CN111398221A

CN111398221A - Method for sensing and measuring diethylstilbestrol based on graphene multiple signal amplification SPR

Info

Publication number: CN111398221A
Application number: CN202010246481.XA
Authority: CN
Inventors: 高志贤; 李双; 白家磊; 彭媛; 宁保安; 王江; 韩殿鹏; 任汉林; 周焕英
Original assignee: Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
Current assignee: Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-07-10
Anticipated expiration: 2040-03-31
Also published as: CN111398221B

Abstract

The invention relates to a method for sensoring and measuring diethylstilbestrol based on graphene multiple signal amplification SPR, which comprises the following steps: s1, sample preparation: using magnetic graphene as an adsorbent to adsorb and enrich diethylstilbestrol in a sample to be detected, then eluting with an eluent, and fixing the volume of the eluted diethylstilbestrol to obtain a sample to be detected; s2, cleaning the surface of the noble metal sheet of the SPR chip and connecting the noble metal sheet with the carboxylated graphene oxide; s3, connecting and fixing DES-BSA by using carboxylated graphene oxide fixed on the surface of the SPR chip noble metal; s4, measuring the concentration of the diethylstilbestrol in the sample to be measured by SPR immunosensing based on an indirect competition method: mixing a sample to be tested containing DES with a solution of monoclonal antibody of DESAnd (3) injecting samples, and jointly competing DES-BSA fixed on the surface of the SPR chip, wherein a signal generated by SPR is inversely proportional to the concentration of DES in a sample to be detected. The diethylstilbestrol content determination method has the detection limit as low as 2.901x10^‑6ng/m L, the recovery rate of the added standard is 94.12-101.22%, the RSD is 3.24-6.59%, and the ultra-trace detection of the diethylstilbestrol can be realized.

Description

Method for sensing and measuring diethylstilbestrol based on graphene multiple signal amplification SPR

Technical Field

The invention relates to the technical field of endocrine disruption detection, in particular to a method for sensing and measuring diethylstilbestrol based on graphene multiple signal amplification SPR.

Background

With the improvement of living standard of people, people pay more and more attention to environmental hormones, and the research on food-borne hormones and human health is more and more concerned. The content of estrogen in the food-derived substances has great influence on the health of human bodies. Especially milk, the main intake of milk is children. Milk contains considerable amounts of estrogen, and some scholars believe that the consumption of milk is significantly increased over 100 years ago. Diethylstilbestrol is one of the estrogen hormones. According to the research, exogenous estrogen can cause the development and the function abnormality of the reproductive system of men, can cause serious diseases such as hypospadias, gonad atrophy, testicular cancer and the like in serious cases, and exogenous estrogen can cause diseases such as irregular menstruation, ovarian atrophy and the like of women, and even can cause diseases such as infertility, hysteromyoma and the like. Estrogen residues also cause premature development in female children, and development of mammary glands in male children tends to be feminized, and because the blood distribution of body tissues and organs of infants does not establish a barrier to prevent or slow down external pollutants, estrogen is much more toxic to infants and juveniles than adults.

In order to ensure the health of the next generation, the content of the diethylstilbestrol in the milk needs to be controlled, so the diethylstilbestrol is a key detection item of the female hormone of the milk at present. Spr (surface Plasmon resonance), is an optical technique used to characterize changes in the refractive index of a surface. The SPR sensing detection chip comprises a gold film and a microfluidic channel on the surface of the gold film (or other noble metal chips such as a silver film and the like), recognition molecules capable of being specifically combined with target molecules are fixed on the surface of the gold film in the microfluidic channel, and a prism is arranged below the gold film. When a sample flows through the microfluidic channel during analysis, target molecules in the sample are combined with recognition molecules, the surface refractive index of the gold film is changed, the SPR angle is finally changed, and information such as the concentration and the affinity of the analyte is obtained by detecting the change of the SPR angle. The SPR sensing measurement of endocrine disturbance has the advantages of simple detection procedure, no need of labeling and real-time rapid dynamic detection, and is one of the most promising technologies for biological trace detection. Because exogenous estrogen has great harmfulness and can be accumulated in a human body, people hope to realize ultra-trace detection on diethylstilbestrol, and how to further reduce the detection limit is a common goal of related researchers.

Disclosure of Invention

Technical problem to be solved

In order to solve the problems in the prior art, the invention provides a method for sensing and measuring diethylstilbestrol based on graphene multiple signal amplification SPR, which is characterized in that the detection limit of diethylstilbestrol in a sample is further reduced and the detection sensitivity is improved by combining an optimized detection program with a modified SPR detection chip and adopting an indirect competition method, so that the ultra-trace amount of diethylstilbestrol in the sample is detected.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

a method for sensoring and measuring diethylstilbestrol based on graphene multiple signal amplification SPR comprises the following steps:

s1 sample preparation

Using magnetic graphene as an adsorbent, carrying out adsorption enrichment on diethylstilbestrol in a sample to be detected, eluting with an eluent, and carrying out constant volume on the eluted diethylstilbestrol to obtain a sample to be detected;

s2, modifying the SPR chip: cleaning the surface of a noble metal chip of the SPR chip, and connecting carboxylated graphene oxide on the surface of the noble metal chip;

s3, connecting and fixing DES-BSA by using carboxylated graphene oxide fixed on the surface of the SPR chip noble metal;

s4, measuring the concentration of the diethylstilbestrol in the sample to be measured by SPR immunosensing based on an indirect competition method: mixing a sample to be detected containing DES and a monoclonal antibody solution of DES, injecting samples, and competing DES-BSA fixed on the surface of an SPR chip together, wherein a signal generated by SPR is inversely proportional to the concentration of DES in the sample to be detected.

