A system, method and device for detecting information of a target in a cell

Abstract

The present invention belongs to but is not limited to the field of medical detection technology, and particularly relates to a system, method and equipment for detecting information of a target in a cell, including: a glass capillary pretreatment module: the nano glass tube used is a borosilicate glass tube with a specification of BF100-58-10, and a capillary puller P-2000 is used to pull a nano pipette with a size of about 200 nanometers; a gold particle nanolayer is modified inside the tip of the nano pipette by a chloroauric acid reduction method; a detection probe design unit module: a controllable light-activated functional DNA probe containing a thiol bond is used to modify the functionalized DNA on the surface of the gold layer inside the nano pipette through the action of a gold-sulfur bond. Based on the probe design unit module, the detection system will feedback different information according to the detected object, thereby realizing the specific detection of the target in the cell; the detection unit module: electrochemical signal detection, fluorescence signal detection and dual signal collection are performed.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of medical detection technology, and in particular relates to a system, method and device for detecting information of a target in a cell. Background Art

[0002] Targets in cells, such as bioinformatic small molecules (ATP), proteins (cytochrome C), and ribonucleic acids (mircoRNA), contain important information, and the detection of this information is an important part of modern biomedical research. Its significance is far-reaching, covering multiple levels such as basic medical research and clinical application. Detection of intracellular targets plays an important role in disease diagnosis and treatment. Many diseases, especially cancer, cardiovascular disease, and neurodegenerative diseases, are often associated with abnormalities in specific target molecules. By detecting these targets, early diagnosis, prognosis assessment, and personalized treatment can be achieved. For example, microRNA (miRNA) is a short-chain (18-22 nucleotide) RNA molecule that participates in the post-transcriptional inhibition of non-coding RNA by mRNA, and is found to be dysregulated in many cancers. Among them, microRNA-21 is especially overexpressed in breast cancer and promotes tumor cell proliferation and metastasis by downregulating tumor suppressor factors5,6. The detection of microRNA-21 plays an increasingly important role in the early diagnosis of breast cancer patients. Its stable structure, specificity, and quantifiability make microRNA-21 an ideal biological marker for detecting breast cancer.

[0003] In addition, the development of targeted therapeutic drugs depends on the accurate identification of specific targets to achieve more effective treatment plans and improve the survival rate and quality of life of patients. Target detection in cells not only provides key tools for basic biological research, but also brings new opportunities for the innovation and development of clinical medicine. Its importance is becoming more and more prominent. In future scientific research and medical practice, target detection will continue to play an indispensable role. At present, there are a series of detection methods for intracellular targets, which mainly include fluorescent dye method, immunofluorescence method, flow cytometry and atomic force microscopy from the detection technology classification. However, the above methods have certain limitations. The fluorescent signal of the fluorescent dye method is easily disturbed by environmental factors, resulting in unstable results, and some fluorescent probes have toxic effects on cells, thereby changing the state of the cells themselves; while the immunofluorescence method requires fixed cells, which limits the real-time observation of dynamic processes; the flow cytometry method requires crushing cells, resulting in damage to cell structure or loss of function, which in turn affects the accuracy of the detection results. Therefore, we urgently need to develop a more adaptable micro-nano electrode information detection and analysis technology and equipment for cell targets.

[0004] In view of the above analysis, the technical problems that urgently need to be solved in the existing technology are: for the detection of intracellular targets, the fluorescent signal is greatly affected by environmental interference, unstable and sensitive to regulation, some fluorescent molecules are toxic to cells, and it is difficult to achieve in situ detection of living cells, which leads to false positive results in the detection of intracellular targets, resulting in medical accidents such as misdiagnosis or untimely medical treatment. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a system, method and device for detecting information of targets in cells.

[0006] The present invention is implemented as follows: a system for detecting information of a target in a cell, comprising:

[0007] Glass capillary pretreatment module: The nano glass tube used is a borosilicate glass tube with a specification of BF100-58-10, and a nanopipette with a size of about 200 nanometers is pulled out using a capillary puller P-2000; a gold particle nanolayer is modified inside the tip of the nanopipette by the chloroauric acid reduction method.

[0008] Detection probe design unit module: The controllable light-activated functional deoxyribonucleic acid (DNA) probe containing a thiol bond is modified on the gold layer surface of the nanopipette through the gold-sulfur bond. Based on the probe design unit module, the detection system will feedback different information according to the detected object, thereby realizing the specific detection of intracellular targets.

[0009] Detection unit module.

