CN110577213B - Dual-functionalized graphene quantum dot and preparation method and application thereof - Google Patents
Dual-functionalized graphene quantum dot and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-functionalized graphene quantum dot and a preparation method and application thereof, and belongs to the technical field of biosensors. According to the invention, citric acid: histidine: and D-penicillamine is in a molar ratio of 1:0.1: 0.01-1: 1:1, and the bifunctional graphene quantum dot DPA-GQD-His is prepared by a hydrothermal method. The double-functionalized graphene quantum dot can be used for quantitatively detecting single or multiple organophosphorus pesticides with high sensitivity and high selectivity, and is simple in preparation method and easy to popularize.
Description
Technical Field
The invention relates to a dual-functionalized graphene quantum dot and a preparation method and application thereof, and belongs to the technical field of biosensors.
Background
Organophosphorus pesticides are typical nerve agents and the mechanism of toxicity of organophosphorus pesticides is generally recognized by those skilled in the art as: the organophosphorus pesticide is combined with acetylcholine (ACh) in cholinergic nerve synapses to inhibit the activity of acetylcholinesterase (AChE) so that ACh cannot be hydrolyzed, and the ACh is accumulated in large quantity in synaptic gaps to generate toxic effect, which is also the main insecticidal mechanism of the organophosphorus pesticide. The common exposure ways of acute poisoning of organophosphorus pesticides include skin contact, oral administration, respiration and the like, the incubation period is different from 30min to 2h, the common clinical symptoms include dizziness, nausea, vomiting, salivation, sweating, blurred vision, dyspnea, muscle tension reduction and the like, and in severe cases, the symptoms of coma, pulmonary edema and liver dysfunction and the like can also appear. Therefore, the development of a method for sensitively and rapidly determining the organophosphorus pesticide has important significance on agricultural safety and environmental monitoring. In fact, a single pesticide may not exceed a threshold level, and the simultaneous exposure of low doses of multiple pesticides may interact in the human body to increase or decrease toxicity with respect to each other, thereby causing a combined effect that is different from the single toxicity. Therefore, the development of the research on the combined toxicity effect of the organophosphorus pesticides is very necessary for the safe use of the pesticides and the guarantee of human health.
The combined toxicity test model mainly comprises an in vivo animal model and an in vitro cell model. In past pesticide toxicology studies, live animals were the most commonly used test model. Among them, mammals such as rats and mice have the advantages of intuitive and visible morphological index changes, convenience for physiological overall function evaluation, similarity of pathological pharmacological metabolism and human bodies (such as genes close to the human bodies), capability of extrapolating test results to the human bodies and the like, so that the method is one of the commonly adopted methods in pesticide combined toxicity evaluation. With the continuous and deep research, people find that not all animal tests can accurately reflect the harm of pesticides to human bodies, particularly in combined toxicity evaluation, a large number of test samples and data are accumulated, and animal tests have the defects of time consumption, labor consumption, high cost, multiple uncontrollable factors and the like. The in vitro cell test is a study which separates cells from the body and is carried out under certain conditions, the in vitro cell culture technology has the advantages of rapidness, simplicity, low cost and the like, and the cell test is used for replacing and verifying animal test results and becomes a common method for evaluating pesticide safety in recent years. At present, ex vivo cell tests are widely used in the research of neurotoxicity, genetic toxicity, immunotoxicity and endocrine disrupting effects of pesticide mixtures.
Photoluminescent nanomaterials such as metal fluorescent nanoclusters, semiconductor Quantum Dots (QDs), and organic fluorescent dyes have become a research hotspot due to their unique optical properties. Semiconductor Quantum Dots (QDs) have outstanding properties such as high fluorescence Quantum Yield (QY), narrow spectrum, good chemical stability, high photobleaching resistance, tunable polychromatic emission and excellent optical properties. However, the QDs generally contain heavy metal elements such as Cd, Pb, and Hg, and their commercial application is limited due to the harmful effects of heavy metals on organisms and the environment; metallic fluorescent nanoclusters and organic fluorescent dyes have high fluorescence, but the poor photobleaching resistance and poor chemical stability of such materials also limit their applications. Graphene Quantum Dots (GQDs) are quasi-zero-dimensional nanomaterials, with particle sizes typically on the order of a few nanometers. GQD has the advantages of excellent optical stability, chemical inertness, photobleaching resistance, low cytotoxicity, biocompatibility and the like, so that GQD is expected to become a promising fluorescent probe in certain chemical and biological analyses.
The prior art reports that an electrochemical luminescence system is constructed by using sulfur and nitrogen co-doped carbon points as a luminophor and a reaction accelerator and is used for detecting the content of atrazine in a water body, but the electrochemical sensing system has high sensitivity, so that the detection result is easily influenced by the detection environment, and the electrochemical signal is unstable. In the prior art, silver nano-ion modified carbon points are used as a substrate for surface enhanced Raman resonance, and a target induces the aggregation of the carbon points to enhance the SERS signal of rhodamine 6G so as to quantitatively detect the atrazine, but the introduction of the silver nano-ions increases the biotoxicity of the carbon points, so that the biocompatibility of the carbon points is weakened, and the method is not suitable for determining the atrazine content in organisms. The prior art also reports that the acetamiprid content in a real sample is successfully and quantitatively detected by utilizing the internal light filtering effect between the nano-gold and the carbon dots, but the detection sensitivity is not enough.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
The graphene quantum dots prepared by the prior art have low fluorescence emission intensity, and the graphene quantum dots are used for optical detection to reduce the detection sensitivity; G-quadruplex/DNase, as a mimetic peroxidase, has a limited intrinsic peroxide activity, thereby limiting the sensitivity of detection.
