CN104745194B - Preparation method of quantum dot@copper nanocluster ratiometric fluorescent probe and its Cu2+ detection application - Google Patents

Abstract

The invention discloses preparation method and the Cu thereof of a kind of quantum dot@copper nano-cluster ratio fluorescent probe²⁺Detection application, belongs to fluorescence sense technical field.To be coupled to, for CuNCs prepared by template, the CdSe QDs@CuNCs ratio fluorescent probe that the CdSe QDs surface of Silica-coated is prepared for having the double fluorescent emission signals of CdSe QDs and CuNCs with polymine.When solution exists Cu²⁺Time, the amino of polymine can be with Cu²⁺Complexation and form copper-amine complex, prevent electronics from amino to the transmission of CuNCs, cause CuNCs fluorescent quenching, and the fluorescence intensity of CdSe QDs is constant.Along with Cu²⁺The increase of concentration, the strength ratio of two fluorescence emission peaks of CuNCs with CdSe QDs is gradually reduced, it is achieved that to Cu²⁺Sensitive fluorescent and Visual retrieval.

Description

Preparation method of quantum dot@copper nanocluster ratiometric fluorescent probe and its Cu2+ detection application

technical field

The invention relates to a preparation method of a quantum dot@copper nano-cluster fluorescent probe and its Cu ²⁺ detection application, belonging to the technical field of fluorescent sensing.

Background technique

Copper in low concentration is an important and essential trace element in animals and plants, and it is also an important coenzyme factor of proteases in many organisms. However, when the concentration of copper ions is too high, it is highly toxic and can cause damage to the central nervous system, as well as some neurological diseases such as Wilson's disease and Alzheimer's disease. High concentrations of copper can induce gastrointestinal disturbances and damage to the liver or kidneys. So far, there are many methods for detecting Cu ²⁺ , including atomic absorption/emission spectrometry, inductively coupled plasma mass spectrometry, electrochemical method, dynamic light scattering method, Raman scattering method and fluorescence method (Fluorescent gold clusters as nanosensors for copper ions in live cells, CVDurgadas, CP Sharma, K. Sreenivasan, Analyst, 2011, 136, 933-940.), etc. Among these developed methods, the fluorescence method has the characteristics of high sensitivity, simplicity, and low instrument cost, which makes the fluorescence-based method show obvious advantages in application prospects. However, the fluorescence response to Cu ²⁺ is mostly based on a single fluorescence quenching probe, which is often limited by the drift of light source or detector or the influence of complex sample environment and other factors. Ratiometric fluorescent probes can avoid these problems and have received great attention in recent years (Ratiometric displacement approach to Cu(II) sensing by fluorescence, M. Royzen, Z. Dai, JW Canary, Journal of the American Chemical Society, 2005, 127, 1612-1613.). The fluorescence intensity ratio of the two probes with separated emission peaks can provide intrinsic correction for environmental interference and eliminate fluctuations in excitation light intensity, thus improving the accuracy of quantitative analysis. Therefore, the development of sensitive Cu ²⁺ detection methods is in great demand in environmental monitoring.

In recent years, metal nanoclusters have attracted great attention due to their unique size-dependent optical and electrical properties that are different from those of atoms and bulk materials, and have shown broad application prospects in many fields as functionalized bridges, such as optoelectronic nanoclusters. Devices, biosensing, nanoelectronics and new catalysis (Onset of catalytic activity of gold clusters on titania with the appearance of nonmetallic properties, M. Valden, X. Lai, DW Goodman, Science, 1998, 281, 1647-1650.) and other fields. So far, a lot of research work has mainly focused on the synthesis of fluorescent gold nanoclusters and silver nanoclusters (Sensitive and selective detection of copper ions with highly stable polyethyleneimine-protected silver nanoclusters, Z. Yuan, N. Cai, Y. Du, Y. He, ES Yeung, Analytical Chemistry, 2014, 86, 419-426.). Compared with Ag (E ⁰ , 0.80 V) and Au (E ⁰ , 1.50 V), the readily oxidizable nature of Cu (E ⁰ , 0.34 V) limits the development of copper nanoclusters (CuNCs) synthesis, especially in solution media Synthesis in . Therefore, due to the difficulty in preparing highly stable and extremely small particles, the synthesis and optical modulation of CuNCs are still in their infancy. However, there is no report on the rapid and environmentally friendly synthesis of CuNCs at room temperature and the construction of ratiometric fluorescent probes for the detection of Cu ²⁺ .

Contents of the invention

The purpose of the present invention is to provide a method for preparing a quantum dot@copper nanocluster fluorescent probe and its Cu ²⁺ detection application, which has the advantages of sensitive detection and good selectivity.

