CN111398221B - Method for sensoring and measuring diethylstilbestrol based on graphene multiple signal amplification SPR - Google Patents
Method for sensoring and measuring diethylstilbestrol based on graphene multiple signal amplification SPR Download PDFInfo
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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, adsorbing and enriching diethylstilbestrol in a sample to be detected, then eluting by using an eluent, and carrying out constant volume on 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 noble metal of the SPR chip; s4, determining the concentration of diethylstilbestrol in the sample to be detected 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 diethylstilbestrol content determination method has the detection limit as low as 2.901x10 ‑6 ng/mL, the standard recovery rate is 94.12-101.22%, the RSD is 3.24-6.59%, and the ultra-trace detection of diethylstilbestrol can be realized.
Description
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 diethylstilbestrol in the milk needs to be controlled, so that diethylstilbestrol is a key detection item of the current milk estrogen. Spr (Surface Plasmon Resonance), is an optical specific 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, determining the concentration of diethylstilbestrol in the sample to be detected by SPR immunosensing based on an indirect competition method: mixing a sample to be detected containing DES and a DES monoclonal antibody solution, 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 invention realizes the multiple amplification of detection signals: the method comprises the steps of enriching diethylstilbestrol in a sample to be detected by using magnetic graphene, eluting and fixing volume to obtain a sample to be detected, wherein the sample to be detected has DES with higher concentration; secondly, modifying the surface of a noble metal chip of the SPR chip by utilizing graphene so as to enhance an SPR signal; thirdly, fixing DES-BSA with higher concentration by utilizing the characteristics that graphene has large specific surface area and the surface of the graphene contains abundant carboxyl, and effectively amplifying SPR signals again. Through the triple signal amplification, the sensitivity of detecting DES from a sample to be detected can be obviously improved, the detection limit of DES is reduced, the ultra-trace detection of DES is realized, and the LOD value is as low as 2.901x10 -6 ng/mL。
According to a preferred embodiment of the present invention, in step S1, the magnetic graphene is prepared according to the following method:
(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 present invention, in step S1: the method comprises the steps of adopting magnetic graphene as an adsorbent, carrying out adsorption enrichment on diethylstilbestrol in a sample to be detected under the condition that the pH value is acidic, wherein the adsorption time is 4-10min, adopting acetonitrile as eluent, eluting the diethylstilbestrol adsorbed by the magnetic graphene, drying with nitrogen and fixing the volume to 100-250 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 a 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 H 2 0 2 :H 2 S0 4 Preparing piranha solution from 1:3, soaking the gold plate of the SPR chip at room temperature to remove organic matters on the surface of the gold plate, cleaning with deionized water and absolute ethyl alcohol, and finally drying with nitrogen; wherein H 2 0 2 Is 30 percent of hydrogen peroxide by mass;
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 the SPR chip and the gold plate into a sensor, and activating carboxyl on the surface of the chip for 5-10min by using EDC (1-ethyl 3- (3-dimethylamino) carbodiimide hydrochloride) solution and NHS (N-hydroxysuccinimide ester) solution; acetate buffer solution with pH =4-5 is used as coupling buffer solution, the coupling buffer solution is used for preparing DES-BSA with certain mass concentration for reaction for 15-30min, ethanolamine is used as confining liquid for reaction for 5-20min, 0.05mol/L NaOH solution is used as regeneration liquid for regeneration for 1-3min, and the steps are repeated for 1-3 times in sequence, so that the DES-BSA is connected with graphene oxide with carboxyl on the surface of a gold sheet of an SPR chip, and the immobilization of the DES-BSA is realized. The method can prepare the solution for standby, and then carry out the immobilization procedure by the aid of relevant software to complete the immobilization of DES-BSA (DES-bovine serum albumin coatingen).
According to the preferred embodiment of the present invention, in step S3, the concentration of EDC is 0.4mol/L, the concentration of NHS is 0.1mol/L, and the activation time is 7min; the pH of the coupling buffer solution is 4.5; 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 a 0.05mol/L NaOH solution is used as a regeneration solution for regeneration for 2min.