[ technical solution ] A
In order to solve the technical problems, the invention provides the bifunctional graphene quantum dot and the preparation method and application thereof.
Firstly, the invention provides a preparation method of a bifunctional graphene quantum dot, which comprises the following steps: according to the weight ratio of citric acid: histidine: the bifunctional graphene quantum dot DPA-GQD-His (D-penicillamine-histidine graphene quantum dot) is prepared by a hydrothermal method according to a molar ratio of 1:0.1: 0.01-1: 1:1 of D-penicillamine.
In one embodiment of the present invention, the operating parameters of the hydrothermal process are: reacting for 0.5-10 h at 150-250 ℃.
In one embodiment of the invention, the method specifically comprises the steps of mixing D-penicillamine, histidine, citric acid and water according to a molar ratio to obtain a mixed solution, transferring the mixed solution into a reaction kettle, and reacting at 150-250 ℃ for 0.5-10 h; after the reaction is finished, adjusting the pH value of the obtained solution to 7.0-8.0, dialyzing in a dialysis bag for 10-72h, and drying the solution in the dialysis bag after the dialysis is finished to obtain DAP-GQD-His.
In one embodiment of the present invention, the dialysis bag has a specification of 1 to 10 KD.
The invention further provides the dual-functionalized graphene quantum dot DPA-GQD-His prepared by the method.
The invention further provides application of the bifunctional graphene quantum dot DPA-GQD-His in detection of acetamiprid and organophosphorus pesticides.
The fourth and the invention provides a method for quantitatively detecting acetamiprid, which comprises the following steps: (1) dissolving DPA-GQD-His in phosphate buffer solution with pH of 6.5-7.4, wherein the mass concentration of DPA-GQD-His is 0.01-1 mg/mL;
(2) mixing 10-100. mu.L of 0.1-10. mu.M DNA sequence shown as sequence SEQ ID NO.1 and 10-100. mu.L of 0.1-10. mu.M DNA sequence shown as sequence SEQ ID NO.2 in 100. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl. sub.2And 0.1mM EDTA) and incubated at room temperature for 20-60 min;
(3) adding acetamiprid with different concentrations into the mixed solution obtained in the step (2), oscillating, and standing at 20-30 ℃ for 20-40 min; adding 10-50 μ L of 25-50mM KCl into the above solution; 30-60min later, adding 25-100 μ L of 0.5-10 μ M hemin, shaking, and incubating at 35-40 deg.C for 40-80 min; then, adding 50-120 μ L of 50-100mM H2O2, 50-100 μ L of 40-140mM O-phenylenediamine OPD and 50-200 μ L of 0.01-1mg/mL DPA-GQD-His dissolved in phosphate buffer saline solution into the mixed solution, incubating for 40-80min at 35-40 ℃, and performing fluorescence spectrum scanning on the mixture, wherein the excitation wavelengths are 360nm and 420nm respectively, and the slit widths are 5-10 nm; and (3) quantitatively detecting the acetamiprid by using the change of the fluorescence intensity.
In one embodiment of the invention, the invention combines a triple helix DNA molecule and G-quadruplex/DNase catalytic oxidation of o-phenylenediamine (OPD) to construct a fluorescent biosensing platform for acetamiprid detection. In the method, an aptamer and a DNA sequence rich in G form a triple helix structure through Watson-Crick base pairing and Hoogsteen hydrogen bond; then in the presence of acetamiprid, the triple-helical DNA molecule is dissociated to release the G-rich DNA sequence, and the free G-rich DNA sequence forms G-quadruplex/DNase under the action of hemin, and the enzyme is subjected to H2O2The catalyst has certain promotion effect on the catalytic oxidation of o-phenylenediamine in the presence of the catalyst; the oxidation product of o-phenylenediamine, 2, 3-Diaminophenazine (DAP), is capable of effectively quenching the fluorescence of DPA-GQD-His; with the increase of the concentration of the acetamiprid, the fluorescence of DPA-GQD-His at 421nm is gradually weakened, the fluorescence of DAP at 565nm is gradually strengthened, and the acetamiprid is quantitatively detected by using the sum of the change of the fluorescence intensity of DPA-GQD-His and DAP at 420nm and the absolute value of the change of the fluorescence intensity at 560 nm.
In one embodiment of the invention, the D-penicillamine is introduced into the D-penicillamine-histidine graphene quantum dot DPA-GQD-His to enhance the fluorescence of the graphene quantum dot, so that the purposes of improving the signal-to-noise ratio of detection and enhancing the sensitivity of a detection system are achieved; the invention relates to G-quadruplex/DNA enzyme (simulating peroxidase, having certain catalytic activity on hydrogen peroxide), and histidine is used for enhancing the catalytic activity of the G-quadruplex/DNA enzyme and accelerating the degradation rate of the hydrogen peroxide so as to improve the fluorescence quenching efficiency of graphene quantum dots.