[0010] (1) Electrochemical signal detection.

[0011] The processed micro-nano electrodes were used to verify the intracellular electrochemical signals through a single-cell instrument; the nanoelectrodes were assembled in 200 mL of the test solution at 4°C overnight, the assembled gold electrodes were sealed with 6-mercapto-1-hexanol for an appropriate time, the micromanipulation system was adjusted, the optical fiber was brought close to the nanoelectrode, 365 nm ultraviolet light was introduced, the tip of the nanoelectrode was irradiated for an appropriate time, and the electrochemical signals at the tip of the nanoelectrode were collected.

[0012] (2) Fluorescence signal detection.

[0013] The processed micro-nano electrodes are used to verify the fluorescence signals in cells through the single-cell instrument. The test droplets are mixed and added to the confocal dish, and clear droplets are observed under a microscope. The position of the optical fiber is adjusted close to the droplets through the three-axis micromanipulation platform of the single-cell instrument. The magnification of the microscope is gradually adjusted to the appropriate magnification, and 365nm light is introduced through the optical fiber to irradiate the droplets. Data is collected at appropriate intervals, and the obtained data is processed and analyzed.

[0014] (3) Dual signal collection.

[0015] Insert the processed micro-nano electrode into the designated position of the cell, extract the cell fluid in a certain organelle, observe the fluorescence changes in the glass tube under a confocal microscope, and collect the current signal with a patch clamp after the reaction stabilizes to obtain the current signal of the target in the object to be tested.

[0016] Furthermore, in the entire detection system, a DNA probe with specific detection is constructed by processing the gold layer and the detection probe design unit module in the micro-nano electrode tube; and modifying the probe in the tube can effectively reduce the interference of the test liquid on the signal, and the nanometer size of the micro-nano probe can reduce the damage to the cells, ensure the activity of the cells, and realize in situ detection.

[0017] Furthermore, the glass capillary pretreatment module needs to adjust the parameters of the drawn capillary to ensure that the size of the capillary is below the nanometer scale to reduce damage to cells.

[0018] Furthermore, in the glass capillary pretreatment module, the thickness of the gold layer needs to be regulated by the reaction time and concentration of chloroauric acid.

[0019] The design of the fluorescent information molecule contained in the DNA in the detection probe design unit module requires regulation of the intensity of the activated laser.

[0020] Furthermore, in the detection unit module shown, the electrochemical processing unit mainly uses an electrochemical workstation and a three-electrode system to detect the electrochemical information in the cell. The 365nm laser is mainly introduced through optical fiber to activate the electrochemical signal probe at the tip of the nanoelectrode. The activated electrochemical signal probe combines with the target molecule in the cell to generate a corresponding electrochemical signal.

[0021] Furthermore, in the detection unit module shown, the fluorescent information molecules mainly activate the DNA probe at the tip of the micro-nano electrode through light in a specific band, and the fluorescent signal is collected using a single cell analyzer test system.

[0022] The tip of the micro-nano electrode modified with a designed DNA probe is inserted into the cell to ensure the activity of the cell and prevent it from being destroyed.

[0023] Another object of the present invention is to provide a method for detecting information of a target in a cell using the system for detecting information of a target in a cell, comprising:

[0024] S1, glass capillary pretreatment module, adjusts the preparation parameters of the capillary puller to pull a nanocapillary with an aperture of about 200 nanometers, and uses the chloroauric acid reduction method to modify a gold particle nanolayer inside the tip of the nanopipette.

[0025] S2, through the action of gold-sulfur bond, the controllable light-activated functional DNA probe is modified on the surface of the gold layer inside the nanopipette; the detection system will feedback different information according to the detected object to achieve specific detection of intracellular targets.

[0026] S3, detection unit module construction, inserting micro-nano electrodes into designated cells, introducing lasers through optical fibers to activate the electrochemical and fluorescent signal probes at the tip of the nanoelectrodes, and releasing electrochemical and fluorescent information when identifying targets in cells to complete target detection.

[0027] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for detecting information of a target in a cell.

[0028] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for detecting information of a target in a cell.

[0029] Another object of the present invention is to provide an information data processing terminal, which includes the information detection system for the target in the cell.

