阅读说明：本技术 基于SiO2核酸探针和杂交链信号放大的Ag+检测方法 (Based on SiO2Nucleic acid probe and hybrid strand signal amplification Ag + detection method ) 是由 宫晓群 李恒轩 常津 王汉杰 陈明慧 于 2019-09-30 设计创作，主要内容包括：本发明属于银离子检测领域,具体涉及一种基于SiO<Sub>2</Sub>核酸探针和杂交链信号放大的Ag<Sup>+</Sup>检测方法,包括下述步骤：1)DNA探针与SiO<Sub>2</Sub>微球的偶联；将氨基化修饰的SiO<Sub>2</Sub>微球与DNA探针P1在偶联剂作用下进行偶联,得到SiO<Sub>2</Sub>-P1；2)定量检测Ag<Sup>+</Sup>；将SiO<Sub>2</Sub>-P1与DNA探针P2,加入到缓冲液中,混匀,然后加入杂交链反应探针H1和荧光探针Cy5-H2,加入待检测目标Ag<Sup>+</Sup>,充分混匀,孵育；在反应完成后,通过使用离心机离心除去上清液,并将沉淀物重悬于水中,测量Cy5荧光信号强度。本发明通过在SiO<Sub>2</Sub>微球上偶联核酸杂交信号,开发了一种简单,高效且灵敏的检测Ag<Sup>+</Sup>的方法。(The invention belongs to the field of silver ion detection, and particularly relates to a method for detecting silver ions based on SiO 2 Nucleic acid probe and hybrid strand signal amplification Ag + The detection method comprises the following steps: 1) DNA probe and SiO 2 Coupling the microspheres; modifying SiO by amination 2 Coupling the microspheres with a DNA probe P1 under the action of a coupling agent to obtain SiO 2 -P1; 2) quantitative determination of Ag + (ii) a Mixing SiO 2 Adding the-P1 and the DNA probe P2 into a buffer solution, mixing uniformly, then adding the hybrid chain reaction probe H1 and the fluorescent probe Cy5-H2, and adding the target Ag to be detected + Fully and evenly mixing, and incubating; after the completion of the reaction, the supernatant was removed by centrifugation using a centrifuge, and the precipitate was resuspended in water, and the Cy5 fluorescence signal intensity was measured. The invention is realized by adding SiO 2 Nucleic acid hybridization signals are coupled on the microspheres, and a simple, efficient and sensitive Ag detection method is developed + The method of (1).)

1. Based on SiO₂Nucleic acid probe and hybrid strand signal amplification Ag⁺The detection method is characterized by comprising the following steps:

1) DNA probe and SiO₂Coupling the microspheres; modifying SiO by amination₂Coupling the microspheres with a DNA probe P1 under the action of a coupling agent to obtain SiO₂-P1；

2) Quantitative determination of Ag⁺(ii) a Mixing SiO₂Adding the-P1 and the DNA probe P2 into a buffer solution, mixing uniformly, then adding the hybrid chain reaction probe H1 and the fluorescent probe Cy5-H2, and adding the target Ag to be detected⁺Is sufficientMixing, and incubating; after the completion of the reaction, the supernatant was removed by centrifugation using a centrifuge, and the precipitate was resuspended in water, and the Cy5 fluorescence signal intensity was measured.

2. SiO-based according to claim 1₂Nucleic acid probe and hybrid strand signal amplification Ag⁺The detection method is characterized in that the DNA probe P1 is: TCAGCCCGGC or AAAAATCAGCCCGGC; the DNA probe P2 is: AGTCTAGGATTCGGCGTGGGTTAAGCCCCCCTGA or AGTCTAGGATTCGGCGTGGGTTAAAAAAAGCCCCCCTGA.

3. SiO-based according to claim 1₂Nucleic acid probe and hybrid strand signal amplification Ag⁺The detection method is characterized in that the buffer solution is a sodium citrate buffer solution.

4. SiO-based according to claim 1₂Nucleic acid probe and hybrid strand signal amplification Ag⁺The detection method is characterized in that the specific steps in the step 2) are as follows: 10 mu L of DNA-SiO connected in the step 1)₂Probe and 0.7 μ L10 μmol/L LDNA probe P2) were added to 25 μ L sodium citrate SSC buffer, mixed well, then 1.5 μ L1 μ M hybrid chain reaction probe H1 and 1.5 μ L1 μmol/L Cy5-H2 were added; finally, target Ag is added⁺Mixing, and incubating at 37 deg.C for 60 min; after the completion of the reaction, the supernatant was removed by centrifugation at 11,000rpm for 10 minutes using a centrifuge, and the precipitate was resuspended in 50 μ L of water, and the intensity of Cy5 fluorescence signal was measured by azure c 600.

Technical Field

The invention belongs to the field of silver ion detection, and particularly relates to a method for detecting silver ions based on SiO₂Nucleic acid probeAnd hybrid strand signal amplification Ag⁺And (3) a detection method.

