Aflatoxin B1 sensor based on liquid crystal microdroplet enhanced hydrogel echo wall mode microcavity and detection method

Abstract

The invention discloses an aflatoxin B1 sensor based on a liquid crystal microdroplet enhanced hydrogel echo wall mode microcavity and a detection method. The detection method comprises the following steps: A1, preparing liquid crystal microdroplet enhanced hydrogel-glass microbubbles; a2, heating and stretching the single-mode optical fiber through flame to obtain a tapered optical fiber, adjusting the relative position of the tapered optical fiber and the liquid crystal microdroplet enhanced hydrogel-glass microbubble, enabling laser emitted by a scanning laser to enter the first single-mode optical fiber through the tapered optical fiber to realize optical field coupling, and obtaining the optical coupling liquid crystal microdroplet enhanced hydrogel-glass microbubble, the laser enters the second single-mode optical fiber through the tapered optical fiber, an aflatoxin B1 solution to be detected is added into the optically coupled liquid crystal microdroplet enhanced hydrogel-glass microbubble, optical information is recorded, and the concentration of the aflatoxin B1 solution is calculated. The problem of low sensitivity caused by naked eye observation or wide spectrum monitoring commonly used in a hydrogel sensor and a liquid crystal sensor is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of fiber optic sensing, and in particular relates to a method for detecting aflatoxin B1 based on a hydrogel whispering gallery mode microcavity enhanced by liquid crystal microdroplets. Background Art

[0002] Chinese herbal medicines are mainly composed of drugs derived from plants, minerals, and animals and have been widely used to treat various diseases. A major problem is the contamination of Chinese medicinal materials by molds, such as aflatoxin B1. The ingestion of aflatoxin B1 poses a significant risk to human health. Therefore, regulatory agencies including China, the United States, and Europe have established strict pharmacopoeias to limit the content of aflatoxin B1 in herbal medicines. Therefore, highly sensitive and specific detection of trace aflatoxin B1 is crucial for ensuring the safety and quality control of traditional Chinese medicines.

[0003] Hydrogels have received extensive attention due to their unique porous structure and tunable physicochemical properties. Their compatibility with various materials enables them to effectively combine aptamers, nanomaterials, polymers, and biomacromolecules. Due to the functionalization and enhancement of these loaded components, hydrogels can provide a versatile platform for a wide range of sensing and medical applications. In addition, hydrogels have optical transparency and tunable refractive indices, which can manipulate the optical path, making them an ideal choice for optical sensing. Therefore, various hydrogel-based optical sensors, such as hydrogel photonic crystal sensors, hydrogel fluorescence sensors, and hydrogel microresonator sensors, have been developed for detection. Although hydrogel optical sensors have developed rapidly, challenges still exist: naked-eye detection is prone to subjectivity and variability in perception; although fluorescence biosensors can perform quantitative analysis, they usually suffer from problems such as broad emission spectra and low signal-to-noise ratios. The complex sensing environment in Chinese herbal medicine extraction solutions exacerbates these limitations, making it difficult to capture the subtle and critical interactions in the analyte fluid.

[0004] Compared with broadband fluorescence, the whispering gallery mode resonator based on evanescent field expansion and boundary total internal reflection provides excellent background suppression and narrow linewidth. Due to the multiple round trips of light, microbubbles with high quality factors allow light to interact with analytes millions of times and exhibit high sensitivity to interface changes. By functionalizing the microbubbles with capture agents, studies have shown that whispering gallery mode microbubbles have the ability to detect analytes with high sensitivity. In addition, liquid crystals (LCs) can serve as a powerful sensing tool to probe the interaction of analytes at their interfaces by functionalizing the interfaces of liquid crystals. Based on the orientation transition and optical anisotropy of liquid crystal molecules, liquid crystal-based sensors have been developed for detecting different targets, including proteins, DNA, small molecules, ions, viruses, and cells. However, most transduction methods of liquid crystal sensors are based on naked-eye observation under a polarized microscope, which limits the further improvement of their sensitivity. By combining the multifunctional properties of liquid crystals with hydrogel whispering gallery mode microresonators, a promising aflatoxin B1 detection platform can be developed, which has important potential in promoting light-stimulated responses and improving CHM analysis (Contextual History Method Analysis). Summary of the Invention

