a few minutes after the reaction is completed (ChemPlusChem.87(2022)e202200090; J.Histochem.Cytochem.47(1999)265 –272). This phenomenon will seriously affect the sensitivity, accuracy and precision of detection. In order to solve this problem, analysts have made many attempts. For example, Li et al. (ACSOmega.4 (2019) 5459–5470) added an appropriate amount of After the alkyl imidazole tetrafluoroborate ionic liquid and the surfactant sodium dodecyl sulfate form micelles, the TMB oxidation product can exist relatively stably in the micelles. However, the introduction of chemicals unrelated to measurement (such as the above-mentioned alkyl imidazole tetrafluoroborate ionic liquid and surfactant sodium dodecyl sulfate) into the reaction system usually makes the properties of the system more complex and the controllability becomes worse. Poor, and the chemicals introduced in this example all have certain cytotoxicity and are not suitable for the detection of components in organisms.

Carbon Dots (CDs) are a type of carbon nanoparticles with a diameter less than 10 nm. Due to the quantum confinement effect and surface/edge state effect, CDs are widely used in dual-mode detection. CDs have abundant surface functional groups, such as polyaromatic ring structures, carboxyl groups, hydroxyl groups, amino groups, and epoxides. Therefore, as photoluminescent probes for dual-mode detection, CDs easily interact with another probe. For example, TMB is easily adsorbed on the surface of CDs (Carbon. 179 (2021) 692–700), and the UV absorption intensity of the solution is significantly reduced after adsorption (J. Mol. Struct. 1268 (2022) 133723). On the other hand, although the properties of the surface functional groups of CDs can be changed through oxidation, acidification, and chemical reaction functionalization, there are currently few reports on the modification of CDs surface groups and their application in dual-mode detection. Since dual-mode detection based on TMB and CDs is widely used in analytical science, modifying the surface of CDs to improve detection performance has good application value.

Contents of the invention

The technical problem solved by the present invention is to provide a method for preparing and processing carbon nanodots. The inventor found that after the carbon nanodots prepared and processed by this method are mixed with the TMB detection system, not only are there no effects on TMB or oxTMB after oxidation, but also Diimine oxTMB produces adsorption and can stabilize the oxidation products of TMB in the solution. In view of this, the present invention provides a method for preparing and processing carbon nanodots, which is characterized by including the following steps:

(1) Chop 15g of mango pulp and mix it with 10mL of triple distilled water. After high temperature reaction at 170℃, centrifuge at 9000 rpm for 10min to remove the precipitate. Then use a 0.45μm filter membrane to filter out large particulate matter. The filtrate is heated and dried at 70℃ to obtain carbon nanodots;

(2) Weigh 4 mg of carbon nanodots and disperse them in 500 μL of NaOH solution. Heat to 70°C and add ozone for treatment. Use an ultrafiltration membrane with a molecular weight cutoff of 50kDa to centrifuge and ultrafiltrate and take the filtrate. Use a filter with a molecular weight cutoff of 3kDa to take the filtrate. After centrifugal ultrafiltration of the ultrafiltration membrane, the upper layer of carbon nanodots is taken.

Preferably, the high-temperature reaction time in step (1) is 3 to 7 hours.

Preferably, the NaOH concentration in the NaOH solution described in step (2) is 50-200mM.

Preferably, the ozone treatment time described in step (2) is 1.5 to 3.5 hours.

Preferably, the flow rate of ozone introduced in step (2) is 4.0-8.0 mL/min.

The invention also provides a ratiometric fluorescence-colorimetric dual-mode detection method. The probe is composed of carbon nanodots and 3,3',5,5'-tetramethylbenzidine. When measured by the ratiometric fluorescence method, the probe is measured at 360 nm. and 500nm electromagnetic wave to excite the measurement solution. The measured emission intensity of the probe at 450nm and 540nm is I ₄₅₀ and I ₅₄₀ respectively. The sample to be measured is quantitatively analyzed with the ratio of fluorescence intensity I ₄₅₀ /I ₅₄₀ ; when measuring by colorimetric method, The UV absorbance A ₄₅₀ of the solution at 450 nm was used for quantitative analysis.

