阅读说明：本技术 基于荧光碳点CDs的镉检测方法 (Cadmium detection method based on fluorescent carbon dots CDs ) 是由 韩芹芹 赵新月 宋玉竹 张金阳 杨亚玲 夏雪山 于 2021-07-27 设计创作，主要内容包括：本发明公开了基于荧光碳点CDs的镉检测方法,该方法采用荧光碳点CDs-核酸适配体偶联物特异性检测镉含量,镉在420nm荧光波长下能使荧光碳点NCDs-核酸适配体偶联物发生荧光猝灭；利用镉标准溶液对荧光碳点NCDs-适配体偶联物进行特异性、灵敏度及实际样品的加标回收检测,实验结果表明该荧光碳点CDs-核酸适配体偶联物具有高特异性、高亲和力的特点,可应用在对实际样品中的镉含量的检测中。(The invention discloses a cadmium detection method based on fluorescent carbon dots CDs, which adopts the specificity of a fluorescent carbon dot CDs-nucleic acid aptamer conjugate to detect the cadmium content, and cadmium can enable the fluorescent carbon dot NCDs-nucleic acid aptamer conjugate to generate fluorescence quenching under the fluorescent wavelength of 420 nm; the cadmium standard solution is used for carrying out specificity and sensitivity on the fluorescent carbon dot NCDs-aptamer conjugate and the labeling recovery detection of an actual sample, and an experimental result shows that the fluorescent carbon dot CDs-aptamer conjugate has the characteristics of high specificity and high affinity and can be applied to the detection of the cadmium content in the actual sample.)

1. A cadmium detection method based on fluorescent carbon dots CDs is characterized in that: the content of cadmium is specifically detected by adopting the fluorescent carbon dot CDs-aptamer conjugate, and the cadmium can enable the fluorescent carbon dot NCDs-aptamer conjugate to generate fluorescence quenching under the fluorescence wavelength of 420 nm.

2. The method for detecting cadmium based on fluorescent Carbon Dots (CDs) as claimed in claim 1, wherein: the fluorescent carbon point CDs-aptamer conjugate is prepared by adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into 9.85mL of a fluorescent carbon point NCDs solution with the concentration of 1-2g/mL at room temperature, wherein the final concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the fluorescent carbon point NCDs solution is 1.2mmol/L, the final concentration of the N-hydroxysuccinimide in the fluorescent carbon point NCDs solution is 1.2mmol/L, adding 150 mu L of amino-modified aptamer with the concentration of 100 mu mol/L after stirring and activating for 1h, and reacting for 2-3h after mixing.

3. The method for detecting cadmium based on fluorescent Carbon Dots (CDs) as claimed in claim 1, wherein: the nucleotide sequence of the amino-modified aptamer CD-4 is 5' -NH₂-GGACTGTTGTGGTATTATTTTTGGTTGTGC-3'。

Technical Field

The invention belongs to the technical field of heavy metal detection, and particularly relates to preparation of a fluorescent carbon dot and aptamer conjugate and a method for establishing specificity detection of cadmium content in pseudo-ginseng based on the fluorescent carbon dot.

Background

Cadmium (Cadmium, Cd) has an atomic number 48, is a non-ferrous metal element, the simple substance is a silvery white metal, can absorb neutrons, is extracted as a byproduct from zinc ore or Cadmium sulfide ore, and is mostly used to protect other metals from corrosion and rust, such as galvanized steel, ironwork, copper, brass, and other alloys. Cadmium exists in a compound state in nature, and when the environment is polluted by cadmium, the cadmium can be enriched in organisms and enter human bodies through food chains to cause chronic poisoning. The pollution source is mainly lead zinc ore and the like. The pollution of cadmium to soil mainly comprises a gas type and a water type. The gas type pollution mainly comes from industrial waste gas, and the pollution range can reach thousands of meters. The water type pollution is mainly caused by the discharge of the beneficiation wastewater of the lead-zinc ores and the wastewater of related industries (electroplating, alkaline batteries and the like) into surface water or the infiltration of underground water. Cadmium can cause irritation to the respiratory tract, and long-term exposure can cause olfaction loss, gingival macula or gradually become yellow circles. After being absorbed by the body through respiration, the liver or kidney-qi can cause damage, especially damage to the kidney, and osteoporosis and malacia.