[0030] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are mainly reflected in the following aspects:

[0031] First, through the design of the detection unit module of the micro-nano electrode, the fluorescent signal and electrochemical signal molecules with controllable light activation function are modified on the nucleic acid probe. By regulating the laser source to activate different signal molecules, the electrochemical signal, fluorescent signal and dual signal can be output separately, and the method is more convenient and easier to operate. In addition, the conformational change of the nucleic acid probe after the functional nucleic acid probe binds to the target in the cavity of the confined space generates the nanopore blocking current and the change of the rectification signal, which will amplify the electrochemical signal and is more conducive to the output of information and accurate judgment.

[0032] The use of a capillary puller can make the capillary tip reach the nanoscale so that the tip can be used as a carrier to modify the fluorescent probe, which solves the problem of the fluorescent probe entering the cell. It does not rely on aptamers to enter the cell and does not harm the cell activity. It can realize the detection of target fluorescence signals of organelles below the cellular level such as the nucleus, Golgi apparatus, chloroplasts, etc.

[0033] The intracavity reaction in the confined space at the tip of the micro-nano electrode reduces signal interference and increases the reaction rate. The detection probe design unit in the confined space of the micro-nano tip can ensure effective binding with the target in the cell, avoiding the generation of interfering signals from different cell subregions. In the electrochemical signal test, the electroosmotic flow can extract the target information molecules in the subcellular region in situ into the confined cavity of the micro-nano electrode, effectively improving the reaction efficiency.

[0034] The technical solution of the present invention combines micro-nano electrode technology with intracellular target electrochemical detection technology and intracellular target fluorescence information detection technology to realize a detection method and test system for the simultaneous output of intracellular target electrochemical signals, fluorescence signals and dual signals by using a micro-nano electrode inner surface modified detection probe unit. It also ensures that the micro-nano electrode does not damage the cells and realizes the in-situ output of target information in living cells, which is of great significance for early screening of diseases and clinical medical diagnosis.

[0035] Second, the technical solution of the present invention overcomes technical prejudice: the invention of the present application can effectively ensure that cell activity is not destroyed, realize in situ detection of target signals in cells, and improve the effectiveness and timeliness of cell signal output. It can effectively overcome the apoptosis of cells caused by methods such as fluorescent dye method, immunofluorescence method, flow cytometry, etc., when the information in the cells cannot be effectively obtained. The method of obtaining cell fluid by crushing cells for detection will cause serious signal interference. The composite nucleic acid probe constructed by the present application, which is processed by electrochemical signal molecules and fluorescent signals, is modified at the tip of the micro-nanoscale capillary, and the nano-sized capillary tip entering the cell will not cause cell apoptosis, which can effectively reduce the specific recognition of probes and targets by multi-component cell fluids, and improve the accuracy of electrochemical and fluorescent signal release.

[0036] Third, the technical solution of the present invention solves the technical problems of the prior art.

[0037] 1) Insufficient sensitivity and specificity of intracellular target detection.

[0038] Problems with existing technologies: In existing methods for detecting intracellular targets, detection sensitivity and specificity are often limited, especially in complex cellular environments, where background noise and nonspecific signal interference reduce the accuracy of detection results.

[0039] The solution of the present invention is to modify the gold particle nanolayer in the nanoglass tube and connect the functionalized DNA probe thereto, which significantly improves the sensitivity and specificity of the detection system. The functionalized DNA probe can specifically bind to the target, reduce the signal interference caused by non-specific binding, and ensure the accuracy of the detection.

[0040] 2) The effect on cell viability and the lack of in situ detection.

[0041] Problems with existing technologies: Traditional intracellular detection technologies, especially when using larger probes or electrodes, are prone to damage cells and affect cell activity, thus limiting the accuracy and reliability of detection. At the same time, existing technologies are difficult to perform in situ detection without destroying cells.

[0042] Solution of the present invention: The nanometer-sized glass tube and probe design used in the present invention significantly reduce the mechanical damage to cells, maintain the activity of cells, and realize in-situ detection in living cells. Through the nanometer-sized design of the micro-nano probe, the probe can be more easily inserted into the cell, and the damage to the cell is minimized, realizing accurate detection in living cells.

[0043] 3) The versatility of the detection system and the signal collection methods are limited.

[0044] Existing technical problems: Many existing cell detection systems are usually limited to the detection of a single signal and cannot detect and analyze multiple signals at the same time. This limitation is particularly evident in cell research that requires multiple signal verification or in complex environments.

[0045] The solution of the present invention: The present invention designs a dual signal detection system that can collect electrochemical signals and fluorescent signals at the same time. By combining electrochemical detection and fluorescent detection, the detection system can analyze the information of the target in a complex environment in multiple dimensions. This dual signal collection method significantly expands the function of the detection system, enabling it to more comprehensively characterize the target in the cell.