According to the preferred embodiment of the invention, in step S3, when fixing DES-BSA, the concentration of DES-BSA is 120 μ g/mL, the fixing amount of DES-BSA on the SPR chip is close to saturation, and the response value of SPR is 625.86m °. In contrast, in general, when the surface of the gold plate of the SPR chip is not modified by graphene oxide, the fixed amount of DES-BSA on the SPR chip is saturated when the concentration of DES-BSA is 30 μ g/mL.
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 regeneration treatments, 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 after multiple regenerations and has good repeatability.
According to the preferred embodiment of the invention, in step S4, after mixing the sample to be tested containing DES and DES-mAb (monoclonal antibody of DES) solution according to the volume ratio of 1:1, incubating at room temperature and adding into a sample cell for sample injection detection, wherein 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 present invention, the step S4 comprises the following steps: mixing DES-mAb 10.00 μ g/mL with DES standard solution with different concentration gradients at equal volume, incubating at room temperature, adding into sample cell, detecting, and monitoring SPR response signal to obtain DES-response signal (SPR angle change m) o ) The calibration curve of (1); according to the calibration curve, mixing the DES-mAb with 10.00 mu g/mL and the sample to be tested containing DES in equal volume, incubating and injecting at room temperature, and determining the concentration of DES in the sample to be tested.
According to the determination method of the invention, the LOD value is reduced to 2.901x10 -6 ng/mL，IC 50 (half inhibitory concentration) 4.520x10 -4 ng/mL, detection range of 1.687 × 10 -5 -1.141×10 -2 ng/mL, 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 the diethylstilbestrol can be realized.
According to a preferred embodiment of the present invention, further comprising step S5, regeneration of SPR: the 0.05mol/L NaOH solution is adopted for 120 min/time and is regenerated for more than 2 times, so that the removal rate of the antibody reaches more than 99.50 percent.
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 graphene material-based method for sensing and measuring diethylstilbestrol by using multiple signal amplification SPR, 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 ELISA method.
(2) The invention uses the gold sheet (silver sheet or platinum sheet) of the graphene modified SPR chip to amplify the SPR signal; meanwhile, due to the fact that the surface of the gold sheet of the SPR chip is provided with the 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/mL, and the SPR response value is 625.86m DEG) with higher concentration can be fixed on the surface of the chip, SPR signals are further effectively amplified, and the minimum detection limit of diethylstilbestrol is 1.826 multiplied by 10 -3 ng/mL，IC 50 The detection range is 0.1710ng/mL, the detection range is 0.009924-5.701ng/mL, and the method has lower detection limit and higher sensitivity compared with the SPR method without graphene oxide amplification and the conventional ELISA 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 LOD value is as low as 2.901x10 -6 ng/mL，IC 50 Is 4.520 × 10 -4 ng/mL, detection range 1.687 × 10 -5 -1.141×10 -2 ng/mL, the recovery rate of the added standard is 94.12-101.22%, 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, and the method has a wider detection range and is particularly suitable for detecting low-concentration samples. Therefore, the invention can realize the detection of low-concentration, trace and ultra-trace samples.
(4) The invention optimizes the fixed concentration of DES-BSA, the sample injection concentration (5.00 mu g/mL) of DES-mAb and the regeneration condition, so that compared with the conventional SPR method and ELISA 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. Mu.g/mL mixed with DES standard solution of different concentration gradient in equal volume injection 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/mL and the DES standard solution of different concentration gradient are mixed and injected with equal volume in example 1 of the present invention.
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 an SPR angular shift curve of indirect competition of comparative example 1 when 15. Mu.g/mL DES-mAb is injected in equal volume mixing with DES standard solutions of different concentration gradients.
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/mL DES-mAb is injected into the sample with mixed volume of DES standard solutions of different concentration gradients.
FIG. 15 is the SPR angle shift curve graph of the present invention in example 2 (pretreatment of introducing magnetic graphene enrichment) when the DES-mAb of 10.00 μ g/mL and DES standard solution of different concentration gradient are mixed and injected in equal volume.
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. Mu.g/mL and DES standard solutions of different concentration gradients are mixed and injected in equal volumes.
