Nucleic acid nano sensor for monitoring dynamic change of ATP (adenosine triphosphate) outside living cells and application of nucleic acid nano sensor

Abstract

The invention relates to the technical field of biosensors, and discloses a nucleic acid nano-sensor for monitoring dynamic change of ATP outside living cells and application of the nucleic acid nano-sensor. The nucleic acid nano sensor comprises a DNA tetrahedron framework formed by four DNA single-stranded base complementation, a cellulose aptamer and an ATP aptamer, one endpoint of the DNA tetrahedron framework is connected with the ATP aptamer for sensing ATP, and the other endpoints are connected with the cellulose aptamer for anchoring plant cell walls. According to the nucleic acid nano-sensor for monitoring the dynamic change of ATP outside the living cells and the application of the nucleic acid nano-sensor, during detection, ATP outside the cells competes with a Q group, so that Cy3 recovers fluorescence, a result is visualized through an instrument, the result is easy and convenient to read, meanwhile, a metabolite is nucleotide and is completely harmless to the cells, and the biological safety is high; the cell wall is anchored for in-situ detection, the sensitivity and the space-time stability are good, and the sensor has extremely high application value in the field of biological signal molecule sensing.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to a nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells and applications thereof. Background Art

[0002] Biosensor is a bioanalytical device developed based on the high-activity fixation and specific biorecognition of related bioactive substances on a certain substrate, combined with appropriate signal conversion methods. Therefore, biosensor is a new analytical detection technology developed based on the intersection of analytical chemistry, life sciences, materials science, electronics and other related disciplines. In biosensing research, various oligonucleotides with specific base sequences can not only be used as a class of signal markers with excellent performance, but also act as nucleic acid aptamers for high-specific recognition of various target analytes. Therefore, on this basis, combining it with various nucleic acid signal amplification strategies developed based on the principle of base complementary pairing can further improve the analytical sensitivity of these biosensing methods.

[0003] In order to study the specific regulation between cells and cell membranes, many different functional materials, including proteins, nucleic acids, nanoparticles and polymers, have been designed and applied to the cell surface. Among them, nucleic acids have the advantages of easy synthesis, predictable structure and high programmability, providing excellent materials for precisely controlling the strength and specificity of interactions. In addition, nucleic acid aptamers are single-stranded nucleic acids screened by in vitro methods. Based on their specific recognition of targets (including living cells), they have expanded the scope of application of nucleic acids in cell biology. In 2021, The Plant Journal reported a cellulose aptamer CELAPT MINI (MN) that can specifically bind to cellulose. Cellulose is a homopolymer of b-(1-4) linked glucose and is the most abundant carbohydrate in plants, usually accounting for 20-30% of the primary cell wall and 50% of the secondary cell wall. The aptamer MN was selected more than ten years ago to specifically bind to cellulose, but it has not been applied. This will be a specific tool for studying the mutual transformation of substances inside and outside plant cells.

[0004] Adenosine triphosphate (ATP) is an important energy currency molecule in organisms, ensuring the energy supply of biochemical reactions and the survival of organisms. Although ATP is produced intracellularly, animal, plant and microbial cells can release ATP from the intracellular to the extracellular matrix. Unlike the role of intracellular ATP as an energy currency molecule, extracellular ATP (eATP) is considered to be a signal molecule that regulates many physiological processes of cells. The physiological functions of eATP in plants have been widely studied. It has been reported that eATP can serve as an early signal for plant defense against pathogen infection and herbivore attack. Many studies have shown that eATP is an important signal that participates in regulating plant responses to various abiotic stresses, such as high salt, cold and heavy metals. In addition, studies have found that eATP can affect plant growth and development, pollen germination and root tropism. Therefore, the monitoring of extracellular ATP is particularly important. However, the current technology for detecting extracellular ATP levels in plants still has some obvious limitations or deficiencies. For example, the ATP content in the extracellular fluid or the liquid medium in which the plant tissue is immersed may actually reflect the amount of eATP released in vitro, rather than the actual eATP level in vivo. Exogenously applied or expressed luciferase will hydrolyze ATP during measurement, increasing the difficulty of continuous real-time monitoring of plant food-derived ATP levels.

