CN110987896A

CN110987896A - Trace amoxicillin detection method taking Ag @ Au as SERS substrate

Info

Publication number: CN110987896A
Application number: CN201911095635.3A
Authority: CN
Inventors: 冯军; 徐娅娟; 程昊; 李利军; 黄文艺; 卢浩
Original assignee: Guangxi University of Science and Technology
Current assignee: Guangxi University of Science and Technology
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2020-04-10
Anticipated expiration: 2039-11-11
Also published as: CN110987896B

Abstract

The invention relates to a trace amoxicillin detection method taking Ag @ Au as an SERS substrate, which adopts a simple seed-mediated method for preparing bimetal Ag @ Au nano particles with stable chemical properties, finally converts triangular AgNPs into disc-shaped Ag @ Au nano particles, and then takes the obtained disc-shaped Ag @ Au nano particles as the SERS substrate for trace amoxicillin detection, thereby realizing trace amoxicillin detection. The invention synthesizes the double-metal Ag @ Au nano particles with uniform appearance, uniform particle size and good dispersibility, and the Ag @ Au nano particles have higher sensitivity, better spectral reproducibility and stronger SERS strength. Experimental results show that the substrate is simple in preparation method, good in dispersity and high in sensitivity, can be used for rapidly detecting foods and medicines, and has wide application prospects.

Description

Trace amoxicillin detection method taking Ag @ Au as SERS substrate

Technical Field

The invention relates to a trace substance detection method, in particular to a trace amoxicillin detection method taking Ag @ Au as an SERS substrate.

Background

The amoxicillin detection method comprises the steps of microbial detection, enzyme-linked immunoassay, high performance liquid chromatography and fluorescence detector, ultraviolet detector and mass spectrum detector combination, high performance liquid chromatography-tandem mass spectrometry, wherein the high performance liquid chromatography has low sensitivity, sample treatment needs derivatization or Solid Phase Extraction (SPE) column purification, and the operation is complicated, the high performance liquid chromatography-mass spectrometry combination is mostly used for detecting β -lactam antibiotics with multiple residues, the amoxicillin detection specificity is not strong, and SPE column purification is needed, the cost is high, and other methods have the defects of complicated sample pretreatment, low sensitivity, time consumption and the like.

Surface Enhanced Raman Scattering (SERS) is one of the most promising analytical techniques, and has become an effective tool for molecular or biological molecule detection in the fields of material science, chemistry, environmental science, etc. Metals commonly used as SERS substrates are gold (Au), silver (Ag), copper (Cu), and the like. Among them, Au and Ag are generally used as SERS substrates because they are more stable than Cu under general environmental conditions. However, AgNPs are thermodynamically unstable, are easily oxidized in general, and have poor chemical stability, which is a major reason why it is limited in practical applications. On the other hand, Ag @ Au core-shell type bimetallic nanoparticles are of great interest because of their improved chemical stability. However, the preparation of the bimetallic Ag @ AuNPs core-shell structure with high sensitivity, good uniformity and good stability still needs to be solved urgently.

Chinese patent application 201810611514.9 discloses a method for preparing an Au @ Ag nanoparticle SERS substrate and a method for detecting glucose using the same, which uses Au @ Ag nanoparticles as SERS substrates, but does not use Ag @ Au nanoparticles as SERS substrates, although the method also uses bimetallic nanoparticles as SERS substrates. Therefore, a technical scheme for detecting trace amoxicillin by adopting Ag @ Au as an SERS substrate is not disclosed.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the method synthesizes double-metal Ag @ Au nano particles with uniform appearance and particle size and good dispersity through a seed-mediated growth process, and the Ag @ Au nano particles have high chemical stability as SERS active substrates and show obvious enhancement effect and good reproducibility in amoxicillin detection.