FIG. 17 shows the indirect competition results of ELISA in comparative example 2: indirect competitive inhibition curve (a) and calibration curve for DES detection (b).
Fig. 18 is a bar graph of experimental results of the specificity assay of example 2 (pretreatment introducing magnetic graphene enrichment) 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 is ultrapure water self-made in a 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, and Phosphate Buffered Saline (1X) 0.0067M (PO) 4 )。
Instrument
An ESPRIT surface plasma resonance biosensor (Auto Lab, netherlands) has an incident wavelength of 670nm, an angle preset range of 62-78 degrees, a dynamic detection range of 4000m degrees, an angle resolution of less than 0.02m degrees and an average baseline fluctuation of 0.1m degrees. The whole reaction temperature is controlled at 25 +/-0.5 ℃. The Data Acquisition software is Autolab ESPRIT Data Acquisition 4.3, and the Data analysis software is Kinetic evaluation5.0.
Microplate reader Microplate spectrometer, BIO-RAD, xmark 0.25 μm filter, BIOFIC.
1. Preparation of magnetic graphene
Mixing nano graphene powder, ferrous sulfate and ferric chloride according to a molar ratio of 4.
Characterization of magnetic graphene materials:
the infrared spectrogram of the graphene and the magnetic graphene is shown as the figure1, 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 vibration. 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 Fe 3 O 4 The 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 Fe 3 O 4 Nanoparticle occupancy.
Fe can be seen from the infrared spectrograms and scanning electron micrographs of the two materials 3 O 4 The 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 2mL of acetonitrile, blow-drying by using 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) Mixing gold flakes with newly prepared piranha solution (30%) 2 0 2 :H 2 S0 4 = 1:3) is soaked for 1-2h at room temperature, organic matters on the surface of the gold sheet are removed, then the gold sheet is cleaned by a large amount of deionized water, cleaned by absolute ethyl alcohol, and finally dried by nitrogen.
(2) Filtering 0.01mol/L MPA ethanol solution, placing the solution in a culture dish, immersing the gold sheet in the solution to avoid bubbles on the gold surface, and 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 (5) drying the gold sheet by nitrogen.
(3) 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 charges in 0.1mg/mL graphene oxide solution 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 after GO modification is 44.98m degrees.
In subsequent experiments, the SPR angle change value after DES-BSA immobilization saturation was 625.86m °, whereas after determining the optimal antibody concentration (5. Mu.g/mL) for binding reaction with DES-BSA, the SPR angle change value was 135.46m °. Therefore, the graphene oxide can be further verified to be successfully adsorbed on the surface of the SPR chip.
4. Fixing DES-BSA on the surface of gold plate of SPR chip
An SPR chip is arranged in a sensor, acetate buffer solution with pH 4.5 is used as coupling buffer solution, 0.4mol/L EDC and 0.1mol/L NHS are used for activating carboxyl on the surface of the chip for 7min, coupling buffer solution is used for preparing DES-BSA with certain mass concentration for reaction for 20min, ethanolamine with pH8.5 is used as blocking solution for reaction for 10min, and 0.05mol/L NaOH solution is used as regeneration solution for regeneration for 2min. According to the DES-BSA immobilization situation and the SPR response value, the method can be repeated for 1-3 times according to the above procedure, and the software is called to carry out the immobilization procedure.
The change value of the SPR angle is increased along with the increase of the mass concentration of the whole antigen, which indicates that the fixed quantity of the whole antigen on the surface of the chip is gradually increased. As shown in FIG. 5, at 120. Mu.g/mL, the fixed amount was close to saturation, and the SPR response was 625.86 m. Therefore, 120. Mu.g/mL was selected as the optimal fixed mass concentration for the whole antigen. When the fixed concentration of the whole antigen is more than 120. Mu.g/mL, the response value of SPR hardly changes any more. Therefore, when the concentration of the whole antigen DES-BSA is 120. Mu.g/mL, the fixed amount is close to saturation.
