CN114878800B - Single-molecule periodic pore modulation and signal detection device based on nanopore technology - Google Patents
Single-molecule periodic pore modulation and signal detection device based on nanopore technology Download PDFInfo
- Publication number
- CN114878800B CN114878800B CN202210298400.XA CN202210298400A CN114878800B CN 114878800 B CN114878800 B CN 114878800B CN 202210298400 A CN202210298400 A CN 202210298400A CN 114878800 B CN114878800 B CN 114878800B
- Authority
- CN
- China
- Prior art keywords
- sample
- detection
- layer
- detected
- groove
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nanotechnology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
本发明公开了基于纳米孔技术的单分子周期性过孔调制及信号检测装置。该装置包括石墨烯纳米孔芯片、微流控顶层、微流控底层、控制电路和检测电路，石墨烯纳米孔芯片中石墨烯层悬空布置在第一支撑部和第二支撑部之间且悬空区域中部设有纳米通孔；微流控顶层包括进样流道、进样微阀和第一凹槽，底层包括第二凹槽、出样流道和出样微阀，第二凹槽与第一凹槽限定出待测样品溶液容纳空间；控制电路提供使待测单分子样品发生周期往复反向易位事件正反调制电压；检测电路采集与参考频率相等的石墨烯纳米孔面向电信号。采用该装置可解决因待测样品信号微弱不易被检测的问题，改善纳米孔检测技术中样品易位行为的随机性。
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
The invention belongs to the technical field of nanopore detection, and relates to a single-molecule periodic via modulation and signal detection device based on a nanopore technology, in particular to a single-molecule periodic via modulation and signal detection device, a detection method and application.
Background
The development of the nanopore detection technology further improves the research efficiency under the single molecular scale, and the translocation event of the sample to be detected can be detected, so that the sample can be quantitatively and qualitatively analyzed, and the nanopore detection technology has great application potential in the aspect of single molecular detection. For example, translocation signals of substances such as proteins, DNA and the like can be counted or identified by a nanopore detection technology, and then the concentration of a sample to be detected (realized by a counting mode) or the characteristics of the sample to be detected (realized by a signal identification mode) can be analyzed. Compared with classical optical and electrochemical single-molecule research techniques, the nanopore detection technique has the advantages of high sensitivity, high detection speed, nondestructive detection, simple operation and the like. The detection principle of the technology is that an external electric field is applied outside the nanopore device to enable a sample to be detected to be translocated, when the sample to be detected passes through a nanopore sensing area, short-time change of detection signals (translocation event of each short-time signal change process, namely, one sample to be detected) can be caused due to the blocking effect of the sample to be detected on the nanopore, and quantitative or qualitative analysis can be carried out on the sample to be detected according to the detected signals. In addition, according to different signal acquisition modes, the detection methods of the nanopore signals can be classified into an ion current detection method, a current-oriented detection method and the like. The ion current detection method mainly detects the ion current in the normal direction of the nano hole, and the current detection method mainly detects the current facing the nano hole device. Compared with ion current detection, the signal amplitude measured by the current detection method is higher, and the signal to noise ratio is improved.
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 nano-pores, the solid nano-pores have the advantages of convenient manufacture, strong stability, good assembly, controllable pore morphology and the like. However, when the detection accuracy of the sample to be detected is extremely high, for example, the resolution of DNA detection is required to reach the single base (0.335 nm) level, graphene has higher spatial resolution compared with the conventional silicon nitride or silicon dioxide nanopore due to its extremely low thickness (0.334 nm).
However, the problem that the signal is weak and difficult to detect generally exists in the aspect of applying the graphene nanopore technology to single-molecule detection is mainly caused by that 1) the randomness of the via event is limited, the high sensitivity of the graphene nanopore is easily affected by other condition changes of a detection area (such as interaction between a material and a sample to be detected, influence of ion transport and the like), and therefore all detected signals are not necessarily caused by the occurrence of a real translocation event of the sample to be detected, 2) the signal-to-noise ratio is limited to be low, and the characteristics of extremely high via speed, low signal amplitude, high noise level and the like of the sample to be detected generally exist in the single-molecule detection technology based on the graphene nanopore, so that the detection signal which really needs to be acquired is easily buried in background noise.
In summary, the existing graphene nanopore detection technology has the advantages of high spatial resolution, high detection speed and the like, but the existing nanopore technology still needs to be further improved in the aspect of structural design of a detection device so as to solve the problem that the signal is weak and difficult to detect.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. It is therefore an object of the present invention to propose a single-molecule periodic via modulation and signal detection device based on nanopore technology. The device can effectively solve the problem that the signal of the sample to be detected is weak and is not easy to detect, improves the randomness of the sample translocation behavior in the nanopore detection technology, realizes the regulation and control of translocation events and the detection of weak signals under a single molecular scale, and has important significance for the development of the graphene nanopore detection technology.