The method realizes multiple amplification of detection signals, wherein the first step is to utilize magnetic graphene to enrich diethylstilbestrol in a sample to be detected, elute and fix the volume to prepare a sample to be detected with higher concentration DES, the second step is to utilize graphene to modify the surface of a noble metal sheet of an SPR chip so as to enhance SPR signals, and the third step is to utilize the characteristics that graphene has large specific surface area and rich carboxyl on the surface, fix higher concentration DES-BSA and effectively amplify the SPR signals again, through the amplification of the triple signals, the sensitivity of DES detection from the sample to be detected can be obviously improved, the detection limit of DES detection is reduced, the ultra-trace detection of DES is realized, and the L OD value is as low as 2.901x10^-6ng/mL。

According to a preferred embodiment of the present invention, in step S1, the magnetic graphene is prepared as follows:

(1) taking nano graphene powder, ferrous iron salt and ferric iron salt as raw materials, wherein the molar ratio of ferrous iron to ferric iron is 1: 2;

(2) adding concentrated hydrochloric acid into the raw material, performing ultrasonic deoxidation, fully dispersing in a mixed solvent of ultrapure water and anhydrous alcohol, mechanically stirring for 12-24h, adding concentrated ammonia water to adjust the pH to be more than 10, continuously stirring under a heating condition to obtain a precipitate, and separating the precipitate;

(3) adding ultrapure water and anhydrous alcohol for cleaning, removing nonmagnetic impurities by using a magnetic separator, dispersing the obtained substance into the anhydrous alcohol, and drying at low temperature to obtain the magnetic graphene.

According to a preferred embodiment of the present invention, in step S1, the ferrous salt is ferrous sulfate or ferrous chloride, and the ferric salt is ferric chloride; adding strong ammonia water to adjust the pH value to be more than 10, stirring at 70-85 ℃ to obtain a precipitate, cleaning with ultrapure water and anhydrous alcohol, and drying at 40-65 ℃ to obtain the magnetic graphene material.

According to the preferred embodiment of the invention, in step S1, magnetic graphene is used as an adsorbent, diethylstilbestrol in a sample to be detected is adsorbed and enriched under the condition that pH is acidic, the adsorption time is 4-10min, acetonitrile is used as an eluent, diethylstilbestrol adsorbed by the magnetic graphene is eluted, and nitrogen is dried and the volume is fixed to be 250 mu L.

According to a preferred embodiment of the present invention, in step S2, the method for modifying the SPR chip comprises the following steps:

step 1: cleaning the noble metal chip of the SPR chip to remove organic matters on the surface of the noble metal chip;

step 2: introducing a group with negative charge on the surface of the noble metal chip of the SPR chip;

and step 3: introducing positively charged groups on the surface of the noble metal chip of the SPR chip by virtue of the electrostatic action of the negatively charged groups;

and 4, step 4: and connecting the carboxylated graphene oxide with negative charges on the surface of the noble metal sheet of the SPR chip by virtue of the electrostatic action of the positively charged groups.

According to the preferred embodiment of the present invention, in step S2, the noble metal chip of the SPR chip is a gold chip, and the specific steps are as follows:

step 1: by volume ratio of H₂0₂:H₂S0₄Preparing piranha solution at the ratio of 1:3, soaking the gold plate of the SPR chip at room temperature, removing organic matters on the surface of the gold plate, cleaning with deionized water and absolute ethyl alcohol, and finally drying with nitrogen; wherein H ₂0₂Is 30 percent of hydrogen peroxide by massWater;

step 2: soaking the gold plate of the SPR chip into an alcohol solution of MPA (mercaptopropionic acid), standing overnight, taking out, washing the gold plate with ethanol to remove redundant MPA, washing with deionized water to remove redundant alcohol, taking out, and drying the gold plate with nitrogen; through soaking, MPA and gold flakes form gold-sulfur bonds and carboxyl with negative charges are introduced;

and step 3: soaking the gold sheet of the SPR chip into PAH (poly (allylamine-hydrochloric acid)) solution, taking out the gold sheet, washing the gold sheet with deionized water, and drying the gold sheet with nitrogen; through soaking and electrostatic interaction, the surface of the gold sheet is connected with PAH with positive charges;

and 4, step 4: and soaking the gold sheet with positive charges in the dispersion liquid of the carboxylated graphene oxide, and connecting the carboxylated graphene oxide to the surface of the gold sheet through the soaking and the electrostatic action of PAH.