The present invention is achieved in this way, a method for preparing a quantum dot@copper nanocluster fluorescent probe, which is characterized in that it comprises the following steps:

(1) Preparation of copper nanoclusters: Dissolve 40 mg of polyethyleneimine in 2 mL of ultrapure water, adjust the pH of the solution to 5 with acetic acid, add 2 mL of 2 mM Cu(NO ₃ ) ₂ under stirring conditions Solution, make Cu ²⁺ complex reaction with the amino ligand in polyethyleneimine for 10 minutes, then add 7 mg of ascorbic acid to the solution and keep stirring for 8 hours, the reaction solution is filtered through an ultrafiltration tube with a molecular weight of 30 K Ultrafiltration at 16000 rpm for 10 minutes to produce copper nanoclusters;

(2) Preparation of quantum dot@copper nanocluster ratio fluorescent probe: Mix the copper nanocluster solution prepared in step (1) with 10 mM boric acid buffer solution and CdSe quantum dot solution, and add 26.4 μg of 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride, shaking reaction for 2 hours; ultrafiltration with a molecular weight of 30 K at 16000 rpm for 5 minutes, the product was dispersed in boric acid buffer solution, prepared Form CdSe quantum dots@copper nanocluster ratio fluorescent probe solution;

Preferably, in step (2), the concentration of the boric acid buffer solution is 10 mM, and the pH is 8.0.

The present invention also relates to the Cu ²⁺ detection application of the quantum dot@copper nanocluster fluorescent probe ^: add 50 μL of Cu ²⁺ with different concentrations to 80 μL of the CdSe quantum dot@copper nanocluster ratio fluorescent probe solution, and use acetic acid -Dilute the total volume of the solution to 230 μL with sodium acetate buffer solution, and incubate at 37 ^o C for 10 minutes, and measure the fluorescence of the solution at an excitation wavelength of 380 nm. With the increase of Cu ²⁺ concentration, the fluorescence intensity ratio F ₄₉₅ /F ₆₂₅ of Cu nanoclusters and CdSe quantum dots gradually decreases, and the Cu ²⁺ concentration and F ₄₉₅ /F ₆₂₅ have a good relationship in the range of 22.22 nM-8.89 μM Linearity with a detection limit of 8.87 nM, which can be used for ultrasensitive detection of Cu ²⁺ .

The technical effect of the present invention is: the present invention uses polyethyleneimine as a template and ascorbic acid as a reducing agent to synthesize CuNCs in one step, and then couples CuNCs to the surface of CdSe QDs to prepare a CdSe QDs@CuNCs ratiometric fluorescent probe. Aiming at the dual fluorescent signals of CdSe QDs and CuNCs, the interference of the environment can be corrected and the fluctuation of excitation light intensity can be eliminated, which improves the accuracy of Cu ²⁺ quantitative analysis.

Description of drawings

Figure 1 is the UV-Vis absorption spectrum and fluorescence spectrum of CuNCs.

Figure 2 is the X-ray photoelectron spectrum of Cu 2p of hPEI-templated CuNCs.

Figure 3 is the fluorescence spectra of (a) CdSe QDs, (b) CuNCs and (c) CdSe QDs@CuNCs.

Figure 4 is the Fourier transform infrared spectra of (a) CdSe QDs, (b) CuNCs and (c) CdSe QDs@CuNCs.

Figure 5 is the TEM images of CdSe QDs (A) and CdSe QDs@CuNCs (B).

Fig. 6 is (A) the fluorescence spectra of CdSe QDs@CuNCs in the presence of different concentrations of Cu ²⁺ at the excitation wavelength of 380 nm. (B) Calibration curve of F ₄₉₅ /F ₆₂₅ versus Cu ²⁺ .

detailed description

The present invention will be further elaborated below in conjunction with accompanying drawing and specific embodiment, and the present invention is not limited thereto;

Example 1

(1) Preparation of copper nanoclusters: Dissolve 40 mg of polyethyleneimine in 2 mL of ultrapure water, adjust the pH of the solution to 5 with acetic acid, add 2 mL of 2 mM Cu(NO ₃ ) ₂ under stirring conditions Solution, make Cu ²⁺ complex reaction with the amino ligand in polyethyleneimine for 10 minutes, then add 7 mg of ascorbic acid to the solution and keep stirring for 8 hours, the reaction solution is filtered through an ultrafiltration tube with a molecular weight of 30 K Ultrafiltration at 16000 rpm for 10 minutes to produce copper nanoclusters;

(2) Preparation of quantum dots@copper nanocluster ratio fluorescent probe: Mix copper nanocluster solution with 10 mM boric acid buffer solution and CdSe quantum dot solution, add 26.4 μg of 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride (EDC), shaking reaction for 2 hours; use ultrafiltration tube with a molecular weight of 30 K at 16000 rpm for 5 minutes, and the product is dispersed in boric acid buffer solution to make CdSe Quantum dot@copper nanocluster ratiometric fluorescent probe solution.