The present application is mainly based on the following problems and findings:
The traditional weak signal processing mode is usually to amplify signals, but the mode amplifies noise at the same time of amplifying signals, so that the signals need to be purified by a filtering means to realize accurate detection of the weak signals. In order to solve the above-mentioned problems, a lock-in amplifier is used as a weak signal detection device having a condition for meeting the detection requirement, and the detection principle is that a periodic reference signal with the same frequency as a signal to be detected is used as a reference to filter out noises different from the reference frequency, thereby extracting useful signal components to be detected. However, since the current flow mode of the sample to be detected for most nanopore detection is a single via, the translocation signal does not have a fixed frequency, and the translocation event cannot be accurately determined by a phase-locked amplifier. Therefore, the condition that the phase-locked amplifier is applied to the nanopore to detect the translocation event of the sample to be detected can be summarized as realizing the controllability of the translocation state of the sample to be detected in the detection process, and the main idea is to control the periodic reciprocating via hole of the sample to be detected so that the detection signal has the frequency equal to that of the via hole of the sample to be detected, and provide an accurate reference signal frequency for the phase-locked amplifier. The existing graphene nanopore technology is commonly used for single via detection of a sample to be detected. The inventor envisages that a relevant micro-fluidic unit is additionally arranged outside a graphene nanopore chip, and a microelectrode is additionally arranged outside the device, further, when the device is in a detection state, periodic positive and negative modulation voltage is externally applied to a microelectrode interface, a sample to be detected can be sealed in a sealed space near a nanopore detection area through control of the micro-fluidic unit, at the moment, the sample to be detected reciprocally shuttles into the nanopore according to the frequency of the externally applied positive and negative modulation voltage, and meanwhile, an external lock-in amplifier takes the frequency of the known positive and negative modulation voltage as the frequency of a reference signal to realize accurate extraction of a detection signal.
In one aspect, the present invention provides a single molecule periodic via modulation and signal detection apparatus. According to an embodiment of the invention, the apparatus comprises:
The graphene nanopore chip comprises a first supporting part, a second supporting part, a graphene layer, a first electrode layer and a second electrode layer, wherein the first supporting part and the second supporting part are arranged at intervals, the first supporting part and the second supporting part 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 supporting part and the second supporting part through the first electrode layer and the second electrode layer, a nanometer through hole is formed in the middle of a suspended area, part of the first electrode layer is connected with the silicon dioxide layer on the first supporting part and the rest part of the first electrode layer is connected with one end of the graphene layer in a covering mode, and the other end of the graphene layer is connected with the silicon dioxide layer on the second supporting part in a covering mode.
The microfluidic top layer comprises a first groove, a sample injection flow channel and a sample injection micro valve for controlling the opening and closing of the sample injection flow channel, the opening of the first groove is downward, and the inner diameter of the first groove is not smaller than the distance between the first electrode layer and the second electrode layer;
The microfluidic substrate comprises a second groove, a sample outlet flow channel and a sample outlet micro valve for controlling the opening and closing of the sample outlet flow channel, the opening of the second groove is upward, the inner diameter of the second groove is not smaller than the size of the graphene nanopore chip, and the second groove and the first groove define an accommodating space of a sample solution to be detected;
the control circuit comprises a first power supply, a signal generator, a first control electrode and a second control electrode, wherein the signal generator is electrically connected with the first power supply, the first control electrode is arranged on the microfluidic top layer and is suitable for being contacted with a sample solution to be tested, the second control electrode is arranged on the microfluidic bottom layer and is suitable for being contacted with the sample solution to be tested, and the control circuit is used for providing a periodic positive and negative modulation voltage for enabling a single-molecule sample to be tested to generate a periodic reciprocating reverse translocation event;
The detection circuit comprises a second power supply, a phase lock amplifier electrically connected with the second power supply, a first detection electrode and a second detection electrode, wherein the first detection electrode is connected with the first electrode layer, the second detection electrode is connected with the second electrode layer, and the detection circuit is used for collecting graphene nanopore facing electric signals equal to a reference frequency.
According to the single-molecule periodic via hole modulation and signal detection device disclosed by the embodiment of the invention, a graphene nanopore technology is combined with a microfluidic technology, and a microfluidic component (namely a control circuit) is packaged outside a traditional nanopore chip, so that a sample to be detected can pass through the nanopore in a fixed-frequency periodic reciprocating manner in the detection process. The device has the advantages that 1) single-layer or less-layer graphene (preferably single-layer graphene) is used as a sensing material to improve the spatial resolution of the nanopore, 2) the graphene nanopore technology is combined with the microfluidic technology, so that a sample to be detected can pass through the nanopore in a fixed-frequency cycle manner in the detection process, the number of samples of translocation behaviors of the sample to be detected is increased, the problem that translocation events have randomness is effectively solved, and 3) because the frequency of the reciprocal via holes of the sample to be detected is known, an accurate reference frequency can be provided for the phase-locked amplifier, and accurate detection and extraction of translocation signals of the sample to be detected by the phase-locked amplifier are facilitated.
In addition, the single-molecule periodic via modulation and signal detection apparatus according to the above embodiment of the present invention may further have the following additional technical features:
in some embodiments of the present invention, the aperture of the nano through hole is 1.1 to 1.6 times of the particle size of the single molecule to be detected.
In some embodiments of the invention, the graphene layer has a thickness of no greater than 1nm.
In some embodiments of the present invention, the bottom wall of the second groove is attached to the lower surfaces of the first support portion and the second support portion, the height of the second groove is not lower than the heights of the first support portion and the second support portion, the lower surface of the first groove is attached to the silicon dioxide layers of the first support portion and the second support portion, and the top wall of the first groove is not lower than the heights of the first electrode layer and the second electrode layer.
In some embodiments of the invention, the output voltage of the first power supply is an ac voltage of no more than 2V.
In some embodiments of the invention, the output voltage is 200-500 mV.