According to the preferred embodiment of the present invention, the method for fixing DES-BSA in step S3 comprises:

loading an SPR chip and a gold plate into a sensor, activating carboxyl on the surface of the chip for 5-10min by EDC (1-ethyl 3- (3-dimethylamino) carbodiimide hydrochloride) solution and NHS (N-hydroxysuccinimide ester) solution, using acetate buffer solution with pH of 4-5 as coupling buffer solution, preparing DES-BSA with certain mass concentration by using the coupling buffer solution for reaction for 15-30min, using ethanolamine as confining solution for reaction for 5-20min, using NaOH solution of 0.05 mol/L as regeneration solution for regeneration for 1-3min, and repeating the cycle for 1-3 times in sequence to connect the DES-BSA with graphene oxide with carboxyl on the surface of the gold plate of the SPR chip to realize the immobilization of the DES-BSA.

According to the preferred embodiment of the invention, in step S3, the concentration of EDC is 0.4 mol/L, the concentration is 0.1 mol/L, the activation time is 7min, and the pH of the coupling buffer solution is 4.5, wherein the coupling buffer solution is used for preparing DES-BSA with a certain mass concentration for reaction for 20min, ethanolamine with the pH of 8.5 is used as a blocking solution for reaction for 10min, and NaOH solution with the concentration of 0.05 mol/L is used as a regeneration solution for regeneration for 2 min.

According to the preferred embodiment of the invention, in step S3, when fixing DES-BSA, the DES-BSA concentration is 120 μ g/m L, the fixing amount of DES-BSA on the SPR chip is nearly saturated, and the response value of SPR is 625.86m degrees, relatively, in general, when the gold surface of the SPR chip is not modified by graphene oxide, the fixing amount of DES-BSA on the SPR chip is saturated when the DES-BSA concentration is 30 μ g/m L.

In addition, after the graphene oxide is modified on the surface of the gold sheet of the SPR chip, the fixed DES-BSA has good stability and repeatability. Even after 35 times of regeneration treatment, the signal value generated by the antibody with the same concentration can still reach more than 90% of the initial signal, which indicates that DES-BSA fixed on the surface of the SPR chip can still keep good activity and good repeatability after being regenerated for many times.

According to the preferred embodiment of the invention, in step S4, a sample to be tested containing DES and a DES-mAb (monoclonal antibody of DES) solution are mixed according to a volume ratio of 1:1, incubated at room temperature and added into a sample cell for sample injection detection, free DES in the sample injection and DES-BSA fixed on a sensor-based chip compete for DES-mAb together, and the SPR response value is determined.

According to the preferred embodiment of the invention, the specific steps of step S4 are mixing DES-mAb of 10.00 μ g/m L with DES standard solution of different concentration gradient in equal volume, incubating at room temperature and adding into sample cell for sample injection detection, monitoring SPR response signal to obtain DES-response signal (change of SPR angle m)^o) According to the calibration curve, mixing DES-mAb of 10.00 mu g/m L with a sample to be tested containing DES in equal volume, incubating at room temperature, injecting sample, and determining the concentration of DES in the sample to be tested.

According to the determination method of the invention, the OD value of L is reduced to 2.901x10^-6ng/mL，IC₅₀(semi-inhibitory concentration) 4.520x10^-4ng/m L, detection range 1.687 × 10^-5-1.141×10^-2ng/m L, the standard recovery rate is 94.12-101.22%, the RSD is 3.24-6.59%, and the accurate detection of the ultra-trace amount of diethylstilbestrol can be realized.

According to the preferred embodiment of the invention, the method further comprises step S5, wherein the regeneration of SPR is carried out by adopting 0.05 mol/L NaOH solution for 120 min/time and for more than 2 times, so that the removal rate of the antibody reaches more than 99.50%.

The regeneration method can remove antibodies specifically bound with the holoantigen and non-specifically adsorbed substances as far as possible on the premise of ensuring the activity of the holoantigen (DES-BSA) fixed on the surface of the SPR chip. After regeneration, the SPR chip can be recycled to determine DES.

(III) advantageous effects

The invention has the beneficial effects that:

(1) the invention provides a method for sensing and measuring diethylstilbestrol by multiple signal amplification SPR based on a graphene material, which has good specificity, can be used for measuring trace or even ultra-trace Diethylstilbestrol (DES) in milk, and has lower detection limit and higher sensitivity than the conventional E L ISA method.

(2) The invention uses the gold sheet (silver sheet or platinum sheet) of the SPR chip modified by graphene to amplify the SPR signal, simultaneously, because the gold sheet surface of the SPR chip has carboxylated graphene oxide, the characteristics of large specific surface area and rich carboxyl of the graphene are utilized, DES-BSA (fixed concentration reaches 120 mu g/m L, the SPR response value is 625.86m DEG) with higher fixed concentration can be fixed on the chip surface, the SPR signal is further effectively amplified, and the minimum detection limit of diethylstilbestrol is 1.826 × 10^-3ng/mL，IC₅₀The detection range is 0.1710ng/m L, and the detection range is 0.009924-5.701ng/m L, and the method has lower detection limit and higher sensitivity than the SPR method without loaded graphene oxide amplification and the conventional E L ISA method.