The invention has the beneficial effects that:
(1) the DPA-GQD-His prepared by the method is high in fluorescence emission intensity, and compared with other quantum dots, the graphene quantum dots are good in biocompatibility, rich in hydrophilic groups on a lamella and strong in water solubility; the D-penicillamine contains abundant electron-donating groups, and when the D-penicillamine is introduced, the electron-donating effect of the groups greatly increases the charge density in the graphene sheet, which is beneficial to improving the luminous efficiency of the graphene quantum dots so as to enhance the fluorescence emission intensity of DPA-GQD-His;
(2) the DPA-GQD-His prepared by the method can improve the catalytic activity of peroxidase, and as for histidine, an imidazole ring of the DPA-GQD-His has strong proton exchange capacity, so that exogenous histidine in the peroxidase serves as a general acid catalyst to improve the catalytic activity, and the degradation rate of hydrogen peroxide is accelerated, so that the fluorescence quenching efficiency of graphene quantum dots is improved;
(3) the DPA-GQD-His prepared by the invention has high sensitivity for quantitative detection of a target, has high fluorescence emission intensity and improves the catalytic activity of G-quadruplex/DNase on hydrogen peroxide, so that the combination of the DPA-GQD-His and a double amplification system enables the detection of the target to have high signal-to-noise ratio and good fluorescence quenching efficiency.
(4) The detection method has high specificity, and the aptamer sequence capable of being specifically combined with the target is introduced into the detection system, so that the acetamiprid can be specifically and quantitatively detected.
(5) Because the DPA-GQD-His prepared by the invention has extremely strong fluorescence emission intensity, when the organophosphorus pesticide, the DPA-GQD-His and the isolated cell are cultured together, only a small amount of DPA-GQD-His enters the cell to emit strong enough fluorescence, thereby improving the determination sensitivity, detecting the organophosphorus pesticide with low concentration and being beneficial to establishing the limit standard of the mixed organophosphorus pesticide.
Drawings
Fig. 1 is a TEM image of the D-penicillamine-histidine graphene quantum dot prepared in example 1 of the present invention.
Fig. 2 shows the thickness of the sheet layer of the D-penicillamine-histidine graphene quantum dot prepared in example 1 of the present invention.
Fig. 3 is an AFM image of the D-penicillamine-histidine graphene quantum dot prepared in example 1 of the present invention.
Fig. 4 is an FTIR spectrum of the D-penicillamine-histidine graphene quantum dot prepared in example 1 of the present invention.
Fig. 5 is a fluorescence excitation and emission spectrum diagram of the D-penicillamine-histidine graphene quantum dot prepared in example 1, where a is a fluorescence excitation spectrum, b is a fluorescence emission spectrum, c is an optical photo of a DPA-GQD-His solution under white light irradiation, and D is an optical photo of a DPA-GQD-His solution under ultraviolet light irradiation.
FIG. 6 is a fluorescence curve of DPA-GQD-His prepared in example 2 at different D-penicillamine ratios.
FIG. 7 is a fluorescence curve of DPA-GQD-His prepared in example 3 at different reaction times.
FIG. 8 is a fluorescence curve of DPA-GQD-His prepared in example 4 at different reaction temperatures.
FIG. 9 is a graph showing the sum of absolute values of the change in fluorescence intensity at 560nm under excitation of 420nm wavelength and the change in fluorescence intensity at 420nm under excitation of 360nm ultraviolet light in example 5 with the second incubation time.
FIG. 10 is the optimization of the o-phenylenediamine concentration in the fluorescence bioassay system in example 5, with the abscissa being the o-phenylenediamine concentration and the ordinate being the sum of the absolute values of the change in fluorescence intensity at 560nm under excitation by 420nm wavelength and the change in fluorescence intensity at 420nm under excitation by 360nm ultraviolet light.
FIG. 11 shows the optimization of hemin concentration in a fluorescence bioassay system, with hemin concentration on the abscissa and the ordinate being the sum of the absolute values of the change in fluorescence intensity at 560nm under excitation by 420nm wavelength and the change in fluorescence intensity at 420nm under excitation by 360nm UV light.
FIG. 12 shows H in fluorescence bioassay system2O2Concentration optimization with abscissa H2O2Concentration, the ordinate is the sum of the absolute values of the change of the fluorescence intensity at 560nm under the excitation of 420nm wavelength and the change of the fluorescence intensity at 420nm under the excitation of 360nm ultraviolet light.