[0005] In view of this, the present invention aims to propose an aflatoxin B1 sensor and detection method based on a liquid crystal microdroplet-enhanced hydrogel whispering gallery mode microcavity to solve at least one technical problem in the background art.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows: A detection method for aflatoxin B1 based on a liquid crystal microdroplet-enhanced hydrogel whispering gallery mode microcavity includes the following steps: A1: Prepare a liquid crystal microdroplet-enhanced hydrogel-glass microbubble; A2: Obtain a tapered fiber by stretching a single-mode fiber through flame heating, adjust the relative position of the tapered fiber and the liquid crystal microdroplet-enhanced hydrogel-glass microbubble prepared in step A1, make the laser emitted by the scanning laser enter, and realize optical field coupling through the tapered fiber into the first single-mode fiber to obtain an optically coupled liquid crystal microdroplet-enhanced hydrogel-glass microbubble. Then the laser enters the second single-mode fiber through the tapered fiber, add the aflatoxin B1 solution to be detected into the liquid crystal microdroplet-enhanced hydrogel-glass microbubble obtained in step A2, record the optical information, and calculate the concentration of the aflatoxin B1 solution to be detected.

[0007] Further, the preparation of the liquid crystal microdroplet-enhanced hydrogel-glass microbubble in step A1 includes the following steps: S1: Use the thermal melting method to taper the microcapillary. Heat the tapered region of the microcapillary with a hydrogen-oxygen flame, pressurize the inside of the microcapillary, and promote the internal expansion of the microcapillary to obtain microbubbles. S2: Prepare cetrimonium bromide-functionalized liquid crystal microdroplets. S3: Dissolve acrylamide, N,N'-methylenebisacrylamide, and polyethylene glycol diacrylate in a buffer solution to obtain a mixed solution. Mix the liquid crystal microdroplets prepared in step S2 with the mixed solution and a photoinitiator to obtain a hydrogel precursor solution. Inject the hydrogel precursor solution into the microbubbles obtained in step S1, cure and rinse, and a liquid crystal microdroplet-reinforced hydrogel-glass microbubble is formed inside the microcapillary.

[0008] Further, in step S1, commercial quartz microcapillaries are used to fabricate microbubbles.

[0009] Further, the preparation of the cetrimonium bromide-functionalized liquid crystal microdroplets in step S2 includes dissolving cetrimonium bromide in chloroform, then adding it to the liquid crystal, drying under a nitrogen stream, ultrasonically mixing the aptamer solution and the blocking oligonucleotide to form functionalized liquid crystal droplets, and keeping the liquid crystal droplets in the aptamer solution at 3 - 5 °C for 10 - 14 hours.

[0010] Further, the sequence of the aptamer in step S2 is SEQ ID NO:1.

[0011] The sequence of SEQ ID NO:1 is: 5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3′.

[0012] Further, the buffer solution in step S3 is a phosphate buffer solution; and / or the photoinitiator in step S3 includes 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

[0013] and / or the curing and rinsing in step S3 includes curing with ultraviolet light for 1.5 - 2.5 s and rinsing with a buffer solution; and / or the wavelength of the ultraviolet light is 350 - 370 nm.

[0014] Further, in step A2, the single-mode optical fiber is stretched by flame heating to obtain a tapered optical fiber, and the waist diameter of the tapered optical fiber is 3 - 4 µm; In step A2, record the optical information in the power meter within 40 - 50 seconds; In step A2, calculate the concentration of aflatoxin B1 according to the moving distance of the spectral resonance wavelength.

