CN114350352A

CN114350352A - Novel carbon material based on coffee beans and method for detecting lead ions and PPi

Info

Publication number: CN114350352A
Application number: CN202111570633.2A
Authority: CN
Inventors: 刘意; 严志红; 姚微; 冯冰洁; 何晓婷; 汪子琨
Original assignee: Meijianji Bioengineering Technology Guangzhou Co ltd; Guangdong Pharmaceutical University
Current assignee: Meijianji Bioengineering Technology Guangzhou Co ltd; Guangdong Pharmaceutical University
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-04-15
Anticipated expiration: 2041-12-21
Also published as: CN114350352B

Abstract

The invention discloses a novel carbon material based on coffee beans and a method for detecting lead ions and PPi, and belongs to the fields of nanoscale carbon material technology and fluorescence detection application thereof. The method comprises the following steps: (1) dissolving coffee bean powder and sodium hydroxide in water to obtain a solution A; (2) carrying out hydrothermal reaction on the solution A at a high temperature to obtain a solution B; (3) dialyzing the solution B to obtain a solution C; (4) filtering and drying the solution C to obtain CQDs. The carbon quantum dots synthesized by the one-step hydrothermal method are used as the fluorescent probe, the fluorescent probe can detect Pb2+ through a fluorescence quenching effect with high sensitivity, and then detect PPi through a fluorescence recovery phenomenon, and the method has the advantages of being green and simple in synthesis method, rapid and convenient to detect, accurate in result and high in application value in the field of biomedicine.

Description

Novel carbon material based on coffee beans and method for detecting lead ions and PPi

Technical Field

The invention belongs to the field of nano-scale carbon material technology and fluorescence detection application thereof, and particularly relates to a novel coffee bean-based carbon material and a method for detecting lead ions and PPi.

Background

Heavy metal pollution seriously threatens the environment and human health, and how to effectively monitor the concentration of toxic heavy metal ions in the environment becomes an increasingly important problem. Lead is one of the most common toxic heavy metals, is widely applied to the fields of pigments, water pipes, storage batteries, anticorrosive coatings, alloys and the like, and is widely present in various fields of the environment. At the same time, due to Pb²⁺Non-biodegradable, Pb when exposed to contaminated air and water sources²⁺Easily accumulate in the human neuro-and cardiovascular system. When Pb is contained in blood²⁺When the concentration is higher than 5. mu.M, anemia, reproductive dysfunction, nervous system dysfunction and developmental disorder may be caused, while too high Pb content may be caused²⁺The concentration may even lead to death. Therefore, it is necessary to establish a rapid and sensitive trace amount of Pb²⁺And (3) a measuring method.

Anions are ubiquitous throughout biological systems and play an important role in a wide range of chemical and biological processes. As an anion, pyrophosphate (PPi) is released upon hydrolysis of adenosine triphosphate to adenosine monophosphate under cellular conditions. It is part of the bioenergy cycle and DNA synthesis and also an inhibitor of several crystallization reactions. Therefore, in recent years, many efforts have been made to develop non-biological sensors of PPi, which have become important in medicine, biology, environment, and nutrition.

Pb²⁺Many conventional methods of determination, such as atomic absorptionSpectroscopy (AAS), Anodic Stripping Voltammetry (ASV), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and inductively coupled plasma mass spectroscopy (ICP-MS). However, these methods typically require expensive and complex instrumentation, time consuming and complex sample pre-treatment procedures. On the other hand, among the numerous PPi sensors reported so far, only a few examples are fluorescent sensors capable of efficiently detecting PPi in a 100% PPi aqueous solution. Most reports focus on their behavior in non-aqueous solvents, and most of these methods are based on the synthesis of small organic molecular probes, are complex and time-consuming, and require organic solvents as reaction media.

In recent years, Carbon Quantum Dots (CQDs) have attracted great interest to researchers as a new kind of fluorescent nanoparticles. The CQDs are generally spherical structures, have particle diameters of less than 10nm, have strong and stable fluorescence, and have excellent water solubility, good light stability, super-strong biocompatibility and low toxicity compared to organic dyes, quantum dots and noble metal nanoparticles, so that the carbon quantum dots can be effectively applied to the practical applications of chemical sensors and biosensors. According to literature research, the preparation of CQDs by hydrothermal method using different raw materials as carbon sources (such as citric acid, leaves, lemon, etc.) has the advantages of simplicity, easy operation, low cost, etc., and is one of the most common methods for preparing CQDs at present, and the synthesized CQDs usually have hydroxyl, carboxyl or amino on the surface, and can coordinate with metal ions and heavy metal ions to cause the change of fluorescence.

