CN114044511A - Preparation method and application of beer-based nitrogen-phosphorus co-doped biomass carbon dots - Google Patents

Abstract

The invention discloses a preparation method of beer-based nitrogen-phosphorus co-doped biomass carbon dots, which comprises the following steps of 1: filtering the beer to obtain beer filtrate; adding beer filtrate into a reaction kettle, carrying out hydrothermal carbonization to obtain a light yellow solution, and naturally cooling to room temperature; step 2: the light yellow solution cooled to room temperature in the step 1 is subjected to membrane filtration and centrifugation, and the centrifuged supernatant is dialyzed in pure water to obtain dialysate; and step 3: and (3) freeze-drying and freeze-drying the dialysate obtained in the step (2) to obtain powdery beer-based nitrogen-phosphorus co-doped biomass carbon dots. The invention also provides an application method of the composition. The preparation method is simple, biomass carbon dots can be obtained by performing one-step hydrothermal carbonization synthesis on the beer filtrate, and the method is simple and easy to implement, low in cost and high in yield, and raw materials are cheap and easy to obtain, and the method is suitable for batch production.

Description

Preparation method and application of beer-based nitrogen-phosphorus co-doped biomass carbon dots

Technical Field

The invention belongs to the fields of fluorescent carbon nanomaterials, biosensing technology and food analysis, and relates to a preparation method and application of beer-based nitrogen-phosphorus co-doped biomass carbon dots.

Background

Iron is a necessary trace element for human bodies, and iron deficiency of the human bodies can cause a plurality of diseases such as anemia and resistance reduction; ascorbic acid is one of the most important water-soluble vitamins, plays an important role in the growth, metabolism and development processes of a human body as an important micronutrient and antioxidant, and can cause severe scurvy due to insufficient intake of the ascorbic acid; ingestion from the diet is an important way to maintain iron and ascorbic acid content in the body; therefore, the method has important significance for monitoring and evaluating the iron element and the ascorbic acid in the diet.

At present, many methods for detecting iron ions and ascorbic acid have been reported, such as atomic absorption method, inductively coupled plasma mass spectrometry/spectrometry, high performance liquid chromatography, electrochemical method, and the like; however, these methods have problems of expensive instruments, complicated operation, long detection time and the like, and limit the application in daily food detection.

The carbon dots are a novel zero-dimensional carbon nano material, have rich functional groups on the surface, have excellent fluorescence performance, and also have the advantages of good biocompatibility, simple and mild preparation steps, easy surface modification and the like; the development of rapid detection technology using carbon dots as fluorescent probes is one of the important research contents in the field of analysis and detection.

The existing carbon dot synthesis methods mainly comprise a laser ablation method, an electrochemical method, a pyrolysis method, an acid oxidation method, a microwave method, an ultrasonic method and a hydrothermal method; however, most of the precursors for preparing the carbon dots are from inorganic materials or organic small molecules, and have the problems of certain toxicity, low environmental friendliness, defects in light-emitting characteristics and the like.

The preparation raw materials of the carbon dots mainly come from carbon nanotube, fullerene, graphene and other large-volume carbon skeleton materials and citric acid, glucose and other organic compounds. However, with the development of society and the advancement of mankind, the introduction of green chemistry concept, the large volume of carbon skeleton material is gradually eliminated, and the use of non-toxic reagents and natural products for preparing carbon dots is explored. As a natural raw material, biomass materials are gradually attracting attention due to their advantages of being renewable, low in pollution, wide in sources, and the like.

However, the untreated pure carbon quantum dots have low fluorescence quantum yield, simple physicochemical properties, and lack functionality, and cannot meet the requirements in practical applications. In order to promote the application, carbon quantum dots doped with heteroatoms are developed; the doping of the heteroatom can effectively introduce a band gap, is beneficial to forming more defects on the surface of the carbon quantum dot, and further adjusts the reactivity, electronic structure and optical property of the carbon quantum dot. Nitrogen, phosphorus, sulfur, silicon, boron, and manganese are common heteroatoms in carbon quantum dot doping; for the doping of heteroatoms in carbon quantum dots, a common strategy is to add a compound containing the atom, such as nitrogen-doped carbon quantum dots, the preparation raw materials of which are usually doped with urea, ammonia water and ethylenediamine; however, achieving doping by adding exogenous compounds would undoubtedly increase the operation and cost of the experiment.