(3) The method also utilizes the magnetic graphene to perform high-power enrichment on diethylstilbestrol in the sample to be detected, and obtains the sample to be detected after elution and constant volume treatment, so that the detected L OD value is reduced to 2.901 × 10^-6ng/mL，IC₅₀Is 4.520 × 10^-4ng/m L, detection Range 1.687 × 10^-5-1.141×10^-2ng/m L, the labeling recovery rate is 94.12-101.22%, the RSD is 3.24-6.59%, the detection limit is further reduced compared with that of the loaded graphene SPR chip, the sensitivity is improved by about 500 times, the detection range is wider, and the method is particularly suitable for detecting low-concentration samplesDetection of amounts and ultra trace samples.

(4) The invention optimizes the fixed concentration of DES-BSA, the sample injection concentration of DES-mAb (5.00 mu g/m L) and the regeneration condition, so that compared with the conventional SPR method and the E L ISA method, the invention has lower detection limit and higher sensitivity, and is beneficial to strictly controlling the content of food-borne estrogen.

Drawings

Fig. 1 is an infrared spectrum of graphene and magnetic graphene in an experimental example of preparing magnetic graphene.

Fig. 2 is a scanning electron microscope image of graphene and magnetic graphene in an experimental example of preparing magnetic graphene.

FIG. 3 is an atomic force microscope of the surface of the gold plate at different stages during the modification of the surface of the gold plate of the SPR chip.

FIG. 4 is a graph showing the SPR angle change of the surface of the gold plate at different stages during the modification of the surface of the gold plate of the SPR chip.

FIG. 5 is a graph of SPR angular shift of different concentrations of DES-BSA immobilized on the surface of an SPR chip (containing graphene modifications).

FIG. 6 is a graph of SPR angular shift of different concentrations of DES-BSA immobilized on the surface of an SPR chip (without graphene modification).

FIG. 7 is a graph showing the effect of regeneration frequency on DES-BSA stability when regenerating SPR chips (containing graphene modifications).

FIG. 8 is a graph of SPR response over the entire course (including regeneration) of an indirect competitive immunoassay using SPR.

FIG. 9 is a graph of SPR angular shift generated by binding of DES-BSA on an SPR chip (with graphene modification) when different concentrations of DES-mAb were injected in example 1 of the present invention.

FIG. 10 is the SPR angle shift curve graph of the DES-mAb of 10.00 μ g/m L injected with the DES standard solution of different concentration gradient in equal volume mixture in example 1 of the present invention.

FIG. 11 shows the indirect competitive inhibition curve (a) and the calibration curve (b) of DES detection when the DES-mAb of 10.00. mu.g/m L and the DES standard solution of different concentration gradient are mixed and injected with equal volume in example 1.

FIG. 12 is a graph of SPR angular shift resulting from binding of DES-BSA on an SPR chip (without graphene modification) with varying concentrations of DES-mAb injected in comparative example 1.

FIG. 13 is the SPR angular shift curve of indirect competition of comparative example 1 when 15 μ g/m L DES-mAb is injected in equal volume mixing with different concentration gradients of DES standard solution.

FIG. 14 shows the indirect competitive inhibition curve (a) and the calibration curve (b) of DES detection in comparative example 1 when 15.00. mu.g/m L DES-mAb is injected into the sample with mixed volume of DES standard solution with different concentration gradient.

FIG. 15 is the SPR angle shift curve graph of the sample injection of the DES-mAb of 10.00 μ g/m L and the DES standard solution with different concentration gradients in the same volume in example 2 (pretreatment of introducing enrichment of magnetic graphene).

FIG. 16 shows an indirect competitive inhibition curve (a) and a calibration curve (b) for DES detection in example 2 (pretreatment for introducing enrichment of magnetic graphene) of the present invention when a DES-mAb of 10.00 μ g/m L and DES standard solutions of different concentration gradients are mixed and injected in equal volumes.

FIG. 17 shows the results of indirect competition of the ISA E L in comparative example 2, i.e., the indirect competition inhibition curve (a) and the calibration curve (b) of DES detection.

Fig. 18 is a bar graph of experimental results of the specificity assay of example 2 (pretreatment introducing enrichment of magnetic graphene) of the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

The reagents, materials and instruments used in the following experiments and examples are described below:

the water used in the experiment was ultrapure water self-made in the laboratory.

0.01M PBS solution with pH 7.4 is used as buffer solution for reaction, is used for diluting DES-mAb and solution to be detected, is mixed with 10% methanol and is used for preparing DES standard solution, Phosphate Buffered Saline (1X)0.0067M (PO)₄)。

Instrument for measuring the position of a moving object

An ESPRIT surface plasma resonance biosensor (Auto L ab, Netherlands), the incident wavelength of the device is 670nm, the angle preset range is 62-78 degrees, the dynamic detection range is 4000m degrees, the angle resolution is less than 0.02m degrees, the average baseline fluctuation is 0.1m degrees, the whole reaction temperature is controlled at 25 +/-0.5 degrees, the data acquisition software is Autolab ESPRIT DataAcquisition 4.3, and the data analysis software is Kinetic Evaluation 5.0.

Microplate reader Microplate spectrometer, BIO-RAD, Xmark 0.25 μm filter, BIOFIC.

Items 1-6 below are a basic overview of the process of the present invention, including: the basic outline of the method comprises the steps of preparing magnetic graphene, treating a sample to be detected by using the magnetic graphene, modifying an SPR chip by using the graphene, fixing DES-BSA on the surface of the SPR chip, performing SPR indirect competitive immunoassay and regenerating the SPR chip. Items 7 to 8 below are the basic outline of the method of the present invention for evaluating the specificity and detecting diethylstilbestrol in a milk sample.

