Tumor marker detection system based on antibody-modified gold nanoparticle periodic linear array

Abstract

This invention discloses a tumor marker detection system based on an antibody-modified gold nanoparticle periodic linear array, belonging to the field of biomolecular detection. In this invention, a primary antibody is modified onto gold nanowires, and a nanolabel of antibody molecules modified onto gold nanoparticles is prepared as a detection tag (secondary antibody) for tumor markers. A test solution mixed with the secondary antibody is drop-coated onto the structure, and after a sufficient reaction time, the entire structure is rinsed. When light is incident perpendicularly on the gold nanowire array, surface plasmon resonance (SPR) occurs. The tumor markers (antigens) present in the solution are specifically adsorbed with the primary and secondary antibodies, forming a double-antibody sandwich structure, which changes the macroscopic color of the structure. By observing whether the color changes in different regions of the nanowire array after detection, the presence of tumor markers in the test solution can be determined. This invention's system can simultaneously detect multiple tumor markers and features short detection time, small size, low cost, high sensitivity, and ease of outdoor observation.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection, and particularly relates to a tumor marker detection system based on antibody-modified gold nanoparticle periodic linear arrays. Background Technology

[0002] Tumor markers are specific antigenic proteins secreted by cells during the carcinogenesis process. They are mainly found in blood, urine, or tumor tissue, and their presence or changes in content can indicate the nature of the tumor, thus aiding in tumor diagnosis, classification, prognosis, and efficacy monitoring. Currently, methods for detecting tumors include traditional chemiluminescence, surface plasmon resonance detection based on prism structures, and polymerase chain reaction (PCR) for detecting tumor genes. Although these methods have certain feasibility, their inherent limitations restrict their application in practical detection, such as: (1) long detection time, numerous and complex procedures, and low detection efficiency; (2) gene detection methods can only perform quantitative analysis and are prone to false positives and false negatives; (3) these methods all require large-scale precision instruments, which are bulky and expensive to manufacture; (4) chemiluminescence requires labeling reagents and involves many steps. Therefore, developing a new technology that can accurately and rapidly detect tumor markers is particularly important.

[0003] In this invention, antibody-modified detection tags are prepared by modifying the surface of metal nanomaterials with antibodies. The specific adsorption of antibodies and antigens forms a double-antibody sandwich structure, which is attached to gold nanowires. This alters the morphology and spacing of the nanowires, affecting the resonance wavelength of the longitudinal surface plasmon resonance and changing the macroscopic color of the structure. By observing the color changes in different regions of the nanowire array after detection, the presence of tumor markers in the test solution can be determined, enabling the simultaneous detection of multiple tumor markers. Furthermore, the concentrations of different types of tumor markers in the test solution can be determined by comparing the measured spectra with those of a calibration reference. The key advantages of this invention are that using nanowires as 3D adsorbents improves detection sensitivity, and the double-antibody sandwich method adsorbed on gold nanoparticles enhances the sensitivity to signal changes. Moreover, the nanowire array excitation mode, sensing the presence and type of tumor markers through color changes, makes the detection method convenient and easy to observe. Summary of the Invention

[0004] To address the shortcomings mentioned in the above technical background, the purpose of this invention is to provide a tumor marker detection system based on antibody-modified gold nanoparticle periodic linear arrays.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] This invention discloses a tumor marker detection system based on an antibody-modified gold nanoparticle periodic linear array. The system's structure comprises: gold and aluminum plating on a glass substrate; forming a porous alumina structure through anodizing; etching to enlarge the pore diameter and filling with gold to form gold nanowires; transferring a layer of periodically ordered polystyrene nanospheres onto the surface of the alumina-gold nanowire film; ion etching to remove the nanowires and alumina not masked by the microspheres, and removing the PS nanospheres; then removing the alumina matrix to obtain a patterned gold nanowire array in air; and finally, performing biomodification on the structure surface by modifying the gold nanowire end faces in different regions with antibodies (primary antibodies) corresponding to different tumor markers.

[0007] Furthermore, the preparation steps of the tumor marker detection system based on antibody-modified gold nanoparticle periodic linear array, according to claim 1, are characterized in that the preparation steps are as follows:

[0008] S1 uses magnetron sputtering to plate gold and aluminum onto a clean, cut glass substrate;

[0009] S2 involves thoroughly anodizing the aluminum film to form a porous aluminum oxide film.

[0010] S3 uses an electrodeposition method to fill the pores of an alumina film with gold nanowires.

[0011] S4 periodic nanowire patterns excite plasmon modes, and PS microsphere monolayer ordered structures are prepared by transfer thin film method as etching masks;

[0012] S5 uses a PS microsphere layer as a mask to form a pattern. The spacing between the microspheres, the array period, and the shape of the array pattern are adjustable, forming a periodic patterned nanowire array that can excite surface plasmons when light is incident perpendicularly. Due to the double-anti-sandwich structure changing the morphology and spacing of the gold nanowires, the resonance wavelength of the longitudinal surface plasmon resonance is affected, and the macroscopic color of the structure is changed.

