A method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles

Abstract

The present invention provides a method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal skeleton and gold nanoparticles, which mainly includes the following steps: S1, amination of a glass slide; S2, thiolation of a glass slide; S3, preparation of a PCN&AuNPs biochip; S4, preparation of an MCH monolayer structure; S5, chip fixing of nucleic acid aptamers; S6, detection of mercury ions. The biochip prepared by the method is simple, fast, low-cost, stable in nature, and can achieve high-throughput detection. At the same time, the present invention uses DSAI fluorescent molecules with AIE characteristics, and the active biological probe interacts with the nucleic acid aptamer to form a fluorescent DNA sequence, thereby improving the sensitivity and accuracy of the sensing platform.

Description

Technical Field

[0001] The invention relates to a method for detecting mercury ions, and more particularly to a method for detecting mercury ions by fixing a DNA sequence on a biochip based on an organic metal skeleton and gold nanoparticles. Background Art

[0002] With the rapid development of economy and industry, heavy metal pollution has become increasingly serious, posing a threat to the ecological environment, sustainable agricultural development, and human health. Heavy metals are widely present in human life through various channels. Common heavy metals with significant biological toxicity include lead, mercury, cadmium, arsenic, etc. Long-term intake of such heavy metals by the human body will pose a serious threat to health. Among the aforementioned heavy metals, mercury and its compounds pose a serious threat to human health and the ecological environment due to their persistence, easy migration, high bioaccumulation and high biological toxicity. Mercury is a liquid metal at room temperature and is easy to evaporate. It mainly causes poisoning in the form of mercury vapor. After mercury enters the human body through the respiratory tract and accumulates to a certain concentration, it damages brain tissue and invades the nervous system. Therefore, the development of highly sensitive and specific mercury ion detection methods has important theoretical significance and practical value for human health and life safety.

[0003] Traditional detection methods for mercury ions mainly include atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry, colorimetry, etc. These traditional detection methods are relatively mature and highly sensitive, but they still have many limitations, such as: expensive instruments, long detection time, complex sample processing, cumbersome operation, high requirements for personnel, and it is difficult to meet the needs of simple, rapid and sensitive detection of mercury ions. In addition to the above traditional detection methods, some new detection methods have been developed, including electrochemical detection, enzyme chain immunoassay, fluorescent probe method, and ion exchange resin method. Among them, nucleic acid aptamers are oligonucleotide fragments that can specifically bind to a certain target and are screened from nucleic acid libraries by exponential enrichment ligand system evolution technology. Nucleic acid aptamers have a stable secondary structure and are artificially synthesized nucleic acids that can specifically bind to the target. There is a large contact area between the aptamer and the target molecule to be detected, and it can be tightly bound to the analyte. Therefore, nucleic acid probes with aptamer structures have good recognition ability and extremely high affinity, but the specificity, sensitivity and selectivity of this detection method are still insufficient in application. Summary of the invention

[0004] In order to solve the shortcomings of the existing technology, a method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles is provided.

[0005] Metal organic frameworks (MOFs) are a class of regular porous crystalline nanomaterials composed of metal ions and organic ligands, with the characteristics of large specific surface area, controllable synthesis, good chemical stability, and rich functional groups. MOFs can convert detection signals by acting as signal probes or as carriers for loaded signal probes, and are widely used in many fields such as adsorption separation, catalysis, biomedicine, and chemical sensing. Due to the inherent advantages of MOFs, MOF-based biosensors usually show excellent detection performance and play an important role in different fields such as disease diagnosis, environmental monitoring, and food safety testing. The porous coordination network structure is a type of MOF nanomaterial, which contains a metal oxide structure of Zr and a photoactive linker, which can have a catalytically active partial oxidation effect on phosphate hydrolysis and thioether.

[0006] Gold nanoparticles (AuNPs) are small particles with diameters ranging from 1nm to 100nm, which have attracted much attention due to their easy surface functionalization and preparation, as well as their biocompatibility, tunable stability, and special optoelectronic and inherent catalytic properties. The surface of AuNPs can be easily functionalized by sulfurized ligands, which bind to the gold surface with high affinity through "Au-S" bonds. The desired ligands self-assemble onto the gold surface to generate functional monolayer protected gold clusters (Au-MPCs) with well-defined regions and significant molecular recognition properties.

