Single molecule scanning, capturing and detecting control method

Abstract

The invention relates to a single molecule scanning, capturing and detecting control method, and belongs to the field of measurement and analysis. According to the method, a nanoscale glass microtube approaches the surface of a sample, and the distance between a probe and the sample is controlled by monitoring the current change in real time. The method comprises the following specific steps: preparation work, initialization of a nano control platform, approaching of a probe to a sample liquid level, approaching of the probe to a sample surface (coarse probe insertion), and approaching of the probe to the sample surface (fine probe insertion). The method can realize rapid capture of a single molecule and accurate detection of via hole current, realizes rapid scanning of sample molecule plane distribution and structure, can longitudinally capture and control a single long-chain molecule, feeds back blocking current of the long-chain molecule in a glass nanopore in real time, realizes structural feature detection of the single long-chain molecule, and has a good application prospect. The detection method can be used for detection of long-chain DNA and RNA molecules. The method has the advantages of simplicity in operation, high detection precision and the like, and has a wide application prospect in the fields of biomolecule detection, single molecule research and the like.

Description

Technical Field

[0001] The invention belongs to the field of measurement and analysis, and relates to a single molecule scanning, capturing and detecting manipulation method. Background Art

[0002] Single molecule detection technology is a technology that can observe, detect and analyze single biological molecules. Traditionally, many biological measurement methods are based on collective measurements, that is, measuring and averaging a large number of molecules. Single molecule detection technology allows researchers to directly observe and analyze the characteristics and reaction processes of individual molecules, thereby providing more detailed and accurate biomolecular information. Compared with traditional collective measurement methods, single molecule detection technology is based on a high-sensitivity, high-resolution dynamic monitoring method to detect and record the signals of individual molecules.

[0003] Scanning ion conductance microscopy (SICM) is an important tool for single-molecule detection. This technology uses nano-glass tubes as probes. When the glass tube probe approaches the sample surface and scans, the surface morphology of the sample to be tested is characterized by real-time recording of the change in conductivity between the electrode in the glass tube and the reference electrode in the electrolytic cell. However, due to the immature negative feedback technology of early SICM, the probe was often broken during the scanning process. In 2009, the jump mode was invented to scan samples with high aspect ratios, but observing the change in sample current is still a great challenge for researchers. When the sample current approaches the threshold, the imaging of the sample will be clearer. Therefore, it is of great significance to study an automatic recognition method for controlling the approach, capture and detection current judgment of the scanning probe, which can improve the resolution of the scanning ion conductivity microscope and realize the capture and feature detection of single molecules. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a single molecule scanning, capture and detection manipulation method. Through the program method and equipment described in this patent, it is possible to quickly capture a single molecule and accurately detect the through-hole current, quickly scan the planar distribution and structure of the sample molecule, capture and manipulate a single long-chain molecule longitudinally, and provide real-time feedback on the blocking current of the long-chain molecule in the glass nanopore, and realize the detection of the structural characteristics of a single long-chain molecule, such as the detection of long-chain DNAF molecules and RNA molecules.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A single molecule scanning, capturing and detecting manipulation method comprises the following steps:

[0007] Preparation: Pull a nanometer-scale glass microtube and inject a solution of specific concentration into the sample cell. Install the glass microtube on top of the nano-control platform and place the electrode in the solution.

[0008] Initialize the nano-control platform: Start the initialization program, select the port parameters, initialize, and record the current nano-stage control axis.

[0009] The probe approaches the sample liquid surface: start the program, adjust the moving distance and speed parameters of the micro-motion stage, the acquisition card begins to collect current and display it in real time. When the real-time current is greater than the initial current, the micro-motion stage stops moving and records the current at this time.

[0010] The probe approaches the sample surface and performs the rough insertion step: adjust the stepping distance of the micro-motion console and the nano-motion console, start the program, and the acquisition card begins to collect and record the current. After the nano-stage descends the set distance, if the current change is greater than the set threshold, the rough insertion step ends; if the target is not achieved, call back the nano-stage and descend the micro-motion stage by the set distance, and repeat the above steps until the target is achieved.

[0011] The probe approaches the sample surface and performs fine needle insertion steps: adjust the micro-stage distance according to the rough needle insertion steps and return the nano-stage to zero, reduce the nano-stage step distance and start needle insertion, the acquisition card collects current, stops the nano-stage movement when the current approaches the threshold, and records the current current and distance.

[0012] Furthermore, when the probe approaches the sample liquid surface, when the real-time current is greater than the initial current, the micro-motion stage stops moving and records the current at this time, and the current is used as a basis for judging whether the probe has entered the liquid surface in subsequent steps.

[0013] Furthermore, in the thick needle insertion step, the threshold is set to the absolute value of the current change.

[0014] Furthermore, in the fine needle insertion step, the current and distance are used as the distance between the probe and the sample surface.

[0015] Furthermore, in the fine needle insertion step, a PID control algorithm is used to adjust the stepping distance of the nanostage to improve the accuracy of sample detection.

