CN107907583B - Method for simultaneously and quantitatively detecting CdTeQDs and degradation or metabolite thereof and application of method - Google Patents

Abstract

The invention relates to the technical field of detection of mixed products of quantum dot degradation and metabolism, in particular to a method for simultaneously and quantitatively detecting CdTe QDs and degradation or metabolism products thereof and application thereof. The detection method of the invention uses 10-40 mmol.L^‑1The phosphate buffer solution with pH of 9.0 is 10-40 mmol.L of electrophoresis buffer solution^‑1Taking borate buffer solution with pH of 9.0 as sample buffer solution, and adopting capillary electrophoresis chromatography to separate Cd in the same medium²⁺And complex thereof, TeO₃ ^2‑Separating from CdTe QDs, and combining with quantitative detection to realize simultaneous quantitative detection of CdTe QDs and their degradation or metabolic products, the detection method is simple, fast and sensitive, and the absolute quantitative detection limit reaches Feike level. Furthermore, a CE-ICP-MS instrument is used in a combined way, so that the rapid separation and the accurate quantification of CdTe QDs and degradation products or metabolic products thereof in complex biological systems such as buffer solution, serum, cell culture solution and the like are realized.

Description

Method for simultaneously and quantitatively detecting CdTeQDs and degradation or metabolite thereof and application of method

Technical Field

The invention relates to the technical field of detection of mixed products of quantum dot degradation and metabolism, in particular to a method for simultaneously and quantitatively detecting CdTeQDs and degradation or metabolism products thereof and application thereof.

Background

Quantum Dots (QDs), also known as semiconductor nanocrystals, are mainly composed of elements of group IIB-VIA or IIIA-VA, and generally have a particle size of 1-10 nm. Due to the quantum size effect, the surface effect, the dielectric confinement effect, the quantum tunneling effect, the coulomb blocking effect and the like, the optical material shows excellent optical characteristics and unique physicochemical properties, such as wide absorption, narrow emission, high fluorescence quantum yield, low photobleaching, adjustable emission wavelength and the like. In 1983, Brus of Bell laboratories in the United states firstly reports that CdS nanocrystals have quantum size effect and other related properties, and the sequence of QDs research is drawn. In 1993, a Bawendi team of Massachusetts institute of technology and technology prepared high-quality CdE QDs (E ═ Se, Te and S) by using TOPO and TOP as solvents, so that the related application of the QDs becomes possible. In 1998, professor Alivisatos and neyming, university of california, applied water-soluble quantum dots to the field of biomedicine, and opened a new field of quantum dot application. QDs are now widely used in the fields of biomedical imaging, disease diagnosis, drug delivery and new target discovery, and quantum dot batteries. With the increasing application of QDs, its environmental and population exposure is increasingly prominent. Because QDs have a nanometer-scale effect or contain toxic components (e.g., Cd, Se, Te, etc.), their potential risks to living beings and the environment have become a focus of researchers' attention. Cell experimental studies show that QDs can cause changes in cell morphology and structure, inhibition of cell growth, mitochondrial dysfunction, DNA damage, apoptosis, and the like; animal experiment research shows that QDs have obvious toxicity to liver, kidney, testis, etc.

In recent years, the mechanism of QDs cytotoxicity and biotoxicity has been studied as the focus of research in this field. Studies have shown that the chemical composition, particle size, surface charge and shell type of QDs affect the toxicity of QDs to some extent. In addition, the absorption, distribution, metabolism and excretion of QDs by cells or living organisms, and the degradation, aggregation and chemical morphology change during the process are all closely related to the toxicity. However, the results of the studies have not been agreed so far due to the complexity of the toxicity-affecting factors and the unmet needs of the research methods and means. Studies have shown that CdSe QDs are degraded under oxidizing or acidic conditions to generate free Cd²⁺Ions, Cd²⁺Ions can induce oxidative stress, which in turn causes a series of cytotoxicity and disruption of calcium signaling. After CdTe QDs are degraded under oxidation or acidic condition or metabolized in living organisms, Cd can be free Cd²⁺Or in the form of a composite thereof, and Te is further oxidized to form TeO₃ ^2-. Therefore, the degradation before QDs infection and the metabolic change after the infection of cells or organisms, i.e. the morphological analysis of QDs and their degradation or metabolic products, become the bottleneck of the toxicity mechanism research or quantum dot quality evaluation。

At present, the conventional research means for QDs morphological analysis mainly includes infrared spectroscopy, X-ray diffraction, TEM, SEM, ICP-MS and the like. The TEM and SEM have large human interference factors, high technical requirements on operators and time and labor waste; the X-ray diffraction energy resolution is low, the signal-to-noise ratio is low, and the requirements on working conditions are strict; ICP-MS generally only can analyze the total amount of elements, and more complex sample pretreatment steps are needed for realizing different morphological analysis, so that larger result errors are brought. None of the above analytical methods can achieve simultaneous, accurate and quantitative analysis of QDs and their degradation or metabolites. Therefore, it is urgently needed to develop a method for analyzing different morphological components of QDs and their degradation or metabolites with high sensitivity, low detection limit, high accuracy, good stability and high analysis speed, which not only provides a new and convenient method for the research of the cytotoxicity and biotoxicity mechanism of QDs, but also helps us to grasp the degradation and aggregation change rule of QDs, and further provides technical support for the quality (purity, absolute quantification, etc.) evaluation and related standard establishment of the commercial QDs.

