Protein photoelectric joint detection platform and detection method based on plasmon nanochannel structure

Abstract

The invention belongs to the technical field of solid-state nanopore protein detection, and particularly relates to a protein photoelectric joint detection platform based on a plasmon nanochannel structure and a detection method.The construction method of the detection platform comprises the steps that surface decontamination treatment is conducted on a silicon-based silicon nitride film; then depositing a gold film on the surface of the silicon-based silicon nitride by using a magnetron sputtering technology, processing a gold film structure by using a focused ion beam, manufacturing a nano-pore channel in the center of the structure, manufacturing a double-bowknot structure around the nano-pore channel, and manufacturing a nano-pore chip; the preparation method comprises the following steps: preparing a nanopore chip, assembling the prepared nanopore chip and a flowcell to obtain a protein photoelectric joint detection platform based on a plasmon nanochannel structure, and constructing a novel plasmon nanochannel device for a trace protein photoelectric joint detection technology by combining a plasmon effect with a solid-state nanopore technology. The sensitivity and the accuracy of protein detection are improved, and richer protein monomolecular structure information is obtained through complementary analysis of optical and electric two-dimensional signals.

Description

Technical Field

[0001] The present invention belongs to the technical field of solid-state nanopore protein detection, and specifically relates to a protein photoelectric combined detection platform and detection method based on a plasmon nanochannel structure. Background Art

[0002] The use of solid-state nanopores to detect proteins is a biosensing technology based on nanoscale pore structures. Nanoscale pores are prepared on solid substrates (such as silicon dioxide, silicon nitride, etc.), and the electric field drives protein molecules to pass through these pores. At the same time, the changes in electrical signals generated when protein molecules pass through the pores (such as current blocking events) are monitored, thereby achieving single-molecule detection of protein molecules. This technology has the advantages of high sensitivity, high throughput, and high spatiotemporal resolution, and has broad application prospects in the fields of biological analysis and disease diagnosis.

[0003] Although traditional solid-state nanopore protein detection technology has many advantages, such as high sensitivity, real-time monitoring and simple signal analysis, it has been used in protein detection, but there are still some limitations in practical applications. Their size needs to be precisely controlled to meet the high-sensitivity detection of protein molecules or amino acid molecules. However, it is a technical challenge to manufacture holes with diameters ranging from sub-nanometer to nanometer levels and maintain their uniformity and stability. At the same time, during the solid-state nanopore detection process, it may be interfered by factors such as impurities from the sample itself and ions in the solution, resulting in increased signal noise and affecting the accuracy of the results. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a protein optoelectronic combined detection platform and detection method based on a plasmon nanochannel structure. By combining the plasmon effect with the solid-state nanopore technology, a new plasmon nanochannel device is constructed for trace protein optoelectronic combined detection, so as to improve the sensitivity and accuracy of protein detection, and obtain more abundant protein single molecule structure information through the complementary analysis of optical and electrical two-dimensional signals.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The purpose of the present invention is to provide a protein optoelectronic combined detection platform based on a plasmon nanochannel structure. The construction method of the detection platform includes: performing surface decontamination treatment on a silicon-based silicon nitride film, then using magnetron sputtering technology to deposit a gold film on the silicon-based silicon nitride surface, using a focused ion beam to process the gold film structure, manufacturing a nanopore in the center of the structure, and manufacturing a double bow-tie structure around the nanopore to make a nanopore chip, and then assembling the prepared nanopore chip with a flowcell to obtain a protein optoelectronic combined detection platform based on a plasmon nanochannel structure.

[0007] The plasmon effect is a special optical phenomenon that occurs on the surface of metal nanostructures. When light hits these nanostructures, the free electrons on the metal surface resonate with the light waves, forming a strong local electromagnetic field enhancement, the so-called "hot spot". This hot spot effect can greatly enhance the interaction between light and matter, including surface enhanced Raman scattering (SERS), light absorption and scattering, thereby achieving highly sensitive optical detection at the nanoscale.