Notoginseng (radix Notoginseng)Panax notoginseng(Burk.) F.H.Chen) is a plant of Araliaceae. Sweet in taste, bitter in taste and warm in nature, has the effects of stopping bleeding, relieving pain, promoting blood circulation and removing blood stasis, and is mainly produced in Yunnan and Guangxi, and then in Guizhou, Sichuan and other places. The Yunnan wenshan of the genuine producing area and the surrounding producing area belong to the mining area environment, and with the increasing serious problems of industrial dust, industrial waste water, tailing sand accumulation and the like, the heavy metals in the pseudo-ginseng exceed the standard frequently, so that the quality and the export of the pseudo-ginseng are seriously restricted. The method for detecting cadmium specified by the national standard is a graphite furnace atomic absorption spectrometry, and has the defects of narrow analysis range, low detection speed, high detection cost and poor detection precision.

Fluorescent Carbon Dots (CDs) are a novel Carbon nano luminescent material with the diameter less than 10nm, and the chemical components of the fluorescent Carbon Dots mainly comprise C, H, O and N. The carbon dots have the advantages of excellent optical property, good water solubility, low toxicity, environmental friendliness, wide raw material source, low cost, good biocompatibility and the like. The preparation method of the carbon dots is mainly divided into a Top-down synthesis method and a Bottom-up synthesis method, and comprises an arc discharge method, a laser ablation method, an electrochemical method, a chemical oxidation method and the like. Are often used in chemical sensors, biosensors, biological imaging, catalysis, and the like.

However, at present, no method or product for specifically detecting cadmium by combining the fluorescent carbon dots with the aptamers exists.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for detecting cadmium based on fluorescent Carbon Dots (CDs). the method adopts the specificity of the fluorescent Carbon Dot (CDs) -aptamer conjugate to detect the cadmium content, and cadmium can enable the fluorescent carbon dot (NCDs) -aptamer conjugate to generate fluorescence quenching under the fluorescence wavelength of 420 nm.

The fluorescent carbon dots CDs are prepared by a conventional one-step hydrothermal method, the fluorescent carbon dots NCDs are characterized to be of a uniformly dispersed spherical structure, the average particle size is 3.6nm, the lattice spacing is 0.23nm, and the fluorescent carbon dots NCDs mainly exist in amorphous carbon; chemical groups existing on the surface of the fluorescent carbon points NCDs are O-H, N-H, C = O, C = C and C-N;

the method for specifically detecting the cadmium content by adopting the fluorescent carbon dot CDs-aptamer conjugate comprises the following steps:

(1) preparing a fluorescent carbon point CDs-aptamer conjugate by adopting a dehydration condensation method, specifically adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into 9.85mL of a fluorescent carbon point NCDs solution with the concentration of 1-2g/mL at room temperature, wherein the final concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the fluorescent carbon point NCDs solution is 1.2mmol/L, the final concentration of the N-hydroxysuccinimide in the fluorescent carbon point NCDs solution is 1.2mmol/L, stirring and activating for 1h, adding 150 mu L of amino-modified aptamer with the concentration of 100 mu mol/L, uniformly mixing, and reacting for 2-3h to obtain the fluorescent carbon point CDs-aptamer conjugate;

the amino modified aptamer CD-4 is a nucleic acid aptamer with a nucleotide sequence shown as SEQ ID NO. 1, wherein the 5' end of the nucleic acid aptamer is modified with amino; namely 5' -NH₂-GGACTGTTGTGGTATTATTTTTGGTTGTGC-3'。