In some embodiments of the present invention, the frequency of the ac voltage is 1 to 20hz.
In some embodiments of the invention, the ground of the first power outlet is grounded and the ground of the second power outlet is grounded.
According to a further aspect of the present invention, a method for performing single molecule sample detection using the single molecule periodic via modulation and signal detection apparatus described above is provided. According to an embodiment of the invention, the method comprises:
(1) Adjusting the output voltage of the first power supply to be a periodic positive and negative modulation voltage;
(2) Adding buffer solution into the sample injection flow channel and the sample outlet flow channel by using a syringe pump, closing the sample outlet micro valve, and closing the sample injection micro valve after a sample to be detected is added into the sample injection flow channel by using the syringe pump;
(3) The flow direction of the fluid of the sample solution to be detected is controlled by the voltage signal output by the control circuit, the output state of the signal generator is ensured to be a periodic reverse electric field, and the detected signal state is monitored by the detection circuit;
(4) And the sample to be detected is driven by the periodic reverse electric field to do periodic reciprocating motion through the graphene layer through hole, and the translocation behavior of the sample molecule to be detected is obtained by analyzing the amplitude and time of the detection signal of the sample molecule to be detected, which is detected by the detection circuit.
Compared with the prior art, the method for detecting the single-molecule sample can realize the periodic modulation and detection of the single-molecule translocation signal, and can also solve the problem that the detection signal is weak and difficult to capture due to the defects of random via events, low signal to noise ratio and the like in the traditional nanopore detection mode.
According to a further aspect of the present invention, the present invention proposes the use of the above-described single molecule periodic via modulation and signal detection device or a method for performing single molecule sample detection using the above-described single molecule periodic via modulation and signal detection device in the field of single molecule detection. Thus, the features and effects described with respect to the above-mentioned single-molecule periodic via modulation and signal detection apparatus and method for single-molecule sample detection are applicable to this application, and will not be described in detail herein.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
fig. 1 is a schematic structural diagram of a single-molecule periodic via modulation and signal detection apparatus according to an embodiment of the present invention.
Fig. 2 is a schematic structural view of a graphene nanopore chip according to one embodiment of the present invention.
Fig. 3 is a schematic structural view of a microfluidic top layer according to one embodiment of the present invention.
Fig. 4 is a schematic structural view of a microfluidic substrate according to one embodiment of the present invention.
Fig. 5 is a schematic structural diagram of a single-molecule periodic via modulation and signal detection apparatus according to still another embodiment of the present invention.
FIG. 6 is a graph showing the comparison of detection effects of sample molecules to be detected without applying a forward and reverse modulation voltage (see FIG. 6 (a)) and without applying a forward and reverse modulation voltage (see FIG. 6 (b)) according to one embodiment of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In the description of the present invention, it should be understood that the terms "length," "thickness," "upper," "lower," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like indicate or are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the present invention and to simplify the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be configured and operated in a particular orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the present invention, unless explicitly stated and limited otherwise, terms such as "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed, mechanically connected, electrically connected, directly connected, or indirectly connected through an intervening medium, or in communication between two elements or in an interaction relationship between two elements, unless otherwise explicitly stated. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances. In the present invention, unless expressly stated or limited otherwise, a first feature "up" or "down" a second feature may be the first and second features in direct contact, or the first and second features in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.
In one aspect, the present invention provides a single molecule periodic via modulation and signal detection apparatus. According to an embodiment of the invention, as understood in conjunction with fig. 1-5, the device comprises a graphene nanopore chip 10, a microfluidic top layer 20, a microfluidic bottom layer 30, a control circuit 40 and a detection circuit 50. According to the device, the graphene nano holes are used as sensing positions to improve the spatial resolution of a nano hole single-molecule detection technology, the structure of a traditional nano hole device is improved through a microfluidic technology, periodic modulation and detection of single-molecule translocation signals are achieved, and the problem that detection signals are weak and difficult to capture due to the defects of random via events, low signal to noise ratio and the like in a traditional nano hole detection mode is solved. The single-molecule periodic via modulation and signal detection device is described below with reference to FIGS. 1-5
Graphene nanopore chip 10
According to an embodiment of the present invention, as shown in fig. 1 and 2, a graphene nanopore chip 10 includes a first supporting portion 11, a second supporting portion 12, a graphene layer 13, a first electrode layer 14 and a second electrode layer 15, where the first supporting portion 11 and the second supporting portion 12 are spaced apart, each of the first supporting portion 11 and the second supporting portion 12 includes a silicon layer a and a silicon dioxide layer b formed on an upper surface of the silicon layer, the graphene layer 13 is suspended between the first supporting portion 11 and the second supporting portion 12 through the first electrode layer 14 and the second electrode layer 15, and a nano through hole 131 is formed in a middle portion of a suspended area, a part of the first electrode layer 14 is connected with the silicon dioxide layer b on the first supporting portion 11 and a remaining part of the first electrode layer is covered with one end connected with the graphene layer 13, a part of the second electrode layer 15 is connected with the silicon dioxide layer b on the second supporting portion 12 and a remaining part of the second electrode layer is covered with another end connected with the graphene layer 13. The first electrode layer 14 and the second electrode layer 15 may be gold electrodes.
