CN113151400A - Pb based on DNA tetrahedral nanostructure mediated HCR signal amplification2+Fluorescence sensing method - Google Patents

Abstract

The invention belongs to the technical field of food safety and environmental monitoring, and particularly relates to Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺The fluorescence sensing method comprises the steps of assembling a DNA tetrahedron three-dimensional nano structure, connecting hairpin DNA at four vertexes, and marking fluorescent groups capable of generating FRET (fluorescence resonance energy transfer) effect on the hairpin. According to the fluorescence sensing method based on the DNA tetrahedron nanostructure mediated HCR signal amplification, the three-dimensional DNA nanostructure is assembled by setting, and the HCR can be carried out towards different directions by virtue of the high stability of the rigid nanostructure and the four vertexes of the DNA tetrahedron in multi-dimensional separation, so that the collision probability is increased, and the constructed sensing system is efficient, sensitive and stable.

Description

Pb based on DNA tetrahedral nanostructure mediated HCR signal amplification2+Fluorescence sensing method

Technical Field

The invention relates to the technical field of food safety and environmental monitoring, in particular to a DNA tetrahedron-based nano-materialPb for structure-mediated HCR signal amplification²⁺A fluorescence sensing method.

Background

With the rapid development of industrial and agricultural production, environmental protection and food safety become important problems restricting the sustainable development of the world, especially China. Heavy metal pollution, one of the major pollution sources, has also attracted widespread social attention. And Pb²⁺Heavy metal contamination is not only a major factor causing environmental deterioration; and the food is difficult to degrade in the environment, is easy to be gradually amplified into a human body through food chain enrichment, and threatens human health. Therefore, heavy metal ion detection has very important significance in a plurality of fields such as environment, food and the like, and is also the research focus of global analysts.

The most reliable and efficient analysis method is currently also the conventional laboratory test method: such as inductively coupled plasma mass spectrometry (ICP-MS), Atomic Absorption Spectrometry (AAS), Atomic Emission Spectrometry (AES), etc., although all of them can achieve high-sensitivity and high-selectivity detection, the above methods mostly depend on a fine and expensive large instrument, require a complex sample pretreatment process and professional technician operation, can only be performed under laboratory conditions, fail to meet the requirements of portable detection, and are difficult to apply to on-site in-situ real-time detection. The existing problems of environmental pollution and food safety are very sudden, time-efficient and frequent, so that higher requirements are put forward on the detection technology. In order to make a response scheme timely and accurately and minimize pollution and injury, people urgently need a convenient, real-time, sensitive and on-site analysis means. As the research on heavy metal detection is realized, the research on heavy metal detection is not limited to laboratories, but gradually develops to the field in situ, the technology gradually develops to simplification, portability and economy, and the equipment gradually develops to miniaturization, portability and integration, and the change becomes a new research hotspot.

The recently newly developed biosensor used for detecting heavy metal ions has the characteristics of simple sample processing process, no need of expensive instruments, simplicity, high efficiency and sensitivity, and provides great feasibility for realizing on-site on-line detection of heavy metal ions. The DNA sensor is one of biosensors, is used for the development of heavy metal ion detection technology and is derived from the discovery of functional nucleic acid, which refers to nucleic acid molecules with functions exceeding the traditional genetic action and comprises nucleic acid aptamers, deoxyribozymes, molecular beacons and the like. The metal ion functional nucleic acid is a nucleic acid molecule which can exhibit a specific function in the presence of a specific ion. The functional nucleic acid has a series of advantages of easy design and modification, good stability, environmental friendliness, high specificity for identifying target molecules and the like, and the characteristics provide various convenient conditions for the design of the biosensor, and are also an important reason for the rapid development of research on the functional nucleic acid sensor in recent years. In this respect, a great deal of research work is carried out, and a series of high-sensitivity and high-specificity metal ion sensors are designed by combining with various signal output modes, such as fluorescence, colorimetry, electrochemistry, magnetic resonance and the like, so that the detection of various heavy metal ions is realized.