(2) Adding 200 mu L of the fluorescent carbon dot CDs-aptamer conjugate and cadmium standard solution into a 15mL tube, diluting to 3mL, wherein the concentration range of cadmium is 0.1-8ng/mL, exciting at 344nm by using a fluorescence spectrometer, measuring an absorption peak at 420nm, and drawing a standard curve by using the concentration of cadmium as a horizontal coordinate and the peak value at 420nm as a vertical coordinate to obtain a regression equation;

(3) pretreating a sample to be treated, adding 200 mu L of the fluorescent carbon dot CDs-aptamer conjugate into a 15mL tube, pretreating the sample, measuring an absorption peak at a position of 420nm, substituting into the regression equation in the step (2), and calculating to obtain the cadmium content in the sample;

(4) and (3) carrying out specificity verification by adopting a cadmium standard solution, a cobalt standard solution, a lithium standard solution, a zirconium standard solution and an arsenic standard solution.

The invention has the following beneficial effects:

compared with the existing heavy metal cadmium detection technology, the cadmium can be identified and combined with high affinity and high specificity by coupling the cadmium specific aptamer and the fluorescent carbon dots, so that the accuracy of the cadmium contained in the pseudo-ginseng is ensured; the cadmium can reduce the fluorescence value of the fluorescent carbon point NCDs-aptamer conjugate, the carbon point material carries fluorescence according to the detection result, and the fluorescence intensity can be observed by naked eyes to estimate the content of the cadmium in the pseudo-ginseng.

Drawings

FIG. 1 is a transmission electron microscope photograph of fluorescent carbon dots NCDs of the present invention;

FIG. 2 is a graph showing the distribution of the particle diameters of the fluorescent carbon dots NCDs of the present invention;

FIG. 3 is an X-ray diffraction pattern of fluorescent carbon dots NCDs of the present invention;

FIG. 4 is a Fourier transform infrared spectrum of the fluorescent carbon dots NCDs of the present invention;

FIG. 5 is a graph of X-ray photoelectron spectroscopy analysis of the fluorescent carbon dots NCDs of the present invention;

FIG. 6 is an analysis of the specificity of the fluorescent carbon dot NCDs-aptamer conjugates for cadmium provided by the examples of the present invention;

FIG. 7 is a sensitivity analysis of fluorescent carbon dot NCDs-aptamer conjugates to cadmium provided by an embodiment of the present invention;

FIG. 8 is a linear relationship of detection of cadmium by the fluorescent carbon dot NCDs-aptamer conjugates provided in the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: preparation and characterization of fluorescent carbon dot NCDs

1. Preparation of fluorescent carbon dot NCDs: fluorescent carbon point NCDs are synthesized by adopting a one-step hydrothermal method

2. Mixing 2.01g of citric acid solid particles and 100 mu L of ethylenediamine, dissolving the mixture in 30mL of deionized water, transferring the mixture into a polytetrafluoroethylene-lined autoclave, heating the mixture at 180 ℃ for 6 hours, taking out the mixture after the autoclave is cooled to room temperature, and centrifuging the mixture at 10000rpm for 10 min; collecting supernatant, and filtering the supernatant through a 0.22 mu m filter membrane; the filtrate was purified again using dialysis membrane (1000 Da) and stored at 4 ℃; exciting at 344nm by using a fluorescence spectrometer, and recording a fluorescence spectrum at 420 nm; 5mL of the fluorescent carbon-point NCDs were frozen in a freeze-dryer and dissolved in water at the time of use to give a concentration of 0.15 g/mL.