Patent document (CN109880619A) discloses a method for detecting fe (iii) ions by using nitrogen-doped lignocellulosic biomass-based carbon quantum dots, but the preparation raw material needs to undergo a series of tedious operations such as milling, screening, enzymolysis and the like; in addition, single nitrogen doping has a certain disadvantage in detection performance compared with multi-element doped carbon dots.

Patent document (CN110791289A) provides a method for preparing nitrogen and phosphorus co-doped biomass-based carbon quantum dots by using defibered absolutely dry bleached needle-leaved pulp as a raw material and adding diammonium hydrogen phosphate; the process requires the additional addition of inorganic diammonium phosphate as a nitrogen source and a phosphorus source.

Beer is a common liquid beverage and contains rich substances such as nitrogen, phosphorus and the like; if the carbon dots are prepared by adopting the beer, the high-temperature carbonization can be directly carried out without a series of complicated pretreatment steps like the solid raw materials; and can directly obtain multi-element doped carbon dots, and can remarkably improve the performance and application value of the carbon dots.

Disclosure of Invention

Aiming at the defects of the existing preparation method, the invention provides a preparation method of beer-based nitrogen-phosphorus co-doped biomass carbon dots, which specifically comprises the following steps:

step 1: filtering the beer to obtain beer filtrate; adding beer filtrate into a reaction kettle, carrying out hydrothermal carbonization to obtain a light yellow solution, and naturally cooling to room temperature;

step 2: the light yellow solution cooled to room temperature in the step 1 is subjected to membrane filtration and centrifugation, and the centrifuged supernatant is dialyzed in pure water to obtain dialysate;

and step 3: and (3) freeze-drying and freeze-drying the dialysate obtained in the step (2) to obtain powdery beer-based nitrogen-phosphorus co-doped biomass carbon dots.

Preferably, the temperature of the hydrothermal carbonization in the step 1 is between 100 ℃ and 250 ℃; the time for hydrothermal carbonization is between 4 hours and 24 hours.

Preferably, the microporous membrane in step 2 is a microporous membrane, and the pore size of the microporous membrane is between 0.1 micron and 0.5 micron.

Preferably, the centrifugation speed of the centrifugation in the step 2 is 5000 to 15000 revolutions per minute; the centrifugation time of the centrifugation is 10 to 20 minutes.

Preferably, the dialysis in the step 2 adopts a dialysis bag of 500Da to 1000Da, and the dialysis time of the dialysis is 12 hours to 18 hours.

Preferably, the temperature for lyophilization and lyophilization in step 3 is from-10 ℃ to-105 ℃.

The invention also provides a method for determining Fe in beer-based nitrogen-phosphorus co-doped biomass carbon dots³⁺And ascorbic acid.

The invention also discloses a method for detecting Fe³⁺The concentration method specifically comprises the following steps:

step S01, adding carbon dots of the beer-based nitrogen-phosphorus co-doped biomass into a phosphate buffer solution with the concentration of 5 mmol to 20 mmol;

step S02, adding the buffer solution obtained in step S01 to Fe³⁺Incubating for 3 to 5 minutes in a standard solution;

step S03, setting the excitation wavelength of a fluorescence spectrometer to be 360-380 nm, and then measuring the fluorescence intensity of the incubated solution in the step S02;

step S04, drawing and fitting the fluorescence intensity data obtained in the step S03 to obtain a linear regression equation, and detecting Fe in the food extracting solution through the linear regression equation³⁺The content of (a).

The invention also discloses a method for detecting the concentration of ascorbic acid, which comprises the following steps:

step S11, adding carbon dots of the beer-based nitrogen-phosphorus co-doped biomass into a phosphate buffer solution with the concentration of 5 mmol to 20 mmol;

step S12, adding the buffer solution obtained in step S01 to Fe³⁺Incubating for 3-5 minutes in a standard solution to prepare the switch type beer-based nitrogen-phosphorus co-doped biomass carbon dot-Fe for detecting the ascorbic acid³⁺A sensor solution;

step S13, adding the sensor solution obtained in the step S12 into an ascorbic acid standard solution, and incubating for 5 to 10 minutes;

step S14, setting the excitation wavelength of a fluorescence spectrometer to be 360-380 nm, and then measuring the fluorescence intensity of the incubated solution in the step S13;

and step S15, drawing and fitting according to the fluorescence intensity data obtained in the step S14 to obtain a linear regression equation, and detecting the content of the ascorbic acid in the food extracting solution through the linear regression equation.