Figure 1 shows the UV-Vis absorption spectrum and fluorescence emission spectrum characterization of CuNCs. The obvious absorption peak at 310 nm indicates the formation of copper nanoclusters. It can be seen from the dependence of the fluorescence spectrum of CuNCs on the wavelength of excitation light. As the wavelength of excitation light changes from When 370 nm increased to 470 nm, the fluorescence emission peak of CuNCs gradually blue-shifted from 508 nm and the fluorescence intensity decreased significantly. Due to the need to maintain a high fluorescence intensity of quantum dots, 380 nm was selected as the excitation wavelength. The characterization results of CuNCs by transmission electron microscopy and atomic force microscopy showed that the average diameter of the synthesized CuNCs was about 1.7 nm and the height was about 2.5 nm. Figure 2 is the X-ray photoelectron spectroscopy (XPS) diagram of the oxidation state of hPEI-templated CuNCs. Two strong peaks are located at 932.2 eV and 952.0 eV, corresponding to the 2p _3/2 and 2p _1/2 electron binding of Cu(0), respectively. can. At the same time, there is a shoulder peak at 942.0 eV, indicating that there is a small amount of unreacted Cu(II) derived from Cu(NO ₃ ) ₂ , due to the electron binding energy of Cu(I) and the 2p _3/2 of Cu(0) The difference in binding energy is only 0.1eV, so it cannot be distinguished in the figure. Cu(I) is mainly caused by the incomplete reduction of Cu(II) by the weak reducing agent. Therefore, the valence states of CuNCs synthesized in the present invention are mainly 0 valence and +1 valence.

Figure 3 shows the fluorescence spectra of CdSe QDs, CuNCs and CdSe QDs@CuNCs. At the excitation wavelength of 380 nm, the maximum fluorescence emission peaks of CdSe QDs and Cu NCs are located at 625 nm and 501 nm, respectively, and the CdSe QDs@CuNCs ratiometric fluorescent probe has dual emission peaks at 625 nm and 495 nm, indicating that CuNCs have been successfully coupled to the surface of CdSe QDs. Figure 4 is the Fourier transform infrared spectrum (FT-IR) of CdSe QDs, CuNCs and CdSe QDs@CuNCs. The FT-IR of CdSe QDs (curve a) shows the characteristics of COO- at 1634 and 1386 cm ^{- 1} The absorption peak, the absorption peak at ³⁴⁴⁹ cm corresponds to the stretching vibration of OH. The absorption peaks at 1563, 1638, and 3452 cm of the FT ^- IR spectrum (curve b) of hPEI-CuNCs correspond to the bending vibration and stretching vibration of NH, respectively, which are caused by hPEI wrapped on the surface of CuNCs. The characteristic absorption peaks at 1642 cm ^-1 and 1558 cm ^-1 of FT-IR of CdSe QDs@CuNCs (curve c) correspond to the absorption of amino I band (C=O) and amino II band (CN and NH), respectively, It shows that hPEI-CuNCs have been coupled to the surface of CdSe QDs through amide bond interaction. TEM results showed that the particle size of carboxyl-functionalized CdSe QDs was about 35 nm (Fig. 5A), and when hPEI-CuNCs were coupled to the surface of CdSe QDs, the edge of the ratiometric fluorescent probe surrounded some CuNCs (Fig. 5B), indicating that through the present invention Methods CdSe QDs@CuNCs were successfully synthesized.

Example 2

Ratiometric fluorescent probes for the detection of Cu ²⁺

(1) Optimization of pH, reaction temperature and reaction time

The pH, reaction temperature and reaction time were optimized. The results show that except for the strong acid environment (pH<4), CdSeQDs@CuNCs have a good response to Cu ²⁺ in the pH range of 4-11, which may be due to the occurrence of amino groups on the surface of CdSe QDs@CuNCs under strong acidic conditions. Deprotonated and unable to complex with Cu ²⁺ , and thus unable to form fluorescence-quenched copper amine complexes. However, the relative fluorescence intensity (F ₄₉₅ /F ₆₂₅ ) of CdSe QDs@CuNCs decreased slightly under alkaline conditions, which may be due to the partial hydrolysis of Cu ²⁺ under alkaline conditions, which inhibited the interaction of Cu ²⁺ with hPEI-CuNCs. Complexation reaction between. Temperature will affect the complexation reaction between Cu ²⁺ and CdSe QDs@CuNCs, and the results of temperature optimization show that 37 ^o C is the optimal reaction temperature. The reaction between Cu ²⁺ and CdSe QDs@CuNCs is very fast, and the reaction equilibrium can be reached within 10 minutes. Therefore, 10 minutes is selected as the optimal reaction time.

