Real-time detection device for protein unfolding dynamics

Abstract

The embodiment of the invention discloses a real-time detection device for protein unfolding dynamics, and relates to the technical field of macromolecule detection. The silicon nitride device is provided with a molybdenum disulfide nano-channel which is used as a channel for protein to flow; firstly, the surface of molybdenum disulfide is irradiated by laser, surface charges of molybdenum disulfide can be changed, so that electrically neutral protein is driven to enter a nano channel under the action of an electroosmotic flow effect, and meanwhile, the protein in the channel is unfolded due to the heat effect of the laser; therefore, the change of the protein structure can be detected in real time according to the change of the current. The surface charge of the molybdenum disulfide material is regulated and controlled by utilizing a laser technology, and the electroosmotic flow intensity and direction in the nano channel are changed, so that the detection of the electrically neutral protein is realized; the temperature in the nano-channel is changed by using the thermal effect of laser, so that the protein is unfolded in the nano-channel, and the whole protein unfolding dynamic process is detected in real time.

Description

Technical Field

[0001] The present invention relates to the technical field of macromolecular detection, and in particular to a real-time detection device for protein unfolding dynamics. Background Art

[0002] There are many types of proteins with different properties and functions. In various fields such as biomedicine, healthcare, microbiology research, life / biological sciences, there are many research links involving protein changes. For example, changes in the pH value, temperature, and ion concentration of the solution may cause changes in the structure of the protein, that is, the transition from folding to unfolding is one of the main forms of structural change.

[0003] If the changes in protein structure are wrong, it will cause many problems. For example, in the field of human medicine, incorrect changes in protein structure can induce a variety of diseases in the human body, such as Alzheimer's disease, Parkinson's disease, etc. Another example: in biological fermentation technology, if the changes in certain protein structures produced by fermentation are abnormal, it will seriously reduce the quality of the final product.

[0004] Therefore, studying the structure and folding of proteins is of great practical significance for research involving protein changes. The commonly used detection method at present is to first increase the temperature to unfold the protein and then conduct structural research. However, these methods cannot detect protein unfolding in real time, especially how to clearly detect the unfolding of electrically neutral proteins, which has become a topic that needs to be studied. Summary of the invention

[0005] The embodiments of the present invention provide a real-time detection device for protein unfolding kinetics, which can be used for real-time unfolding detection of the unfolding kinetics of electrically neutral proteins.

[0006] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0007] A real-time detection device for protein unfolding dynamics, the components of the real-time detection device include: a lower liquid pool (1), an upper liquid pool (2), a laser source (3), a silicon nitride device (5), a thin film layer (4) covering the silicon nitride device (5); the thin film layer (4) is provided with a micron-scale pore, the middle of the micron-scale pore is connected to a nanochannel, wherein silicon in the silicon nitride device is etched, the silicon nitride film is suspended on the silicon substrate, and the silicon nitride film has a micron hole connected to the micron hole on the film. The upper surface of the lower liquid pool (1) is provided with a mounting groove, the size and shape of the mounting groove match the bottom of the upper liquid pool (2), the upper liquid pool (2) is embedded and installed in the mounting groove so that the bottom of the upper liquid pool (2) extends into the lower liquid pool (1), and the extension depth is 2 to 10 mm. The bottom of the upper liquid pool (2) is provided with a through hole, and the through hole is used to connect the upper liquid pool (2) and the lower liquid pool (1).

[0008] The real-time detection device for protein unfolding dynamics provided by the embodiment of the present invention makes a molybdenum disulfide nanochannel on a silicon nitride device, then fixes it on an upper liquid pool, and finally irradiates the upper surface of the nanochannel with a laser. After the molybdenum disulfide nanochannel is manufactured, the silicon nitride device is placed on the small hole of the upper liquid pool, and the device and the liquid pool are sealed with AB glue, and then the upper liquid pool is placed on the edge of the lower liquid pool, and the salt solution containing protein is poured into the liquid pool. Under the action of the photoelectric effect, the electroosmotic flow in the nanochannel is enhanced, driving the protein into the nanochannel. Under the action of the thermal effect, the protein unfolds in the nanochannel, thereby detecting the changes in the unfolding dynamics of the protein in real time. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0010] Figure 1 A schematic diagram of the overall structure provided by an embodiment of the present invention;

[0011] Figure 2 A schematic diagram of a silicon nitride device provided by an embodiment of the present invention;

[0012] Figure 3 Schematic diagram of micrometer-scale pores and nanometer channels provided in an embodiment of the present invention.

