Preparation method and application of dual-color fluorescence co-localization system for detecting silver ions

Abstract

The present invention discloses a preparation method and application of a dual-color fluorescence co-localization system for silver ion detection. This system uses quantum dots with an emission wavelength of 525 nm and Cy5 fluorescent molecules with an emission wavelength of 660 nm, which are respectively conjugated with silver ion-specific DNA strands rich in cytosine bases. When there are silver ions, free nucleic acid aptamers perform base complementary pairing through the C-Ag+-C complex structure, and dual-color super-resolution imaging is carried out under a fluorescence microscope to perform dual-color fluorescence co-localization of quantum dots and Cy5 molecules. At the same time, the positions where the quantum dot and Cy5 fluorescence signals can be detected are the binding sites that effectively capture silver ions. By using the method of dual-color fluorescence super-resolution co-localization, non-specific binding sites are excluded, greatly improving the specificity of silver ion detection, being easy to distinguish, having high sensitivity and good reliability. By calculating the co-localization coefficient of the quantum dot and Cy5 fluorescence signals, quantitative detection of silver ion concentration is achieved.

Description

Technical Field

[0001] The invention relates to the field of silver ion detection and two-color fluorescence co-localization, and in particular to a preparation method and application of a two-color fluorescence co-localization system for silver ion detection. Background Art

[0002] Silver has the advantages of thermal conductivity, electrical conductivity, soft texture and high reflectivity, so it is widely used in production and life. However, with the development of human society, a large amount of silver ions enter the environment, and silver ions can be enriched through the food chain, only transferred but not degraded. After entering the human body, silver ions react with proteins and enzymes, making them inactive and ultimately endangering human health. WHO stipulates that silver is safe for the human body only when it is below 0.05ppm, and the limit of silver ions in drinking water is 0.05mg / L. Therefore, detecting the concentration of silver ions in the water environment is crucial to protecting human life and health.

[0003] At present, the methods for detecting silver ions mainly include atomic absorption spectroscopy, atomic emission spectroscopy, ultraviolet visible spectrophotometry, inductively coupled plasma mass spectrometry and electrochemistry. These methods have good stability and reliability for silver ion detection within a certain concentration range. However, for silver ion detection with extremely low concentration, its sensitivity and specificity are difficult to meet the requirements. In addition, these methods have cumbersome operating steps, and require complex processing processes such as concentration enrichment, extraction or interference suppression during measurement, which makes the real-time, efficient and highly sensitive analysis of silver ions subject to certain restrictions.

[0004] Co-localization analysis based on optical microscopic images shows great application potential in biological detection. Super-resolution microscopy technology breaks through the diffraction limit of visible light and can perform fluorescence imaging of single molecules. Quantum dots have excellent fluorescence properties such as wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, adjustable color, high photochemical stability, and long fluorescence lifetime. They are widely used as fluorescent probes. Cy5 fluorescent molecules are widely used in the labeling and detection of proteins, antibodies, nucleic acids and other biological molecules. Selecting suitable quantum dots and Cy5 fluorescent dyes, their excitation spectra and emission spectra can be distinguished from each other, and multi-channel imaging can be achieved under super-resolution microscopy. Cytosine can be used to form C-Ag with silver ions. + Based on the characteristics of the -C composite structure, a silver ion-specific DNA chain rich in cytosine bases was designed to couple to quantum dots and Cy5 fluorescent molecules to construct a two-color fluorescence co-localization system for specific identification of silver ions.

[0005] The present application uses quantum dots with an emission wavelength of 525nm and Cy5 fluorescent molecules with an emission wavelength of 660nm, on which silver ion-specific DNA chains rich in cytosine bases are coupled respectively, to obtain a two-color fluorescence co-localization system that can specifically identify silver ions, and performs two-color super-resolution imaging under a fluorescence microscope to perform two-color fluorescence co-localization of quantum dots and Cy5 molecules to specifically detect silver ions. This method utilizes two-color fluorescence super-resolution co-localization to exclude nonspecific binding sites, greatly improves the specificity of silver ion detection, is easy to distinguish, has high sensitivity, and has good reliability. By calculating the co-localization coefficient of quantum dots and Cy5 fluorescence signals, quantitative detection of silver ion concentration is achieved. Summary of the invention

[0006] Purpose of the invention: In order to overcome the problems of complex detection steps and low sensitivity encountered in silver ion detection, the present invention provides a two-color fluorescence co-localization system to achieve quantitative detection of silver ion concentration. The present invention also provides a preparation method and application of the two-color fluorescence co-localization system.