3. Characterization of fluorescent carbon dots NCDs

(1) The morphological characteristics of the fluorescent carbon dot NCDs are characterized by utilizing transmission electron microscope images, as shown in figures 1 and 2, the NCDs are uniformly dispersed spherical structures, the average diameter is 3.6nm, and the lattice spacing is 0.23 nm;

(2) the existence form of the fluorescent carbon dot NCDs was analyzed by X-ray diffraction pattern, as shown in fig. 3, there was only one diffraction peak around 22 °, indicating that the fluorescent carbon dot NCDs exist mainly in the form of amorphous carbon;

(3) the functional groups on the surfaces of the fluorescent carbon dots NCDs were analyzed by Fourier transform infrared spectroscopy, and as shown in FIG. 4, the O-H and N-H vibrational elongations were 3488 cm, respectively⁻¹And 3360 cm⁻¹Has obvious peak values, C = O, C = C and C-N are 1715, 1580 and 1425 cm⁻¹Respectively has obvious suctionCollecting peaks;

(4) and the components of the fluorescent carbon point NCDs were analyzed by X-ray photoelectron spectroscopy, as shown in fig. 5, there were three dominant peaks at 284.5, 399.4 and 531.5 eV, respectively belonging to C, N and O elements, indicating that the synthesized NCD contains C, N and O.

Example 2: preparation of fluorescent carbon-dot NCDs-aptamer conjugates

Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into 9.85mL of a fluorescent carbon point NCDs solution with the concentration of 1.5g/mL at room temperature, wherein the final concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the fluorescent carbon point NCDs solution is 1.2mmol/L, the final concentration of the N-hydroxysuccinimide in the fluorescent carbon point NCDs solution is 1.2mmol/L, after stirring and activating for 1h, adding 150 μ L of amino-modified aptamer with the concentration of 100 μmol/L, uniformly mixing, reacting for 2h to obtain the product, transferring the product into a 15mL centrifuge tube, and storing the product in a refrigerator at 4 ℃; wherein the amino-modified aptamer CD-4 is prepared by modifying amino at the 5' end of the aptamer with the nucleotide sequence shown in SEQ ID NO:1 (synthesized by Kunming division, Biotech, Inc., Beijing Ongzhike).

Example 3: specific detection of cadmium with fluorescent carbon-point NCDs-aptamer conjugates

To verify whether the conjugate specifically recognizes cadmium, cobalt (Co), lithium (Li), zirconium (Zr) and arsenic (As) were used As controls in this experiment

1. Taking 6 empty 15mL centrifuge tubes, adding 200 muL of the prepared fluorescent carbon dot NCDs-aptamer conjugate, wherein 2800 muL of deionized water is directly added into one centrifuge tube and is marked as a blank group; respectively adding a cadmium (Cd) standard solution, cobalt chloride, lithium nitrate, zirconium chloride and arsenic trioxide solution into the other 5 centrifuge tubes, then adding deionized water to a constant volume of 3mL, uniformly mixing metal ions with the concentration of 1 mu g/mL in a 3mL system, and incubating for 30min in a shaking table at 37 ℃;

2. the fluorescence spectrum is recorded at 420nm after being excited at 344nm by adopting a fluorescence spectrometer, and the result is shown in figure 6, and the result shows that cadmium can enable the fluorescence quenching of the fluorescent carbon dot NCDs-aptamer conjugate, so that the fluorescent carbon dot-containing CDs-aptamer conjugate system has the function of specifically identifying cadmium.

Example 4: sensitivity analysis of fluorescent carbon dot NCDs-aptamer conjugates to cadmium

1. Taking 9 15mL centrifuge tubes, adding 200 μ L of the fluorescent carbon dot NCDs-aptamer conjugate prepared in example 1 and 1mg/mL of cadmium (Cd) standard solution, adding deionized water to the centrifuge tubes to a constant volume of 3mL, wherein the concentrations of cadmium (Cd) in a 3mL system of each tube are 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.3ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 4ng/mL and 8ng/mL respectively, and performing shake-bed incubation at 37 ℃ for 30 min;

2. fluorescence spectra were recorded at 420nm with excitation at 344nm using a fluorescence spectrometer. The results are shown in FIG. 7, from which it can be seen that the detection limit of cadmium (Cd) by the fluorescent carbon dot CDs-aptamer conjugate is 0.1 ng/mL.