According to the embodiment of the invention, when the graphene nanopore chip is prepared, a supporting part consisting of silicon dioxide and silicon can be firstly prepared, then single-layer graphene is prepared above the supporting part, a gold electrode is deposited, then the supporting part is etched to realize a suspended layer of graphene, and finally an ion beam is utilized to punch the center of the graphene layer. Specifically, a supporting layer can be formed by silicon dioxide (the thickness can be 300nm and the like) deposited on a silicon wafer (the length x width x height can be 15mm x 500 μm and the like) by a chemical vapor deposition method, a layer of photoresist (the thickness can be 300nm and the like) of an electron beam is spin-coated on the bottom surface of the supporting layer, an etching window is formed on the photoresist by using an electron beam lithography technology, the supporting layer is placed in a xenon difluoride gas reactor, a silicon part of the supporting layer is etched and a trapezoid groove is formed, the silicon dioxide layer is suspended on the silicon surface, a single-layer graphene is prepared by the chemical vapor deposition method and is transferred to the surface of the silicon dioxide supporting layer, a layer of photoresist (the thickness can be 300nm and the like) of an electron beam is spin-coated on the surface of the graphene, a submicron-wide photoresist strip is formed by using an electron beam lithography technology, the part is exposed in plasma for a period of time (such as 30 seconds) to etch the single-layer graphene which is not covered by the photoresist, a layer of photoresist is spin-coated on the surface of the graphene which is left, the photoresist (the thickness can be 300nm and the like), a film is formed by the electron beam lithography technology, the pattern such as 20 μm is formed on the surface of the target electrode structure, the whole is then soaked in dimethylacetamide solution at 45 deg.c for 10 min to eliminate photoresist in non-electrode area and deposited gold, one photoresist layer with thickness of 300nm is spun onto the surface of non-graphene covered silicon dioxide as protecting layer, and the photoresist layer is rinsed in deionized water and then soaked in hydrofluoric acid buffering solution for 5 min to eliminate silicon dioxide in the bottom of graphene, and the whole is then transferred into deionized water and dropped into alcohol in low speed. And (3) transferring the whole body into ethanol and amyl acetate with the concentration of 100% until the concentration of the ethanol reaches about 90%, drying the part by a supercritical point drying method to obtain a suspended single-layer graphene structure with electrodes at two ends, and punching a through hole (for example, the diameter can be not more than 10nm and the like) on a central window of the part by utilizing helium ion focusing ion beams to obtain the suspended graphene nanopore chip structure.
According to the embodiment of the invention, before the graphene nanopore chip is assembled, the wafer is required to be pretreated to remove impurities of the chip and hydrophilized, and the process mainly comprises the steps of cleaning the wafer with acetone and ethanol to remove particles, impurities and the like on the surface of the wafer, flushing the wafer with deionized water and placing the wafer in a clean container for standby, and further treating the wafer with NanoStrip solution (consisting of sulfuric acid and hydrogen peroxide), for example, a 100mL container can be used for holding about 50mL of NanoStrip solution, the container can be placed on a 100 ℃ hot plate for preheating for 10 minutes, then the nanopore chip is placed in the NanoStrip solution, meanwhile, the temperature of the hot plate is reduced to 80 ℃, and the temperature is kept constant and the wafer is heated for 2 hours. The temperature of the hot plate is gradually reduced to reduce the temperature before the chip is taken out.
According to the embodiment of the invention, the pore size of the nano through hole 131 can be flexibly selected according to the actual conditions such as the size of the single-molecule sample to be detected, specifically, the pore size of the nano through hole 131 can be slightly larger than the size of the single-molecule sample to be detected, preferably the pore size of the nano through hole 131 can be 1.1-1.5 times of the particle size of the single-molecule sample to be detected, for example, can be 1.1-1.2 times, 1.3 times, 1.4 times or 1.5 times of the particle size of the single-molecule sample to be detected, and the like, thereby not only ensuring that a single sample to be detected can smoothly pass through the through hole of the graphene layer without blocking, but also avoiding confusion of detected signals caused by a plurality of molecules to be detected simultaneously passing through the nano through hole, and further being beneficial to accurate detection and extraction of translocation signals of the sample to be detected.
According to the embodiment of the invention, the thickness of the graphene layer 13 may be not more than 1nm, and preferably may be a single-layer graphene layer, so that high detection sensitivity and high spatial resolution when a single-molecule sample to be detected is through-hole can be further ensured.
Microfluidic top layer 20
As understood with reference to fig. 1 and 3, according to an embodiment of the present invention, the microfluidic top layer 20 includes a first groove 21, a sample injection flow channel 22, and a sample injection micro valve 23 for controlling the opening and closing of the sample injection flow channel 22, the first groove 21 is opened downward and the inner diameter of the first groove 21 is not smaller than the distance between the first electrode layer 14 and the second electrode layer 15. 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 opposite ends of the first electrode 14 and the second electrode 15. Wherein the first groove 21 is used for fixing the upper part of the graphene nanopore chip 10. In addition, the microfluidic top layer 20 may use PDMS as a material, and may print the microfluidic top layer substrate portion by using a 3D printing technology, to implement the arrangement of the micro flow channels and the groove structures, and set the micro valve structures in the sample injection flow channels.
Microfluidic bottom layer 30
According to an embodiment of the present invention, as will be understood with reference to fig. 1 and 4, the microfluidic bottom layer 30 includes a second groove 31, a sample outlet flow channel 32, and a sample outlet micro valve 33 for controlling the opening and closing of the sample outlet flow channel, where the second groove 32 is opened upwards and the inner diameter of the second groove 31 is not smaller than the size of the graphene nanopore chip 10, and the second groove 31 and the first groove 21 define a receiving space for a sample solution to be measured. The microfluidic bottom layer 30 may also use PDMS as a material, and may print the substrate portion of the microfluidic bottom layer by using a 3D printing technology, so as to implement the arrangement of the micro flow channel and the groove structure.