Based on the urgent need of preparing novel carbon quantum dots, the method takes coffee beans as raw materials, interacts with alkali (NaOH) with certain concentration, synthesizes the carbon quantum dots by a one-step hydrothermal method, and takes the carbon quantum dots as a fluorescent probe to rapidly detect Pb²⁺And PPi.

Disclosure of Invention

The invention aims to provide a novel carbon material based on coffee beans and detection of Pb²⁺PPi method, in particular for detecting Pb based on the fluorescent 'on-off' effect²⁺And PPi method, which uses coffee bean as raw material, and uses carbon quantum dot synthesized by one-step hydrothermal method as fluorescent probeThe needle can detect Pb with high sensitivity by the fluorescence quenching effect²⁺And PPi is detected by fluorescence recovery, so that the method has the advantages of green and simple synthesis method, rapid and convenient detection and accurate result, and solves the problems of high equipment cost, complex operation and the like of the traditional lead ion and pyrophosphate detection method.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, the present invention provides a method for preparing a novel carbon material based on coffee beans, comprising the steps of:

(1) dissolving coffee bean powder and sodium hydroxide in water to obtain a solution A;

(2) reacting the solution A at a high temperature to obtain a solution B;

(3) dialyzing the solution B to obtain a solution C;

(4) filtering and drying the solution C to obtain CQDs.

Preferably, in step (1), the coffee bean powder is obtained by pulverizing coffee beans by means of grinding, crushing, micronizing, jet milling, etc.

Preferably, in the step (1), the mass ratio of the coffee bean powder to the sodium hydroxide is 2-4:7.2 to 16.8.

In this reaction, the action of the sodium hydroxide solution as a catalyst and a decomposing agent is crucial for the preparation of the final product. The sodium hydroxide not only can degrade macromolecules (such as protein, cellulose and the like) in the coffee beans under alkaline conditions so as to provide raw materials for generating carbon quantum dots, but also participates in a series of chemical reactions of carbon sources for generating quanta. The process is that polymers (polysaccharides, proteins and the like) contained in coffee can be catalytically decomposed to form amine and carboxylic acid micromolecule carbon-containing compounds under the high-temperature condition containing sodium hydroxide, and the obtained micromolecule carbon-containing substances react under the high-temperature and high-pressure condition to form carbon quantum dots.

Preferably, in the step (1), the mass ratio of the coffee bean powder to the water is 1: 50-200.

Preferably, in step (1), the water is pure water.

Preferably, in step (1), it is sufficiently dissolved by sonication.

Preferably, in the step (2), the reaction temperature is 150-200 ℃, and the reaction time is 240-480 min.

Preferably, step (2) is performed in a polytetrafluoroethylene lining in the reaction stainless steel kettle.

Preferably, in the step (3), the dialysis is performed in a dialysis bag of pure water and 1000-12000D, and the dialysis time is 24-72 h.

Preferably, in step (4), the drying manner is freeze drying.

In still another aspect, the present invention provides a novel coffee bean-based carbon material prepared according to the above preparation method.

In still another aspect, the present invention provides the novel carbon material based on coffee beans as described above for detecting Pb²⁺And use in PPi.

In another aspect, the present invention provides a method for rapidly detecting Pb²⁺The method comprises the following steps:

s1, dissolving the CQDs in ultrapure water to obtain solution D, mixing the solution D with ultrapure water, and testing the fluorescence intensity of the solution D to be F₀；

S2, mixing Pb with different concentrations²⁺Respectively mixing the solution with the solution D to obtain mixed solutions, and performing fluorescence test on the mixed solutions to respectively obtain fluorescence intensity values F of the mixed solutions;

with Pb²⁺Concentration is abscissa, and fluorescence quenching rate is 1-F/F₀Linear fitting is performed for the ordinate to obtain a regression equation y₁＝k₁x₁+b₁Wherein y is₁As fluorescence quenching rate, x₁Is Pb²⁺Concentration, k₁The value is the slope, b₁The value is the intercept;

s3, mixing the sample to be detected with the solution D to obtain a mixed solution; testing the fluorescence intensity value of the obtained mixed solution to further obtain the fluorescence quenching rate, and substituting the fluorescence quenching rate into the linear regression equation y in the step S2₁＝k₁x₁+b₁Obtaining Pb²⁺The concentration of (c).