1. Preparation of magnetic graphene

Mixing nano graphene powder, ferrous sulfate and ferric chloride at a molar ratio of 4:3:4 by adopting a chemical coprecipitation method, adding 0.8m L37% of concentrated hydrochloric acid, performing ultrasonic deoxidation, fully dispersing in a solution of 200m L ultrapure water and absolute ethyl alcohol, mechanically stirring at 1500rpm for 1000min, adding concentrated ammonia water to adjust the pH to be more than 10, stirring at 80 ℃ for 40min, respectively cleaning with the ultrapure water and the absolute ethyl alcohol by using a magnetic separator for 5 times, removing nonmagnetic impurities, dispersing in the absolute ethyl alcohol, and drying at 60 ℃ to obtain the magnetic graphene material.

Characterization of magnetic graphene materials:

the infrared spectrogram of graphene and magnetic graphene is shown in figure 1 at 1600cm ^-1 and 3430cm^-At position 1, both materials have absorption peak of 3430cm^-The absorption peak of moderate intensity at 1 is caused by O-H bond stretching vibration, 1600cm^-The weaker absorption peak at 1 is caused by C ═ C bond stretching vibrations. While the magnetic graphene is 617cm^-1 part has a strong absorption peak which belongs to the stretching vibration of Fe-O bonds, and the spectrogram of the graphene has no absorption peak, which shows that Fe₃O₄The nanoparticles are immobilized on a thin layer of graphene.

The scanning electron micrograph of the observed graphene and the magnetic graphene is shown in fig. 2, the graphene is in a clear lamellar shape and has a smooth surface, and particles with different numbers are coated on the surface of the magnetic graphene lamellar layer, which indicates that the sites on the surface of the graphene are coated by Fe₃O₄Nanoparticle occupancy.

Fe can be seen from the infrared spectrograms and scanning electron micrographs of the two materials₃O₄The magnetic core is coated on the graphene, and the material is successfully prepared.

2. Enrichment method of magnetic graphene on sample to be detected

Optimizing enrichment conditions by adopting high performance liquid chromatography, adsorbing diethylstilbestrol in a sample to be detected for 5min by using 30mg of magnetic graphene under the condition that the pH value is acidic, eluting for 5min by using 2m L acetonitrile, drying by nitrogen and fixing the volume to 200 mu L to be used as a sample to be detected.

3. Method for modifying surface of gold sheet of SPR chip by graphene

(1) The gold flakes were treated with a newly formulated piranha solution (30% H)₂0₂:H₂S0₄1:3) soaking for 1-2h at room temperature, removing organic matters on the surface of the gold flakes, then washing with a large amount of deionized water, washing with absolute ethyl alcohol, and finally drying with nitrogen.

(2) Filtering 0.01 mol/L MPA ethanol solution, placing in a culture dish, immersing gold plate in the solution to avoid bubbles on the gold surface, standing overnight, washing the gold plate with ethanol for three times to remove excessive MPA, washing with deionized water for three times to remove excessive alcohol, and blowing the gold plate with nitrogen.

(3) And soaking the MPA modified gold sheet (with carboxyl) in 1% PAH solution for 1h, washing with water and drying with nitrogen, wherein the surface of the gold film is provided with positive charge PAH.

(4) And then soaking the gold film with positive charge in a graphene oxide solution of 0.1mg/m L for 1h, and fixing the graphene oxide with carboxyl on the surface of the gold sheet through the electrostatic interaction between PAH and the graphene oxide with carboxyl.

Characterization of the functional modification of the chip surface:

as can be seen from FIG. 3a, the bare gold sheet surface is smooth; after introduction of-COOH, the roughness is increased (fig. 3 b); as can be seen from fig. 3c and fig. 3b, the smoothness is improved, which indicates that after-COOH is introduced and positive PAH activation treatment is performed, graphene oxide is successfully modified on the surface of the chip.

The change in the SPR signal value is based on the change in the refractive index of the medium on the surface of the chip and the amount of fixturing the surface of the chip. Therefore, examining the change in SPR signal value allows one to examine whether the chip surface has been successfully modified. As can be seen in FIG. 4, the SPR angle of the MPA self-assembly process was changed by about 39.18m °; the SPR angle change after PAH positive electrification is 277.09m degrees, and the SPR angle change value after GO modification is 44.98m degrees.

In subsequent experiments, after DES-BSA immobilization saturation, the SPR angle change value is 625.86m degrees, and after determining that the optimal antibody concentration (5 mu g/m L) is subjected to DES-BSA binding reaction, the SPR angle change value is 135.46m degrees.

4. Fixing DES-BSA on the surface of gold plate of SPR chip

Loading an SPR chip into a sensor, taking acetate buffer solution with pH 4.5 as coupling buffer solution, taking 0.4 mol/L EDC and 0.1 mol/L NHS to activate carboxyl on the surface of the chip for 7min, using the coupling buffer solution to prepare DES-BSA with certain mass concentration for reaction for 20min, taking ethanolamine with pH8.5 as blocking solution for reaction for 10min, taking 0.05 mol/L NaOH solution as regeneration solution for regeneration for 2min, repeating the steps for 1-3 times according to DES-BSA fixing condition and SPR response value, and calling software to perform an immobilization procedure.