According to the embodiment of the 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. And finally, depositing a control/detection electrode at a corresponding point of the device, and arranging a micro-valve structure in a sample inlet channel and a sample outlet channel.
According to an embodiment of the present invention, as understood with reference to fig. 1, the bottom wall of the second groove 31 is bonded to the lower surfaces of the first support 11 and the second support 12, the height of the second groove 31 is not lower (preferably higher) than the heights of the first support 11 and the second support 12, the lower surface of the first groove 21 is bonded to the silicon dioxide layer b of the first support 11 and the second support 12, and the top wall of the first groove 21 is not lower (preferably higher) than the heights of the first electrode layer 11 and the second electrode layer 12. Therefore, the method is more beneficial to forming a closed accommodating space capable of accommodating the sample solution to be measured 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 they can cooperate with the graphene nanopore chip to define a closed accommodating space capable of accommodating a sample solution to be measured, for example, the first groove 21 may be a cylindrical groove, the second groove 31 may be a square groove, further, a substrate structure of the microfluidic top layer and the microfluidic bottom layer may be designed by drawing software, wherein a single-layer graphene layer (thickness 0.334 nm) may be adopted, the size of the microfluidic top layer may be 200mm×200mm×20mm (length×width×height), the diameter of a sample injection channel may be 50 μm, the diameter of the cylindrical groove may be 8mm, the depth of the groove may be 25 μm, the size of the microfluidic bottom layer may be 200mm×200mm×35mm (length×width×height), the diameter of a sample outlet channel may be 50 μm, the size of the square groove may be 15mm×15mm×500.3 μm (length×width×height), and the size of a control electrode communicating with an external control circuit and a detection circuit may be 5×5mm×5 mm.
Control circuit 40
According to an embodiment of the present invention, as understood with reference to fig. 1, the control circuit 40 includes a first power source 41, a signal generator 42 electrically connected to the first power source 41, a first control electrode 43 and a second control electrode 44, the first control electrode 43 is disposed on the microfluidic top layer 20 and adapted to be in contact with a sample solution to be tested, the second control electrode 44 is disposed on the microfluidic bottom layer 30 and adapted to be in contact with the sample solution to be tested, and the control circuit is used for providing a periodic forward and reverse modulation voltage for causing a periodic reciprocal reverse translocation event of a single molecule sample to be tested, i.e. for controlling a translocation state of the sample to be tested, and its main element is the signal generator 42. In the detection process, the ground pin of the first power supply 41 socket should be properly connected with the ground to avoid interference.
According to the embodiment of the present invention, the output voltage of the first power source 41 may be an ac voltage not greater than 2V, for example, may be 0.2V, 0.4V, 0.6V, 0.8V, 1.2V or 1.5V, etc., and the inventor finds that if the output voltage is too large, it may cause damage to related devices, and in the present invention, by adopting the output ac voltage not greater than 2V, not only the normal use of the devices may be ensured, but also the forward and reverse modulation voltage may be provided for the periodic reciprocal reverse translocation event occurring in the sample to be tested. Preferably, the output voltage of the first power supply 41 may be an ac voltage of 200-500 mv, and the normal use of the device may be further ensured by adopting the output voltage. Further, the frequency of the ac voltage outputted from the first power source 41 may be 1 to 20Hz, for example, 1Hz, 3Hz, 5Hz, 7Hz, 9Hz, 12Hz, 15Hz or 18Hz, which is found by the inventor that if the frequency of the ac voltage is too low, the via frequency of the sample molecule to be detected is affected, and if the frequency of the ac voltage is too fast, although the detection resolution is improved, the detection accuracy is affected, and part of the sample to be detected with the via may not be available yet, i.e. the via is pushed back by the reverse voltage, so that the periodic variation of the via translocation is inaccurate. In some embodiments of the invention, the ground of the first power outlet is grounded and the ground of the second power outlet is grounded.
Detection circuit 50
According to an embodiment of the present invention, as understood with reference to fig. 1, 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, where the first detection electrode 53 is connected to the first electrode layer 14, the second detection electrode 54 is connected to the second electrode layer 15, and the detection circuit is used for collecting graphene nano (through) hole facing electrical signals equal to a reference frequency, and the main element 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 earth to avoid interference.
According to the embodiment of the invention, the detection process of executing a certain sample to be detected by using the single-molecule periodic via hole modulation and signal detection device of the embodiment of the invention needs to be communicated with an external control circuit and a detection circuit, wherein the main element of the control circuit is a signal generator, the part is used for providing periodic positive and negative modulation voltage for enabling the sample to be detected to generate periodic reciprocating reverse translocation events, the main element of the detection circuit is a phase-locked amplifier, and the part is used for collecting graphene nanopore facing electric signals with the same reference frequency. When the device is in a detection state, the sample injection micro valve and the sample discharge micro valve are in a closed state, and periodic positive and negative modulation voltage is generated in the vertical direction of the device through the signal generator so as to realize that a sample to be detected periodically and reversely passes through the nanopore sensing area. When a single via event occurs to a sample to be tested, a corresponding via current signal is generated, so that the physical occupation caused by the event can change the facing electrical signal collected by an external detection circuit.