Disclosure of Invention

The invention aims to provide a method for simultaneously and quantitatively detecting CdTeQDs and degradation or metabolic products thereof.

Meanwhile, the invention also provides an application of the method for simultaneously and quantitatively detecting the CdTeQDs and degradation or metabolic products thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for simultaneously and quantitatively detecting CdTe QDs and degradation or metabolic products thereof comprises the steps of measuring 10-40 mmol.L^-1The phosphate buffer solution with pH of 9.0 is an electrophoresis buffer solution, and 10-40 mmol.L^-1And (3) taking a borate buffer solution with the pH value of 9.0 as a sample buffer solution, separating the target object in the sample to be detected by adopting capillary electrophoresis chromatography, and then quantitatively detecting the separated target object.

Optionally, the phosphate buffer comprises: including Na₂HPO₄And NaH₂PO₄。

Optionally, the borate buffer comprises: including Na₂B₄O₇And H₃BO₃。

Optionally, the separated target is quantitatively detected by connecting a capillary electrophoresis analyzer with an ICP-MS (inductively coupled plasma-mass spectrometry).

Optionally, the capillary electrophoresis analyzer has the following working conditions: uncoated fused silica capillary: i.d.75 μm; o.d.360 μm; length 100 cm; detection wavelength: 214 nm; sample introduction pressure: 35 mbar; sample introduction time: 5 s; separation voltage: 16 kV; separation temperature: 25 ℃; pre-running a program: 1.0N NaOH, 180s → H₂O, 180s → running buffer, 300 s; sheath fluid type: 1ppm In internal standard aqueous solution; flow rate of sheath fluid: 0.5 ml/min; the split ratio is as follows: 1:100.

Optionally, the ICP-MS operating conditions are: incident power: 1550W; sampling depth: 8.0 mm; flow rate of carrier gas: 1.20-1.40L/min; flow rate of auxiliary gas: 0.8-1.0L/min; plasma gas flow rate: 13-16L/min; compensating the airflow speed: 1.0-1.3L/min; rotation speed of peristaltic pump: 0.5 to 2.0 rps; temperature of the atomization chamber: 2-5 ℃; and (3) detecting mass number:¹¹¹Cd，¹²⁵te; detecting an internal standard:¹¹⁵in; sampling mode: TRA; integration time:¹¹¹cd and¹²⁵the Te is 0.1s, and the content of the lead is,¹¹⁵in was 0.05 s.

The detection method is applied to the aspect of simultaneously detecting CdTe QDs in serum, cell culture solution or buffer solution and degradation or metabolic products thereof.

Since commercially available CdTe QDs are degraded during actual production or storage to produce free Cd²⁺、TeO₃ ^2-The above-mentioned detection method can be used to detect the components of commercially available CdTe QDs and further evaluate the quality thereof.

The detection method of the invention uses 10-40 mmol.L^-1The phosphate buffer solution with pH of 9.0 is 10-40 mmol.L of electrophoresis buffer solution^-1Using borate buffer solution with pH of 9.0 as sample buffer solution, and successfully separating Cd in the same medium by adopting capillary electrophoresis chromatography²⁺And complex thereof, TeO₃ ^2-Separating from CdTe QDsThe method is simple, convenient and rapid, has high sensitivity, low cost of reagents and low cost, and the absolute quantitative detection limit reaches Feike (fg) level.

Furthermore, the invention adopts the CE-ICP-MS instrument for combined use, thereby realizing the rapid separation and the accurate quantification of the CdTe QDs and the degradation products or the metabolic products thereof in complex biological systems such as buffer solution, serum, cell culture solution and the like.

Drawings

FIG. 1 is a schematic diagram of the synthetic principle of L-GSH/L-cysteine-capped CdTe QDs;

FIG. 2 the UV-VISIBLE absorption, fluorescence and excitation spectra of L-GSH/L-cysteine-capped CdTe QDs;

FIG. 3 TEM image of L-GSH/L-cysteine-capped CdTe QDs;

FIG. 4 is a separation detection spectrum of a sample to be detected in example 1;

FIG. 5 Cd in example 1²⁺And TeO₃ ^2-Separating and detecting a spectrum by mixing standard solutions;

FIG. 6 is a separation detection spectrum of a sample to be detected in example 2;

FIG. 7 Cd in example 2²⁺And TeO₃ ^2-Separating and detecting a spectrum by mixing standard solutions;

FIG. 8 is a separation detection spectrum of a sample to be detected in example 3;

FIG. 9 Cd in example 3²⁺And TeO₃ ^2-Separating and detecting a spectrum by mixing standard solutions;

FIG. 10 commercial CdTe-NH in example 6₂-560 an isolated detection profile of the stock solution;

FIG. 11 separation detection spectrum of stock solution of commercially available CdTe-COOH-560 in example 6;

FIG. 12 is a graph of the isolated test spectra of the sera from rats infected with the virus at different sampling times in example 7;

FIG. 13 is a graph of the isolated test spectra of the sera from rats infected with the virus at different sampling times in example 7;

FIG. 14 Standard Curve as drawn in example 1.