The technical scheme for solving the technical problems is as follows: a trace amoxicillin detection method taking Ag @ Au as an SERS substrate comprises the following steps:

(1) preparation of AgNPs: 0.01M AgNO₃Mixing 4mL of the aqueous solution with 6mL of a 0.1M trisodium citrate solution, adding water to a final volume of 40mL, magnetically stirring the mixture for 10min, and adding 4mL of freshly prepared 100mM NaBH₄A solution; subsequently, 1mL of H₂O₂Quickly injecting into the solution, wherein the color of the reaction mixture is changed into light yellow, red and green within 3-4min, and finally changed into blue, and the final product AgNPs is collected after being centrifuged at 10000rpm for about 30 minutes, and then repeatedly washed by deionized water for later use;

(2) preparation of Ag @ Au nanoparticles: preparing an Ag @ Au core-shell nano compound at room temperature by using AgNPs as template particles and adopting a seed-mediated growth method; that is, 100mg of AA and 66.6mg of PVP were dissolved in 15mL of water under magnetic stirring, then 5mL of AgNPs and 0.5mL of 0.2M aqueous NaOH solution were added to the reaction mixture in order, the pH of the reaction mixture was raised to 11 by adding the NaOH solution, and then 0.1M HAuCl was slowly added₄Changing the color of the mixture solution from dark blue to pink by 100 mu L of the solution to show that Ag @ Au nano particles are formed, centrifuging the solution at 10000rpm for 15min, washing the solution by using deionized water, and re-dispersing the obtained product in the deionized water for later use;

(3) respectively preparing 5-6 amoxicillin standard solutions with different concentration gradients by using hydrochloric acid aqueous solutions, mixing and stirring the standard solutions and the Ag @ Au nanoparticles prepared in the step (2) for 0.5-1 hour, dripping the mixture on a glass slide, and performing Raman signal detection by using a Raman spectrometer to obtain an SERS spectrogram with corresponding concentration;

(4) taking a corresponding SERS substrate to independently perform Raman spectrum test to obtain a substrate background Raman signal;

(5) normalizing the surface enhanced Raman spectrum of the amoxicillin standard solution by using a substrate background Raman signal as an internal standard;

(6) establishing a contrast working curve of the relative intensity-concentration standard of the Raman spectrum line of the amoxicillin standard solution;

(7) detecting the concentration of an unknown sample: preparing a sample to be measured with unknown concentration into a solution by using a hydrochloric acid aqueous solution, mixing and stirring the solution and the Ag @ Au nano particles prepared in the step (2) for 0.5-1h, then testing by using a laser Raman spectrometer, and performing spectral peak intensity normalization treatment to obtain an amoxicillin surface enhanced Raman spectrum with unknown concentration; and calculating the concentration of amoxicillin by a formula.

HAuCl in step (2)₄The addition rate of the solution was 10. mu.L/min.

The concentration change value of 5-6 amoxicillin standard solutions with different concentration gradients is 10^-3mol/L to 10^-11Between mol/L.

The invention adopts a simple method, and synthesizes the bimetal Ag @ Au nano particles with uniform appearance, uniform particle size and good dispersity through a seed-mediated growth process. Compared with AgNPs, the prepared Ag @ Au nano particles have obvious chemical stability, the SERS performance of the Ag @ Au nano particles is greatly improved, and the enhancement factor is as high as 3.9 multiplied by 10⁵. In addition, the Ag @ Au nano particles have higher sensitivity, and the linear range of amoxicillin detection is 10^-3mol/L to 10^-11Between mol/L, the detection limit is 10^-11mol/L, Relative Standard Deviation (RSD) is 6.4%, and the SERS has good spectral reproducibility and strong SERS intensity. At the same time, the sum of the concentration of Amoxicillin (AMC) and 1139cm^-1The characteristic peak intensity is subjected to linear fitting, the linear relation is good, and r is 0.996. Experimental results show that the substrate is simple in preparation method, good in dispersity and high in sensitivity, can be used for rapidly detecting foods and medicines, and has wide application prospects.

