Preparation method of iron-nitrogen co-doped graphene quantum dot fluorescent probe and rapid detection method of imipenem based on the same

Abstract

The present invention discloses a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe, wherein an iron compound, graphene, citric acid, and m-phenylenediamine are used as precursors, and the iron-nitrogen co-doped graphene quantum dot fluorescent probe is synthesized by hydrothermal reaction. The present invention also discloses a rapid detection method for imipenem based on this fluorescent probe, wherein the iron-nitrogen co-doped graphene quantum dot fluorescent probe is used as a detection molecule, a phosphate buffer solution is used as a buffer solution, and different concentrations of imipenem are added; a standard linear curve is drawn. The Fe-doped graphene quantum dot fluorescent probe prepared by the present invention has good selectivity and high sensitivity, and can accurately and specifically identify the imipenem concentration in the imipenem-cilastatin sodium drug. This method can be used for real-time analysis and detection of imipenem content in the blood of critically ill patients such as clinical sepsis. Compared with the existing high-performance liquid chromatography, it can realize rapid bedside detection by dropping blood, and 5-10 samples can be measured per minute.

Description

Technical Field

[0001] The invention belongs to the field of functional material preparation, relates to the preparation of Fe-doped graphene quantum dots and the application of Fe-doped graphene quantum dots in imipenem drug detection, and specifically relates to a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe. Background Art

[0002] There are two problems in the use of antibiotics in ICU patients with severe infection and septic shock: (1) rapid analysis of antibiotic concentrations in the blood to provide timely feedback on the anti-infection effect and avoid overuse of drugs and sequelae of poisoning; (2) appropriate dosage for different patients with severe infection, and the pharmacokinetics of antibiotics for different cases are required; antibiotics commonly used in intensive care units include Tylenol, which mainly contains imipenem and cilastatin sodium. Among them, imipenem is a carbapenem antibiotic, an atypical β-lactam antibiotic with a broad antibacterial spectrum and strong antibacterial activity. It is mainly used for the treatment of multidrug-resistant Gram-negative bacilli infections, severe mixed infections of aerobic and anaerobic bacteria, and severe infections with unidentified pathogens. With the increasing number of fungal infections, their clinical manifestations lack specificity, which can easily lead to misdiagnosis and missed diagnosis, delaying the best treatment time, or excessive medical treatment due to fungal preventive treatment. The blood concentration of imipenem is measured in patients with severe infection. The main measurement methods currently include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. These methods have strong specificity and are currently the most reported methods for measuring the blood concentration of imipenem. However, these methods have high requirements for instruments and operators, have long measurement cycles, and cannot achieve real-time monitoring. Imipenem has become one of the important drugs for the treatment of severe infections. It is necessary to establish a simple, rapid, stable, and reliable high-throughput analytical detection method to achieve therapeutic drug monitoring of imipenem during the treatment of patients with severe infections, adjust the dosage in a timely manner, and ensure the safety and effectiveness of the medication.

[0003] Graphene quantum dots (GQDs) usually have a lateral size of less than 100 nm and a thickness of less than 10 nm. This nanoscale size produces a size-dependent quantum confinement effect. Affected by the edge effect, when combined with the surrounding chemical functional groups, stable photoluminescence can be produced. This results in GQDs not only having optical properties, but also a series of excellent physical and chemical properties such as chemical inertness, low cell toxicity, and good biocompatibility. Compared with semiconductor quantum dot materials, it is less harmful to the environment. At the same time, the surface chemical structure can be changed by element doping, thereby exhibiting specific molecular recognition properties. It has been widely studied in the fields of optical sensing, biomedicine, photocatalysis, etc.

[0004] The current method for detecting the concentration of imipenem in patients has problems in practical applications, such as long measurement time and inability to obtain test results quickly. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe, using Fe and N co-doped quantum dots to obtain a fluorescent probe molecule with obvious selective recognition ability for imipenem. By using the fluorescence quenching mechanism of Fe, N-GQDs probe molecules, the blood drug imipenem concentration in the blood of critically ill patients can be simply and quickly detected, which is of great significance for clinical medical detection.