Detailed Description

The technical solution of the present invention will be described in detail by specific examples.

The instruments and reagents used in the following examples:

capillary electrophoresis analyzers (Agilent Technology, aldbernn, Germany); fused silica capillary (Fengjinchromatograph devices, Inc. in Yongyecounty, Hebei); an inductively coupled plasma mass spectrometer (8800ICP-MS Triple Quad, Agilent Technology, Tokyo, Japan); a CE-ICP-MS interface (CE ESI Sprayer II, Agilent Technology, Germany) is used for connecting the capillary electrophoresis analyzer with the inductively coupled plasma mass spectrometer to form CE-ICP-MS analysis equipment;

transmission electron microscopy (TEM, JEOL EM2010, Tokyo, Japan); ultraviolet-visible spectrophotometers (UV-2600, Shimadzu, Japan); molecular fluorescence spectrophotometer (RF-5301pc, Shimadzu, Japan); a pH meter (PB-10, Sartorius,

germany); zeta-potential analyzers (Malvern, Worcestershire, United Kingdom); digital display intelligent temperature control magnetic stirrers (SZCL-3B, Vol instruments Limited liability company, Hill City);

water-soluble L-GSH/L-cysteine-capped CdTe QDs (synthesized by the laboratory); 2 kinds of CdTe QDs (CdTe-NH)₂560 + -5 CdTe-COOH-560 + -5, made in China); potassium borohydride (KBH)₄Not less than 95%), cadmium chloride (CdCl)₂·2.5H₂O, > 99.0%), L-glutathione (reduced form, BR), L-cysteine (BR), tellurium powder (Te, high purity grade) are all purchased from national drug group, Inc.; sodium tellurite (Na)₂TeO₃99.7%, Aladdin, shanghai); sodium hydroxide, borax (Na)₂B₄O₇·10H₂O) and boric acid (Sigma-Aldrich) were purchased from Sigma-Aldrich; disodium hydrogen phosphate (Na)₂HPO₄·7H₂O), sodium dihydrogen phosphate (NaH)₂PO₄·H₂O) and tween 20 were purchased from amresco (Ohio, America);

1g/L of Cd²⁺The standard solution is prepared from cadmium chloride; 1g/L of TeO₃ ^2-Prepared from sodium tellurite (calculated as Te); 1g/L of In standard solution (containing 1mol/L of HNO)₃) Purchased from the national center for standards; human serum albumin (HSA, Sigma, USA); cell culture fluid (DMEM, corning cellgro, USA); australian fetal bovine serum (FBS, Corning CellGro, USA).

All solutions were prepared with Milli-Q ultrapure water (resistivity ≥ 18.2M Ω. cm), and glassware for the experiment was HNO-treated₃After soaking overnight, the mixture is washed clean by pure water.

Wherein the synthesis principle of the water-soluble L-GSH/L-cysteine-capped CdTe QDs is shown in figure 1, and the specific synthesis method comprises the following steps:

(1) preparation of CdTe QDs precursor KHTe:

respectively weighing 0.13g of tellurium powder and 0.17g of potassium borohydride in a reaction bottle, putting in magnetons, sealing, injecting 1-2 mL of ultrapure water by using an injector, protecting with nitrogen, and reacting on a magnetic stirrer for 1h to obtain a CdTe QDs precursor, wherein the reaction formula is as follows:

4KBH4+2Te+7H₂O→2KHTe+Na₂BO₇+14H₂↑

(2) cadmium chloride reaction System preparation

0.47g of CdCl was weighed out separately₂·2.5H₂O, 0.10-0.19 g of Glutathione (GSH) and 0.15-0.24 g of Cysteine (Cysteine), sequentially adding the materials into a 500ml three-neck flask which is placed in a magneton in advance, adding 400ml of ultrapure water, placing the mixture into an electric heating sleeve type temperature-controlled magnetic stirrer, uniformly stirring, dropwise adding 1.0mol/L NaOH solution, gradually changing the solution from white emulsion into colorless transparent solution, and finally adjusting the pH value of the solution to 9.0 by using 1.0mol/L NaOH;

(3) preparation of CdTe QDs

Introducing nitrogen into one side of a three-neck flask for 15min, injecting the CdTe QDs precursor reaction solution prepared in the step (1) into the cadmium chloride reaction system in the step (2), refluxing for 1h under the protection of nitrogen at 96 ℃, cooling, and storing in a dark place, wherein the product is CdTe QDs with GSH and Cysteine groups modified on the surface, namely L-GSH/L-Cysteine-capped CdTe QDs. The reaction formula is as follows: cd [ Cd ]²⁺+HTe^-+OH^-→CdTe+H₂O。