FIG. 13 shows the linear range of the fluorescence biosensor platform in the present invention, the acetamiprid concentration is 1.0 × 10-15～1.0×10-9M, A is fluorescence emission spectrum of biosensing system with excitation wavelength of 420nm (acetamiprid concentration is from top to bottom according to peak position)Is 1.0X 10-9，7.5×10-10，2.5×10-10，1×10-10,7.5×10-11,1×10-11,7.5×10-12,2.5×10-12,1×10-12,7.5×10-13,5×10-13,2.5×10-13,1×10-13,7.5×10-14,5×10-14,2.5×10-14,1×10-14,7.5×10-15,5×10-15,2.5×10-15,1.0×10-15M); b is fluorescence emission spectrum of biosensing system with excitation wavelength of 360nm (acetamiprid concentration is 1.0 × 10 from top to bottom according to the position of the first peak-15,2.5×10-15,5×10-15,7.5×10-15,1×10-14,2.5×10-14,5×10-14,7.5×10-14,1×10-13,2.5×10-13,5×10-13,7.5×10-13,1×10-12,2.5×10-12,7.5×10-12,1×10-11,7.5×10-11,1×10-10,2.5×10-10,7.5×10-10And 1.0X 10-9M); the concentration of acetamiprid is 1.0 × 10-15～1.0×10-9When M is higher, C is a curve of the change of the fluorescence intensity at 560nm along with logC under the excitation of 420nm wavelength; d is a curve of the change of logC along with the sum of the change value of the fluorescence intensity at 560nm under the excitation of 420nm wavelength and the change value of the fluorescence intensity at 420nm under the excitation of 360nm ultraviolet light.
FIG. 14 is the selectivity of the biosensing system. Wherein the concentration of the organophosphorus pesticide is 1.0 multiplied by 10-13M, the abscissa is different organophosphorus pesticides, and the ordinate is the sum of absolute values of the change of the fluorescence intensity at 560nm under the excitation of 420nm wavelength and the change of the fluorescence intensity at 420nm under the excitation of 360nm ultraviolet light.
FIG. 15: the fluorescence emission spectrum of DAP at 560nm under the existence of different graphene quantum dots in the biosensor is shown on 15A, the fluorescence emission spectrum of the quantum dots per se at 420nm under the existence of different graphene quantum dots in the biosensor is shown on 15A, and the solid line-acetamiprid concentration is 1 multiplied by 10-13M, dotted line-acetamiprid concentration 0; at 560nm on 15B corresponding to the DAP fluorescence spectrum on 15AA value corresponding to the maximum emission intensity, corresponding to a value corresponding to the maximum emission intensity at 420nm of the fluorescence spectra of different graphene quantum dots at 15A at 15B; wherein, the concentration of the quantum dots is 1 mg/mL.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
The equipment and materials used in the invention:
cary Escrips fluorescence photometer (Warran, USA); JEM-2100(HR) transmission electron microscope (JEOL Ltd., Japan); nicolet iS50 FT-IR Fourier Infrared Spectroscopy (Sammer Feishell science, USA); TU-1901 double-beam UV-visible spectrophotometer (Beijing Pujingyo general instruments, Inc.); MuLtimode 8 atomic force microscope (brueck technologies ltd, germany).
Citric acid monohydrate (analytically pure), sodium chloride (analytically pure), sodium dihydrogen phosphate (analytically pure), disodium hydrogen phosphate (analytically pure), potassium chloride (analytically pure), sodium hydroxide (analytically pure), disodium ethylenediaminetetraacetate (99.0%) were purchased from national pharmaceutical group chemical reagent company; histidine (99%), o-phenylenediamine (99.5%), D-penicillamine (Tg > 98%) were purchased from Aladdin reagents, Inc. The sequence of SEQ ID NO.1 is: 5'-CTCTCTCTCTCTGACACCATATTATGAAGATCTCTCTCTC-3', SEQ the sequence of ID NO.2 is: 5'-GGGTTTTGGGTAGGAGAGAGAGA GA TCCTTGGGTTTTGGG-3', DNA the DNA sequence was purified by HPLC from Shanghai Biometrics Ltd, and the DNA sequence was heated at 95 ℃ for 5min before use, then gradually cooled to room temperature and stored in a refrigerator for use.
EXAMPLE 1 preparation of DPA-GQD-His
Weighing a certain amount of citric acid monohydrate in a beaker, adding water for ultrasonic dissolution, and then mixing the citric acid monohydrate: histidine: d-penicillamine and histidine were weighed into a beaker at a molar ratio of 1:1:0.5 and dissolved ultrasonically. After complete dissolution, the mixture was quantitatively transferred to a high pressure reactor and placed in a forced air drying oven to react for 1 hour at 200 ℃. After the reaction was completed, the pH of the resulting solution was adjusted to neutral, and then placed in a dialysis bag of 3.5KD for dialysis for 48 hours. And after dialysis is finished, taking the solution in the bag, and freeze-drying to obtain D-penicillamine-histidine bifunctional graphene quantum dot (DAP-GQD-His) solid powder. The D-penicillamine-histidine double-graphene quantum dots prepared in this example were characterized, and as can be seen from the TEM image in fig. 1, the D-penicillamine-histidine bifunctional graphene quantum dots DPA-GQD-His consist of 2D graphene sheets, the size is between 1nm and 6nm, and the average size is 3.6 nm.
The HRTEM image of fig. 2 and AFM image of fig. 3 show that the average height of DPA-GQD-His is about 0.9 nm. It is demonstrated that most DPA-GQD-His consists of 1-2 graphene layers.
FIG. 4 shows FTIR spectra of DPA-GQD-His, which shows 3300--1Characteristic IR absorption of stretching vibration of OH and NH bonds in the wave number range, 3000--1Is the absorption peak of C-H bond stretching vibration, 1700cm-1The absorption peak at (B) is attributed to stretching vibration of carbonyl C ═ O bond, 1380cm-1Is a C-H bending vibration absorption peak, is a characteristic absorption peak of a methyl group, and is 1600cm-1Is the characteristic absorption peak for the C ═ C double bond. The results of IR analysis indicated the presence of-OH, -NH-, -CH-in DPA-GQD-His3C ═ C-, -C ═ O bonds, which can demonstrate the formation of nano-graphene sheets and the introduction of functional groups in D-penicillamine and histidine.
