CN110006722B - Preparation method and application of ferroferric oxide/gold structure nanoparticles - Google Patents
Preparation method and application of ferroferric oxide/gold structure nanoparticles Download PDFInfo
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Abstract
The invention discloses a preparation method and application of ferroferric oxide/gold structure nanoparticles. According to the ferroferric oxide/gold structure nano-particle, 3',5,5' -Tetramethylbenzidine (TMB) is selected as a Raman reporter molecule and is combined on the surface of gold through a gold-sulfur (Au-S) covalent bond to generate Surface Enhanced Raman Spectroscopy (SERS). Due to the catalytic action of ferroferric oxide on particles, 3',5,5' -tetramethylbenzidine can be rapidly oxidized by hydrogen peroxide in detection, and the change of SERS signals before and after oxidation of 3,3',5,5' -tetramethylbenzidine molecules can realize H2O2Direct measurement of concentration and indirect measurement of glucose concentration. The method has the advantages of high sensitivity, strong specificity, no interference of other components in the sample during detection, no need of pretreatment and simpler operation steps. Furthermore, the method is further upgraded and can be applied to detecting H in single cells2O2And (4) concentration.
Description
Technical Field
The invention relates to the technical field of optical measuring instruments, and relates to a preparation method and application of ferroferric oxide/gold structure nanoparticles.
Background
The advent of nanoscience and the development of nanotechnology provide new opportunities for the design and construction of nanomaterial-based mimic enzymes. Because the mimic enzyme has the activity of natural enzyme and is easy to be usedThe preparation and purification of the mimic enzyme have more tenacious resistance to temperature, acidity, inhibitors and the like, and have the advantages of high efficiency, strong specificity and low cost, so that the mimic enzyme becomes the focus of research of scientists in recent years. Fe3O4The nanoparticles have similar enzymatic activity to native horseradish peroxidase, such that they are Fe-based3O4Nanoparticle mimetic enzymes have become a hot area of scientific research.
Raman spectroscopy is the "fingerprint" spectrum of a substance, making detection based on raman spectroscopy generally have excellent specificity; when the raman-active molecule is on the surface of a noble metal, the intensity of its raman signal can be increased by many orders of magnitude, which is Surface Enhanced Raman Spectroscopy (SERS). The SERS signal intensity is high, so that the detection based on SERS has high sensitivity, which is a unique advantage of SERS compared with the traditional detection method. The detection technology based on SERS can even realize single molecule detection of substances, so the method is widely applied to the fields of chemistry, materials, biomedicine and the like.
Conventional methods for detecting hydrogen peroxide and glucose concentrations include electrochemical methods, spectrophotometry, fluorimetry, and the like. In 2018, the Xie topic group adopts a cobalt nitride nano array as an electrochemical sensor to detect hydrogen peroxide and glucose, and H is detected2O2The detection limit of (2) reaches 1. mu.M, and the detection limit of glucose reaches 50nM, but the method is susceptible to interference by proteins in blood, blood color, and electrode contamination. In 2017, Chamaraja et al used sulfosalicylic acid as a chromogenic probe and used spectrophotometry to measure hydrogen peroxide for H2O2The detection limit of (2) reached 73 nM. However, the method is easily interfered by other colored components, and the purity of the measured object is required to be higher; in 2018, the Liu project group developed a fluorescent probe based on iminocoumarin derivatives using aromatic boronates as sensing units for H2O2The detection limit of (2) reached 60 nM. While fluorometry can overcome the problems of the two previous methods, it is susceptible to interference from background autofluorescence. In view of the above disadvantages, it is necessary to develop a method for rapidly and highly sensitively detecting hydrogen peroxide concentrationAnd (4) a method of measuring.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention develops a method for quickly detecting the concentrations of hydrogen peroxide and glucose by SERS (surface enhanced Raman scattering) ultrasensitivity based on ferroferric oxide/gold structure nanoparticles. The particle size of the ferroferric oxide/gold structure nano aged developed by the invention is between 30nm and 60nm, the particle size of gold nanoparticles loaded on the surface of the ferroferric oxide through physical adsorption is between 10nm and 15nm, and the distance between adjacent gold nanoparticles on the surface of the same ferroferric oxide particle is between 0nm and 10 nm.
Under physiological conditions, H2O2 is a half-life molecule, the concentration is usually low, the invention utilizes the catalytic property of Fe3O4 mimic enzyme to realize the rapid detection of the concentration of hydrogen peroxide, and the detection time is between 5min and 20 min.