Characterization of L-GSH/L-cysteine-capped CdTe QDs:

in the specific embodiment of the invention, the ultraviolet-visible absorption spectrum and the fluorescence spectrum of the L-GSH/L-cysteine-capped CdTe QDs synthesized by the aqueous phase direct synthesis method are shown in figure 2, the characterization result of a Transmission Electron Microscope (TEM) is shown in figure 3, and the maximum absorption wavelength and the maximum fluorescence wavelength of the CdTe QDs are shown in figure 2 and are 560nm and 578nm (lambda)_ex450nm), a smaller full width at half maximum (FWHM 36nm) indicates a narrower particle size distribution of the synthesized quantum dots. TEM results show that the synthesized CdTe QDs has particle size of 4.8 +/-0.5 nm and homogeneous distribution. In addition, Zeta potential analysis shows that the Zeta potential value of the synthesized CdTe QDs is- (42.6 +/-4.8) mV, which indicates that the surface of the quantum dot has negative charges and has good stability, thereby meeting the use requirements of experiments.

The self-synthesized L-GSH/L-cysteine-capped CdTe QDs needs to be purified before use, and the treatment method comprises the following steps: taking appropriate amount of L-GSH/L-cysteine-entrapped CdTe QDs synthetic system stock solution, adding 3 times volume of anhydrous ethanol, mixing well, centrifuging at 5000rpm for 5min at 4 deg.C, removing supernatant, precipitating with 10mM Na equal to the stock solution₂HPO₄(pH9.0), fully dissolving, adding 3 times of volume of absolute ethyl alcohol, fully mixing uniformly, performing ultrasonic treatment for 5min, centrifuging at 4 ℃ for 5min at 5000rpm, removing supernatant, performing repeated treatment on the precipitate according to the steps once, and removing supernatant after final centrifugation to obtain the precipitate, namely the purified L-GSH/L-cysteine-capped CdTe QDs.

The sample buffer solution in the following examples is 10 to 40 mmol.L^-1pH9.0 Borate buffer with Na as component₂HPO₄And NaH₂PO₄。

The electrophoresis buffer solution is 10-40 mmol.L^-1The phosphate buffer solution with pH of 9.0 is electrophoresis buffer solution, and the component is Na₂B₄O₇And H₃BO₃。

Example 1

The embodiment provides a method for simultaneously and quantitatively detecting CdTe QDs and degradation or metabolic products thereof in a buffer solution system, which comprises the following specific operation steps:

1) dissolving the purified L-GSH/L-cysteine-capped CdTe QDs 40nmoL in 960 μ L of sample buffer solution, and adding 40 μ L of HSA (50g/L) as a sample to be detected;

2) adding the sample to be detected prepared in the step 1) into a CE sample chamber of a CE-ICP-MS, adopting automatic sample injection, detecting to obtain a detection map, as shown in figure 4, showing CdTe QDs and TeO₃ ^2-、Cd²⁺Baseline separation can be achieved;

the operating conditions for CE were: uncoated fused silica capillary: i.d.75 μm; o.d.360 μm; length 100 cm; detection wavelength: 214 nm; sample introduction pressure: 35 mbar; sample introduction time: 5 s; separation voltage: 16 kV; separation temperature: 25 ℃; pre-running a program: 1.0N NaOH, 180s → H₂O, 180s → running buffer, 300 s; sheath fluid type: 1ppm In internal standard aqueous solution; flow rate of sheath fluid: 0.5 ml/min; the split ratio is as follows: 1: 100;

the ICP-MS working conditions are as follows: incident power: 1550W; sampling depth: 8.0 mm; flow rate of carrier gas: 1.20-1.40L/min; flow rate of auxiliary gas: 0.8-1.0L/min; plasma gas flow rate: 13-16L/min; compensating the airflow speed: 1.0-1.3L/min; rotation speed of peristaltic pump: 0.5 to 2.0 rps; temperature of the atomization chamber: 2-5 ℃; and (3) detecting mass number:¹¹¹Cd，¹²⁵te; detecting an internal standard:¹¹⁵in; sampling mode: TRA; integration time:¹¹¹cd and¹²⁵the Te is 0.1s, and the content of the lead is,¹¹⁵in was 0.05 s.

Taking Cd with known concentration²⁺And TeO₃ ^2-Mixing the standard solution, adding sample buffer and HSA, performing CE-ICP-MS separation analysis on the standard solution under the same conditions as step 2), and obtaining the result shown in FIG. 5, wherein FIG. 5 shows that Cd is present under the conditions²⁺Splitting of peaks (Cd with shorter migration time)²⁺Peak is known as Cd²⁺[1]Cd with longer migration time²⁺Peak is known as Cd²⁺[2]) And Cd²⁺[1]The retention time of (D) is consistent with the peak of CdTe QDs, which means that Cd is superposed in the signal of CdTe QDs in FIG. 4²⁺[1]A signal. Analysis of Cd²⁺[1]The reason for the signal is that HSA is added and a part of Cd²⁺Binding to form HSA-Cd²⁺Complexes having migration times consistent with CdTe QDs resulting inThe two substances cannot be separated, i.e. in the separation diagram of the sample to be detected, a part of HSA-Cd is superposed in the Cd signal of CdTe QDs peak²⁺Cd signal of (1).