[0007] Technical solution: The dual-color fluorescence co-localization system described in the present invention is characterized by using quantum dots with an emission wavelength of 525nm and Cy5 fluorescent molecules with an emission wavelength of 660nm, to which silver ion-specific DNA chains rich in cytosine bases are coupled respectively. When silver ions are introduced into the system, the free nucleic acid aptamers specifically recognize the silver ions, and the silver ions are detected by C-Ag. + -C complex structure for base complementary pairing, dual-color super-resolution imaging under a fluorescence microscope, and dual-color fluorescence co-localization of quantum dots and Cy5 molecules. Only the sites where quantum dots and Cy5 fluorescence signals are detected simultaneously are silver ion-specific reaction sites, thereby excluding non-specific fluorescence signals and performing quantitative detection of silver ions through the co-localization coefficient value. The specific process is shown in Figure 1 .

[0008] The preparation method of the above-mentioned two-color fluorescence co-localization system comprises the following steps:

[0009] (1) NHS and EDC were added to the carboxyl water-soluble ZnCdSe / ZnS quantum dot solution, mixed evenly, and then the amino-modified nucleic acid aptamer (NH2-DNA) was added, and the reaction was shaken at room temperature. The reactant was ultrafiltered and purified by an ultrafiltration tube, and dispersed in PBS buffer to obtain a QD@NH2-DNA solution;

[0010] NHS is N-hydroxysuccinimide and EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, which are used to activate the carboxyl group.

[0011] (2) Add the nucleic acid aptamer (Cy5-DNA) labeled with the Cy5 fluorescent group to the QD@NH2-DNA solution obtained in step (1), and mix them evenly to obtain QD@NH2-DNA@Cy5-DNA, which is a dual-color fluorescence co-localization system that can specifically recognize silver ions.

[0012] In step (1), the molar ratio of the carboxyl water-soluble ZnCdSe / ZnS quantum dots to EDC is 1:2000-5000.

[0013] In step (1), the molar ratio of NHS to EDC is 1:4-10.

[0014] In step (1), the molar ratio of the carboxyl water-soluble ZnCdSe / ZnS quantum dots to NH2-DNA is 1:100.

[0015] In step (1), the NH2-DNA sequence is 5'-NH2 C6-GTCCACCCAAGG-3'.

[0016] In step (2), the molar ratio of the Cy5-DNA to the carboxyl water-soluble ZnCdSe / ZnS quantum dots is 1:2.

[0017] In step (2), the Cy5-DNA sequence is 5'-CCTTGCCTGGAC-Cy5-3'.

[0018] The above two-color fluorescence co-localization system is applied to the detection of silver ions. The method is as follows: (1) adding silver nitrate solutions of different concentrations to the QD@NH2-DNA@Cy5-DNA solution, mixing evenly, shaking the solution at room temperature, and ultrafiltration purifying the reactant with an ultrafiltration tube to obtain QD@NH2-DNA@Ag + @Cy5-DNA, disperse it in PBS buffer; (2) fix the sample treated in step (1) on an eight-well plate, perform single-molecule localization super-resolution imaging under a fluorescence microscope, the excitation light wavelengths are 405nm and 642nm, the corresponding filters are BP 495-575 and LP 655, perform dual-color fluorescence co-localization on the generated quantum dot channel fluorescence signal and Cy5 channel fluorescence signal, and detect the positions of quantum dots and Cy5 fluorescence signals at the same time, effectively capturing silver ions. Calculate the co-localization coefficient of quantum dots and Cy5 fluorescence signals, and use the co-localization coefficient value to quantitatively detect the silver ion concentration.

[0019] The above two-color fluorescence co-localization system is used to detect silver ions based on the TIRF imaging method of co-localization of quantum dots and Cy5 fluorescent groups. The specific method is as follows:

[0020] Take 100 μL QD@NH2-DNA@Cy5-DNA solution, add AgNO3 solution and phosphate buffer (PBS, pH 7.4, 10 mM) to keep the total volume of the solution at 500 μL, the silver ion concentrations are 0, 0.002 nM, 0.02 nM, 0.2 nM, 2 nM, 20 nM, 200 nM, mix well, shake at room temperature for 12 h, and then use a 100 Kda ultrafiltration tube to centrifuge at 6300 round / min for 10 min for ultrafiltration purification to obtain QD@NH2-DNA@Ag + @Cy5-DNA was dispersed in 400 μL PBS buffer; 300 μL 0.5% PEI was added to the eight-well plate, left to stand for 15 min, taken out, and washed twice with PBS solution; 400 μL QD@NH2-DNA@Ag + @Cy5-DNA solution was allowed to stand for 15 minutes, taken out, and washed twice with PBS solution; finally, PBS solution was added, and the sample was placed on a fluorescence microscope. Super-resolution imaging was performed using TIRF illumination mode. The excitation light wavelengths were 405nm and 642nm, respectively, and the corresponding filters were BP 495-575 and LP 655, respectively. The generated quantum dot channel fluorescence signals and Cy5 channel fluorescence signals were co-localized with dual-color fluorescence. At the same time, the positions of the quantum dots and Cy5 fluorescence signals were detected as the binding sites that effectively captured the silver ions. The co-localization coefficient of the quantum dots and Cy5 fluorescence signals was calculated to achieve quantitative detection of the silver ion concentration.