However, most of the current metal ion sensors based on functional nucleic acids are still in the laboratory stage. If the on-site and on-line detection of heavy metal ions in systems such as environment and food is to be realized, many problems still need to be solved, wherein two outstanding problems are as follows: the response speed and detection sensitivity of the sensor are to be further improved. In order to further improve the response speed and detection sensitivity of the sensor, a three-dimensional DNA nanostructure-mediated non-enzymatic isothermal nucleic acid amplification mechanism is created based on the Watson-Crick base complementary pairing principle and the nucleic acid controllable self-assembly principle, a high-efficiency, sensitive and stable sensing system is constructed based on the nucleic acid amplification signal amplification technology, the space constraint effect and the effective collision theory, and the method is further applied to Pb in samples such as water, food and the like²⁺Detection of (3).

Disclosure of Invention

Based on the prior technical problem, the invention provides Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺A fluorescence sensing method.

The invention provides Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺The fluorescence sensing method comprises the following steps of firstly, assembling a DNA tetrahedron three-dimensional nano structure, connecting hairpin DNA at four vertexes, and marking fluorescent groups capable of generating FRET effect on the hairpin;

step two, passing Pb²⁺Catalytic cleavage of DNAZyme controls HCR reaction;

step three, based on FRET fluorescent signal to Pb²⁺And (4) establishing a standard curve according to the concentration.

Preferably, the tetrahedral DNA nanostructure consists of four oligonucleotide strands, the hairpin structure is two HA8 and HB8, Pb²⁺Selecting Pb from DNAZyme²⁺Specifically recognized GR5 sequence.

Preferably, the hairpin structure HA8 labeled fluorophore cy3, HB8 labeled fluorophore cy5 and GR5 sequence comprise a polymerase chain (GR5) and a substrate chain (S3838)_TM)。

Preferably, the tetrahedral DNA nanostructure is in the buffers Tris-HCl and MgCl₂Heating at 95 deg.C for 5min, and keeping the temperature at 4 deg.C for 30 min.

Preferably, the Pb is²⁺Specifically recognized GR5 sequence substrate chain (S)_TM) Inducing HCR reaction to generate fluorescence resonance energy transfer FRET, adding Pb²⁺After cutting into S_TAnd S_MThe HCR reaction cannot be initiated.

Preferably, the Pb based on the DNA tetrahedral three-dimensional nanostructure mediated HCR signal amplification technology²⁺The fluorescence sensing method can complete detection within 15 min;

preferably, the method is mainly divided into self-assembly of nanostructures and Pb²⁺And identifying and detecting two parts. First, synthesis of DNA tetrahedrally assembled hairpin complexes, shown in FIG. 1A, self-assembly by nucleic acid hybridization reaction to form DNA tetrahedron structures, with four vertices connecting hairpin DNA required for HCR reaction. Followed by Pb²⁺The substrate strand (S) of GR5 DNAzyme is shown as B in FIG. 1_TM) Can be used as a primer to trigger HCR reaction, and the fluorescent groups cy3 and cy5 are drawn close, so that FRET fluorescence is generatedA signal. And at Pb²⁺When present, Pb²⁺Can specifically recognize GR5 DNAzyme and convert S_TMThe reaction is cut into two sections, thereby inhibiting the HCR reaction and failing to generate FRET signal. Thus Pb²⁺The change of concentration can be converted into the change of FRET fluorescent signal, and Pb can be realized by monitoring the fluorescent signal²⁺And (4) carrying out quantitative detection. In this method, the four vertices of the tetrahedron provide multidimensional spatial orientation for the HCR reaction; and a single tetrahedron assembles multiple hairpin structures, thereby concentrating the local concentration of hairpins. The synergistic effect of the two can effectively increase the collision probability, thereby greatly improving the reaction rate and leading Pb to be²⁺The detection can be completed within 15min, and the detection limit can reach 0.5 pM. Meanwhile, the sensing method is used for Pb in substrates such as water, vegetables, grains and the like²⁺And (3) detecting to obtain a result consistent with the traditional ICP-MS method, and indicating the feasibility of the sensing method applied to a complex matrix.