[0005] DNA tetrahedron structures have attracted extensive attention in the field of interface engineering. In this work, we developed a series of amphiphilic (tetrahedral) DNA probes for cell walls using DNA self-assembly, aiming to build an effective, stable, biocompatible and universal platform for specific monitoring of changes in extracellular ATP levels. These amphiphilic tetrahedrons are self-assembled from four DNA oligonucleotides, with a pendant DNA probe at one vertex and cell wall-bound DNA tetrahedrons with different numbers of aptamer MNs attached to the other three vertices, which can be effectively anchored to the cell wall, and the trivalent tetrahedron has the best anchoring effect, so as to monitor changes in extracellular ATP.

[0006] Chlorella is a common single-cell green algae of the genus Chlorella in the Chlorophyta. It is a spherical single-cell freshwater algae with a diameter of 3 to 8 μm. It is one of the earliest life on Earth, appearing more than 2 billion years ago. It is an efficient photosynthetic plant that grows and reproduces by photosynthesis and autotrophy. It is widely distributed and contains rich nutrients such as polyunsaturated fatty acids, proteins, polysaccharides, vitamins and minerals. It is widely used in the food industry. The cultivation conditions of Chlorella are simple, and single cells are easier to observe, which is conducive to the study of extracellular ATP sensing and monitoring. Summary of the invention

[0007] The purpose of the present invention is to provide a nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells and its application. During detection, extracellular ATP competes with the Q group to restore the fluorescence of Cy3. The result is visualized by an instrument, and the result is simple and convenient to read. At the same time, the metabolites are nucleotides, which are completely harmless to cells and have high biological safety. It is anchored on the cell wall for in situ detection, has good sensitivity and spatiotemporal stability, and has extremely high application value in the field of biological signal molecule sensing.

[0008] To achieve the above-mentioned purpose, the present invention provides a nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells, comprising a DNA tetrahedron framework formed by the complementary bases of four single-stranded DNA strands, a cellulose aptamer and an ATP aptamer, wherein one end of the DNA tetrahedron framework is connected to the ATP aptamer for sensing ATP, and the remaining ends are connected to the cellulose aptamer for anchoring the plant cell wall.

[0009] Furthermore, the ATP aptamer is connected to the fluorescent molecule Cy3 through a covalent bond, and is connected to the fluorescent quencher chain through base complementary pairing, and the fluorescent quencher chain contains a fluorescent quencher group Q.

[0010] Furthermore, the cellulose aptamers are arranged in 1, 2 or 3 strips, and the remaining end points of the DNA tetrahedron framework are connected to 1 cellulose aptamer to form a monovalent nucleic acid nanosensor, the remaining end points of the DNA tetrahedron framework are respectively connected to 2 cellulose aptamers to form a divalent nucleic acid nanosensor, and the remaining end points of the DNA tetrahedron framework are respectively connected to 3 cellulose aptamers to form a trivalent nucleic acid nanosensor.

[0011] Furthermore, the anchoring efficiency of the nucleic acid nanosensor on the cell surface is 59%-71%, wherein the anchoring efficiency of the monovalent nucleic acid nanosensor on the cell surface is 59%, the anchoring efficiency of the divalent nucleic acid nanosensor on the cell surface is 67%, and the anchoring efficiency of the trivalent nucleic acid nanosensor on the cell surface is 71%.

[0012] Furthermore, the synthesis method of the DNA tetrahedral framework is: placing the four single strands of the DNA tetrahedral framework in 1×PBS buffer, then heating to 95°C for 8 to 10 minutes, and then lowering the temperature to room temperature for 30 to 60 minutes to obtain the DNA tetrahedral framework.