The invention adopts a simple seed-mediated method for preparing bimetal Ag @ Au nano particles with stable chemical properties, and finally converts triangular AgNPs into disc-shaped Ag @ Au nano particles. Compared with the prior method for preparing the rod-shaped Ag @ Au nano material (the method disclosed in the patent application No. 201810501598.0), the method is characterized in that: 1. the nano-materials have different structures, appearances and particle sizes: the invention prepares disc-shaped Ag @ Au nano material, while the prior art prepares rod-shaped Au @ Ag nano material; 2. the preparation method of the disc-shaped Ag @ Au nano material is simple and can be finished at room temperature, and the rod-shaped Au @ Ag nano material needs a water bath at 60 ℃ and reaction conditions of about 3 hours; 3. the application fields of the nano materials are different: the rod-shaped Au @ Ag nano material is invented to better understand the influence of a surfactant on overgrowth kinetics and find a proper surfactant for more reasonable molecular design; the disc-shaped Ag @ Au nano material is prepared to be used as a Surface Enhanced Raman Scattering (SERS) substrate and is used for detecting food and medicines.

Compared with the Chinese patent application 201810611514.9, the difference of the invention is that: 1. SERS substrates differ: in the patent application, Au @ Ag nanoparticles are used as an SERS active substrate, while the SERS substrate is Ag @ Au nanoparticles, and the prepared shapes have different particle sizes; 2. the method for preparing Au @ Ag nano particles is complex (such as heating to boiling, drying at 80 ℃, reacting for 12-20 hours and the like), and the method can be completed only under the conditions of normal temperature and stirring; 3. the detection target is different: the invention relates to detection of amoxicillin, which is used for detecting glucose.

Drawings

FIG. 1 is a perspective electron micrograph of AgNPs, Ag @ AuNPs and an EDS map; wherein a is a perspective electron microscope image of AgNPs, b is an EDS image of AgNPs, c is a perspective electron microscope image of Ag @ AuNPs, and d is an EDS image of Ag @ AuNPs.

FIG. 2 is a UV spectrum of AgNP, AuNPs, Ag @ Au-AMC; wherein a is an ultraviolet spectrum of AgNP, b is an ultraviolet spectrum of AuNPs, c is an ultraviolet spectrum of Ag @ AuNPs, and d is an ultraviolet spectrum of Ag @ Au-AMC.

FIG. 3 is SERS spectra of different substrates, wherein (a) is amoxicillin standard; (b) is AcoxibHydrochloric acid aqueous solution of forest (10)^-9moL/L); (c) is Ag-amoxicillin; (d) is Ag @ Au-amoxicillin.

FIG. 4 is SERS graph of AgNPs, Ag @ AuNPs at different time periods, and the bar graph shows (0-3 months) amoxicillin (10 months)^-9mol/L) solution at 1139cm^-1(ii) raman peak at, error bars represent standard deviations of three measurements; wherein a and b are AgNPs, and c and d are Ag @ AuNPs.

FIG. 5(A) is a SERS spectrum of amoxicillin at different concentrations on a substrate of Ag @ AuNPs; (B) is 1139cm^-1And (3) a linear relation graph of SERS spectrum signals and amoxicillin concentration logarithm.

In FIG. 6(A) is eight groups of amoxicillin (10)^-10mol/L) SERS spectra; (B) is eight groups of oxacillin (10)^- ¹⁰mol/L) solution at 1139cm^-1Raman peak at (a), error bars indicate the standard deviation of three measurements.

Detailed Description

Example 1: a trace amoxicillin detection method taking Ag @ Au as an SERS substrate comprises the following steps:

(1) preparation of AgNPs: 0.01M AgNO₃4mL of aqueous (silver nitrate) solution and 6mL of trisodium citrate 0.1M solution were mixed together, water was then added to bring the final volume of the reaction solution to 40mL, the mixture was magnetically stirred for 10min, and then 4mL of freshly prepared 100mM NaBH was added₄(sodium borohydride) solution; subsequently, 1mL of H₂O₂(hydrogen peroxide) is quickly injected into the solution, the color of the reaction mixture is changed into light yellow, red and green within 3-4min, finally the reaction mixture is changed into blue, and the final product AgNPs (nano silver) is collected after being centrifuged at 10000rpm for about 30 minutes and then is repeatedly washed by deionized water for standby;