The beneficial effects of the invention are as follows:

1. by arranging and assembling the three-dimensional DNA nano structure, with the help of the high stability of the rigid nano structure, the HCR can be carried out towards different directions by four vertexes separated in a multi-dimensional way in the DNA tetrahedron, so that the collision probability is increased, and the constructed sensing system is efficient, sensitive and stable.

2. The non-enzymatic nucleic acid amplification technology is introduced into the sensor, so that nucleic acid amplification reaction can be initiated in the presence of a quantitative target object, a large number of signal output units are enriched, detection signals are amplified, the detection sensitivity is improved, the detection can be carried out at constant temperature even at room temperature under mild reaction conditions, and the sensor is more suitable for on-site on-line analysis and detection.

3. The proposed sensing platform is for Pb by setting up HCR accelerated by three-dimensional nanostructures²⁺The detection shows extremely fast response and high sensitivity, and the analysis process can be completed within 15min, so that the sensor can be suitable for Pb in some water bodies, food and other complex matrixes²⁺Rapid and sensitive detection.

Drawings

FIG. 1 is a DNA tetrahedron-based nanostructure according to the present inventionPb mediating HCR signal amplification²⁺A schematic of a fluorescence sensing method;

FIG. 2 shows Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺Synthesizing an electrophoresis chart by using a three-dimensional nano structure of a fluorescence sensing method;

FIG. 3 shows Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺Pb of fluorescence sensing method²⁺Atomic force microscopy characterization of HCR product in presence;

FIG. 4 shows Pb mediated HCR signal amplification based on DNA tetrahedral nanostructure²⁺Pb of fluorescence sensing method²⁺Atomic force microscopy characterization of HCR product in the absence;

FIG. 5 shows Pb mediated HCR signal amplification based on DNA tetrahedral nanostructure²⁺Presence or absence of Pb in fluorescent sensing method²⁺Particle size distribution profile of HCR product when present;

FIG. 6 shows Pb mediated HCR signal amplification based on DNA tetrahedral nanostructure²⁺Fluorescence signal ratio F of fluorescence sensing method_A/F_DFollowing Pb²⁺Graphs of the concentration (0-200 nM);

FIG. 7 shows Pb mediated HCR signal amplification based on DNA tetrahedral nanostructure²⁺Signal response plot of non-target ions for fluorescence sensing method.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Referring to FIGS. 1-7, a Pb based on DNA tetrahedral nanostructure mediated HCR signal amplification²⁺The fluorescence sensing method comprises the following steps of firstly, assembling a DNA tetrahedron three-dimensional nano structure, connecting hairpin DNA at four vertexes, and marking fluorescent groups capable of generating FRET effect on the hairpin;

further, step two, passing Pb²⁺Catalytic cleavage of DNAZyme controls HCR reaction, thereby controlling FRET efficiency;

further, step three, based on FRET fluorescent signal to different Pb²⁺Response of concentration, establishing standard curve, and realizing Pb²⁺The quantitative detection of (3);

the tetrahedral DNA nano structure consists of four oligonucleotide chains, the hairpin structure HAs two types of HA8 and HB8, Pb²⁺Selecting Pb from DNAZyme²⁺The GR5 sequence is specifically recognized, the hairpin structure HA8 marks a fluorescent group cy3, HB8 marks a fluorescent group cy5, and the GR5 sequence comprises a polymerase chain (GR5) and a substrate chain (S5)_TM) Tetrahedral DNA nanostructures in buffer Tris-HCl and MgCl₂The reaction temperature is 95 ℃ for 5min, the temperature is 4 ℃ for 30min, and Pb is contained²⁺Specifically recognized GR5 sequence substrate chain (S)_TM) Inducing HCR reaction to generate fluorescence resonance energy transfer FRET, adding Pb²⁺After cutting into S_TAnd S_MPb based on DNA tetrahedral three-dimensional nanostructure-mediated HCR signal amplification technology without initiating HCR reaction²⁺The fluorescence sensing method can complete detection within 15 min;