Compared with the prior art, the invention has the beneficial effects that:

1. the carbon dots prepared by the method have the advantages of green, environmental protection and low biological toxicity; and the fluorescence intensity and the fluorescence quantum yield are high, and a selective and sensitive switch type fluorescence sensing system is provided and can be used for Fe³⁺And a novel method for detecting ascorbic acid.

2. The synthetic method is simple; the beer biomass carbon dots can be obtained by performing one-step hydrothermal carbonization synthesis on the beer filtrate, and the method is simple and easy to implement, low in cost and high in yield, and raw materials are cheap and easy to obtain, and the method is suitable for batch production.

3. The invention has low cytotoxicity and high fluorescence quantum yield: the biomass carbon dots prepared from the beer filtrate are green and environment-friendly raw materials, so that the carbon dots have low cytotoxicity and high biocompatibility; in addition, the biomass carbon dots are rich in surface functional groups and high in fluorescence intensity and fluorescence quantum yield.

4. The biomass carbon dot has good fluorescence emission property, and Fe is used for detecting the ascorbic acid based on the biomass carbon dot³⁺When added into biomass carbon point solution, Fe³⁺The fluorescence of the biomass carbon dots is quenched due to the electrostatic effect of the adsorption to the surfaces of the biomass carbon dots, and the intensity of the fluorescence quenching is matched with the Fe³⁺The concentration of (A) is in a linear relationship and can be used for Fe³⁺Sensitive and selective detection of; at biomass carbon point-Fe³⁺Ascorbic acid is added into the composite system, and the ascorbic acid converts Fe due to oxidation-reduction reaction³⁺Reduction to Fe²⁺So that the fluorescent particles are separated from the surface of the biomass carbon dots to cause the fluorescent recovery of the biomass carbon dots, the degree of the fluorescent recovery of the biomass carbon dots is gradually enhanced along with the increase of the concentration of the ascorbic acid, and the fluorescent intensity of the biomass carbon dots and the concentration of the ascorbic acid are in a linear relation, so that the method can be used for sensitive and selective detection of the ascorbic acid.

5. The invention has low detection cost and easy popularization; the method does not need to be marked, does not modify carbon points at all, simplifies the synthesis steps, reduces the detection cost, and has the advantages of simple preparation process, mild reaction conditions and easy popularization.

6. The invention has high detection sensitivity and high selectivity: the invention adopts a switch-type fluorescence detection mode, and utilizes carbon dots and Fe³⁺The system can obviously improve the detection sensitivity by strong specific combination; can effectively shield other interfering substances in the detection of the ascorbic acid, and has high selectivity.

7. The invention has strong practicability: the detection method disclosed by the invention is simple to prepare, convenient to operate and practical in practical application.

Drawings

FIG. 1 is a TEM photograph of a carbon dot of biomass prepared in example 1 of the present invention.

FIG. 2 shows fluorescence excitation and fluorescence emission spectra of biomass carbon dots prepared in example 1 of the present invention.

FIG. 3 shows the carbon points of biomass at different concentrations of Fe³⁺Fluorescence spectrum of (2).

FIG. 4 is a fluorescence spectrum of a complex of biomass carbon dots and iron with ascorbic acid at various concentrations.

FIG. 5 is a graph of selectivity for ascorbic acid detection.

Detailed Description

The technical solution of the present patent will be described in further detail with reference to the following embodiments.