[0015] Aflatoxin B1 sensor based on a liquid crystal droplet - enhanced hydrogel whispering gallery mode microcavity, using the above - mentioned detection method for aflatoxin B1 based on a liquid crystal droplet - enhanced hydrogel whispering gallery mode microcavity, includes a liquid crystal droplet - enhanced hydrogel - glass microbubble, a tunable scanning laser, a tapered fiber, a power meter, and a bracket; The first single - mode fiber and the second single - mode fiber are connected through a tapered fiber; the position of the tapered fiber is adjusted by the bracket; the tunable scanning laser is set at one end of the first single - mode fiber; the power meter is set at one end of the second single - mode fiber; The laser emitted by the scanning laser is incident, enters the tapered fiber through the first single - mode fiber, an evanescent field is generated in the tapered fiber, and the bracket adjusts the relative position between the tapered fiber and the liquid crystal droplet - enhanced hydrogel - glass microbubble to achieve optical field coupling to obtain a photo - coupled liquid crystal droplet - enhanced hydrogel - glass microbubble, which returns to the second single - mode fiber through the tapered fiber, and the corresponding optical signal is detected by the power meter. By measuring the redshift of the spectral resonance wavelength, high - resolution sensing of aflatoxin B1 concentration is achieved.

[0016] Compared with the prior art, the aflatoxin B1 sensor and detection method based on a liquid crystal droplet - enhanced hydrogel whispering gallery mode microcavity of the present invention have the following advantages: 1. The present application proposes an aptamer - enhanced hydrogel whispering - gallery - mode microresonator for aflatoxin B1 detection. A microbubble is used as the structural framework of the hydrogel to form a whispering - gallery - mode resonance. To achieve the sensing function, aptamer - functionalized liquid crystal droplets are integrated into the hydrogel, and then the hydrogel is modified inside the microbubble to achieve aflatoxin B1 capture. After aflatoxin B1 binds, the interaction between the aptamer and aflatoxin B1 triggers the release of cetyltrimethylammonium bromide (CTAB) in the aptamer - CTAB complex, and at the same time causes the orientation of the liquid crystal molecules to change from bipolar to radial arrangement. This change leads to an increase in refractive index, resulting in a redshift of the spectral resonance wavelength. By monitoring this wavelength shift, the concentration of aflatoxin B1 can be accurately analyzed.

[0017] 2. The present application can detect aflatoxin B1. Utilizing the liquid crystal orientation change and the birefringence effect in its optical properties, combined with the light - matter interaction in the whispering - gallery - mode microresonator, it provides an opportunity for sensing and amplifying subtle detection target binding events. The spectral resonance wavelength redshift is used as a parameter to determine the content of aflatoxin B1 bound to the hydrogel whispering - gallery - mode microbubble. It solves the problem of low sensitivity caused by naked - eye observation or broad - spectrum monitoring commonly used in hydrogel sensors and liquid crystal sensors. Description of the Drawings

[0018] The accompanying drawings, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 Schematic diagram of an aflatoxin B1 sensor based on a liquid crystal microdroplet-enhanced hydrogel whispering gallery mode microcavity according to Embodiment 1 of the present invention; Figure 2 Cross-sectional schematic diagram of a light-coupled liquid crystal microdroplet-enhanced hydrogel-glass microbubble with a whispering gallery mode according to Embodiment 1 of the present invention; Figure 3 Schematic diagram of the liquid crystal microdroplet orientation change according to Embodiment 1 of the present invention; Figure 4 Schematic diagram of the microbubble spectral response according to Embodiment 1 of the present invention (A is the schematic diagram of the microbubble spectral response under the action of 1 ng / ml aflatoxin B1, and B is the schematic diagram of the microbubble spectral response under the action of 1 pg / ml aflatoxin B1).