The fixed quantity of the conventional chip without graphene oxide modification is close to saturation at 30 mu g/mL, and as shown in FIG. 6, the response value of SPR is only 250.74 degrees. Therefore, after the carboxylated graphene oxide is introduced, the fixed amount (fixed concentration) of the whole antigen DES-BSA on the surface of the chip is obviously increased.
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 carboxylated graphene oxide modified surface plasmon resonance biosensor 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 of
Regeneration requires that antibodies specifically binding to the whole antigen and non-specifically adsorbed substances be removed as much as possible while ensuring the activity of the whole antigen immobilized on the surface of the chip. At the time of study, reagents as listed in table 2 were selected: 0.1mol/L hydrochloric acid solution, 0.1mol/L hydrochloric acid +0.1% SDS solution, 0.1mol/L hydrochloric acid +0.1% Triton X-100 solution, 0.05mol/L pH2.0 glycine-hydrochloric acid solution, 0.05mol/L NaOH solution was regenerated once; 0.05mol/L NaOH solution regenerated 2 times of antibody removal. 88.73%, 92.54%, 87.75%, 32.28%, 96.36%, 99.59% of the bound antibody (table 2) was removed, respectively.
TABLE 2
Therefore, the optimum regeneration conditions are 0.05mol/L NaOH solution 120 min/regeneration 2 times.
FIG. 7 shows the effect of the regeneration times on the DES-BSA activity of the 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 still can 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 has good repeatability after multiple regenerations.
After regeneration, in order to investigate the influence of nonspecific adsorption on the SPR chip surface, the antibody was replaced with a 12.5. Mu.g/mL BSA solution, and the response value of SPR was monitored without changing other reaction conditions and reaction processes. Repeat 7 times, no change in SPR angle was observed, indicating: the non-specific adsorption on the regenerated chip surface is 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. The magnetic graphene enrichment and SPR indirect competition method is adopted for detection, and the concentration of the magnetic graphene is different (1 multiplied by 10) in high, medium and low within the detection range -4 ng/mL；1×10 -3 ng/mL；1×10 -2 ng/mL), six replicates of each concentration were used to calculate spiked recovery.
The result of the normalized recovery is shown in table 3, the normalized recovery is 94.12% -101.22%, and the Relative Standard Deviation (RSD) is 3.24% -6.59%, which indicates that the method is accurate and reliable, and if the mass concentration of the sample DES to be detected is low, the steps of magnetic graphene enrichment and concentration can be adopted to detect DES residues.
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
(1) Gold tablets were reconstituted with piranha solution (30% H) 2 0 2 :H 2 S0 4 = 1:3) is soaked for 1-2h at room temperature, organic matters on the surface of the gold sheet are removed, then the gold sheet is cleaned by a large amount of deionized water, cleaned by absolute ethyl alcohol, and finally dried by nitrogen.
(2) Filtering 0.01mol/L MPA ethanol solution, placing the solution in a culture dish, immersing the gold sheet in the solution to avoid bubbles on the gold surface, and 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 (5) drying the gold sheet by nitrogen.
(3) 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 charges in 0.1mg/mL graphene oxide solution 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.
Step two: fixing DES-BSA on the surface of gold plate of SPR chip
An SPR chip is loaded into a sensor, acetate buffer solution with pH 4.5 is used as coupling buffer solution, 0.4mol/L EDC and 0.1mol/L NHS are used for activating carboxyl on the surface of the chip for 7min, 120 mu g/mL DES-BSA binding reaction is prepared by using the coupling buffer solution for 20min, ethanolamine with pH8.5 is used as confining liquid for reaction for 10min, and 0.05mol/L NaOH solution is used as regeneration liquid for regeneration for 2min. Repeat 1 time according to the procedure described above. The SPR angle change value after DES-BSA fixation saturation is 625.86m degrees.
Step three: SPR indirect competitive immunoassay
The concentration of the DES-mAb antibody is determined prior to determining the DES content in the sample. As shown in FIG. 9, the SPR angle shift curve generated by binding to DES-BSA on the SPR chip (with graphene modification) was monitored by injecting DES-mAb with different concentrations, and it was found that the binding of the antibody on the chip surface was not saturated when the concentration of DES-mAb antibody was less than 5.00. Mu.g/mL. Therefore, the optimal antibody concentration is 5.00. Mu.g/mL.