[0013] The present invention also provides the use of the above-mentioned nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells in extracellular ATP monitoring. The nucleic acid nanosensor is anchored outside a plant cell with a cell wall structure and is used for in situ real-time monitoring of ATP.

[0014] Furthermore, when applied, the detection principle is: before the nucleic acid nanosensor detection, the quenching group Q connected to the ATP aptamer quenches the fluorescence of Cy3. During the detection, ATP binds to the ATP aptamer, the quenching group Q is replaced, and the fluorescence of Cy3 is restored. The results are observed by fluorescence spectroscopy and laser confocal microscopy to monitor extracellular ATP.

[0015] The present invention also provides the use of the nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells in monitoring the extracellular ATP of Chlorella.

[0016] Further, when applied, the magnesium ion concentration is 2.5-7.5 mM, the incubation time is 5-35 min, and the OD of Chlorella vulgaris is 680 =1, final concentration of cellulose aptamer 50 nM, nucleic acid nanosensor anchored on the surface of Chlorella.

[0017] The present invention also provides the use of the nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells in monitoring the extracellular ATP of Chlorella under salt stress.

[0018] Furthermore, the concentration of NaCl for salt stress was 200 mM.

[0019] Furthermore, Chlorella was cultured in a light incubator (temperature, 25°C; light intensity, 1500 lx; ​​12 hours of day and 12 hours of night) using BG-11 medium. The flask was manually shaken three times a day. The OD 680 ) The concentration of Chlorella was determined by absorbance method.

[0020] The advantages and positive effects of the nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells and its application described in the present invention are:

[0021] 1. In the present invention, an ATP aptamer labeled with Cy3 is connected to the top of the DNA tetrahedral framework, and then a quenching group Q is connected to quench the fluorescence of Cy3. Extracellular ATP competes with the Q group to expose Cy3, and ATP is sensed by fluorescence recovery. The results are visualized by fluorescence spectroscopy and laser confocal microscopy. The results are easy and convenient to read, and have extremely high application value in the field of biological signal molecule sensing.

[0022] 2. In the present invention, a cellulose aptamer is connected to the bottom of the DNA tetrahedral framework. The cellulose aptamer specifically binds to cellulose and is used to anchor on the cell wall of the plant to achieve in situ real-time monitoring of extracellular ATP.

[0023] 3. The preparation method of the ATP sensor of the present invention is simple and can be prepared in a short time (within 3 hours). The metabolites of the sensor are nucleotides, which are completely harmless to cells and have high biological safety.

[0024] 4. The ATP sensor of the present invention performs in situ detection of eATP by anchoring to the cell wall, and has good sensitivity and temporal and spatial stability.

[0025] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthetic route map of the nucleic acid nanosensor in the embodiment of the present invention;

[0027] Figure 2 Agarose gel electrophoresis of synthesized DNA tetrahedron and synthesized multivalent DNA tetrahedron in the embodiments of the present invention;

[0028] Figure 3 The atomic force microscope image of the multivalent DNA tetrahedron in the embodiment of the present invention, wherein A is TDN0, B is TDN1, C is TDN2, and D is TDN3;

[0029] Figure 4 This is a dynamic light scattering particle size detection diagram in an embodiment of the present invention;

[0030] Figure 5 The binding effect between the cellulose aptamer and phosphocellulose in the embodiment of the present invention;

[0031] Figure 6 The optimization results of the best incubation parameters of cellulose and Chlorella in the embodiment of the present invention, wherein A is the optimization of the best magnesium ion concentration, and B is the optimization of the best incubation time;

[0032] Figure 7 is the binding efficiency of sensors with different valence states on the cell wall surface in the embodiments of the present invention;

[0033] Figure 8 is the surface potential of TDN3 in the embodiment of the present invention;

[0034] Fig. 9 This is a laser confocal image of sensors with different valence states anchored to Chlorella in an embodiment of the present invention;

[0035] Fig.10 The response of the nucleic acid nanosensor to the external ATP in the embodiment of the present invention;

[0036] Fig.11 This is the response of the nucleic acid nanosensor to salt stress in an embodiment of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0038] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental instruments, equipment and reagents without source indication in the following examples are all commercially available raw materials.

