A fabrication process for a SERS microfluidic chip for nucleic acid detection

Abstract

The invention belongs to the technical field of nucleic acid detection, and specifically is a manufacturing process of a SERS microfluidic chip for nucleic acid detection. The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows: step one: firstly select a polymer plastic as a substrate, perform a drying treatment after ultrasonic cleaning on the substrate, and use a vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate, and the film thickness is 1-3 microns; step two: use a mask plate with a pattern designed in advance according to a microfluidic reaction chamber and a flow channel to cover the surface of the substrate, and use the vacuum evaporation method again to construct a multilayer film nanostructure spike of Au and Co, and the thickness of each layer is 150-250 nanometers. The SERS substrate prepared by the invention has good SERS hot spot stability, high sensitivity, good reproducibility, and can effectively overcome the disadvantage of the small size of the existing SERS substrate, can realize the detection of multiple proteases and small molecules, and has flexible application level expansion.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid detection, and in particular to a manufacturing process of a SERS microfluidic chip for nucleic acid detection. Background Art

[0002] Microfluidic chip technology integrates the basic operation units of sample preparation, reaction, separation, detection, etc. in the biological, chemical, and medical analysis process into a micron-scale chip to automatically complete the entire analysis process. Due to its great potential in the fields of biology, chemistry, and medicine, it has developed into a new research field that intersects biology, chemistry, medicine, fluids, electronics, materials, and other disciplines. Surface-enhanced Raman spectroscopy (SERS) is an emerging spectral detection technology with great potential application prospects in the field of high-sensitivity biochemical detection. Among them, on some specially prepared metal good conductor surfaces or sols, in the excitation region, the Raman scattering signal of the adsorbed molecules is greatly enhanced compared to the ordinary Raman scattering signal due to the enhancement of the electromagnetic field on the sample surface or near the surface, which overcomes the disadvantage of low sensitivity of Raman spectroscopy and can obtain structural information that is not easy to obtain with conventional Raman spectroscopy.

[0003] Nucleic acid detection is a detection method that uses deoxyribonucleic acid / ribonucleic acid (DNA / RNA) as a marker, which can be applied to early virus and pathogen detection. Among various nucleic acid detection methods, it mainly uses negatively charged resins and polymer films to detect nucleic acids by selectively binding to nucleic acid molecules. Generally speaking, it is necessary to perform qualitative or quantitative measurements on various antigens or antibodies. For the quantitative detection of specific proteins of the human body or virus, the SERS detection sample based on multiple antibody labels is generally amplified by a fluorescence or enzymatic reaction, and the liquid droplets are dropped onto a glass sheet modified with a specific antibody, and then the SERS probe labeled with a specific antibody is dropped onto the corresponding SERS substrate, and then scanned by Raman spectroscopy. Generally speaking, the capture antibody (Ab1) of the target analyte is fixed on a solid phase Raman enhanced substrate (generally a composite nanoparticle composed of gold, silver nanoparticles or other non-polar materials), and the signal antibody (Ab2) of the target analyte is also fixed on another nanoparticle of similar material. When the positive sample forms a "ternary sandwich complex" with Ab1 and Ab2, the Raman spectrum signal is greatly enhanced when the near surface of the nanoparticles and the slits between the nanoparticles that are close to each other form a "hotspot structure". The more target analytes, the more ternary sandwich complexes are formed and the more corresponding SERS hotspots.

[0004] Since the self-assembled structure on the surface of nanoparticles is consistent and uncontrollable, SERS hot spots are easily affected. It is difficult to achieve consistency in the hot spots and states formed by each immune response, so the final measured Raman signal fluctuates greatly, which seriously affects the quantitative detection performance of SERS. In recent years, through the preparation of a variety of precious metal nanostructured SERS substrates, through semiconductor mask process technology, coupled with nanoimprint technology and evaporation technology, a larger area of ​​nano-spike structure can be prepared, and through micro-channel process design, the measured liquid is placed in the matrix of nano-spikes, thereby ensuring uniform distribution of hot spots.

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a large-area SERS substrate prepared on a PC film substrate. First, the substrate is cleaned by ultrasonic cleaning and then dried. A basic AuCo alloy film is constructed on the surface by vacuum evaporation technology. A semiconductor mask technology is used to secondary evaporate and construct magnetic nano spikes on the substrate surface to form a microstructure for loading capture antibody Ab1 and signal antibody Ab2 molecules. According to measurement, the substrate has high sensitivity, and the detection limit for some molecules can reach 10-9M. The substrate has high sensitivity, good reproducibility, and a simple preparation process. Through different antibody combinations, the substrate can realize the detection of molecules in different systems, and has broad application prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a manufacturing process of a SERS microfluidic chip for nucleic acid detection, so as to solve the problem that the self-assembly structure of the nanoparticle surface proposed in the above background technology is consistent and uncontrollable, the SERS hotspot is easily affected, and the hotspot and state formed by each immune reaction are difficult to be consistent, so the Raman signal finally measured fluctuates greatly, which seriously affects the quantitative detection performance of SERS.

[0007] To achieve the above object, the present invention provides the following technical solution: a process for manufacturing a SERS microfluidic chip for nucleic acid detection, the specific steps of the process for manufacturing a SERS microfluidic chip for nucleic acid detection are as follows:

[0008] Step 1: First, select a polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use a vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate with a film thickness of 1-3 microns;

[0009] Step 2: Using a mask plate with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel, covering the surface of the above substrate, and again using the vacuum evaporation method to construct a multilayer film nanostructure spike of Au and Co, each layer having a thickness of 150-250 nanometers;

[0010] Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 150-250 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position to form a SERS hotspot.