Example one

The embodiment provides a preparation method of a beer-based nitrogen-phosphorus co-doped biomass carbon dot, which specifically comprises the following steps:

filtering beer with microporous membrane of 0.22 μm in 100 ml of commercially available beer to obtain beer filtrate;

adding 50 ml of beer filtrate into a Teflon-lined stainless steel high-pressure reaction kettle, sealing the reaction kettle, putting the reaction kettle into a forced air drying oven, performing hydrothermal carbonization for 6 hours at 160 ℃ to obtain a light yellow solution, and naturally cooling to room temperature;

passing the light yellow solution through a 0.22 micron microporous membrane, and centrifuging; dialyzing the centrifuged supernatant in pure water for 12 hours, wherein the molecular cut-off is 500Da, and obtaining dialysate;

and freeze-drying the obtained dialysate at-80 ℃ to obtain powdery beer-based nitrogen-phosphorus co-doped biomass carbon dots.

Referring to fig. 1, the effect of the prepared product under an electron microscope is shown.

Referring to fig. 2, the prepared product has a good luminous effect.

Under the condition that other conditions are not changed, only the temperature of hydrothermal carbonization is changed, and the temperature is respectively as follows: obtaining fluorescence intensity data (excitation wavelength: 370 nm; emission wavelength: 450nm) of corresponding biomass carbon dots at 110 ℃, 130 ℃, 150 ℃, 170 ℃ and 190 ℃, specifically:

as can be seen from the above table, the temperature is controlled between 160 ℃ and 170 ℃, preferably.

Under the condition that other conditions are not changed, only the hydrothermal carbonization time is changed, and the hydrothermal carbonization time is respectively as follows: 4 hours, 5 hours, 7 hours, 8 hours, 12 hours, corresponding yield data were obtained, specifically:

as can be seen from the above table, the reaction time is controlled to about 6 hours, preferably about 6 hours

Example two

The embodiment discloses a method for detecting Fe by using beer-based nitrogen-phosphorus co-doped biomass carbon dots³⁺The method specifically comprises the following steps: adding 1 ml of beer-based nitrogen-phosphorus co-doped biomass carbon point dialysate prepared in the second example and 300 microliters of phosphate buffer solution (with the concentration of 10 mmol and the pH value of 7.4) into a centrifugal tube in sequence, and uniformly mixing;

preparing Fe at a concentration of 0 to 400 micromoles per liter³⁺Adding the mixed buffer solution into Fe with different concentrations³⁺Removing the standard solution, shaking uniformly, and standing for 5 minutes;

the solution fluorescence spectrum was measured for each standard solution with an excitation wavelength of 370 nm.

FIG. 3 shows different concentrations of Fe³⁺And (3) quenching effect of the standard solution on the fluorescence intensity of the beer-based nitrogen-phosphorus co-doped biomass carbon dots. The linear regression equation is obtained as y-0.0013 x +0.4431, and the linear correlation coefficient R²0.9931 for Fe³⁺The detection limit is 0.14 micromole per liter, and Fe can be realized³⁺Sensitive detection of (3).

EXAMPLE III

The embodiment discloses a method for detecting ascorbic acid by using a beer-based nitrogen-phosphorus co-doped biomass carbon dot, which specifically comprises the following steps:

adding 1 ml of beer-based nitrogen-phosphorus co-doped biomass carbon point dialysate prepared in the second example and 300 microliters of phosphate buffer solution (with the concentration of 10 mmol and the pH value of 7.4) into a centrifugal tube in sequence, and uniformly mixing;

preparing Fe at a concentration of 0 to 500 micromoles per liter³⁺Several parts of standard solution as fluorescence quenching agent, adding the above mixed buffer solution into Fe with different concentrations³⁺Removing the standard solution, shaking uniformly, and standing for 5 minutes;

preparing a plurality of portions of ascorbic acid standard solution with concentration of 0-300 micromole per liter to be used as a fluorescence recovery agent, adding the solution after fluorescence quenching into ascorbic acid standard solutions with different concentrations after the carbon point fluorescence quenching of the beer-based nitrogen-phosphorus co-doped biomass reaches balance, shaking uniformly, and incubating for 5-10 minutes at room temperature;

the excitation wavelength was set at 370nm for fluorescence measurement.

