CN108872169B - Method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue - Google Patents
Method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
Abstract
The invention provides a method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissues. The method comprises the following steps: cleaning and drying plant roots, and coating a mixed component CdS/ZnS quantum dot solution with different surface ligands; carrying out laser-induced nanosecond time-resolved fluorescence spectrum detection on the epidermis of the plant root to obtain a fluorescence spectrum; and scanning the fluorescence spectra of different quantum dots to obtain derivative fluorescence spectra, and establishing standard curves of the fluorescence intensity and the concentration of different quantum dots in the epidermis of the plant root. According to the method, the nanosecond time-resolved fluorescence spectroscopy and the constant matrix derivative synchronous fluorescence spectroscopy are combined for use, so that the interference of the autofluorescence signal of the plant root surface can be effectively eliminated, the in-situ quantitative analysis of the mixed component quantum dots adsorbed by the plant root epidermis can be realized, the method is high in accuracy and stability, and the recovery rate and the selectivity are excellent; meanwhile, the method of the invention does not cause damage to plants and can carry out plant living body detection.
Description
Technical Field
The invention relates to the field of chemical detection, in particular to a method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissues.
Background
Over the past 30 years, nanoscience has achieved significant achievements. Quantum Dots (QDs) are used as typical Engineering Nanomaterials (ENMs), and due to the characteristics of small particle size (2-10nm), Quantum confinement and discrete energy level structures of electrons and holes and the like, the Quantum dots have unique optical properties and are widely applied to the fields of single molecule detection, single cell tracking, animal living body imaging and the like.
With the expansion of the application area of QDs, the production volume and the amount of access to the environment increase year by year, and thus whether new environmental pollutants are brought and new environmental problems are caused, and the concern of researchers is caused. Previous studies have demonstrated that QDs are responsible for a wide variety of classesOrganisms (algae, microorganisms, plants, animals, etc.) are potentially harmful. For example, Wu et al examined the toxic effect of 3-mercaptopropionic acid-coated water-soluble CdTe QDs on Caenorhabditis elegans (Caenorhabditis elegans), and confirmed that such QDs can induce the larvae to generate a large amount of active oxygen, and inhibit the activities of transporters and receptors of glutamic acid, 5-hydroxytryptamine and dopamine at the gene level, thereby causing the organisms to generate behavior defects and damaging the learning and memory behaviors; modlitbova et al show that CdS/ZnS QDs coated with two groups of glutathione (GSH-) and 3-mercaptopropionic acid (MPA-) have similar toxicity to duckweed (Lemna minor L.), but slightly lower than CdCl at the same Cd molar concentration2Toxicity of the solution.
In order to better control the health risks, the global circulation of the QDs environment, the process and mechanism of environment return need to be deeply understood. A large number of research results prove that the QDs are actively exchanged on the environment-plant root interface, and the QDs absorbed by the plant root system can be transmitted and enriched in the food chain, so that the QDs are one of important ways for the environmental exposure of animals and even human beings. Al-Salim et Al, conducted as early as 2011, around plant root uptake QDs, demonstrated that roots of Lolium perenne (Lolium perenne L.), Allium cepa (Allium cepa L.), and Chrysanthemum morifolium (Chrysanthemum chemomum L.) all can be enriched to varying degrees with CdSe/ZnS QDs that are rich in various ligands (glycine, cysteine, amino, and mercaptosuccinic acid). Subsequently, Das et al examined the process of absorbing N-acetylcysteine-coated manganese-doped CdS/ZnS QDs from the roots of living sugar-pod peas (Pisum sativum L.) using a combination of Atomic Force Microscopy (AFM) and Raman imaging (RM) techniques, and showed that most of such QDs adsorbed to the root epidermis and only a small portion of the QDs migrated to the pea seeds, although the absorption process was different between the ex vivo and living gramineous plants. Thwala et al introduced the progress of the work on the interaction between ENMs (including QDs) and higher plants in a review form and concluded that the process of enrichment and migration of metal QDs on the root surface (superficial and superficial) of plants is an important step in the access of deposit/water to the plant body in both free-soluble and bound QDs. Meanwhile, the ENMs (including QDs) are usually present as mixed components in the near-shore estuary region environment, and competitive adsorption and migration processes between different components may exist. Therefore, the research on the adsorption and migration processes of the mixed components QDs of different surface ligands on the surface (surface layer and superficial layer) of the plant root is of great significance for comprehensively researching the process mechanism of the plant root system for absorbing the QDs.
