Single-molecule periodic pore modulation and signal detection device based on nanopore technology

Abstract

The present invention discloses a single-molecule periodic through-hole modulation and signal detection device based on nanopore technology. The device includes a graphene nanopore chip, a microfluidic top layer, a microfluidic bottom layer, a control circuit and a detection circuit. In the graphene nanopore chip, the graphene layer is suspended between the first support part and the second support part, and a nano-through hole is provided in the middle of the suspended area; the microfluidic top layer includes an inlet flow channel, an inlet microvalve and a first groove, and the bottom layer includes a second groove, an outlet flow channel and an outlet microvalve. The second groove and the first groove define a space for accommodating the sample solution to be tested; the control circuit provides positive and negative modulation voltages to cause the single-molecule sample to be tested to undergo a periodic reciprocating reverse translocation event; and the detection circuit collects the graphene nanopore-oriented electrical signal equal to the reference frequency. The use of this device can solve the problem that the sample signal to be tested is weak and difficult to be detected, and improve the randomness of the sample translocation behavior in the nanopore detection technology.

Description

Technical Field

[0001] The present invention belongs to the field of nanopore detection technology, and relates to a single-molecule periodic via-hole modulation and signal detection device based on nanopore technology, specifically, a single-molecule periodic via-hole modulation and signal detection device, a detection method and use. Background Art

[0002] The development of nanopore detection technology has further improved the research efficiency at the single-molecule scale. It can detect the translocation event of the sample to be tested and then conduct quantitative and qualitative analysis of the sample, and has great application potential in single-molecule detection. For example, nanopore detection technology can realize the counting or identification of translocation signals of molecular-level substances such as proteins and DNA, and then analyze the concentration of the sample to be tested (by counting) or its own characteristics (by signal recognition). Compared with the classical optical and electrochemical single-molecule research techniques, nanopore detection technology has the advantages of high sensitivity, fast detection speed, non-destructive detection and simple operation. The detection principle of this technology is: an external electric field is applied to the outside of the nanopore device to cause the sample to be tested to translocate. When the sample to be tested passes through the nanopore sensing area, the blocking effect of the sample to be tested on the nanopore will cause a short-term change in the detection signal (each short-term signal change process is a translocation event of the sample to be tested). According to the detected signal, the sample to be tested can be quantitatively or qualitatively analyzed. In addition, according to the different signal acquisition methods, the detection methods of nanopore signals can be divided into ion current detection method and current-oriented detection method. Among them, the ion current detection method mainly detects the ion current in the normal direction of the nanopore, and the face current detection method mainly detects the face current of the nanopore device. Compared with the ion current detection, the signal amplitude measured by the face current detection method is higher, which has more advantages in improving the signal-to-noise ratio.

[0003] In recent years, nanopore technology represented by solid-state nanopores and biological nanopores has been widely used in the field of sensing detection. Compared with biological nanopores, solid-state nanopores have the advantages of easy manufacturing, strong stability, good assembly and controllable pore morphology. However, when the detection accuracy of the sample to be tested is extremely high, for example, the resolution of DNA detection is required to reach the single base (0.335nm) level, graphene has higher spatial resolution than traditional silicon nitride or silicon dioxide nanopores due to its extremely low thickness (0.334nm).

[0004] However, the application of graphene nanopore technology in single-molecule detection generally has the problem of weak signals that are difficult to detect. The main reasons are: 1) Limited by the randomness of the through-hole event: Due to the high sensitivity of the graphene nanopore, it is easily affected by changes in other conditions in the detection area (such as the interaction between the material and the sample to be tested, the influence of ion transport, etc.), so the detected signals are not necessarily all caused by the real translocation event of the sample to be tested; 2) Limited by the low signal-to-noise ratio: The single-molecule detection technology based on graphene nanopores usually has the characteristics of extremely fast through-hole speed of the sample to be tested, low signal amplitude and high noise level. Therefore, the detection signal that really needs to be collected is easily buried in the background noise.

[0005] In summary, the existing graphene nanopore detection technology has the advantages of high spatial resolution and fast detection speed, but the existing nanopore technology still needs to be further improved in the structural design of the detection device to solve the problem that its signal is weak and difficult to detect. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one purpose of the present invention is to propose a single-molecule periodic perforation modulation and signal detection device based on nanopore technology. The use of this device can effectively solve the problem that the signal of the sample to be tested is weak and difficult to be detected, and improve the randomness of the sample translocation behavior in the nanopore detection technology, so as to achieve the regulation of translocation events at the single-molecule scale and the detection of weak signals, which is of great significance to the development of graphene nanopore detection technology.