[0013] The outermost layer of the S6 three-layer sandwich is modified with antibody molecules on the outer layer of gold nanoparticles.

[0014] Furthermore, in step S1, the gold film thickness is 7 nm and the aluminum film thickness is 200 nm.

[0015] Furthermore, in step S2, the aperture of the holes is 70 nm, the spacing is 100 nm, and the height is 200 nm.

[0016] Furthermore, the height of the gold nanowires filled in step S3 is 200 nm.

[0017] Furthermore, in step S6, the diameter of the gold nanoparticles is approximately 20 nm.

[0018] The beneficial effects of this invention are:

[0019] The structure prepared in this invention is as follows: gold and aluminum are plated on a glass substrate, and a porous alumina structure is formed through anodizing. The diameter of the pores is enlarged by etching and then filled with gold to form gold nanowires. A layer of periodically ordered polystyrene nanospheres is then transferred onto the surface of the alumina-gold nanowire film. Ion etching is used to remove the nanowires and alumina not masked by the microspheres, and the PS nanospheres are also removed. The alumina matrix is ​​then removed, yielding a patterned gold nanowire array in air. When light is incident perpendicularly on the gold nanowire array, surface plasmon resonance (SPR) occurs. Tumor markers (antigens) present in the solution are specifically adsorbed with primary and secondary antibodies to form a double-antibody sandwich structure, altering the morphology and spacing of the nanowires, affecting the resonance wavelength of the longitudinal SPR, and changing the macroscopic color of the structure. By observing whether the color changes after detection in different regions of the nanowire array, the presence of tumor markers in the test solution can be determined, enabling the simultaneous detection of multiple tumor markers. Furthermore, the concentrations of different types of tumor markers in the test solution can be determined by comparing the measured spectra with the calibrated reference spectra. The system of this invention can simultaneously detect multiple tumor markers and features short detection time, small size, low cost, high sensitivity, and ease of outdoor observation. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a cross-sectional schematic diagram of the present invention.

[0022] Figure 2 This is a top view of the present invention.

[0023] Figure 3 This is a schematic diagram of the aluminum anodizing process.

[0024] Figure 4 This is a schematic diagram of the fabrication process of the gold nanoparticle periodic linear array in the tumor marker detection system of the present invention.

[0025] Figure 5 This is a schematic diagram illustrating the principle of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses a tumor marker detection system based on an antibody-modified gold nanoparticle array. The system mainly comprises a patterned gold nanoparticle array on a glass substrate. The main fabrication process is as follows: gold and aluminum are plated on a glass substrate; a porous alumina structure is formed through anodizing; the pore diameter is enlarged by etching and then filled with gold to form gold nanowires. A periodically ordered polystyrene nanosphere film is then transferred onto the surface of the alumina-gold nanowire film. Ion etching is used to remove the nanowires and alumina not covered by the microspheres, and the PS nanospheres are also removed. The alumina matrix is ​​then removed to obtain the patterned gold nanoparticle array in air. Finally, the surface is biomodified by modifying the gold nanowire end faces in different regions with antibodies (primary antibodies) corresponding to different tumor markers.

[0028] The preparation steps of this invention are as follows:

[0029] (1) A 10 nm thick tantalum pentoxide binder layer and a 7 nm thick gold film as a weakly conductive layer were deposited on a glass substrate by sputtering tantalum using a 20% oxygen / 80% argon mixture. A 200 nm thick aluminum film was then deposited on the multilayer glass substrate by magnetron sputtering. Figure 4 As shown in (a).

[0030] (2) A porous alumina structure was synthesized by two-step anodizing in 0.3M oxalic acid at 40V; after the initial anodizing process, the poorly ordered porous layer was removed by etching in a solution of H3PO4 (3.5%) and CrO3 (20 g L⁻¹) at 70°C; a second anodizing was performed, resulting in pores with a diameter of approximately 70 nm, a spacing of approximately 100 nm, and a height of approximately 200 nm. Figure 4 As shown in (b).

[0031] (3) The sample was etched in a 30mM NaOH solution to enlarge the diameter of the hole and remove the barrier layer.

[0032] (4) Gold nanowires with a height of approximately 200 nm were formed by electrodeposition of gold using a non-cyanide solution via a three-electrode system. Figure 4 As shown in (c).

[0033] (5) Transfer a periodically ordered polystyrene nanosphere film onto the surface of an alumina gold nanowire film, such as Figure 4As shown in (d).

[0034] (6) Ion etching removes gold nanowire structures not masked by microspheres, such as Figure 4 As shown in (e).

[0035] (7) Remove the PS nanospheres and etch the remaining alumina matrix in 30 mM NaOH solution to form an air-patterned gold nanoarray, such as... Figure 4 As shown in (f).

[0036] (8) Biomodification of the structural surface, such as antibody modification onto gold nanowires, for example... Figure 4 As shown in (g).

[0037] (9) The test solution is dropped onto the detection device, where the primary antibody, secondary antibody, and antigen are adsorbed to form a double-antibody sandwich structure, such as... Figure 4 As shown in (h).

[0038] The resulting double-antibody sandwich structure alters the morphology and spacing of the gold nanowires, affecting the resonance wavelength of the longitudinal surface plasmon resonance, such as... Figure 5 As shown.