Preferably, the ratio of the mass concentrations of carbon nanodots and 3,3',5,5'-tetramethylbenzidine in the measured solution is (8-32): 1

Preferably, the pH value of the measurement solution is 1-2.

When the ratiometric fluorescence-colorimetric dual-mode detection probe constructed using the carbon nanodots provided by the invention is used for sarcosine detection, the detection limits are 0.046 and 0.0014 μM respectively, which is far superior to the existing technology.

Description of the drawings

Figure 1 TEM image of CDs.

Figure 2 CDs particle size distribution chart.

Figure 3 XPS spectrum of CDs before ozone treatment.

Figure 4 XPS spectrum of CDs after ozone treatment.

Figure 5 Fluorescence spectra of CDs before ozone treatment.

Figure 6 Fluorescence emission spectrum of CDs after ozone treatment.

Figure 7 (A) UV absorption spectra of measurement system 1 at different times; (B) Corresponding absorbance change curve with time.

Figure 8 (A) Measurement system 2 UV absorption spectra at different times; (B) Corresponding absorbance change curve with time.

Figure 9 (A) UV absorption spectra of measurement system 3 at different times; (B) Corresponding absorbance change curve with time.

Figure 10 (A) Fluorescence response of the system to different concentrations of sarcosine when excited at 360nm; (B) Fluorescence response of the system to different concentrations of sarcosine when excited at 500nm; (C) Corresponding ratio fluorescence linear relationship diagram.

Figure 11 (A) Colorimetric response of the system to different concentrations of sarcosine; (B) Corresponding linear relationship graph.

Detailed ways

The invention provides a method for preparing and processing carbon nanodots, which includes the following two steps:

(1) Chop 15g of mango pulp and mix it with 10mL of triple distilled water. After high temperature reaction at 170℃, centrifuge at 9000 rpm for 10min to remove the precipitate. Then use a 0.45μm filter membrane to filter out large particulate matter. The filtrate is heated and dried at 70℃ to obtain carbon nanodots;

(2) Weigh 4 mg of carbon nanodots and disperse them in 500 μL of NaOH solution. Heat to 70°C and add ozone for treatment. Use an ultrafiltration membrane with a molecular weight cutoff of 50kDa to centrifuge and ultrafiltrate and take the filtrate. Use a filter with a molecular weight cutoff of 3kDa to take the filtrate. After centrifugal ultrafiltration of the ultrafiltration membrane, the upper layer of carbon nanodots is taken.

Wherein, the high-temperature reaction time in step (1) is 3-7h; the NaOH concentration in the NaOH solution in step (2) is 50-200mM, the ozone treatment time is 1.5-3.5h, and the ozone treatment time is 1.5-3.5h. The flow rate of ozone is 4.0~8.0mL/min.

In addition, the present invention also provides a ratiometric fluorescence-colorimetric dual-mode detection method. The probe is composed of carbon nanodots and 3,3',5,5'-tetramethylbenzidine. When measured by the ratiometric fluorescence method, respectively The measurement solution is excited by electromagnetic waves of 360nm and 500nm, and the emission intensities of the probe at 450nm and 540nm are measured to be I ₄₅₀ and I ₅₄₀ respectively. The sample to be tested is quantitatively analyzed with the ratio of fluorescence intensity I ₄₅₀ /I ₅₄₀ ; when measuring by colorimetry, Quantitative analysis was performed by measuring the UV absorbance A ₄₅₀ of the solution at 450 nm.

The ratio of the mass concentrations of carbon nanodots to 3,3',5,5'-tetramethylbenzidine in the measurement solution is (8-32):1; the pH value of the measurement solution is 1-2.

In order to further understand the present invention, the preparation, processing method and application of the carbon nanodots provided by the present invention will be described in detail below with reference to the examples. The protection scope of the present invention is not limited by the following examples.