[0007] Biochip, also known as DNA or protein microarray, is a miniaturized bioanalysis system that uses probe biomolecules attached to a solid matrix to identify target biomolecules, and then uses various methods (radioactivity, fluorescence or electrochemical technology) to screen biorecognition elements. It combines the advantages of biology and engineering, and has rapidly developed into a new detection method for biomolecules with its simplicity, repeatability, and cost-effectiveness.

[0008] The present invention prepares a biochip by modifying PCN&AuNPs on a mercapto glass slide, and then fixes DNA modified with DSAI fluorescent group to achieve Hg 2+ Highly sensitive detection.

[0009] The specific plan is as follows:

[0010] A method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles comprises the following steps:

[0011] S1. Aminated glass slide: Aminated glass slide is modified to obtain amino-modified glass slide;

[0012] S2, thiolation glass slide: thiolation-modify the amino-modified glass slide obtained in S1 to obtain a thiolation glass slide;

[0013] S3, preparing PCN&AuNPs biochip: adding PCN&AuNPs to the surface of the thiolated glass slide obtained in S2 for reaction to obtain a biochip, wherein the concentration of PCN&AuNPs is 20-140 μg / mL;

[0014] S4, preparing an MCH monolayer: adding MCH to the surface of the biochip obtained in S3 for reaction, the reaction time is 3-10 minutes, and a monolayer structure is prepared;

[0015] S5. Immobilizing nucleic acid aptamers on the chip: adding thiol-modified nucleic acid aptamers to react on the basis of S4, the reaction temperature is 0-55°C, the reaction time is 6-48h, and the nucleic acid aptamers are immobilized on the chip surface through covalent interaction with the chip surface;

[0016] S6. Detection of mercury ions: Add Hg to the surface of the chip obtained in S5 2+ The reaction was carried out at 30-40°C for 2 hours, and a corresponding linear relationship graph was drawn based on the obtained fluorescence value and the concentration of mercury ions.

[0017] Preferably, the specific steps of aminated glass slides in S1 are: mixing H2SO4 and H2O2 to obtain solution A, soaking the glass slides in solution A for 2-10 hours, and washing and drying after soaking; mixing ethanol and APTES to obtain solution B, soaking the dried glass slides in solution B for 10 minutes to 120 minutes, and washing and drying after soaking to obtain the aminated modified glass slides.

[0018] Preferably, the specific steps of performing thiol modification in S2 are: adding MPA to a mixed solution containing NHS and EDC, reacting at room temperature, and obtaining activated MPA after the reaction; mixing PBS with the activated MPA to obtain solution C, immersing the amino-modified glass slide obtained in S1 in solution C, and washing and drying after immersion to obtain the thiol-modified glass slide.

[0019] Preferably, the sequence of the thiol-modified nucleic acid aptamer in S4 is: 5′-SH-TTTTTTGGGTGGGTGGGTGGGTTTTTTT-3′.

[0020] Preferably, the concentration of PCN&AuNPs in S3 is 80 μg / mL, the reaction temperature is 25°C, and the reaction time is 12 h.

[0021] Preferably, the temperature for fixing DNA on the chip in S5 is 4° C., and the reaction time is 12 h.

[0022] Preferably, DSAI is used to label the nucleic acid aptamer in S5, the final concentration of DSAI is 10 μM, the concentration of the nucleic acid aptamer is 20 nM, and the ammonium cations in DSAI combine with the phosphate anions in the nucleic acid aptamer so that the biosensor carries stable fluorescence.

[0023] Preferably, the concentration of MCH in S4 is 1 mM and the reaction time is 5 min.

[0024] Preferably, Hg in S6 2+ The concentration was 0.02-2 μM and the reaction temperature was 37°C.

[0025] Beneficial effects:

[0026] Compared with traditional sensors, biochips can not only maintain characteristics such as specificity and high sensitivity, but also achieve high-throughput detection, meeting the requirements of portability and efficiency.