Further, the Cd in the mixed standard solution is changed²⁺The concentration and the HSA concentration are kept unchanged, CE-ICP-MS separation analysis is carried out under the same condition, and Cd in the separation spectrum of mixed standard solutions with different concentrations is analyzed²⁺[1]Peak and Cd²⁺[2]The ratio of the signal size (peak area) of the peak remains unchanged, so Cd can be used²⁺[2]Signal pair Cd in sample to be detected²⁺[1]The signal is quantified, which lays a solid theoretical foundation for the accurate quantification of different components in the buffer solution system.

The specific quantitative method comprises the following steps:

drawing a standard curve:

for Cd with different concentrations²⁺And TeO₃ ^2-Carrying out CE-ICP-MS separation analysis on the mixed standard solution according to the method to obtain a separation map;

te standard curve: separating TeO in the spectra of the mixed standard solutions with different concentrations₃ ^2-Concentration of (2) and corresponding TeO₃ ^2-Performing linear fitting on the peak area of the sample to draw a standard curve between the peak area and the concentration of Te, and obtaining a relational expression 1 between the peak area and the concentration of Te as shown in FIG. 14;

standard curve of Cd: cd in mixed standard solution separation spectrum²⁺[1]And Cd²⁺[2]The sum of the concentrations is Cd in the standard solution²⁺Concentration of Cd²⁺[1]And Cd²⁺[2]The size of the total peak area and Cd in the standard solution²⁺Performing linear fitting on the concentration, drawing a standard curve between the peak area and the Cd concentration, and obtaining a relational expression 2 between the peak area and the Cd concentration as shown in FIG. 14;

in addition, Cd is caused by²⁺[1]And Cd²⁺[2]The peak area is a fixed value, and then Cd in the chromatogram is separated according to the mixed standard solution²⁺[1]And Cd²⁺[2]Calculating Cd according to the peak area size ratio²⁺[1]Corresponding Cd concentration and Cd²⁺[2]Corresponding Cd concentration, respectively adding Cd²⁺[1]The peak area size of the Cd is linearly fitted with the corresponding Cd concentration to draw the Cd²⁺[1]Standard curve between peak area and Cd concentration, as shown in FIG. 14, Cd²⁺[1]The relationship between the peak area of (a) and the concentration of Cd is 3; adding Cd into the solution²⁺[2]The peak area size of the Cd is linearly fitted with the corresponding Cd concentration to draw the Cd²⁺[2]Standard curve between peak area and Cd concentration, as shown in FIG. 14, Cd²⁺[2]The relationship between the peak area of (a) and the concentration of Cd is 4;

and (3) quantitative calculation:

quantification of Te: separating TeO in the atlas of the sample to be detected₃ ^2-The peak area is substituted into the relation 1, so that the degradation of CdTe QDs in the sample to be measured and TeO in the metabolite can be directly calculated₃ ^2-The concentration of (2); and substituting the peak area of Te in CdTe QDs in the separation map of the sample to be detected into a relation formula 1, and calculating the content of Te in CdTe QDs.

Quantification of Cd: separating Cd from the spectrum according to a sample to be detected²⁺[2]Peak area of (with ACd)²⁺[2]Expressed), is brought into the relation 4 to obtain the total Cd generated by the degradation or metabolism of CdTe QDs²⁺The content or concentration; separating Cd from the chromatogram of the sample to be tested²⁺[2]Peak area of (with ACd)²⁺[2]Expression) calculation of HSA-Cd²⁺Cd of complexes²⁺Peak area (using ACd)²⁺[1]Expressed), was substituted into relation 3, HSA-Cd was calculated²⁺Cd in the Complex²⁺The concentration of (c); ACd subtracted from the Cd peak area at the CdTe QDs peak²⁺[1]The peak area of Cd in CdTe QDs (expressed by AQDs-Cd), and the concentration of Cd in the CdTe QDs is the content or concentration of Cd in the CdTe QDs by substituting the AQDs-Cd into the relational expression 2.

Example 2

The embodiment provides a method for simultaneously and quantitatively detecting CdTe QDs and degradation or metabolic products thereof in a cell culture solution, which comprises the following specific operation steps:

1) taking 500 mu L of DMEM containing 1% double antibody and 10% Fetal Bovine Serum (FBS), adding 40nmoL of purified L-GSH/L-cysteine-capped CdTe QDs and 500 mu L of sample buffer solution to serve as a sample to be detected;

2) separating and analyzing the sample to be detected in the step 1) according to the same working conditions as the step 2) of the embodiment to obtain a detection map, which is shown in figure 6;

in this embodiment, Cd²⁺And TeO₃ ^2-Adding DMEM containing 1% double antibody and 10% Fetal Bovine Serum (FBS) and sample buffer solution into the mixed standard solution, and performing separation analysis under the same working conditions of the step 2) to obtain a detection map, as shown in FIG. 7;

from FIG. 6, CdTe QDs and TeO₃ ^2-、Cd²⁺Baseline separation can be achieved; from FIG. 7, Cd can be seen²⁺The peak will also appear bimodal (with Cd therein)²⁺[1]Migration time same as CdTe QDs). Further verification, different concentrations of Cd²⁺Standard solution, Cd²⁺[1]Peak sum Cd²⁺[2]The signal ratio of the peak is not changed, so the same quantitative method in the embodiment 1 can be adopted to realize the simultaneous quantitative detection of CdTe QDs in the cell culture solution and the degradation or metabolic products thereof.