[0007] This application is mainly based on the following questions and findings:

[0008] The traditional weak signal processing method is usually to amplify the signal, but this method also amplifies the noise while amplifying the signal. Therefore, it is necessary to purify the signal by filtering to achieve accurate detection of such weak signals. In view of the above phenomenon, the phase-locked amplifier, as a weak signal detection device, has the conditions to cope with the detection demand. Its detection principle is: using a periodic reference signal with the same frequency as the signal to be tested as a reference, filtering out noise different from the reference frequency, thereby extracting useful components of the signal to be tested. However, since the flow mode of the sample to be tested in most nanopore detections is a single pass, its translocation signal does not have a fixed frequency, and its translocation event cannot be accurately determined by the phase-locked amplifier. Therefore, the conditions for applying the phase-locked amplifier to the translocation event of the sample to be tested in the nanopore detection can be attributed to the controllability of the translocation state of the sample to be tested during the detection process. The main idea is to control the periodic reciprocating pass of the sample to be tested so that the detection signal has a frequency equal to the pass of the sample to be tested, and provide an accurate reference signal frequency for the phase-locked amplifier. The existing graphene nanopore technology is generally a single pass detection of the sample to be tested. The inventors envision that relevant microfluidic units can be added to the outside of the graphene nanopore chip, and microelectrodes can be added to the outside of the device; further, when the device is in a detection state, a periodic positive and negative modulation voltage will be applied externally at the microelectrode interface, and the sample to be tested can be enclosed in a closed space near the nanopore detection area through the control of the microfluidic unit. At this time, the sample to be tested will shuttle back and forth in the nanopore according to the frequency of the applied positive and negative modulation voltage; at the same time, the external phase-locked amplifier will use the known positive and negative modulation voltage frequency as the frequency of the reference signal to achieve accurate extraction of the detection signal.

[0009] In one aspect of the present invention, the present invention provides a single-molecule periodic via modulation and signal detection device. According to an embodiment of the present invention, the device includes:

[0010] A graphene nanopore chip, the graphene nanopore chip comprising a first support portion, a second support portion, a graphene layer, a first electrode layer and a second electrode layer, the first support portion and the second support portion are spaced apart, the first support portion and the second support portion both comprise a silicon layer and a silicon dioxide layer formed on the upper surface of the silicon layer, the graphene layer is suspended between the first support portion and the second support portion through the first electrode layer and the second electrode layer, a nano-through hole is provided in the middle of the suspended region, a portion of the first electrode layer is connected to the silicon dioxide layer on the first support portion and the remaining portion thereof covers one end connected to the graphene layer, a portion of the second electrode layer is connected to the silicon dioxide layer on the second support portion and the remaining portion thereof covers the other end connected to the graphene layer;

[0011] A microfluidic top layer, the microfluidic top layer comprising a first groove, an injection channel and an injection microvalve for controlling the opening and closing of the injection channel, the first groove opening faces downward and the inner diameter of the first groove is not less than the distance between the first electrode layer and the second electrode layer;

[0012] A microfluidic bottom layer, the microfluidic bottom layer comprising a second groove, a sample outlet flow channel and a sample outlet microvalve for controlling the opening and closing of the sample outlet flow channel, the second groove opening is upward and the inner diameter of the second groove is not less than the size of the graphene nanopore chip, and the second groove and the first groove define a storage space for a sample solution to be tested;

[0013] A control circuit, the control circuit comprising a first power supply and a signal generator electrically connected to the first power supply, a first control electrode and a second control electrode, the first control electrode being arranged on the microfluidic top layer and being suitable for contacting with the sample solution to be tested, the second control electrode being arranged on the microfluidic bottom layer and being suitable for contacting with the sample solution to be tested, the control circuit being used to provide a periodic positive and negative modulation voltage to cause a periodic reciprocating reverse translocation event to occur in the single-molecule sample to be tested;

[0014] A detection circuit, the detection circuit includes a second power supply and a phase-locked amplifier electrically connected to the second power supply, a first detection electrode and a second detection electrode, the first detection electrode is connected to the first electrode layer, the second detection electrode is connected to the second electrode layer, and the detection circuit is used to collect a graphene nanopore-oriented electrical signal equal to a reference frequency.

[0015] The single-molecule periodic perforation modulation and signal detection device of the above embodiment of the present invention combines graphene nanopore technology with microfluidics technology, and encapsulates microfluidics components (i.e., control circuits) outside the traditional nanopore chip, so that the sample to be tested can achieve a fixed frequency periodic reciprocating through the nanopore during the detection process. The device has at least the following beneficial effects: 1) using a single layer or a few layers of graphene (preferably a single layer) as a sensing material to improve the spatial resolution of the nanopore; 2) combining graphene nanopore technology with microfluidics technology, so that the sample to be tested can achieve a fixed frequency periodic reciprocating through the nanopore during the detection process, thereby increasing the number of samples of the translocation behavior of the sample to be tested, and effectively improving the problem of randomness of the translocation event; 3) since the frequency of the reciprocating perforation of the sample to be tested is known, it can provide an accurate reference frequency for the phase-locked amplifier, which is convenient for the phase-locked amplifier to accurately detect and extract the translocation signal of the sample to be tested.

[0016] In addition, the single-molecule periodic via-hole modulation and signal detection device according to the above embodiment of the present invention may also have the following additional technical features:

[0017] In some embodiments of the present invention, the pore size of the nanopore is 1.1 to 1.6 times the particle size of a single molecule to be detected.

