Aflatoxin b1 label-free detection method based on aptamer functionalized terahertz metasurface

Abstract

The application discloses an aflatoxin B1 label-free terahertz detection method based on aptamer functionalized gold super surface, and belongs to the technical field of terahertz biosensing. The method constructs a thiol-modified DNA aptamer recognition interface on a gold super surface terahertz resonance structure, and combines a surface sealing strategy to effectively reduce non-specific adsorption interference. When aflatoxin B1 specifically combines with the aptamer, the aptamer conformation changes and the local dielectric constant is disturbed, so that the terahertz resonance peak is caused to have a measurable frequency shift. The target object is quantitatively detected by comparing the terahertz transmission spectrum changes before and after combination. Further, through interface dissociation regeneration treatment, the sensor can be repeatedly used, and the detection stability and service life are improved. The application utilizes the coupling mechanism of the aptamer molecular recognition and the terahertz super surface high-Q resonance enhancement effect, and realizes high-sensitivity, strong-specificity and repeatable label-free detection of aflatoxin B1.

Description

Technical Field

[0001] This invention relates to the fields of terahertz detection technology, biosensing technology and food safety detection technology, and in particular to a label-free detection method for aflatoxin B1 based on an aptamer-functionalized terahertz metasurface. Background Technology

[0002] Aflatoxins are a class of secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus. Among them, aflatoxin B1 is highly toxic and carcinogenic, and is widely found in corn, peanuts, rice, wheat, nuts, and their processed foods, posing a serious threat to human health and food safety. Therefore, developing rapid and highly sensitive detection methods for aflatoxin B1 is of great significance.

[0003] Currently, the main methods for detecting aflatoxin B1 include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), thin-layer chromatography (TLC), and enzyme-linked immunosorbent assay (ELISA). While these methods offer high accuracy, they generally suffer from drawbacks such as complex sample pretreatment, long detection cycles, expensive equipment, high operator skill requirements, and difficulty in achieving rapid on-site detection.

[0004] Terahertz waves are electromagnetic waves with frequencies between microwaves and infrared. They possess characteristics such as low photon energy, non-contact and non-destructive testing, and sensitivity to molecular conformation, making them promising for applications in biomedicine, food safety, and agricultural product quality testing. However, because aflatoxin B1 is a small molecule toxin, its direct response in the terahertz frequency band is weak, making conventional terahertz time-domain spectroscopy techniques insufficient for trace detection.

[0005] Terahertz metasurfaces are composed of subwavelength artificial microstructures that can generate strong electromagnetic resonances and localized electromagnetic field enhancement effects in specific terahertz frequency bands. Their resonant frequency, transmission amplitude, and quality factor are highly sensitive to changes in the surface dielectric environment. If an aflatoxin B1-specific aptamer is immobilized on the surface of the terahertz metasurface's metallic structure, the conformational changes and localized dielectric property changes caused by the aptamer's binding to aflatoxin B1 can be amplified by the localized electromagnetic field of the terahertz metasurface, manifesting as a frequency shift of the resonant peak or a change in transmission intensity.

[0006] Therefore, it is necessary to propose a label-free detection method for aflatoxin B1 based on aptamer-functionalized terahertz metasurfaces to solve the problems of complex procedures, slow detection speed, and insufficient sensitivity of direct terahertz detection of small molecules in existing detection methods. Summary of the Invention

[0007] The purpose of this invention is to provide a label-free detection method for aflatoxin B1 based on an aptamer-functionalized terahertz metasurface, aiming to solve the problems of complex detection process, high detection cost, insufficient real-time performance, and weak terahertz response of small molecule toxins in the existing technology for aflatoxin B1 detection.

[0008] To achieve the above objectives, this invention employs a label-free detection method for aflatoxin B1 based on an aptamer-functionalized terahertz metasurface, comprising the following steps:

[0009] A DNA aptamer capable of specifically recognizing aflatoxin B1 was immobilized on the surface of a gold supersurface terahertz sensor, and the unoccupied gold surface sites were blocked to obtain an aptamer-functionalized gold supersurface terahertz sensor. The baseline terahertz transmission spectrum of the aptamer-functionalized gold supersurface terahertz sensor was measured. A test sample containing aflatoxin B1 was applied to the surface of the aptamer-functionalized gold supersurface terahertz sensor, causing aflatoxin B1 to specifically bind to the aptamer. After cleaning and drying, the aptamer-functionalized gold supersurface terahertz sensor was measured again. The terahertz transmission spectrum after application was compared with the baseline terahertz transmission spectrum to obtain the resonance peak frequency shift, and label-free detection of aflatoxin B1 was achieved based on the resonance peak frequency shift.