For a long time, UV-Vis absorption Spectroscopy has been the most convenient, efficient and rapid method of analysis in the laboratory for determining QDs content. However, the levels of ENMs (including QDs) in real environments are extremely low, far beyond the detection limits of the UV-Vis method. In order to make up for the deficiency of the analysis capability of the method, researchers measure the element composition of metal QDs (CdS/ZnS QDs, CdSe/ZnS QDs, CdTe QDs and the like) by an Inductively coupled plasma mass spectrometry (ICP-MS) method, and further calculate the content of the QDs in environmental samples (water, sediments and plants). However, although the ICP-MS method has high sensitivity and selectivity of analyzing metal elements, it requires a series of steps such as extraction, purification and measurement, which makes it impossible to quantitatively analyze the content of QDs on the root surface (surface layer and superficial layer) of a plant. In addition, under the actual environment or simulated ecological conditions, the phenomenon of QDs metal release appears on the root surface of the plant, and the ICP-MS method as a method for measuring total metal elements is difficult to effectively distinguish the QDs body from the released metal elements. In order to research the environmental processes of enrichment, migration and the like of the QDs on the plant root surface, the problems need to be overcome firstly, and a related method with the capability of in-situ quantitative analysis of the metal QDs on the plant root surface is constructed.
In view of the fact that such QDs have high fluorescence quantum yield, conventional fluorescence emission spectroscopy is suitable for detecting trace amounts of QDs in water and other organic solvents as an important means of in-situ quantitative analysis, however, unlike other liquid substrates, strong autofluorescence signal interference exists on plant root surfaces (surface layer and shallow surface layer), which makes the method unable to accurately obtain the fluorescence spectrum of the plant root surface QDs. Research shows that the autofluorescence signal mainly comes from rhizosphere secretion adsorbed on the surface of plant roots, root microorganisms capable of emitting fluorescence and the like, and the fluorescence lifetime of the autofluorescence signal is extremely short, usually less than 10ns, unlike the fluorescence signal of QDs. Based on this, researchers have recently preliminarily constructed an in-situ quantitative analysis method of plant root surface QDs by subtracting short-lived fluorescent signals by using Nanosecond time-resolved fluorescence spectroscopy (NTFS), and the sensitivity reaches the level of ng/g. However, up to now, such methods have not been able to effectively resolve fluorescence emission spectra of multi-component QDs for in situ quantitative analysis of mixed components QDs. Therefore, the defect that the fluorescence spectrum signals of the mixed components QDs cannot be analyzed by an NTFS method is overcome, and the in-situ quantitative analysis method for constructing the mixed components QDs on the root surface of the plant is one of the core problems to be solved urgently.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for quantitatively measuring mixed component CdS/ZnS quantum dots in the epidermal tissue of a plant root.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue, the method comprising:
(a) cleaning and drying the roots of the model plants, and then coating a solution of mixed component CdS/ZnS quantum dots with different surface ligands;
(b) performing laser-induced nanosecond time-resolved fluorescence spectrum detection on the epidermis of the model plant root coated with the quantum dots to obtain a corresponding fluorescence spectrum;
scanning the fluorescence spectra of different quantum dots by the same path of the matrix fluorescence signal to obtain derivative fluorescence spectra, and establishing standard curves of the fluorescence intensity and concentration of different quantum dots in the epidermis of the plant root.
Preferably, in the step (a) of the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the model plant is obtained by culturing the plant embryonic axis and/or the plant seed under the condition of a pollution-free substrate.
Preferably, in the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the cultivation is sand cultivation.
Preferably, in the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the culture is carried out in a constant-temperature illumination incubator; wherein the illumination intensity is 200 mu mol/m2S, light/dark cycle time 14/10 h.
Preferably, in the step (a) of the method for quantitatively determining mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the root of the model plant is a main root with a meristematic region and an elongation region.
Preferably, in the step (a) of the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the mixed component CdS/ZnS quantum dots with different surface ligands include: with oleic acid, mercaptoethylamine, PEG-COOH, PEG-NH2、MPA-NH2Or at least two of the CdS/ZnS quantum dots of the MPA-COOH ligand.