by arranging and assembling the three-dimensional DNA nano structure, the high stability of the rigid nano structure and the four vertexes of the DNA tetrahedron in multi-dimensional separation enable HCRs to be carried out towards different directions, so that the collision probability is increased, and the constructed sensing system is efficient, sensitive and stable;

the non-enzymatic nucleic acid amplification technology is introduced into the sensor, so that nucleic acid amplification reaction can be initiated in the presence of a quantitative target object, a large number of signal output units are enriched, detection signals are amplified, the detection sensitivity is improved, the detection can be carried out at constant temperature even at room temperature under mild reaction conditions, and the sensor is more suitable for on-site on-line analysis and detection;

the proposed sensing platform is for Pb by setting up HCR accelerated by three-dimensional nanostructures²⁺The detection shows extremely fast response and high sensitivity, and the analysis process can be completed within 15min, so that the sensor can be suitable for Pb in some water bodies, food and other complex matrixes²⁺Rapid and sensitive detection.

Example one

Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺A fluorescence sensing method comprises the following steps of A, material synthesis preparation and assembly methods and detection processes of a sensor:

(1) 4 oligonucleotides (a, b, c and d) were added at the same molar concentration in 10mM MgCl₂And 20mM Tris-HCl buffer (pH 7.4), and the mixture was heated to 95 ℃ for 5min and then cooled to 4 ℃ within 1 min. The prepared TDN is directly used or stored at 4 ℃ for later use.

(2) Hairpin DNA preparation by addition of HA8cy3 or HB8cy5, respectively, to a buffer (20mM Tris-HCl,10mM MgCl)₂pH 7.4). The mixture was heated at 95 ℃ for 5min and 25 ℃ for 10min to form HA8cy3-hairpin or HB8cy5-hairpin, respectively. The prepared hairpin DNA is used directly or stored at 4 ℃ for further use.

(3) TDN Assembly hairpin preparation of TDN and hairpin DNA (HA8cy3-hairpin or HB8cy5-hairpin) at a concentration of 1:4 containing 10mM MgCl₂Was mixed with 20mM Tris-HCl buffer (pH 7.4). Unless otherwise stated, the concentration of tetrahedral DNA was 50nM and the hairpin DNA (HA8cy3-hairpin or HB8cy5-hairpin) was 100nM, respectively.

(4) Incubation at 25 ℃ for 30min resulted in formation of TDNHA8cy3 and TDNHB8cy 5.

Prepared TDN_HA8cy3And TDN_HB8cy5It can be used directly or stored at 4 deg.C for use.

(5) First 50nM GR5 and 50nM S_TMMixed in buffer (20mM Tris-HCl,10mM MgCl)₂50mM NaCl, pH 7.4), heated to 95 ℃ for 5min and 25 ℃ for 30min to form GR5-DNAzyme structures. Then adding Pb with different concentrations²⁺Stock solution, vortex for 1min, ensure S_TMAnd (4) cracking. Next, 50nM cGR5 was added to the above mixture and heated to 95 deg.C for 5min and 25 deg.C for 30 min. Finally, the above mixture is added to the prepared solution (TDN)_HA8cy3And TDN_HB8cy5Mixed at the same molar concentration), the FRET fluorescent signal of the solution was recorded after standing at room temperature for 15 min.