[0027] (1) The present invention designs a biochip based on an organic metal framework and gold nanoparticles to fix DNA sequences for detecting mercury ions. PCN & AuNPs are added to a glass slide carrying a thiol group (-SH), so that the -SH of the glass slide binds to the AuNPs to form a stable "Au-S" bond. Then, thiol-modified DNA bound to DSAI is added. The DNA is also fixed on the surface of the glass slide by the action of the "Au-S" bond. Finally, Hg is added 2+ , achieving high-sensitivity detection, and detecting Hg in the fields of environment and food safety 2+ Brought great convenience.

[0028] (2) The present invention prepares a corresponding biochip by modifying PCN&AuNPs on the surface of a glass slide, and then uses the action of the "Au-S" bond to fix DNA on the chip surface to achieve mercury ion detection, which has the advantages of high throughput, simplicity and speed.

[0029] (3) The present invention uses DSAI fluorescent molecules with AIE properties, which interact with nucleic acid aptamers in active biological probes to form fluorescent DNA sequences, generating a more sensitive and accurate "on" fluorescent signal for the sensing platform, and providing a new exploration direction for label-free fluorescent aptamer sensors.

[0030] (4) In order to achieve the best detection effect of mercury ions by the sensing platform, the concentration of PCN&AuNPs, the reaction temperature of the biochip, and the reaction time of the biochip in the technical solution were optimized. Finally, it was determined that the concentration of PCN&AuNPs was 80 μg / mL, the optimal reaction temperature of the biochip was 4°C, and the optimal reaction time of the biochip was 12 h.

[0031] (5) The method for preparing the biochip in the present invention is simple, low-cost and has stable properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a technical roadmap for constructing and applying biochips in the embodiments;

[0033] Figure 2 is the concentration optimization diagram of PCN&AuNPs a;

[0034] Figure 3 is the concentration optimization diagram of PCN&AuNPs;

[0035] Figure 4 Figure a is the effect of reaction temperature on the biochip;

[0036] Figure 5 Figure b is the effect of reaction temperature on the biochip;

[0037] Figure 6 Figure a shows the effect of different reaction times on the biochip;

[0038] Figure 7 Figure b is the effect of different reaction times on the biochip;

[0039] Figure 8 It is a comparison of the sensitivity to different concentrations of Hg2+.

[0040] Fig. 9 It is the sensitivity comparison chart b for different concentrations of Hg2+;

[0041] Fig.10 It is the sensitivity comparison chart c for different concentrations of Hg2+;

[0042] Fig.11 This is a comparison chart of the fluorescence recovery rate changes for different types of ions;

[0043] Fig.12 This is a comparison chart of the fluorescence recovery rate changes for different types of ions; DETAILED DESCRIPTION

[0044] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0045] Example:

[0046] In this scheme, the glass slide is treated with amino and thiol groups in sequence to make the glass slide carry thiol groups. On this basis, PCN&AuNPs are added to make the thiol groups on the glass slide bind to AuNPs to form a stable "Au-S" bond. Then, thiol-modified DNA bound to DSAI is added to fix the DNA on the surface of the glass slide by using the "Au-S" bond. Finally, Hg 2+ , use a scanner to detect the chip, Figure 1 This is the technical roadmap for constructing and applying the biochip of the present invention: The specific technical scheme is as follows:

[0047] (1) Amino-modified glass slides: Mix concentrated H2SO4 and H2O2 in a volume ratio of 7:3, soak the glass slides in it for at least 2 hours, wash with ultrapure water three times and dry. Then mix 95% ethanol and 2%-3% APTES, soak the dried glass slides in 100 mL of the mixture for 30 minutes, then wash with ultrapure water three times and dry to obtain amino-modified glass slides;

[0048] (2) Thiolation of glass slides: 2 mL of MPA was added to 2 mL of a mixture containing 80 mM NHS and 32 mM EDC, and the mixture was allowed to react at room temperature for 12 h to obtain activated MPA. PBS and the activated MPA were then mixed to obtain a mixed solution with a total volume of 100 mL. The amino glass slides obtained above were then washed for 12 h, washed with ultrapure water for 3 times, and dried to obtain thiolated glass slides.

[0049] (3) Preparation of PCN&AuNPs biochip: Add 100 μL of 20-140 μg / mL PCN&AuNPs to the surface of the thiolated glass slide obtained in S2, react at 25°C for 12 h, then remove the incompletely fixed PCN&AuNPs, wash with ultrapure water and dry.

