Chiral symmetric Fano resonance biosensor and manufacturing method

Abstract

The present invention provides a chiral symmetric Fano resonance biosensor, a manufacturing method, and a use method, including: a supersurface chip, the supersurface chip includes a sapphire substrate and multiple groups of chiral symmetric double N structures, the chiral symmetric double N structures are arranged on the upper surface of the sapphire substrate, the supersurface chip is soaked and adsorbed by MUA to form a self-assembled layer, and incubated with a specified tumor marker antibody solution to form a biosensor substrate; a microfluidic channel, the biosensor substrate is placed in the microfluidic channel; the tumor marker antibody linked to the chiral symmetric double N structure is combined with the tumor antigen in the patient's serum, and the tumor antigen concentration data in the patient's serum is obtained through the change of the refractive index. The chiral symmetric double "N" gold nanostructure supersurface provided by the present invention improves the detection sensitivity of tumor markers, and tumors can be effectively and quickly diagnosed.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor detection, and in particular relates to a chiral symmetric Fano resonance biosensor and a manufacturing method thereof. Background Art

[0002] Nowadays, malignant tumors are still an important factor threatening human health. The three stages of malignant tumors can be roughly divided into early, middle and late stages. The treatment in the early stage is the simplest and has the best effect. In this stage, local methods such as surgery and radiotherapy can generally be selected for radical cure. After surgery, radiotherapy and chemotherapy can be supplemented according to the condition, and the treatment effect and prognosis are the best. The middle stage is an important treatment period. If surgical treatment is given priority to those who meet the surgical indications, supplemented by radiotherapy and chemotherapy, the cure rate can be improved and the quality of life can be improved. This shows that the earlier the malignant tumor is discovered, the greater the survival rate of the patient can be.

[0003] However, traditional tumor detection methods such as tissue biopsy, X-ray, endoscopy, magnetic resonance imaging, etc., not only cause varying degrees of pain to patients, but are also not used in screening. There is currently a lack of accurate and convenient ways to detect tumors. Summary of the invention

[0004] The present invention provides a chiral symmetric Fano resonance biosensor and a manufacturing method thereof, aiming to solve the problem of lack of a method for accurately and conveniently detecting tumors.

[0005] The present invention is implemented as follows: a chiral symmetric Fano resonance biosensor, comprising:

[0006] A metasurface chip, comprising a sapphire substrate and a plurality of sets of chiral symmetric double N structures, wherein the chiral symmetric double N structures are arranged on the upper surface of the sapphire substrate,

[0007] The super surface chip is soaked in a self-assembled layer formed by adsorbing MUA (11-mercaptoundecanoic acid), and incubated with a specified tumor marker antibody solution to form a biosensor substrate, wherein the tumor marker antibody is linked to a chiral symmetric double N structure through MUA;

[0008] A microfluidic channel, wherein the biosensor substrate is placed in the microfluidic channel;

[0009] After the patient serum to be tested is injected into the microfluidic channel, the tumor marker antibody linked to the chiral symmetric double N structure combines with the tumor antigen in the patient serum, and the tumor antigen concentration data in the patient serum is obtained through the change of the refractive index.

[0010] Preferably, the chiral symmetric double N structure is composed of two groups of N-shaped structures, the N-shaped structure is 1100 nm long, 250 nm wide, and 30 nm high, the spacing between the two groups of N-shaped structures is 100 nm, and the distance between adjacent chiral symmetric double N structures is 100 nm.

[0011] Preferably, the active sites on the surface of the ultrasurface chip bind to the tumor marker antibody during immersion in the tumor marker antibody solution, and the active sites that have not completed binding with the tumor marker antibody are blocked by bovine serum albumin.

[0012] Preferably, the microfluidic channel is provided with an Inset port and an Outset port, and a channel is provided between the Inset port and the Outset port, so that the patient serum to be tested enters the channel from the Inset port and leaves from the Outset port after passing through the channel.

[0013] Preferably, the biosensor substrate is placed in a channel connecting the Inset port and the Outset port.