[0046] The present invention achieves significant technical progress.

[0047] 1) Improved detection sensitivity and specificity.

[0048] The present invention successfully improves the sensitivity and specificity of the detection system by modifying gold particles and functionalized DNA probes inside the nanoglass tube, and can accurately identify and detect targets in a complex cell environment, thus solving the detection accuracy problem in the prior art.

[0049] 2) Realizes in situ detection of cells with low damage.

[0050] By using nanometer-sized micro-nano probes, the present invention can perform in-situ detection without significantly damaging cells, thus maintaining cell activity. This technological advancement makes it possible to perform precise in-situ detection in living cells, greatly improving the accuracy and reliability of detection.

[0051] 3) Expanded the functionality of the detection system.

[0052] The present invention significantly enhances the versatility of the system by designing a detection system with dual signal collection capabilities, which can simultaneously detect electrochemical and fluorescent signals. This multi-signal detection capability not only improves the accuracy of detection, but also expands the application range of the system in complex biological environments and provides more comprehensive target information.

[0053] 4) Simplified the detection process and enhanced system stability.

[0054] By modifying the gold layer and DNA probe inside the nano glass tube, the present invention simplifies the assembly and operation process of the detection system and reduces the potential sources of error in the detection step. In addition, the stability of the probe and the high sensitivity of the nano electrode ensure the long-term stability of the system, making it suitable for experimental requirements with high repeatability.

[0055] The present invention successfully solves the key problems of detection sensitivity, specificity, cell damage and multi-signal detection capability in the prior art through innovative nanoglass tube modification and functionalized DNA probe design, achieving significant technological progress. This technology has important application prospects and value in the fields of biomedical research and intracellular target detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a flow chart of the information detection method of the target in the cell provided by the embodiment of the present invention.

[0057] Figure 2 This is a schematic diagram of information detection of a target in a cell by a functional nucleic acid probe modified with light response activation in a micro-nano electrode provided by an embodiment of the present invention.

[0058] Figure 3 This is a FESEM image of the glass capillary of the micro-nano electrode provided in an embodiment of the present invention.

[0059] Figure 4 This is a FESEM image of a cross section of a glass capillary of a micro-nano electrode provided in an embodiment of the present invention.

[0060] Figure 5 This is a microscope image of the micro-nano electrode provided in an embodiment of the present invention detecting a target in a cell.

[0061] Figure 6 This is a microscope image of the fluorescent signal of the micro-nano electrode provided in the embodiment of the present invention detecting the target in the cell.

[0062] Figure 7 It is a data diagram of electrochemical information of intracellular target detection by the micro-nano electrode provided in an embodiment of the present invention.

[0063] Figure 8It is a structural diagram of the information detection system of the target in the cell provided by the embodiment of the present invention.

[0064] Fig. 9 It is a comparison diagram of the changes in electrochemical and fluorescent signals before and after the capillary tip modified with the designed nucleic acid probe provided by the example of the present invention is inserted. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0066] A new generation of information technology mentioned in the present invention, MicroRNA (miRNA) is a short-chain (18-22 nucleotide) RNA molecule that participates in the post-transcriptional inhibition of mRNA and non-coding RNA, and is found to be dysregulated in a variety of cancers. In particular, microRNA-21 is overexpressed in breast cancer and promotes tumor cell proliferation and metastasis by downregulating tumor 5,6 inhibitory factors. The detection of microRNA-21 plays an increasingly important role in the early diagnosis of breast cancer patients. Taking the detection of microRNA-21 in cells as an example, the following is a specific embodiment.

[0067] (1) Preparation of glass capillaries.

[0068] The nano glass tube used is a borosilicate glass tube with a specification of BF100-58-10, and a nanopipette with a size of about 200 nanometers is pulled using a capillary puller P-2000.

[0069] (a) Cleaning of sample tubes: Soak the sample tubes in anhydrous ethanol for 30 minutes in a fume hood, pour out the anhydrous ethanol, dry them in an oven at 70°C, and take them out after 25 minutes. Cut the sample tubes of both specifications into appropriate lengths.

[0070] (b) Soak the glass capillary tube in anhydrous ethanol and ultrasonicate for 30 minutes. Then soak it in piranha solution (98% H2SO4:30% H2O2=3:1) for 2 hours to remove organic matter on the surface of the glass tube and hydroxylate the surface of the glass tube.