By utilizing the SERS technology based on the nano gold particles, H pair is realized2O2And high-sensitivity detection of glucose concentration. 3,3',5,5' -Tetramethylbenzidine (TMB) was chosen as the Raman reporter molecule, which was covalently bound to the gold surface via gold-sulfur (Au-S) bonds to generate Surface Enhanced Raman Spectroscopy (SERS). Due to the catalytic action of ferroferric oxide on particles, 3',5,5' -tetramethylbenzidine can be rapidly oxidized by hydrogen peroxide in detection, and the change of SERS signals before and after oxidation of 3,3',5,5' -tetramethylbenzidine molecules can realize H2O2The direct detection of concentration and the indirect detection of glucose concentration have the advantages that as ferroferric oxide on the particles is a magnetic material, the particles are gathered to generate a coupling effect under the action of an external magnetic field, and a stronger local electric field hot point is formed, so that the sensitivity of SERS detection is further improved, and the detection limit can reach 10-9An order of magnitude.
Raman spectra are "fingerprint" spectra of matter, and TMB produces a wavenumber of 1195cm-1Under the catalysis of ferroferric oxide, hydrogen peroxide and 3,3',5,5' -tetramethyl benzidine undergo redox reaction to generate oxidized 3,3',5,5' -tetramethyl benzidine (TMB), the wave number is 1195cm through SERS detection-1The intensity of Raman characteristic peak at the position is reduced, and the wave number is 685cm-1A new Raman characteristic peak can be generated, and hydrogen peroxide detection based on Raman spectrum has excellent specificity according to the change of SERS signal intensity before and after oxidation of 3,3',5,5' -tetramethyl benzidine molecules.
In addition, during detection, under the action of a magnetic field, the ferroferric oxide/gold structure nano-sized particle can be separated from a complex sample for SERS detection, so that interference of other components in the sample is avoided.
The detection particles designed and prepared by the invention do not need pretreatment, and the operation steps are simpler. Furthermore, the method is further upgraded and can be applied to detecting H in single cells2O2And (4) concentration.
The technical scheme adopted by the invention is as follows:
the preparation method of the ferroferric oxide/gold structure nano particles comprises the following specific steps:
(1)Fe3O4preparation of nanoparticle solution:
sequentially adding a polyethyleneimine solution, a ferrous sulfate solution, a potassium nitrate solution and a sodium hydroxide solution in a nitrogen-introduced water bath environment, reacting for 1-3h, settling the particles by using a magnet, and washing by using deionized water to obtain Fe3O4Nanoparticle solution of said Fe3O4The particle size of the nano particles is between 30nm and 60 nm;
(2) preparing a gold nano seed solution:
preparing a mixed solution A of sodium borohydride and sodium citrate; preparing a chloroauric acid solution with a certain concentration, adding a sodium citrate solution under magnetic stirring, adding a mixed solution A, reacting for a period of time to obtain a gold nano-seed solution, wherein the particle size of the gold nano-seeds is 3-5 nm;
(3) preparing ferroferric oxide/gold nanoparticles:
fe prepared in the step (1)3O4Mixing the nano-particle solution with the gold nano-seed solution prepared in the step (2), fully stirring, reacting for 1-3h, performing magnetic separation, washing with deionized water to remove excessive gold nano-seeds, adding a polyethyleneimine solution, reacting for 0.5-2h at 55-65 ℃,preparing ferroferric oxide/gold nanoparticles, washing the ferroferric oxide/gold nanoparticles for 1-2 times by using deionized water, wherein the maximum gap between gold nanoparticles on the surface of the same ferroferric oxide particle is 27 nm;
(4) once growing of ferroferric oxide/gold nanoparticles:
uniformly mixing a hexadecyl trimethyl ammonium chloride solution, a chloroauric acid solution, a potassium bromide solution and an AA solution to prepare a gold growth solution; adding a hexadecyl trimethyl ammonium chloride solution into the ferroferric oxide/gold seed nano particle solution prepared in the step (3), uniformly stirring, adding a gold growth solution, and reacting for 15-20 min;
(5) and (2) repeatedly regrowing ferroferric oxide/gold nanoparticles:
adding chloroauric acid solution again into the reaction solution in the step (4), then adding AA solution, reacting for 15-20min, and growing gold particles again; and repeating the step of growing again for 2-5 times generally until a coupled LSPR peak is generated in the scattering spectrum of the ferroferric oxide/gold nanoparticles, washing the coupled LSPR peak for 1-2 times by using deionized water for later use, wherein the particle size of the repeatedly regrown ferroferric oxide/gold nanoparticles is 40-75 nm.
In the step (1), the water bath heating temperature is 80-100 ℃, and the molar ratio of the polyethyleneimine, the ferrous sulfate heptahydrate, the potassium nitrate and the sodium hydroxide is 1:115:500: 250.