(2) preparation of Ag @ Au nanoparticles: preparing an Ag @ Au core-shell nano compound at room temperature by using AgNPs as template particles and adopting a seed-mediated growth method; that is, 100mg of AA (ascorbic acid) and 66.6mg of PVP (polyvinylpyrrolidone) were dissolved in 15mL of water under magnetic stirring, and then 5mL of AgNPs and 0.5mL of 0.2M aqueous NaOH (sodium hydroxide) were added to the reaction mixture in this order, and NaOH solution was added to allow the reaction to proceedThe pH of the mixture was raised to 11 and then slowly added to a solution containing 0.1M HAuCl₄Changing the color of the mixture solution from dark blue to pink by 100 mu L of (chloroauric acid) solution to show that Ag @ Au nano particles are formed, centrifuging at 10000rpm for 15min, washing with deionized water, and re-dispersing the obtained product in the deionized water for later use;

In this example, HAuCl was used in the step (2)₄The addition rate of the solution was 10. mu.L/min. In the step (3), the concentration change values of 5-6 amoxicillin standard solutions with different concentration gradients are 10^-3mol/L to 10^-11Between mol/L.

The development process of the invention is as follows.

Preparing a sample and collecting a Raman spectrum.

4mg of amoxicillin was accurately weighed at room temperature and made 10 with aqueous hydrochloric acid (pH 2.0)^-3mol/L and diluting to the required concentration for standby. Respectively sucking 100 mu L of AgNPs into a 1.5mL centrifuge tubeAnd adding Ag @ Au sol into 400 mu L of amoxicillin with different concentrations, performing ultrasonic treatment for 1h, dripping the mixed solution on a quartz glass sheet applied with an external magnetic field, and drying, wherein each sample is subjected to Raman spectrum collection for 3 times. The parameters of the spectra are: the wavelength was 638nm, the integration time was 10s, and the average number of times was 1.

TEM and EDS characterization.

FIGS. 1(a-d) are TEM and EDS images of AgNPs and Ag @ AuNPs, respectively, and triangular AgNPs having an edge length of 20-30nm were synthesized according to the method of example 1 of the present invention (FIG. 1 a). It can be seen from the figure that the prepared AgNPs have good dispersibility and uniform size, only the Ag element spectrum peak appears in the energy spectrum (figure 1b), and other impurity peaks do not appear. The Ag @ Au core-shell nanocomposite was obtained by depositing reduced Au atoms onto triangular Ag NPs seeds and further controlling the oxidative dissolution of the tips using polyvinylpyrrolidone (PVP) as surfactant and AA as reducing agent, as shown in figure (1 c). It can be seen from the figure that the reduced Au nanoparticles are uniformly dotted on the surface of the triangular AgNPs, and as the reaction proceeds, the Ag @ AuNPs (50nm) with the triangular structure gradually evolves into a circular disc-shaped structure. Structures that exhibit strong coupling to analyte molecules due to their relatively large surface area have highly enhanced SERS signals. The pH of the reaction solution was adjusted to 11 by adding NaOH (0.2M) solution, followed by slow addition of HAuCl₄And (3) solution. Relevant studies have shown that the addition of HAuCl at higher pH (pH 11)₄The solution can significantly reduce the reduction potential of gold ions, thereby enabling uniform deposition of Au nanoparticles on Ag seeds. Meanwhile, a spectrum peak of the Au element appears in an energy spectrum (figure 1d), and further proves that the Ag @ AuNPs can be successfully prepared.

And 2, UV characterization.

FIGS. 2(a-d) are Ultraviolet (UV) spectra of AgNP, AuNPs, Ag @ AuNPs, and Ag @ Au-AMC, respectively, and it can be found by comparing curves (2a) and (2c) that the maximum absorption wavelength after the deposition of Au on Ag nanoparticles is blue-shifted (63nm) and is almost close to the maximum absorption wavelength of Au nanoparticle subgraph (2b), indicating that Au nanoparticles are successfully deposited on the surface of Ag particles. As can be seen, the maximum absorption wavelength of (Ag @ Au-AMC) conjugated with amoxicillin is significantly red-shifted (52nm) compared to Ag @ AuNPs. Because amoxicillin has active groups such as hydroxyl, benzene ring and methyl, the groups can be easily adsorbed on the surface of the metal nano-particles. After the amoxicillin conjugate is conjugated with an SERS active substrate (Ag @ AuNPs), the maximum absorption wavelength is obviously red-shifted, and the successful combination of the amoxicillin and the SERS active substrate is proved.