FIG. 5 is a graph of fluorescence excitation and emission spectra of DPA-GQD-His. Wherein, the curve a is fluorescence excitation spectrum, which is mainly distributed in the wavelength range of 300-400nm, the maximum excitation wavelength is 360nm, the excitation wavelength is obviously larger than that of the small molecular organic compound, and the graphene sheet is proved to have large electronic delocalization range. DPA-GQD-His can generate green fluorescence emission under 360nm ultraviolet light irradiation. Curve b shows the quasi-symmetrical shape of the corresponding fluorescence emission spectrum, with the maximum emission peak at 421 nm. Optical photographs of the DPA-GQD-His solution under white light and ultraviolet light irradiation are shown in fig. 5(c) and (d), respectively.
EXAMPLE 2 preparation of DPA-GQD-His
When the reaction temperature in example 1 was 200 ℃ and the reaction time was 1h, the citric acid: the molar ratio of histidine is 1:1, the molar ratios of citric acid and D-penicillamine are adjusted to make the molar ratios of D-penicillamine and citric acid respectively 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, and 0.7:1, and the other operation steps are in accordance with example 1, to prepare different DPA-GQD-His D-penicillamine-histidine bifunctional graphene quantum dots.
The fluorescence intensity of the DPA-GQD-His obtained by the preparation was measured, and the lines in fig. 6 correspond to, from bottom to top, the fluorescence intensities of the DPA-GQD-His obtained by the preparation, wherein the molar ratios of D-penicillamine to citric acid were 0.1:1, 0.2:1, 0.3:1, 0.7:1, 0.4:1, 0.6:1, and 0.5:1, respectively. It can be found that the fluorescence intensity of DPA-GQD-His gradually increases with the increase of the incorporation ratio of D-penicillamine, and the fluorescence intensity of the quantum dots is maximum when the molar ratio of the D-penicillamine to the citric acid is 0.5: 1.
EXAMPLE 3 preparation of DPA-GQD-His
When the reaction temperature in example 1 is 200 ℃, the molar ratio of citric acid, histidine and D-penicillamine is 1:1: and when the time is 0.5 hour, the hydrothermal reaction time is controlled to be 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours respectively, the other operation steps are consistent with those of the example 1, and different DPA-GQD-His D-penicillamine-histidine bifunctional graphene quantum dots are prepared.
The fluorescence intensity of the prepared DPA-GQD-His was measured, and the results are shown in fig. 7, where the lines in fig. 7 correspond to the fluorescence intensities of the prepared DPA-GQD-His with reaction times of 6h, 5h, 4h, 3h, 0.5h, 2h, and 1h, respectively, from bottom to top, respectively. It was found that the fluorescence intensity of DPA-GQD-His reached a maximum at a reaction time of 1 h.
Example 4 preparation of DPA-GQD-His
When the reaction time in example 1 is 1h, the molar ratio of citric acid, histidine and D-penicillamine is 1:1: at 0.5, controlling the temperature of the hydrothermal reaction as follows: the other operation steps are the same as those in the example 1 at the temperature of 170 ℃, 180 ℃, 190 ℃, 200 ℃, 220 ℃ and 250 ℃, and different DPA-GQD-His D-penicillamine-histidine bifunctional graphene quantum dots are prepared.
The fluorescence intensity of the DPA-GQD-His obtained by the preparation was measured, and the results are shown in fig. 8, where the lines in fig. 8 correspond to the fluorescence intensities of the DPA-GQD-His obtained by the preparation at the reaction temperatures of 170 ℃, 180 ℃, 190 ℃, 250 ℃, 220 ℃ and 200 ℃ from bottom to top, respectively. It was found that the fluorescence intensity of DPA-GQD-His reached a maximum at a reaction temperature of 200 ℃.
Example 5 quantitative detection of acetamiprid
1. Optimization of the second incubation time
(1) The DPA-GQD-His solid prepared in example 1 is dissolved in PBS buffer solution (pH 6.8), and the mass concentration of the DPA-GQD-His is 0.1 mg/mL;
(2) mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. Adding 1X 10-13And respectively adding the acetamiprid solution of M into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of 5. mu.M hemin was added, shaken well and incubated for 1h at 37 ℃. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270 microliter of 70mM OPD and 100 microliter of 0.1mg/mL DPA-GQD-His dissolved in PBS buffer solution are incubated at 37 ℃ for 10min, 20min, 60min, 80min and 100min respectively, and fluorescence spectrum scanning is carried out on the mixture, wherein the excitation wavelengths are 360nm and 420nm respectively, and the slit width is 10 nm. The absolute values of the fluorescence intensity changes at 420nm and 560nm under the excitation of the wavelengths of 360nm and 420nm are used for condition screening, and the result is shown in fig. 9, which shows that the change value of the fluorescence intensity reaches the maximum when the second incubation time of the DPA-GQD-His and the OPD in the system is 60 min.