As the mass concentration of the whole antigen increases, the change value of the SPR angle is larger, which indicates that the fixing amount of the whole antigen on the surface of the chip is gradually increased, as shown in FIG. 5, the fixing amount is close to saturation at 120 mu g/m L, and the response value of the SPR is 625.86m degrees, so that 120 mu g/m L is selected as the optimal fixing mass concentration of the whole antigen.

The immobilized amount of the traditional chip without graphene oxide modification is close to saturation at 30 mu g/m L, and as shown in FIG. 6, the response value of SPR is only 250.74 degrees.

5. SPR indirect competitive immunoassay

And mixing the antibody with a certain mass concentration and DES solutions with different concentrations in an equal volume, incubating at room temperature, adding into a sample pool, allowing free DES and DES-BSA fixed on a sensing base chip to compete for DES-mAb together, and determining SPR response values respectively. The DES-mAb bound to DES-BSA was washed away by the regeneration solution to regenerate the chip, and the reaction process is shown in FIG. 8. To ensure the repeatability of the test results, 3 measurements were made for each experiment.

As shown in fig. 8, the surface plasmon resonance biosensor modified by carboxylated graphene oxide detects DES by an indirect competition method, and the change process of the SPR angle value includes a binding stage, a dissociation stage and a regeneration stage.

6. Regeneration

In the research, reagents listed in Table 2, namely 0.1 mol/L hydrochloric acid solution, 0.1 mol/L hydrochloric acid + 0.1% SDS solution, 0.1 mol/L hydrochloric acid + 0.1% Triton X-100 solution, 0.05 mol/L pH2.0 glycine-hydrochloric acid solution, 0.05 mol/L solution regenerated once, and 0.05 mol/L NaOH solution regenerated 2 times, are selected to remove 88.73%, 92.54%, 87.75%, 32.28%, 96.36% and 99.59% of the bound antibody (Table 2) respectively.

TABLE 2

Therefore, the optimum regeneration conditions are 0.05 mol/L NaOH solution 120 min/regeneration 2 times.

FIG. 7 shows the effect of the number of regenerations on the DES-BSA activity of a graphene oxide-modified chip surface. The graph shows that after 35 times of regeneration, the signal value generated by the antibody with the same concentration can still reach more than 90% of the initial signal, which indicates that DES-BSA fixed on the surface of the SPR chip can still keep good activity and good repeatability after multiple regenerations.

After regeneration, to investigate the effect of nonspecific adsorption on the SPR chip surface, the antibody was replaced with a 12.5. mu.g/m L BSA solution, the other reaction conditions and reaction process were unchanged, and the SPR response was monitored for 7 repetitions without any change in SPR angle, indicating that the nonspecific adsorption on the regenerated chip surface was negligible.

7. Specificity of

Selecting structural and functional analogues of DES, namely bisphenol A, estradiol, dienestrol and estrone, respectively preparing different concentration gradients, carrying out experiments by using an indirect competition method, and carrying out specificity evaluation by calculating the inhibition rate of the reaction.

In order to evaluate the specificity of the constructed magnetic graphene enriched graphene oxide amplification determination method, diethylstilbestrol, five structural and functional analogues of bisphenol A, estradiol, diethylstilbestrol, dienestrol and estrone are respectively determined. The results of the specificity experiments are shown in fig. 18, where the inhibition rate was less than 6% for all analogues, indicating that they hardly bound to the antibody. Therefore, other analogues had little effect on the detection of DES, indicating that the method is very specific.

8. Detection of milk samples

The milk samples were purchased from local supermarkets, verified to be free of DES by high performance liquid chromatography analysis, and subjected to a standard recovery experiment. Enrichment with magnetic grapheneThe SPR indirect competition method is used for detection, and the concentration is set to be different in high, medium and low within the detection range (1 × 10)^-4ng/mL；1×10^-3ng/mL；1×10^-2ng/m L), six replicates for each concentration, and normalized recovery was calculated.

The result of the standard addition recovery is shown in table 3, the standard addition recovery is between 94.12% and 101.22%, and the Relative Standard Deviation (RSD) is between 3.24% and 6.59%, which indicates that the method is accurate and reliable, and in practical application, if the mass concentration of the DES of the sample to be detected is low, the steps of enrichment and concentration of magnetic graphene can be adopted to detect the DES residue.

TABLE 3

The features and technical effects of the present invention will be further described below with reference to specific examples and specific comparative examples of the present invention.

Example 1

This example does not include a pretreatment step of enriching diethylstilbestrol in the sample to be tested with magnetic graphene. The present embodiment includes the following steps:

the method comprises the following steps: method for modifying surface of gold sheet of SPR chip by graphene

Step two: fixing DES-BSA on the surface of gold plate of SPR chip

The SPR chip is loaded into a sensor, acetate buffer solution with pH 4.5 is used as coupling buffer solution, EDC with 0.4 mol/L and NHS with 0.1 mol/L are used for activating carboxyl on the surface of the chip for 7min, DES-BSA binding reaction with 120 mu g/m L is prepared by using the coupling buffer solution for 20min, ethanolamine with pH8.5 is used as blocking solution for reaction for 10min, NaOH solution with 0.05 mol/L is used as regeneration solution for regeneration time of 2min, the procedures are repeated for 1 time, and the change value of the SPR angle after the fixation saturation of DES-BSA is 625.86m degrees.