[0071] (c) Wash with secondary water several times until it is neutral and the aqueous solution does not remain in the strands, then wash with anhydrous ethanol several times, put it in anhydrous ethanol, ultrasonicate for 30 minutes, and dry it in a 70℃ oven before use.

[0072] (d) Adjust the parameters of the laser capillary drawing instrument to draw nanometer diameter capillaries.

[0073] (2) Modification of detection probes inside nanoglass capillaries.

[0074] (a) A gold nanopipette tip was modified with a gold nanoparticle layer by chloroauric acid reduction.

[0075] (b) Leave the tube at room temperature for 30 min to allow the chloroauric acid solution to fully react with the tube wall. Take out the solution with a pipette, rinse it with anhydrous ethanol several times, and place it in a 70°C oven to dry.

[0076] (c) A series of light-controlled functional nucleic acid recognition units that can specifically interact with the target substance microRNA-21 are designed. According to the principle of base complementary pairing, the nucleic acid design software (NUPACK software) is used to design the series of functional nucleic acid recognition units.

[0077] (d) A liquid containing the designed detection DNA probe is added into the nano glass capillary. Through the interaction between the thiol bonds of the gold particles and DNA, the designed DNA probe is modified inside the glass capillary to form a micro-nano electrode test unit.

[0078] (3) Use micro-nano electrodes to detect electrochemical information within cells.

[0079] Insert the micro-nano electrode into the cell to be detected, and activate the designed detection DNA composite probe by irradiating it with light of a specific wavelength. After the probe is combined with the target, the structure of the composite DNA probe modified in the tip of the glass capillary changes, causing the current signal to change, thereby outputting the changed electrochemical signal. At this time, the rectification ratio data information of the electrochemical signal can be used to quantify the microRNA-21 in the cell.

[0080] (4) Use micro-nano electrodes to detect fluorescence information inside cells.

[0081] Using a micromanipulation system that is adjustable all around and equipped with a stepper motor with an accuracy of 20nm, the micro-nano electrodes are precisely inserted into the specific measurement area of ​​a single cell. The micro-nano electrodes can be precisely positioned in a specific subcellular area and then the microRNA-21 signal is extracted in situ using electroosmotic flow. The fluorescence changes in the glass tube are observed under a confocal microscope. After the reaction is stable, the fluorescence information in the glass tube is collected using a confocal microscope to evaluate the intensity of the fluorescence signal in the cell and the microRNA-21 content in the cell.

[0082] like Figure 1 As shown, the present invention provides a method and system for detecting information of intracellular targets by using a functional DNA composite probe modified in a micro-nano electrode, and the preparation method comprises the following steps:

[0083] Step 1: In the glass capillary pretreatment module, the preparation parameters of the capillary puller are adjusted to pull a nanocapillary with an aperture of about 200 nanometers, and a gold particle nanolayer is modified inside the tip of the nanopipette using the chloroauric acid reduction method.

[0084] Step 2: Through the gold-sulfur bond, a controllable light-activated functional DNA probe is modified on the gold layer surface of the nanopipette. The detection system will feedback different information according to the detected object to achieve specific detection of intracellular targets.

[0085] Step three: construct the detection unit module, insert the micro-nano electrode into the designated cell, introduce the laser through the optical fiber to activate the electrochemical and fluorescent signal probes at the tip of the nanoelectrode, and release the electrochemical and fluorescent information when identifying the target in the cell to complete the target detection.

[0086] like Figure 2 As shown, a schematic diagram of a method and system for detecting information of a target in a cell by modifying a functional nucleic acid probe activated by light response in a micro-nano electrode provided by an embodiment of the present invention; the present invention modifies two functional molecules with electrochemical signals and fluorescent signals on nucleic acid molecules through DNA probe design, and modifies the designed DNA molecules on the tip of a glass capillary of a micro-nano result. The micro-nano electrode is inserted into the cell without damage, and 365nm light is introduced to the surrounding of a designated single cell through a micro-nano optical fiber to selectively activate the electrochemical signal functional nucleic acid probe in the single cell, identify the target unit, and express the electrochemical signal and fluorescent signal.

[0087] Figure 3 This is a FESEM image of a glass capillary of a micro-nano electrode provided in an embodiment of the present invention; the glass capillary is successfully drawn by adjusting the drawing parameters in a capillary drawer to obtain a glass capillary tip with a micro-nano structure. As can be seen from the figure, the tip of the glass capillary is flat, and gold nanoparticles can be modified on the inner surface of the capillary tip by chloroauric acid reduction, and the designed DNA molecular chain can be successfully modified on the inner surface of the tip by capillary action, thereby realizing the preparation of a micro-nano electrode.