[0008] The present invention combines the plasmon effect with solid-state nanopore technology to construct a new type of plasmon nanochannel device for trace protein photoelectric detection. Among them, the plasmon nanostructure is cleverly integrated around the solid-state nanopore, and the hot spot effect and light capture effect generated by the plasmon effect are used to enhance the optical signals of protein molecules near the nanopore (such as surface enhanced Raman, light absorption scattering and other optical signals).

[0009] Furthermore, the surface decontamination treatment method for the silicon-based silicon nitride film is as follows: after the silicon-based silicon nitride film is treated with an acetone solution, it is washed with ethanol and ultrapure water in sequence to remove organic and inorganic pollutants on the surface of the film.

[0010] Furthermore, a method for depositing a gold film on the surface of silicon-based silicon nitride using magnetron sputtering technology is as follows: the treated silicon-based silicon nitride film is fixed in a magnetron sputtering coating machine with conductive glue, and in a vacuum environment, the inert gas is ionized, the magnetic field is enhanced, and the gold target is bombarded with ions, and the gold target falls off to form a layer of gold film on the silicon nitride film.

[0011] Furthermore, the thickness of the gold film is 20-50 nm.

[0012] Furthermore, the method for processing the gold film structure using a focused ion beam is as follows: etching is performed using a focused ion beam system with a voltage of 30 keV and a current of 5 pA.

[0013] Furthermore, the nanopores prepared on the silicon-based silicon nitride core are 3-10nm.

[0014] Furthermore, the double bow-tie structure is composed of four symmetrical isosceles triangles.

[0015] Furthermore, the prepared nanopore chip was assembled with the flowcell, and the two chambers of the flowcell were connected to the e-One-HS micro patch clamp through Ag / AgCl electrodes and equipped with a Raman spectrometer. The Raman spectrometer was used to collect Raman signals, and the patch clamp was used to record the electrical signals of protein molecules.

[0016] The present invention also aims to provide a detection method for a protein optoelectronic combined detection platform based on a plasmon nanochannel structure. The protein detection method comprises: preparing a test solution and a complex of the test solution and nanogold particles, adding the test solution to the cis side of the flowcell, and adding only a blank electrolyte to the trans side. When starting the test, first, only the eOne-HS micro-patch clamp is turned on, a 100mV bias is applied, and the electrical signal is tested; secondly, only the Raman spectrometer is turned on to collect the Raman signal of the protein molecule; finally, the patch clamp and the Raman spectrometer are turned on at the same time, and their electrical signals and optical signals are tested at the same time. Subsequently, the complex of the test solution and nanogold particles is added to the cis side of the flowcell, and only a blank electrolyte is added to the trans side, and the above test operation is repeated.

[0017] After combining the plasmon effect with solid-state nanopore technology, the protein molecules are driven through the nanopore by the electric field to achieve the joint detection of optical and electrical signals. This comprehensive technology can not only improve the sensitivity of protein detection, but also obtain more abundant protein single molecule structure information, such as conformational changes, intermolecular interactions, etc., through the complementary analysis of optical and electrical two-dimensional signals.

[0018] Compared with the prior art, the beneficial technical effects of the present invention are:

[0019] 1. The present invention is based on single-molecule solid-state nanochannel electrical detection technology and optical microscopy technology, using silicon-based silicon nitride film with a gold double bow-tie plasmon structure deposited on the surface as a nanopore carrier material to perform single-molecule detection of proteins in electrical and optical dimensions. By analyzing the size and duration of the blocking current generated by the protein entering the nanopore under bias, basic information such as the volume and surface charge of the protein can be obtained through electrical methods.