[0016] Furthermore, in the fine needle insertion step, when the current exceeds a threshold value, the movement of the nanostage is stopped, and the current and distance at this time are recorded to prevent the probe from being too close to the sample surface and causing damage.

[0017] The beneficial effects of the present invention are:

[0018] (1) The present invention combines a piezoelectric drive platform and a three-dimensional micrometer motion platform, and the two platforms can be controlled separately in the same program;

[0019] (2) The current value can be monitored and recorded in real time, making it easier for experimenters to understand the relative position between the current probe and the sample.

[0020] (3) The probe position can be set and the position data of the probe when it enters the sample pool and approaches the sample can be recorded, which is convenient for parameter setting in the next experiment.

[0021] (4) The present invention combines a PID control algorithm. After the current threshold generated when the nanotube approaches the sample is calculated by a formula, the result will be input into the PID algorithm. By adjusting the PID parameters to ensure that no overshoot occurs, the accuracy of sample detection can be further improved.

[0022] (5) When the probe enters the sample pool and approaches the sample surface, the present invention can automatically detect an excessive current gap and stop immediately, thereby facilitating the experimenter to make the next parameter adjustment. In addition, the tool can also stop the probe at an appropriate position to prevent it from getting too close to the glass tube and causing damage, or from being too far away and affecting the accuracy of the detection.

[0023] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 It is a schematic diagram of the system structure of the present invention;

[0026] Figure 2 This is a schematic diagram of the probe entering the sample cell;

[0027] Figure 3 This is a flow chart of the first stage of the procedure of the present invention;

[0028] Figure 4 This is a flow chart of the second stage of the program of the present invention.

[0029] Figure numerals: nano-scale glass microtube 1, piezoelectric drive platform 2, current amplifier 3, three-dimensional micrometer motion stage 4, control system 5, computer and control software 6, glass nanotube 7, current feedback circuit 8, sample pool 9. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0031] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0032] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0033] like Figure 1 As shown, in order to realize the scanning, capturing and detection manipulation of single molecules, the present invention designs a single molecule scanning, capturing and detection manipulation device based on a piezoelectric drive platform and a low-noise current detection probe. The device structure includes a nano-scale glass microtube 1, a piezoelectric drive platform 2, a current amplifier 3, a three-dimensional micron motion stage 4, a control system 5, a computer and control software 6. The present invention focuses on the control method for realizing single molecule scanning, capturing and detection in the control software.

[0034] like Figure 2 The schematic diagram of the nanotube entering the sample pool is shown in FIG. The present invention is realized by the following design scheme:

[0035] (1) A glass microtube 1 is drawn and a buffer solution is injected, which is then mounted on the upper three-dimensional micrometer motion stage 4 through a glass microtube holder and connected to an upper electrode, which is connected to a feedback current.

[0036] By setting electrodes in a glass microtube filled with a buffer solution and a sample cell filled with a configuration solution, we can generate an ion current. This distance-dependent current signal can be used as a feedback signal to control the distance between the tip of the glass tube and the sample cell. When the nanotube approaches the sample surface, the substrate hinders the flow of ions, thereby reducing the current intensity. After passing through the current amplifier, we are able to achieve non-contact control between the tip of the nanoglass tube and the sample. At the same time, by combining the glass tube resistance and contact resistance and calculating the ion current, we can determine a current drop threshold, thereby effectively controlling the position of the glass tube. This method ensures that the sample or nanoglass tube is not damaged during the experiment and can improve the measurement accuracy.

[0037] (2) Design and purchase a piezoelectric drive platform 2. Place a sample pool on the piezoelectric drive platform 2, and put a solution and a sample to be tested into the pool.

[0038] The piezoelectric drive platform 2 mainly has Z-axis control performance, and a sample pool is installed above the platform. It is mainly responsible for realizing fine needle insertion for sample approach after the three-dimensional micron motion platform 4 completes entering the liquid surface and completing the work of approaching the sample.

[0039] (3) Design and purchase a three-dimensional micrometer motion stage 4. The three-dimensional micrometer motion stage 4 has a glass microtube 1 installed on it and a piezoelectric drive stage 2 at the bottom.

[0040] The three-dimensional micrometer motion stage 4 has three-axis control performance, which is mainly used to adjust the XY axis position of the piezoelectric drive platform 2, and is responsible for position control when the probe has not yet entered the liquid surface and when it enters the liquid surface and approaches the sample. By cooperating with the piezoelectric drive platform, more accurate single molecule detection can be achieved.

[0041] (4) Prepare the solution and place it in the sample cell.

[0042] The solution in the experimental sample pool is 2 mol lithium chloride solution and 10 mmol Tris buffer with a pH of 7.4. The reason is that when potassium chloride is used for the experiment, crystals may precipitate, but the conductivity of lithium chloride with a lower concentration is insufficient, so 2 mol lithium chloride solution is used for the experiment.

[0043] (5) Design computer software to control the entire single-molecule detection process.