2. Optimization of o-phenylenediamine concentration
(1) The DPA-GQD-His solid prepared in example 1 is dissolved in PBS buffer solution (pH 6.8), and the mass concentration of the DPA-GQD-His is 0.1 mg/mL;
(2) mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. Adding 1X 10-13Of MRespectively adding the acetamiprid solution into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of 5. mu.M hemin was added, shaken well and incubated for 1h at 37 ℃. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270. mu.L of o-phenylenediamine and 100. mu.L of 0.1mg/mL DPA-GQD-His in PBS buffer at concentrations of 10mM, 30mM, 50mM, 70mM, 90mM and 140mM, respectively, were incubated at 37 ℃ for 1 hour and then subjected to fluorescence spectrum scanning, with excitation wavelengths of 360nm and 420nm, respectively, and a slit width of 10nm, respectively. The results of screening the OPD concentrations using the absolute values of the change in fluorescence intensity at 420nm and 560nm under excitation at wavelengths of 360nm and 420nm are shown in FIG. 10, and it is found that the change in fluorescence intensity of the system is maximized at an o-phenylenediamine concentration of 70 mM.
3. Optimization of hemin concentration
(1) The DPA-GQD-His solid prepared in example 1 is dissolved in PBS buffer solution (pH 6.8), and the mass concentration of the DPA-GQD-His is 0.1 mg/mL;
(2) mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. Adding 1X 10-13And respectively adding the acetamiprid solution of M into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of hemin with a concentration of 0.05. mu.M, 0.1. mu.M, 0.25. mu.M, 0.5. mu.M, 0.75. mu.M, 1. mu.M, were added, shaken well and incubated at 37 ℃ for 1 h. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270 μ L of 70mM OPD and 100 μ L of 0.1mg/mL DPA-GQD-His in PBS buffer solution were incubated at 37 ℃ for 1h, followed by fluorescence spectrum scanning, with excitation wavelengths of 360nm and 420nm, respectively, and a slit width of 10 nm. The hemin concentration was optimized using the absolute values of the change in fluorescence intensity at 420nm and 560nm under excitation at 360nm and 420nm, and the results are shown in FIG. 11, which shows that when the hemin concentration was 0.5. mu.M, the change in fluorescence intensity of the systemThe value reaches the maximum; at hemin concentrations below 0.5. mu.M, the segmented G-quadruplexes in the system did not all form G-quadruplexes/DNase, which is at H2O2The catalytic oxidation of OPD in the presence of this is limited and does not quench the fluorescence of DPA-GQD-His to the maximum extent. However, at hemin concentrations above 0.5 μ M, all segmented G-quadruplexes in the system have completely formed G-quadruplexes/DNase, the fluorescence quenching degree of DPA-GQD-His has reached a maximum, and if a large amount of hemin exists in the system, significant background fluorescence will be generated, which is not favorable for later analysis and detection.
4、H2O2Optimisation of concentration
(1) The DPA-GQD-His solid prepared in example 1 is dissolved in PBS buffer solution (pH 6.8), and the mass concentration of the DPA-GQD-His is 0.1 mg/mL;
(2) mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. Adding 1X 10-13And respectively adding the acetamiprid solution of M into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of 5. mu.M hemin was added, shaken well and incubated for 1h at 37 ℃. Thereafter, 90. mu.L of H was added to the mixed solution at concentrations of 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, respectively2 O 270 μ L of 70mM OPD and 100 μ L of 0.1mg/mL DPA-GQD-His in PBS buffer were incubated at 37 ℃ for 1 h. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270 μ L of 70mM OPD and 100 μ L of 0.1mg/mL DPA-GQD-His in PBS buffer solution were incubated at 37 ℃ for 1h, followed by fluorescence spectrum scanning, with excitation wavelengths of 360nm and 420nm, respectively, and a slit width of 10 nm. The hemin concentration was optimized by using the absolute values of the fluorescence intensity changes at 420nm and 560nm under the excitation of the wavelengths of 360nm and 420nm, and the results are shown in FIG. 12, which shows that when H is2O2When the concentration is 75mM, the change value of the fluorescence intensity of the system reaches the maximum; if H is2O2At concentrations below 75mM, the OPD in the system is not sufficiently oxidized to produce large amounts of DAP, and thus the fluorescence of DPA-GQD-His is not significantly quenched, i.e., the change in the fluorescence intensity value is not significant. If H is2O2At concentrations above 75mM, the OPD in the system was sufficiently oxidized that no more DAP could be generated to quench the fluorescence of DPA-GQD-His.
In conclusion, the optimal conditions are finally selected as follows: the second incubation time of DPA-GQD-His and OPD in the system is 60min, the concentration of o-phenylenediamine is 70mM, the concentration of hemin is 0.5 mu M and H2O2The concentration was 75 mM.