[0010] Preferably, the gold metasurface terahertz sensor includes a substrate layer and a metal resonant structure layer disposed on the surface of the substrate layer, wherein the metal resonant structure layer is composed of periodically arranged subwavelength metal units. The metal resonant structure may be an open-loop resonant structure, an asymmetric double-opening structure, a ring structure, an interdigitated structure, or other metasurface structures capable of generating a high Q-value resonant response in the terahertz frequency band.

[0011] Preferably, the aptamer can be a DNA aptamer that specifically recognizes aflatoxin B1, as disclosed in the prior art. This existing aptamer, after being modified by introducing a thiol linker at its 5′ end, is used to construct a recognition interface immobilized on a gold supersurface.

[0012] Preferably, the aptamer is a DNA aptamer modified with a thiol linker arm at its 5′ end, wherein the thiol linker arm comprises a Thiol-TEG modified structure. The Thiol group is used to connect to the gold supersurface structure via an Au–S bond, and the TEG linker arm is used to increase the spatial freedom of the aptamer on the gold surface, reduce the conformational restriction of the aptamer by the gold surface, and enhance the change in the local dielectric environment before and after aflatoxin B1 binding.

[0013] The innovation of this invention lies not in the core recognition sequence of the aptamer itself, but in the immobilization construction method of the aptamer on the surface of a gold superconducting terahertz sensor after modification with a 5′ end thiol linker, and the method for label-free detection of aflatoxin B1 based on aptamer conformational changes.

[0014] Preferably, the sealing treatment uses 6-mercapto-1-hexanol to seal the gold surface sites not occupied by the aptamer, thereby reducing non-specific adsorption.

[0015] Preferably, the terahertz response characteristics include resonant peak frequency shift Δf, transmission amplitude change ΔT, quality factor change ΔQ, or a combination thereof. The quantitative relationship between the aflatoxin B1 concentration and the terahertz response characteristics can be established using standard curves, linear regression, partial least squares regression, support vector machines, random forests, or other machine learning methods.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] First, the present invention uses an aflatoxin B1 specific aptamer as a recognition element, which has high selectivity and specificity;

[0018] Second, this invention utilizes the high Q-value resonance and local electromagnetic field enhancement effect of terahertz metasurfaces to improve the response capability to weak dielectric changes of small molecule toxins.

[0019] Third, the present invention improves the spatial conformational freedom of the aptamer on the gold surface through the Thiol-TEG modified structure, which is beneficial to improving the consistency of the aptamer binding response.

[0020] Fourth, this invention achieves label-free detection of aflatoxin B1 without the need for fluorescent, enzyme, or nanolabels;

[0021] Fifth, the present invention can improve detection reliability through bare gold metasurface comparison, sensor regeneration and multi-parameter analysis, and can be extended to terahertz label-free detection of other small molecule pollutants or biomarkers. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic flowchart of a label-free detection method for aflatoxin B1 based on an aptamer-functionalized terahertz metasurface according to the present invention.

[0024] Figure 2 This is a schematic diagram illustrating the principle by which the aptamer of this invention specifically binds to aflatoxin B1 and causes a change in the local dielectric environment.

[0025] Figure 3 Schematic diagram of the frequency shift of the terahertz transmission spectrum resonance peak under the action of different concentrations of aflatoxin B1;

[0026] Figure 4 Schematic diagram of the quantitative modeling process between terahertz response characteristics and aflatoxin B1 concentration in this invention Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] This invention provides a label-free detection method for aflatoxin B1 based on an aptamer-functionalized terahertz metasurface. The method involves immobilizing an aflatoxin B1-specific aptamer on the surface of a gold metasurface sensor and using a terahertz time-domain spectroscopy system to detect the change in resonant frequency before and after the aptamer binds to aflatoxin B1, thereby achieving label-free detection of aflatoxin B1.

[0029] Example 1

[0030] After fabricating the gold metasurface sensor, it was cleaned.

[0031] The aflatoxin B1 specific aptamer was folded, dropped onto the surface of the gold superstructure, and fixed.

[0032] After fixation, a sealing process is performed to reduce non-specific adsorption. This yields an aptamer-functionalized gold metasurface sensor, which can be used for subsequent terahertz detection.

[0033] Example 2: Preparation of aflatoxin B1 standard solution

[0034] Aflatoxin B1 standard solution was prepared using acetonitrile-water solution as a uniform solvent system. Standard solutions of different concentrations were obtained through serial dilution for subsequent detection and quantitative analysis.

[0035] Example 3: Bare Sensor Comparison Detection

[0036] After cleaning the bare gold metasurface sensor, its terahertz response under different concentrations of aflatoxin B1 was measured to obtain nonspecific background signals.

[0037] This control group was used to evaluate the specific detection performance of the aptamer-functionalized sensor.

[0038] Example 4: Detection of aptamer-functionalized sensors

[0039] The sample was dropped onto the surface of the aptamer-functionalized sensor and wet-incubated to allow aflatoxin B1 to specifically bind to the aptamer.