Preferably, in the step S1, the mass concentration of the solution D is 5-10 mg/ml, and the mixing ratio of the solution D and the ultrapure water is 1: 39.

Preferably, in step S2, the Pb is²⁺The concentration range of the solution is 12-260 mu M.

Preferably, in step S2, Pb²⁺The solutions were mixed with solution D at a ratio of 39:1, respectively.

Preferably, in step S3, the mass ratio of the sample to be tested to the solution D is 29-49: 1, and most preferably 39: 1.

Finally, the invention provides a method for rapidly detecting PPi, which comprises the following steps:

s1, dissolving the CQDs obtained by the preparation method in ultrapure water to obtain solution D.

S2, adding Pb²⁺Mixing the solution with the solution D to obtain a mixed solution E, and performing fluorescence test on the mixed solution E to obtain a fluorescence intensity value F₀(ii) a Adding PPi with different concentrations into the mixed solution E, performing fluorescence test to obtain a fluorescence intensity value F, performing linear fitting by taking the concentration of PPi as a horizontal coordinate and the fluorescence growth rate, namely F/F0-1, as a vertical coordinate to obtain a regression equation y₂＝k₂x₂+b₂Wherein y is₂As the rate of increase in fluorescence, x₂Is the concentration of PPi, k₂The value is the slope, b₂The value is the intercept;

s3, mixing the sample to be tested with Pb²⁺Mixing the solution and the solution D to obtain a mixed solution, testing the fluorescence intensity value of the mixed solution, and substituting the fluorescence intensity value into the linear regression equation y in the step S2₂＝k₂x₂+b₂The concentration of PPi was obtained.

Preferably, in the step S1, the mass concentration of the solution D is 5-10 mg/ml.

Preferably, in step S2, Pb is²⁺The concentration of the solution is 12-260 mu M.

Preferably, in step S2, the concentration range of the PPi with different concentrations is 37.5-125 μ M.

Preferably, in step S2, Pb²⁺Solutions andsolution D was mixed at a ratio of 39: 1.

Preferably, in step S3, the sample to be tested and Pb²⁺The solution and the solution D were mixed at a ratio of 38:1: 1.

Preferably, in step S3, the Pb is²⁺The concentration was 10 mM.

The invention has the beneficial effects that:

1. the invention is used for Pb based on the carbon quantum dot 'on-off' fluorescence effect²⁺And the detection scheme of PPi content has the advantages of high sensitivity, good selectivity, less spectral interference, lower cost and the like, and the carbon quantum dots are synthesized by taking coffee beans as raw materials, so that the synthesis scheme is green and environment-friendly, has good biocompatibility and has the potential of being used for biological imaging.

2. The carbon quantum dots prepared by the technical scheme of the invention have the advantages of excellent dynamic and thermodynamic properties, good physical and chemical stability, good water solubility, good biocompatibility, small harm to the environment, high sensitivity to lead ions and the like.

3. According to the invention, coffee beans are used as raw materials, carbon quantum dots synthesized by a one-step hydrothermal method are used as fluorescent probes, and the synthesis is green and simple.

Drawings

FIG. 1 CQDs of products of various examples and comparative examples were examined for Pb²⁺A contrast plot of sensitivity;

FIG. 2 FTIR plots of CQDs that are products of example 1;

FIG. 3 is a histogram of hemolysis rates of CQDs;

FIG. 4 depicts detection of Pb by CQDs before pretreatment²⁺A histogram of selectivity of;

FIG. 5 is detection of Pb by CQDs after pretreatment²⁺A histogram of selectivity of;

FIG. 6 shows the change in fluorescence intensity of CQDs with Pb²⁺A graph of the relationship between concentrations;

FIG. 7 is a graph of the intensity change in recovery of fluorescence from CQDs versus PPi concentration;

FIG. 8 is a bar graph of the selectivity of the CQDs detection PPi.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. The following is merely an exemplary illustration of the scope of the invention as claimed, and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which also fall within the scope of the invention as claimed.

The present invention will be further described below by way of specific examples. The various chemicals used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.

In the following examples, the coffee bean powder was obtained by grinding coffee beans.

Example 1

1) Dissolving 3g of coffee bean powder in 30mL of pure water, and adding 7.2g (6mM) of sodium hydroxide to obtain a mixed solution A;

2) transferring the solution A into a polytetrafluoroethylene lining, and then putting the polytetrafluoroethylene lining into a stainless steel reaction kettle to react for 480min at 200 ℃ to obtain a solution B;

3) transferring the solution B into a 12000D dialysis bag, and dialyzing for 48h to obtain a solution C;

4) and (3) carrying out suction filtration on the solution C by using an organic filter membrane with the diameter of 0.22 mu m, freezing for 4h at the temperature of minus 60 ℃, extracting, vacuum drying for 12h to obtain brown-black powdery CQDs, and storing for later use at the temperature of 2-8 ℃.