[0014] A method for manufacturing the chiral symmetric Fano resonance biosensor as described above comprises:

[0015] First, the substrate made of sapphire material is ultrasonically cleaned with an acetone solution, a photoresist is spin-coated, and an electron beam exposure technique is used for exposure to form a super surface structure profile; a 5nm Cr adhesion layer and a 30nm Au layer are evaporated by vapor deposition, and the super surface structure profile formed by EBL is peeled off to form a chiral symmetric double "N" gold nanostructure;

[0016] Step 2: Preparation of biosensor substrate

[0017] The supersurface chip is immersed in MUA to allow the surface layer of the supersurface to adsorb a self-assembled layer formed by MUA, and then immersed in a NHS / EDC mixed solution; the activated supersurface chip is then incubated in a designated tumor marker antibody solution, and then in a BSA (bovine serum albumin) solution to block the active sites of the unbound antibodies. After incubation, the preparation of the biosensor substrate is completed;

[0018] Step 3. Preparation of microfluidic channels

[0019] A microfluidic mold is made by photolithography technology, and then a PDMS (polydimethylsiloxane) substrate is made by molding; PDMS is poured on the mold and cured; then the biosensor substrate is installed in the PDMS microfluidic channel to complete the preparation of the biosensor.

[0020] Preferably, the Inset port is connected to different Outset ports by shunt pipes, and the patient serum entering from the Inset port flows to different Outset ports through the shunt pipes respectively; the metasurface chip is arranged in the shunt pipes, and the metasurface chips in different shunt pipes are placed in different tumor marker antibody solutions for incubation in advance, so that the metasurface chips in different shunt pipes can be combined with a single tumor marker antibody.

[0021] Preferably, the concentration ratio of the NHS / EDC mixed solution is 1:4.

[0022] A method for using the chiral symmetric Fano resonance biosensor as described above comprises:

[0023] Step 1: Inject the patient's serum into the microfluidic channel through the Inset port, and intercept the tumor antigens in the patient's serum on the biosensor through the biosensor; intercept the patient's serum in the sensor channel. Since different antibodies will only intercept corresponding antigens, different types of antigens will be intercepted in different channels;

[0024] Step 2: After completing the patient serum perfusion operation, use near-infrared light to illuminate the biosensor through the microfluidic channel. By observing the peak shift of various transmission spectra collected by the spectrometer, the combined detection of multiple tumor markers can be achieved.

[0025] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0026] 1. The chiral symmetric Fano resonance biosensor and manufacturing method provided by the present invention constructs a chiral symmetric double "N" gold nanostructure supersurface with sapphire as the substrate, and uses this structure to improve the detection sensitivity of tumor markers, so that tumors can be diagnosed effectively and quickly.

[0027] 2. The chiral symmetric Fano resonance biosensor and manufacturing method provided by the present invention carry antibodies corresponding to different tumor markers on each group of metasurface chips, and after being evenly divided in the microfluidic channel, different metasurface chips are calibrated and recorded for different tumor markers. By observing the peak shift of the transmission spectra of different metasurface chips collected by the spectrometer, the combined detection of multiple tumor markers can be achieved, thereby diagnosing the type of tumor the patient suffers from. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural schematic diagram of a chiral symmetric Fano resonance biosensor and a manufacturing method provided by the present invention.

[0029] Figure 2 It is a schematic diagram of a microfluidic channel structure of a chiral symmetric Fano resonance biosensor and a manufacturing method provided by the present invention.

[0030] Figure 3 The present invention provides a chiral symmetric Fano resonance biosensor and a manufacturing method thereof, which is a structural schematic diagram of the complexation process of a tumor marker antibody and a tumor marker antigen.

[0031] Figure 4 This is a diagram of FDTD simulation supersurface results of a chiral symmetric Fano resonance biosensor and a manufacturing method provided by the present invention.