[0018] In some embodiments of the present invention, the thickness of the graphene layer is no greater than 1 nm.

[0019] In some embodiments of the present invention, the bottom wall of the second groove is in contact with the lower surfaces of the first supporting portion and the second supporting portion, the height of the second groove is not lower than the height of the first supporting portion and the second supporting portion, the lower surface of the first groove is in contact with the silicon dioxide layer of the first supporting portion and the second supporting portion, and the top wall height of the first groove is not lower than the first electrode layer and the second electrode layer.

[0020] In some embodiments of the present invention, the output voltage of the first power supply is an AC voltage not greater than 2V.

[0021] In some embodiments of the present invention, the output voltage is 200-500 mV.

[0022] In some embodiments of the present invention, the frequency of the AC voltage is 1-20 Hz.

[0023] In some embodiments of the present invention, a ground wire of the first power socket is grounded, and a ground wire of the second power socket is grounded.

[0024] According to another aspect of the present invention, the present invention proposes a method for implementing single-molecule sample detection using the above-mentioned single-molecule periodic via-hole modulation and signal detection device. According to an embodiment of the present invention, the method includes:

[0025] (1) adjusting the output voltage of the first power supply to a periodic positive and negative modulation voltage;

[0026] (2) adding a buffer solution to the sample inlet flow channel and the sample outlet flow channel by using a syringe pump and closing the sample outlet microvalve, and closing the sample inlet microvalve after adding the sample to be tested to the sample inlet flow channel by using a syringe pump;

[0027] (3) controlling the flow direction of the sample solution fluid to be tested by the voltage signal output by the control circuit, ensuring that the output state of the signal generator is a periodic reverse electric field, and monitoring the detected signal state by the detection circuit;

[0028] (4) The sample to be tested performs periodic reciprocating motion through the through hole of the graphene layer under the driving force of the periodic reverse electric field, and the translocation behavior of the sample to be tested molecules is obtained by analyzing the amplitude and time of the detection signal of the sample to be tested molecules detected by the detection circuit.

[0029] Compared with the prior art, the single-molecule sample detection method of the above embodiment of the present invention can not only realize the periodic modulation and detection of the single-molecule translocation signal, but also improve the problem of weak detection signal that is difficult to capture due to the drawbacks of the traditional nanopore detection method such as random through-hole events and low signal-to-noise ratio.

[0030] According to another aspect of the present invention, the present invention proposes the use of the above-mentioned single-molecule periodic via-hole modulation and signal detection device or the method for implementing single-molecule sample detection using the above-mentioned single-molecule periodic via-hole modulation and signal detection device in the field of single-molecule detection. Therefore, the features and effects described for the above-mentioned single-molecule periodic via-hole modulation and signal detection device and the method for single-molecule sample detection are applicable to this use and will not be described one by one here.

[0031] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0033] Figure 1 Schematic diagram of the structure of a single-molecule periodic via modulation and signal detection device according to an embodiment of the present invention.

[0034] Figure 2 Schematic diagram of the structure of a graphene nanopore chip according to an embodiment of the present invention.

[0035] Figure 3 Schematic diagram of the structure of a microfluidic top layer according to an embodiment of the present invention.

[0036] Figure 4 Schematic diagram of the structure of a microfluidic substrate according to an embodiment of the present invention.

[0037] Figure 5 4 is a schematic structural diagram of a single-molecule periodic via-hole modulation and signal detection device according to another embodiment of the present invention.

[0038] Figure 6 According to one embodiment of the present invention, the sample molecules to be tested are not subjected to positive and negative modulation voltages (see Figure 6 (a)) and applying positive and negative modulation voltage (see Figure 6 (b)) Detection effect comparison chart. DETAILED DESCRIPTION

[0039] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0040] In the description of the present invention, it should be understood that the terms "length", "thickness", "upper", "lower", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are 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 position, be constructed and operated in a specific position, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the present invention, unless otherwise clearly specified and defined, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral body; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0041] In one aspect of the present invention, the present invention provides a single molecule periodic via modulation and signal detection device. According to an embodiment of the present invention, in combination with Figures 1 to 5It is understood that the device includes: a graphene nanopore chip 10, a microfluidic top layer 20, a microfluidic bottom layer 30, a control circuit 40 and a detection circuit 50. The device uses graphene nanopores as sensing parts to improve the spatial resolution of nanopore single molecule detection technology, and improves the structure of traditional nanopore devices through microfluidic technology to achieve periodic modulation and detection of single molecule translocation signals, thereby improving the problem of weak detection signals that are difficult to capture due to the drawbacks of random through-hole events and low signal-to-noise ratio in traditional nanopore detection methods. Figures 1 to 5 The single molecule periodic through-hole modulation and signal detection device