Example 2

The same as in example 1, except that 2g of coffee bean powder was added.

Example 3

Preparation method of carbon quantum dots referring to example 1, the amount of sodium hydroxide was varied, and the specific steps were as follows:

the same as in example 1, except that 12g of sodium hydroxide was added.

Example 4

In contrast to example 1, solution A was transferred to a Teflon liner and placed in a stainless steel reactor to react at 150 ℃ for 480min to give solution B, the rest being the same.

Example 5

The dialysis bag had a specification of 1000D, which was the same as that of example 1.

Comparative example 1

The same as in example 1, except that no sodium hydroxide was added.

And (3) testing the sensitivity:

the CQDs, the products of the above examples and comparative examples, were prepared as a 10mg/mL D solution, the D solution and pure water were mixed at a ratio of 1:39, and the initial fluorescence intensity was measured and recorded as F₀Then the solution D is mixed with 250 μ M Pb²⁺The solutions were mixed at a ratio of 1:39 and the fluorescence intensity after quenching was measured and recorded as F. By F/F₀The value of (A) is a detection index of sensitivity (FIG. 1), i.e., F/F₀The lower the value of (A), the higher the degree of quenching and the higher the sensitivity. It can be seen that the CQDs prepared in comparative example 1 were less effective in implementation. The results were combined to use the product CQDs of example 1 as the detection material of the subsequent detection example 1 and detection example 2.

CQDs detection of Pb²⁺The selectivity test of (2):

the final concentration of the various metal ions involved in the experiment was designed to be 250 μ M for selectivity testing, and it can be seen that Cu²⁺、Hg²⁺、Fe³⁺For Pb²⁺Has interference, but Cu is treated with glutathione and ascorbic acid as chelating agents and reducing agents²⁺、Hg²⁺、Fe³⁺The post-interference disappeared, with a final concentration of metal ions of 50. mu.M, glutathione of 0.08mg/mL and ascorbic acid of 0.5 mg/mL. FIGS. 4-5 show that CQDs vs. Pb²⁺The detection effect is stable and not affected, and the selectivity is high.

Detection example 1

The CQDs obtained in the above example 1 of the present invention are used for detecting the lead ion content by fluorescence spectroscopy, and the method comprises the following steps:

1) dissolving powder CQDs in ultrapure water to obtain 10mg/mL D solution, mixing the D solution and pure water at a ratio of 1:39, and testing fluorescence intensity of 10100.5 and F₀；

2) The concentration is 12, 35, 55, 86, 110, 130, 170, 210, 260. mu.M Pb²⁺Respectively mixing the solution with the solution D according to the ratio of 39:1 to respectively obtain mixed solutions, and carrying out fluorescence test on the mixed solutions to obtain fluorescence intensity values of 9578.3, 8856.2, 8415.5, 8171.5, 6977.9, 6860.2, 5649.7, 4660.6 and 3270.5 which are marked as F;

with Pb²⁺Concentration as abscissa, fluorescence quenching rate (1-F/F)₀) Linear fitting is performed for the ordinate to obtain a regression equation y₁＝0.00241x₁-0.0321, wherein y₁As fluorescence quenching rate, x₁Is Pb²⁺The concentration of (c);

3) mixing a lake water sample to be detected containing lead ions (50 mu M) with a certain concentration with the solution D according to the ratio of 39:1 to obtain a mixed solution; performing fluorescence test, and substituting the obtained fluorescence intensity value into the linear regression equation y₁＝0.00241x₁0.0321, the average concentration of lead ions was calculated to be 51.7 μ M (n ═ 3), and the average recovery was calculated to be 103.4% (n ═ 3). The excitation wavelength of fluorescence in the fluorescence test used was 370 nm.

