Preparation method of gold nanobipyramid and application of gold nanobipyramid in optical fiber biosensing

Abstract

The present application relates to the technical field of nanomaterials, and particularly relates to a preparation method of gold nanobipyramids and application of the gold nanobipyramids in optical fiber biosensing. The gold nanobipyramids prepared by the present application have high yield and good uniformity, provide more possibilities for optical fiber biosensing and treatment in the near-infrared II band, and have simple operation, short process, low cost of consumables, and low overall preparation cost. When applied to optical fiber biosensing, the gold nanobipyramids loaded with WS2 are modified on the surface of a conical micro-nano optical fiber, and compared with a micro-nano optical fiber sensor without interface modification, the optical fiber biosensing has higher sensitivity, can detect different concentrations of prostate cancer cell exosomes (even in the whole serum of a prostate cancer patient), significantly improves the sensitivity and accuracy of early diagnosis and screening of prostate cancer cells, and has simple preparation method, non-toxic raw materials, and high safety in biological application.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a method for preparing gold nanobipyramidal structures and their application in fiber optic biosensing. Background Technology

[0002] Noble metal nanoparticles possess abundant localized surface plasmon resonance (LSPR) properties. Their surface plasmon resonance peaks typically redshift with increasing refractive index of the surrounding environment, and their surface plasmon wavelengths are highly sensitive to the surrounding refractive index, forming the basis of LSPR spectroscopy. Therefore, ultrasensitive plasmon sensing can be achieved using gold nanoparticles.

[0003] In recent years, the synthesis of anisotropic nanoparticles has attracted widespread attention in the biomedical field due to their unique properties in spectroscopy, optoelectronics, catalytic sensing, and drug delivery. A large number of potential applications lie primarily in their interesting local surface plasmon resonance (LSPR) properties, with anisotropic metallic nanoparticles (rods, bipyramids, and decahedrons) exhibiting two LSPR modes corresponding to the transverse and longitudinal directions, thus possessing more prominent advantages. Gold bipyramid nanoparticles have attracted considerable attention due to their superior optical properties, larger extinction cross section, and more significant local electric field enhancement than gold nanorods. Unlike the rounded ends of gold nanorods, gold bipyramids have two sharper vertices at their intersections. Studies have shown that, due to their high absorbance in the near-infrared (NIR) window and biocompatibility in humans, gold bipyramids have been used as a photothermal conversion agent for cancer ablation. Compared to the LSPR properties of gold nanorods, the sharp-edge structure of gold bipyramids is more sensitive to local changes in the dielectric environment and exhibits stronger local electric field enhancement. Gold nanobipyramidal structures not only possess a horizontal SPR peak similar to that of gold nanoparticles, allowing absorption of visible light (530 nm), but also exhibit a higher longitudinal SPR peak in the near-infrared region (λ>700 nm). Furthermore, the longitudinal plasmon resonance wavelengths (LPRWs) of gold nanobipyramidal structures can be tuned to match the excitation wavelength, thereby improving the sensitivity of fiber optic micro / nano biosensors. However, currently available gold nanobipyramidal structures can only match excitation wavelengths in the near-infrared I region (NIR-I, 700-950 nm) and the visible light band, with an aspect ratio of approximately 100 nm. In contrast, near-infrared II region (NIR-II, 1000-1700 nm) lasers have minimal tissue dispersion and deeper tissue penetration (up to 15 nm). Gold nanobipyramidal structures matched to this wavelength range can serve as materials for photothermal therapy. Therefore, it is extremely important to fabricate gold nanobipyramidal structures with different aspect ratios to match local plasmon resonance in higher wavelength bands (especially the near-infrared II region). Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a method for preparing a gold nanobipyramidal solution and a method for modifying and characterizing gold nanobipyramidal solutions using two-dimensional materials, applicable to the field of fiber optic biosensing.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing gold nanobipyramidal structures includes the following steps:

[0007] S101. Add HAuCl4 aqueous solution and sodium citrate solution to deionized water, stir, and then add NaBH4 solution to obtain an orange-red gold seed solution;

[0008] S102. Aging the orange-red gold seed solution at room temperature for at least 2 hours to completely hydrolyze the unreacted NaBH4 and form a pink seed solution;

[0009] S103. Add HAuCl4 aqueous solution, AgNO3 solution, HCl and ascorbic acid to CTAB solution in sequence, and stir the solution until it becomes colorless to obtain the growth solution;

[0010] S104. Inject the pink seed solution obtained in step S102 into the growth solution and let it stand in a 30°C water bath for 20-24 hours until the solution turns into a purplish-red solution;

[0011] S105. The purple-red solution is centrifuged using density gradient centrifugation to remove impurities and obtain a purified gold nanoparticle bipyramidal solution, which is the gold nanoparticle bipyramidal solution.

[0012] In the above method, the aspect ratio of the gold nanobipyramidal structure is controlled by adjusting the amount of pink seed solution added in step S104.