[0088] Figure 4 This is a FESEM image of a cross section of a glass capillary of a micro-nano electrode provided in an embodiment of the present invention; by adjusting the drawing parameters, the controllable preparation of the tip aperture of the glass capillary is achieved. The tip aperture size affects the deposition of gold particles on the inner wall of the tip and the modification of the designed DNA probe. When piercing into cells, it also affects cell activity. The smaller the aperture of the tip, the less damage it causes to cells, but the small aperture increases the difficulty of DNA probe modification. Therefore, the tip aperture size is an important factor in balancing the difficulty of DNA probe modification and cell activity.

[0089] Figure 5This is a microscope image of the detection of targets in cells by the micro-nano electrodes provided in the embodiment of the present invention; as shown in the figure, the micro-nano electrodes are inserted into cells in the single-cell instrument operating system without damaging the cells, thereby realizing the output of in-situ information of the cells. The designed DNA molecular probes are used to identify the targets inserted into the cells, and two corresponding signals are obtained using the receiving devices of the electrochemical signals and the fluorescent signals.

[0090] Figure 6 This is a microscope image of the fluorescent signal of the micro-nano electrode provided by an embodiment of the present invention detecting a target in a cell; as can be seen from the figure, in the single-cell instrument operating system, the micro-nano electrode is inserted into a specified cell, and a 365nm laser is introduced through a tiny optical fiber to activate the DNA fluorescent signal probe at the tip of the nanoelectrode, which releases a fluorescent signal after identifying the target in the cell.

[0091] Figure 7 This is a data graph of electrochemical information of intracellular target detection by the micro-nano electrodes provided by the embodiment of the present invention. The figure shows the electrochemical signals obtained by inserting the micro-nano electrodes of the blank control group and the experimental group into the cells, and introducing the electrochemical signal probe at the tip of the 365nm laser-activated nanoelectrode through the optical fiber. Compared with the electrochemical signal of the control group, the current of the micro-nano electrodes of the experimental group changed after the probe was activated by ultraviolet light after being inserted into the cells; when there was no laser activation of the probe, it was just identifying the target, and the current also had a slight change; however, when the laser activated the probe to identify a specific target, the electrochemical signal would change significantly, thereby obtaining clear electrochemical information.

[0092] like Figure 8 As shown, an embodiment of the present invention provides a system for detecting information of a target in a cell, comprising:

[0093] Glass capillary pretreatment module: The nano glass tube used is a borosilicate glass tube with a specification of BF100-58-10, and a capillary puller P-2000 is used to pull a nanopipette with a size of about 200 nanometers; a gold particle nanolayer is modified inside the tip of the nanopipette by the chloroauric acid reduction method.

[0094] Detection probe design unit module: The controllable light-activated functional DNA probe containing thiol bonds is modified on the gold layer surface of the nanopipette through the gold-sulfur bond. Based on the probe design unit module, the detection system will feedback different information according to the detected object, thereby realizing the specific detection of intracellular targets.

[0095] Detection unit module:

[0096] (1) Electrochemical signal detection.

[0097] The processed micro-nano electrodes were used to verify the intracellular electrochemical signals through a single-cell instrument; the nanoelectrodes were assembled in 200L of test solution at 4°C overnight, the assembled gold electrodes were sealed with 6-mercapto-1-hexanol for an appropriate time, the micromanipulation system was adjusted, the optical fiber was brought close to the nanoelectrode, 365nm ultraviolet light was introduced, the tip of the nanoelectrode was irradiated for an appropriate time, and the electrochemical signals at the tip of the nanoelectrode were collected.

[0098] (2) Fluorescence signal detection.

[0099] The processed micro-nano electrodes are used to verify the fluorescence signals in cells through the single-cell instrument. The test droplets are mixed and added to the confocal dish, and clear droplets are observed under a microscope. The position of the optical fiber is adjusted close to the droplets through the three-axis micromanipulation platform of the single-cell instrument. The magnification of the microscope is gradually adjusted to the appropriate magnification, and 365nm light is introduced through the optical fiber to irradiate the droplets. Data is collected at appropriate intervals, and the obtained data is processed and analyzed.

[0100] (3) Dual signal collection.

[0101] Insert the processed micro-nano electrode into the designated position of the cell, extract the cell fluid in a certain organelle, observe the fluorescence changes in the glass tube under a confocal microscope, and collect the current signal with a patch clamp after the reaction stabilizes to obtain the current signal of the target in the object to be tested.