Regeneration was performed 2 times with 0.05M NaOH solution, and DES was determined.
In the determination, 10.00 u g/mL DES-mAb and different concentration gradient of DES standard solution were mixed in equal volume, monitoring the response signal, the response signal value is shown in figure 10. As shown in FIG. 11, the LOD (limit of detection) value was 0.001826ng/mL, IC50 was 0.1710ng/mL, and detection range was 0.009924-5.701ng/mL.
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) Gold tablets were reconstituted with piranha solution (30% H) 2 0 2 :H 2 S0 4 = 1:3) soaking at room temperature for 1-2h to remove organic matter on the surface of the gold sheet, and then washing with a large amount of deionized water withoutWashing with water and ethanol, and finally blowing with nitrogen.
(2) Filtering 0.01mol/L MPA ethanol solution, placing the solution in a culture dish, immersing the gold sheet in the solution to avoid bubbles on the gold surface, and 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 (5) drying the gold sheet by nitrogen.
(3) An SPR chip is loaded into a sensor, acetate buffer solution with pH 4.5 is used as coupling buffer solution, 0.4mol/L EDC and 0.1mol/L NHS are used for activating carboxyl on the surface of the chip for 7min, the coupling buffer solution is used for preparing DES-BSA with the concentration of 30 mu g/mL, the binding reaction is carried out for 20min, ethanolamine with pH8.5 is used as blocking solution for reaction for 10min, and 0.05mol/L NaOH solution is used as regeneration solution for regeneration time of 2min. Repeat 1 time following the above procedure.
DES-BSA with different concentrations is fixed on the surface of the SPR chip without graphene modification in advance, and an SPR angular shift curve graph is monitored. As shown in FIG. 6, it was confirmed that the optimal immobilization concentration of DES-BSA was 30. Mu.g/mL, and the amount of immobilization was close to saturation (the SPR response did not change as the concentration of DES-BSA increased).
Step 2: SPR indirect competitive immunoassay
The concentration of the DES-mAb antibody is determined prior to determining the DES content in the sample. As shown in FIG. 12, the injection of DES-mAb with different concentrations was used to monitor the SPR angle shift curve generated by binding to DES-BSA on the SPR chip (without graphene modification), and finally the concentration of DES-mAb antibody was determined to be 2.50. Mu.g/mL.
Regeneration was performed 2 times using 0.05M NaOH solution, and DES was determined.
In the assay, 5.00. Mu.g/mL of DES-mAb was mixed with varying concentration gradients of DES standard solution in equal volumes and the response signal was monitored and the values of the response signal are shown in FIG. 13. As shown in FIG. 14, the LOD (limit of detection) value was 0.01617ng/mL, the IC50 was 0.3240ng/mL, and the detection range was 0.07279-5.302ng/mL. Compared with example 1 and comparative example 1, the LOD of the detection limit of comparative example 1 is 8.8 times that of example 1, and the IC is higher than that of comparative example 1 50 Which 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 the 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 2mL of 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.
The experimental results show that: after the pretreatment step of magnetic graphene enrichment is introduced, DES is detected by using a carboxylated graphene oxide modified surface plasmon resonance biosensor indirect competition method, and a response signal is monitored, wherein the value of the response signal is shown in fig. 15. The results are shown in FIG. 16, where the LOD value is 2.901 × 10 -6 ng/mL，IC 50 Is 4.520 × 10 -4 ng/mL, detection range 1.687 × 10 -5 -1.141×10 -2 ng/mL。
Compared to example 1, example 1 had a LOD value 630 times that of example 2, IC 50 378 times that of example 2.
Comparative example 2
This comparative example uses an indirect competitive enzyme-linked immunosorbent assay (ELISA) method to determine the DES content. DES-BSA was used as the coating antigen, DES-mAb was the primary antibody, igG: HRP is used as a secondary antibody, OVA is used as a confining liquid, and TMB solution is used as a color developing agent. The optimal dilution factor for DES-BSA and DES mAb was 1/8100 (1.130. Mu.g/ml) and 1/8000 (0.5454. Mu.g/ml), respectively.