[0050] (4) Preparing an MCH monolayer: adding MCH to the surface of the biochip obtained in S3 for a reaction time of 3-10 min to prepare a monolayer structure;

[0051] (5) Immobilizing nucleic acid aptamers on the chip: Add thiol-modified nucleic acid aptamers to S4 for reaction at a temperature of 0-55°C for a reaction time of 6-48 hours, and immobilize the nucleic acid aptamers on the chip surface through covalent interaction with the chip surface;

[0052] (6) Detection of mercury ions: 20 nM DSAI fluorescently modified DNA was added to the PCN&AuNPs chip, reacted at 4°C for 12 h, unreacted DNA was removed, and the chip was washed with PBS and dried. Finally, Hg 2+The reaction was carried out at 37°C for 2 hours, the reaction solution was removed, and then washed with PBS and dried. A linear relationship diagram was drawn based on the obtained fluorescence value and the concentration of mercury ions.

[0053] In order to achieve the best detection effect of mercury ions by the sensing platform, the above reaction conditions were optimized.

[0054] 1. Optimizing the concentration of PCN&AuNPs

[0055] Add 100 μL of different concentrations of PCN&AuNPs (0, 20, 40, 60, 80, 100, 120 and 140 μg / mL) to the prepared thiol glass slide and react at 25°C in the dark for 12 hours. Immerse the chip in 1 mM MCH solution for 5 minutes to prepare an MCH monolayer on the chip surface. Add DNA with a final concentration of 20 nM and 10 μΜ DSAI and react at 4°C in the dark for 12 hours, then wash and dry, and finally select the concentration of PCN&AuNPs by the fluorescence intensity measured by the scanner. Figure 2 , 3 is the concentration optimization diagram of PCN&AuNPs. Figure 2 , 3 It can be seen that the fluorescence intensity measured by the chip corresponding to 80 μg / mL PCN&AuNPs is the highest, which means the effect of DNA fixation is the best. 80 μg / mL PCN&AuNPs is selected as the optimal experimental reaction concentration.

[0056] 2. Optimize the reaction temperature of the biochip

[0057] After adding DNA, the chip was placed at different reaction temperatures (0, 4, 20, 37 and 55°C) for experiments. Figure 4 , 5 is the effect of reaction temperature on the biochip; Figure 4 , 5 It can be seen that when the temperature is 4°C, the fluorescence intensity of the chip is the highest, that is, the best immobilization effect is achieved when the reaction temperature is 4°C.

[0058] 3. Optimizing the reaction time of the biochip

[0059] The chip was scanned at different reaction times (6, 12, 18, 24, 36 and 48 h) after adding DNA. Figure 6 , 7 This is the effect of different reaction times on the biochip. As shown in the figure, the fluorescence of the scanned chip basically reaches a stable state after 12 hours, so 12 hours is selected as the time for chip fixation of DNA.

[0060] The above three groups of optimization time are obtained:

[0061] (1) The optimal reaction concentration of PCN&AuNPs was 80 μg / mL.

[0062] (2) The optimal reaction temperature of the biochip is 4°C.

[0063] (3) The optimal time for fixing DNA on the biochip is 12 hours.

[0064] The optimized technical solution is shown below:

[0065] Steps (1) and (2) are the same as above.

[0066] (3) Biochip preparation: Add 100 μL of 60 μg / mL PCN&AuNPs to the thiolated glass slide and react at 25°C for 12 h. Then remove the incompletely fixed PCN&AuNPs, wash with ultrapure water and dry.

[0067] (4) Preparation of MCH monolayer structure on the chip surface: DNA with thiol end groups can covalently bind to the gold on the chip surface and thus be fixed on the chip surface. MCH molecules can compete with weaker nonspecific interactions and block the nonspecific adsorption between DNA and the chip surface, thereby improving the sensitivity of detection.

[0068] (5) Chip detection of mercury ions: After the corresponding chip was prepared, 20 nM DSAI fluorescently modified DNA was added to the PCN&AuNPs chip, reacted at 4°C for 12 h, the unreacted DNA was removed, and the chip was washed with PBS and dried. Finally, Hg 2+ The reaction was carried out at 37°C for 2 h, the reaction solution was removed, and the cells were washed with PBS and dried.