According to the embodiment of the invention, the method for carrying out single-molecule detection by utilizing the single-molecule periodic via hole modulation and signal detection device comprises the following steps of 1) connecting an external circuit, wherein the external circuit comprises a control circuit for driving a sample to be detected to flow and a detection circuit for collecting detection signals, the two circuits are connected with the device in a corresponding mode before detection, 2) dropwise adding the sample to be detected, firstly ensuring that micro valves of the device are in an open state, secondly dropwise adding quantitative buffer solution into a sample injection flow channel of a microfluidic top layer and a sample outlet flow channel of a bottom layer by utilizing a syringe pump, immediately closing the sample outlet micro valves, then dropwise adding quantitative sample to be detected into the sample injection flow channel of the microfluidic top layer, immediately closing all the micro valves after the process of dropwise adding the sample to be detected is finished, 3) controlling the sample to be detected, namely opening a signal generator to enable the signal generator to generate periodic forward and backward modulation voltage, enabling the sample to be detected sealed in the detection area to reciprocally shuttle in a sensing area of a nano hole (namely a through hole in the middle of a graphene layer), and 4) collecting signals, and taking the frequency of the periodic modulation voltage as the frequency of the phase-locked amplifier to realize extraction of the forward and backward modulation signal. Therefore, the problem that translocation events have randomness can be effectively solved, and accurate detection and extraction of translocation signals of samples to be detected by the lock-in amplifier can be realized.
In summary, in the single-molecule periodic via modulation and signal detection device of the embodiment of the invention, the microfluidic technology has the characteristics of accurately controlling and manipulating micro-scale fluid, combines the micro-scale fluid with the graphene nanopore technology, encapsulates a microfluidic component (i.e. a control circuit) outside a traditional nanopore chip, and enables a sample to be detected to pass through the nanopore periodically at a fixed frequency in the detection process, and has at least the following beneficial effects that 1) the spatial resolution of the nanopore is improved by taking single-layer or less graphene (preferably single-layer) as a sensing material, 2) the application of the vertical periodic forward and reverse modulation voltage of the device is realized by the arrangement of microelectrodes (i.e. the control electrode), so that the sample to be detected periodically passes through the nanopore in the detection process to increase the via detection times of the sample to be detected, the sample to be detected is enlarged to improve the high-sensitivity detection authenticity of the graphene nanopore, and effectively improve the problem that the translocation event has randomness, 3) the sample to be detected periodically passes through the nanopore by the application of the periodic forward and reverse voltage, and the amplifier can provide a phase-locked signal for the frequency of the sample to be detected to the amplifier, and the accurate phase-locked signal can be conveniently provided for the accurate and the accurate phase-locked channel to pass through the signal to be detected by the microchannel is ensured, and the accurate phase-locked channel is provided by the amplifier is convenient for the accurate and the detection channel to pass through the sample to be detected.
According to a further aspect of the present invention, a method for performing single molecule sample detection using the single molecule periodic via modulation and signal detection apparatus described above is provided. The method comprises the steps of (1) adjusting output voltage of a first power supply to be periodic positive and negative modulation voltage, (2) adding buffer solution into a sample injection flow channel and a sample outlet flow channel by using an injection pump, closing a sample outlet micro valve after adding a sample to be detected into the sample injection flow channel by using the injection pump, and closing the sample injection micro valve, (3) controlling flow direction of fluid of the sample to be detected by using a voltage signal output by a control circuit, ensuring that the output state of a signal generator is a periodic reverse electric field, and monitoring the detected signal state by a detection circuit, (4) enabling the sample to be detected to periodically reciprocate through a graphene layer through hole under the drive of the periodic reverse electric field, and obtaining translocation behavior of molecules of the sample to be detected by analyzing amplitude and time of a detection signal of the sample molecule to be detected by the detection circuit. Compared with the prior art, the method for detecting the single-molecule sample not only can realize the periodical modulation and detection of the single-molecule translocation signal, but also can solve the problem that the detection signal is weak and difficult to capture due to the defects of random via events, low signal to noise ratio and the like in the traditional nano-hole detection mode. It should be noted that the features and effects described in the foregoing description of the single-molecule periodic via modulation and signal detection apparatus are applicable to the method, and are not described herein in detail.