[0040] The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0041] Unless otherwise defined or described, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0042] Synthesis and identification of eATP detection elements:

[0043] 1. Synthesis:

[0044] The four single strands of the tetrahedron are named A, B, C, and D. The four single strands with interfaces are named A-ATP, Bc, Cc, and Dc. The 5' end of A-ATP is connected to Cy3, named A-ATP-Cy3, and the quenching chain is named ATP-Q. It is connected to the 5' end of A-ATP-Cy3 to quench the fluorescence of Cy3. When ATP binds to the ATP aptamer part in A-ATP, ATP-Q is replaced and the fluorescence of Cy3 is restored; the other three DNA single strands can be connected to the cellulose aptamer MN through the interface to achieve the effect of binding to cellulose, thereby anchoring on the cell wall surface. The sequences are shown in Table 1.

[0045] Table 1 Sequence

[0046]

[0047]

[0048] (Table Notes: The bolded sequences are complementary sequences. Single light underline Some are connection points)

[0049] 1 μL of each of the four single-stranded B, C, D, and A-ATP with a concentration of 10 μM and 1 μL of 4 mM NaCl were added to 15 μL of 1× PBS buffer, and the entire 20 μL system was heated to 95°C for 8 minutes, and then cooled to room temperature for 1 hour. The synthesized volume was 20 μL, and the concentration was 500 nM. Zero-valent DNA tetrahedron, referred to as TDN0.

[0050] 1 μL of each of the four single-stranded Bc, C, D, and A-ATP with a concentration of 10 μM and 1 μL of 4 mM NaCl were added to 14 μL of 1× PBS buffer, and the whole system was heated to 95°C for 8 minutes, and then cooled to room temperature for 1 hour. 1 μL of 10 μM CELAPTMINI cellulose aptamer was added and connected to the Bc chain to synthesize a monovalent DNA tetrahedron with a volume of 20 μL and a concentration of 500 nM, referred to as TDN1.

[0051] The divalent DNA tetrahedron is referred to as TDN2. The synthesis method is the same as TDN1. The synthetic chains are Bc, Cc, D, and A-ATP. 2μL of 10μM MN cellulose aptamer is added and connected to the Bc and Cc chains. The trivalent DNA tetrahedron is referred to as TDN3. The synthesis method is the same as TDN1. The synthetic chains are Bc, Cc, Dc, and A-ATP. 3μL of 10μM MN cellulose aptamer is added and connected to the Bc, Cc, and Dc chains.

[0052] Connecting quenching group Q: Take the synthesized multivalent DNA tetrahedron (20μL 0.5μM) and add it to the buffer (300mM NaCl, 5mM MgCl2, 25mM Tris-HCl, pH=8.3) containing ATP-Q (20μL 0.5μM) chain, and place it at 4℃ for 24h. The synthetic route of TDN1, TDN2, and TDN3 is shown in Figure 1 shown.

[0053] 2. Identification:

[0054] Characterization of eATP in situ detection components:

[0055] 1) Agarose gel electrophoresis:

[0056] Prepare 2.5% agarose gel: add 0.5 g agarose to 20 mL TBE buffer solution.

[0057] Sample loading and electrophoresis: Mix 2 μL of 5× loading buffer with 8 μL of sample and marker, and then add them to the corresponding electrophoresis tank. Perform electrophoresis for 35 minutes at a constant voltage of 120V.

[0058] The results are as follows Figure 2 As shown, the TDN plate is a synthetic TDN0 panel. From lane 1 to lane 4 of the panel, DNA single strands are gradually added. Lane M is Marker, lane 2 is S9, lane 3 is S9+S6, lane 3 is S9+S6+S7, and lane 4 is S9+S6+S7+S8. Lanes M to 4 of the TDN-n plate are Marker, TDN0, TDN1, TDN2, and TDN3, respectively. The electrophoresis bands involving tetrahedrons have very bright high molecular weight bands, indicating that the tetrahedrons are successfully assembled.