[0020] 2. The present invention provides a stronger light-excitation hot spot effect than the traditional structure through the gold double bow-tie-shaped plasmon structure deposited on the surface, which can slow down the perforation of protein molecules and improve the capture rate and time resolution of molecular perforation. In addition, plasmon resonance can improve the spatial resolution of optical signals, including Raman spectral signals and changes in light absorption and scattering signals caused by molecular perforation. Thus, optical dimension detection is introduced, which corroborates with the information obtained by electrochemistry, increases the accuracy and sensitivity of device detection, and realizes high spatiotemporal resolution analysis of single-molecule optical and electrical two-dimensional signals.

[0021] 3. The present invention establishes a high-sensitivity trace analysis platform for proteomics by monitoring the conformation of single-molecule proteins and changes in the molecular structure of omics. The combination of plasmon effect and solid-state nanopore technology provides new ideas and methods for high-sensitivity trace analysis of proteomics, clinical proteomics monitoring of major diseases, component evolution tracking analysis, and mining of early diagnostic markers. It is expected to play an important role in clinical monitoring and early diagnosis of major diseases.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic structural diagram of the gold double bow-tie plasmon on the silicon nitride film of the present invention.

[0024] Figure 2 It is a schematic diagram of the protein principle of the photoelectric combined detection of the present invention.

[0025] Figure 3 This is a diagram of the electrical original signal trajectory of the AFP protein passing through the plasmon nanopore in Example 4 of the present invention.

[0026] Figure 4 This is a statistical diagram of the electrical signal analysis of the AFP protein molecule perforation in Example 4 of the present invention.

[0027] Figure 5 This is an optical Raman scattering signal diagram of the protein molecule in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0029] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The present invention firstly performs surface decontamination treatment on the silicon-based silicon nitride film by cleaning with acetone solution and oxygen plasma, then uses magnetron sputtering technology to deposit a gold film with a thickness of 20-50nm on the surface of the silicon-based silicon nitride, uses a focused ion beam to process the gold film structure, and manufactures a nanochannel (3-10nm) with a stable aperture in the center of the structure, and manufactures a double bow-tie structure around the nanopore. Afterwards, the prepared nanopore chip is assembled with a customized flowcell, and the protein to be detected is added to the cis side of the flowcell. At the same time, the patch clamp and Raman spectrometer are connected, so that the electrical and optical signals of the protein can be detected respectively.

[0031] The silicon-based silicon nitride nanochip (SiN x) was purchased from Suzhou Nanopore Company (film thickness: ~20nm, window: ~4μm 2 ).

[0032] Example 1

[0033] A method for constructing a protein photoelectric combined detection platform based on a plasmon nanochannel structure is as follows:

[0034] Step 1: Silicon nitride chip pretreatment and solid-state nanopore preparation

[0035] S N x Before use, the chip was treated with hot acetone solution and then cleaned with ethanol and ultrapure water to remove organic and inorganic pollutants on the membrane surface to obtain clean nanopores. x The film is fixed in a magnetron sputtering coating machine with conductive glue. In a vacuum environment, the inert gas is ionized to enhance the magnetic field. The gold target is bombarded with ions, and the gold target falls off to form a 20nm thick gold film on the silicon nitride film. Then, a focused ion beam (FIB) system is used to etch with a voltage of 30keV and a current of 5pA to prepare a 5nm nanochannel in the center of the silicon nitride chip. With this as the center, the gold film is milled out using the edited program. Figure 1 The double bow-tie structure shown. The bow-tie structure consists of four symmetrical isosceles triangles, and its four tips can jointly provide a hot spot effect to significantly enhance the Raman signal.

[0036] Step 2: Assembly of the nanopore and optoelectronic detection platform

[0037] SiN with double bow-tie plasmon structure x The nanopore chip was transferred to the detection pool, and the two chambers of the flowcell were connected to the e-One-HS micro-patch clamp through Ag / AgCl electrodes. At the same time, the Raman signal was collected using a Raman spectrometer with a 60x water immersion objective and a 785nm laser beam. The patch clamp and Raman spectrometer recorded the electrical signal and Raman signal of the protein molecule respectively.