[0006] To achieve the above-mentioned purpose, the technical solution of the present invention is to design a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe, which includes the following preparation steps performed in sequence: using iron compounds, graphene, citric acid, and m-phenylenediamine as precursors, and synthesizing the iron-nitrogen co-doped graphene quantum dot fluorescent probe by hydrothermal reaction after ultrasonic dispersion.

[0007] A further technical solution is to centrifuge and filter the hydrothermal synthesized product through a filter membrane to obtain an iron-nitrogen co-doped graphene quantum dot probe molecule solution.

[0008] A further technical solution is that the iron compound is ferric chloride, ferric nitrate or ferric acetate.

[0009] A further technical solution is that the concentration ratio of each component in the precursor is: iron compound, graphene, citric acid, and meta-phenylenediamine is (1-5): (10-15): (1-2): (1-2) mg / ml.

[0010] A further technical solution is that the ultrasonic dispersion time is 30 minutes, the hydrothermal reaction is carried out in a reactor, the hydrothermal reaction temperature is 180° C., and the hydrothermal reaction time is 12 hours.

[0011] A further technical solution is that the rotation speed of the centrifugal treatment is 10000 rpm and the centrifugal time is 3 minutes; the filter membrane used in the filtration treatment is a sterile microbial analysis filter membrane, the filter membrane sheet diameter is 47 mm, and the pore size of the filter membrane is 0.22 μm.

[0012] The present invention also provides a technical solution, which is a method for rapid detection of imipenem by a fluorescent probe prepared by a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe, comprising the following detection steps: using the iron-nitrogen co-doped graphene quantum dot fluorescent probe as a detection molecule, and using 0.1 mol.L -1Phosphate buffer solution was used as the buffer solution, and different concentrations of imipenem were added; 330nm was used as the excitation wavelength, the PL fluorescence intensity was measured at 431nm, and a standard linear curve was drawn; the concentration of the iron-nitrogen co-doped graphene quantum dot fluorescent probe was 1-100mg / ml, and the measured linear range of imipenem concentration was 2-60μg / ml. After the blood sample to be tested was centrifuged, 500μl of plasma was taken and added to the PBS solution (1.5ml) of the iron-doped graphene quantum dot fluorescent probe molecule, and oscillated for 5s, and the PL fluorescence intensity was measured under the same conditions. Three measurements were performed to calculate the relative standard deviation (RSD)

[0013] A further technical solution is that the pH of the buffer solution is 7.4, and the preparation method of the buffer solution is: weigh 8g NaCl, 0.2g KCl, 12g Na2HPO4, 3.63g H2O, and 0.24g KH2PO4, dissolve them in 900mL distilled water, and add water to make up to 1L.

[0014] The advantages and beneficial effects of the present invention are: high efficiency, sensitivity, rapidity, and rapid bedside detection can be achieved, and 5-10 samples can be measured per minute. The Fe-doped graphene quantum dot fluorescent probe prepared by the present invention has good selectivity and high sensitivity, and can accurately and specifically identify the imipenem concentration in the imipenem-cilastatin sodium drug. This method can be used for real-time analysis and detection of imipenem content in the blood of critically ill patients such as clinical sepsis. Compared with the existing high-performance liquid chromatography, it can achieve rapid bedside detection by dropping blood, and 5-10 samples can be measured per minute. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a fluorescence emission spectrum of iron-nitrogen co-doped graphene quantum dots prepared in Example 1 of a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe of the present invention;

[0016] Figure 2 is a standard curve diagram of imipenem concentration-fluorescence intensity in Example 1 of the present invention;

[0017] Figure 3 is a standard curve diagram of imipenem concentration-fluorescence intensity in Example 2 of the present invention;

[0018] Figure 4 is a graph showing the change in PL intensity of the imipenem solution according to Example 3 of the present invention as a function of storage time;

[0019] Figure 5 This is a mechanism diagram of the present invention using iron-nitrogen co-doped graphene quantum dot fluorescent probe molecules to achieve fluorescence quenching for recognition of the imipenem drug. DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention. Embodiment 1