The experimental methods described in the following examples, unless otherwise specified, are all conventional methods; the reagents and materials, unless otherwise specified, can be purchased from commercial sources.

Example 1: Preparation of carbon nanodots

Peel off the skin of ripe mangoes purchased from the supermarket, remove the mango core, cut the mango flesh into small pieces of about 5 mm × 5 mm, and place them in a vacuum drying oven at 70°C at constant temperature until constant weight. Take 15.0g of mango pulp and put it into the inner tank of a 50mL polytetrafluoroethylene high-pressure reactor (Xi'an Instrument Factory, Xi'an), add 10mL of triple distilled water, tighten the high-pressure reactor, put it in an oven and heat it to 170°C, react for 3 hours and then naturally cool to room temperature. . Remove the contents with a medicine spoon and filter with gauze. Add the filtrate into a centrifuge tube to remove the precipitate, centrifuge at 9000 rpm for 10 min, take the supernatant, filter the supernatant with a 0.45 μM filter membrane (Jiuding High-tech, Beijing), and heat and dry the filtrate at 70°C to obtain the CDs product.

Example 2: Ozone treatment of carbon nanodots

Weigh 4 mg of carbon nanodots and disperse them in 500 μL of 50 mM NaOH solution. Put the solution into a 2 mL polypropylene centrifuge tube, place it in a 70°C water bath, and turn on the GN-S2S ozone generator (Anze Electronics, Anhui Province) to generate ozone was passed into the carbon nanodot solution at a flow rate of 4.0 mL/min. After treatment for 2 hours, perform ultrafiltration at 14,000 rpm. Ultrafiltration is performed in two steps. First, use an ultrafiltration membrane with a molecular weight cutoff of 50kDa to centrifuge and ultrafiltrate, then take the filtrate, and then filter the filtrate with an ultrafiltration membrane with a molecular weight cutoff of 3kDa. After membrane centrifugation and ultrafiltration, the upper layer of carbon nanodots was collected.

Example 3: Transmission electron microscopy characterization of carbon nanodots

The morphology of carbon nanodots was measured by a Talos F200S transmission electron microscope (TEM, Thermo Fisher Scientific Co., Ltd., USA). Before the test, the CDs product was dissolved in triple distilled water, treated with ultrasonic for 20 min, and then dropped on the ultra-thin carbon film. Analysis was performed after drying overnight at room temperature. The TEM results show that CDs are spherical (Figure 1). From the particle size distribution diagram (Figure 2), the particle size ranges from 4 to 11 nm, with an average particle size of 7.5 nm.

Example 4: X-ray photoelectron spectroscopy characterization of carbon nanodots

X-ray photoelectron spectroscopy (XPS) of carbon nanodots was characterized using an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific, Inc., USA). The XPS spectra of CDs before and after ozone treatment are shown in Figures 3 and 4 respectively. In the two spectra C1s spectrum, four peaks appeared at 284.8, 286.4, 287.9 and 289.3eV, corresponding to C-C or C-H(C1), C-O(C2), C=O or O-C-O(C3) and O-C=O(C4 ). After ozone treatment, the content of C1 increased from 33.3% to 39.3%, while the content of C2 increased from 51.2% to 46.5%, indicating that a small part of the hydroxyl groups on the surface of CDs were oxidized to C=O or O-C-O; in addition, the results showed that CDs The C4 content corresponding to the number of carboxyl groups on the surface decreases, indicating that ozone treatment causes the decomposition of oxygen-containing functional groups on the surface of carbon nanodots.