[0059] 2) Atomic force microscopy:

[0060] Method: DNA sample (5 μL, 50 nM) was dropped onto freshly cleaved mica and immediately incubated with 1 mM Mg 2+ After rinsing, the samples were blown dry with an ear bulb, and the samples were imaged using Multimode 8 (Bruker / USA) and ScanAsyst mode, and the data were analyzed using Nanoscopeanalysis software.

[0061] The results are as follows Figure 3 As shown, the scale bar is 1 μm (original image); 200 nm (enlarged view), Figure 3 It can be seen that from A to D are TDN0, TDN1, TDN2, and TDN3, and the sizes gradually increase.

[0062] 3) Dynamic light scattering:

[0063] Method: Take TDN0, TDN1, TDN2, and TDN3 (1 mL, 5 nM) sample solutions and place them in a dynamic light scattering detector for detection.

[0064] The results are as follows Figure 4 As shown, TDN0, TDN1, TDN2, and TDN3, the size of the tetrahedron increases gradually with the increase of the valence state.

[0065] In the following examples, Chlorella is selected as a representative of cell wall cells for illustration.

[0066] Example 1 Cellulose aptamer anchoring cellulose

[0067] 1) Synthesis of PASC (phosphoric acid swollen cellulose):

[0068] Take 0.1 g of microcrystalline cellulose and dissolve it in 2.5 mL of 85% ice-cold phosphoric acid. The dissolved cellulose is stirred at 4°C for 1 hour, 47.5 mL of cold water is added for precipitation, and then washed with 50 mL of ice water, 50 mL of 2 mM sodium bicarbonate solution and 250 mL of 50 mM sodium hydrogen phosphate buffer, and finally treated with a homogenizer to obtain PASC.

[0069] 2) PASC combined with MN:

[0070] Take 200 μL PASC, add cellulose aptamer MN to make the final concentration of MN 50 nM, add 5 mM Mg 2+ , incubate for 30 min, centrifuge, and observe the fluorescence of the precipitate. The control group is any single strand of DNA labeled with FAM.

[0071] The results are as follows Figure 5 As shown, the fluorescence reduction of the supernatant in the cellulose aptamer MN group was significantly higher than that in the control group, indicating that the cellulose aptamer can anchor cellulose well.

[0072] Example 2 Anchoring of MN on the cell wall surface

[0073] 1) Screening of the optimal incubation magnesium ion concentration for the combination of MN and Chlorella:

[0074] Chlorella was cultured in BG-11 medium at a concentration of 1 (OD 680 =1), take 100 μL of Chlorella, add MN to make the final concentration of MN 50 nM, set the magnesium ion concentration gradient to 0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM MgCl2, incubate for 30 min, centrifuge, take the supernatant, and measure the percentage of fluorescence reduction. The control group was without Chlorella.

[0075] The results are as follows Figure 6 As shown in A, when the magnesium ion concentration is 5 mM, the fluorescence reduction percentage is the highest, so the optimal magnesium ion concentration is 5 mM.

[0076] 2) Screening of the optimal incubation time for the combination of MN and Chlorella:

[0077] Take 100 μL of Chlorella vulgaris (OD 680 =1), add MN to make the final concentration of MN 50nM, add 5mM MgCl2. Incubate, centrifuge, take the supernatant, measure the percentage of fluorescence reduction, the control group is without Chlorella. Set the incubation time gradient to 5min, 15min, 25min, 35min.

[0078] The results are as follows Figure 6 As shown in B, when the incubation time was 35 min, the fluorescence reduction percentage was the highest, so the optimal incubation time was 35 min.