[0038] Example 2

[0039] A method for constructing a protein photoelectric combined detection platform based on a plasmon nanochannel structure is as follows:

[0040] Step 1: Silicon nitride chip pretreatment and solid-state nanopore preparation

[0041] S N x Before use, the chip was treated with hot acetone solution and then cleaned with ethanol and ultrapure water to remove organic and inorganic pollutants on the membrane surface to obtain clean nanopores. xThe film is fixed in a magnetron sputtering coating machine with conductive glue. In a vacuum environment, the inert gas is ionized to enhance the magnetic field. The gold target is bombarded with ions, and the gold target falls off to form a 30nm thick gold film on the silicon nitride film. Then, a focused ion beam (FIB) system is used to etch with a voltage of 30keV and a current of 5pA to prepare a 10nm nanochannel in the center of the silicon nitride chip. With this as the center, the gold film is milled out using the edited program. Figure 1 The double bow-tie structure shown. The bow-tie structure consists of four symmetrical isosceles triangles, and its four tips can jointly provide a hot spot effect to significantly enhance the Raman signal.

[0042] Step 2: Assembly of the nanopore and optoelectronic detection platform

[0043] SiN with double bow-tie plasmon structure x The nanopore chip was transferred to the detection pool, and the two chambers of the flowcell were connected to the e-One-HS micro-patch clamp through Ag / AgCl electrodes. At the same time, the Raman signal was collected using a Raman spectrometer with a 60x water immersion objective and a 785nm laser beam. The patch clamp and Raman spectrometer recorded the electrical signal and Raman signal of the protein molecule respectively.

[0044] Example 3

[0045] A method for constructing a protein photoelectric combined detection platform based on a plasmon nanochannel structure is as follows:

[0046] Step 1: Silicon nitride chip pretreatment and solid-state nanopore preparation

[0047] S N x Before use, the chip was treated with hot acetone solution and then cleaned with ethanol and ultrapure water to remove organic and inorganic pollutants on the membrane surface to obtain clean nanopores. x The film is fixed in a magnetron sputtering coating machine with conductive glue. In a vacuum environment, the inert gas is ionized to enhance the magnetic field. The gold target is bombarded with ions, and the gold target falls off to form a 50nm thick gold film on the silicon nitride film. Then, a focused ion beam (FIB) system is used to grind with a voltage of 30keV and a current of 5pA to prepare a 10nm nanochannel in the center of the silicon nitride chip. With this as the center, the gold film is milled out using the edited program. Figure 1 The double bow-tie structure shown. The bow-tie structure consists of four symmetrical isosceles triangles, and its four tips can jointly provide a hot spot effect to significantly enhance the Raman signal.

[0048] Step 2: Assembly of the nanopore and optoelectronic detection platform

[0049] SiN with double bow-tie plasmon structure x The nanopore chip was transferred to the detection pool, and the two chambers of the flowcell were connected to the e-One-HS micro-patch clamp through Ag / AgCl electrodes. At the same time, the Raman signal was collected using a Raman spectrometer with a 60x water immersion objective and a 785nm laser beam. The patch clamp and Raman spectrometer recorded the electrical signal and Raman signal of the protein molecule respectively.

[0050] As shown in the following Example 4, the detection platform constructed in Example 1 is used to detect the liver cancer marker alpha-fetoprotein as an example. It should be understood that the detection platform of the present invention can also be used to detect other proteins.

[0051] Example 4

[0052] Detection of the photoelectric signal of the nanopore

[0053] 1. Test solution and sample preparation

[0054] Prepare 20-100 nM AFP test solution: dissolve the AFP mother solution in a buffer solution of 2M LiCl, 5mM CaCl2, 10mM Tris, pH 7.4 to obtain a protein sample of the desired concentration.

[0055] Prepare AFP and nanogold complex solution with a size of ~20 nm: Mix 20-100 nM AFP solution and nanopore aqueous solution in a buffer solution of 2M LiCl, 5mM CaCl2, 10mM Tris, pH 7.4 under light-proof environment.