[0013] Preferably, in step S101, the volume ratio of HAuCl4 aqueous solution, sodium citrate solution, deionized water, and NaBH4 solution is 0.125:0.25:9.625:0.15; the concentrations of the HAuCl4 aqueous solution, sodium citrate solution, and NaBH4 solution are all 0.01M. Under these concentration and ratio conditions, morphologically stable gold nanocrystals can be produced.

[0014] Preferably, in step S103: the concentration of CTAB solution is 0.1M, the concentration of HAuCl4 aqueous solution is 0.01M, the concentration of AgNO3 solution is 0.01M, the concentration of HCl is 1M, and the concentration of ascorbic acid is 0.1M; the volume ratio of CTAB solution, HAuCl4 aqueous solution, AgNO3 solution, HCl, and ascorbic acid is 40∶2∶0.4∶0.5∶0.32. Under this ratio, the solution growth environment results in small size differences in gold nanoparticles, exhibiting advantages of high reproducibility and high yield. Precise control of gold nanoparticles can be achieved by changing the ratio without altering the volume ratio.

[0015] Preferably, step S105 specifically includes:

[0016] S1051. Add the purple-red solution to deionized water at a ratio of 1:3. Centrifuge at 9000 rpm for 10 min, remove the supernatant, and obtain the precipitate.

[0017] S1052. Five density gradient solutions were prepared using ethylene glycol and CTAB solution, respectively, with the volume ratios of ethylene glycol to CTAB solution in the five density gradient solutions being 50%, 60%, 70%, 80%, and 90%, respectively.

[0018] S1053. Add the five density gradient solutions to the centrifuge tube one by one, with the ethylene glycol concentration decreasing from top to bottom.

[0019] S1054. The precipitate obtained in S1051 was redispersed in CTAB solution, and then the dispersion was added dropwise to five density gradient solutions in a centrifuge tube. The mixture was centrifuged at 8000 rpm for 20 min to obtain the centrifuged layered solution.

[0020] S1055. Extract the gold nanoparticle bipyramidal centroid from the layered solution and redisperse it in deionized water to obtain the purified gold nanoparticle bipyramidal solution.

[0021] Compared to unpurified gold nanoparticle bipyramidal solution, purified gold nanoparticle bipyramidal solution has higher yield, fewer impurities, and better dispersibility, with the gold nanoparticle bipyramidal content reaching over 70%.

[0022] Preferably, in step S103, the pH value of the growth solution is controlled between 3 and 5 by adjusting the solution ratio. Within this pH range, the grown gold nanoparticles exhibit uniform and regular morphology, high repeatability, and high stability.

[0023] As can be seen from the above description, this preparation method has the following advantages:

[0024] 1. This method employs seed-mediated growth, utilizing NaBH4 to reduce Au.3+ Au single crystals were generated, and the Au single crystals were grown into five-fold twins by controlling the ripening temperature to prepare a gold seed solution. Then, AgNO3 was added to change the redox potential of the gold seeds. Finally, ascorbic acid was added to promote the growth of gold seeds into gold nanoparticles. The pH environment was then changed by changing the amount of HCl. By controlling the amount of seed solution added to the growth solution, the gold nanoparticles were grown into different aspect ratios.

[0025] 2. When this method is applied, gold nanobicones with different aspect ratios can be prepared by changing the amount of different gold stellates and hydrochloric acid. The gold nanobicones have high yield and good uniformity, which provides more possibilities for fiber optic biosensing and therapy in the near-infrared II band. In addition, the preparation method is simple to operate, has a short process, low consumable prices, and low overall preparation cost.

[0026] To achieve the above-mentioned technical objectives, another technical solution provided by the present invention is:

[0027] A micro / nano fiber optic biosensor, wherein the micro / nano fiber optic biosensor has a gold nanobipyramid obtained by the aforementioned gold nanobipyramid preparation method, and performs sensing and detection by modifying the gold nanobipyramid and combining it with two-dimensional materials.

[0028] Preferably, the two-dimensional material is WS2.

[0029] As can be seen from the above description, this micro / nano fiber optic biosensor has the following advantages:

[0030] The addition of two-dimensional material WS2 allows for the formation of a composite interface between the WS2 and gold nanoparticles on the fiber surface, thereby enhancing the fiber's interface sensitivity. After combination, WS2 acts as a conductor, enhancing the local electric field. Compared to other two-dimensional materials, WS2 possesses abundant hydrophilic functional groups (-O and -OH), superior electromagnetic properties, and a wider plasmon gap. Due to the interaction between interband transitions and boundary effects, it offers the possibility of directly tuning the plasma frequency across a broad spectral range from near-infrared (NIR) to mid-infrared (MIR). Therefore, combining WS2 with micro / nano-fibers can significantly improve the evanescent field electromagnetic properties of micro / nano-fibers, enhance the interaction between gold nanoparticles and biomolecules, improve the sensitivity of fiber-optic biosensors, and the composite interface has a wider specific surface area, providing more binding sites and a larger effective contact area for biomolecules, thus increasing the adsorption capacity and sensitivity of biomolecules.

[0031] To achieve the above-mentioned technical objectives, another technical solution provided by the present invention is:

[0032] A method for fabricating a micro / nano fiber optic biosensor includes the following steps:

[0033] S201. Prepare silica micro / nano optical fibers. Clean the bare silica micro / nano optical fibers with piranha solution to fully expose the hydroxyl groups on the surface of the micro / nano optical fibers.