[0102] An application embodiment of the present invention provides a computer device, which includes a memory and a processor. The memory stores a computer program. When the computer program is executed by the processor, the processor executes the steps of a method for detecting information of a target in a cell.

[0103] An application embodiment of the present invention provides a computer-readable storage medium storing a computer program. When the computer program is executed by a processor, the processor executes the steps of a method for detecting information of a target in a cell.

[0104] An application embodiment of the present invention provides an information data processing terminal, which includes an information detection system for a target in a cell.

[0105] The present invention relates to the construction of a system, method and equipment for detecting information of a target in a cell. Detection technology plays an important role in the development of new drugs. It not only provides a scientific basis for the effectiveness and safety of drugs, but also greatly improves the efficiency of research and development. The detection technology of the present application can be applied to the drug screening stage. In this step, researchers can use the detection technology of the present application to dynamically detect the content of the target to enter a large number of candidate compounds into cells, and determine their potential biological activity. Through the high-throughput detection and screening technology of the present application, researchers can test thousands of compounds at the same time, thereby quickly identifying drugs with therapeutic potential. The application of this technology significantly reduces the research cycle and lays the foundation for subsequent drug optimization.

[0106] The technology of this invention has outstanding performance in the application field of early screening of clinical diseases. With the continuous advancement of medical science and technology, the role of detection technology in early screening of diseases is becoming increasingly important. Early screening can effectively detect potential diseases, thereby improving the success rate of treatment, reducing mortality, and improving the quality of life of patients. The detection technology of this application is modified on the tip of the capillary by designing a nucleic acid probe unit, which can enter the cell without damaging the cell, quickly identify the target and release multiple signals such as electrochemistry and fluorescence. The signal receiving system can amplify the signal in the cell, thereby achieving effective and accurate screening of early diseases. For example, the detection of the microRNA-21 target in the example is an ideal biological marker for detecting breast cancer.

[0107] In the combined example, the present application designs a nucleic acid probe to be modified on the inner surface of the capillary tip, and uses the three-axis micromanipulation system of the single-cell instrument to adjust the position of the laser fiber to penetrate the cell close to the capillary tip, and introduces 365nm light to the nucleic acid probe through the optical fiber to activate the electrochemical and fluorescent signal molecules, and the data can be detected ( Fig. 9 ) It can be seen that after the fluorescent signal molecule is activated, the nucleic acid probe will emit a fluorescent signal after binding to the intracellular target, and the corresponding fluorescent signal can be obtained through the signal receiving system; at the same time, the electrochemical signal molecule is also activated. Under ultraviolet light, the current between the nucleic acid probe and the intracellular target will change significantly, and a clear electrochemical signal can be obtained through the signal receiving system. Therefore, combining the two types of electrochemical and fluorescent information can accurately determine the content of the target in living cells, thereby determining the lesions in the cells, achieving early screening of the disease, early detection and early treatment, and reducing the economic burden of patients' medical costs and doctors' diagnostic costs.

[0108] It should be noted that the embodiments of the present invention can be implemented by hardware, software, or a combination of software and hardware. The hardware part can be implemented using dedicated logic; the software part can be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated design hardware. It can be understood by a person of ordinary skill in the art that the above-mentioned devices and methods can be implemented using computer executable instructions and / or contained in a processor control code, such as a carrier medium such as a disk, CD or DVD-ROM, a programmable memory such as a read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. Such code is provided on the carrier medium. The device and its modules of the present invention can be implemented by hardware circuits such as very large-scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable hardware devices such as field programmable gate arrays, programmable logic devices, etc., can also be implemented by software executed by various types of processors, and can also be implemented by a combination of the above-mentioned hardware circuits and software, such as firmware.