5. Quantitative detection of acetamiprid
mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. The concentrations are respectively 1.0X 10-15,2.5×10-15,5×10-15,7.5×10-15,1×10-14,2.5×10-14,5×10-14,7.5×10-14,1×10-13,2.5×10-13,5×10-13,7.5×10-13,1×10-12,2.5×10-12,7.5×10-12,1×10-11,7.5×10-11,1×10-10,2.5×10-10,7.5×10-10And 1.0X 10-9And respectively adding the acetamiprid solution of M into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of 5. mu.M hemin was added, shaken well and incubated for 1h at 37 ℃. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270 μ L of 70mM OPD and 100 μ L of 0.1mg/mL DPA-GQD-His in PBS buffer solution were incubated at 37 ℃ for 1h, followed by fluorescence spectrum scanning, with excitation wavelengths of 360nm and 420nm, respectively, and a slit width of 10 nm. With the increase of the concentration of the acetamiprid, the fluorescence of DPA-GQD-His at 421nm is gradually weakened, the fluorescence of DAP at 565nm is gradually strengthened, and the acetamiprid is quantitatively detected by using the change of the fluorescence intensity values of the DPA-GQD-His and the DAP. The results are shown in the figure13A and 13B show that the fluorescence of DPA-GQD-His at 421nm is obviously reduced with the increase of the concentration of acetamiprid, and the fluorescence of DAP at 565nm is continuously enhanced. At acetamiprid concentration of 1 × 10-16～1×10-9In the mol/L range, the fluorescence intensity F increases linearly with the increasing logarithm of the acetamiprid concentration (FIG. 13C and FIG. 13D), and the calculation formula of the acetamiprid concentration obtained from FIG. 13C and FIG. 13D is: f560＝105.01×logC+1701.1，ΔF560+420145.27 × logC +2183, wherein F560Is the fluorescence intensity at 560nm,. DELTA.F560+420Is the sum of absolute values of fluorescence intensity changes at 560nm and 420nm, and C is the acetamiprid concentration in mol/L. Therefore, the detection limit of the method is as low as 3.8 multiplied by 10 on the basis of the signal to noise ratio of 3-16mol/L。
In addition, the method of the invention is compared with other fluorescence methods for detecting the content of the acetamiprid reported in the literature (Table 1). Compared with other existing fluorescence detection systems, the analysis method for quenching DPA-GQD-His fluorescence by using the triple helix DNA molecule and the combination of the amount of the G-quadruplex/DNAse catalytic oxidation OPD has ultrahigh sensitivity and selectivity. The main reason why the method has high sensitivity and selectivity is that firstly, DPA-GQD-His used in the detection method has extremely strong fluorescence emission, and the detection sensitivity can be obviously improved; secondly, the detection system of the invention adopts the DNA sequence for specifically identifying the acetamiprid, which can obviously improve the selectivity of the analysis method.
Table 1 shows the effect of comparing different fluorescent probes for acetamiprid detection on detection sensitivity
The measurement accuracy of the method is measured by using the method for adding the standard, and the result is shown in table 2, so that the method provided by the invention has the standard adding recovery rate of more than 94% for the micro acetamiprid.
TABLE 2 recovery of spiked samples from the detection method of this example
mu.L of a 1. mu.M DNA sequence of the sequence SEQ ID No.1 and 25. mu.L of 1. mu. M G-DNA were mixed in 250. mu.L of PBS buffer (pH 6.8,300mM NaCl,2.5mM MgCl)2And 0.1mM EDTA). The solution was incubated at room temperature for 30min to promote the production of triple-helical DNA molecules. Adding the mixture to the solution with the concentration of 2.8 multiplied by 10-14Respectively adding the acetamiprid solution of mol/L into the mixed solution, shaking uniformly, and standing at room temperature for 20 min. 25 μ L of 50mM KCl was added to the above solution to promote the formation of the G-quadruplex structure. After 30min, 50. mu.L of 5. mu.M hemin was added, shaken well and incubated for 1h at 37 ℃. Thereafter, 90. mu.L of 75mM H was added to the mixed solution2O270 μ L of 70mM OPD and 100 μ L of 0.1mg/mL DPA-GQD-His in PBS buffer were incubated at 37 ℃ for 1h, respectively. Fluorescence intensity values at 420nm and 560nm were obtained at 602.58 and 274.96, respectively, under excitation at wavelengths of 360nm and 420 nm.
Will be 3X 10-13The mol/L is respectively substituted into the linear regression equation:
F560when C is 2.63 × 10, obtained by 105.01logC +1701.1-14mol/L, and the addition concentration is 2.8X 10-14Compared with the mol/L ratio, the method can accurately measure the acetamiprid to be as low as 10-14Concentration of mol/L.
Example 6 detection of organophosphorus pesticides
(1) SH-SY5Y cells in logarithmic growth phase are collected, and cell suspension is adjusted to 1 × 104The concentration of individual cells/well was seeded in 96-well cell culture plates at a volume of 100. mu.L per well of cytosol. Transfer the plates to CO2In a constant temperature incubator at 37 ℃ and 5% CO2And culturing under saturated humidity condition to make the cells adhere to the wall, and culturing for 24 h. Adding a mixed solution of acetamiprid and chlorpyrifos at a concentration ratio of 1:1 to a culture plate to make the final concentration of pesticide in the mixed solution 0.2. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, exposing and culturing for 48h, aspirating the supernatant, adding each wellmu.L of fresh culture medium (DMEM/F12(1: 1)) culture medium (containing phenol red) + 5% of imported fetal bovine serum (5% being the mass fraction of the culture medium) + 0.5% penicillin-streptomycin solution (100X)) and 10. mu.L of PBS buffer solution of DPA-GQD-His, and culturing is continued for 4 hours, so that the DPA-GQD-His can sufficiently enter the cell. The culture was then terminated by aspirating the supernatant, adding a certain amount of PBS buffer solution to each well, and shaking for 10min to disperse as many cells as possible on the cell culture plate in the solution.