[0042] Graphene nanopore chip10

[0043] According to an embodiment of the present invention, Figure 1 and 2 As shown, the graphene nanopore chip 10 includes a first support portion 11, a second support portion 12, a graphene layer 13, a first electrode layer 14, and a second electrode layer 15. The first support portion 11 and the second support portion 12 are arranged at intervals. The first support portion 11 and the second support portion 12 both include a silicon layer a and a silicon dioxide layer b formed on the upper surface of the silicon layer. The graphene layer 13 is suspended between the first support portion 11 and the second support portion 12 through the first electrode layer 14 and the second electrode layer 15, and a nano-through hole 131 is provided in the middle of the suspended area. Part of the first electrode layer 14 is connected to the silicon dioxide layer b on the first support portion 11 and the remaining part thereof covers one end connected to the graphene layer 13, and part of the second electrode layer 15 is connected to the silicon dioxide layer b on the second support portion 12 and the remaining part thereof covers the other end connected to the graphene layer 13. Among them, the first electrode layer 14 and the second electrode layer 15 can be gold electrodes.

[0044] According to an embodiment of the present invention, when preparing a graphene nanopore chip, a support portion composed of silicon dioxide and silicon can be first made, and then a single layer of graphene is prepared on the support portion and a gold electrode is deposited. The support portion is then etched to realize a suspended layer of graphene, and finally an ion beam is used to drill a hole in the center of the graphene layer. Specifically, a support layer can be formed by depositing silicon dioxide (thickness can be 300 nm, etc.) on a silicon wafer (length×width×height dimensions can be 15 mm×15 mm×500 μm, etc.) by chemical vapor deposition; a layer of electron beam photoresist (thickness can be 300 nm, etc.) is spin-coated on the bottom surface of the support layer, and the photoresist is exposed and developed using electron beam lithography to form an etching window; the support layer is placed in a xenon difluoride gas reactor, the silicon part of the support layer is etched to form a trapezoidal groove, so that the silicon dioxide layer is suspended on the silicon surface; a single layer of graphene is prepared by chemical vapor deposition, and transferred to the surface of the silicon dioxide support layer; a layer of electron beam photoresist (thickness can be 300 nm, etc.) is spin-coated on the graphene surface, and the photoresist is exposed and developed using electron beam lithography to form a sub-micron wide photoresist strip; the above part is exposed to plasma for a period of time. time (such as 30 seconds) to etch the single-layer graphene not covered by the photoresist; spin-coat a layer of photoresist (thickness can be 300nm, etc.) on the retained graphene surface again, and form a pattern matching the target electrode structure through electron beam lithography technology; deposit a gold film (such as 20μm thick) on the surface of the above-mentioned part, and then soak the whole in a 45°C dimethylacetamide solution (such as 10 minutes) to remove the photoresist in the non-electrode area and the gold deposited thereon; spin-coat a layer of photoresist (thickness can be 300nm, etc.) on the silicon dioxide surface not covered by graphene as a protective layer; rinse the above-mentioned part in deionized water and transfer it to a hydrofluoric acid buffer solution for soaking (such as soaking in a hydrofluoric acid buffer solution with a hydrofluoric acid concentration of 37wt% for 5 minutes) to remove the silicon dioxide on the bottom of the graphene; then transfer the whole to deionized water, and drip ethanol at a low speed. After the ethanol concentration reaches about 90%, the whole is transferred to 100% ethanol and amyl acetate; the above part is dried by a supercritical point drying method to obtain a suspended single-layer graphene structure with electrodes at both ends; a through hole (for example, the diameter can be no more than 10 nm, etc.) is punched in the central window of the above part using a helium ion focused ion beam to obtain a suspended graphene nanopore chip structure.

[0045] According to an embodiment of the present invention, before assembling the graphene nanopore chip, it is necessary to pre-treat it to remove impurities from the chip and perform hydrophilic treatment on the chip. The process mainly includes the following steps: cleaning the silicon wafer with acetone and ethanol to remove particles and impurities on the surface of the silicon wafer; rinsing the silicon wafer with deionized water and placing the silicon wafer in a clean container for standby; further treating the silicon wafer with NanoStrip solution (composed of sulfuric acid and hydrogen peroxide), for example, a 100mL container can be used to hold about 50mL of NanoStrip solution, and the container can be placed on a hot plate at 100°C for preheating for 10 minutes, and then the nanopore chip can be placed in the NanoStrip solution, while the temperature of the hot plate is reduced to 80°C, and the temperature is kept constant for 2 hours. Before taking out the chip, gradually reduce the temperature of the hot plate to cool it down.

[0046] According to an embodiment of the present invention, the pore size of the nanopore 131 can be flexibly selected according to actual conditions such as the size of the single molecule sample to be detected. Specifically, the pore size of the nanopore 131 can be slightly larger than the size of the single molecule sample to be detected. Preferably, the pore size of the nanopore 131 can be 1.1 to 1.5 times the particle size of the single molecule to be detected, for example, it can be 1.1 to 1.2 times, 1.3 times, 1.4 times or 1.5 times the particle size of the single molecule to be detected. In this way, it can not only ensure that a single molecular sample to be detected can pass through the through hole of the graphene layer smoothly without being blocked, but also avoid confusion of detected signals caused by multiple molecules to be detected passing through the nanopore at the same time, which can further facilitate the accurate detection and extraction of the translocation signal of the sample to be detected.