[0019] Figure 5 Schematic diagram of the microbubble spectral response according to Comparative Example 1 of the present invention.

[0020] Explanation of reference numerals: 1. Tunable scanning laser; 2. Tapered fiber; 3. Liquid crystal microdroplet-enhanced hydrogel-glass microbubble; 4. Power meter; 5. Bracket; 6. First single-mode fiber; 7. Second single-mode fiber. Detailed implementation manners

[0021] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0022] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0023] Embodiment 1 An aflatoxin B1 sensor based on a whispering gallery mode microresonator of a liquid crystal microdroplet-enhanced hydrogel. The sensor is composed of a tunable scanning laser 1, a tapered fiber 2, a liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3, and a power meter 4 connected by a single-mode fiber; wherein, the position of the tapered fiber is controlled and adjusted by a bracket 5 (the bracket 5 is a three-dimensional displacement bracket table).

[0024] The laser emitted by the scanning laser 1 is incident, enters the tapered fiber 2 through the first single-mode fiber 6, an evanescent field is generated and coupled to the liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3. Subsequently, the light energy is coupled out of the liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3, returns to the second single-mode fiber 7 through the tapered fiber 2. Therefore, this structure can form a whispering gallery mode microresonator.

[0025] The present invention discloses a measurement method of an aflatoxin sensor based on a liquid crystal microdroplet-enhanced hydrogel whispering gallery mode optical resonator, and the method includes the following steps: The laser emitted by the scanning laser 1 passes through the first single-mode optical fiber 6. Adjust the relative position of the tapered optical fiber 2 and the liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3 prepared in step A1. The laser emitted by the scanning laser is incident, and enters the first single-mode optical fiber 6 through the tapered optical fiber 2 to achieve optical field coupling, obtaining an optically coupled liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3. The laser passes through the tapered optical fiber 2 and enters the second single-mode optical fiber 7. The aflatoxin B1 solution to be detected is added into the optically coupled liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3, and the corresponding optical signal is detected by the power meter 5. By measuring the WGM spectral shift (Whispering Gallery Mode spectral shift), high-resolution sensing of the aflatoxin B1 concentration is achieved.

[0026] The liquid crystal microdroplet-enhanced hydrogel-glass microbubble 3, as the main sensitive element, can achieve the detection of aflatoxin B1. An aptamer (sequence: 5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3′) and CTAB are modified on the liquid crystal microdroplet interface, and the liquid crystal microdroplets are contained in the hydrogel, enabling specific detection of aflatoxin B1. Based on the interaction between aflatoxin B1 and the aptamer, the structure of the aptamer changes, promoting the release of CTAB and the transformation of the liquid crystal orientation arrangement. The evanescent field passes through the microbubble and enters the liquid crystal microdroplet-hydrogel region and undergoes total reflection. Due to the increase in the liquid crystal refractive index, it first shows a spectral red shift in the WGM spectrum. Finally, the spectral red shift response is used as a sensing parameter to achieve highly sensitive sensing of aflatoxin B1 ultimately.

[0027] Example 2 Step 1: Use a commercial quartz microcapillary with an outer radius of 175 μm and an inner radius of 125 μm to fabricate a microbubble, and use a centimeter-scale oxyhydrogen flame to achieve the taper of the microcapillary by the thermal melting method; Step 2: Use a millimeter-scale oxyhydrogen flame to heat the tapered region of the microcapillary. At the same time, pressurize the inside of the microcapillary to promote the expansion inside the microcapillary and form a microbubble with a diameter of 210 μm; Step 3: To prepare cetyltrimethylammonium bromide (CTAB)-functionalized LC microbubbles, CTAB is dissolved in chloroform and added to 200 μL LC, and dried under a nitrogen stream. A 100 μM aptamer solution and a 200 μM blocking oligonucleotide are ultrasonically mixed to form functionalized LC droplets. Then, the LC droplets are kept in the aptamer solution at 4°C for 12 hours.