(3) And (3) centrifugally washing the obtained solution, removing the inactive DPA-GQD-His, and detecting the fluorescence emission intensity at 421nm under the excitation of 360nm by using a fluorescence spectrometer. The fluorescence emission intensity at 421nm of the zero-adjustment group (the zero-adjustment group is 90. mu.L of fresh culture solution and 10. mu.L of 1mg/mLDPA-GQD-His PBS buffer solution) and the fluorescence emission intensity at 421nm of the control group (the experimental group is not added with the organophosphorus pesticide group) are simultaneously measured. And finally, the following steps are carried out: the cell survival rate was calculated as (fluorescence intensity of treatment group-fluorescence intensity of zero adjustment group)/(fluorescence intensity of control group-fluorescence intensity of zero adjustment group) × 100%. The cell survival rate can be used for detecting the damage of the organophosphorus pesticide to the cells under the concentration, thereby being beneficial to establishing the limit standard of the mixed organophosphorus pesticide.
Comparative example 1
When D-penicillamine is not added in the preparation process of the quantum dot, the molar ratio of histidine to citric acid monohydrate is 1:1, and the other preparation methods are consistent with those in example 1, so that the histidine graphene quantum dot is prepared. As shown in fig. 15, it is obvious that the fluorescence intensity of DPA-GQD-His is far higher than that of His-GQD when the content of acetamiprid is measured by using the histidine graphene quantum dots prepared in comparative example 1, and the sensitivity of analysis and detection is seriously affected because the fluorescence intensity of the single histidine functionalized graphene quantum dots is too low.
Comparative example 2
When no histidine is added in the preparation process of the quantum dot, the molar ratio of the D-penicillamine to the citric acid monohydrate is 0.5:1, and the other preparation methods are consistent with those in example 1, so that the D-penicillamine graphene quantum dot is prepared. As shown in fig. 15, when the content of acetamiprid is measured by using the D-penicillamine graphene quantum dots prepared in comparative example 2, the change values of fluorescence intensities at 420nm and 560nm are obviously lower than those of DPA-GQD-His as can be seen from the bar chart in fig. 15B. The introduction of histidine in DPA-GQD-His increases the variation of fluorescence signals, thereby improving the detection sensitivity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. The method for quantitatively detecting the acetamiprid is characterized in that the acetamiprid is quantitatively detected by utilizing dual-functionalized graphene quantum dots or a biological detection sensor containing the dual-functionalized graphene quantum dots;
the preparation method of the bifunctional graphene quantum dot comprises the following steps: according to the weight ratio of citric acid: histidine: and D-penicillamine is in a molar ratio of 1:0.1: 0.01-1: 1:1, and the bifunctional graphene quantum dot DPA-GQD-His is prepared by a hydrothermal method.
2. The method for quantitatively detecting acetamiprid according to claim 1, wherein the operational parameters of the hydrothermal method are as follows: reacting for 0.5-10 h at 150-250 ℃.
3. The method for quantitatively detecting acetamiprid according to claim 1 or 2, wherein the method comprises the steps of mixing D-penicillamine, histidine, citric acid and water according to a molar ratio to obtain a mixed solution, transferring the mixed solution to a reaction kettle, and reacting at 150-250 ℃ for 0.5-10 h; after the reaction is finished, adjusting the pH value of the obtained solution to 7.0-8.0, dialyzing in a dialysis bag for 10-72h, and drying the solution in the dialysis bag after the dialysis is finished to obtain DAP-GQD-His.
4. The method for quantitatively detecting acetamiprid according to claim 3, wherein the dialysis bag has a specification of 1-10 KD.
5. The method for quantitatively detecting acetamiprid according to claim 1, which comprises the following steps:
(1) dissolving DPA-GQD-His in phosphate buffer solution with pH of 6.5-7.4, wherein the mass concentration of DPA-GQD-His is 0.01-1 mg/mL;
(2) mixing 10-100 μ L of 0.1-10 μ M DNA sequence shown as sequence SEQ ID NO.1 and 10-100 μ L of 0.1-10 μ M DNA sequence shown as sequence SEQ ID NO.2 in 100-300 μ L PBS buffer solution, and incubating at room temperature for 20-60 min;
(3) adding acetamiprid with different concentrations into the mixed solution obtained in the step (2), oscillating, and standing at 20-30 ℃ for 20-40 min; adding 10-50 μ L of 25-50mM KCl into the above solution; 30-60min later, adding 25-100 μ L of 0.5-10 μ M hemin, shaking, and incubating at 35-40 deg.C for 40-80 min; then, 50-120. mu.L of 50-100mM H was added to the mixed solution2O250-100 mu L of 40-140mM o-phenylenediamine OPD and 50-200 mu L of 0.01-1mg/mL DPA-GQD-His dissolved in phosphate buffer saline solution, incubating for 40-80min at 35-40 ℃, and then performing fluorescence spectrum scanning on the mixture, wherein the excitation wavelengths are 360nm and 420nm respectively, and the slit widths are 5-10 nm; and (3) quantitatively detecting the acetamiprid by using the change of the fluorescence intensity.
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