Example 3

The embodiment provides a method for simultaneously and quantitatively detecting CdTe QDs in animal serum and degradation or metabolic products thereof, which comprises the following specific operation steps:

1) adding 40nmoL of purified L-GSH/L-cysteine-capped CdTe QDs into 50 mu L of rat serum, and adding 500 mu L of sample buffer solution and 450 mu L of pure water to serve as a sample to be detected;

2) separating and analyzing the sample to be detected in the step 1) according to the same working conditions as in the step 2) of the embodiment to obtain a detection map, which is shown in figure 8;

in this embodiment, Cd²⁺And TeO₃ ^2-Adding rat serum and sample buffer solution into the mixed standard solution, and performing separation analysis according to the same working conditions in the step 2) to obtain a detection map, wherein the detection map is shown in figure 9;

as can be seen from FIGS. 8 and 9, the CdTe QDs and TeO are produced according to the method of this example in the environment of animal serum₃ ^2-、Cd²⁺Baseline separation can be achieved and FIG. 9 shows Cd²⁺The peak will also appear bimodal (with Cd therein)²⁺[1]Migration with CdTe QDsThe same time). Further verification, different concentrations of Cd²⁺Standard solution, Cd²⁺[1]Peak sum Cd²⁺[2]The signal ratio of the peak is not changed, so the quantitative detection of the CdTe QDs in the animal serum and the degradation or metabolic products thereof can be realized simultaneously by adopting the same quantitative method in the example 1.

Example 4

In the embodiment, the standard curve, the detection limit, the accuracy and the precision of the detection method in the embodiment 1 to 3 are calculated and verified

Respectively taking buffer salt solution, cell culture solution (DMEM) and rat serum as matrixes to prepare a series of Cd with different concentrations²⁺And TeO₃ ^2-After the standard series mixed solution is analyzed by CE-ICP-MS by adopting the detection methods described in the embodiment 1, the embodiment 2 and the embodiment 3, respectively, a standard curve is drawn by taking the concentration as a horizontal coordinate and the chromatographic peak area as a vertical coordinate, and an equation relation between the chromatographic peak area and the concentration is obtained, and the result is shown in the following table 1;

the detection limit is calculated according to the concentration of the component to be detected corresponding to 3 times of the baseline noise, namely D is 3N/S (wherein D is the detection limit, N is the noise, and S is the sensitivity), and the result is shown in Table 1;

in addition, a certain amount of L-GSH/L-cysteine-capped CdTe QDs which are independently synthesized in the specific embodiment are respectively added into a buffer salt solution, a cell culture solution (DMEM) and a rat serum matrix, 2 parts (total 6 parts) are respectively prepared, one part of Cd is added into each system, and Cd is added into one part of each system²⁺And TeO₃ ^2-The standard solution was diluted with a sample buffer (pH9.0) as appropriate to prepare 6 sample solutions for different systems. The injection measurement was repeated 3 times, and the recovery rate and the Relative Standard Deviation (RSD) were calculated, and the results are shown in Table 1.

TABLE 1 Cd in different systems²⁺And TeO₃ ^2-Standard curve equation, detection limit, spiked recovery and method relative standard deviation of

Example 5 method feasibility verification

Studies have shown that the quantification of different components in large particles generally does not correspond to their content from a free ion standard curve, because particles have a lower atomization efficiency than free ions in solution. As the particle size of QDs is small (generally about 5 nm), the CE analysis sample introduction amount is small (nano-upgrading), samples are fully dispersed in electrophoresis buffer solution and sheath solution, and the atomization efficiency is basically consistent with that of free ions, the quantitative analysis can be directly carried out on the samples. In order to verify the feasibility of the basis and the quantitative method, the independently synthesized L-GSH/L-cysteine-blocked CdTe QDs are taken as experimental objects, two parts of the independently synthesized L-GSH/L-cysteine-blocked CdTe QDs with the same amount of purified L-GSH/L-cysteine-blocked CdTe QDs are taken, and one part of the L-GSH/L-cysteine-blocked CdTe QDs is converted into Cd by nitric acid hydrolysis and oxidation²⁺And TeO₃ ^2-And the other part is not subjected to any pretreatment, then CE-ICP-MS analysis is carried out under the optimal analysis condition, sample injection is repeated for 3 times, and the measurement result is subjected to statistical analysis by using SPSS17.0, and the result is shown in Table 2.