[0021] The dual-color fluorescence co-localization system of the present invention utilizes functional nucleic acids to specifically identify silver ions, and quantum dots and Cy5 fluorescent groups are co-localized under a TIRF microscope to achieve the detection of silver ions.

[0022] Beneficial effects: The present invention constructs a dual-color fluorescence co-localization system for detecting silver ions in water, using quantum dots with an emission wavelength of 525nm and Cy5 fluorescent molecules with an emission wavelength of 660nm, on which silver ion-specific DNA chains rich in cytosine bases are coupled respectively. When no silver ions are added, the hydrogen bond force between the two non-complementary base pairs is offset by the strong electrostatic repulsion, and the two DNA chains are in a free state; when silver ions are added, the two DNA chains are separated by C-Ag + -C is specifically bound, two-color super-resolution imaging is performed under a fluorescence microscope, and two-color fluorescence co-localization of quantum dots and Cy5 molecules is performed, and the positions of quantum dots and Cy5 fluorescence signals can be detected at the same time as binding sites that effectively capture silver ions. The present invention utilizes the method of two-color fluorescence super-resolution co-localization to exclude non-specific binding sites, greatly improve the specificity of silver ion detection, and is easy to distinguish, with high sensitivity and good reliability. By calculating the co-localization coefficient of quantum dots and Cy5 fluorescence signals, quantitative detection of silver ion concentration is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the dual-color fluorescence co-localization system for detecting silver ions;

[0024] Figure 2 TIRF images of quantum dots and Cy5 in the dual-color fluorescence co-localization system of the present invention; (a) TIRF image of quantum dots when the silver ion concentration is 0; (b) TIRF image of Cy5 when the silver ion concentration is 0; (c) TIRF image of quantum dots when the silver ion concentration is 0.2 nM; (d) TIRF image of Cy5 when the silver ion concentration is 0.2 nM; Scale bar: 5 μm;

[0025] Figure 3 A graph showing the relationship between the colocalization coefficient of quantum dots and Cy5 molecules and the concentration of silver ions in the dual-color fluorescence colocalization system of the present invention. DETAILED DESCRIPTION

[0026] 1. Source of raw materials

[0027] 1. PBS buffer is pH=7.4, concentration is 10mM PBS buffer;

[0028] 2. Carboxyl water-soluble ZnCdSe / ZnS quantum dots were provided by Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.;

[0029] 3. DNA chain was provided by Sangon Biotech (Shanghai) Co., Ltd.;

[0030] 4. The remaining materials were commercially available.

[0031] 2. Preparation of dual-color fluorescence co-localization system

[0032] Example: Take 100 μL of 0.1 μM carboxyl water-soluble ZnCdSe / ZnS quantum dots, add 4 μL of 10 mM EDC and 1 μL of 10 mM NHS, mix well, and then add 10 μL of 100 μM NH2-DNA solution. After shaking for 12 hours at room temperature, the reactant is ultrafiltered and purified at 6300 round / min for 10 minutes using a 100 KDa ultrafiltration tube to remove excess NH2-DNA, and it is dispersed in 400 μL PBS buffer to obtain QD@NH2-DNA solution; 5 μL of 1 μM Cy5-DNA is added to the QD@NH2-DNA solution to obtain QD@NH2-DNA@Cy5-DNA solution.

[0033] 3. TIRF imaging dual-color fluorescence co-localization detection of silver ions

[0034] Example: AgNO3 solution and PBS were added to the prepared QD@NH2-DNA@Cy5-DNA sample, and the sample volume was maintained at 500 μL so that the silver ion concentrations were 0, 0.002 nM, 0.02 nM, 0.2 nM, 2 nM, 20 nM, and 200 nM, respectively. After shaking for 12 hours at room temperature, the reactants were purified by ultrafiltration using a 100KDa ultrafiltration tube at 6300 round / min for 10 minutes, and then dispersed in 400μL PBS buffer; 300μL of 0.5% PEI was added to the eight-well plate, allowed to stand for 15 minutes, taken out, and washed twice with PBS solution; 400μL of the reactant solution was added to each well, allowed to stand for 15 minutes, taken out, and washed twice with PBS solution; finally, PBS solution was added, placed on a fluorescence microscope, and super-resolution imaging was performed using TIRF illumination mode. The excitation wavelengths were 405nm and 642nm, respectively, and the corresponding filters were BP 495-575 and LP 655, respectively. The generated quantum dot channel fluorescence signal and Cy5 channel fluorescence signal were subjected to dual-color fluorescence co-localization imaging, and the co-localization coefficient of the quantum dot and Cy5 fluorescence signals was calculated. Results are shown in Figure 2 and Figure 3 .