Preferably, in the step (b), the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root further comprises the step of positioning and fixing the root of the model plant coated with the quantum dots on a sample rack, and then carrying out laser-induced nanosecond time-resolved fluorescence spectrum detection.
Preferably, in the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, a quartz plate with scales is fixedly arranged at the center of the top of the sample rack; wherein, the roots of the model plants coated with the quantum dots are fixed on the quartz plate.
Preferably, in the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the size of the sample holder is as follows; (160-170) mmx (70-80) mmx (100-110) mm; and/or the quartz plate has the following dimensions: (75-80) mmx (23-26) mmx (1-2) mm.
Preferably, in the method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root, the conditions of laser-induced nanosecond time-resolved fluorescence spectrum detection are as follows: objective lenses 0X/0.8 and 40X/0.65DIC dry lenses; a delay time of 30 ns; cycle number 24; excitation light wavelength is 405 nm; the pulse frequency is 40 MHz; the single signal accumulation time is 5.00 s; image resolution 256 × 256 pixels; the pixel size was 49.7 nm.
Compared with the prior art, the invention has the beneficial effects that:
in the method, a nanosecond time-resolved fluorescence spectrum testing method is combined with a constant matrix derivative synchronous fluorescence spectrum method for use, so that the interference of the autofluorescence signal of the plant root surface can be effectively eliminated, the in-situ quantitative analysis of the mixed component quantum dots adsorbed by the plant root epidermis can be realized, and the method has high accuracy, good stability, excellent recovery rate and excellent selectivity;
meanwhile, the method of the invention does not cause damage to plants and can carry out plant living body detection.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In view of the fact that the existing detection method can not realize single quantitative detection on the mixed component quantum dots attached to the epidermis of the plant root, the invention particularly provides a method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermis tissue of the plant root so as to solve the defects in the prior art.
The detection method provided by the invention comprises the following steps:
(a) cleaning and drying the roots of the model plants, and then coating a solution of mixed component CdS/ZnS quantum dots with different surface ligands;
in this step, the model plant is preferably Kandelia candel (K.obovata) or wheat (Triticum assivnm L.);
in order to avoid inaccurate detection results caused by pollution of model plants, in the invention, preferably, the method takes the eggplant embryonic axis and wheat seeds collected from an uncontaminated area as base materials, cleans and dries the base materials, and then plants the base materials in a constant-temperature illumination incubator for 6 months by sand culture to obtain a plurality of groups of the Kandelia candel and wheat plants.
Before the mixed component CdS/ZnS quantum dots are polluted and exposed, the roots of wheat seedlings and Kandelia candel plants need to be cleaned to remove impurities stained to the roots; after drying, the primary root containing the meristematic region and the elongation region is selected as the experimental substrate, and preferably, the primary root (rooted plant) having a size of about 0.5cm × 2.0cm (diameter × length) is selected as the experimental sample.
Then, coating a solution (preferably acetone solution) of mixed-component CdS/ZnS quantum dots with different surface ligands on the surface of the Kandelia candel or wheat root sample;
in the invention, the preferable mixed component of three different CdS/ZnS quantum dots with surface ligands of oleic acid, PEG-COOH (carboxyl modified PEG) and MPA-COOH is taken as the raw material of the quantum dots to be tested;
meanwhile, preferably, in order to establish a standard curve of the fluorescence intensity of different quantum dots in the epidermis of the plant root and the corresponding concentration thereof, in the invention, a mixed component CdS/ZnS quantum dot solution with different concentrations (the total concentration of the quantum dots and the concentrations of the different quantum dots can be adjusted) is subjected to multiple groups of parallel experiments under the same condition, so that the corresponding standard curve and the linear corresponding relation are obtained.
(b) Performing laser-induced nanosecond time-resolved fluorescence spectrum detection on the epidermis of the model plant root coated with the quantum dots to obtain a corresponding fluorescence spectrum;
unlike plant leaves, plant roots are cylindrical in shape. Therefore, in the fluorescence measurement, especially in the parallel pollution experiment process of the mixed CdS/ZnS quantum dots with different concentrations of a plurality of groups of plant roots, the conventional fixing means is adopted, so that the plant roots measured at each time are difficult to be ensured to be at the same position, and the accuracy and the repeatability of the detection result are influenced.