Example 5: Fluorescence spectrum characterization of carbon nanodots

The fluorescence properties were studied using a FS5 fluorescence spectrometer (Edinburgh Instruments Ltd., UK). CDs were dissolved in triple distilled water. The excitation wavelength range was 250 nm to 550 nm, and the emission wavelength range was 300 nm to 700 nm. Both the incident slit and the exit slit were 5nm. The experimental results (Figure 5 and Figure 6) show that the wavelength of the fluorescence emission spectrum of CDs does not change significantly before and after ozone treatment, and the shapes of the peaks are very similar. The difference is that the emission intensity increases significantly after treatment. The above phenomenon shows that the lattice defects on the surface of CDs increase after ozone treatment. It also shows that when CDs after ozone treatment are used as fluorescent probes, the detection sensitivity will be greatly improved.

Example 6: Effect of ozone treatment on dual-mode detection performance

This example takes the detection of sarcosine as an example, using CDs as the fluorescent probe and 3,3',5,5'-tetramethylbenzidine (TMB) as the colorimetric probe to illustrate the synthesis and processing of the present invention. Performance of CDs.

Synthesis of CDs: The steps are the same as in Example 1, except that during synthesis, the oven is heated to 170°C, and after 7 hours of reaction, it is naturally cooled to room temperature to obtain the initial CDs of this example.

For the CDs without ozone treatment used in this example, the obtained initial CDs need to be subjected to ultrafiltration at 14,000 rpm. The ultrafiltration is performed in two steps. First, use an ultrafiltration membrane with a molecular weight cutoff of 50kDa, centrifuge and ultrasonic. After filtration, take the filtrate, centrifuge and ultrafiltrate the filtrate with an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and then take the upper layer of carbon nanodots.

For the ozone-treated CDs used in this example, the initial CDs were treated with ozone. The steps were the same as in Example 2. The difference was that the NaOH concentration in the NaOH solution was 200mM. During ozone treatment, the flow rate was 8.0mL/min. The processing time is 1h.

Measurement system 1: Mix 150 μL of 10 μM sarcosine solution (dissolved in 50 mM phosphate buffer solution, pH 7.4) and 20 μL of 20 U/mL sarcosine oxidase solution, incubate at 37°C for 10 min, cool to room temperature, and then add 10 μL of 2mM sarcosine oxidase solution. TMB solution and 5 μL of 30 U/mL horseradish peroxidase solution were incubated at room temperature for 5 min in the dark, then 5 M hydrochloric acid was added to adjust the pH to 1.0 to terminate the reaction.

Measurement system 2: Add 5 μL of CDs without ozone treatment to the final solution of measurement system 1. At this time, the ratio of the mass concentrations of carbon nanodots to 3,3',5,5'-tetramethylbenzidine in the solution is 8:1.

Measurement system 3: Add 5 μL of ozone-treated CDs to the final solution of measurement system 1. At this time, the mass concentration ratio of carbon nanodots to 3,3',5,5'-tetramethylbenzidine in the solution is 8: 1.

The results of measurement system 1 show that diimine oxTMB, the final product of TMB oxidation, produces an obvious absorption peak at 450 nm (Figure 7A). However, the absorbance gradually decreases with time, from 0.17 at 0 min to 0.16 at 60 min (Figure 7B) .

The results of measurement system 2 show that after adding CDs without ozone treatment to the final solution of measurement system 1, the absorption peak at 450 nm of diimine oxTMB, the final product of TMB oxidation, is still clearly discernible (Figure 8A). However, the absorbance increases with the The burning time decreased rapidly, gradually decreasing from 0.14 at 0 min to 0.112 at 60 min (Figure 8 Figure 7B).

The results of measurement system 3 show that after adding ozone-treated CDs to the final solution of measurement system 1, the absorption peak at 450nm of diimine oxTMB, the final product of TMB oxidation, is still clearly discernible (Figure 9A), and the absorbance basically does not vary. It changes with time and remains between 0.157 and 0.158 in the time range from 0 min to 60 min (Figure 9B). It can be considered that there is no change within the allowable range of the measurement error.

The above experimental results show that when the CDs prepared by the present invention are used for fluorescence-colorimetric dual-mode detection after ozone treatment, they not only do not interfere with the detection of the contrastive colorimetric method, but also stabilize diimineoxTMB, the final product of TMB oxidation, thus extremely Dramatically improve detection performance.