[0034] S202. First, immerse the cleaned silica micro / nano optical fiber in deionized water and anhydrous ethanol solution in sequence;

[0035] S203. Then immerse the silica micro / nano optical fiber in APTES solution to amination the surface of the optical fiber;

[0036] S204. Next, the silica micro / nano optical fiber is sequentially immersed in anhydrous ethanol solution and WS2 solution;

[0037] S205. Then, the silica micro / nano optical fiber is successively immersed in deionized water and gold nanobipyramidal solution. The amino group and the carboxyl group on the gold nanobipyramidal undergo a covalent reaction, and the gold nanobipyramidal gradually binds to the surface of the optical fiber, thus completing the modification.

[0038] S206. Finally, the modified silica micro / nano optical fiber is dried in an oven to obtain the micro / nano optical fiber biosensor.

[0039] As can be seen from the above description, this preparation method has the following advantages:

[0040] The surface of micro- and nano-fibers is silanized by immersing them in a piranha solution, anhydrous ethanol, and APTES solution. Then, gold nanobipyramids are covalently coupled through Au-S bonds. The preparation method is simple, the raw materials are non-toxic, and the biological application is highly safe.

[0041] To achieve the above-mentioned technical objectives, the present invention provides two other technical solutions:

[0042] Application of the micro / nano fiber optic biosensor in the preparation of products for diagnosing prostate cancer.

[0043] Using the aforementioned micro / nano fiber optic biosensor, spectral shifts caused by the binding of exosomes and aptamers to prostate cancer cells were detected in vitro.

[0044] As can be seen from the above description, both of the above-mentioned micro / nano fiber optic biosensing detection methods have the following advantages:

[0045] 1. The sensor uses modified gold nanobipyramidal particles to detect the spectral shift caused by the binding of exosomes and aptamers in prostate cancer cells. It has high detection sensitivity and can be directly applied to the detection of exosomes in prostate cancer cells with low detection limits.

[0046] 2. When using micro-nano optical fibers for sensing, the sensitivity of the micro-nano optical fibers can be enhanced. By utilizing the evanescent wave on the side of the tapered micro-nano optical fiber to the changes in the external environment, the conformational changes of aptamers caused by the specific binding of prostate cancer exosomes and aptamers can be detected. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the WS2-supported gold nanobipyramidal interface modified micro / nano optical fiber prepared according to the present invention.

[0048] Figure 2 This is a scanning electron microscope (SEM) image of the gold nanobipyramidal combined with WS2 modified on the surface of micro / nano optical fiber prepared in this invention.

[0049] Figure 3 This is a schematic diagram illustrating the principle of detecting prostate cancer exosomes using a gold nanobiconical interface-modified micro / nano optical fiber prepared in this invention.

[0050] Figure 4 This is a schematic diagram of the fiber optic biosensing system of the present invention;

[0051] Figure 5 The gold nanobipyramidal modified with WS2 prepared in this invention is used in a micro / nano fiber optic biosensor to detect spectral drift patterns of prostate cancer exosomes at different concentrations of 10% bovine serum.

[0052] Figure 6 The spectral drift bar chart shows the gold nanobipyramidal modified with WS2 prepared in this invention for use in a micro / nano fiber optic biosensor to detect serum from prostate cancer patients.

[0053] Figure labels: 1-prostate cancer cell exosomes, 2-specific aptamers, 3-gold nanobicones, 4-micro / nano fiber core, 5-micro / nano fiber cladding, 6-tungsten disulfide. Detailed Implementation

[0054] Combination Figures 1 to 5 The embodiments of the present invention will be described in detail, but the claims of the present invention will not be limited in any way.

[0055] Example 1

[0056] A method for preparing a gold nanoparticle bipyramidal solution includes the following steps:

[0057] 1. Preparation of gold nanobipyramidal structures:

[0058] 1.1 Preparation of gold nanoseeds: HAuCl4 aqueous solution (0.01M, 0.125mL) and sodium citrate solution (0.01M, 0.25mL) were added to deionized water (9.625mL). Then, under vigorous stirring, freshly prepared NaBH4 solution (0.01M, 0.15mL) that had been refrigerated at 3°C ​​for 15 minutes was added to obtain an orange-red gold seed solution. The orange-red gold seed solution was aged at room temperature for at least 2 hours to completely hydrolyze the unreacted NaBH4, forming a pink seed solution.

[0059] 1.2 Preparation of growth solution: Prepare 40 ml of 0.1 M CTAB solution. After cooling the solution to room temperature, first add 2 ml of 0.01 M HAuCl4 aqueous solution, then add AgNO3 (0.01 M, 0.4 mL), followed by HCl (1.0 M, 0.5 mL) and ascorbic acid (0.1 M, 0.32 mL), and stir the solution vigorously until it becomes colorless (this is because after adding AA, due to Au...). 3+ Restore to Au + A growth solution is obtained by controlling the pH value of the growth solution between 3 and 5 during this process.

[0060] 1.3 Preparation of gold nanobipyramidal solution: The pink seed solution was injected into the growth solution and left to stand overnight in a 30°C water bath (approximately 20-24 hours) until the solution changed from colorless to purplish-red, indicating the formation of gold nanobipyramidal solution.

[0061] 2. Purification process

[0062] Since the gold nanoparticle bipyramidal solution obtained by the above method is a mixed solution containing gold nanoparticle bipyramidal particles and gold nanospheres, it contains impurities. In order to further improve the purity of the gold nanoparticle bipyramidal solution, density gradient centrifugation is used to purify the obtained purple-red gold nanoparticle bipyramidal solution. The specific steps include:

[0063] 2.1 The prepared gold nanoparticle bipyramidal solution was added to deionized water at a ratio of 1:3 and centrifuged (9000 rpm for 10 min). The supernatant was then aspirated with a dropper, leaving Au NBP precipitate.

[0064] 2.2 Based on the principle of density gradient method, five density gradient solutions were prepared using ethylene glycol (purity ≥98.5%) and CTAB (10mM), respectively. The volume ratios of ethylene glycol to CTAB solution in the five density gradient solutions were 50%, 60%, 70%, 80%, and 90%, respectively.

[0065] 2.3 The prepared 5-layer density gradient solution was added layer by layer along the longitudinal direction of the centrifuge tube in a manner with gradually decreasing density (lower ethylene glycol concentration) to form 5 layers of density gradient;

[0066] 2.4 The Au NBP precipitate was redispersed in CTAB (10 mM 0.5 mL) solution. 0.25 mL of the redispersed Au NBP solution was added to the 5-layer density gradient in the centrifuge tube, and then centrifuged (8000 rpm, 20 minutes) to obtain the centrifuged layered solution.

[0067] 2.5 Manually extract the layered solution using a micropipette, and then redisperse the extracted gold nanoparticle bipyramidal centrifuge in deionized water to obtain a uniformly dispersed gold nanoparticle bipyramidal solution with few impurities.

[0068] The gold nanobipyramidal solution prepared according to the above method has a long aspect ratio and good uniformity.

[0069] When implementing the above method, the following improvements can be made:

[0070] 1. In some embodiments, polyethylene glycol (PEG) can also be used to cap the gold nanoparticles. 20 mg of PEG is added to each milliliter of gold nanoparticle solution, and then the solution is dispersed evenly by ultrasonic vibration to obtain a gold nanoparticle solution that no longer grows, which is convenient for subsequent applications.

[0071] 2. In some embodiments, the amount of pink seed solution added to the growth solution in the step of preparing the gold nanobipyramidal solution can be changed (i.e., the amount of gold seeds can be changed) to effectively control the aspect ratio of the final obtained gold nanobipyramidal solution.

[0072] 3. In some embodiments, the amount of HCl added in the preparation of the growth solution can be changed to control the pH value of the solution to be stable at around 3, so as to affect the morphology of the final gold nanobipyramidal nanoparticles, making their morphology regular and uniform, and the yield considerable.

[0073] 4. In some embodiments, the amount of AgNO3 added in the preparation of the growth solution can be changed to regulate the growth environment of the gold seeds, thereby affecting the morphology of the final gold nanobipyramidal nanoparticles and making them more uniform.

[0074] Example 2

[0075] The gold nanobicone solution prepared according to the above method will be applied to the field of fiber optic biosensors, specifically micro-nano fiber optic biosensors.

[0076] Due to the unique anisotropic structure of gold nanobipyramidal structures, they possess two surface plasmon resonances corresponding to electronic oscillations along the longitudinal and transverse directions. Longitudinal surface plasmon resonances (LSPRs) exhibit strong optical extinction and can be easily tunable from the visible to near-infrared regions by changing their aspect ratio. Therefore, tunable LSPR coupling strategies can enhance the interfacial sensitivity of micro / nano fiber optic biosensors.

[0077] The specific method involves modifying and characterizing gold nanobipyramidal structures, and then fabricating micro / nano fiber optic biosensors using tungsten disulfide (WS2), a two-dimensional material. The fabricated fiber optic biosensors can be observed using scanning electron microscopy (SEM).

[0078] Because SEM requires a clean sample substrate, free of dust, organic solvents, or other unwanted reagent residues, and the sample must be conductive (if non-conductive, gold sputtering is necessary), the gold nanoparticle bipyramidal solution obtained according to Example 1 requires pretreatment.

[0079] Since the gold nanobipyramidal particles prepared in this invention are coated with a layer of CTAB, which affects the SEM characterization of the samples, the prepared gold nanobipyramidal particles need to be treated. The specific treatment steps are as follows:

[0080] 1. Take out the gold nano biconical solution and centrifuge it (8000 rpm, 10 min). Remove the supernatant with a dropper to obtain the precipitate. Then add anhydrous ethanol to the precipitate and then put it into an ultrasonic cleaner for ultrasonic cleaning until the solution is completely mixed to obtain a mixed solution.

[0081] 2. Centrifuge the completely mixed solution again (8000 rpm, 10 min). Remove the supernatant with a dropper to obtain a precipitate. Add anhydrous ethanol to the precipitate and then ultrasonically clean it until the solution is completely mixed, obtaining a secondary mixed solution.

[0082] 3. After adding deionized water to the secondary mixed solution, centrifuge it again (8000 rpm, 10 min). Remove the supernatant with a dropper to obtain the final precipitate, which is the gold nanoparticle biconical solution for modification and characterization.

[0083] like Figure 1 As shown, a two-dimensional material was used to modify and characterize the gold nanoparticle biconical solution to prepare a micro / nano fiber optic biosensor. The method is as follows:

[0084] 1. Prepare silica micro / nano optical fibers (preferably tapered micro / nano optical fibers, as tapered micro / nano optical fibers have higher sensitivity than ordinary straight micro / nano optical fibers). Clean the bare silica micro / nano optical fibers with piranha solution to fully expose the hydroxyl groups on the surface of the micro / nano optical fibers.

[0085] 2. First, immerse the cleaned silica micro / nano optical fiber in deionized water and anhydrous ethanol solution for 2 minutes each. Then, immerse it in 99% APTES solution for 30 minutes and then immerse it in anhydrous ethanol for 2 minutes. After that, immerse it in WS2 solution for 20 minutes. Finally, immerse it in deionized water for 2 minutes and then immerse it in gold nanobipyramidal solution for 30 minutes. The modification is then complete.

[0086] 3. The modified micro-nano optical fibers were dried in an oven (drying temperature 57℃, time 8h) to obtain the micro-nano optical fiber biosensor.

[0087] like Figure 2 As shown, SEM observation revealed that sheet-like WS2 and gold nanobipyramidal particles with a length of about 250 nm were uniformly dispersed and self-assembled layer by layer on the surface of micro-nano optical fibers.

[0088] In the above technical solution:

[0089] 1. The surface of micro- and nano-fibers is silanized by immersing them in piranha solution, anhydrous ethanol, and APTES solution. Then, gold nanobipyramids are covalently coupled through Au-S bonds. The preparation method is simple, the raw materials are non-toxic, and the biological application is highly safe.

[0090] 2. First, the interaction between the evanescent wave of the optical fiber and the surface material is used to sense the refractive index change caused by the binding of target molecules. The evanescent field energy is enhanced by the localized surface plasmon resonance effect of gold nanoparticles falling in the light source region. This utilizes the sensitivity of the evanescent wave on the side of the tapered micro / nano fiber to changes in the external environment to enhance the fiber's sensitivity. Then, the addition of the two-dimensional material WS2 allows it to combine with the gold nanoparticles on the fiber surface, forming a composite interface that further improves the fiber's interface sensitivity. After combining the two, the WS2 material, acting as a conductor, enhances the local electric field, thereby strengthening the interaction between the gold nanoparticles and biomolecules, improving the sensitivity of the fiber optic biosensor. Furthermore, the composite interface has a wider specific surface area, providing more binding sites and a larger effective contact area for biomolecules, increasing the adsorption capacity and sensitivity. Therefore, the modification with the two-dimensional material WS2 further optimizes the enhancement of the evanescent field by the plasmon resonance effect and increases the ability of the fiber surface to bind specific aptamers, resulting in higher sensitivity compared to micro / nano fiber sensors without interface modification.

[0091] Example 3

[0092] The following describes the application of the gold nanobipyramidal solution preparation method from Example 1 and the micro / nano fiber optic biosensor from Example 2 to the detection of prostate cancer exosomes in bovine serum. Figure 3 As shown, by adjusting the LSPR peak of the high aspect ratio gold nanobipyramidal structure to match the operating wavelength of the micro / nano fiber optic biosensor in the communication band, prostate cancer cell aptamers bound to the surface of the micro / nano fiber recognize and capture dissociated prostate cancer exosomes. Subsequently, the target component enters the evanescent field of the micro / nano fiber. With the formation of prostate cancer exosome-aptamer complexes on the surface of the micro / nano fiber, the refractive index RI increases. As a refractive index sensor, the increase in refractive index of the micro / nano fiber biosensor reflects the redshift of the transmission spectrum of the micro / nano fiber, thus enabling highly sensitive quantitative detection of prostate cancer cells.

[0093] The specific implementation plan is as follows:

[0094] 1. Reagent preparation

[0095] 1.1 Preparation of gold nanoparticle bipyramidal solution: A high aspect ratio gold nanoparticle bipyramidal solution was prepared according to the method in Example 1, and the gold nanoparticle bipyramidal solution was subjected to characterization pretreatment according to the method mentioned in Example 2 to meet the characterization requirements;

[0096] 1.2 Preparation of WS2 solution: Add anhydrous ethanol to a WS2·NH2 dispersion with a concentration of 1 mg / mL at a ratio of 1:2;

[0097] 1.3 Preparation of prostate cancer aptamer solution: Dilute 100 μg of the initial prostate cancer cell aptamer solution with a concentration of 1 mg / mL using TE buffer to dilute it to a concentration of 10 μM.

[0098] 1.4 Preparation of 10% bovine serum (BSA) solution: The prostate cancer exosomes, after cell culture and ultracentrifugation, were diluted with PBS buffer to a concentration of 10%. 9 The original bovine serum solution (cells / mL) was first diluted to a 10% volumetric concentration, and then prostate cancer exosomes were prepared one by one based on the 10% bovine serum solution, with concentrations of 10% and 10% respectively. 0 -10 8 Bovine serum solution with cells / mL.

[0099] 2. Functionalization of Micro / Nano Fiber Optic Biosensor Interfaces

[0100] 2.1 First, use piranha solution to clean the exposed silica micro / nano optical fibers, so that the hydroxyl groups on the surface of the micro / nano optical fibers are fully exposed;

[0101] 2.2 The cleaned silica micro / nano optical fiber was then immersed in deionized water and anhydrous ethanol solution for 2 minutes each, and then immersed in 99% APTES solution for 30 minutes to aminate the fiber surface. It was then immersed in anhydrous ethanol for 2 minutes and WS2 solution for 20 minutes, and finally immersed in deionized water for 2 minutes and high aspect ratio gold nanobipyramidal solution for 30 minutes. Due to the covalent reaction between the amino groups and the carboxyl groups on the gold nanobipyramidal fibers, the gold nanobipyramidal fibers gradually bonded to the fiber surface.

[0102] 2.3 Finally, the prostate cancer cell aptamers with carboxyl groups were immobilized on the surface of the micro-nano optical fiber by immersing them in a prostate cancer cell aptamer solution for 45 minutes. The micro-nano optical fiber modification was completed, and the micro-nano optical fiber biosensor was obtained.

[0103] 3. Detection of bovine serum prostate cancer exosomes

[0104] Modified micro / nano optical fibers are integrated as sensors into fiber optic biosensing systems. For example... Figure 4 As shown, the entire sensing system includes at least a sensor, an ultra-wideband light source, and a spectrometer. The ultra-wideband light source is connected to the sensor via a single-mode fiber, and then connected to the spectrometer via another single-mode fiber. The sensor (i.e., the micro / nano fiber) is immersed in light sources with concentrations ranging from 10... 0 -10 8 This system enables the detection of prostate cancer exosomes in bovine serum solutions containing [number] cells / mL, achieving ultrasensitive analysis of prostate cancer exosomes. The sensing system operates as follows: an ultra-wideband light source emits a 1250-1650nm laser beam, which, after passing through a single-mode optical fiber, excites the LSPR effect in the micro-nano sensing region to detect changes in the fiber's surface refractive index. The optical signal is then transmitted to a spectrometer, which converts the optical signal into an electrical signal, displaying a transmission spectrum. Changes in the spectrum are then used for further refractive index analysis of the target analyte solution or analysis of liquid biological samples.

[0105] In the above scheme, based on a tunable LSPR coupling strategy, gold nanobipyramids loaded with WS2 are first prepared and dispersed on the surface of micro / nano optical fibers, which are then used as an ultrasensitive fiber optic biosensing platform. Then, specific aptamer molecules for prostate cancer exosomes are attached to the modified gold nanobipyramids and WS2 structures. The LSPR on the gold nanobipyramids plays a near-infrared absorption role, exciting electron-hole pairs and converting them into hot carriers. These hot carriers are then injected into the WS2 two-dimensional nanosheets. Therefore, the transient electromagnetic field at the micro / nano optical fiber interface is enhanced. Due to the specific binding of the aptamer and exosome, the target component enters the evanescent field of the micro / nano optical fiber. As the aptamer-exosome complex forms on the surface of the micro / nano optical fiber, the refractive index increases, resulting in different spectral drift patterns.

[0106] Observing the changes in the spectrum reveals that the sensor's transmission spectrum differs under different exosome solution concentrations, and a regular and obvious red shift is observed as the exosome solution concentration increases. Figure 5 As shown, the sensitivity is as high as 1.506 nm / log 10 (particles / mL).

[0107] Example 4

[0108] The following describes the application of the gold nanobipyramidal solution preparation method in Example 1 and the micro / nano fiber optic biosensor in Example 2 to the detection of prostate cancer exosomes in human serum, with the principle being similar to that in Example 3.

[0109] The specific implementation plan is as follows:

[0110] 1. Reagent preparation

[0111] 1.1 Preparation of gold nanoparticle bipyramidal solution: A high aspect ratio gold nanoparticle bipyramidal solution was prepared according to the method in Example 1, and the gold nanoparticle bipyramidal solution was subjected to characterization pretreatment according to the method mentioned in Example 2 to meet the characterization requirements;

[0112] 1.2 Preparation of WS2 solution: Add anhydrous ethanol to a WS2·NH2 dispersion with a concentration of 1 mg / mL at a ratio of 1:2;

[0113] 1.3 Preparation of prostate cancer aptamer solution: Dilute the initial aptamer solution (specific recognition molecule) with 100 μg and a concentration of 1 mg / mL using TE buffer to obtain a 10 μM aptamer solution;

[0114] 1.4 Prepare human serum solution from prostate cancer patients. Usually, whole serum solution from prostate cancer patients is obtained from the hospital.

[0115] 2. Functionalization of Micro / Nano Fiber Optic Biosensor Interfaces

[0116] 2.1 First, use piranha solution to clean the exposed silica micro / nano optical fibers, so that the hydroxyl groups on the surface of the micro / nano optical fibers are fully exposed;

[0117] 2.2 The cleaned silica micro / nano optical fiber was then immersed in deionized water and anhydrous ethanol solution for 2 minutes each, followed by immersion in 99% APTES solution for 30 minutes to aminate the fiber surface. It was then immersed in anhydrous ethanol for 2 minutes and WS2 solution for 20 minutes, and finally in deionized water for 2 minutes and high aspect ratio gold nanobipyramidal solution for 30 minutes. Due to the covalent reaction between the amino groups and the carboxyl groups on the gold nanobipyramidal fibers, the gold nanobipyramidal fibers gradually bonded to the fiber surface.

[0118] 2.3 Finally, the prostate cancer cell aptamers with carboxyl groups were immobilized on the surface of the micro-nano optical fiber by immersing them in a prostate cancer cell aptamer solution for 45 minutes. The micro-nano optical fiber modification was completed, and the micro-nano optical fiber biosensor was obtained.

[0119] 3. Detection of prostate cancer exosomes in human serum

[0120] The modified micro-nano optical fiber is integrated as a sensor into the optical fiber biosensing system. The entire sensing system includes at least a sensor, an ultra-wideband light source, and a spectrometer. The architecture and working principle of the sensing system are the same as in Example 3. The micro-nano optical fiber is immersed in human serum solution for detection, which enables ultrasensitive analysis of prostate cancer exosomes.

[0121] In the above scheme, WS2-loaded gold nanobipyramids are first prepared and dispersed on the surface of micro / nano optical fibers, which are then used as an ultrasensitive fiber optic biosensing platform. Specific aptamer molecules for prostate cancer exosomes are then attached to the modified gold nanobipyramids and WS2 structure. The LSPR on the gold nanobipyramids acts as a near-infrared absorber, exciting electron-hole pairs and converting them into hot carriers. These hot carriers are then injected into the WS2 two-dimensional nanosheets. Therefore, the transient electromagnetic field at the micro / nano optical fiber interface is enhanced. Due to the specific binding of the aptamer and exosome, the target component enters the evanescent field of the micro / nano optical fiber. As the aptamer-exosome complex forms on the surface of the micro / nano optical fiber, the refractive index increases, resulting in different spectral drift patterns, thus distinguishing between normal human serum and prostate cancer patient serum.

[0122] Observing the changes in the spectrum reveals a shift in the sensor's transmission spectrum. As exosomes in the serum solution gradually bind to aptamers on the optical fiber, a distinct redshift spectrum is observed. This allows for sensitive detection of prostate cancer exosomes in whole human serum. Finally, data on the spectral shift in serum from 10 groups of prostate cancer patients were obtained and statistically analyzed. Figure 6 As shown.

[0123] In summary, the high aspect ratio gold nanobicones prepared by this invention have the following beneficial effects on fiber optic biosensing:

[0124] 1. This invention employs a seed-mediated growth method, utilizing NaBH4 to reduce Au. 3+Au single crystals are generated and grown into five-fold twins by controlling the ripening temperature to prepare a gold seed solution. AgNO3 is then added to change the redox potential of the gold seeds. Finally, ascorbic acid is added to promote the growth of gold seeds into gold nanobipyramidal nanoparticles. The pH environment is then changed by altering the amount of HCl. By controlling the amount of seed solution added to the growth solution, gold nanobipyramidal nanoparticles with different aspect ratios can be grown. Furthermore, by changing the amount of different gold seeds and hydrochloric acid, gold nanobipyramidal nanoparticles with different aspect ratios can be prepared. The gold nanobipyramidal nanoparticles have high yield and good uniformity, providing more possibilities for fiber optic biosensing and therapy in the near-infrared II band. Moreover, the preparation method is simple to operate, has a short process, low consumable prices, and low overall preparation cost.

[0125] 2. When the high aspect ratio gold nanobicones prepared in this invention are applied to fiber optic biosensing, by modifying the surface of tapered micro / nano-fibers with WS2-loaded gold nanobicones, the refractive index change caused by the binding of target molecules is sensed by the interaction between the evanescent wave of the fiber and the surface material. The evanescent field energy is enhanced by the local surface plasmon resonance effect of the gold nanobicones whose resonance peak falls in the light source region. The enhancement of the evanescent field by the plasmon resonance effect is optimized by the modification of the two-dimensional material WS2, thereby increasing the ability of the fiber surface to bind specific aptamers. Compared with micro / nano-fiber sensors without interface modification, the technology proposed in this invention achieves an enhancement of fiber optic sensing sensitivity.

[0126] 3. The micro-nano fiber optic biosensor prepared in this invention is silanized on the surface of the micro-nano fiber by immersing it in piranha solution, anhydrous ethanol, and APTES solution. After being modified with a specifically bound aptamer by covalently coupling gold nanobipyramids with Au-S bonds, it can detect prostate cancer cell exosomes at different concentrations (even in 30% concentration in patient serum), significantly improving the sensitivity and accuracy of early diagnosis and screening of prostate cancer cells. Moreover, its preparation method is simple, the raw materials are non-toxic, and it has high safety for biological applications.