[0056] Blank electrolyte: 2M LiCl, 5mM CaCl2, 10mM Tris, pH 7.4 buffer solution.

[0057] 2. Protein (AFP and its complex with gold nanoparticles) testing

[0058] Add 20nM AFP test solution to the cis side of the flowcell, and only add blank electrolyte to the trans side. When starting the test, first, only open the eOne-HS micro patch clamp, apply a 100mV bias, and test the electrical signals of the protein and the complex with the gold nanoparticles; second, only turn on the Raman spectrometer to collect the Raman signal of the protein; finally, turn on the patch clamp and the Raman spectrometer at the same time, and test their electrical and optical signals at the same time. Subsequently, add the AFP and gold nanoparticle complex test solution to the cis side of the flowcell, and only add blank electrolyte to the trans side. Repeat the above operation. When starting the test, first, only open the eOne-HS micro patch clamp, apply a 100mV bias, and test the electrical signals of the protein and the complex with the gold nanoparticles; second, only turn on the Raman spectrometer to collect the Raman signal of the protein; finally, turn on the patch clamp and the Raman spectrometer at the same time, and test their electrical and optical signals at the same time.

[0059] 3. Analyze and compare data

[0060] The difference in electrical signals of the AFP protein and its gold nanoparticle complex when only tested by patch clamp at the same pore size was compared, and their respective translocation time, blocking amplitude and pore resistance were analyzed; then the enhanced Raman signal of the AFP protein and gold nanoparticle complex was analyzed when only tested by Raman spectrometer; finally, when the patch clamp and Raman spectrometer were turned on at the same time, the synchronous correspondence between the electrical signal and the optical signal of the AFP protein and its gold nanoparticle complex was analyzed.

[0061] Note: All the above measurements were performed in a dark Faraday cage.

[0062] Figure 3 This is the electrical original signal trajectory diagram of AFP protein passing through the plasmon nanopore. The test results in the figure show that the protein molecule perforation has good capture and signal-to-noise ratio.

[0063] Figure 4 This is a statistical diagram of the electrical signal analysis of the AFP protein molecule perforation. In the figure, A is the perforation blocking time, in the figure, B is the perforation ion current blocking amplitude, and in the figure, C is the blocking rate.

[0064] Figure 5 This is the Raman scattering signal image of the protein after optical enhancement. Under the enhancement effect of the substrate plasmon structure, some structural information of the protein can be preliminarily obtained. In the future, the plasmon structure needs to be optimized to obtain a stronger molecular characteristic Raman scattering signal.

[0065] The present invention discloses a single-molecule protein optoelectronic combined detection platform and method based on a plasmon nanochannel structure. With its unique innovative fusion of double-butterfly plasmon nanostructure and solid-state nanopore, it demonstrates great commercial application potential in the fields of bioinformatics and clinical diagnosis. The core advantage of this technology is that it can utilize the efficient light excitation hot spot effect and light capture ability of the double-bow-knot plasmon structure to greatly enhance the detection sensitivity of optical signals such as surface enhanced Raman spectroscopy (SERS) and light absorption and scattering of single protein molecules, and significantly improve the detection accuracy of optical signals at the single-molecule level. The promotion and application of this new structural device will not only promote biomolecule detection technology to move towards higher sensitivity and higher resolution, but also open up new horizons for life science research.

[0066] At the same time, the plasmon nanochannel device of the present invention can be widely used in multiple fields such as disease diagnosis, drug development and personalized medicine. Specifically, by real-time monitoring of the conformational changes of single protein molecules and the dynamic evolution of the molecular structure of the group, the device can construct a highly sensitive and accurate proteomics analysis platform. This platform can achieve accurate capture and early warning of trace biomarkers in patients in the clinical monitoring of major diseases (such as early cancer screening, neurodegenerative diseases, etc.), providing a scientific basis for early diagnosis of the disease, disease assessment and optimization of treatment plans. In addition, this technology also shows great value in drug screening and evaluation, which can accelerate the process of new drug development and improve the accuracy of drug targeting and efficacy evaluation.