[0039] Because the pattern is formed by using PS microspheres as a mask, the spacing between the microspheres is adjustable, the array period is adjustable, and the shape of the array pattern is adjustable, forming a periodic patterned nanowire array that can excite surface plasmons when light is incident perpendicularly. Due to the double-anti-sandwich structure changing the morphology and spacing of the gold nanowires, the resonance wavelength of the longitudinal surface plasmon resonance is affected, thus changing the macroscopic color of the structure.

[0040] Taking CEA (carcinoembryonic antigen) as an example, the steps for modifying CEA antibodies on gold nanowires are as follows:

[0041] (1) Thiol self-assembly: Mercaptoundecanoic acid (HS(CH2)11COOH) was used as the linker layer. The thiol end of mercaptoundecanoic acid interacted with the gold nanowires on the end face of the detection device, formed a bond, and was stably adsorbed on the gold film. The carboxyl end served as an active group and could be linked to the antibody protein. The cleaned detection device was immersed in a 10 μM mercaptoundecanoic acid ethanol solution and reacted fully at 25 °C for 20 minutes.

[0042] (2) EDC / NHS activation: The detection device after thiol self-assembly was ultrasonically cleaned with ethanol, dried with nitrogen, and then immersed in a mixture of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-carboxysuccinimide) (EDC 40 mg / ml, NHS 110 mg / ml) to activate the carboxyl end of the thiol. The reaction was carried out at 25°C for 20 minutes.

[0043] (3) Antibody protein fixation: Antibody corresponding to carcinoma antigen (anti-CEA) was used. After rinsing the activated detection device with ultrapure water, it was immersed in a 40ug / mL protein solution. After reacting at 25°C for 2 hours, it was incubated at 4°C for 12 hours to form a stable monomolecular antibody layer on the gold film surface of the detection device.

[0044] (4) BSA blocking: After rinsing with phosphate-buffered saline (PBS, pH 7.4), the detection device was immersed in 0.1 mg / mL bovine serum albumin (BSA) solution to block any groups that failed to connect. After BSA blocking for 2 hours, the device was rinsed with PBS buffer and dried with nitrogen to complete the fixation of protein antibodies on the surface of the SPR sensor.

[0045] (5) Specific binding of tumor markers (antigens) to antibodies: CEA (carcinoembryonic antigen) solutions with concentrations of 100 pg / ml, 200 pg / ml, 300 pg / ml and 400 pg / ml were dropped onto the detection device, gold nanoparticles modified with a second antibody were added, and the mixture was allowed to react fully at 37°C for 30 minutes. The mixture was then rinsed with PBS buffer and dried with nitrogen.

[0046] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing a tumor marker detection system based on antibody-modified gold nanoparticle periodic linear arrays, characterized by, The preparation method comprises the following steps: S1, using a magnetron sputtering method to plate gold and aluminum on a cut and cleaned glass substrate; S2, performing thorough anodic oxidation on the aluminum film to form a porous aluminum oxide film; S3, using an electrodeposition method to fill gold nanowires in the pores of the aluminum oxide film; S4, periodically exciting plasmon modes by using a nanowire pattern, and using a transfer film method to prepare a PS microsphere single-layer ordered structure as an etching mask; S5, using the PS microsphere layer as a mask to form a pattern, the microsphere spacing is adjustable, the array period is adjustable, the array pattern shape is adjustable, a nanowire array with a periodic pattern is formed, which can excite surface plasmons when light is perpendicular to the incident, and due to the double-antibody sandwich structure changing the morphology and gap of the gold nanowires, the resonance wavelength of the longitudinal surface plasmon resonance is affected, and the color of the structure macroscopically is changed; S6, the outermost layer of the three-layer sandwich is modified by an antibody molecule on the outer layer of the gold nanoparticles; In step S2, the pore diameter of the porous aluminum oxide film is 70 nm, the spacing is 100 nm, and the height is 200 nm; in step S3, the gold nanowires filled have a height of 200 nm; in step S6, the gold nanoparticles have a diameter of 20 nm; In step S1, the thickness of the gold film is 7 nm, and the thickness of the aluminum film is 200 nm.

2. A tumor marker detection system based on antibody-modified gold nanoparticle periodic linear arrays, characterized by The system is prepared by the preparation method of the tumor marker detection system based on the antibody-modified gold nanometer periodic line array of claim 1, and the structure of the system is that gold and aluminum are plated on a glass substrate, a porous aluminum oxide structure is formed by an anodic oxidation process, the diameter of the pores is expanded and filled with gold to form gold nanowires, then a layer of periodically ordered polystyrene nanospheres is transferred on the surface of the aluminum oxide gold nanowire film, ion etching is used to remove the nanowires and aluminum oxide that are not masked by the microspheres, and the PS nanospheres are removed, then the aluminum oxide matrix is removed, an air-patterned gold nanowire array is obtained, and finally, biological modification is performed on the surface of the structure, and different tumor marker corresponding antibodies are modified on the end faces of the gold nanowires in different regions.

Images