In order to solve the problem, the method of the invention is characterized in that a novel sample holder is adopted, and the size of the sample holder is preferably 167.5mm multiplied by 74.2mm multiplied by 150.0mm (length multiplied by width multiplied by height);
meanwhile, a quartz plate with millimeter scales is fixedly arranged at the center of the top of the sample holder (the quartz plate can be symmetrically arranged along the axis of the sample holder and is kept horizontal), and the size of the quartz plate is preferably 76.2mm multiplied by 25.4mm multiplied by 1.2mm (length multiplied by width multiplied by height);
furthermore, after the model plant roots after the quantum dot pollution exposure (quantum dot coating) are fixed on the quartz plate, the positions of the plant roots in each experiment can be adjusted through scale reading on the quartz plate, so that the plant root samples in multiple experiments can be placed at the same positions, the experiment conditions of each group are kept the same, and the stability and the repeatability of the method are improved.
Taking oleic acid (oleic acid) -CdS/ZnS QDs adsorbed on K.obovata root samples as an example, after the root samples are fixed by the sample rack, the maximum emission peak position (lambda) is obtainedex＝405nm，λem455nm) from 10.26% to 2.63% (n 9), the reproducibility is significantly improved, and the use of the sample holder is also one of the important reasons that the measurement method of the present invention can be realized.
After the model plant roots coated with the quantum dots are fixed on a quartz plate, preferably, the position of a light ray probe is also adjusted, so that the distance between the light ray probe and the sample roots is kept to be about 4.0 mm;
then, the objective lenses are 0X/0.8 and 40X/0.65DIC dry lenses; the delay time is 30 ns; the cycle number is 24; the excitation light wavelength is 405 nm; the pulse frequency is 40 MHz; the single signal accumulation time is 5.00 s; the image resolution is 256 × 256 pixels; and (3) detecting fluorescence spectra (laser-induced nanosecond time-resolved fluorescence spectra) under the detection condition that the pixel size is 49.7nm, and obtaining the fluorescence spectra corresponding to different quantum dots.
Then, drawing the fluorescence spectra of the oleic acid-CdS/ZnS quantum dots, the PEG-COOH-CdS/ZnS quantum dots and the MPA-COOH-CdS/ZnS quantum dots into the same contour map (which can be completed by utilizing origin and other software), selecting the scanning path with the same matrix fluorescence signal, scanning the fluorescence spectrum (laser-induced nanosecond time-resolved fluorescence spectrum), and then obtaining a derivative fluorescence spectrum through calculation (such as program or manual calculation);
and then, establishing a standard curve of the fluorescence intensity and the concentration of different quantum dots in the epidermis of the plant root according to the application concentration of the mixed component quantum dots in the experiment and fluorescence intensity data obtained by corresponding calculation.
As can be seen from the steps of the detection method, the method provided by the invention is a constant matrix derivative nanosecond time-resolved fluorescence spectrum in-situ analysis method which combines the laser-induced nanosecond time-resolved fluorescence spectrum and the derivative fluorescence spectrum.
Meanwhile, through the steps (a) and (b), an experimental system for determining the nanosecond time-resolved fluorescence spectrum in-situ analysis method of the constant matrix derivatives of the mixed components of the plant root epidermis, such as Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs, MPA-COOH-CdS/ZnS QDs and the like, can be established, and the concentration of the three or more quantum dots adsorbed in the plant root epidermis to be detected can be determined by adopting the system;
similarly, by adjusting the quantum dots used in the experiment, an experiment system of a constant matrix derivative nanosecond time-resolved fluorescence spectrum in-situ analysis method for detecting the concentration of other types of quantum dots can be established, and the adsorption concentration of the corresponding quantum dots on the roots of the plants can be measured.
The steps of the detection method for the concentration of the quantum dots adsorbed in the plant roots to be detected can be referred to as follows:
(c) cleaning the roots of the plants to be detected, and selecting root regions (namely main roots with a meristematic region and a rooting region, the size of which is about 0.5cm multiplied by 2.0 cm) which are the same as the model plants;
then, according to the same treatment method of the model plant, the plant root is fixed on the sample rack provided by the invention, and the fixed position of the sample rack is the same as that of the model plant;
then, carrying out laser-induced nanosecond time-resolved fluorescence spectrum detection to obtain a corresponding fluorescence spectrum;
and then scanning the fluorescence spectra of different quantum dots by the same path of the matrix fluorescence signal to obtain a derivative fluorescence spectrum, and obtaining the concentration of the corresponding quantum dots in the epidermal tissue of the plant root to be detected according to a standard curve.