[0067] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0068] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A protein optoelectronic combined detection platform based on plasmon nanochannel structure, characterized in that: The method for constructing the detection platform includes: surface decontamination treatment of the silicon-based silicon nitride film, then using magnetron sputtering technology to deposit a gold film on the silicon-based silicon nitride surface, using a focused ion beam to process the gold film structure, creating a nanopore in the center of the structure, and creating a double bow-tie structure around the nanopore to make a nanopore chip, and then assembling the prepared nanopore chip with a flowcell to obtain a protein optoelectronic combined detection platform based on a plasmon nanochannel structure.

2. A protein photoelectric combined detection platform based on a plasmon nanochannel structure as claimed in claim 1, characterized in that: The method for surface decontamination treatment of silicon-based silicon nitride film is as follows: after treating the silicon-based silicon nitride film with an acetone solution, the film is washed with ethanol and ultrapure water in sequence to remove organic and inorganic pollutants on the film surface.

3. A protein photoelectric combined detection platform based on a plasmon nanochannel structure as claimed in claim 1, characterized in that: The method of depositing a gold film on the surface of silicon-based silicon nitride using magnetron sputtering technology is as follows: the treated silicon-based silicon nitride film is fixed in a magnetron sputtering coating machine with conductive glue, in a vacuum environment, the inert gas is ionized, the magnetic field is enhanced, and the gold target is bombarded with ions, and the gold target falls off to form a layer of gold film on the silicon nitride film.

4. A protein photoelectric combined detection platform based on a plasmon nanochannel structure as claimed in claim 3, characterized in that: The thickness of the gold film is 20-50 nm.

5. The protein photoelectric combined detection platform based on plasmon nanochannel structure according to claim 1, characterized in that: The method for processing the gold film structure using a focused ion beam is as follows: etching is performed using a focused ion beam system with a voltage of 30 keV and a current of 5 pA.

6. The protein photoelectric combined detection platform based on plasmon nanochannel structure according to claim 1, characterized in that: The nanopores prepared on the silicon-based silicon nitride core are 3-10nm.

7. The protein photoelectric combined detection platform based on plasmon nanochannel structure according to claim 1, characterized in that: The double bow-tie structure consists of four symmetrical isosceles triangles.

8. The protein photoelectric combined detection platform based on plasmon nanochannel structure according to claim 1, characterized in that: The prepared nanopore chip was assembled with the flowcell, and the two chambers of the flowcell were connected to the e-One-HS micro patch clamp through Ag / AgCl electrodes and equipped with a Raman spectrometer. The Raman spectrometer was used to collect Raman signals, and the patch clamp was used to record the electrical signals of protein molecules.

9. A detection method for a protein optoelectronic combined detection platform based on a plasmon nanochannel structure according to any one of claims 1 to 8, characterized in that: The protein detection method includes: preparing a test solution and a complex of the test solution and nanogold particles, adding the test solution to the cis side of the flowcell and only adding a blank electrolyte to the trans side. When starting the test, first, only opening the eOne-HS micro patch clamp, applying a 100mV bias, and testing the electrical signal; second, only opening the Raman spectrometer to collect the Raman signal of the protein molecule; finally, opening the patch clamp and the Raman spectrometer at the same time, and testing the electrical signal and the optical signal at the same time. Subsequently, adding the complex of the test solution and nanogold particles to the cis side of the flowcell and only adding a blank electrolyte to the trans side. When starting the test, first, only opening the eOne-HS micro patch clamp, applying a 100mV bias, and testing the electrical signal; second, only opening the Raman spectrometer to collect the Raman signal of the protein molecule; finally, opening the patch clamp and the Raman spectrometer at the same time, and testing the electrical signal and the optical signal at the same time.

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