[0040] After incubation, the cells are gently cleaned and dried, and then the resonant frequency change is measured using a terahertz time-domain spectroscopy system.

[0041] The presence and concentration of aflatoxin B1 can be determined by calculating the frequency shift Δf. The sensor can be regenerated after each concentration detection to enable repeated detection.

[0042] Example 5: Data Processing and Quantitative Analysis

[0043] By comparing the terahertz responses of bare sensors and aptamer-functionalized sensors at different concentrations, the frequency shift of the resonance peak, changes in transmission amplitude, and changes in quality factor were calculated. Standard curves or regression models were established to achieve rapid, label-free quantitative detection of aflatoxin B1.

[0044] The specificity and anti-interference ability of the method can be verified by detecting blank samples, random sequence aptamer samples, and similar toxin samples.

[0045] Through the above embodiments, this invention transforms the molecular recognition event between aflatoxin B1 and its aptamer into a change in the dielectric environment of the near-field region of a gold metasurface, and utilizes the high Q-value resonance characteristics of the terahertz metasurface to amplify this change into a detectable resonant frequency shift signal, thereby achieving rapid, highly sensitive, and label-free detection of aflatoxin B1.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces, characterized in that, The process includes the following steps: immobilizing a DNA aptamer capable of specifically recognizing aflatoxin B1 onto the surface of a gold supersurface terahertz sensor, and sealing the surface sites not occupied by the aptamer to obtain an aptamer-functionalized gold supersurface terahertz sensor; measuring the terahertz transmission spectrum of the aptamer-functionalized gold supersurface terahertz sensor under no-load conditions as a baseline spectrum; and applying a test sample containing aflatoxin B1 to the surface of the aptamer-functionalized gold supersurface terahertz sensor, allowing aflatoxin B1 to interact with the DNA aptamer. The ligands bind specifically; after cleaning and drying the aptamer-functionalized gold metasurface terahertz sensor, its terahertz transmission spectrum is measured as the test spectrum; the test spectrum is compared with the baseline spectrum to obtain the frequency shift of the sensor's resonant peak, and label-free detection of aflatoxin B1 is achieved based on the frequency shift; the aptamer-functionalized gold metasurface terahertz sensor that has completed detection is regenerated to dissociate the bound aflatoxin B1 from the aptamer surface, so as to achieve repeated detection by the sensor.

2. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The method for obtaining the resonant peak frequency shift includes the following steps: measuring the baseline terahertz transmission spectrum of the aptamer-functionalized gold metasurface terahertz sensor under no-load conditions; applying the sample to be tested to the surface of the aptamer-functionalized gold metasurface terahertz sensor; cleaning and drying the sensor after application, and measuring its terahertz transmission spectrum. The frequency shift of the sensor resonance peak before and after aflatoxin B1 treatment was obtained by comparing the test spectrum with the baseline spectrum.

3. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The gold metasurface terahertz sensor is a terahertz metasurface chip with a metal resonant structure on its surface. The metal resonant structure can generate a resonant response in the terahertz frequency band and is sensitive to changes in the local dielectric environment of the surface.

4. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The DNA aptamer is a DNA aptamer modified with a thiol linker arm at the 5′ end. The thiol is used to fix the aptamer to the surface of the gold supersurface terahertz sensor via Au–S bonds.

5. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 4, characterized in that, The connecting arm is a TEG connecting arm, which is used to increase the spatial degree of freedom of the aptamer on the gold surface, reduce the restrictive effect of the gold surface on the conformation of the aptamer, and enhance the terahertz response caused by the change in local dielectric environment before and after aflatoxin B1 binding.

6. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The sample to be tested is applied to the surface of the aptamer-functionalized gold metasurface terahertz sensor by drop-addition and wet incubation. After the sample is applied, it is washed with an aqueous solution containing acetonitrile, rinsed with deionized water and dried before terahertz transmission spectrum measurement.

7. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, Aflatoxin B1 binds to the aptamer, causing a change in the spatial conformation of the aptamer, which in turn leads to a change in the effective dielectric constant of the near-field region of the gold supersurface and causes a frequency shift in the terahertz resonance peak.

8. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The regeneration process uses an acidic dissociation solution to dissociate the bound aflatoxin B1 from the aptamer surface. The sensor is then cleaned, dried, and the baseline is remeasured. When the baseline returns to near the initial baseline, the next detection is performed.

9. The label-free terahertz detection method for aflatoxin B1 based on aptamer-functionalized gold metasurfaces as described in claim 1, characterized in that, The detection method further includes measuring the non-specific response of the bare gold metasurface terahertz sensor to aflatoxin B1, and comparing the non-specific response with the response of the aptamer-functionalized gold metasurface terahertz sensor to distinguish between the non-specific response and the aptamer-specific recognition response.

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