[0013] Figure 4 Schematic diagram of protein passing through the nanochannel;

[0014] The numbers therein represent: lower liquid pool-1, upper liquid pool-2, laser source-3, thin film layer-4, silicon nitride device-5, electrode-6, silicon plate-501, silicon nitride film-502, lower molybdenum disulfide film-401, upper molybdenum sulfide film-402, polymer PMMA film-8, silicon nitride hole-9, molybdenum disulfide nanochannel-10, bi-conical micron channel-11, lower molybdenum disulfide film-401, molybdenum disulfide isolation zone-12. DETAILED DESCRIPTION

[0015] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention will be described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention. It can be understood by those skilled in the art that, unless specifically stated, the singular forms "one", "one", "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present invention refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. It should be understood that when we say that an element is "connected" or "coupled" to another element, it can be directly connected or coupled to other elements, or there may also be intermediate elements. In addition, the "connection" or "coupling" used here may include wireless connection or coupling. The term "and / or" used herein includes any unit and all combinations of one or more associated listed items. It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as generally understood by those of ordinary skill in the art to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless defined as herein.

[0016] The present invention provides a real-time detection device for protein unfolding kinetics. Figure 1 , 2As shown, the components of the real-time detection device include: a lower liquid pool (1), an upper liquid pool (2), a laser source (3), a silicon nitride device (5), and a thin film layer (4) covering the silicon nitride device (5); optionally, the material of the thin film layer (4) is molybdenum disulfide or tungsten disulfide. In this embodiment, the rigid silicon nitride device (5) is made of a composite of silicon and silicon nitride, or a composite of silicon, silicon nitride and silicon dioxide, wherein a layer of silicon nitride or silicon nitride and silicon dioxide is grown on a silicon plate.

[0017] The thin film layer (4) is provided with micron-sized pores, wherein the middle of the micron-sized pores is connected to a nano-channel;

[0018] The upper surface of the lower liquid pool (1) is provided with a mounting groove, the size and shape of which match the bottom of the upper liquid pool (2). The upper liquid pool (2) is embedded and installed in the mounting groove so that the bottom of the upper liquid pool (2) extends into the lower liquid pool (1), and the extension depth is 2 to 10 mm. A through hole is provided at the bottom of the upper liquid pool, and the silicon nitride device (5) is installed on the through hole, and the diameter of the through hole is 1 to 3 mm. It should be noted that the symbol "~" used in describing the numerical range in this embodiment means greater than or equal to the value on the left side of the interval, and less than or equal to the value on the right side of the interval. For example, the extension depth is 2 to 10 mm, which can be understood as: 2 mm ≤ extension depth ≤ 10 mm, or as extension depth ∈ [2 mm, 10 mm]. The through hole is used to connect the upper liquid pool (2) and the lower liquid pool (1).

[0019] In this embodiment, the silicon nitride device (5) comprises: a silicon plate (501) and a silicon nitride film (502) grown on the silicon plate (501); in the silicon nitride device (5), the silicon nitride film (502) is suspended on the silicon plate (501), and a silicon nitride hole (9) is provided on the silicon nitride film (502). The silicon nitride device (5) is rigid and not easy to bend, and can well protect the nanochannel without deformation. The thickness of the silicon nitride film (502) is 100nm to 300nm.