[0047] According to an embodiment of the present invention, the thickness of the graphene layer 13 may be no greater than 1 nm, and may preferably be a single-layer graphene layer, thereby further ensuring high detection sensitivity and high spatial resolution when a single molecule sample to be detected passes through the hole.

[0048] Microfluidic Top Layer 20

[0049] According to an embodiment of the present invention, referring to Figure 1 and Figure 3 It is understood that the microfluidic top layer 20 includes a first groove 21, an injection channel 22 and an injection microvalve 23 for controlling the opening and closing of the injection channel 22, and the first groove 21 opens downward and the inner diameter of the first groove 21 is not less than the distance between the first electrode layer 14 and the second electrode layer 15. It should be noted that the distance between the first electrode layer 14 and the second electrode layer 15 in the present invention refers to the maximum distance between the two ends of the first electrode 14 and the second electrode 15 facing back to back. Among them, the first groove 21 is used to fix the upper part of the graphene nanopore chip 10. In addition, the microfluidic top layer 20 can be made of PDMS, and the microfluidic top layer substrate part can be printed by 3D printing technology to realize the setting of the microchannel and groove structure, and set the microvalve structure in the injection channel.

[0050] Microfluidic Substrate 30

[0051] According to an embodiment of the present invention, referring to Figure 1 and Figure 4 It is understood that the microfluidic bottom layer 30 includes a second groove 31, a sample outlet channel 32 and a sample outlet microvalve 33 for controlling the opening and closing of the sample outlet channel, the second groove 32 opens upward and the inner diameter of the second groove 31 is not less than the size of the graphene nanopore chip 10, and the second groove 31 and the first groove 21 define a storage space for the sample solution to be tested. Among them, the microfluidic bottom layer 30 can also be made of PDMS, and the microfluidic bottom layer substrate can be printed by 3D printing technology to achieve the setting of the microchannel and groove structure.

[0052] According to an embodiment of the present invention, after the microfluidic top layer 20 and the microfluidic bottom layer 30 are processed, the processed graphene nanopore chip 10 can be placed in the second groove 31 of the microfluidic bottom layer 30, and the microfluidic top layer 20 and the microfluidic bottom layer 30 are bonded together. Finally, the control / detection electrodes are deposited at the corresponding points of the device and the microvalve structures are set in the sample inlet and sample outlet channels.

[0053] According to an embodiment of the present invention, referring to Figure 1It is understood that the bottom wall of the second groove 31 is in contact with the lower surface of the first support part 11 and the second support part 12, the height of the second groove 31 is not lower than (preferably higher than) the height of the first support part 11 and the second support part 12, the lower surface of the first groove 21 is in contact with the silicon dioxide layer b of the first support part 11 and the second support part 12, and the top wall height of the first groove 21 is not lower than (preferably higher than) the first electrode layer 11 and the second electrode layer 12. Thus, it is more conducive to forming a closed accommodation space that can accommodate the sample solution to be tested after the microfluidic top layer and the microfluidic bottom layer are bonded. The shapes of the first groove 21 and the second groove 31 are not particularly limited, as long as the two can cooperate with the graphene nanopore chip to define a closed accommodation space that can accommodate the sample solution to be tested. For example, the first groove 21 can be a cylindrical groove, and the second groove 31 can be a cubic groove. Further, the matrix structure of the microfluidic top layer and the microfluidic bottom layer can be designed by drawing software, wherein a single-layer graphene layer (thickness 0.334nm) can be used, and the size of the microfluidic top layer can be 200mm×200mm×20mm (length×width× The diameter of the sample inlet channel can be 50 μm, the diameter of the cylindrical groove can be 8 mm, and the groove depth can be 25 μm; the size of the microfluidic bottom layer can be 200 mm × 200 mm × 35 mm (length × width × height), the diameter of the sample outlet channel can be 50 μm, and the size of the cube groove can be 15 mm × 15 mm × 500.3 μm (length × width × height); the sizes of the control electrode and the detection electrode connected to the external control circuit and the detection circuit can be 5 mm × 5 mm × 500 μm (length × width × height).

[0054] Control circuit 40

[0055] According to an embodiment of the present invention, referring to Figure 1 It is understood that the control circuit 40 includes a first power supply 41 and a signal generator 42 electrically connected to the first power supply 41, a first control electrode 43 and a second control electrode 44, the first control electrode 43 is arranged on the microfluidic top layer 20 and is suitable for contacting the sample solution to be tested, and the second control electrode 44 is arranged on the microfluidic bottom layer 30 and is suitable for contacting the sample solution to be tested, and the control circuit is used to provide a periodic positive and negative modulation voltage that causes the single molecule sample to be tested to undergo a periodic reciprocating reverse translocation event, that is, to control the translocation state of the sample to be tested, and its main component is the signal generator 42. Among them, during the detection process, the ground pin of the first power supply 41 socket should be properly connected to the ground to avoid interference.