As can be seen from FIG. 4, the fluorescence intensity of B-N, P-CQDs is gradually increased with the increase of the ascorbic acid concentration, the right graph is a linear relationship graph between the ascorbic acid concentration and the fluorescence intensity, and the ascorbic acid concentration is in a good linear relationship with the fluorescence enhancement intensity of the system in the range of 1 to 200 micromole per liter. The linear regression equation is that y is 1.201+0.0153x, and the linear correlation coefficient R²At 0.995, the detection line for ascorbic acid was 0.84 micromoles per liter, allowing sensitive detection of ascorbic acid.

FIG. 5 shows the interference effect of some other common amino acids (Arg, Val, Pro, Gly, GSH, Ser, His) and organic substances (AA, TA, Cys) on the system, and it can be seen that the fluorescence intensity of the system is not affected by these interference substances, which indicates that the method of the present invention has good selectivity for ascorbic acid detection.

Claims (9)

1. A preparation method of beer-based nitrogen and phosphorus co-doped biomass carbon dots is characterized by comprising the following steps:

step 1: filtering the beer to obtain beer filtrate; adding beer filtrate into a reaction kettle, carrying out hydrothermal carbonization to obtain a light yellow solution, and naturally cooling to room temperature;

step 2: the light yellow solution cooled to room temperature in the step 1 is subjected to membrane filtration and centrifugation, and the centrifuged supernatant is dialyzed in pure water to obtain dialysate;

and step 3: and (3) freeze-drying and freeze-drying the dialysate obtained in the step (2) to obtain powdery beer-based nitrogen-phosphorus co-doped biomass carbon dots.

2. The method of claim 1, wherein:

the temperature of hydrothermal carbonization in the step 1 is between 100 ℃ and 250 ℃; the time for hydrothermal carbonization is between 4 hours and 24 hours.

3. The method of claim 1, wherein:

in the step 2, the microporous membrane body is a microporous membrane, and the pore size of the microporous membrane is between 0.1 and 0.5 microns.

4. The method of claim 1, wherein:

the centrifugal speed of the centrifugation in the step 2 is 5000 to 15000 revolutions per minute; the centrifugation time of the centrifugation is 10 to 20 minutes.

5. The method of claim 1, wherein:

in the step 2, a dialysis bag of 500Da to 1000Da is adopted for dialysis, and the dialysis time of the dialysis is 12 hours to 18 hours.

6. The method of claim 1, wherein:

the temperature for freeze-drying and freeze-drying in the step 3 is-10 ℃ to-105 ℃.

7. The method for determining Fe of carbon dots of beer-based nitrogen-phosphorus co-doped biomass obtained by the preparation method according to claims 1 to 6³⁺And ascorbic acid.

8. Detecting Fe³⁺A method of concentration characterized by:

step S01, adding carbon dots of the beer-based nitrogen-phosphorus co-doped biomass into a phosphate buffer solution with the concentration of 5 mmol to 20 mmol;

step S02, adding the buffer solution obtained in step S01 to Fe³⁺Incubating for 3 to 5 minutes in a standard solution;

step S03, setting the excitation wavelength of a fluorescence spectrometer to be 360-380 nm, and then measuring the fluorescence intensity of the incubated solution in the step S02;

step S04, drawing and fitting the fluorescence intensity data obtained in the step S03 to obtain a linear regression equation, and detecting Fe in the food extracting solution through the linear regression equation³⁺The content of (a).

9. A method of detecting ascorbic acid concentration, comprising:

step S11, adding carbon dots of the beer-based nitrogen-phosphorus co-doped biomass into a phosphate buffer solution with the concentration of 5 mmol to 20 mmol;

step S12, adding the buffer solution obtained in step S01 to Fe³⁺Incubating for 3-5 minutes in a standard solution to prepare the switch type beer-based nitrogen-phosphorus co-doped biomass carbon dot-Fe for detecting the ascorbic acid³⁺A sensor solution;

step S13, adding the sensor solution obtained in the step S12 into an ascorbic acid standard solution, and incubating for 5 to 10 minutes;

step S14, setting the excitation wavelength of a fluorescence spectrometer to be 360-380 nm, and then measuring the fluorescence intensity of the incubated solution in the step S13;

and step S15, drawing and fitting according to the fluorescence intensity data obtained in the step S14 to obtain a linear regression equation, and detecting the content of the ascorbic acid in the food extracting solution through the linear regression equation.

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