SERS performance study.

3.1 SERS of amoxicillin.

FIG. 3 shows SERS spectra of amoxicillin standards, aqueous hydrochloric acid of amoxicillin, Ag-amoxicillin and Aa @ Au-amoxicillin. As can be seen from FIG. 3a, 902cm^-1The absorption peak is C-C in-plane deformation vibration and O-CO-CH on benzene ring₃The in-plane deformation vibration of; 1107cm^-1Is O-CO-CH₃Asymmetric stretching vibration and CH₃The asymmetric in-plane deformation vibration of; 1139cm^-1Is the in-plane deformation vibration of CH on the benzene ring; 1205cm^-1Is Ph-OCOCH₃Stretching vibration and in-plane deformation vibration of CH on the benzene ring; 1301cm^-1In-plane deformation vibration of OH and CH stretching vibration on a benzene ring; 1474cm^-1Is an in-plane deformation vibration of a stretching vibration CH on the C-C benzene ring; comparing the curves b and c, the AgNPs serving as the SERS active substrate can cause stretching vibration of certain functional groups of amoxicillin molecules, and the stretching vibration is obviously enhanced by 902cm and 1139cm^-1The absorption peak at (c). However, it can be seen from the curves c and d that the enhancement effect of Ag @ AuNPs as the SERS active substrate graph (3d) is significantly higher than that of the AgNPs graph (3c) as the SERS active substrate. Matrix-based plasmonic nanostructure assembly helps to create uniform and more intense "hot spots" that can enhance the raman signal of an analyte. And the defects that AgNPs are easy to agglomerate and oxidize when being used as an SERS active substrate are overcome, so that the Ag @ AuNPs can be used as an ideal SERS active substrate.

3.2Ag and Ag @ Au stability investigation.

In order to examine the stability of different substrates, newly prepared AgNPs and Ag @ AuNPs active substrates are stored, and amoxicillin optical SERS under the AgNPs and Ag @ AuNPs active substrates is detected by selecting different time periods (newly prepared, 1 month, 2 months and 3 months)Spectra (4a, 4c) are respectively 4 groups of amoxicillin SERS spectrograms obtained by AgNPs and Ag @ AuNPs substrate test, and the spectra are 1139cm^-1A histogram of the intensity values of the SERS signal at the location is shown in the figures (4b, 4 d). The relative standard deviation of the SERS signal intensity in 0, 1, 2 and 3 months is 4.8% and 3.9%, respectively. The uniformity is good, the stability is good, however, the characteristic peak intensity of the substrate Ag @ AuNPs is not changed greatly with the passage of time, the SERS signal of the substrate AgNPs in 3 months is reduced obviously, because the silver ions are easy to be oxidized and easy to agglomerate, and the further agglomeration and oxidation of silver are prevented after the gold nanoparticles are deposited outside, so that the stability of the substrate is improved obviously, compared with the substrate AgNPs, the stability of the substrate Ag @ AuNPs is improved obviously.

3.3 sensitivity investigation.

FIG. 5(A) shows different concentrations (10)^-3～10^-11mol/L) of amoxicillin on an Ag @ AuNPs substrate, and from the graph, 1139cm of SERS spectrogram can be seen along with the reduction of the concentration of amoxicillin^-1The intensity of the characteristic peak at (a) is gradually reduced. The reason is that as the concentration of the amoxicillin solution is reduced, the amoxicillin molecules adsorbed by the Ag @ AuNPs are reduced, and the amoxicillin molecules irradiated by laser in a unit area are gradually reduced, so that the signals of Raman spectra are gradually weakened. When the amoxicillin is 10^-11The intensity of the characteristic peak at mol/L is extremely weak, and thus 10 can be determined^-11And mol/L is the detection limit of amoxicillin. Meanwhile, the spectrum of the amoxicillin SERS is 1139cm^-1The peak intensity and its concentration (10)^-3～10^-11mol/L) was fitted linearly to fig. 5(B), and a good linear relationship was found between the two, with the linear equation y being 414.8x +5319.6 and r being 0.996. Thus, 1139cm was selected^-1The peak at (B) is used as an index for amoxicillin quantitative analysis. Next, the SERS Enhancement Factor (EF) pair was calculated by 1139cm^-1The SERS signal is quantified by the Raman peak, and the calculation formula of the Raman enhancement factor EF is as follows:

wherein I_ers-characteristic peaks of amoxicillin molecules absorbed on the Ag @ AuNPs substrate;