[0079] Example 3 Anchoring of multivalent sensor elements on the cell wall surface

[0080] 3) Anchoring efficiency of sensors with different valence states on the surface of Chlorella:

[0081] Take 100 μL of Chlorella vulgaris (OD 680 =1), TDN1, TDN2, and TDN3 were added respectively to make the final concentration of TDN 50 nM, 5 mM magnesium ion was added, and the mixture was incubated for 35 min. Centrifugation was performed, and the supernatant was taken to measure the percentage of fluorescence reduction. The control group was without adding Chlorella.

[0082] The results are as follows Figure 7 As shown, with the increase of cellulose aptamers, the percentage of fluorescence reduction gradually increases (the anchoring efficiency of the monovalent state is 59%, the anchoring efficiency of the divalent state is 67%, and the anchoring efficiency of the trivalent state is 71%). The sensor with the best binding efficiency is the trivalent state, that is, the trivalent nucleic acid nanosensor.

[0083] 4) Measurement of Zeta potential:

[0084] Chlorella, TDN3, Chlorella + Mg 2+ , Chlorella + Mg 2+ The potential of +TND3 is detected.

[0085] The results are as follows Figure 8 As shown, the potential of Chlorella is about -20mV, the potential of TDN3 is about -12mV, and the potential of Chlorella+Mg 2+ The potential is about -14mV, Chlorella + Mg 2+ The potential of +TND3 was about -13 mV, and the potential increased compared with that of Chlorella alone, indicating that TND3 could better anchor Chlorella.

[0086] 5) Laser confocal:

[0087] Take the Chlorella with different valence sensors (without quenching group Q) anchored on it, drop it on the slide, and observe the fluorescence intensity of Chlorella and sensor under 63 times oil lens. The instrument model used is Nikon AXR NSPARC super-resolution laser scanning confocal microscope, and the image analysis is performed using NIS-Elements Viewer software. No sensor is added to the control group.

[0088] The results are as follows Fig. 9 As shown, the fluorescence of the trivalent state sensor Cy3 channel is the strongest, indicating that the anchoring efficiency of TDN3 is the highest.

[0089] Example 4 Sensor response to ATP

[0090] 1) Sensor response to applied ATP:

[0091] Take the Chlorella with the trivalent sensor (bound to the quenching group Q) anchored, and add different concentration gradients of ATP, 200nM, 500nM, 1000nM, 2000nM. Drop it on the slide, and observe the fluorescence intensity of Chlorella and the sensor under the 63x oil lens. The instrument model used is Nikon AXR NSPARC super-resolution laser scanning confocal microscope, and the image analysis is performed using NIS-Elements Viewer software. No ATP is added to the control group.

[0092] The results are as follows Fig.10 As shown, the fluorescence of the Cy3 channel has recovered, and the higher the ATP concentration, the more obvious the fluorescence recovery is, which indicates that the nucleic acid sensor of the present invention can be better anchored on the surface of Chlorella and perform ATP monitoring.

[0093] 2) Sensor response to salt stress and release of ATP:

[0094] Take the Chlorella with the anchored trivalent sensor (bound to the quenching group Q), add NaCl to make the final concentration 200mM, drop it on the slide, and observe the fluorescence intensity of Chlorella and the sensor under a 63x oil lens. The instrument model used is a Nikon AXR NSPARC super-resolution laser scanning confocal microscope, and the image analysis is performed using NIS-Elements Viewer software. No NaCl was added to the control group.

[0095] The results are as follows Fig.11 As shown, the acquisition time for the same field of view is 1min, 2min, 5min, and 8min. As the acquisition time increases, the fluorescence of the Cy3 channel gradually recovers, indicating that under salt stress conditions, the nucleic acid sensor can still be better anchored on the surface of Chlorella for ATP monitoring.