Experimental example 1
K.obovata hypocotyls from the mangrove forest protected area (24 ° 36 'N, 118 ° 14' E) in the northern sea of Guangxi and purchased wheat (Triticum aestivnm L.) seeds were collected. After the sample is washed with tap water for three times, the sample is naturally dried, placed in a constant-temperature illumination incubator and cultivated in sand for 6 months. The culture conditions were: the illumination intensity is as follows: 200. mu. mol/m2s1(ii) a Light/dark cycle time: 14/10 h; temperature: 298.15 +/-1K; humidity: 70 percent.
Wheat seedlings (Triticum austivnm L.) and K.obovata roots were washed 3 times with distilled water to remove debris such as sand, sediment and sludge, and after natural air-drying, a main root having a meristematic region and an elongation region was selected and sized 0.5cm × 2.0cm (diameter × length).
Acetone solutions of varying concentrations of Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs were slowly applied to K.obovata or wheat root samples using a 20 μ L micropipette.
And (3) placing the plant root samples polluted and exposed by CdS/ZnS QDs with different concentrations on a quartz plate of a sample rack, and adjusting the position of the optical fiber probe to enable the optical fiber probe to be about 4.0mm right above the center of the plant root sample. And then, measuring the relevant fluorescence spectra (n is 9) of the plant root epidermis Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs in situ by adopting the instrument condition of the laser-induced nanosecond time-resolved fluorescence spectrometer.
And drawing the fluorescence spectra of the three into the same contour map, and selecting the scanning path with the same matrix (background) fluorescence signal. And finally, performing corresponding nanosecond time resolution fluorescence spectrum scanning, and obtaining a corresponding derivative fluorescence spectrum.
The results of the measurements are shown in Table 1 below.
TABLE 1 analytical characterization of the method of the invention
aThe detection limit calculation method of the method is that three times of relative standard deviation is divided by the slope;by represents the fluorescence intensity value of CdS/ZnS QDs in the epidermis of the plant root;cx represents CdS/ZnS QDs content in root epidermis
As can be seen from the data of the detection results in Table 1, the relative fluorescence intensity of the compounds increases linearly (R.obovata. root epidermis Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs) with the increase of the concentration of the mixed components2Respectively as follows: 0.9922, 0.9901, and 0.9915). The absorption to the root coat CdS/ZnS QDs mixed component of wheat (Triticum assivnm L.) resulted in a similar result.
Further, in the detection method provided by the invention, the linear ranges of the mixed components adsorbed to K.obovata and wheat (Triticum assivnm L.) root epidermis Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs measured in situ are respectively as follows: oleic acid-CdS/ZnS QDs, 28.9-1230ng/g and 20.2-1350 ng/g; PEG-COOH-CdS/ZnS QDs, 41.3-1195ng/g and 33.7-1180 ng/g; MPA-COOH-CdS/ZnS QDs 35.8-1200ng/g and 23.3-1285 ng/g.
Therefore, the method has the capability of quantitatively determining the CdS/ZnS QDs mixed component of the plant root epidermis in situ.
Examination of the detection method of the present invention
(1) In order to verify the analytical characteristics of the established method for measuring the CdS/ZnS QDs mixed component of the plant root epidermis, a standard recovery rate experiment is carried out, and the results are shown in the following table 2:
TABLE 2 recovery of the process of the invention
From the detection results in table 2, the result of the standard recovery rate experiments of the plant root epidermis Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs is that the recovery rate ranges of the three in the K.obovata root epidermis are respectively: 91.3%, 115.8% and 92.6%, respectively, the recovery in wheat (Triticum aestivum L.) root epidermis is: 93.2%, 109.5% and 94.7%.
(2) In actual environment, Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs not only exist in a mixture form generally, but also the ratios of Oleic acid-CdS/ZnS QDs adsorbed by the plant root epidermis, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs in different regions are obviously different. Therefore, to ensure the accuracy of the analysis method, an interference experiment was performed, and the results are shown in table 3.
TABLE 3 interference experiments with the method of the invention
As can be seen from the experimental results shown in Table 3 above, the concentrations of PEG-COOH-CdS/ZnS QDs adsorbed to plant roots and MPA-COOH-CdS/ZnS QDs were fixed at an intermediate concentration of 400.0ng/g in the linear range, and the change in the concentration of Oleic acid-CdS/ZnS QDs had no effect on the measurement thereof. This phenomenon also exists for the other two substances.