[0032] Figure 5 The present invention provides a chiral symmetric Fano resonance biosensor and a manufacturing method thereof, and shows a sensor sensitivity curve diagram when the environmental refractive index n changes. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0034] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0035] The present invention provides a chiral symmetric Fano resonance biosensor and a manufacturing method thereof. Figure 1-Figure 5 As shown, the chiral symmetric Fano resonance biosensor comprises:

[0036] A metasurface chip, comprising a sapphire substrate and a plurality of groups of chiral symmetric double-N structures, wherein the chiral symmetric double-N structures are arranged on the upper surface of the sapphire substrate;

[0037] The super surface chip is soaked in a self-assembled layer formed by adsorbing MUA, and incubated with a specified tumor marker antibody solution to form a biosensor substrate, wherein the tumor marker antibody is linked to a chiral symmetric double N structure through MUA;

[0038] The biosensor substrate is placed in a microfluidic channel.

[0039] The patient serum to be tested is injected into the microfluidic channel, and the tumor marker antibody linked to the chiral symmetric double N structure combines with the tumor antigen in the patient serum, and the tumor antigen concentration data in the patient serum is obtained through the change of refractive index.

[0040] In this embodiment, a supersurface with a chiral symmetric double "N" gold nanostructure is provided on the sapphire substrate, and then the thiol group at one end of 11-mercaptoundecanoic acid (MUA) is linked to the supersurface through a coordination bond, and the carboxyl group at the other end is activated to an aldehyde group and then connected to a tumor marker antibody to prepare a biosensor substrate;

[0041] Surface plasmons (SPs) are electromagnetic waves that are localized in a specific area on the surface of a metal or semiconductor in a resonant state. When the frequency of the light field matches the resonant frequency of the surface plasmon, the interaction between photons and surface plasmons is enhanced. This strong coupling leads to a highly localized electromagnetic field enhancement of energy near the metal surface. The local field enhancement can make the interaction between light and molecules stronger, leading to enhanced sensor sensitivity and a series of nonlinear optical effects, such as second harmonic generation, enhanced Raman scattering, photoluminescence, etc. However, traditional surface plasmon resonance (SPR) has the problems of low quality factor and limited field intensity enhancement, resulting in poor performance in applications. When coherent coupling occurs between a discrete localized state (dark mode) and a continuous state (bright mode), a sharp and asymmetric resonance line shape can be generated, which is the Fano resonance.

[0042] Fano resonance can suppress radiation dissipation in plasmonic nanostructures, concentrating and storing energy more efficiently in the structure. This effect of suppressing radiation loss enables the structure to obtain a high Q factor, thereby increasing sensitivity to changes in structural parameters and the refractive index of the surrounding environment.

[0043] The high Q factor and high sensitivity formed by the special structure of the metasurface chip are used to improve the detection sensitivity of tumor markers. Since the refractive index of most substances is between 1.30-1.55, the refractive index around the metasurface is changed from 1.30 to 1.55 through FDTD. The frequency shift of the resonance position can be fitted into a straight line, and the slope is its sensitivity. The sensitivities of the two resonance positions are different, so the sensitivity range is between 652.2nm / RIU-796.5nm / RIU.

[0044] In this embodiment, the microfluidic channel is manufactured by photolithography technology, a PDMS substrate is manufactured by molding, a polydimethylsiloxane PDMS substrate is cast on a mold and cured, and then the biosensor substrate is installed in the polydimethylsiloxane PDMS microfluidic channel; the active sites on the surface of the super surface chip are bound to the tumor marker antibody during the immersion process of the tumor marker antibody solution, and the active sites that have not completed the binding with the tumor marker antibody are blocked by bovine serum albumin;

[0045] As a preferred implementation of this embodiment, the microfluidic channel is provided with an Inset port and an Outset port, and a channel is provided between the Inset port and the Outset port, so that the patient serum to be tested enters the channel from the Inset port and leaves from the Outset port after passing through the channel; the biosensor substrate is placed in the channel connecting the Inset port and the Outset port, and the tumor marker antibody binds to the antigen in the patient serum to be tested to form a complex;

[0046] In this embodiment, the patient serum containing tumor antigens is injected into the microfluidic channel through the inset port, the antigen is trapped in the channel, the antibody and the antigen specifically bind to form a complex complex, which causes the refractive index around the metasurface chip to change, and then the resonance position moves. Within a certain limit, the greater the antigen concentration, the more it binds to the antibody, and the more the refractive index changes. When the biosensor is irradiated with near-infrared light, the peak of the transmission spectrum collected by the spectrometer moves with the change of antigen concentration, which can realize the detection of tumor marker concentration.