[0069] Based on the above technical solutions, different Hg 2+ The sensitivity of the biochip under different concentrations. 2+ (0-2μM), react at 37℃ in the dark for 2h, wash and dry, and measure different concentrations of Hg with a scanner 2+ caused by the change in fluorescence intensity. Figure 8 , 9 , 10 are for different concentrations of Hg 2+ The sensitivity comparison chart shows that as Hg 2+ As the concentration of Hg increases, the fluorescence intensity measured by the chip forms an upward trend and gradually flattens. 2+ When the concentration is 0-100nM, the fluorescence intensity is related to Hg 2+ The concentration showed an obvious linear relationship.

[0070] In this embodiment, several Hg 2+Different ions (Na + Mg 2+ , Mn 2+ , K + 、Zn 2+ , Ca 2+ 、Ni 2+ , Cu 2+ , Fe 2+ , Pb 2+ ) The selectivity of the sensor was evaluated on the chip. Metal ions with a final concentration of 100 nM were added to the system; the fluorescence intensity was measured to evaluate the selectivity of the sensor. Fig.11 , 12 This is a comparison of the fluorescence recovery rates of different types of ions. It can be seen from the figure that Hg 2+ The change in fluorescence intensity caused by the method is the most obvious, which is clearly distinguished from several other substances and has significant differences, indicating that the sensor of the present invention has good selectivity.

[0071] As a further improvement, the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles, characterized in that: The following steps are involved: S1. Aminated glass slides: H2SO4 and H2O2 are mixed to obtain solution A, and the glass slides are immersed in solution A for 2-10 hours, and then washed and dried; ethanol and APTES are mixed to obtain solution B, and the dried glass slides are immersed in solution B for 10-120 minutes, and then washed and dried to obtain amino-modified glass slides; S2, thiolation of glass slides: adding MPA to a mixture containing NHS and EDC, reacting at room temperature, and obtaining activated MPA after the reaction; mixing PBS with the activated MPA to obtain solution C, and soaking the amino-modified glass slide obtained in S1 in solution C, and washing and drying after soaking to obtain thiolation of the glass slide; S3, preparing PCN&AuNPs biochip: adding PCN&AuNPs to the surface of the thiolated glass slide obtained in S2 for reaction to obtain a biochip, wherein the concentration of PCN&AuNPs is 20-140 μg / mL; S4, preparing a MCH monolayer structure: adding MCH to the surface of the biochip obtained in S3 for reaction, the reaction time is 3-10 minutes, and a monolayer structure is prepared; S5. Immobilizing nucleic acid aptamers on the chip: adding thiol-modified nucleic acid aptamers to react on the basis of S4, the reaction temperature is 0-55°C, and the reaction time is 6-48h; S6. Detection of mercury ions: Add Hg to the surface of the chip obtained in S5 2+ The reaction was carried out at 30-40°C for 2 hours, and a linear relationship diagram was drawn based on the fluorescence value obtained and the concentration of mercury ions; In S5, DSAI fluorescent molecules with AIE properties were used to label the nucleic acid aptamers. The final concentration of DSAI was 10 μM and the concentration of the nucleic acid aptamer was 20 nM.

2. The method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles according to claim 1, characterized in that: The sequence of the thiol-modified nucleic acid aptamer in S4 is: 5´-SH-TTTTTTGGGTGGGTGGGTGGGTTTTTTT-3´.

3. The method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles according to claim 1, characterized in that: The concentration of PCN&AuNPs in S3 was 80 μg / mL, the reaction temperature was 25 °C, and the reaction time was 12 h.

4. The method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles according to claim 1, characterized in that: The temperature for fixing DNA on the chip in S5 is 4° C., and the reaction time is 12 h.

5. The method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles according to claim 1, characterized in that: The concentration of MCH in S4 was 1 mM and the reaction time was 5 min.

6. The method for detecting mercury ions by fixing DNA sequences on a biochip based on an organic metal framework and gold nanoparticles according to claim 1, characterized in that: Hg in S6 2+ The concentration was 0.02-2 μM and the reaction temperature was 37°C.

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