[0021] The present invention is a method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe. Ferric chloride, graphene, citric acid, and m-phenylenediamine are weighed and dissolved in 30 ml of distilled water. The mass concentration ratio is 1:10:1:1, and the mass concentration unit is mg / ml, that is, the masses of ferric chloride, graphene, citric acid, and m-phenylenediamine are 30 mg, 300 mg, 30 mg, and 30 mg, respectively. After ultrasonic dispersion for 30 minutes, the mixture is added to a reactor and subjected to hydrothermal reaction at 180°C for 12 hours. The product is centrifuged (centrifugal speed 10,000 rpm, centrifuged for 3 minutes), and filtered with a filter membrane (sterile microbial analysis filter membrane, Φ47 mm×0.22 μm) to obtain an iron-nitrogen co-doped graphene quantum dot probe molecular solution. The fluorescence emission spectrum of the prepared iron-nitrogen co-doped graphene quantum dots is shown in the attached figure. Figure 1 (EX is the excitation spectrum, 310nm is the excitation spectrum, the curve on the right side of the figure is the emission spectrum, the excitation spectrum only provides one curve, and the others are emission spectra obtained after different excitation spectra). Its PL spectrum shows that with 330nm as the excitation wavelength, the PL fluorescence intensity is the strongest at 431nm.

[0022] Take 20 μl of the above iron-nitrogen co-doped graphene quantum dot fluorescent probe molecular solution and add 10 ml of PBS buffer solution {with 0.1 mol.L -1 Phosphate buffer solution (PBS) was used as the buffer solution (pH = 7.4, weighed 8g NaCl, 0.2g KCl, 12g Na2HPO4, 3.63g H2O, 0.24g KH2PO4, dissolved in 900mL distilled water, and fixed to 1L with water)}, and different concentrations of imipenem were added. With 330nm as the excitation wavelength, the PL fluorescence intensity was measured at 431nm, and the standard linear curve was drawn, see the attached Figure 2 . Its linear range is 2-60μg / ml, and the linear correlation coefficient is 0.998. After centrifuging the blood sample to be tested, 500μl of the upper plasma clear liquid was taken and added to the PBS solution (1.5ml) of iron-nitrogen co-doped graphene quantum dot fluorescent probe molecules, and the PL fluorescence intensity was measured under the same conditions. The concentration of imipenem in the blood sample to be tested was calculated to be 10.3μg / ml. The recovery rate test was carried out using a spiked sample of 10μg / ml, and the recovery rate was 97.3%. The same sample was measured using HPLC (High Performance Liquid Chromatography\HPLC), and the concentration of imipenem was 9.7μg / ml.

[0023] Determination principle: Fe ions in the iron-nitrogen co-doped graphene quantum dot fluorescent probe molecules are used to induce terminal imino complexation reaction, produce fluorescence quenching effect, and realize the recognition of imipenem drugs. See the schematic diagram in the instruction manual Figure 5 . Embodiment 2

[0024] The difference from Example 1 is that iron nitrate, graphene, citric acid, and m-phenylenediamine are weighed and dissolved in 30 ml of distilled water in a mass ratio of 5:15:2:2 mg / ml. After ultrasonic dispersion, they are added to a reactor and subjected to hydrothermal reaction at 180°C for 12 h. The product is centrifuged and filtered to obtain an iron-doped graphene quantum dot probe molecule solution.

[0025] 50 μl of the above iron-doped graphene quantum dot probe molecule solution was taken, 10 ml PBS was added as buffer solution, and different concentrations of imipenem were added. The PL fluorescence intensity was measured at 431 nm with an excitation wavelength of 330 nm, and a standard linear curve was drawn. The linear range was 1-40 μg / ml, and the linear correlation coefficient was 0.996, see attached Figure 3 . Blood samples from patients undergoing intestinal resection were centrifuged and 500 μl of the upper plasma clear layer was added to a PBS solution (1.5 ml) of iron-nitrogen co-doped graphene quantum dot fluorescent probe molecules. The PL fluorescence intensity was measured under the same conditions. The imipenem concentration in the blood sample to be tested was calculated to be 37 μg / ml. The recovery rate was tested using a spiked sample of 20 μg / ml, and the recovery rate was 95.3%. Embodiment 3

[0026] The difference from Example 1 is that iron acetate, graphene, citric acid, and m-phenylenediamine are weighed and dissolved in 30 ml of distilled water in a mass ratio of 2:12:1:2 mg / ml. After ultrasonic dispersion, they are added to a reactor and subjected to hydrothermal reaction at 180°C for 12 h. The product is centrifuged and filtered with a filter membrane to obtain an iron-nitrogen co-doped graphene quantum dot probe molecule solution.