[0047] A method for manufacturing the chiral symmetric Fano resonance biosensor as described above, characterized by comprising:

[0048] Step 1: Preparation of metasurface

[0049] First, the sapphire substrate was ultrasonically cleaned with an acetone solution, and a photoresist was spin-coated, and electron beam lithography (EBL) was used to expose the supersurface structure profile. A 5nm Cr adhesion layer and a 30nm Au layer were evaporated by vapor deposition, and then cleaned in an acetone solution. The supersurface structure profile formed by the electron beam lithography was peeled off to form a chiral symmetric double "N" gold nanostructure;

[0050] Step 2: Preparation of biosensor substrate

[0051] The supersurface chip is immersed in 11-mercaptoundecanoic acid (MUA) to allow the surface layer of the supersurface to adsorb a self-assembled layer formed by MUA, and then immersed in a mixed solution of NHS / EDC with a concentration ratio of 1:4; the activated supersurface chip is then incubated in a designated tumor marker antibody solution, and then in a bovine serum albumin (BSA) solution to block the active sites of the unbound antibodies. After incubation, the preparation of the biosensor substrate is completed;

[0052] Step 3. Preparation of microfluidic channels

[0053] A microfluidic mold is made by photolithography technology, and then a PDMS (polydimethylsiloxane) substrate is made by molding; PDMS is poured on the mold and cured; then the biosensor substrate is installed in the PDMS microfluidic channel to complete the preparation of the biosensor.

[0054] In this embodiment, the Inset port is connected to different Outset ports by a shunt pipe, a liquid shunt is provided at the rear end of the Inset port inlet, the front end of the liquid shunt is connected to the Inset port, and the rear end of the liquid shunt is connected to the shunt pipe, and the patient serum entering from the Inset port flows to different Outset ports through the shunt pipes respectively; the metasurface chip is arranged in the shunt pipe, and the metasurface chips in different shunt pipes are placed in advance incubated with different tumor marker antibody solutions, so that the metasurface chips in different shunt pipes are only combined with one tumor marker antibody, but the tumor marker antibodies combined with the metasurface chips in different shunt pipes are different, and one group of shunt pipes tests one tumor marker data, and multiple groups of parallel methods are used to achieve the purpose of joint testing;

[0055] The patient serum flowing into the Inset port will be evenly distributed to the shunt pipes, and the patient serum in the shunt pipes will flow out from the Outset port. Since the tumor marker antibodies bound to the super-surface chips in different shunt pipes are different, after the patient serum is shunted to different shunt pipes, different tumor marker antibodies in different shunt pipes capture tumor antigens. Each group of shunt pipes only captures the corresponding tumor antigens, and tumor marker information is obtained by summarizing and analyzing the shunt pipes.

[0056] A method for using the chiral symmetric Fano resonance biosensor as described above, characterized by comprising:

[0057] Step 1: Inject the patient's serum into the microfluidic channel through the Inset port, and intercept the tumor antigens in the patient's serum on the biosensor through the biosensor; intercept the patient's serum in the sensor channel. Since different antibodies will only intercept corresponding antigens, different types of antigens will be intercepted in different channels;

[0058] Step 2: After completing the patient serum perfusion operation, use near-infrared light to illuminate the biosensor through the microfluidic channel. By observing the peak shift of various transmission spectra collected by the spectrometer, the combined detection of multiple tumor markers can be achieved.