As can be seen from Table 2, the measurement results of the two methods are basically consistent, and the difference is not statistically different (P > 0.05), further proving that the method of the invention is completely suitable for accurate quantitative analysis of CdTe QDs and degradation or metabolic products thereof.

TABLE 2 CE-ICP-MS quantitative analysis results of CdTe QDs by different sample pretreatment methods (n is 3)

Note: CdTe QDs-Cd represents the content of Cd in CdTe QDs; CdTe QDs-Te represents Te content in CdTe QDs

Example 6 evaluation of quality of commercially available CdTe QDs

Two commercially available CdTe QDs surface modified with carboxyl or amino groups were analyzed as described in example 1, FIG. 10And FIG. 11, the results of the quantitative analysis are shown in Table 3. From Table 3, CdTe-NH₂The stock solution of-560 contained 64.1% free Cd²⁺And 47.4% TeO₃ ^2-The stock solution of CdTe-COOH-560 contains free Cd 39.3%²⁺And 3.3% of TeO₃ ^2-It shows that the CdTe QDs are degraded to a certain degree.

TABLE 3 CE-ICP-MS quantitative analysis results of commercial CdTe QDs (n ═ 3)

Note: CdTe-NH₂560 is CdTe QDs with surface modified with amino group and emission wavelength of 560 nm; CdTe-COOH-560 is CdTe QDs with carboxyl group modified on the surface and 560nm emission wavelength; CdTe QDs-Cd refers to the Cd content in QDs, and CdTe QDs-Te refers to the Te content in QDs.

Example 7 detection of animal serum samples

After the self-synthesized L-GSH/L-cysteine-capped CdTe QDs are purified in the embodiment of the invention, Wistar male rats are infected with poison through tail veins, blood is taken at different time, the detection method described in the embodiment 1 of the invention is utilized to analyze serum samples, as shown in fig. 10 and fig. 11, it can be seen that the method successfully detects the CdTe QDs and free Cd in serum²⁺But TeO was not detected₃ ^2-. On one hand, the reagent may be related to the fact that the contamination dose is too small and is lower than the detection limit of the instrument, and on the other hand, the possible reason is that CdTe QDs are not easy to degrade in serum, but free Cd detected in the experiment²⁺Is Cd in rat serum^{2 +}The background value of (1) and Cd generated after the degradation of CdTe QDs²⁺The sum of the values.

As is clear from the results of the examination in the above examples, the concentration of the surfactant is 10 to 40 mmol.L^-1The phosphate (PB, 9.0) is 10-40 mmol/L of electrophoresis buffer^-1Borate buffer (Na)₂B₄O₇+H₃BO₃pH9.0) is used as a sample buffer solution, and a CE-ICP-MS instrument combined analysis method is adopted to realize CdTe in the buffer solution, the cell culture solution and the animal serumQDs and their degradation or metabolites (Cd)²⁺And TeO₃ ^2-Etc.) and simultaneously accurate quantification. The method has the advantages of high sensitivity (the absolute quantitative detection limit reaches fg level), high accuracy, wide linear range, good correlation and good repeatability, and the used reagents are conventional and cheap. The method is used for evaluating the quality of the commercialized CdTe QDs modified with different groups on the surface, and detecting the serum of rats infected with the CdTe QDs, thereby obtaining satisfactory results. The method provides a method approach for researching the cytotoxicity and animal toxicity mechanism of the CdTe QDs, and provides related technical support for the quality evaluation of the commercialized CdTe QDs. In addition, the successful construction of the method also provides a thought for the quality evaluation and toxicity mechanism research of other types of QDs.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (3)

1. A method for simultaneously and quantitatively detecting CdTe QDs and degradation or metabolic products thereof, which is characterized in that,

(1) dissolving the purified L-GSH/L-cysteine-capped CdTe QDs in a sample buffer solution, and adding Human Serum Albumin (HSA) to obtain a sample to be detected; in an amount of 10 to 40 mmol/L^-1The phosphate buffer solution with pH =9.0 is an electrophoresis buffer solution, and 10-40 mmol.L^-1Using a borate buffer solution with the pH =9.0 as a sample buffer solution, and separating and quantitatively detecting a target object in a sample to be detected by adopting a capillary electrophoresis and inductively coupled plasma mass spectrometry (CE-ICP-MS) combined technology; the phosphate buffer had the composition: including Na₂HPO₄And NaH₂PO₄The borate buffer had the composition: including Na₂B₄O₇And H₃BO₃(ii) a The working conditions of the capillary electrophoresis analyzer are as follows: uncoated fused silica capillary: i.d.75 μm; o.d.360 μm; length 100 cm; detection wavelength: 214 nm; sample introduction pressure: 35 mbar; sample introduction time: 5 s; separation voltage: 16 kV; separation temperature: 25 ℃; pre-running a program: 1.0N NaOH, 180s → H₂O, 180s → running buffer, 300 s; sheath fluid type: 1ppm In internal standard aqueous solution; flow rate of sheath fluid: 0.5 ml/min; the split ratio is as follows: 1: 100;