[0044] The computer software includes a processing software module that can control the piezoelectric drive platform and enable it to move along the z-axis; a three-dimensional micrometer motion stage module that enables it to achieve three-axis motion; a current detection module that enables it to detect current changes in real time; and a central control system module that uses obvious current changes found after current detection as a reference for nano-stage control and piezoelectric stage control, thereby forming a more accurate single-molecule scanning, capture and detection control method.

[0045] The single molecule detection tool equipment and method include nano-scale glass microtube 1, piezoelectric drive platform 2, current amplifier 3, three-dimensional micrometer motion platform 4, control system 5, computer and control software 6. The system provides a new single molecule detection method, using nano glass tube to approach the test sample to control the movement of the experimental platform according to the feedback current, so that the single molecule detection greatly saves time and greatly shortens the detection time, and the current information can be recorded in real time on the system.

[0046] Reference Figure 3 Including preparation work, initializing the nano control platform and the control platform to allow the probe to enter the sample pool liquid surface, including the following steps

[0047] S1: Pull a 10nm nano-glass microtube and inject a 2mol LiCl solution into the sample cell. Install the glass microtube on the nano-control platform and place the electrode in the solution.

[0048] S2: Start the initialization program of the nano-control platform, initialize after selecting the port parameters, and record the current nano-stage control axis.

[0049] S3: The first stage of the probe approaches the sample liquid surface: start the program, at this time the initial current I0 is approximately equal to zero, adjust the moving distance and speed parameters of the micro-motion stage, and the motor starts. At this time, the acquisition card starts to collect current and displays it in real time. The current collected by the acquisition card is recorded as I1. When the real-time current I1 is greater than the initial current I0, it means that the probe has entered the liquid surface, the micro-motion stage stops moving and records the current I1 at this time. The first stage ends.

[0050] Reference Figure 4 The probe approaches the experimental sample from the liquid surface and records the detection current, including the following steps:

[0051] S1: In the second stage, the probe approaches the sample surface from the liquid surface (coarse needle insertion): adjust the stepping distance h of the micro-motion console and the nano-motion console, start the program, and the acquisition card begins to collect current and record it as I2. After the nano-stage descends a distance of h, if |I2-I1|>Ith, the coarse needle insertion step is ended. If the goal is not achieved, call back the nano-stage and descend the micro-motion stage a distance of h, and then repeat the above steps until the goal is achieved.

[0052] S2: The second stage of the probe approaches the sample surface from the liquid surface (fine needle insertion): adjust the micro-stage distance according to S1 and return the nano-stage to zero, reduce the nano-stage step distance and start needle insertion, and the acquisition card collects current. When the current approaches the threshold, the nano-stage stops moving. The current current and distance are recorded, and the fine needle insertion is completed.

[0053] 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 the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A single molecule scanning, capture and detection manipulation method, characterized in that: The following steps are involved: Preparation: Pull a nanometer-scale glass microtube and inject a solution of specific concentration into the sample cell. Install the glass microtube on top of the nano-control platform and place the electrode in the solution. Initialize the nano-control platform: Start the initialization program, select the port parameters, initialize, and record the current nano-stage control axis. The probe approaches the sample liquid surface: start the program, adjust the moving distance and speed parameters of the micro-motion stage, the acquisition card begins to collect current and display it in real time. When the real-time current is greater than the initial current, the micro-motion stage stops moving and records the current at this time. The probe approaches the sample surface and performs the rough needle insertion step: adjust the stepping distance of the micro-motion console and the nano-motion console, start the program, and the acquisition card starts to collect and record the current. After the nano-stage descends the set distance, if the current change is greater than the set threshold, the rough needle insertion step ends; If the target is not reached, the nanostage is called back and the microstage is lowered by a set distance, and the above steps are repeated until the target is reached. The probe approaches the sample surface and performs fine needle insertion steps: adjust the micro-stage distance according to the rough needle insertion steps and return the nano-stage to zero, reduce the nano-stage step distance and start needle insertion, the acquisition card collects current, stops the nano-stage movement when the current approaches the threshold, and records the current current and distance.

2. A single molecule scanning, capture and detection manipulation method according to claim 1, characterized in that: When the probe approaches the sample liquid surface, when the real-time current is greater than the initial current, the micro-motion stage stops moving and records the current at this time, which is used as a basis for judging whether the probe has entered the liquid surface in subsequent steps.

3. A single molecule scanning, capture and detection manipulation method according to claim 1, characterized in that: In the thick needle insertion step, the threshold is set to the absolute value of the current change.

4. A single molecule scanning, capture and detection manipulation method according to claim 1, characterized in that: In the fine needle insertion step, the current and distance are used as the distance between the probe and the sample surface.

5. A single molecule scanning, capture and detection manipulation method according to claim 1, characterized in that: In the fine needle insertion step, a PID control algorithm is used to adjust the stepping distance of the nanostage to improve the accuracy of sample detection.

6. A single molecule scanning, capture and detection manipulation method according to claim 1, characterized in that: In the fine needle insertion step, when the current exceeds a threshold value, the movement of the nanostage is stopped, and the current and distance at this time are recorded to prevent the probe from being too close to the sample surface and causing damage.

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