[0109] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A system for detecting information of a target in a cell, characterized in that: The gold nanopipette was used to modify the gold nanoparticle layer by chloroauric acid reduction, and the functionalized thiol bond DNA probe was modified on the gold layer surface by gold-sulfur bond connection inside the nanopipette. Combined with electrochemical and fluorescence signal detection, a micro-nano electrode system capable of specifically detecting intracellular targets was constructed; including: Glass capillary pretreatment module: The nano glass tube used is a borosilicate glass tube with a specification of BF100-58-10, and a nano pipette with a size of 200 nanometers is pulled out by a capillary puller P-2000; a gold particle nanolayer is modified inside the tip of the nano pipette by chloroauric acid reduction method; Detection probe design unit module: a controllable light-activated functional DNA probe containing a thiol bond is used to modify the functionalized DNA on the surface of the gold layer inside the nanopipette through the action of gold-sulfur bonds; based on the probe design unit module, the detection system will feedback different information according to the detected object, thereby realizing the specific detection of intracellular targets; Detection unit module: (1) Electrochemical signal detection The processed micro-nano electrodes were used to verify the intracellular electrochemical signals through a single cell analyzer; the nanoelectrodes were assembled in 200L of the test solution at 4°C overnight, the assembled gold electrodes were sealed with 6-mercapto-1-hexanol for a suitable time, the micromanipulation system was adjusted, the optical fiber was brought close to the nanoelectrode, 365nm ultraviolet light was introduced, the tip of the nanoelectrode was irradiated for a suitable time, and the electrochemical signals of the tip of the nanoelectrode were collected; (2) Fluorescence signal detection Through the single cell instrument, the processed micro-nano electrodes are used to verify the fluorescence signal in the cells; the test droplets are mixed and added to the confocal dish, and clear droplets are observed under the microscope. The position of the optical fiber is adjusted close to the droplets through the three-axis micro-manipulation platform of the single cell instrument, and the magnification of the microscope is gradually adjusted to the appropriate multiple. 365nm light is introduced through the optical fiber to irradiate the droplets, and data is collected at appropriate intervals, and the obtained data is processed and analyzed; (3) Dual signal collection Insert the processed micro-nano electrode into the designated position of the cell, extract the cell fluid in a certain organelle, observe the fluorescence changes in the glass tube under a confocal microscope, and collect the current signal with a patch clamp after the reaction stabilizes to obtain the current signal of the target in the object to be tested.

2. The information detection system for a target in a cell according to claim 1, characterized in that: In the entire detection system, a DNA probe with specific detection is constructed by processing the gold layer and the detection probe design unit module in the micro-nano electrode tube; modifying the probe inside the tube can effectively reduce the interference of the test liquid on the signal, and the nanometer size of the micro-nano probe can reduce the damage to the cells, ensure the activity of the cells, and realize in situ detection.

3. The information detection system for a target in a cell according to claim 1, characterized in that: The glass capillary pretreatment module needs to adjust the parameters of the drawn capillary to ensure that the size of the capillary is below the nanometer scale to reduce damage to cells.

4. The information detection system for a target in a cell according to claim 1, characterized in that: In the glass capillary pretreatment module, the thickness of the gold layer needs to be regulated by the reaction time and concentration of chloroauric acid; The DNA in the detection probe design unit module contains the design of fluorescent information molecules.

5. The information detection system for a target in a cell according to claim 1, characterized in that: In the detection unit module shown, the electrochemical processing unit mainly uses an electrochemical workstation and a three-electrode system to detect intracellular electrochemical information. The 365nm laser is mainly introduced through optical fiber to activate the electrochemical signal probe at the tip of the nanoelectrode. The activated electrochemical signal probe combines with the target molecule in the cell to generate a corresponding electrochemical signal.

6. The information detection system for a target in a cell according to claim 1, characterized in that: In the detection unit module shown, the fluorescent information molecules mainly activate the DNA probe at the tip of the micro-nano electrode through light in a specific wavelength band, and the fluorescent signal is collected by the single cell analyzer test system; The tip of the micro-nano electrode modified with a designed DNA probe is inserted into the cell to ensure the activity of the cell and prevent it from being destroyed.

7. A method for detecting information of a target in a cell using the system for detecting information of a target in a cell as claimed in any one of claims 1 to 6, characterized in that: include: S1, glass capillary pretreatment module, adjusts the preparation parameters of the capillary puller to pull a nanocapillary with an aperture of 200 nanometers, and uses the chloroauric acid reduction method to modify the inner part of the tip of the nanopipette with a nanolayer of gold particles; S2, through the action of gold-sulfur bonds, a controllable light-activated functional DNA probe is modified on the surface of the gold layer inside the nanopipette; the detection system will feedback different information according to the detected object to achieve specific detection of intracellular targets; S3, detection unit module construction, inserting micro-nano electrodes into designated cells, introducing lasers through optical fibers to activate the electrochemical and fluorescent signal probes at the tip of the nanoelectrodes, and releasing electrochemical and fluorescent information when identifying targets in cells to complete target detection.

8. A computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to execute the steps of the method for detecting information of a target in a cell as claimed in claim 7.

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