[0020] If molybdenum disulfide is used to make the thin film layer (4), the molybdenum disulfide is transferred to the silicon nitride film (502) by dry transfer technology to form a lower molybdenum disulfide film (401), and the lower molybdenum disulfide film (401) is provided with a bi-conical micron channel (11) and a nanochannel (10), thereby completing the processing of the molybdenum disulfide nanochannel.

[0021] Specifically, after the molybdenum disulfide is transferred to the silicon nitride film (502) by dry transfer technology, an ion beam is used to bombard the lower molybdenum disulfide film (401) to form a bi-conical micro-channel (11), and the inner wall of the bi-conical micro-channel (11) covers the lower molybdenum disulfide film (401). Figure 3 As shown, two conical micrometer-sized channels of the same shape form a double-conical micrometer channel (11), and the double-conical micrometer channel (11) is tightened toward the center position to facilitate the opening of a nanometer channel (10). After the double-conical micrometer channel (11) is formed by ion beam bombardment, a hole is punched at the center position of the double-conical micrometer channel (11) by dielectric perforation technology to form a nanometer channel (10). Furthermore, the nanometer channel can also be formed by using transition metal two-dimensional materials such as molybdenum diselenide. Compared with graphene two-dimensional materials, transition metal two-dimensional materials have more charges on their surfaces, and the charges are more easily regulated.

[0022] Specifically, a silicon nitride substrate can be manufactured by low-pressure chemical vapor deposition, and then a suspended silicon nitride film can be obtained by reactive ion etching. Then, a micron hole can be machined in the silicon nitride film by ion beam processing. Molybdenum sulfide can be transferred to the silicon nitride film by a wet transfer method, and then a nanochannel can be machined in the molybdenum disulfide by ion beam. Finally, another layer of molybdenum disulfide can be transferred to the processed nanochannel as a cover plate, thereby forming a closed nanochannel.

[0023] In a preferred embodiment, the width of the large end of the tapered micrometer-sized pore is 1 to 5 μm, and the width of the small end is 0.02 to 0.1 μm; the diameter of the nanochannel (10) is 1 to 10 nm. The height of the nanochannel is 1 to 10 nm. When the height of the nanochannel is less than 1 nm, the nanochannel will collapse, thereby affecting the inability of proteins to pass through the nanochannel; when the height of the nanochannel is higher than 10 nm, the electroosmotic flow effect of the nanochannel will not be obvious, thereby failing to drive electrically neutral proteins into the nanochannel. The length of the nanochannel (10) is greater than 20 nm or less than 50 nm; when the length of the nanochannel is less than 20 nm, the nanochannel is difficult to process and is easy to break; when the length of the nanochannel is greater than 50 nm, the protein permeation signal will be weak.

[0024] In this embodiment, an upper molybdenum disulfide film (402) with a thickness of 50nm to 100nm is covered on the upper surface of the lower molybdenum disulfide film (401), and a polymer PMMA film (8) with a thickness of 300nm to 1μm is covered on the upper surface of the upper molybdenum disulfide film (402).

[0025] Furthermore, the method applied to the real-time detection device includes:

[0026] The silicon nitride device (5) is mounted on a mounting hole provided at the bottom of the upper liquid pool (2), and the contact point between the silicon nitride device (5) and the mounting hole is sealed using AB glue;

[0027] Pour the salt solution containing protein into the lower liquid pool (1) and the upper liquid pool (2), remove the bubbles in the solution, and finally insert an electrode (6) into the lower liquid pool (1) and the upper liquid pool (2) respectively;

[0028] The laser source (3) aims at the nanochannel (10) to irradiate the laser, and at the same time, the electrode (6) is energized to form an electric field, and the protein (7) is driven to pass through the nanochannel (10) under the action of electroosmotic flow, and the protein unfolding change is obtained through the change of current.

[0029] An ion beam is used to etch a stepped channel on a molybdenum disulfide film step by step in both directions, and finally a double-conical micrometer channel is formed, and a molybdenum disulfide isolation zone (12) with a length of 20 to 50 nm is reserved between the two channels. The breakdown voltage of the molybdenum disulfide nanochannel manufactured by dielectric breakdown is 10 to 30 V, and the diameter of the nanochannel is 1 to 10 nm. The wavelength of the laser emitted by the laser source (3) is 300 to 700 nm, the laser intensity is 1 to 10 mW, and the laser spot diameter is 0.5 to 2 μm.