Background

Silver element is one of the components of human body, and a small amount of Ag⁺Is nontoxic but takes up excessive Ag in human body⁺Is harmful, the World Health Organization (WHO) recommends a maximum concentration of 0.1mg/L (about 927nM) as allowable in drinking water, and the United states Environmental Protection Agency (EPA) recommends a maximum concentration of 0.05mg/L (464 nM). Ag, Ag⁺The toxic action mainly comprises the following points: firstly, the protein and various enzymes of the human body are denatured. When excess Ag⁺When transported in blood, due to Ag⁺Can be combined with sulfydryl to ensure that the corresponding sulfydryl-containing enzyme fails, thereby influencing the metabolism of human bodies and seriously harming the health of the human bodies. II, due to Ag⁺Strong oxidizing property of, Ag entering human body⁺Symptoms such as visceral edema can be caused and even death can result. Excessive intake of Ag due to the absence of effective silver-expelling mechanism⁺It is deposited in tissues or organs such as gums, skin, liver, etc. And Ag⁺Poisoning currently has no effective antidote, so that the establishment of Ag with high selectivity and high sensitivity is urgently needed⁺The identification and detection method has important significance for environmental monitoring, medical health and the like.

Conventional Ag⁺The detection method mainly comprises inductively coupled plasma mass spectrometry, atomic absorption spectrometry, stripping voltammetry and the like. These methods are Ag in a particular age⁺Detection has made a great contribution, and they have been used up to now because of their high sensitivity and stable detection means. However, due to the non-portability of these methods, high instrument costs, complex material preparation and the need for professional manipulation, their range of application is also highly limited. Therefore, there is a need for a simple, fast and inexpensive method for detecting Ag⁺The method can make up for the defects of the traditional method. Recently, Ag has been found⁺Can form a C-Ag < + > -C special structure through the specific combination of coordination and cytosine to realize the mismatching between DNAs, and the phenomenon is widely applied to Ag⁺Detection of (3). With the advance of science and technologyWith the development of processes and productivity, there are more and more methods and means for detecting trace or trace substances, such as colorimetry, fluorescence, electrochemical analysis, and the like. Compared with the traditional method, the methods are faster and more convenient, are very reliable in sensitivity and specificity, do not need complicated preparation stages and expensive instruments and facilities, and better meet the requirements of current world scientific experiments.

Hybrid Chain Reaction (HCR) is an enzyme-free isothermal nucleic acid amplification technique discovered and reported for the first time in 2004 by Pierce and Dirks, and is a nucleic acid amplification technique that can be performed in vitro, and is widely applied to high-sensitivity detection of substances such as specific DNA sequences, proteins, small molecules, and the like. The biggest advantage of the hybridization chain reaction is that the enzyme is not needed to participate, so the Polymerase Chain Reaction (PCR) is not needed to be subjected to the cycle of three steps of deformation, annealing and extension, the phenomenon that the experiment is influenced by nonspecific amplification rarely occurs, and the false negative and false positive results are greatly avoided. The hybrid chain reaction is easy to control, the whole reaction condition is mild, and the amplified short-chain DNA can be quickly obtained by only one-step reaction. The signal amplification technology of the hybrid chain reaction can be matched with various detection and analysis methods well. Based on the above advantages, researchers often use the hybrid chain reaction to improve the sensitivity of detection of biomolecules such as proteins and nucleic acids, as well as heavy metals and other small molecules. Compared with other nucleic acid chain amplification reactions, the hybridization chain reaction has the greatest advantage for researchers that no enzyme participates in the whole process, which brings great convenience for later work.

Disclosure of Invention

The invention aims to provide a SiO-based material₂Nucleic acid probe of microsphere and Ag for amplifying hybridization chain signal⁺And (3) a detection method.

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

based on SiO₂Nucleic acid probe and hybrid strand signal amplification Ag⁺The detection method is characterized by comprising the following steps:

1) DNA probe and SiO₂Coupling the microspheres; modifying SiO by amination₂Coupling the microspheres with a DNA probe P1 under the action of a coupling agent to obtain SiO₂-P1；

2) Quantitative determination of Ag⁺(ii) a Mixing SiO₂Adding the-P1 and the DNA probe P2 into a buffer solution, mixing uniformly, then adding the hybrid chain reaction probe H1 and the fluorescent probe Cy5-H2, and adding the target Ag to be detected⁺Fully and evenly mixing, and incubating; after the completion of the reaction, the supernatant was removed by centrifugation using a centrifuge, and the precipitate was resuspended in water, and the Cy5 fluorescence signal intensity was measured.

The DNA probe P1 is: TCAGCCCGGC or AAAAATCAGCCCGGC; the DNA probe P2 is: AGTCTAGGATTCGGCGTGGGTTAAGCCCCCCTGA or AGTCTAGGATTCGGCGTGGGTTAAAAAAAGCCCCCCTGA.

The buffer solution is sodium citrate buffer solution.