Detection example 2

The CQDs obtained in the above example 1 of the present invention are used for detecting the PPi content by fluorescence spectroscopy, and the method comprises the following steps:

1) dissolving powdery CQDs in ultrapure water to prepare a 10mg/mL solution D;

2) 250 μ M of Pb²⁺Mixing the solution with the solution D according to the ratio of 39:1 to obtain a mixed solution E, and performing fluorescence test on the mixed solution E to obtain a fluorescence intensity value of 2070.2, which is recorded as F₀(ii) a Mixing the PPi solution and the solution E at a ratio of 1:39 of 37.5, 50, 62.5, 75, 87.5, 100, 112.5 and 125 mu M, and performing fluorescence test to obtain fluorescence intensity values of 2243.3, 2733.5, 3227.4, 3676.8, 4360.9, 4866.4, 5358.7 and 5679.2, wherein the fluorescence intensity values are represented as F, the PPi concentration is used as an abscissa, and the fluorescence increase rate (F/F) is used as an abscissa₀-1) is the ordinate, linear fitting is performed to obtain the regression equation y₂＝0.02051x₂+0.7250 wherein y₂As the rate of increase in fluorescence, x₂Is the concentration of PPi;

3) the solution containing PPi to be tested (50 μ M) and Pb²⁺Mixing the solution (10mM) and the solution D according to the ratio of 38:1:1 to obtain PPi-Pb-CQDs mixed solution; performing fluorescence test to obtain fluorescence intensity value, substituting into linear regression equation y₂＝0.02051x₂+0.7250, the average PPi concentration was calculated to be 50.6. mu.M with an average recovery of 101.2%.

It can be seen that the method of the present invention has the advantages of accurate and reliable test results and convenient and rapid test steps.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of a novel carbon material based on coffee beans is characterized by comprising the following steps:

(2) reacting the solution A at a high temperature to obtain a solution B;

(3) dialyzing the solution B to obtain a solution C;

(4) filtering and drying the solution C to obtain CQDs.

2. The production method according to claim 1, wherein in the step (1), the mass ratio of the coffee bean powder to the sodium hydroxide is 2-4:7.2-16.8, and the mass ratio of the coffee bean powder to the water is 1: 50-200.

3. The method as claimed in claim 1, wherein the step (2) is performed in a polytetrafluoroethylene lining in the reaction stainless steel reactor, the temperature of the reaction is 150 ℃ and 200 ℃, and the reaction time is 240 ℃ and 480 min.

4. The method as claimed in claim 1, wherein the dialysis in step (3) is carried out in a dialysis bag of pure water, 1000-12000D for a dialysis time of 24-72 hours.

5. A novel coffee bean-based carbon material produced by the production method according to any one of claims 1 to 4.

6. Use of the novel coffee bean-based carbon material according to claim 5 for detecting Pb²⁺And use in PPi.

7. Rapid detection Pb²⁺The method is characterized by comprising the following steps:

s1, dissolving CQDs prepared by the preparation method of any one of claims 1 to 4 in ultrapure water to obtain solution D, mixing the solution D and ultrapure water, and testing the fluorescence intensity of the solution D as F₀；

8. The method according to claim 7, wherein in the step S1, the mass concentration of the solution D is 5-10 mg/ml, and the mixing ratio of the solution D and the ultrapure water is 1: 39; in step S2, Pb is²⁺The concentration range of the solution is 12-260 mu M; in step S2, Pb²⁺Mixing the solution with the solution D at a ratio of 39: 1; in step S3, the sample and the solution to be testedD, mass ratio of the mixed materials is 29-49: 1.

9. a method for rapidly detecting PPi is characterized by comprising the following steps:

s1, dissolving CQDs prepared by the preparation method of any one of claims 1 to 4 in ultrapure water to obtain solution D;

s2, adding Pb²⁺Mixing the solution with the solution D to obtain a mixed solution E, and performing fluorescence test on the mixed solution E to obtain a fluorescence intensity value F₀(ii) a Adding PPi with different concentrations into the mixed solution E, performing fluorescence test to obtain fluorescence intensity value F, and taking the concentration of PPi as abscissa, wherein the fluorescence growth rate is F/F₀-1 is a vertical coordinate, linear fitting is carried out to obtain a regression equation y₂＝k₂x₂+b₂Wherein y is₂As the rate of increase in fluorescence, x₂Is the concentration of PPi, k₂The value is the slope, b₂The value is the intercept;

10. The method according to claim 9, wherein in step S1, the mass concentration of solution D is 5-10 mg/ml; in step S2, Pb is²⁺The concentration of the solution is 12-260 mu M; in step S2, the concentration range of the PPi with different concentrations is 37.5-125 μ M; in step S2, Pb²⁺Mixing the solution with the solution D at a ratio of 39: 1; in step S3, the sample to be tested and Pb²⁺Mixing the solution and the solution D according to the ratio of 38:1: 1; in step S3, Pb is²⁺Is 10 mM.