(2) Under optimized experimental conditions, CdSe QDs@CuNCs ratiometric fluorescent probes were used for quantitative detection of Cu ²⁺ . Add 50 μL of different concentrations of Cu ²⁺ to 80 μL of CdSe QDs@CuNCs solution, dilute the total volume of the solution to 230 μL with acetic acid-sodium acetate buffer solution, incubate at 37 ^o C for 10 min, and measure the fluorescence of the solution. It can be seen from Fig. 6 that under the excitation of 380 nm, the emission peaks of CuNCs and CdSe QDs appeared at 495 nm and 625 nm, respectively. With the increase of Cu ²⁺ concentration, the fluorescence of CuNCs gradually weakened, while the fluorescence of CdSe QDs remained unchanged, so that the ratio of fluorescence intensity F ₄₉₅ /F ₆₂₅ of CuNCs to CdSe QDs gradually decreased, and the ratio of Cu ²⁺ concentration to F ₄₉₅ / F ₆₂₅ showed good linearity in the range of 22.22 nM-8.89 μM, and the detection limit was 8.87 nM.

(3 ⁾ In terms of the selectivity of CdSe QDs@CuNCs to Cu ²⁺ detection, other metal ions include Cr ³⁺ , Fe ²⁺ , Ni ²⁺ , Co ²⁺ , K ⁺ , Ti ²⁺ , Mn ²⁺ , Mg ^{2 +} , Ca ²⁺ , Sn ²⁺ , Al ³⁺ , Cd ²⁺ , Pb ²⁺ , Hg ²⁺ , Fe ³⁺ and Ag ⁺ did not interfere with the detection of Cu ²⁺ , indicating that CdSe QDs@ ^CuNCs have a strong The detection has good selectivity.

Claims (3)

1. the preparation method of quantum dot@copper nano-cluster ratio fluorescent probe, it is characterised in that described preparation method includes following step Rapid:

(1) preparation of copper nano-cluster: 40 mg polymine are dissolved in 2 mL ultra-pure waters, regulate solution ph with acetic acid It is 5, adds the Cu (NO of 2 mL 2 mM under agitation₃)₂Solution, makes Cu²⁺Occur with the amino ligands in polymine Complex reaction 10 minutes, with adding 7 mg ascorbic acid continuously stirred 8 hours, by reaction solution molecular weight in backward solution It is super filter tube ultrafiltration 10 minutes under 16000 rpm rotating speeds of 30 K, i.e. makes copper nano-cluster；

(2) preparation of quantum dot@copper nano-cluster ratio fluorescent probe: copper nano-cluster solution prepared by step (1) and 10 mM boron Acid buffering solution and the mixing of CdSe quantum dot solution, add 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 26.4 μ g Hydrochlorate, concussion reaction 2 hours；With the super filter tube that molecular weight is 30 K ultrafiltration 5 minutes under 16000 rpm rotating speeds, product divides Dissipate in borate buffer solution, make CdSe quantum dot@copper nano-cluster ratio fluorescent probe solution.

The preparation method of quantum dot@copper nano-cluster ratio fluorescent probe the most according to claim 1, it is characterised in that step (2) in, the concentration of described borate buffer solution is 10 mM, and pH is 8.0.

3. the Cu of the quantum dot@copper nano-cluster ratio fluorescent probe as prepared by claim 1²⁺Detection application, it is characterised in that Cu by 50 μ L variable concentrations²⁺Join in 80 μ L CdSe quantum dot@copper nano-cluster ratio fluorescent probe solutions, with acetic acid- The cumulative volume of sodium acetate buffer dilute solution is to 230 μ L, and 37^oC is hatched 10 minutes, and measuring excitation wavelength is 380 The fluorescence of solution during nm；Along with Cu²⁺The increase of concentration, the ratio F of copper nano-cluster and the fluorescence intensity of CdSe quantum dot₄₉₅/F₆₂₅By The least, Cu²⁺Concentration and F₄₉₅/F₆₂₅22.22 in the range of nM-8.89 μM in good linear, detection is limited to 8.87 nM, Can be used for Cu²⁺Super sensitivity detection.