(2) drawing a standard curve for Cd with different concentrations²⁺And TeO₃ ^2-Mixing standard solutions, adding a sample buffer solution and HSA, and performing CE-ICP-MS separation analysis according to the same conditions in the step (1) to obtain a separation map; wherein Cd²⁺Splitting into Cd²⁺[1]And Cd²⁺[2]Two peaks, Cd²⁺[1]Peak is HSA-Cd²⁺Complex peak, short retention time, Cd when analyzing the sample to be tested²⁺[1]The peak overlaps with the CdTe QDs peak; cd [ Cd ]²⁺[2]Peak is free Cd²⁺Peak and retention time is long, and Cd is obtained when a sample to be detected is analyzed^{2 +}[2]Peaks with CdTe QDs peaks and TeO₃ ^2-The peak can be completely separated; changing Cd in mixed standard solution²⁺The concentration of HSA and the concentration of the HSA are kept unchanged, CE-ICP-MS separation analysis is carried out under the same condition, and Cd in separation patterns of mixed standard solutions with different concentrations²⁺[1]Peak and Cd²⁺[2]The peak area ratio of the peak is kept unchanged, and Cd can be used²⁺[2]Signal pair Cd in sample to be detected²⁺[1]Quantifying the signal;

te standard curve, separating TeO in spectrum of mixed standard solution with different concentration₃ ^2-Concentration and corresponding TeO₃ ^2-Linear fitting is carried out on peak area to draw TeO₃ ^2-Obtaining a standard curve between the peak area and the Te concentration to obtain TeO₃ ^2-Relation 1 between peak area and Te concentration;

② standard curve of Cd, separating Cd in mixed standard solution with different concentrations²⁺[1]And Cd²⁺[2]Total peak area of (2) and corresponding Cd²⁺Linear fitting is carried out on the concentration to draw Cd²⁺[1]And Cd²⁺[2]The standard curve between the total peak area and the concentration of Cd is obtained to obtain Cd²⁺[1]And Cd²⁺[2]The relation 2 between the total peak area and the Cd concentration; wherein Cd with short migration time²⁺Peak is known as Cd²⁺[1]Cd with longer migration time²⁺Peak is known as Cd²⁺[2]；

Thirdly, separating Cd in the mixed standard solution by using a separation spectrum²⁺[1]The peak area of the Cd is linearly fitted with the corresponding Cd concentration to draw the Cd²⁺[1]The standard curve between the peak area and the corresponding Cd concentration is obtained to obtain Cd²⁺[1]The relationship between the peak area of (a) and the concentration of Cd is 3;

fourthly, mixing Cd²⁺[2]The peak area of the Cd is linearly fitted with the corresponding Cd concentration to draw the Cd²⁺[2]The standard curve between the peak area and the concentration of Cd is obtained to obtain Cd²⁺[2]The peak area of (a) and the corresponding concentration of Cd is in a relational expression 4;

(3) quantitative calculation

Firstly, separating TeO in a sample to be detected₃ ^2-Substituting the peak area into the relation 1 to directly calculate the degradation of CdTe QDs in the sample to be measured and TeO in the metabolite₃ ^2-The concentration of (c); substituting the peak area of Te in CdTe QDs in the separation map of the sample to be detected into the relation formula 1 to obtain the concentration of Te in CdTe QDs;

② separating Cd in the atlas of the sample to be measured²⁺[2]Substituting the peak area into the above relation 4 to obtain total Cd generated by CdTe QDs degradation or metabolism²⁺Concentration; separating Cd from the chromatogram of the sample to be tested²⁺[2]Calculating HSA-Cd by peak area²⁺Cd in composites²⁺Peak area, i.e. Cd²⁺[1]Substituting the peak area into the above relational expression 3 to obtain HSA-Cd²⁺Cd in the Complex²⁺The concentration of (c); cd was subtracted from the Cd peak area at the CdTe QDs peak²⁺[1]The peak area is the peak area of Cd in the CdTe QDs, and then the peak area of CdTeQDs-Cd is substituted into the relational expression 2 to obtain the concentration of Cd in the CdTeQDs.

2. The method for simultaneously and quantitatively detecting CdTe QDs and their degradation or metabolic products according to claim 1, wherein the ICP-MS working conditions are as follows: incident power: 1550W; sampling depth: 8.0 mm; flow rate of carrier gas: 1.20-1.40L/min; flow rate of auxiliary gas: 0.8-1.0L/min; plasma gas flow rate: 13-16L/min; compensating the airflow speed: 1.0-1.3L/min; rotation speed of peristaltic pump: 0.5 to 2.0 rps; temperature of the atomization chamber: 2-5 ℃; and (3) detecting mass number:¹¹¹Cd，¹²⁵te; detecting an internal standard:¹¹⁵in; sampling mode: TRA; integration time:¹¹¹cd and¹²⁵the Te is 0.1s, and the content of the lead is,¹¹⁵in was 0.05 s.

3. The application of the detection method as defined in claim 1 in quality evaluation of CdTe QDs, characterized in that the detection method as defined in claim 1 is used for detecting the components in CdTe QDs to be evaluated.

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