In order to facilitate understanding of the above-mentioned single-molecule periodic via modulation and signal detection device and the method for implementing single-molecule sample detection using the device, the following uses a double-stranded lambda-DNA sample to be detected as an example to describe the translocation behavior in detail, and the operation steps are as follows:
1) Connecting the external circuit, namely building an external control circuit and a detection circuit, wherein the detection circuit is shown in the embodiment of the invention in the schematic diagram of figure 1. The connection method of the control circuit comprises connecting the power line of the signal generator to 220V,50HZ AC power supply, and connecting the control electrode of the device to the circuit, wherein the ground wire pin of the power socket should be properly connected with the earth to avoid interference, each output knob on the panel should be rotated to minimum before starting up;
the connection method of the detection circuit comprises connecting the power line of the lock-in amplifier to the second power supply, and connecting the detection electrode of the device to the circuit;
2) Firstly, adding EDTA (ethylenediamine tetraacetic acid) solution with the concentration of 1mmol/L and Tris-HCl (composed of Tris (hydroxymethyl) aminomethane and hydrochloric acid) solution with the concentration of 10mmol/L into a sample injection flow channel and a sample discharge flow channel by using an injection pump, immediately closing a sample discharge micro valve, and then injecting DNA solution with the concentration of 2 mug/mL into the sample injection flow channel, and immediately closing the sample injection micro valve;
3) And controlling the sample to be detected, namely controlling the flow direction of fluid through the voltage signal output by the control circuit in the early stage of dripping the sample to be detected, ensuring the output state of the signal generator to be a periodic reverse electric field after the process of dripping the sample to be detected is finished, and monitoring the detection signal state of the device through the detection circuit. When the frequency and amplitude area of the signal to be detected is stable, whether the sample to be detected stably shuttles back and forth in the nanopore detection area or not can be judged;
4) And analyzing signals, namely, periodically reciprocating the DNA molecules in a nanopore detection area under the drive of periodic positive and negative modulation voltage, wherein when the DNA molecules pass through the nanopore once, the physical occupation causes the detection signals of the device to change, corresponding via hole time is generated, and the translocation behavior of double-chain lambda-DNA molecules in a sample to be detected can be obtained by analyzing the amplitude and time of the detection signals. FIG. 6 is a graph showing the comparison of the detection effect of the DNA molecules of the sample to be detected when the forward and reverse modulation voltages are not applied (as shown in FIG. 6 (a)) and when the forward and reverse modulation voltages are applied (as shown in FIG. 6 (b)).
According to a further aspect of the present invention, the present invention proposes the use of the above-described single molecule periodic via modulation and signal detection device or a method for performing single molecule sample detection using the above-described single molecule periodic via modulation and signal detection device in the field of single molecule detection. Thus, the features and effects described with respect to the above-mentioned single-molecule periodic via modulation and signal detection apparatus and method for single-molecule sample detection are applicable to this application, and will not be described in detail herein.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (7)
1. The method for detecting the single-molecule sample by adopting the single-molecule periodic via hole modulation and signal detection device is characterized in that the single-molecule sample is DNA or protein,
The single-molecule periodic via modulation and signal detection device comprises:
The graphene nanopore chip comprises a first supporting part, a second supporting part, a graphene layer, a first electrode layer and a second electrode layer, wherein the first supporting part and the second supporting part are arranged at intervals, the first supporting part and the second supporting part 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 supporting part and the second supporting part through the first electrode layer and the second electrode layer, a nanometer through hole is formed in the middle of a suspended area, part of the first electrode layer is connected with the silicon dioxide layer on the first supporting part and the rest part of the first electrode layer is connected with one end of the graphene layer in a covering mode, and the other end of the graphene layer is connected with the silicon dioxide layer on the second supporting part in a covering mode.
The microfluidic top layer comprises a first groove, a sample injection flow channel and a sample injection micro valve for controlling the opening and closing of the sample injection flow channel, the opening of the first groove is downward, and the inner diameter of the first groove is not smaller than the distance between the first electrode layer and the second electrode layer;
The microfluidic substrate comprises a second groove, a sample outlet flow channel and a sample outlet micro valve for controlling the opening and closing of the sample outlet flow channel, the opening of the second groove is upward, the inner diameter of the second groove is not smaller than the size of the graphene nanopore chip, and the second groove and the first groove define an accommodating space of a sample solution to be detected;
the control circuit comprises a first power supply, a signal generator, a first control electrode and a second control electrode, wherein the signal generator is electrically connected with the first power supply, the first control electrode is arranged on the microfluidic top layer and is suitable for being contacted with a sample solution to be tested, the second control electrode is arranged on the microfluidic bottom layer and is suitable for being contacted with the sample solution to be tested, and the control circuit is used for providing a periodic positive and negative modulation voltage for enabling a single-molecule sample to be tested to generate a periodic reciprocating reverse translocation event;
And
The detection circuit comprises a second power supply, a phase lock amplifier electrically connected with the second power supply, a first detection electrode and a second detection electrode, wherein the first detection electrode is connected with the first electrode layer, the second detection electrode is connected with the second electrode layer, the detection circuit is used for collecting graphene nanopore facing electric signals with the same reference frequency,
Wherein,
The aperture of the nano through hole is 1.1-1.6 times of the particle size of the single molecule to be detected;
the thickness of the graphene layer is not more than 1nm;
the method comprises the following steps:
(1) Adjusting the output voltage of the first power supply to be a periodic positive and negative modulation voltage;
(2) Adding buffer solution into the sample injection flow channel and the sample outlet flow channel by using a syringe pump, closing the sample outlet micro valve, and closing the sample injection micro valve after a sample to be detected is added into the sample injection flow channel by using the syringe pump;
(3) The flow direction of the fluid of the sample solution to be detected is controlled by the voltage signal output by the control circuit, the output state of the signal generator is ensured to be a periodic reverse electric field, and the detected signal state is monitored by the detection circuit;
(4) And the sample to be detected is driven by the periodic reverse electric field to do periodic reciprocating motion through the graphene layer through hole, and the translocation behavior of the sample molecule to be detected is obtained by analyzing the amplitude and time of the detection signal of the sample molecule to be detected, which is detected by the detection circuit.
2. The method of claim 1, wherein a bottom wall of the second groove is bonded to lower surfaces of the first support portion and the second support portion, a height of the second groove is not lower than a height of the first support portion and the second support portion, a lower surface of the first groove is bonded to silicon dioxide layers of the first support portion and the second support portion, and a top wall of the first groove is not lower than a height of the first electrode layer and the second electrode layer.