[0056] According to an embodiment of the present invention, the output voltage of the first power supply 41 can be an AC voltage not greater than 2V, for example, it can be 0.2V, 0.4V, 0.6V, 0.8V, 1.2V or 1.5V, etc. The inventors have found that if the output voltage is too large, it may cause damage to related devices. In the present invention, by adopting an output AC voltage not greater than 2V, not only can the normal use of the device be guaranteed, but also positive and negative modulation voltages can be provided for periodic reciprocating reverse translocation events of the sample to be tested. Preferably, the output voltage of the first power supply 41 can be an AC voltage of 200 to 500mV, and the use of this output voltage can further ensure the normal use of the device. Further, the frequency of the AC voltage output by the first power supply 41 can be 1 to 20 Hz, for example, 1 Hz, 3 Hz, 5 Hz, 7 Hz, 9 Hz, 12 Hz, 15 Hz or 18 Hz, etc. The inventors have found that if the frequency of the AC voltage is too low, it will affect the frequency of the through-holes of the sample molecules to be tested; and if the frequency of the AC voltage is too fast, although the detection resolution can be improved, it will affect the detection accuracy. Some samples to be tested that are to be through-holes may be pressed back by the reverse voltage before they can pass through the holes, resulting in inaccurate periodic changes in the through-hole translocation. In the present invention, by controlling the frequency of the AC voltage output by the first power supply to be within the above range, it is more conducive to improving the detection resolution and detection accuracy. In some embodiments of the present invention, the ground wire of the first power socket is grounded, and the ground wire of the second power socket is grounded.

[0057] Detection circuit 50

[0058] According to an embodiment of the present invention, referring to Figure 1 It is understood that the detection circuit 50 includes a second power supply 51 and a phase-locked amplifier 52 electrically connected to the second power supply 51, a first detection electrode 53 and a second detection electrode 54, the first detection electrode 53 is connected to the first electrode layer 14, and the second detection electrode 54 is connected to the second electrode layer 15. The detection circuit is used to collect the graphene nano (through) hole facing electrical signal equal to the reference frequency, and the main component is the phase-locked amplifier. In the detection process, the ground pin of the second power supply 51 socket should be properly connected to the ground to avoid interference.

[0059] According to the embodiment of the present invention, the detection process of a certain sample to be tested using the single-molecule periodic through-hole modulation and signal detection device of the above embodiment of the present invention needs to be connected to the external control circuit and the detection circuit. The main component of the control circuit is the signal generator, which is used to provide a periodic positive and negative modulation voltage that causes the sample to be tested to undergo a periodic reciprocating reverse translocation event; the main component of the detection circuit is a phase-locked amplifier, which is used to collect the graphene nanopore-oriented electrical signal equal to the reference frequency. When the device is in the detection state, the sample inlet microvalve and the sample outlet microvalve are both in the closed state, and the signal generator generates a periodic positive and negative modulation voltage in the vertical direction of the device to achieve the periodic reciprocating reverse passage of the sample to be tested through the nanopore sensing area. When a single through-hole event occurs in the sample to be tested, a corresponding through-hole current signal will be generated, so the physical occupation caused by the event will cause the oriented electrical signal collected by the external detection circuit to change.

[0060] According to an embodiment of the present invention, a method for performing single molecule detection using the single molecule periodic through-hole modulation and signal detection device of the above embodiment of the present invention includes the following steps: 1) connecting an external circuit: the external circuit includes a control circuit for driving the flow of the sample to be tested and a detection circuit for collecting detection signals. Before detection, the devices corresponding to the two circuits need to be connected to the device; 2) dripping the sample to be tested: first, ensure that the microvalves of the device are in an open state, and then use a syringe pump to drip a quantitative buffer solution in the sample inlet flow channel of the top layer of the microfluidic control and the sample outlet flow channel of the bottom layer and immediately close the sample outlet microvalve, and then drip a quantitative sample to be tested into the sample inlet flow channel of the top layer of the microfluidic control, and immediately close all microvalves after the process of dripping the sample to be tested is completed; 3) controlling the sample to be tested: turning on the signal generator to generate a periodic positive and negative modulation voltage, so that the sample to be tested enclosed in the nanopore detection area shuttles back and forth in the nanopore (i.e., the through hole in the middle of the graphene layer) sensing area; 4) collecting signals: using the frequency of the periodic positive and negative modulation voltage as the reference frequency of the phase-locked amplifier to extract the detection signal. In this way, the randomness of the translocation event can be effectively improved, and the phase-locked amplifier can also accurately detect and extract the translocation signal of the sample to be tested.