Example 7: Ratio fluorescence-colorimetric dual-mode detection of sarcosine

Mix 150 μL of sarcosine solution of different concentrations (dissolved in 50mM phosphate buffer solution, pH 7.4) and 20 μL of 20 U/mL sarcosine oxidase solution, incubate at 37°C for 10 min, cool to room temperature, and then add 10 μL of 2mM sarcosine oxidase solution. TMB solution and 5 μL of 30 U/mL horseradish peroxidase solution were incubated for 5 minutes at room temperature in the dark, then 5 M hydrochloric acid was added to adjust the pH to 2.0 to terminate the reaction, and then 20 μL of ozone-treated CDs were added to obtain a measurement solution. At this time, the measurement solution The mass concentration ratio of carbon nanodots to 3,3',5,5'-tetramethylbenzidine is 32:1.

Quantitative analysis: When measuring by the ratiometric fluorescence method, the measurement solution is excited by electromagnetic waves of 360nm and 500nm respectively. The measured emission intensities of the probe at 450nm and 540nm are I ₄₅₀ and I ₅₄₀ respectively. The sample to be tested is measured based on the ratio of fluorescence intensity I ₄₅₀ /I ₅₄₀ . For quantification; when measuring by colorimetry, measure the UV absorbance A ₄₅₀ of the solution at 450nm for quantification.

The experimental results show that when the sarcosine concentration increases from 0 to 30 μM, I ₄₅₀ and I ₅₄₀ gradually decrease (Figure 10A, Figure 10B), and the fluorescence ratio I ₄₅₀ /I ₅₄₀ has a good linear relationship with the concentration (Figure 10C). The detection limit of the fluorescence ratiometric method is 0.046 μM. During colorimetric detection, the absorbance of the system at 450nm gradually increased as the sarcosine concentration increased (Figure 11A), showing a good linear relationship in the range of 0 to 30 μM. The detection limit of the colorimetric method was 0.0014 μM.

After literature search, the detection limit of sarcosine of the present invention is better than the current reports (Table 1).

Table 1 Detection limits of sarcosine in the prior art

Note, the corresponding references in the table:

[1]X.Zhang,W.Xiao,S.Xie,G.Fan,X.Shi,H.Meng,H.Yang,Low-density Ptnanoarray-based hydrogen peroxide sensing platform and its application intrace sarcosine detection,Electrochimica Acta .443(2023)141952.https://doi.org/10.1016/j.electacta.2023.141952.

[2] L.P.T.Carneiro, A.M.F.R.Pinto, M.G.F.Sales, Development of aninnovative flexible paper-based methanol fuel cell (PB-DMFC) sensing platform–Application to sarcosine detection, Chemical Engineering Journal.452(2023)139563.https://doi .org/10.1016/j.cej.2022.139563.

[3]Porphyrin-Containing Metallacage with Precise Active Sites andSuper Long-Term Stability as a Specific Peroxidase Mimic for VersatileAnalyte Determination|Analytical Chemistry,(n.d.).https://pubs.acs.org/doi/10.1021/acs.analchem. 2c03206 (accessed June 5, 2023).

[4] M.Wang, L.Zhang, X.Zhou, J.Zhang, C.Zhou, 340188. https://doi.org/10.1016/j.aca.2022.340188.

[5]Q.Liu,S.Cao,Q.Sun,C.Xing,W.Gao,X.Lu,X.Li,G.Yang,S.Yu,Y.Chen,Aperylenediimide modified SiO2@TiO2 yolk- shell light-responsive nanozyme: Improved peroxidase-like activity for H2O2 and sarcosine sensing, Journal of Hazardous Materials.436(2022)129321.https://doi.org/10.1016/j.jhazmat.2022.129321.

[6]X.Lin,M.Tian,C.Cao,T.Shu,Y.Wen,L.Su,X.Zhang,Using bimetallic Au/Cunanoplatelets for construction of facile and label-free inner filter effect-based photoluminescence sensing platform for sarcosine detection,AnalyticaChimica Acta.1192(2022)339331.https://doi.org/10.1016/j.aca.2021.339331.