[0027] Pipette 100μl of the above iron-nitrogen co-doped graphene quantum dot fluorescent probe molecule solution, add 10ml PBS as buffer solution, and add different concentrations of imipenem-cilastatin sodium. With 330nm as the excitation wavelength, the PL fluorescence intensity is measured at 431nm, and a standard linear curve is drawn. The linear range is 1-20μg / ml, and the linear correlation coefficient is 0.916. Take another blood sample from a patient with small intestinal perforation tumor and centrifuge it. Take 500μl of the upper plasma clear liquid and add it to the PBS solution (1.5ml) of iron-nitrogen co-doped graphene quantum dot fluorescent probe molecule, and measure the PL fluorescence intensity under the same conditions. The change of PL intensity of this mixed solution within 5 minutes is shown in the attached figure. Figure 4(IMI in the figure is the standard imipenem solution with a concentration of 5μg / ml), indicating that the quenching of PL fluorescence intensity by imipenem-cilastatin sodium responds quickly and remains stable within 300s. The imipenem concentration in the blood sample to be tested was calculated to be 12μg / ml. The recovery rate test was performed using a spiked sample of 10μg / ml, and the recovery rate was 96.9%.

[0028] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe, characterized in that: The method comprises the following preparation steps which are carried out in sequence: using iron compound, graphene, citric acid and m-phenylenediamine as precursors, performing ultrasonic dispersion and hydrothermal reaction to synthesize iron-nitrogen co-doped graphene quantum dot fluorescent probes.

2. The method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to claim 1, characterized in that: The hydrothermal synthesized product is centrifuged and filtered through a filter membrane to obtain an iron-nitrogen co-doped graphene quantum dot probe molecule solution.

3. The method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to claim 2, characterized in that: The iron compound is ferric chloride, ferric nitrate or ferric acetate.

4. The method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to claim 3, characterized in that: The concentration ratio of each component in the precursor is: iron compound, graphene, citric acid, and m-phenylenediamine is (1-5): (10-15): (1-2): (1-2) mg / ml.

5. The method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to claim 4, characterized in that: The ultrasonic dispersion time is 30 min, the hydrothermal reaction is carried out in a reactor, the hydrothermal reaction temperature is 180° C., and the hydrothermal reaction time is 12 h.

6. The method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to claim 5, characterized in that: The rotation speed of the centrifugal treatment is 10000 rpm and the centrifugal time is 3 minutes; the filter membrane used in the filtration treatment is a sterile microbial analysis filter membrane, the filter membrane sheet diameter is 47 mm, and the pore size of the filter membrane is 0.22 μm.

7. A method for rapid detection of imipenem using a fluorescent probe prepared by the method for preparing an iron-nitrogen co-doped graphene quantum dot fluorescent probe according to any one of claims 1 to 6, characterized in that: The detection steps include: using iron-nitrogen co-doped graphene quantum dot fluorescent probe as the detection molecule, and using 0.1 mol.L -1 A phosphate buffer solution was used as the buffer solution, and different concentrations of imipenem were added; 330 nm was used as the excitation wavelength, the PL fluorescence intensity was measured at 431 nm, and a standard linear curve was drawn; wherein the concentration of the iron-nitrogen co-doped graphene quantum dot fluorescent probe was 1-100 mg / ml, and the measured linear range of imipenem concentration was 2-60 μg / ml.

8. The rapid detection method for imipenem according to claim 7, characterized in that: The pH of the buffer solution is 7.

4. The preparation method of the buffer solution is as follows: weigh 8 g of NaCl, 0.2 g of KCl, 12 g of Na2HPO4, 3.63 g of H2O, and 0.24 g of KH2PO4, dissolve them in 900 mL of distilled water, and add water to make the volume to 1 L.

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