[0028] To block unbound CTAB, 200 μM blocking oligonucleotides were used.

[0029] Step 4: Prepare a hydrogel precursor solution by dissolving 475 mg of acrylamide, 25 mg of N,N - methylenebisacrylamide, and 5 μL of polyethylene glycol diacrylate in 500 μL of PBS buffer (Phosphate - Buffered Saline). Subsequently, add 60 mg of photoinitiator and 100 μL of LC microdroplets to the hydrogel precursor solution to achieve subsequent liquid crystal microdroplet - hydrogel polymerization. After injecting the precursor solution into the microbubble through a syringe, perform ultraviolet light curing for 2 seconds. Rinse the inside of the microbubble with PBS buffer using a syringe to remove the unreacted precursor solution. An aptamer for detecting aflatoxin B1 was formed and enhanced. All the above - mentioned treatments were carried out by directly injecting the solution into the microbubble.

[0030] Step 6: Use a single - mode optical fiber and fabricate a tapered optical fiber with a waist diameter of 3 - 4 μm by the heat - melting technique and fix it on the three - dimensional displacement support stage 5. Adjust the three - dimensional displacement support stage 5 so that the tapered optical fiber realizes optical field coupling with the microresonator; Step 7: Pump the aflatoxin B1 solution into the microbubble through a syringe pump and record the optical signal in the power meter within 40 seconds. The power meter directly detects Figure 4 the signal, the displacement at the resonance wavelength (valley) position of the spectrum within 40 seconds, and the concentration of the aflatoxin B1 solution is y. The calculation method is as described below: y = 0.01174x + 0.02259 y is the concentration, with the unit of pg / ml; x is the wavelength displacement, with the unit of nm.

[0031] Comparative Example 1 Compared with Example 1, no liquid crystal microdroplets were added, and the rest were the same.

[0032] As Figure 5 shown, for the detection example of 40 pg / ml of aflatoxin B1, the group with added liquid crystal has an obvious spectral response (0.456 nm), while the group without liquid crystal enhancement only produces a spectral response of 0.002 nm. According to the criterion of feedbacking the aflatoxin B1 content information based on the degree of spectral response, adding liquid crystal is beneficial to amplifying the spectral change information and realizing highly sensitive detection.

[0033] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection method of aflatoxin B1 based on a liquid crystal microdroplet enhanced hydrogel whispering gallery mode microcavity, characterized in that: The method includes the following steps: A1: Prepare a liquid crystal droplet-enhanced hydrogel-glass microbubble; A2: A single-mode optical fiber is stretched by flame heating to obtain a tapered optical fiber. Adjust the relative position between the tapered optical fiber and the liquid crystal droplet-enhanced hydrogel-glass microbubble prepared in step A1. The laser emitted by the scanning laser is incident, enters the first single-mode optical fiber through the tapered optical fiber to achieve optical field coupling, and a photo-coupled liquid crystal droplet-enhanced hydrogel-glass microbubble is obtained. The laser then enters the second single-mode optical fiber through the tapered optical fiber. The aflatoxin B1 solution to be detected is added into the photo-coupled liquid crystal droplet-enhanced hydrogel-glass microbubble, and the optical information is recorded to calculate the concentration of the aflatoxin B1 solution to be detected.

2. The detection method of aflatoxin B1 for a hydrogel whispering gallery mode microcavity based on liquid crystal droplet enhancement according to claim 1, wherein: The preparation of the liquid crystal microdroplet-enhanced hydrogel-glass microbubble in step A1 includes the following steps: S1: Use the thermal melting method to taper a microcapillary. Heat the tapered area of the microcapillary with a hydrogen-oxygen flame, pressurize the inside of the microcapillary, and promote the internal expansion of the microcapillary to obtain a microbubble; S2: Prepare cetyltrimethylammonium bromide-functionalized liquid crystal droplets; S3: Dissolve acrylamide, N,N'-methylenebisacrylamide, and polyethylene glycol diacrylate in a buffer solution to obtain a mixed solution. Mix the liquid crystal droplets prepared in step S2 with the mixed solution and a photoinitiator to obtain a hydrogel precursor solution. Inject the hydrogel precursor solution into the microbubble obtained in step S1, cure and rinse, and a liquid crystal droplet-enhanced hydrogel-glass microbubble is formed inside the microcapillary.