Example 5: linear relationship analysis for detecting cadmium by using fluorescent carbon dot CDs-aptamer conjugate

1. Taking 11 15mL centrifuge tubes, adding 200 μ L of the fluorescent carbon dot CDs-aptamer conjugate prepared in example 1 and 1mg/mL of cadmium (Cd) standard solution, adding deionized water to the centrifuge tubes to fix the volume to 3mL, wherein the concentration of cadmium (Cd) in each tube in a 3mL system is 0ng/mL, 20ng/mL, 35ng/mL, 40ng/mL, 45g/mL, 50ng/mL, 55ng/mL, 60ng/mL, 1 μ g/mL, 2 μ g/mL and 5 μ g/mL respectively, and performing shake-bed incubation at 37 ℃ for 30 min;

2. exciting at 344nm by using a fluorescence spectrometer, and recording a fluorescence spectrum at 420 nm; the peak value at 420nm is recorded, and the result is shown in figure 8, and the linear relation is obvious when the concentration of cadmium is 0 ng/mL-60 ng/mL, and R²=0.992。

Example 6: labeling, recovering and detecting panax notoginseng samples by using fluorescent carbon dot CDs-aptamer conjugates

1. Sample pretreatment

(1) Taking two different pseudo-ginseng powders, and respectively dissolving 0.1g of pseudo-ginseng powder in 10mL of deionized water as a detection sample, wherein the concentration is 0.01 g/mL;

(2) centrifuging the sample solution at 4000rpm for 20min, sucking supernatant, filtering the supernatant with 0.45 μm membrane to obtain sample 1 and sample 2, and storing in a refrigerator at 4 deg.C;

2. performing labeling recovery detection and recovery rate calculation on Notoginseng radix sample

(1) Taking 6 15mL centrifuge tubes, adding 200 μ L of the fluorescent carbon dot CDs-aptamer conjugate prepared in example 1, adding 200 μ L of sample 1 to three tubes, and adding 200 μ L of sample 1 to the other three tubes;

(2) adding 1mg/mL of cadmium (Cd) standard solution into a three-tube containing a sample 1 respectively, adding deionized water to a constant volume of 3mL, wherein the concentration of cadmium (Cd) in a 3mL system is 20ng/mL, 40ng/mL and 60ng/mL respectively; sample 1 for sample 2; incubate at 37 ℃ for 30min with a shaker.

(3) Exciting at 344nm by using a fluorescence spectrometer, and recording a fluorescence spectrum at 420 nm; substituting the obtained result into the linear equation of the embodiment 5 to obtain the corresponding concentration of cadmium;

according to the formula: the recovery P = [ (measured value of spiked sample-measured value of sample) ]/spiked amount ] × 100% gives the spiked recovery, with the results: the recovery rate of the sample 1 containing 20ng/mL cadmium is 86.50%, the recovery rate of the sample 1 containing 40ng/mL cadmium is 99.30%, and the recovery rate of the sample 1 containing 60ng/mL cadmium is 96.67%; the recovery rate of the sample 2 containing 20ng/mL cadmium is 89.49%, the recovery rate of the sample 2 containing 40ng/mL cadmium is 104.09%, and the recovery rate of the sample 2 containing 60ng/mL cadmium is 103.56%.

Meanwhile, an ICP-MS method is adopted to detect the added standard sample, and the results show that the recovery rate of the sample 1 containing 20ng/mL cadmium is 44.10%, the recovery rate of the sample 1 containing 40ng/mL cadmium is 102.80%, and the recovery rate of the sample 1 containing 60ng/mL cadmium is 72.43%; the recovery rate of the sample 2 containing 20ng/mL cadmium is 94.20%, the recovery rate of the sample 2 containing 40ng/mL cadmium is 62.68%, and the recovery rate of the sample 2 containing 60ng/mL cadmium is 103.80%.

The preparation method of the fluorescent carbon dot CDs-nucleic acid aptamer conjugate is simple, the concentration of cadmium and the fluorescence absorption peak value are in a linear relation, and the cadmium content in a sample is detected with good specificity and sensitivity.

Sequence listing

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