Secondly, the processing process of the sensing system before the sample detection:

river water was collected from a small clear river (Tianjin southern open area) and then filtered through a 0.45 μm membrane to remove larger insoluble particles and algae. Pulverizing 0.5g of grains and 5g of vegetables respectively with a grinder, and mixing with 10mL of HNO₃And soaking overnight. Placed in a porous graphite digestion furnace and heated to 120 ℃ until the solution is approximately 2 mL. After cooling, 2mL of H was added₂O₂And digestion was continued at 120 ℃ until the volume was below 1.0mL, with ddH₂O diluted to a volume of 25mL, shaken, left overnight, and then centrifuged (5000rmp, 2 min). Finally, 10. mu.L of each of the above samples was incorporated into the circular DNase products to trigger the cleavage and amplification process, and the detection procedure was the same as described above.

The invention has high sensitivity and short reaction time, and provides the Pb in foods such as grains, vegetables, fruits and the like, which is suitable for detecting the environmental water with low ion concentration²⁺And (3) a detection method.

Thirdly, the structure and the performance of the sensing system prepared by the invention are explained in detail in the following with the attached drawings

(1) Electrophoretic analysis

First, the reaction solution was separated by 2% agarose gel electrophoresis (100V, 30 minutes). The gel was stained with GelGreen beforehand and visualized with an imaging system. As shown in FIG. 1, by the sequential addition of oligonucleotide strands to hairpins (1-5: a, b, c, d and HA8cy3), the location of the bright band continuously increased and the electrophoretic mobility continuously decreased, indicating that the tetrahedral structure and the TDNHA8 structure were successfully assembled.

(2) Atomic force microscopy analysis

The present invention observes the structure of a reaction solution with an Atomic Force Microscope (AFM) in the absence of a target Pb²⁺In the case of (2), the dendritic linked products were observed to aggregate together (in FIG. 2), demonstrating that the HCR reaction occurred. And at Pb²⁺In the presence, it can be clearly seen that the characterization results are some short chain structures and tetrahedral hairpin structures in a dispersed state.

(3) Particle size detection analysis

When HCR reaction is free of Pb²⁺When the structure length of DNA reaches micron level, the reaction bodyIn which Pb is present²⁺At this time, most of the structures in solution were 200-300nm long, indicating the presence of Pb²⁺The HCR reaction can not occur in the solution, the tetrahedral structure is provided with hairpin TDNHA8 and TDNHB8, the broken substrate short chain is dispersed in the solution, and almost no HCR reaction exists, and the characterization results prove the correctness of the invention.

(4) Sensitivity of sensing system

To investigate this method for Pb²⁺The sensitivity of the detection, the ratio F of the fluorescence signals of the detection system at 660 nm (cy5 fluorescence) and 560nm (cy3 fluorescence) is recorded_A/F_DFollowing Pb²⁺The results are shown in FIG. 5, for the concentration (0-200 nM). When Pb is contained in the system²⁺F when the concentration increased from 0.001nM to 0.02nM_A/F_DWith Pb²⁺The concentration is in a good linear relationship (R)²0.9963), Pb was calculated from 3 σ/slope (σ is the standard deviation of multiple determinations on blank samples, slope is the slope of the analytical standard curve)²⁺Has a detection limit of 0.5 pM. Far below the drinking water lead safety limit (72nM) specified by the U.S. environmental protection agency. To determine the method detects Pb²⁺Reproducibility of (2) selecting Pb²⁺Was 0.02nM, and the 10-group experiment was repeated at the same time, giving a detected intra-group bias of 2.9%. The deviation between groups obtained by repeating the 10-group experiment at different times was 3.5%. Deviations of less than 5% between groups or groups indicate Pb detection by this method²⁺Has good reproducibility.