3. The detection method of aflatoxin B1 based on a liquid crystal microdroplet enhanced hydrogel whispering gallery mode microcavity according to claim 2, wherein: In step S1, a commercial quartz microcapillary is used to manufacture the microbubble.

4. The detection method of aflatoxin B1 based on a liquid crystal microdroplet-enhanced hydrogel whispering gallery mode microcavity according to claim 2, wherein: The preparation of the cetyltrimethylammonium bromide-functionalized liquid crystal droplets in step S2 includes dissolving cetyltrimethylammonium bromide in chloroform, then adding it to the liquid crystal, drying under a nitrogen stream, ultrasonically mixing the aptamer solution and the blocking oligonucleotide to form functionalized liquid crystal droplets, and keeping the liquid crystal droplets in the aptamer solution at 3-5 °C for 10-14 hours.

5. The detection method of aflatoxin B1 based on a liquid crystal microdroplet enhanced hydrogel whispering gallery mode microcavity according to claim 4, characterized in that: The sequence of the aptamer in step S2 is SEQ ID NO:

1.

6. The detection method of aflatoxin B1 based on a liquid crystal microdroplet enhanced hydrogel whispering gallery mode microcavity according to claim 4, characterized in that: The buffer solution in step S3 is a phosphate buffer solution; and / or the photoinitiator in step S3 includes 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone; and / or the curing and rinsing in step S3 includes curing with ultraviolet light for 1.5-2.5 s and rinsing with a buffer solution; and / or the wavelength of the ultraviolet light is 350-370 nm.

7. The detection method of aflatoxin B1 based on a hydrogel whispering gallery mode microcavity enhanced by liquid crystal microdroplets according to claim 1, characterized in that: In step A2, a single-mode optical fiber is stretched by flame heating to obtain a tapered optical fiber, and the waist diameter of the tapered optical fiber is 3-4 µm; In step A2, the optical information in the power meter is recorded within 40-50 seconds; In step A2, the concentration of aflatoxin B1 is calculated according to the moving distance of the spectral resonance wavelength.

8. Aflatoxin B1 sensor based on a liquid crystal droplet-enhanced hydrogel whispering gallery mode microcavity, using the detection method of aflatoxin B1 based on a liquid crystal droplet-enhanced hydrogel whispering gallery mode microcavity according to any one of claims 1-7, characterized in that: It includes a liquid crystal droplet-enhanced hydrogel-glass microbubble, a tunable scanning laser, a tapered optical fiber, a power meter, and a bracket; The first single-mode optical fiber and the second single-mode optical fiber are connected through a tapered optical fiber; the position of the tapered optical fiber is adjusted by the bracket, The tunable scanning laser is arranged at one end of the first single-mode optical fiber; The power meter is arranged at one end of the second single-mode optical fiber; The laser emitted by the scanning laser enters through the first single-mode optical fiber into the tapered optical fiber, an evanescent field is generated in the tapered optical fiber, the bracket adjusts the relative position between the tapered optical fiber and the liquid crystal microdroplet-enhanced hydrogel-glass microbubble to achieve optical field coupling to obtain the optically coupled liquid crystal microdroplet-enhanced hydrogel-glass microbubble, and it returns to the second single-mode optical fiber through the tapered optical fiber. The corresponding optical signal is detected by a power meter, and by measuring the redshift of the spectral resonance wavelength, high-resolution sensing of the concentration of aflatoxin B1 is achieved.

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