[0035] Figure 2 TIRF images of quantum dots and Cy5. Figure 2 (a) and Figure 2 (b) TIRF images of quantum dots and Cy5 when the silver ion concentration is 0, respectively; Figure 2 (c) and Figure 2 (d) TIRF images of quantum dots and Cy5 at a silver ion concentration of 0.2 nM. Figure 2 It can be seen that when the silver ion concentration is 0, the Cy5 molecules are freely distributed, and the quantum dots and the Cy5 fluorescent groups are not co-localized; when the silver ion concentration is 0.2nM, the fluorescence signals of the quantum dots and Cy5 channels are co-localized, indicating that the dual-color fluorescence co-localization system specifically captures silver ions.

[0036] Figure 3 The relationship curve between the colocalization coefficient of the dual-color fluorescence colocalization system and the silver ion concentration is shown in Figure 1. As can be seen, with the increase of silver ion concentration, the colocalization coefficient of quantum dots and Cy5 molecules gradually increases in an "S"-shaped curve.

Claims

1. A two-color fluorescence co-localization system, characterized in that: Quantum dots with an emission wavelength of 525 nm and Cy5 fluorescent molecules with an emission wavelength of 660 nm were used to couple silver ion-specific DNA chains rich in cytosine bases. When silver ions were introduced into the system, the free nucleic acid aptamers specifically recognized the silver ions. + -C complex structure for base complementary pairing, dual-color super-resolution imaging under a fluorescence microscope, and dual-color fluorescence co-localization of quantum dots and Cy5 molecules; only the sites where quantum dots and Cy5 fluorescence signals are detected at the same time are silver ion-specific reaction sites, thereby excluding non-specific fluorescence signals, and quantitative detection of silver ions is performed through the co-localization coefficient value; The method for preparing the dual-color fluorescence co-localization system comprises the following steps: (1) NHS and EDC were added to the carboxyl water-soluble ZnCdSe / ZnS quantum dot solution, mixed evenly, and then the amino-modified nucleic acid aptamer NH2-DNA was added. The reaction was shaken at room temperature, and the reactants were ultrafiltered and purified using an ultrafiltration tube, and dispersed in PBS buffer to obtain QD@NH2-DNA solution; (2) Adding the nucleic acid aptamer Cy5-DNA labeled with a Cy5 fluorescent group to the QD@NH2-DNA solution obtained in step (1), and mixing them evenly, to obtain QD@NH2-DNA@Cy5-DNA, which is a dual-color fluorescence co-localization system that specifically recognizes silver ions; In step (1), the molar ratio of the carboxyl water-soluble ZnCdSe / ZnS quantum dots to EDC is 1:2000-5000; In step (1), the molar ratio of NHS to EDC is 1:4-10; In step (1), the molar ratio of the carboxyl water-soluble ZnCdSe / ZnS quantum dots to NH2-DNA is 1:100; In step (1), the NH2-DNA sequence is 5'-NH2 C6-GTCCACCCAAGG-3'; In step (2), the molar ratio of Cy5-DNA to carboxyl water-soluble ZnCdSe / ZnS quantum dots is 1:2; In step (1), the Cy5-DNA sequence is 5'-CCTTGCCTGGAC-Cy5-3'.

2. Application of the dual-color fluorescence co-localization system according to claim 1 in silver ion detection, characterized in that: The method of application is: (1) Add silver nitrate solution of different concentrations to the QD@NH2-DNA@Cy5-DNA solution, mix well, shake at room temperature, and purify the reactant by ultrafiltration to obtain QD@NH2-DNA@Ag + @ Cy5-DNA, disperse it in PBS buffer; (2) The sample treated in step (1) is fixed on an eight-well plate, and single-molecule localization super-resolution imaging is performed under a fluorescence microscope. The excitation light wavelengths are 405 nm and 642 nm, respectively, and the corresponding filters are BP 495-575 and LP 655, respectively. The generated quantum dot channel fluorescence signal and Cy5 channel fluorescence signal are subjected to dual-color fluorescence co-localization, and the positions of the quantum dots and Cy5 fluorescence signals can be detected at the same time, which effectively captures the silver ions; the co-localization coefficient of the quantum dots and Cy5 fluorescence signals is calculated, and the silver ion concentration is quantitatively detected by the co-localization coefficient value.

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