Step three: SPR indirect competitive immunoassay

As shown in FIG. 9, the DES-mAb injection samples with different concentrations were used to monitor the SPR angle shift curve generated by binding to DES-BSA on the SPR chip (with graphene modification), and it was found that when the DES-mAb antibody concentration was below 5.00. mu.g/m L, the binding of the antibody to the chip surface did not reach saturation, therefore, the optimal antibody concentration was 5.00. mu.g/m L.

Regeneration was performed 2 times with 0.05M NaOH solution, and DES was determined.

In the determination, 10.00 mu g/m L DES-mAb and different concentration gradient DES standard solution were mixed in equal volume, response signal was monitored, and the value of the response signal is shown in FIG. 10, as shown in FIG. 11, L OD (detection limit) value is 0.001826ng/m L50 is 0.1710ng/m L, and the detection range is 0.009924-5.701ng/m L.

Comparative example 1

Comparative example 1 is based on example 1, the first step is removed, that is, the surface of the gold sheet of the SPR chip is not modified by graphene. This comparative example comprises the steps of:

step 1: fixing DES-BSA on the surface of gold plate of SPR chip

(1) The gold flakes were treated with a newly formulated piranha solution (30% H)₂0₂:H₂S0₄1:3) at room temperatureSoaking for 1-2h, removing organic matters on the surface of the gold sheet, then cleaning with a large amount of deionized water, cleaning with absolute ethyl alcohol, and finally drying with nitrogen.

(3) The SPR chip is loaded into a sensor, acetate buffer solution with pH 4.5 is used as coupling buffer solution, EDC with 0.4 mol/L and NHS with 0.1 mol/L are used for activating carboxyl on the surface of the chip for 7min, DES-BSA with the concentration of 30 mu g/m L is prepared by the coupling buffer solution, binding reaction is carried out for 20min, ethanolamine with pH8.5 is used as blocking solution for reaction for 10min, NaOH solution with 0.05 mol/L is used as regeneration solution for 2min, and the regeneration time is 1 time according to the above procedure.

The results are shown in FIG. 6, and the fixing amount is close to saturation when the optimal fixing concentration of DES-BSA is 30 μ g/m L (the SPR response value does not change along with the increase of the concentration of DES-BSA).

Step 2: SPR indirect competitive immunoassay

As shown in figure 12, the DES-mAb injection with different concentrations is adopted, the SPR angle shift curve generated by combination of the DES-mAb injection with DES-BSA on an SPR chip (without graphene modification) is monitored, and finally the concentration of the DES-mAb antibody is determined to be 2.50 mug/m L.

Regeneration was performed 2 times using 0.05M NaOH solution, and DES was determined.

In the determination, 5.00 mu g/m L DES-mAb and different concentration gradient DES standard solution are mixed in equal volume, response signal is monitored, and the value of response signal is shown in FIG. 13. As shown in FIG. 14, L OD (detection limit) is 0.01617ng/m L50 is 0.3240ng/m L, and detection range is 0.07279-5.302ng/m L. compared with example 1 and comparative 1, the detection limit L OD of comparative 1 is 8.8 times that of example 1, IC is 8 times that of comparative 1₅₀Which is 2 times that of example 1.

Example 2

In this embodiment, on the basis of embodiment 1, magnetic graphene is further used to perform pretreatment of enriching diethylstilbestrol in a sample to be detected, and a sample to be detected is obtained after the pretreatment, where the pretreatment specifically is:

optimizing enrichment conditions by adopting high performance liquid chromatography, adsorbing diethylstilbestrol in a diluted DES standard sample for 5min by using 30mg of magnetic graphene under the condition that the pH value is 5-6, eluting the magnetic graphene for 5min by using 2m L acetonitrile after filtering, drying the magnetic graphene by using nitrogen, and fixing the volume to 200 mu L to be used as a sample to be detected.

The other steps and conditions were carried out with reference to example 1.

Experimental results show that after the pretreatment step of magnetic graphene enrichment is introduced, the surface plasma resonance biosensor modified by the carboxylated graphene oxide is used for detecting DES by an indirect competition method, response signals are monitored, the values of the response signals are shown in figure 15, the experimental results are shown in figure 16, and the L OD value is 2.901 × 10^-6ng/mL，IC₅₀Is 4.520 × 10^-4ng/m L, detection Range 1.687 × 10^-5-1.141×10^-2ng/mL。

Compared to example 1, example 1 had an L OD 630 times greater than example 2, IC₅₀378 times that of example 2.

Comparative example 2

The optimal dilution times of the DES-BSA and the DES monoclonal antibody are 1/8100(1.130 mu g/ml) and 1/8000(0.5454 mu g/ml) respectively.

The results are shown in FIG. 17, wherein the OD value of L is 0.045ng/m L₅₀The detection range is 0.172ng/m L and 0.048-0.437ng/m L.

Several indirect competitive immunoassays as described above use the same whole antigen and antibody.