[7]Z.Ramezani,M.Safdarian,A.A.Ghadiri,Metal-coded hydrogel magneticmolecularly imprinted polymer for preconcentration and cleanup of sarcosine:Determination in urine; coupled to on-column capillary electrophoresis,Talanta.230(2021)122309.https: //doi.org/10.1016/j.talanta.2021.122309.

[8]H.Shen,H.Shi,B.Feng,C.Ding,S.Yu,A versatile biomimetic multienzymecascade nanoplatform based on boronic acid-modified metal–organic framework for colorimetric biosensing,J.Mater.Chem.B.10 (2022)3444–3451. https://doi.org/10.1039/D2TB00158F.

[9] M. Masumoto, S. Ohta, M. Nakagawa, Y. Hiruta, D. Citterio, Colorimetric paper-based sarcosine assay with improved sensitivity, Anal Bioanal Chem. 414 (2022) 691–701. https://doi. org/10.1007/s00216-021-03682-0.

[10]J. de Cássia LRda Rocha,TBCapelari,MCPrete,PNAngelis,MGSegatelli,CRTTarley,Design and performance of novelmolecularly imprinted biomimetic adsorbent for preconcentration of prostatecancer biomarker coupled to electrochemical determination by using multi-walled carbon nanotubes//> /Ni(OH)2-modified screen-printed electrode,Journal of Electroanalytical Chemistry.878(2020)114582.https://doi.org/10.1016/j.jelechem.2020.114582.

The above embodiments are only used to help understand the method and its core idea of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (8)

1. A method for preparing and treating carbon nanodots, comprising the steps of:

(1) Cutting 15g mango pulp, mixing with 10mL of triple distilled water, reacting at 170 ℃ for 10min, removing sediment by 9000 r/m separating core, filtering large-particle substances by using a 0.45 mu m filter membrane, and heating and drying filtrate at 70 ℃ to obtain carbon nano-dots;

(2) 4mg of carbon nano dots are weighed and dispersed in 500 mu L of NaOH solution, ozone is introduced for treatment after the solution is heated to 70 ℃, the filtrate is obtained after centrifugal ultrafiltration by an ultrafiltration membrane with the molecular weight cutoff of 50kDa, and the upper layer of carbon nano dots is obtained after centrifugal ultrafiltration of the filtrate by an ultrafiltration membrane with the molecular weight cutoff of 3 kDa.

2. The method according to claim 1, wherein the high temperature reaction time in step (1) is 3 to 7 hours.

3. The method according to claim 1, wherein the NaOH concentration in the NaOH solution in step (2) is 50-200 mM.

4. The method of claim 1, wherein the ozone is introduced in step (2) for a treatment time of 1 to 2 hours.

5. The method of claim 1, wherein the flow rate of ozone introduced in step (2) is 4.0-8.0 mL/min.

6. A ratio fluorescence-colorimetry dual-mode detection method comprises a probe consisting of carbon nanodots and 3,3', 5' -tetramethylbenzidine, wherein when the ratio fluorescence method is used for measuring, electromagnetic waves of 360nm and 500nm are respectively used for exciting measuring solutions, and the emission intensities at 450nm and 540nm of the probe are respectively I ₄₅₀ And I ₅₄₀ At a ratio of fluorescence intensities I ₄₅₀ /I ₅₄₀ Quantitatively analyzing a sample to be detected;

in colorimetric measurement, the ultraviolet absorbance A at 450nm of the solution is measured ₄₅₀ Quantitative analysis was performed.

7. The probe of claim 6, wherein the ratio of the mass concentration of the carbon nanodots to the mass concentration of 3,3', 5' -tetramethylbenzidine in the measurement solution is (8 to 32): 1.

8. the probe of claim 6, wherein the pH of the measurement solution is 1 to 2.