The experimental results are examined by combining the detection methods, so that the recovery rate, the sensitivity and the selectivity of the analysis method provided by the invention all meet the requirements of in-situ measurement of the added amount of Oleic acid-CdS/ZnS QDs, PEG-COOH-CdS/ZnS QDs and MPA-COOH-CdS/ZnS QDs in the epidermis of the plant root.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (9)
1. A method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissues is characterized by comprising the following steps:
(a) cleaning and drying the roots of the model plants, fixing the model plants on a sample rack, and coating a solution of mixed components CdS/ZnS quantum dots with different surface ligands;
(b) performing laser-induced nanosecond time-resolved fluorescence spectrum detection on the epidermis of the model plant root coated with the quantum dots to obtain a corresponding fluorescence spectrum;
scanning the fluorescence spectra of different quantum dots by the same path of the matrix fluorescence signal to obtain derivative fluorescence spectra, and establishing standard curves of the fluorescence intensity and the concentration of different quantum dots in the epidermis of the plant root;
a quartz plate with millimeter scales is fixed at the center of the top of the sample frame, after the model plant roots exposed by quantum dot pollution are fixed on the quartz plate, the positions of the plant roots in the experiments are adjusted through scale reading on the quartz plate, so that plant root samples in different experiments can be placed at the same position, and the experiment conditions are ensured to be the same;
(c) the steps of the detection method for the concentration of the quantum dots adsorbed in the plant roots to be detected can be referred to as follows:
cleaning the roots of the plants to be detected, and selecting root areas identical to the model plants;
then, according to the same treatment method of the model plant, fixing the plant roots on a sample rack, and enabling the fixed position of the plant roots to be the same as that of the model plant;
then, carrying out laser-induced nanosecond time-resolved fluorescence spectrum detection to obtain a corresponding fluorescence spectrum;
scanning the fluorescence spectra of different quantum dots by the same path of the matrix fluorescence signal to obtain a derivative fluorescence spectrum, and obtaining the concentration of the corresponding quantum dots in the epidermal tissue of the plant root to be detected according to a standard curve;
the ligands with different surfaces are oleic acid and sulfydrylEthylamine, PEG-COOH, PEG-NH2、MPA-NH2Or MPA-COOH.
2. The method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue according to claim 1, wherein in step (a), the model plant is obtained by culturing plant hypocotyls and/or plant seeds under the condition of non-pollution matrix.
3. The method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue according to claim 2, wherein the culturing is sand-culture planting.
4. The method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue according to claim 2, wherein the culturing is carried out in a constant temperature illumination incubator;
wherein the illumination intensity is 200 mu mol/m2S, light/dark cycle time 14/10 h.
5. The method for quantitatively determining mixed component CdS/ZnS quantum dots in epidermal tissue of plant root as claimed in claim 1, wherein in step (a), the root of said model plant is the main root with meristematic and elongation regions.
6. The method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root as claimed in claim 1, wherein the step (b) further comprises the step of performing laser-induced nanosecond time-resolved fluorescence spectroscopy detection after the root of the model plant coated with quantum dots is positioned and fixed on a sample holder.
7. The method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root as claimed in claim 6, wherein a quartz plate with scales is fixedly arranged at the center of the top of the sample holder;
wherein, the roots of the model plants coated with the quantum dots are fixed on the quartz plate.
8. The method for quantitatively determining mixed component CdS/ZnS quantum dots in plant root epidermal tissue according to claim 7, wherein the size of the sample holder is; (160-170) mmx (70-80) mmx (100-110) mm
And/or the quartz plate has the following dimensions: (75-80) mmx (23-26) mmx (1-2) mm.
9. The method for quantitatively determining the mixed component CdS/ZnS quantum dots in the epidermal tissue of the plant root as claimed in claim 1, wherein the conditions for laser-induced nanosecond time-resolved fluorescence spectroscopy detection are as follows:
objective lenses 0X/0.8 and 40X/0.65DIC dry lenses; a delay time of 30 ns; cycle number 24; the wavelength of the exciting light is 405 nm; the pulse frequency is 40 MHz; the single signal accumulation time is 5.00 s; image resolution 256 × 256 pixels; the pixel size was 49.7 nm.
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