[0030] In practical applications, the method for real-time detection of protein unfolding using the device of this embodiment includes:

[0031] First, a MoS2 nanochannel is made on a silicon nitride device. After the MoS2 nanochannel is made, it is encapsulated in a small hole at the upper end of a liquid pool, and then fixed on the upper liquid pool. Finally, a laser is irradiated on the upper surface of the nanochannel. Under the action of the photoelectric effect, the electroosmotic flow in the nanochannel is enhanced, driving the protein into the nanochannel. Under the action of the thermal effect, the protein unfolds in the nanochannel, thereby detecting the changes in the unfolding kinetics of the protein in real time. Figure 1 As shown, the upper end of the liquid pool is placed on the edge of the lower end of the liquid pool, and a solution containing protein (potassium chloride or sodium chloride solution) is placed in the liquid pool. The solution in the upper liquid pool is separated from the solution in the lower liquid pool by a silicon nitride device. Then electrodes are inserted into the upper and lower liquid pools, and both ends of the electrodes are connected to a power supply. Under the action of the voltage, the protein enters the molybdenum disulfide nanochannel. Finally, a white light-laser confocal system is placed at the upper end of the liquid pool. After determining the position of the molybdenum sulfide nanochannel, the intensity and wavelength of the laser are adjusted to change the surface charge of the molybdenum disulfide film layer, thereby increasing the electroosmotic flow intensity in the nanochannel. Under the action of an external electric field, the protein 7 in the solution enters the molybdenum disulfide nanochannel. At the same time, under the thermal effect of the laser, the protein entering the nanochannel is unfolded, resulting in a change in the ion current. As shown Figure 4 As shown in FIG. 1 , when no protein passes through the nanochannel, the current in the nanochannel is the reference current. When the protein passes through the nanochannel, the current in the channel changes, thereby generating a blocking current, ΔI(t) = I0-I pore(t), where ΔI(t) is the current change at time t, I0 is the baseline current in the absence of protein, and I pore (t) is the current when the protein is in the nanopore, which depends on the conformation and position of the protein. Therefore, the change of blocking current can be used to analyze the structure and dynamic changes of the protein in real time.

[0032] For example: in actual use, a salt solution containing protein can be placed at both ends of the liquid pool, and then the position of the molybdenum disulfide nanochannel can be determined by a white light-laser confocal system. The laser is irradiated on the nanochannel, and the energy of the laser is adjusted. Under the irradiation of the laser, the protein in the nanochannel unfolds. An electric field is added at both ends of the liquid pool, and the protein is driven through the nanochannel under the action of electroosmosis. The changes in protein unfolding are obtained through changes in current.

[0033] Compared with the prior art, the advantages of this embodiment are: protein unfolding kinetics detection can detect not only charged proteins, but also electrically neutral proteins; the nanochannels used use double-tapered micron channels to connect the nanochannels, which can ensure that the proteins can pass through the nanochannels smoothly and ensure that the unfolding signals of the proteins in the nanochannels are more obvious; laser irradiation of the nanochannels can cause the proteins to unfold in the nanochannels, thereby realizing real-time observation of the kinetic behavior of the proteins.

[0034] The nanochannel in this embodiment can replace molybdenum disulfide with other transition sulfide metal compounds, and can also achieve real-time detection of protein structure and unfolding dynamics.