Preferably, the specific steps of step 2) are as follows: 10 mu L of DNA-SiO connected in the step 1)₂Probe and 0.7. mu.L of 10. mu. mol/L DNA probe P2) were added to 25. mu.L of sodium citrate SSC buffer, mixed well, and then 1.5. mu.L of 1. mu.M hybrid chain reaction probe H1 and 1.5. mu.L of 1. mu. mol/L Cy5-H2 were added; finally, target Ag is added⁺Mixing, and incubating at 37 deg.C for 60 min; after the completion of the reaction, the supernatant was removed by centrifugation at 11,000rpm for 10 minutes using a centrifuge, and the precipitate was resuspended in 50 μ L of water, and the intensity of Cy5 fluorescence signal was measured by azure c 600.

Compared with the prior art, the invention has the beneficial effects that:

the invention is realized by adding SiO₂Nucleic acid hybridization signals are coupled on the microspheres, and a simple, efficient and sensitive Ag detection method is developed⁺The method of (1). Due to C-Ag⁺the-C structure has strong and specific binding interaction, so that a DNA probe with a plurality of cytosine bases at two ends is designed for detecting Ag⁺This can significantly improve the specificity of the sensor. Meanwhile, by means of enzyme-free isothermal nucleic acid amplification of HCR, Ag can be achieved by detecting the fluorescent signal of Cy5 labeled on the component H2⁺High detection sensitivity andand (4) a large analysis range. Finally, we also verified the specificity of the sensor and its detectability in real samples and compared it with standard Ag⁺Detection method ICP-MS was compared. The sensor based on the design can realize low-cost, high-specificity and ultra-sensitive detection of Ag⁺The ability of the cell to perform. The invention firstly prepares C-Ag⁺The combination of the-C structure and the hybridization chain reaction can detect the Ag with pM level⁺Realize Ag⁺High efficiency and quick detection.

Description of the drawings:

FIG. 1 shows Ag⁺Detecting a schematic diagram;

FIG. 2 shows SiO-based₂Nucleic acid probe and hybrid strand signal amplification Ag⁺A sensitivity fitting graph of the detection method;

FIG. 3 shows SiO-based preparation₂Nucleic acid probe and hybrid strand signal amplification Ag⁺A detection method condition optimization experimental graph;

FIG. 4 shows SiO-based preparation₂Nucleic acid probe and hybrid strand signal amplification Ag⁺Specific experimental diagram of detection method.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and preferred embodiments.

The technical method realizes the aim of aligning Ag⁺The detection with high efficiency, rapidness, sensitivity and specificity is specifically explained as follows: C-Ag⁺the-C structure is a recently discovered strong and specific binding interaction, and can be applied to Ag⁺Detection of (3). However, the signal of the nucleic acid thus converted is difficult to detect, and therefore a means for amplifying the detection signal is required. In recent years, the development of simple, rapid, efficient and inexpensive signal amplification techniques has become important in the fields of biochemical analysis, biomedicine and the like. Currently, the signal amplification mechanism can be divided into two categories, namely temperature-variable signal amplification (such as polymerase chain reaction, ligase chain reaction, etc.) and isothermal signal amplification (such as rolling circle replication, DNA strand substitution reaction, etc.). Variable temperature signal amplifierCompared with the prior art, the isothermal signal amplification method avoids a complicated temperature changing procedure, is simple and easy to operate, and is favored by the majority of scientific researchers. The hybridization chain reaction is an isothermal signal amplification technology based on DNA chain substitution reaction. In an HCR system, a target molecule initiates the alternate ring opening of two DNA stem-loops, and a linear double-stranded DNA nano structure containing a large number of repeating units is obtained by self-assembly, so that the method has the advantages of constant temperature, enzyme immunity, high amplification efficiency and the like. The invention is based on the use of SiO₂Coupling hybrid chain reaction signal on microsphere, and reacting C-Ag⁺The nucleic acid signal of-C conversion is further amplified, so that the Ag can be detected with low cost, high specificity and ultra-sensitivity⁺The method of (1) is achieved.

The detection strategy of the present invention is specifically as shown in fig. 1: (1) SiO 2₂Microspheres were modified with P1 probe as detection platform. Both P1 and P2 have three consecutive cytosines. In Ag⁺When present, P1 and P2 may be modified by the formation of C-Ag⁺-C structural bonding. Since the latter half of P2 is the initiation chain of hybridization chain reaction, SiO is obtained₂The P1-P2 coupler can continuously open H1 and H2 stem-loop structures to initiate HCR, so that the whole HCR product is connected with SiO₂On the microspheres. Cy5 is a fluorescent dye labeled on H2. Thus, after centrifugation, in the presence of Ag⁺In the case of (2), a fluorescence signal of Cy5 was detected in the precipitate, and the fluorescence intensity was correlated with Ag⁺Is proportional to the concentration of (c). By analyzing the fluorescence intensity, the Ag can be quantitatively detected⁺The concentration of (c). When Ag is present⁺In the absence of SiO₂No HCR product was coupled to the microspheres, so that the Cy5 fluorescent signal could not be detected. (2) In this invention, we used azure c600 to quantify the fluorescence intensity, so that the detection limit reached the pM level, and the invention was specifically tested, demonstrating its good specificity.