(5) Specificity of the sensing System

To prove that this method detects Pb²⁺With good selectivity, we investigated the sensor for other non-target ions (Ca)²⁺、Mn²⁺、Cd²⁺、Ba²⁺、Zn²⁺、Ni²⁺、Cu²⁺、 Cr³⁺、Hg²⁺) The signal response of (c). The results are shown in FIG. 6. Wherein, only Pb is present²+ addition to disable FRET effect, F_A/F_DNo change, no influence of other ions on HCR reaction, and obvious FRETA signal. The above results show that our method has good selectivity for other non-target ions.

All DNA and RNA oligonucleotides were purchased from Sangon Biotech co.ltd. (shanghai, china) and purified by high performance liquid chromatography. Tris (hydroxymethyl) aminomethane (Tris) was obtained from Sigma-Aldrich. MgCl for use in the present invention₂And other reagents are analytically pure and can be directly used without further purification. Deionized water (resistor) was used throughout the process>18M Ω · cm). 1000 mug/mL lead ion standard stock solution (GSB04-1742-2004) and other standard solutions of metal ions were obtained from the national center for analysis and test of nonferrous metals and electronic materials (Beijing, China).

All fluorescence measurements were performed on a Shimadzu RF-5301PC fluorescence spectrometer (Shimadzu Ltd., Japan) using 540nm as the excitation wavelength and a wavelength range of 550-800 nm. Incubation or annealing was performed on an XP cycler (Hangzhou Borui technologies, Inc., Hangzhou, China). UV spectral measurements were performed on a Shimadzu UV-2450 spectrometer (Shimadzu ltd., Japan). AFM testing was performed on Agilent Technologies 5500 (USA) to observe the morphology of the samples. Dynamic Light Scattering (DLS) experiments were performed on Malvern Zetasizer Nano ZS90(Malvern Instruments, ltd., Worcestershire, UK).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (6)

1. Pb based on DNA tetrahedral nano-structure mediated HCR signal amplification²⁺A fluorescence sensing method, characterized by: assembling a DNA tetrahedron three-dimensional nano structure, connecting hairpin DNA at four vertexes, and marking fluorescent groups capable of generating FRET effect on the hairpin;

step two, passing Pb²⁺Catalytic cleavage of DNAZyme controls HCR reaction;

step three, based on FRET fluorescent signal to different Pb²⁺And (5) responding to the concentration, and establishing a standard curve.

2. The DNA tetrahedral nanostructure-based Pb-mediated HCR signal amplification of claim 1²⁺A fluorescence sensing method, characterized by: the tetrahedral DNA nano structure consists of four oligonucleotide chains, the hairpin structure comprises two types of HA8 and HB8, and Pb²⁺Selecting Pb from DNAZyme²⁺Specifically recognized GR5 sequence.

3. The DNA tetrahedral nanostructure-based Pb-mediated HCR signal amplification of claim 1²⁺A fluorescence sensing method, characterized by: the hairpin structure HA8 labeled fluorophore cy3, HB8 labeled fluorophore cy5 and GR5 sequence comprise a polymerase chain (GR5) and a substrate chain (S)_TM)。

4. The DNA tetrahedral nanostructure-based Pb-mediated HCR signal amplification of claim 1²⁺A fluorescence sensing method, characterized by: the tetrahedral DNA nanostructure is in buffer Tris-HCl and MgCl₂Heating at 95 deg.C for 5min, and keeping the temperature at 4 deg.C for 30 min.

5. The DNA tetrahedral nanostructure-based Pb-mediated HCR signal amplification of claim 1²⁺A fluorescence sensing method, characterized by: the Pb²⁺Specifically recognized GR5 sequence substrate chain (S)_TM) Initiating HCR reaction to generate fluorescence resonance energy transfer, adding Pb²⁺After cutting into S_TAnd S_MThe HCR reaction cannot be initiated.

6. The DNA tetrahedral nanostructure-based Pb-mediated HCR signal amplification of claim 1²⁺A fluorescence sensing method, characterized by: pb based on DNA tetrahedral three-dimensional nanostructure mediated HCR signal amplification technology²⁺The fluorescence sensing method can complete detection within 15 min.

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