I_bulk-common raman intensity of amoxicillin molecules;

N_ersthe number of amoxicillin molecules irradiated by laser on the Ag @ AuNPs substrate;

N_bulkthe number of molecules of amoxicillin molecules irradiated with laser light.

The invention is as follows 10^-31139cm at mol/L concentration^-1The intensity of the Raman peak at (A) is taken as the I value,

at 10³Substituting into formula to calculate the enhancement factor of Ag @ AuNPs basal amoxicillin to be 3.9 multiplied by 10⁵。

3.4 Spectrum reproducibility study.

In order to examine the reproducibility of the amoxicillin SERS spectrum, eight groups of amoxicillin solutions (10) were prepared in parallel^- ¹⁰mol/L) were subjected to SERS detection (FIG. 6(A)), and it can be seen that amoxicillin of the eight groups was 1139cm^-1The intensity of SERS peaks is not very different and almost the same, and 1139cm is calculated^-1(fig. 6B) Relative Standard Deviation (RSD) of peak intensities to evaluate SERS reproducibility of the substrate. RSD ═ SD/I_mWhere SD is the standard deviation of the peak intensity, I_mIs the average raman peak intensity of the most dominant peak. The results show that: the relative standard deviation of the amoxicillin solutions prepared in parallel in the eight groups was 6.4%, while the relative standard deviation with silver as the SERS-active substrate was 13.2%. The Ag @ AuNPs serving as the SERS active substrate has good reproducibility.

Claims

1. A trace amoxicillin detection method taking Ag @ Au as an SERS substrate is characterized in that: the method comprises the following steps:

(1) preparation of AgNPs: 0.01M AgNO₃Mixing 4mL of the aqueous solution with 6mL of 0.1M trisodium citrate solution, adding water to make the final volume of the reaction solution reach 40mL, magnetically stirring the mixture for 10min, and adding 4mL of fresh sodium citratePrepared 100mM NaBH₄A solution; subsequently, 1mL of H₂O₂Quickly injecting into the solution, wherein the color of the reaction mixture is changed into light yellow, red and green within 3-4min, and finally changed into blue, and the final product AgNPs is collected after being centrifuged at 10000rpm for about 30 minutes, and then repeatedly washed by deionized water for later use;

(2) preparation of Ag @ Au nanoparticles: preparing an Ag @ Au core-shell nano compound at room temperature by using AgNPs as template particles and adopting a seed-mediated growth method; that is, 100mg of AA and 66.6mg of PVP were dissolved in 15mL of water under magnetic stirring, then 5mL of AgNPs and 0.5mL of 0.2M aqueous NaOH solution were added to the reaction mixture in order, the pH of the reaction mixture was raised to 11 by adding the NaOH solution, and then 0.1M HAuCl was slowly added₄Changing the color of the mixture solution from dark blue to pink by 100 muL of the solution to show that Ag @ Au nano particles are formed, centrifuging the solution at 10000rpm for 15min, washing the solution by using deionized water, and re-dispersing the obtained product in the deionized water for later use;

2. The method for detecting trace amoxicillin by taking Ag @ Au as the SERS substrate as claimed in claim 1, characterized in that: HAuCl in step (2)₄The addition rate of the solution was 10 μ L/min.

3. The method for detecting trace amoxicillin by taking Ag @ Au as the SERS substrate according to claim 1 or 2, characterized in that: the concentration change value of 5-6 amoxicillin standard solutions with different concentration gradients is 10^-3mol/L to 10^-11Between mol/L.