[0035] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the device embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment. The above is only a specific implementation method of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A real-time detection device for protein unfolding kinetics, characterized in that: The components of the real-time detection device include: a lower liquid pool (1), an upper liquid pool (2), a laser source (3), a silicon nitride device (5), and a thin film layer (4) covering the silicon nitride device (5); The thin film layer (4) is provided with micron-sized pores, wherein the middle of the micron-sized pores is connected to a nano-channel; The upper surface of the lower liquid tank (1) is provided with a mounting groove, the size and shape of which match the bottom of the upper liquid tank (2), and the upper liquid tank (2) is embedded and mounted in the mounting groove so that the bottom of the upper liquid tank (2) extends into the lower liquid tank (1); A through hole is provided at the bottom of the upper liquid pool (2), and the through hole is used to connect the upper liquid pool (2) and the lower liquid pool (1).

2. The device according to claim 1, characterized in that The material of the thin film layer (4) is molybdenum disulfide or tungsten disulfide.

3. The device according to claim 2, characterized in that The silicon nitride device (5) comprises: a silicon plate (501) and a layer of silicon nitride film (502) grown on the silicon plate (501); If molybdenum disulfide is used to make the thin film layer (4), the molybdenum disulfide is transferred to the silicon nitride film (502) by dry transfer technology to form a lower molybdenum disulfide film (401), and the lower molybdenum disulfide film (401) is provided with a bi-conical micron channel (11) and a nanochannel (10), thereby completing the processing of the molybdenum disulfide nanochannel; wherein the thickness of the silicon nitride film (502) is 100nm to 300nm.

4. The device according to claim 3, characterized in that After the molybdenum disulfide is transferred onto the silicon nitride film (502) by dry transfer technology, ion beam bombardment is used to form a bi-conical micron channel (11), at which time the inner wall of the bi-conical micron channel (11) covers the lower molybdenum disulfide film (401).

5. The device according to claim 4, characterized in that A double-conical micron-sized channel (11) is formed by two conical micron-sized channels of the same shape, and the double-conical micron-sized channel (11) is tightened toward the center position to facilitate the opening of a nanochannel (10); After forming a double-conical micron-hole (11) by ion beam bombardment, a hole is punched at the center of the isolation zone (12) between the double-conical micron-holes (11) by dielectric perforation technology to form a nanochannel (10), and at this time, the inner wall of the nanochannel (10) is also covered with the lower molybdenum disulfide film (401).

6. The device according to claim 5, characterized in that The width of the large end of the tapered micrometer-scale channel is 1 to 5 μm, and the width of the small end is 0.02 to 0.1 μm; the diameter of the nanochannel (10) is 1 to 10 nanometers.

7. The device according to claim 6, characterized in that The length of the nanochannel (10) is greater than 20 nm or less than 50 nm.

8. The device according to claim 3, characterized in that The upper surface of the lower molybdenum disulfide film (401) is covered with an upper molybdenum disulfide film (402) with a thickness of 50nm to 100nm, and the upper surface of the upper molybdenum disulfide film (402) is covered with a polymer PMMA film (8) with a thickness of 300nm to 1μm.

9. The device according to claim 1, characterized in that A method applied to the real-time detection device, comprising: The silicon nitride device (5) is mounted on a through hole provided at the bottom of the upper liquid pool (2), and the contact point between the silicon nitride device (5) and the through hole is sealed using AB glue; Pour the salt solution containing protein into the lower liquid pool (1) and the upper liquid pool (2), remove the bubbles in the solution, and finally insert an electrode (6) into the lower liquid pool (1) and the upper liquid pool (2) respectively; The laser source (3) aims at the nanochannel (10) to irradiate the laser, and at the same time, the electrode (6) is energized to form an electric field, and the protein (7) is driven to pass through the nanochannel (10) under the action of electroosmotic flow, and the protein unfolding change is obtained through the change of current.

10. The device according to claim 1, characterized in that The protein unfolding change is obtained by the change of the current, including: The current in the nanochannel (10) when no protein passes through is taken as a reference current; When the protein passes through the nanochannel (10), the current in the channel changes and a blockage occurs, which is expressed as: ΔI(t) = I0-I pore (t), where ΔI(t) is the current change with time t, I0 is the baseline current in the absence of protein, and I pore (t) is the current when the protein is in the nanopore. The conformation and position of the protein will affect I pore The value of (t).

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