[0096] In the present invention, a cellulose aptamer and an ATP aptamer are combined to obtain a multivalent in situ eATP sensor anchored on the cell wall. Using Chlorella as a cell wall plant model, the sensor can quickly and effectively detect eATP under 200mM salt stress conditions. Moreover, under 5mM magnesium ions and an incubation time of 35min, the sensor has the best anchoring efficiency on the surface of Chlorella, and sensors of different valence states have different anchoring efficiencies on algae, among which the trivalent state sensor has the highest anchoring efficiency.

[0097] Therefore, the present invention adopts the above-mentioned nucleic acid nanosensor for monitoring the dynamic changes of ATP outside living cells and its application. During detection, the extracellular ATP competes with the Q group to restore the fluorescence of Cy3. The result is visualized by the instrument, and the result is simple and convenient to read. At the same time, the metabolites are nucleotides, which are completely harmless to cells and have high biological safety. It is anchored on the cell wall for in situ detection, has good sensitivity and spatiotemporal stability, and has extremely high application value in the field of biological signal molecule sensing.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells, characterized in that: It includes a DNA tetrahedron framework formed by complementary bases of four DNA single strands, a cellulose aptamer and an ATP aptamer. One end of the DNA tetrahedron framework is connected to the ATP aptamer for sensing ATP, and the other ends are connected to the cellulose aptamer for anchoring the plant cell wall.

2. A nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells according to claim 1, characterized in that: The ATP aptamer is connected to the fluorescent molecule Cy3 through a covalent bond and to the fluorescent quencher chain through base complementary pairing. The fluorescent quencher chain contains the fluorescent quencher group Q.

3. A nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells according to claim 1, characterized in that: The cellulose aptamers are arranged in 1, 2 or 3 strips, and the remaining end points of the DNA tetrahedron framework are connected to 1 cellulose aptamer to form a monovalent nucleic acid nanosensor, the remaining end points of the DNA tetrahedron framework are respectively connected to 2 cellulose aptamers to form a divalent nucleic acid nanosensor, and the remaining end points of the DNA tetrahedron framework are respectively connected to 3 cellulose aptamers to form a trivalent nucleic acid nanosensor.

4. A nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells according to claim 1, characterized in that: The anchoring efficiency of nucleic acid nanosensors on the cell surface was 59%-71%.

5. A nucleic acid nanosensor for monitoring dynamic changes of ATP outside living cells according to claim 1, characterized in that: The synthesis method of the DNA tetrahedral framework is as follows: four single strands of the DNA tetrahedral framework are placed in 1×PBS buffer, then heated to 95°C for 8 to 10 minutes, and then the temperature is lowered to room temperature for 30 to 60 minutes to obtain the DNA tetrahedral framework.

6. Use of a nucleic acid nanosensor for monitoring dynamic changes of extracellular ATP according to any one of claims 1 to 5 in monitoring extracellular ATP, characterized in that: Nucleic acid nanosensors anchored outside plant cells with cell wall structures are used for in situ real-time monitoring of ATP.

7. The use according to claim 6, characterized in that: When used, the detection principle is: before the nucleic acid nanosensor detection, the quenching group Q connected to the ATP aptamer quenches the fluorescence of Cy3. During detection, ATP binds to the ATP aptamer, the quenching group Q is replaced, and the fluorescence of Cy3 is restored. The results are observed by fluorescence spectroscopy and laser confocal microscopy to monitor extracellular ATP.

8. Use of a nucleic acid nanosensor for monitoring dynamic changes of extracellular ATP in living cells as claimed in any one of claims 1 to 5 in monitoring extracellular ATP in Chlorella.

9. The use according to claim 8, characterized in that: When applied, the magnesium ion concentration was 2.5-7.5 mM, the incubation time was 5-35 min, and the OD of Chlorella vulgaris was 680 =1, final concentration of cellulose aptamer 50 nM, nucleic acid nanosensor anchored on the surface of Chlorella.

10. Use of a nucleic acid nanosensor for monitoring dynamic changes of extracellular ATP in living cells according to any one of claims 1 to 5 in monitoring extracellular ATP in Chlorella under salt stress.

Images