The experimental results of the four detection methods of example 1, example 2, comparative example 1 and comparative example 2 are recorded in table 4, and comparing the results of the conventional SPR experiment without graphene modification with the E L ISA, it can be seen that the SPR detection method has a lower detection limit and a wider detection range than the E L ISA method, and has the disadvantage of a larger amount of antibody used, whereas when the chip surface is modified with graphene oxide, the detection limit of SPR is further reduced, the sensitivity is improved by about 1.895 times, and the detection range is wider.

TABLE 4

It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for sensoring and measuring diethylstilbestrol based on graphene multiple signal amplification SPR is characterized by comprising the following steps:

s1 sample preparation

2. The method according to claim 1, wherein in step S1, the magnetic graphene is prepared according to the following method: taking nano graphene powder, ferrous iron salt and ferric iron salt as raw materials, wherein the molar ratio of ferrous iron to ferric iron is 1: 2; adding concentrated hydrochloric acid into the raw material, performing ultrasonic deoxidation, fully dispersing in a mixed solvent of ultrapure water and anhydrous alcohol, mechanically stirring for 12-24h, adding concentrated ammonia water to adjust the pH to be more than 10, continuously stirring under a heating condition to obtain a precipitate, and separating the precipitate; adding ultrapure water and anhydrous alcohol for cleaning, removing nonmagnetic impurities by using a magnetic separator, dispersing the obtained substance into the anhydrous alcohol, and drying at low temperature to obtain the magnetic graphene.

3. The method according to claim 1, wherein in step S1, the magnetic graphene is used as an adsorbent, the diethylstilbestrol in the sample to be tested is adsorbed and enriched under the acidic condition of pH, the adsorption time is 4-10min, acetonitrile is used as an eluent, the diethylstilbestrol adsorbed by the magnetic graphene is eluted, and nitrogen is blown dry and the volume is fixed to 100-250 μ L.

4. The method of claim 1, wherein the method of modifying the SPR chip in step S2 comprises the steps of:

5. The method of claim 4, wherein in step S1, in step S2, the noble metal chip of the SPR chip is gold chip, and the method comprises the following steps:

step 1: by volume ratio of H₂0₂:H₂S0₄Preparing piranha solution at the ratio of 1:3, soaking the gold plate of the SPR chip at room temperature, removing organic matters on the surface of the gold plate, cleaning with deionized water and absolute ethyl alcohol, and finally drying with nitrogen; wherein H₂0₂Is 30 percent of hydrogen peroxide by mass;

step 2: soaking the gold sheet of the SPR chip into an alcohol solution of MPA, standing overnight, taking out, washing the gold sheet with ethanol to remove redundant MPA, washing with deionized water to remove redundant alcohol, taking out, and drying the gold sheet with nitrogen; through soaking, MPA and gold flakes form gold-sulfur bonds and carboxyl with negative charges are introduced;

and step 3: soaking the gold sheet of the SPR chip into a PAH solution, taking out the gold sheet, washing the gold sheet with deionized water, and drying the gold sheet with nitrogen; through soaking and electrostatic interaction, the surface of the gold sheet is connected with PAH with positive charges;

6. The method of claim 1, wherein the method for immobilizing DES-BSA in step S3 is:

loading an SPR chip and a gold plate into a sensor, activating carboxyl on the surface of the chip for 5-10min by EDC solution and NHS solution, taking acetate buffer solution with pH of 4-5 as coupling buffer solution, preparing DES-BSA with a certain mass concentration by the coupling buffer solution for reaction for 15-30min, taking ethanolamine as confining solution for reaction for 5-20min, taking NaOH solution of 0.05 mol/L as regeneration solution for regeneration for 1-3min, and repeating the cycle for 1-3 times in sequence to connect the DES-BSA with graphene oxide with carboxyl on the surface of the gold plate of the SPR chip, thereby realizing the immobilization of the DES-BSA.

7. The method of claim 6, wherein in step S3, the EDC concentration is 0.4 mol/L concentration is 0.1 mol/L, the activation time is 7min, and the pH of the coupling buffer solution is 4.5, wherein the coupling buffer solution is used for preparing DES-BSA with a certain mass concentration for 20min, the ethanolamine with a pH of 8.5 is used as a blocking solution for 10min, and the NaOH solution with a concentration of 0.05 mol/L is used as a regeneration solution for 2 min.

8. The method of claim 6, wherein in step S3, when DES-BSA is immobilized, the DES-BSA concentration is 120 μ g/m L, and the SPR has a response value of 625.86m °.

9. The method of claim 1, wherein in step S4, the DES-containing sample to be tested and the DES-mAb solution are mixed at a volume ratio of 1:1, incubated at room temperature, added into a sample cell, injected for detection, free DES and DES-BSA fixed on a sensor-based chip compete for DES-mAb together in the injection, and SPR response values are determined, wherein the DES-mAb concentration of the DES-mAb solution is 10.00 μ g/m L, and the DES-mAb concentration of the injection is 5.00 μ g/m L.

10. The method of claim 1, further comprising a step S5, wherein the regeneration of SPR is performed by using 0.05 mol/L NaOH solution 120 min/time, and the regeneration is performed more than 2 times, so that the antibody removal rate is more than 99.50%.