[0059] It should be noted that, for the above-mentioned embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the described order of actions, because according to the present invention, some steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A chiral symmetric Fano resonance biosensor, characterized in that: include: A metasurface chip, comprising a sapphire substrate and a plurality of sets of chiral symmetric double N structures, wherein the chiral symmetric double N structures are arranged on the upper surface of the sapphire substrate, The super surface chip is soaked in a self-assembled layer formed by adsorbing MUA (11-mercaptoundecanoic acid), and incubated with a specified tumor marker antibody solution to form a biosensor substrate, wherein the tumor marker antibody is linked to a chiral symmetric double N structure through MUA; A microfluidic channel, wherein the biosensor substrate is placed in the microfluidic channel; After the patient serum to be tested is injected into the microfluidic channel, the tumor marker antibody linked to the chiral symmetric double N structure combines with the tumor antigen in the patient serum, and the tumor antigen concentration data in the patient serum is obtained through the change of the refractive index.

2. A chiral symmetric Fano resonance biosensor according to claim 1, characterized in that: The chiral symmetric double N structure is composed of two groups of N-shaped structures, the N-shaped structure is 1100 nm long, 250 nm wide, and 30 nm high. The spacing between the two groups of N-shaped structures is 100 nm, and the distance between adjacent chiral symmetric double N structures is 100 nm.

3. A chiral symmetric Fano resonance biosensor as claimed in claim 2, characterized in that: The active sites on the surface of the ultrasurface chip are combined with the tumor marker antibody during immersion in the tumor marker antibody solution, and the active sites that have not completed the combination with the tumor marker antibody are blocked by bovine serum albumin.

4. A chiral symmetric Fano resonance biosensor as claimed in claim 3, characterized in that: The microfluidic channel is provided with an Inset port and an Outset port, and a channel is provided between the Inset port and the Outset port. The patient serum to be tested enters the channel from the Inset port and leaves from the Outset port after passing through the channel.

5. A chiral symmetric Fano resonance biosensor as claimed in claim 4, characterized in that: The biosensor substrate is placed in a channel connecting the Inset port and the Outset port.

6. A method for manufacturing a chiral symmetric Fano resonance biosensor as claimed in claim 5, characterized in that: include: Step 1: Preparation of metasurface First, the substrate made of sapphire material is ultrasonically cleaned with acetone solution, and photoresist is spin-coated, and EBL (electron beam exposure technology) is used for exposure to form the super surface structure profile; a 5nm Cr adhesion layer and a 30nm Au layer are evaporated by vapor deposition, and then cleaned in acetone solution, and the super surface structure profile formed by EBL is peeled off to form a chiral symmetric double "N" gold nano-microelement structure; Step 2: Preparation of biosensor substrate The metasurface chip is immersed in MUA to allow the surface of the metasurface to adsorb a layer of self-assembled layer formed by MUA, and then immersed in a NHS / EDC mixed solution; The activated supersurface chip is then placed in a designated tumor marker antibody solution for incubation, and then placed in a BSA (bovine serum albumin) solution to block the active sites of the unbound antibodies. After incubation, the preparation of the biosensor substrate is completed; Step 3. Preparation of microfluidic channels A microfluidic mold is made by photolithography technology, and then a PDMS (polydimethylsiloxane) substrate is made by molding; PDMS is poured on the mold and cured; then the biosensor substrate is installed in the PDMS microfluidic channel to complete the preparation of the biosensor.

7. The method for manufacturing a chiral symmetric Fano resonance biosensor according to claim 6, characterized in that: The Inset port is connected to different Outset ports by shunt pipes, and the patient's serum entering from the Inset port flows to different Outset ports through the shunt pipes.

8. The method for manufacturing a chiral symmetric Fano resonance biosensor according to claim 7, characterized in that: The super surface chip is arranged in the shunt channel, and the super surface chips in different shunt channels are placed in advance in different tumor marker antibody solutions for incubation, so that the super surface chips in different shunt channels are combined with a single tumor marker antibody.

9. The method for manufacturing a chiral symmetric Fano resonance biosensor according to claim 8, characterized in that: The concentration ratio of the NHS / EDC mixed solution is 1:4.

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