3. The method of claim 1, wherein the output voltage of the first power source is an ac voltage of no more than 2V.
4. The method of claim 3, wherein the output voltage is 200-500 mv.
5. A method according to claim 3, wherein the ac voltage has a frequency of 1 to 20hz.
6. The method of claim 1 or 4, wherein the ground of the first power outlet is grounded and the ground of the second power outlet is grounded.
7. Use of the method of any one of claims 1-6 in the field of single molecule detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210298400.XA CN114878800B (en) | 2022-03-24 | 2022-03-24 | Single-molecule periodic pore modulation and signal detection device based on nanopore technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210298400.XA CN114878800B (en) | 2022-03-24 | 2022-03-24 | Single-molecule periodic pore modulation and signal detection device based on nanopore technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114878800A CN114878800A (en) | 2022-08-09 |
| CN114878800B true CN114878800B (en) | 2025-01-07 |
Family
ID=82667823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210298400.XA Active CN114878800B (en) | 2022-03-24 | 2022-03-24 | Single-molecule periodic pore modulation and signal detection device based on nanopore technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114878800B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116127288B (en) * | 2023-04-14 | 2023-09-15 | 南京邮电大学 | Nanopore sensing signal noise removal method and device based on independent component analysis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901763A (en) * | 2012-09-25 | 2013-01-30 | 清华大学 | Deoxyribonucleic acid (DNA) sequencing device based on graphene nanopore-microcavity-solid-state nanopore and manufacturing method |
| CN109196343A (en) * | 2016-06-03 | 2019-01-11 | 株式会社日立高新技术 | Biomolecule measuring device |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9921181B2 (en) * | 2014-06-26 | 2018-03-20 | International Business Machines Corporation | Detection of translocation events using graphene-based nanopore assemblies |
| CN104713932B (en) * | 2015-03-24 | 2017-03-29 | 清华大学 | A kind of multiparameter nanometer pore single-molecule analyser of AC mode |
| CN110346579B (en) * | 2019-07-24 | 2023-07-07 | 中国科学院重庆绿色智能技术研究院 | In vitro HIV protease detection instrument and method based on nanopore |
| CN114715840B (en) * | 2022-04-11 | 2023-09-05 | 清华大学 | Differential suspension single-layer graphene nanopore sensor and preparation method and application thereof |
-
2022
- 2022-03-24 CN CN202210298400.XA patent/CN114878800B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901763A (en) * | 2012-09-25 | 2013-01-30 | 清华大学 | Deoxyribonucleic acid (DNA) sequencing device based on graphene nanopore-microcavity-solid-state nanopore and manufacturing method |
| CN109196343A (en) * | 2016-06-03 | 2019-01-11 | 株式会社日立高新技术 | Biomolecule measuring device |
Non-Patent Citations (1)
| Title |
|---|
| Molecular Dynamics Simulation of a Single Carbon Chain through an Asymmetric Double-Layer Graphene Nanopore for Prolonging the Translocation Time;Yaolong Zhou, et al.;ACS Omega;20220506;第7卷(第19期);16422-16429 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114878800A (en) | 2022-08-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9989516B2 (en) | Electro-diffusion enhanced bio-molecule charge detection using electrostatic interaction | |
| CA2872602C (en) | Fabrication of nanopores using high electric fields | |
| US7777476B2 (en) | Dynamic modulation for multiplexation of microfluidic and nanofluidic based biosensors | |
| US10145846B2 (en) | Digital protein sensing chip and methods for detection of low concentrations of molecules | |
| JP2016502644A (en) | Integrated circuit having sensing transistor array, sensing device, and measuring method | |
| CN114878800B (en) | Single-molecule periodic pore modulation and signal detection device based on nanopore technology | |
| RU2638132C2 (en) | Integrated circuit with nano-conductive sensors in field transistors, manufactured by chemical method, sensor device, method of measurement and method of manufacture | |
| Leyden et al. | Fabrication and characterization of carbon nanotube field-effect transistor biosensors | |
| Kumar et al. | Progresses in nanopores fabrications and nanopore sequencing | |
| Xu et al. | Single-Molecule Detection Based on Graphene Cage-like Nanopores | |
| Liu et al. | Integrated Nanopore Device for Electronic Single Molecule Trapping in Femtolitre Cavities Fabricated by Self-Aligned Etching | |
| Bouhamidi et al. | Characterizing Surface Charge Density of Solid-State Nanopore Sensors for Improved Biosensing Applications | |
| Sacco et al. | Transfer-Free Integration of Graphene on Suspended Micro-Hotplates for No2 Sensing | |
| Handziuk | Low-noise cost-efficient nanowire structures for biosensing applications | |
| Wang et al. | Fabrication of Multi-Size Nanopores Auto-Positioning on Pyramidal Membrane Tip by Multi-Level Voltage Pulses | |
| US20130040313A1 (en) | Nanofluidic biochemical sensors based on surface charge modulated ion current | |
| Yu | Nanomaterials characterization and bio-chemical sensing using microfabricated devices | |
| CN105866398B (en) | Nano channel for detecting specific binding of antigen and antibody, and preparation method and detection method thereof | |
| Zulfiqar | Novel Diagnostic Method for Personalized Treatment of Cancer | |
| Yemenicioglu | Stability and bandwidth investigation of alternative structures for nanopore sensors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |