CN115165807A - FOLSPR aptamer sensor based on AuNPs polymer and preparation method and application thereof - Google Patents

Abstract

The invention discloses a FOLSPR biosensor based on AuNPs multimer and a preparation method and application thereof. The AuNPs polymer is successfully constructed on the end face of the optical fiber by a two-step assembly method of the AuNPs, and then the preparation of the sensor can be completed by connecting the aptamer. The AuNPs are arranged at the nano-interval, so that the interaction between optical nano-structures can be effectively increased, and the AuNPs polymer structure has a large number of nano-gaps, so that the electromagnetic field intensity of a limited area can be remarkably enhanced through a hot spot effect, and the sensitivity of the FOLSPR sensor is effectively improved. The vomitoxin is used as a detection object to be tested, and the performance of the FO LSPR sensor based on the AuNPs monomer is compared, and the result shows that the LSPR signal intensity of the sensor based on the AuNPs multimer is improved by about 4.0 times, the LSPR signal deviation value is improved by about 3.4 times, and the sensitivity is improved by about 7.8 times.

Description

FOLSPR aptamer sensor based on AuNPs polymer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of FOLSPR (fiber localized Surface plasma Resonance) detection, and particularly relates to a FOLSPR aptamer sensor based on an AuNPs polymer, and a preparation method and application thereof.

Background

The vomitoxin is technically named as Deoxynivalenol (DON) and is the B-type compound of the most main trichothecenes produced by fusarium graminearum. DON is soluble in organic agents, slightly soluble in water, can bind to ribosomes, inhibit the synthesis of proteins, RNA and DNA, and induce apoptosis. Long-term ingestion of low concentrations of DON can damage the health of humans and animals, and when excessive amounts of DON are ingested, toxic reactions such as vomiting, diarrhea, anorexia, nausea, neurological disorders, and the like can occur, and even the hematopoietic system can be damaged and death can result. DON has higher chemical stability, high temperature resistance and weak acid resistance, can stably exist in the processes of processing and storing feed and food without being degraded, but can reduce the toxicity of DON in an alkaline environment. A large number of researches prove that DON has similar heat resistance, pressure resistance and other characteristics with other fungus toxins, and the researches show that DON is very stable and has very high concentration when being baked at 170-350 ℃ in the food processing process. DON is less toxic than aflatoxin, but it must be considered an important food safety issue because DON is a mycotoxin that is common in wheat, corn, barley, and other food and derivatives, and is prevalent in food and feed producing crops throughout the world.

At present, the determination of DON and the derivative thereof in food adopts isotope dilution liquid chromatography-tandem mass spectrometry, immunoaffinity chromatography purification high performance liquid chromatography, thin layer chromatography determination and enzyme linked immunosorbent assay screening. However, the conventional methods for detecting DON often require professional technicians, expensive reagents and instruments, complicated sample preparation processes, long detection period, and capability of being completed only in a laboratory. The fiber LSPR sensor has the characteristics of small volume, low cost, high sensitivity, no need of marking and the like, and becomes a hotspot for research in the field of analysis and detection.

Most of traditional FOLSPR sensors take precious metal nanoparticle monomers as sensing materials, and take FOLSPR sensors based on AuNPs monomers as examples, such sensors have the defects that AuNPs on the end face of an optical fiber are easy to agglomerate or fall off, LSPR signal strength is low, DON signal response is weak, and the like, and the generation of LSPR offset signals is seriously interfered. In recent years, plasmon coupling caused by interference between noble metal nanoparticles is one of effective methods to improve sensitivity. AuNPs are arranged at a distance of several nanometers to form nano-gaps, and the nano-gaps can effectively enhance the interaction between light and a nano structure, and the strength of electromagnetic field intensity of a limited area is remarkably enhanced through a 'hot spot' effect. Stronger plasmon electromagnetic enhancement can produce more absorption, which is very sensitive to changes in refractive index around the nanoparticle. A large number of nano gap structures can be generated by constructing the AuNPs multimer, so that signal amplification is realized and the sensitivity of the sensor is improved.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing the FOLSPR sensor based on the AuNPs multimer, and realizing high-sensitivity detection on DON.

The invention aims to provide a preparation method and application of an AuNPs polymer-based FOLSPR sensor.

The technology to be finally solved by the invention is to provide a DON toxin detection method, which is based on FOLSPR sensor and 'hot spot' effect of AuNPs polymer and can sensitively detect DON in food.

The technical scheme is as follows: in view of the above technical problems to be solved, the present invention provides a FOLSPR biosensor based on AuNPs multimers, which is obtained by immobilizing a sulfhydryl-modified DON aptamer onto an AuNPs multimer through a gold-sulfur bond.

Wherein the sequence of the DON aptamer is 5'- (SH) - (CH 2) 6-GCA TCA CTA CAG TCA TTA CGC ATC GTA GGG GGG ATC GTT AAG GAA GTG CCC GGA GGC GGT ATC GTG TGA AGT GCT GTC CC-3'.

Wherein the particle size of the AuNPs polymer is 16-28nm.

The invention also discloses a preparation method of the FOLSPR biosensor based on the AuNPs multimer, which comprises the following steps:

1) Fiber assembly of AuNPs multimers:

2) And (3) fixing the sulfhydryl modified DON aptamer on the optical fiber assembled with the AuNPs multimer.

5. The method for preparing an AuNPs multimer-based FOLSPR biosensor according to claim 4, wherein said assembling the AuNPs multimer-based fiber in step 1) comprises the steps of:

1.1 Preparing AuNPs;

1.2 Cleaning the end face of the optical fiber and modifying the end face by hydroxylation;

1.3 Step 1.2) immersing the modified optical fiber into an APTES solution;

1.4 Step 1.3) modifying the modified optical fiber into AuNPs through a gold-ammonia bond;

1.5 Step 1.4) the modified optical fiber is treated by PETMP to realize the modification of sulfydryl on the AuNPs on the optical fiber;

1.6 Step 1.5) the treated fiber is assembled by Au-S bond to obtain the fiber of AuNPs polymer.

Wherein, the concentration of the APTES solution in the step 1.3) is 1.0 percent, the APTES treatment time is 2-18 h, the treatment time of the AuNPs to the optical fiber in the step 1.4) is 1-8 h, the treatment time of the PETMP in the step 1.5) is 1-5 h, and the treatment time in the step 1.6) is 1-10 min.

Wherein the concentration of the DON aptamer of the step 2) is 1-25 mu M.

The invention relates to a preparation method of a FOLSPR sensor based on AuNPs multimers, which specifically comprises the following steps:

1) The bare fiber is cleaned by ultrapure water and dried by nitrogen. Then, the bare optical fiber is immersed in the piranha solution, treated for 40min at the temperature of 80 ℃, washed by ultrapure water, cleaned by ultrasonic waves and dried by nitrogen, so that the end face of the optical fiber is hydroxylated.

2) The optical fiber is immersed in an APTES solution (volume ratio: APTES: ethanol: water = 1: 98) for treatment, then the optical fiber is repeatedly washed by ethanol and ultrapure water in sequence, and is dried by nitrogen gas, so that the end face of the optical fiber is provided with abundant amino groups.

3) And (3) immersing the optical fiber sensor into AuNPs solution for treatment, and washing with ultrapure water. And then soaking the optical fiber into a PETMP solution (ethanol is used as a solvent) for treatment, washing the optical fiber with ethanol and ultrapure water in sequence, and drying the optical fiber with nitrogen. And then immersing the optical fiber into the AuNPs solution again for treatment, washing with ultrapure water, and drying with nitrogen, thus assembling the AuNPs polymer on the end face of the optical fiber.

4) And (3) immersing the optical fiber assembled with the AuNPs polymer into DON aptamer solution for treatment, incubating overnight, repeatedly washing with aptamer buffer solution to remove non-covalently bound DON aptamer, and drying by nitrogen to finish the preparation of the FOLSPR biosensor based on the AuNPs polymer.

The invention further discloses the application of the FOLSPR biosensor based on the AuNPs multimer in DON toxin detection.

The invention also comprises a method for detecting DON toxin, which comprises the following steps:

1) Scanning the FOLSPR biosensor line spectrum, and recording the peak position of the LSPR peak of the sensor;

2) Immersing the FOLSPR biosensor in DON toxin solutions with different concentrations, incubating, performing spectral scanning on the FOLSPR biosensor again, and calculating the deviation value of the LSPR peak positions twice;

3) And calculating the concentration of the DON sample by using the obtained concentration relation between the LSPR peak deviation value and the DON.

Wherein the concentration of the DON toxin is 0.05-200 ng/mL.

The present invention also compares the response of AuNPs based FO LSPR sensors based on AuNPs monomers to those based on AuNPs multimers to DON. And (3) comparing the signal deviation degrees of the DON standard solutions by detecting the DON standard solutions with different concentrations, and analyzing the sensitivity of the DON standard solutions.

The invention provides an AuNPs polymer-based FOLSPR aptamer biosensor, the sensor takes the end face of a multimode bare optical fiber as a sensing platform, the AuNPs polymer as a sensing material, a DON aptamer as a specific capture element and a reflection spectrum transmitted through a Y-type optical fiber, and the working mechanism of the sensor is shown in figure 1. The AuNPs are connected to the end face of an optical fiber in two steps to form an AuNPs polymer, then the mercapto-modified DON aptamer is fixed on the AuNPs polymer through a gold-sulfur bond (Au-S) to complete the construction of the optical fiber sensor, and the AuNPs processing time, the PETMP processing time, the AuNPs processing time, the APTES processing time, the DON aptamer concentration and the time required by detection in the first step are optimized. The specific binding of DON and the aptamer enables the LSPR peak of the optical fiber sensor to shift, and the shifting degree is related to the concentration of DON, so that the purpose of quantitatively detecting the DON is achieved.

The installation and connection of the fiber optic sensing system is shown in fig. 2. The high-flux multimode bare fiber is used for constructing the FO LSPR sensor, and the Y-shaped fiber is used for signal transmission among the light source, the sensor and the spectrometer. Tungsten halogen lamps (HL-2000, ocean optics, USA) are used to provide 400-1000nm light. Use of a CCD spectrometer (i-trometer, BW Tek, newark, DE, USA) for receiving the reflected light of the FO LSPR sensor with reception from 99% Spectralon ^TM Reflectance standards (Labsphere inc., north Sutton, USA) interface to reflectance spectra.

If the optical fiber sensor is damaged, the damaged part can be cut, and the end face can be polished to realize recycling.

Has the advantages that: compared with the prior art, the invention has the following advantages: the detection method is rapid and convenient; the detection work can be carried out on site; the requirement on the operation skill of the detection personnel is low; the mass use of the sensor of the invention can realize the detection of a large amount of samples in a short time. The optical fiber LSPR sensor prepared by the invention has high detection sensitivity on the DON toxin, the lowest detection limit is 0.05ng/mL, the DON toxin in the food is detected with high specificity, and the detection result is accurate and reliable.

Drawings

FIG. 1 is a schematic diagram of a FO LSPR biosensor detecting DON;

FIG. 2, construction of AuNPs multimers and "Hot Point Effect";

FIG. 3, FO LSPR sensing system;

fig. 4, characterization of AuNPs, (a) appearance of AuNPs solution; (B) TEM image of AuNPs; (C) the UV-VIS absorption spectra of AuNPs; (D) the particle size distribution of AuNPs;

FIG. 5, characterization of FOLSPR sensor, (A) AuNPs monomer based optical fiber under scanning electron microscope; (B) AuNPs polymer of the end face of the optical fiber under a scanning electron microscope; (C) absorption peaks of optical fibers based on AuNPs monomers; (D) absorption peaks of optical fibers with AuNPs multimers;

figure 6 condition optimization of FO LSPR sensors, (a) effect of first connection time on AuNPs multimer formation; (B) Effect of PETMP treatment time on AuNPs multimers (C) effect of second ligation time on AuNPs multimer formation (D) 1% effect of aptes treatment time on AuNPs multimer formation (E) effect of different concentrations of DON aptamers on LSPR shift response value of 10ng/ml DON solution (F) effect of incubation time on LSPR shift;

FIG. 7, detection of DON toxin by fiber-optic LSPR sensor; (A) LSPR spectra of DON solutions of different concentrations; (B) a non-linear relationship between LSPR red shift amount and DON concentration; (C) a linear relationship between LSPR red shift and DON concentration logarithm;

FIG. 8, selective analysis of fiber LSPR sensor;

figure 9, response of the AuNPs multimer-based and AuNPs monomer-based FO LSPR sensors to different concentrations of DON.

Detailed Description

The following examples further illustrate details of the preparation of the composite material of the present invention and the preparation and electrochemical performance of the electrode material thereof.

Example 1 preparation and characterization of AuNPs

The entire reaction procedure for synthesizing AuNPs was stirred and refluxed, and 100mL of chloroauric acid (0.01% wt) was added to a 250mL three-necked flask and heated to boiling. And then, quickly dropwise adding 3mL of 1% sodium citrate solution with mass concentration into the reaction solution, continuously heating and refluxing, continuously reacting for 15min, stopping heating, waiting for the solution to be cooled to room temperature of 25 ℃, collecting the prepared AuNPs solution, sealing and storing at 4 ℃. As shown in fig. 4A, the synthesized AuNPs appeared wine red, and the ultraviolet-visible absorption spectrum of the AuNPs solution was measured using a fiber optic sensing system, with the absorption peak at 524nm (fig. 4C). Microscopic characterization was performed using transmission electron microscopy (fig. 4B), and the particle size distribution was measured using a particle size distribution analyzer (fig. 4D), it was found that the distribution of the synthesized AuNPs of the present invention was mainly around 21 nm.

Example 2 preparation of FOLSPR sensor

And (4) cleaning the bare optical fiber with a smooth end face by using ultrapure water, and drying by using nitrogen. And then immersing the end face of the bare optical fiber into 2-3cm of piranha solution (prepared by 98% sulfuric acid and 30% hydrogen peroxide according to the volume ratio of 7: 3), treating for 40min at 80 ℃, then flushing with ultrapure water, immersing the end face of the bare optical fiber into the ultrapure water, ultrasonically cleaning for 5min, taking out, and drying with nitrogen to obtain the hydroxylated optical fiber sensor.

The subsequent treatment and detection of the optical fiber are carried out at 25 ℃. Immersing the hydroxylated fiber endface in a solution of 3-aminopropyltriethoxysilane ((3-aminopropyl) triethoxy silane, APTES) in a concentration of 1.0% by about 1-2cm (volume ratio: APTES: ethanol: water = 1: 98), treating for 12h, connecting APTES to the fiber endface through hydrolytic condensation, forming abundant amino groups on the fiber endface, then repeatedly rinsing with ethanol and ultra-pure water in sequence, and drying with nitrogen to obtain the aminated fiber sensor.

Immersing the aminated optical fiber sensor into AuNPs solution for about 0.5cm, treating for 5h, modifying the AuNPs to the end face of the optical fiber through gold-ammonia bonds, washing with ultrapure water to remove the non-covalently bonded AuNPs, and drying with nitrogen to obtain the optical fiber with the sparsely distributed AuNPs monomers.

Then, the optical fiber was immersed in 4mg/mL pentaerythritol 3-mercaptopropionate (3-mercaptoxanthonate), PETMP (ethanol as a solvent) for 3 hours, washed with ethanol and ultra-pure water in this order, and dried with nitrogen. PETMP contains four sulfydryl groups, and part of the thiol groups are firmly anchored on AuNPs through Au-S bonds, so that a layer of abundant sulfydryl groups on the surface of the AuNPs can be combined with additional AuNPs. And immersing the optical fiber into the AuNPs solution again for treatment for 5min, and carrying out rinsing with ultrapure water and blowing with nitrogen for drying. AuNPs on the end face of the optical fiber and AuNPs in the solution form a structure of AuNP- (PETMP-AuNPs) n through Au-S bond action, and then AuNPs polymer can be assembled on the end face of the optical fiber (figure 2).

The optical fiber assembled with the AuNPs multimer was treated by immersing in 15.0 μ M thiol-modified DON aptamer solution for 12h, and repeatedly washed with aptamer buffer to remove non-covalently bound DON aptamer. The DON aptamer is connected to the surface of AuNPs through sulfydryl, the structure of the fiber-AuNPs polymer-DON aptamer is finally formed, and the preparation of the DON FO LSPR biosensor based on the AuNPs polymer is completed. Ensuring that the fiber-optic endface is clean and not abraded during the fabrication of the sensor is critical to success.

Example 3 characterization and comparison of Signal Strength of Sensors based on AuNPs multimers and AuNPs monomers

The signal of the biosensor based on the fiber LSPR is closely related to the distribution of the AuNPs on the end face of the fiber. The optical fiber directly connected to the AuNPs and the optical fiber connected to the AuNPs multimer were characterized by scanning electron microscopy, and the results are shown in fig. 5A and 5B. The optical fiber connected with AuNPs, the AuNPs are randomly distributed on the end face of the optical fiber, the method can realize the enhancement of the signal intensity of the LSPR by directly increasing the density of AuNPs monomers on the end face of the optical fiber, and the defect is that the AuNPs are easy to aggregate when the processing time is longer (as shown in figure 5A). The AuNPs polymer is constructed on the end face of the optical fiber through a two-step connection method of AuNPs, the AuNPs polymer is formed by coupling a plurality of AuNPs through PETMP, the polymer is randomly distributed on the end face of the optical fiber and has higher density, and the AuNPs in a single polymer are obviously not aggregated at intervals. The ultraviolet-visible spectra of the AuNPs monomer and AuNPs multimer at the end face of the fiber were measured by a fiber sensing system (see fig. 5C and 5D). The randomly assembled AuNPs monomers also have a nanogap structure, but the generation of the hot spot effect depends on the gap distance between two nanoparticles. The test result shows that compared with the direct connection method, the two-step assembly method has shorter processing time, is easier to construct an effective nano-gap structure during actual operation, and improves the intensity of an LSPR signal by about 4.0 times.

Example 4 Condition optimization of sensor preparation

Firstly, the time for connecting AuNPs in the first step is optimized, and the AuNPs are used for processing optical fibers for 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h. The results shown in FIG. 6A are the absorbance of the optical fiber surface polymer at 520nm as a function of the treatment time, and show that the fiber LSPR signal at 5h in the first step of AuNPs treatment time is saturated when the second step of AuNPs treatment time is 5 min. After 5h, auNPs monomers randomly distributed on the end face of the optical fiber continue to increase, aggregation does not occur in the first-step connection, but the AuNPs polymers formed on the end face of the optical fiber have higher density and are easy to aggregate when the second-step connection is performed. The absorbance continued to increase for the next three hours because the AuNPs multimer at the end face of the fiber aggregated and the absorption peak increased around 600 nm. Therefore 5h was chosen as the connection time for the first step.

Next we optimized the 4mg/mL PETMP treatment times for 1h, 2h, 3h, 4h, 5h (FIG. 6B). The PETMP with proper concentration can form a stable polymer structure when the second-step connection is carried out, the polymer structure formed when the concentration is too low is poor in stability and easy to damage, the surface sulfydryl of AuNPs connected to the end face of the optical fiber in the first step is increased when the concentration is too high, aggregation is easy to occur when the AuNPs are connected in the second step, and finally the optical fiber sensor which can obtain stable and strong LSPR signals within 3h of processing time is obtained.

We then optimized the second AuNPs treatment time (1 min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10 min) (fig. 6C). At 1-5min, auNPs polymer increases along with the increase of processing time, and at the subsequent 5min, then increases, the AuNPs polymer aggregates due to excessive bound AuNPs, the hot spot effect disappears, the absorbance of the polymer at 520nm is reduced, and then the AuNPs aggregate in large quantity at the end face of the optical fiber, and then the absorbance increases in small quantity at 520 nm.

The fibers were treated with 1% APTES for different times (2 h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18 h), the AuNPs multimers at the fiber end face increased with increasing APTES treatment time and reached a maximum at 12h, and then increasing the treatment time had no significant effect on absorbance at 520nm, with values that tended to be smooth, thus 1% APTES treatment time optimization resulted in 12h (fig. 6D).

The coverage rate of the AuNPs polymer surface DON aptamer (5 '- (SH) - (CH 2) 6-GCA TCA CTA CAG TCA TTA CGC ATC GTA GGG GGG ATC GTT AAG GAA GTG CCC GGA GGC GGT ATC GTG TGA AGT GCT GTC CC-3', prepared by Shanghai bioengineering Co., ltd.) is important to the aspects of detection limit, sensitivity, response time and the like of the sensor. For achieving an optimal sensing performance, the density of DON aptamers needs to reach a higher level, but when the concentration of the DON aptamers is too high, the density of the DON aptamers on the surface of the AuNPs polymer is extremely high, so that a steric effect is generated, and the combination of DON and DON aptamers is difficult. The results in FIG. 6E show that the LSPR shift of the sensor to a 10ng/mL DON solution reaches a maximum when the DON aptamer concentration of the AuNPs multimer is treated to 15 μm/L (FIG. 6E).

Example 5 optimization of sensor response time

The incubation time required by the sensor is optimized by adopting the processing time of 1h, 4h, 8h, 12h, 16h, 20h and 24h, the optical fiber continuously captures DON molecules through a DON aptamer within 0-12 h, the LSPR deviation value continuously rises, the DON aptamer of the sensor is saturated after 12h, and DON in the solution can not be captured, so that the incubation time of the sensor is set to be 12h (figure 6F).

Example 6 detection of DON toxin by FOLSPR sensor

DON standard solutions with different concentrations (0.05 ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200 ng/mL) were detected by using the optimized LSPR sensing system. When DON aptamer captures DON, the refractive index of AuNPs polymer under local environment is increased, so that LSPR peak generates red shift, and in addition, the hot spot effect of the polymer enhances the red shift, and the red shift value is in positive correlation with DON concentration. Fig. 7A is a plot of normalized LSPR spectra after treatment with increasing DON concentration. The degree of bathochromic shift versus DON concentration is shown in FIG. 7B, where the bathochromic shift increases with increasing DON concentration and levels off as higher concentrations are reached. The relationship between LSPR shift and concentration is log-dependent, but linearly dependent on the log of DON concentration (fig. 7C). The concentration logarithm value is in a linear relation (R) within the range of 0.05-200.0 ng/mL ² ＝0.9934，LOD＝0.04ng/mL，LOQ＝0.05n/mL)。

EXAMPLE 7 Selective analysis of the sensor

In order to examine the selectivity of the constructed LSPR biosensor, LSPR shift of different toxins was analyzed by using fiber LSPR sensors to detect solutions with the concentrations of 10ng/mL DON, AFB1, FB1, OTA and T-2 respectively under the same conditions (FIG. 8). The results show that only DON is significantly red-shifted by 11.0nm. Of these, AFB1, FB1, T-2 produced red-shifts of 0.9, 1.1 and 1.3nm, while OTA produced a blue-shift of 1.7nm, which is reasonable because the analyte concentrations used for the specific assay were relatively high. This indicates that the capture of DON and the generation of signal by this sensor are not affected by interfering molecules, which depend on specific binding of DON to DON aptamers.

Example 8 comparison of Performance of Sensors based on AuNPs multimers with AuNPs monomers

The optimized FOLSPR sensor based on AuNPs monomer and the optimized FOLSPR sensor based on AuNPs polymer are used for detecting DON standard solutions with different concentrations (0.05, 1, 10, 100 ng/mL). Both FOLSPR sensors produce a red-shifted response by the binding of DON to the aptamer, but the FOLSPR sensors based on AuNPs show a higher and more sensitive red-shifted response. In 0.05ng/mL DON standard solution, the AuNPs monomer based fiber produced a negative shift, similar to the test results for 0ng/mL DON solution, but the AuNPs multimer based fiber still produced a positive LSPR shift. The results of the two sensors tested on 1, 10, 100ng/mL DON solutions showed that the LSPR signal offset value of the AuNPs-based multimeric structure was improved by about 3.4 times compared to the FOLSPR sensor based on AuNPs monomeric structure. The LOD test result of the FOLSPR sensor based on the AuNPs monomer structure is 0.32ng/mL, while the LOD of the FOLSPR sensor based on the AuNPs multimer is 0.04ng/mL, and the sensitivity is improved by about 7.8 times.

Example 9 detection of corn samples by Sensors

The method comprises the steps of carrying out reagent detection and standard adding recovery test by taking dry corns as samples, setting three standard adding concentrations of 0.2mg/kg, 0.4mg/kg and 1mg/kg for analysis, and setting three times for each concentration and blank; and the feasibility of the method was verified by ELISA. The test results are shown in table 1, and the method has good accuracy (the recovery rate is 105.2-113.6%) on the DON quantitative detection result; in addition, compared with the experimental result of the existing ELISA detection method, P is more than 0.05, which shows that the two detection methods have no significant difference and can be used for detecting DON in actual samples.

TABLE 1

Claims (10)

1. A FOLSPR biosensor based on AuNPs multimers, the FOLSPR biosensor being obtained by immobilizing a sulfhydryl-modified DON aptamer onto an AuNPs multimer via a gold-sulfur bond.

2. The AuNPs multimer-based FOLSPR biosensor of claim 1, wherein the sequence of said DON aptamer is 5'- (SH) - (CH 2) 6-GCA TCA CTA CAG TCA TTA CGC ATC GTA GGG GGG ATC GTT AAG GAA GTG CCC GGA GGC GGT ATC GTG TGA AGT GCT GTC CC-3'.

3. The AuNPs multimer-based FOLSPR biosensor of claim 1, wherein the AuNPs multimer has a particle size of 16-28nm.

4. The method for preparing the FOLSPR biosensor based on the AuNPs multimer, as claimed in any one of claims 1 to 3, comprising the following steps:

1) Fiber assembly of AuNPs multimers:

2) And (3) fixing the sulfhydryl modified DON aptamer on the optical fiber assembled with the AuNPs multimer.

5. The method for preparing an AuNPs multimer-based FOLSPR biosensor according to claim 4, wherein said assembling the AuNPs multimer-based fiber in step 1) comprises the steps of:

1.1 Preparing AuNPs;

1.2 Cleaning the end face of the optical fiber and modifying the end face by hydroxylation;

1.3 Step 1.2) immersing the modified optical fiber into an APTES solution;

1.4 Step 1.3) the modified optical fiber is modified with AuNPs through gold-ammonia bond;

1.5 Step 1.4) the modified optical fiber is treated by PETMP to realize modification of sulfydryl on AuNPs on the optical fiber;

1.6 Step 1.5) the treated fiber is assembled by Au-S bond to obtain the fiber of AuNPs polymer.

6. The method for preparing the FOLSPR biosensor based on the AuNPs multimer according to claim 5, wherein the concentration of the APTES solution in step 1.3) is 1.0%, the APTES treatment time is 2 to 18h, the treatment time of the AuNPs on the optical fiber in step 1.4) is 1 to 8h, the treatment time of the PETMP in step 1.5) is 1 to 5h, and the treatment time in step 1.6) is 1 to 10min.

7. The method for preparing an AuNPs multimer-based FOLSPR biosensor according to claim 4, wherein the concentration of DON aptamer of step 2) is 1-25 μ M.

8. Use of the AuNPs multimer-based FOLSPR biosensor of any one of claims 1-3 in the detection of a DON toxin.

9. A method for detecting DON toxin is characterized by comprising the following steps:

1) Scanning the FOLSPR biosensor line spectrum, and recording the peak position of the LSPR peak of the sensor;

2) Immersing the FOLSPR biosensor in DON toxin solutions with different concentrations, incubating, performing spectral scanning on the FOLSPR biosensor again, and calculating the deviation value of the LSPR peak positions twice;

3) And calculating the concentration of the DON sample by using the obtained concentration relation between the LSPR peak deviation value and the DON.

10. The method for detecting the DON toxin according to claim 9, wherein the concentration of the DON toxin is 0.05 to 200ng/mL.

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