The results are shown in FIG. 17, where the LOD value is 0.045ng/mL, IC 50 The concentration is 0.172ng/mL, and the detection range is 0.048-0.437ng/mL.
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 as shown in table 4, and the comparison of the experimental results of ELISA and the conventional SPR without graphene modification can be seen: the SPR detection method has lower detection limit and wider detection range than the ELISA method, and has the defect of larger using amount of the antibody. When the surface of the chip is modified by using the 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. According to the invention, after the enrichment step of the pretreatment of the magnetic graphene is further introduced, the detection limit is reduced by more than 5500 times, and the method is particularly suitable for detecting the content of DES in a low-concentration sample.
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 (7)
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
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; the noble metal chip of the SPR chip is a gold chip and comprises the following specific steps:
step 1: by volume ratio of H 2 O 2 :H 2 SO 4 Preparing piranha solution from 1:3, soaking the gold plate of the SPR chip at room temperature to remove organic matters on the surface of the gold plate, cleaning with deionized water and absolute ethyl alcohol, and finally drying with nitrogen; wherein H 2 O 2 Is prepared fromHydrogen peroxide with the content of 30 percent;
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 MPA modified gold sheet with carboxyl into PAH solution, taking out, washing 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: soaking a gold sheet with positive charges in a dispersion liquid of 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;
s3, connecting and fixing DES-BSA by using carboxylated graphene oxide fixed on the surface of the noble metal of the SPR chip;
the method for fixing DES-BSA comprises the following steps: loading an SPR chip and gold sheets into a sensor, activating carboxyl on the surface of the chip for 5-10min by using EDC solution and NHS solution, taking acetate buffer solution with pH =4-5 as coupling buffer solution, preparing DES-BSA with a certain mass concentration by using the coupling buffer solution for reaction for 15-30min, taking ethanolamine as confining liquid for reaction for 5-20min, taking NaOH solution of 0.05mol/L as regeneration liquid for regeneration for 1-3min, and repeating the cycle for 1-3 times in sequence to connect the DES-BSA with the oxidized graphene with carboxyl on the surface of the gold sheet of the SPR chip so as to realize the immobilization of DES-BSA;
s4, determining the concentration of diethylstilbestrol in the sample to be detected by SPR immunosensing based on an indirect competition method: mixing a sample to be detected containing DES and a DES-mAb solution of a monoclonal antibody of DES, injecting a sample, wherein DES in the sample to be detected and DES-BSA fixed on the surface of an SPR chip compete for DES-mAb together, and a signal generated by SPR is inversely proportional to the concentration of DES in the sample to be detected.
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, characterized in that in step S1: the method comprises the steps of adopting magnetic graphene as an adsorbent, carrying out adsorption enrichment on diethylstilbestrol in a sample to be detected under the condition that the pH value is acidic, wherein the adsorption time is 4-10min, adopting acetonitrile as eluent, eluting the diethylstilbestrol adsorbed by the magnetic graphene, drying with nitrogen and fixing the volume to 100-250 mu L.
4. The method of claim 1, wherein in step S3, the concentration of EDC is 0.4mol/L, the concentration of NHS is 0.1mol/L, and the activation time is 7min; the pH of the coupling buffer solution is 4.5; 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 a 0.05mol/L NaOH solution is used as a regeneration solution for regeneration for 2min.
5. The method of claim 1, wherein in step S3, when DES-BSA is immobilized, the DES-BSA concentration is 120 μ g/mL, and the SPR has a response value of 625.86m °.
6. The method according to claim 1, characterized in that, in step S4, the sample to be tested containing DES and the DES-mAb solution are mixed in 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; wherein the DES-mAb concentration of the DES-mAb solution is 10.00. Mu.g/mL, and the DES-mAb concentration in the injection sample is 5.00. Mu.g/mL.
7. The method of claim 1, further comprising step S5, regeneration of SPR: the 0.05mol/L NaOH solution is adopted for 120 min/time and is regenerated for more than 2 times, so that the removal rate of the antibody reaches more than 99.50 percent.
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