[0061] In summary, in the single-molecule periodic perforation modulation and signal detection device of the above-mentioned embodiment of the present invention, the microfluidic technology has the characteristics of being able to accurately control and manipulate microscale fluids. It is combined with the graphene nanopore technology, and the microfluidic components (i.e., the control circuit) are packaged outside the traditional nanopore chip, so that the sample to be tested can achieve a fixed frequency periodic reciprocating passage through the nanopore during the detection process, which has at least the following beneficial effects: 1) using a single layer or a few layers of graphene (preferably a single layer) as a sensing material to improve the spatial resolution of the nanopore; 2) by setting the microelectrode (i.e., the control electrode), the application of periodic positive and negative modulation voltages in the vertical direction of the device is achieved, so that the sample to be tested can be detected during the detection process. The method comprises the following steps: 1) periodically reciprocating through the nanopore to increase the number of times the sample to be tested passes through the nanopore, expanding the sample of the translocation behavior of the sample to be tested to improve the authenticity of the high-sensitivity detection of the graphene nanopore, and effectively improving the problem of randomness of the translocation event; 2) the sample to be tested is caused to undergo periodic translocation behavior by applying periodic positive and reverse voltages. Since the frequency of the sample to be tested reciprocating through the nanopore is known, an accurate reference signal frequency can be provided for the phase-locked amplifier, which facilitates the phase-locked amplifier to accurately detect and extract the translocation signal of the sample to be tested; 3) the microchannel and microvalve are set to provide an effective closed activity space for the behavior of the sample to be tested repeatedly passing through the nanopore, thereby ensuring the effectiveness of the detection.

[0062] According to another aspect of the present invention, the present invention proposes a method for implementing single-molecule sample detection using the above-mentioned single-molecule periodic through-hole modulation and signal detection device. According to an embodiment of the present invention, the method includes: (1) adjusting the output voltage of the first power supply to a periodic positive and negative modulation voltage; (2) using a syringe pump to add a buffer solution to the sample inlet and sample outlet channels and close the sample outlet microvalve, and using a syringe pump to add the sample to be tested to the sample inlet channel and close the sample inlet microvalve; (3) controlling the flow direction of the sample solution fluid to be tested by the voltage signal output by the control circuit, ensuring that the output state of the signal generator is a periodic reverse electric field, and monitoring the detected signal state by the detection circuit; (4) the sample to be tested performs a periodic reciprocating motion through the through-hole of the graphene layer under the drive of the periodic reverse electric field, and the translocation behavior of the sample to be tested molecule is obtained by analyzing the amplitude and time of the detection signal of the sample to be tested molecule detected by the detection circuit. Compared with the prior art, the single-molecule sample detection method can not only realize the periodic modulation and detection of the single-molecule translocation signal, but also improve the problem of weak detection signals that are difficult to capture due to the drawbacks of the traditional nanopore detection method such as random through-hole events and low signal-to-noise ratio. It should be noted that the features and effects described for the above-mentioned single-molecule periodic through-hole modulation and signal detection device are applicable to this method and will not be described one by one here.

[0063] To facilitate understanding of the above-mentioned single-molecule periodic via-hole modulation and signal detection device and the method for implementing single-molecule sample detection using the device in the present invention, the translocation behavior of a double-stranded λ-DNA sample to be tested is described in detail using the device as an example. The operation steps are as follows:

[0064] 1) Connect external circuit: Build external control circuit and detection circuit. Figure 1 Schematic diagram of the detection circuit of the embodiment of the present invention. The connection method of the control circuit is as follows: connect the power cord of the signal generator to a 220V, 50HZ AC power supply, and connect the control electrode of the device to the circuit. It should be noted that the ground pin of the power socket should be properly connected to the ground to avoid interference; before turning on the machine, the output knobs on the panel should be turned to the minimum; in order to obtain sufficient frequency stability, preheating is required; by adjusting the parameters, it outputs a periodic positive and negative modulation voltage with a frequency of 1Hz and an amplitude of 200mV;

[0065] The detection circuit is connected as follows: the power line of the lock-in amplifier is connected to the second power supply, and the detection electrode of the device is connected to the circuit;

[0066] 2) Add the sample to be tested: First, use the syringe pump to add 1mmol / L EDTA (ethylenediaminetetraacetic acid) and 10mmol / L Tris-HCl (composed of tris(hydroxymethyl)aminomethane and hydrochloric acid) solution into the sample inlet and sample outlet channels, and then immediately close the sample outlet microvalve. Then, inject a DNA solution with a concentration of 2μg / mL into the sample inlet channel and then close the sample inlet microvalve.

[0067] 3) Control the sample to be tested: In the early stage of dripping the sample to be tested, the flow direction of the fluid is controlled by the voltage signal output by the control circuit. After the process of dripping the sample to be tested is completed, the output state of the signal generator is ensured to be a periodic reverse electric field, and the detection signal state of the device is monitored by the detection circuit. When the frequency and amplitude region of the signal to be tested are stable, it can be determined whether the sample to be tested is stably shuttling back and forth in the nanopore detection region;

[0068] 4) Analyze the signal: Driven by the periodic positive and negative modulation voltage, the DNA molecule performs periodic back-and-forth motion in the nanopore detection area. When the DNA molecule passes through the nanopore once, the physical occupancy will cause the detection signal of the device to change and generate a corresponding pore-passing time. By analyzing the amplitude and time of the detection signal, the translocation behavior of the double-stranded λ-DNA molecule in the sample to be tested can be analyzed. Figure 6 The DNA molecules of the sample to be tested are not subjected to positive and negative modulation voltages (such as Figure 6 (a)) and applying positive and negative modulation voltages (as shown in Figure 6 Detection effect comparison chart shown in (b)).