[0127] It is understood that the above detailed description of the present invention is for illustrative purposes only and is not intended to limit the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effects; as long as the usage requirements are met, they are all within the protection scope of the present invention.

Claims

1. A method for fabricating a micro / nano fiber optic biosensor, characterized in that, The micro-nano fiber optic biosensor has gold nanobicones, and sensing and detection are performed by modifying the gold nanobicones and combining them with a two-dimensional material, wherein the two-dimensional material is WS2. The preparation method includes the following steps: S201. Prepare silica micro / nano optical fibers, and clean the bare silica micro / nano optical fibers with piranha solution to fully expose the hydroxyl groups on the surface of the micro / nano optical fibers. S202. First, immerse the cleaned silica micro / nano optical fibers in deionized water and anhydrous ethanol solutions in sequence; S203. Then immerse the silica micro / nano optical fiber in APTES solution to amination the fiber surface; S204. Next, the silica micro / nano optical fiber is immersed in anhydrous ethanol solution and WS2 solution in sequence; S205. Then, the silica micro / nano optical fiber is successively immersed in deionized water and gold nanobipyramidal solution. The amino groups and carboxyl groups on the gold nanobipyramidal undergo a covalent reaction, and the gold nanobipyramidal gradually binds to the surface of the optical fiber, thus completing the modification. S206. Finally, the modified silica micro / nano optical fiber is dried in an oven to obtain the micro / nano optical fiber biosensor. The gold nanobipyramidal structure was prepared by the following method: S101. Add HAuCl4 aqueous solution and sodium citrate solution to deionized water, stir, and then add NaBH4 solution to obtain an orange-red gold seed solution; S102. Aging the orange-red gold seed solution at room temperature for at least 2 hours to completely hydrolyze the unreacted NaBH4 and form a pink seed solution; S103. Add HAuCl4 aqueous solution, AgNO3 solution, HCl and ascorbic acid to CTAB solution in sequence, and stir the solution until it becomes colorless to obtain the growth solution; S104. Inject the pink seed solution obtained in step S102 into the growth solution and let it stand in a 30°C water bath for 20-24 hours until the solution turns into a purplish-red solution; S105. The purple-red solution was centrifuged using density gradient centrifugation to remove impurities and obtain a purified gold nanoparticle bipyramidal solution, i.e., gold nanoparticle bipyramidal solution was obtained. In the above method, the aspect ratio of the gold nanobipyramidal structure is controlled by adjusting the amount of pink seed solution added in step S104.

2. The method of claim 1, wherein the micro- or nano-fiber biosensor is prepared by the steps of: In step S101: The volume ratio of HAuCl4 aqueous solution, sodium citrate solution, deionized water, and NaBH4 solution is 0.125:0.25:9.625:0.15; The concentration of the HAuCl4 aqueous solution is 0.01M, the concentration of the sodium citrate solution is 0.01M, and the concentration of the NaBH4 solution is 0.01M.

3. The method of claim 2, wherein the micro- or nano-fiber biosensor is prepared by the steps of: In step S103: The volume ratio of CTAB solution, HAuCl4 aqueous solution, AgNO3 solution, HCl, and ascorbic acid is 40:2:0.4:0.5:0.

32. The concentrations of CTAB solution, HAuCl4 aqueous solution, AgNO3 solution, HCl, and ascorbic acid were 0.1M, 0.01M, 0.01M, 1M, and 0.1M respectively.

4. The method for fabricating the micro / nano fiber optic biosensor according to claim 3, characterized in that, Step S105 specifically includes: S1051. Add the purple-red solution to deionized water at a ratio of 1:3, centrifuge at 9000 rpm for 10 min, remove the supernatant, and obtain the precipitate; S1052. Five density gradient solutions were prepared using ethylene glycol and CTAB solution, with volume ratios of ethylene glycol to CTAB solution of 50%, 60%, 70%, 80%, and 90%, respectively. S1053. Add the five density gradient solutions to the centrifuge tube one by one, with the ethylene glycol concentration decreasing from top to bottom. S1054. The precipitate obtained in S1051 was redispersed in CTAB solution, and then the dispersion was added dropwise to five density gradient solutions in a centrifuge tube. The mixture was centrifuged at 8000 rpm for 20 min to obtain the centrifuged layered solution. S1055. Extract the gold nanoparticle bipyramidal centroid from the layered solution and redisperse it in deionized water to obtain the purified gold nanoparticle bipyramidal solution.

5. The method for fabricating a micro / nano fiber optic biosensor according to claim 1, characterized in that, In step S103, the pH value of the growth solution is controlled at 3-5 by adjusting the solution ratio.

6. The application of the micro / nano fiber optic biosensor prepared by the method according to claim 1 in the preparation of products for diagnosing prostate cancer.

7. A micro / nano fiber optic biosensing detection method, characterized in that, Using the micro / nano fiber optic biosensor prepared according to claim 1, the spectral shift caused by the binding of exosomes and aptamers of prostate cancer cells was detected in vitro.

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