[0011] Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area;

[0012] Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites plasmon resonance conditions to enhance SERS hotspots.

[0013] Preferably, the polymer plastic in step one includes one or more of polyvinyl alcohol, polyacrylate, and polyacrylic acid.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Using a large area of ​​polymer substrate as a centralized source, and using vacuum coating technology to construct a surface film of Au on the substrate surface, a growth substrate suitable for Au / Co magnetic multilayer film can be provided. Using semiconductor mask technology, sufficient positioning can be provided for Au / Co multilayer film patterning and array formation, and the process of preparing precious metal nano spikes can be effectively simplified, shortening the preparation cycle. The Au / Co magnetic nano spikes formed by the substrate can quickly load molecular probes by using their own magnetic driving ability and controlling the size of the nanostructure, and can form a self-assembled monomolecular film for rapid identification of small molecule particles in the detection unit. The SERS substrate prepared by the invention has good SERS hotspot stability, high sensitivity, and high reproducibility, and can effectively overcome the shortcomings of the existing SERS substrates with small sizes, and can realize the detection of a variety of proteases and small molecules, with flexible expansion of application levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Create a flow chart for the substrate process;

[0017] Figure 2 is a schematic diagram of the structure of the mask 1;

[0018] Figure 3 is a schematic diagram of the structure of the mask 2;

[0019] Figure 4Schematic diagram of Au / Co multilayer magnetic nanospikes;

[0020] Figure 5 Schematic diagram of loading capture antibody Ab1 and signal antibody Ab2. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0023] See also Figure 1-5 The present invention provides a technical solution: a manufacturing process of a SERS microfluidic chip for nucleic acid detection. The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows:

[0024] Step 1: First, select a polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use a vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate with a film thickness of 1-3 microns;

[0025] Step 2: Using a mask plate with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel, covering the surface of the above substrate, and again using the vacuum evaporation method to construct a multilayer film nanostructure spike of Au and Co, each layer having a thickness of 150-250 nanometers;

[0026] Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 150-250 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position to form a SERS hotspot.

[0027] Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area;

[0028] Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites plasmon resonance conditions to enhance SERS hotspots.

[0029] The polymer plastic in step one includes one or more of polyvinyl alcohol, polyacrylate, and polyacrylic acid.

[0030] Embodiment 1:

[0031] The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows:

[0032] Step 1: First, select polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate, with a film thickness of 1 micron;

[0033] Step 2: Use a mask with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel to cover the surface of the above substrate, and use the vacuum evaporation method again to construct a multilayer film nanostructure spike of Au and Co, with each layer having a thickness of 150 nanometers;

[0034] Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 150 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position, forming a SERS hotspot.

[0035] Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area;

[0036] Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites the plasma exciton resonance condition to enhance the SERS hotspot.

[0037] Embodiment 2:

[0038] The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows:

[0039] Step 1: First, select polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate, with a film thickness of 2 microns;

[0040] Step 2: Use a mask with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel to cover the surface of the above substrate, and use the vacuum evaporation method again to construct a multilayer film nanostructure spike of Au and Co, with each layer having a thickness of 200 nanometers;

[0041] Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 200 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position to form a SERS hotspot.

[0042] Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area;

[0043] Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites the plasma exciton resonance condition to enhance the SERS hotspot.

[0044] Embodiment 3:

[0045] The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows:

[0046] Step 1: First, select polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate, with a film thickness of 3 microns;

[0047] Step 2: Use a mask with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel to cover the surface of the above substrate, and use the vacuum evaporation method again to construct a multilayer film nanostructure spike of Au and Co, with each layer having a thickness of 250 nanometers;

[0048] Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 250 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position to form a SERS hotspot.

[0049] Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area;

[0050] Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites plasmon resonance conditions to enhance SERS hotspots.

[0051] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

[0052] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for manufacturing a SERS microfluidic chip for nucleic acid detection, characterized in that: The specific steps of the manufacturing process of the SERS microfluidic chip for nucleic acid detection are as follows: Step 1: First, select a polymer plastic as the substrate, clean the substrate ultrasonically and then dry it, and use a vacuum evaporation method to construct a uniform Au surface film on the surface of the substrate with a film thickness of 1-3 microns; Step 2: Using a mask plate with a pattern designed in advance according to the microfluidic reaction chamber and the flow channel, covering the surface of the above substrate, and again using the vacuum evaporation method to construct a multilayer film nanostructure spike of Au and Co, each layer having a thickness of 150-250 nanometers; Step 3: Using another mask, vacuum evaporation method is used to construct multilayer nanostructure spikes of Au and Co and form an array. The thickness of each layer is 150-250 nanometers. Magnetic nanospikes are constructed in the molecular convergence area. Magnetic force is applied from directly below the detection unit so that the signal antibody and the detection particles in this area are closely arranged at the predetermined position to form a SERS hotspot. Step 4: Place the capture antibody. Use the pipetting method to place 4-mercaptobenzoic acid MBA as the capture antibody Ab1 in various areas of the molecular capture area, and a self-assembled monolayer can be formed in the nanomagnetic spike area; Step 5: Place the signal antibody. Use the pipetting method to place 4-aminothiophenol ATP as a signal antibody in the molecular confluence area. It can form a metal-molecule-metal sandwich structure with the Au / Co nanomagnetic spike area. Under laser irradiation, it excites plasmon resonance conditions to enhance SERS hotspots.

2. The process for manufacturing a SERS microfluidic chip for nucleic acid detection according to claim 1, characterized in that: The polymer plastic in step one includes one or more of polyvinyl alcohol, polyacrylate, and polyacrylic acid.

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