[0069] According to another aspect of the present invention, the present invention proposes the use of the above-mentioned single-molecule periodic via-hole modulation and signal detection device or the method for implementing single-molecule sample detection using the above-mentioned single-molecule periodic via-hole modulation and signal detection device in the field of single-molecule detection. Therefore, the features and effects described for the above-mentioned single-molecule periodic via-hole modulation and signal detection device and the method for single-molecule sample detection are applicable to this use and will not be described one by one here.

[0070] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0071] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for implementing single-molecule sample detection using a single-molecule periodic via-hole modulation and signal detection device, characterized in that: The single molecule sample is DNA or protein, The single-molecule periodic via-hole modulation and signal detection device comprises: A graphene nanopore chip, the graphene nanopore chip comprising a first support portion, a second support portion, a graphene layer, a first electrode layer and a second electrode layer, the first support portion and the second support portion are spaced apart, the first support portion and the second support portion both comprise a silicon layer and a silicon dioxide layer formed on the upper surface of the silicon layer, the graphene layer is suspended between the first support portion and the second support portion through the first electrode layer and the second electrode layer, a nano-through hole is provided in the middle of the suspended region, a portion of the first electrode layer is connected to the silicon dioxide layer on the first support portion and the remaining portion thereof covers one end connected to the graphene layer, a portion of the second electrode layer is connected to the silicon dioxide layer on the second support portion and the remaining portion thereof covers the other end connected to the graphene layer; A microfluidic top layer, the microfluidic top layer comprising a first groove, an injection channel and an injection microvalve for controlling the opening and closing of the injection channel, the first groove opening faces downward and the inner diameter of the first groove is not less than the distance between the first electrode layer and the second electrode layer; A microfluidic bottom layer, the microfluidic bottom layer comprising a second groove, a sample outlet flow channel and a sample outlet microvalve for controlling the opening and closing of the sample outlet flow channel, the second groove opening is upward and the inner diameter of the second groove is not less than the size of the graphene nanopore chip, and the second groove and the first groove define a storage space for a sample solution to be tested; A control circuit, the control circuit comprising a first power supply and a signal generator electrically connected to the first power supply, a first control electrode and a second control electrode, the first control electrode being arranged on the microfluidic top layer and being suitable for contacting with the sample solution to be tested, the second control electrode being arranged on the microfluidic bottom layer and being suitable for contacting with the sample solution to be tested, the control circuit being used to provide a periodic positive and negative modulation voltage to cause a periodic reciprocating reverse translocation event to occur in the single-molecule sample to be tested; as well as, A detection circuit, the detection circuit comprising a second power supply and a phase-locked amplifier electrically connected to the second power supply, a first detection electrode and a second detection electrode, the first detection electrode being connected to the first electrode layer, the second detection electrode being connected to the second electrode layer, the detection circuit being used to collect a graphene nanopore-oriented electrical signal having a frequency equal to a reference frequency, in, The pore size of the nanopore is 1.1 to 1.6 times the particle size of the single molecule to be detected; The thickness of the graphene layer is no more than 1 nm; The method comprises: (1) adjusting the output voltage of the first power supply to a periodic positive and negative modulation voltage; (2) adding a buffer solution to the sample inlet flow channel and the sample outlet flow channel by using a syringe pump and closing the sample outlet microvalve, and closing the sample inlet microvalve after adding the sample to be tested to the sample inlet flow channel by using a syringe pump; (3) controlling the flow direction of the sample solution fluid to be tested by the voltage signal output by the control circuit, ensuring that the output state of the signal generator is a periodic reverse electric field, and monitoring the detected signal state by the detection circuit; (4) The sample to be tested performs periodic reciprocating motion through the through hole of the graphene layer under the driving force of the periodic reverse electric field, and the translocation behavior of the sample to be tested molecules is obtained by analyzing the amplitude and time of the detection signal of the sample to be tested molecules detected by the detection circuit.

2. The method according to claim 1, characterized in that The bottom wall of the second groove is in contact with the lower surfaces of the first supporting part and the second supporting part, the height of the second groove is not lower than the height of the first supporting part and the second supporting part, the lower surface of the first groove is in contact with the silicon dioxide layer of the first supporting part and the second supporting part, and the top wall height of the first groove is not lower than the first electrode layer and the second electrode layer.

3. The method according to claim 1, characterized in that The output voltage of the first power supply is an AC voltage not greater than 2V.

4. The method according to claim 3, characterized in that The output voltage is 200~500mV.

5. The method according to claim 3, characterized in that: The frequency of the AC voltage is 1-20 Hz.

6. The method according to claim 1 or 4, characterized in that: The ground wire of the first power socket is grounded, and the ground wire of the second power socket is grounded.

7. Use of the method according to any one of claims 1 to 6 in the field of single molecule detection.

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