In the step (2), preparing a mixed solution A containing 0.075% by mass of sodium borohydride and sodium citrate; and (3) mixing the chloroauric acid aqueous solution and the sodium citrate solution according to the mass concentration of 1:2 under magnetic stirring, and adding the solution A to prepare the gold nano seed solution.
In the step (3), mixing the ferroferric oxide nano particle solution and the gold nano seed solution according to the volume ratio of 1:45, fully shaking and uniformly mixing, reacting for 2 hours, washing for 3-5 times by using deionized water, adding deionized water with the same volume as the gold nano seed solution, fully shaking and mixing, adding 0-4g/L polyethyleneimine solution, reacting for 1 hour at 55-65 ℃, and if aggregation occurs during the reaction, performing ultrasonic dispersion, and washing for 3-5 times by using deionized water to obtain the ferroferric oxide/gold nano particles.
In the step (4), preparing a gold growth solution according to the mass ratio of the hexadecyl trimethyl ammonium chloride, the chloroauric acid, the potassium bromide and the AA of 5000:1.25:1:20, and fully shaking up; and (4) adding a hexadecyl trimethyl ammonium chloride solution with the same amount of substances into the ferroferric oxide/gold seed nano particle solution prepared in the step (3), adding the gold growth solution into the ferroferric oxide/gold seed nano particle solution, and performing ultrasonic treatment for 3min to fully react.
In the step (5), adding a chloroauric acid solution with the same volume as the chloroauric acid solution in the step (4) into the product obtained in the step (4), adding an AA solution with the same volume as the AA solution in the step (4), performing ultrasonic treatment for 1min, and fully reacting.
The invention provides an ultra-sensitive detection H by SERS (surface enhanced Raman scattering) of ferroferric oxide/gold structure nanoparticles2O2And (3) concentration method.
The application uses ferroferric oxide/gold structure nano particles as probes and 3,3',5,5' -tetramethyl benzidine as signal molecules, and the specific method is as follows:
(1) functionalization of ferroferric oxide/gold nanoparticle probes:
preparing 3,3',5,5' -tetramethylbenzidine aqueous solution with the substance quantity concentration of 80 mu mol/mL, adding ferroferric oxide/gold structure nanoparticle solution into the solution, wherein the volume ratio of the ferroferric oxide/gold structure nanoparticle solution to the 3,3',5,5' -tetramethylbenzidine solution is 100:1, reacting at normal temperature for 40-60min, separating by using a magnet, washing twice by using deionized water, and adding deionized water for heavy suspension to obtain the SERS probe;
(2) SERS probe for detecting H with different concentrations2O2Solution:
configuring a certain concentration gradient (1-10)-9mol/L)H2O2And (3) adding 100 mu L of SERS probe prepared in the step (1) into the solution, reacting for 5-20min, magnetically sucking the probe, and dropwise adding the probe into an analytically pure aluminum sheet for Raman testing. Wherein the wavelength of the Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
The invention provides a method for detecting glucose concentration by SERS (surface enhanced Raman scattering) of ferroferric oxide/gold structure nanoparticles.
The application uses ferroferric oxide/gold structure nano particles as probes and 3,3',5,5' -tetramethyl benzidine as signal molecules, and the specific method is as follows:
(1) functionalization of ferroferric oxide/gold nanoparticle probes:
preparing 3,3',5,5' -tetramethylbenzidine aqueous solution with the substance quantity concentration of 80 mu mol/mL, adding ferroferric oxide/gold structure nanoparticle solution into the solution, wherein the volume ratio of the ferroferric oxide/gold structure nanoparticle solution to the 3,3',5,5' -tetramethylbenzidine solution is 100:1, reacting at normal temperature for 40-60min, separating by using a magnet, washing twice by using deionized water, and adding deionized water for heavy suspension to obtain the SERS probe;
(2) enzyme treatment of glucose solutions of different concentrations:
configuring a certain concentration gradient (1-10)-9mol/L), adding 20 mu L of 20mg/mL glucose oxidase GOx solution into 1mL of glucose solution with different concentrations, and carrying out water bath reaction at 37.5 ℃ for 30 min;
(3) SERS detects glucose solutions of different concentrations:
and adding 100 mu L of the SERS probe into glucose solutions with different concentrations, reacting for 5-20min, magnetically absorbing the probe, and dropwise adding the probe onto an analytically pure aluminum sheet for Raman testing. Wherein the wavelength of the Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
The invention has the following positive effects:
the detection probe developed by the invention takes 3,3',5,5' -tetramethyl benzidine as a signal molecule and ferroferric oxide as a catalyst to catalyze the reaction of oxidizing 3,3',5,5' -tetramethyl benzidine by hydrogen peroxide, and on the other hand, Fe is generated when a magnetic field is applied3O4The Au-structured nanoparticles can be gathered to generate coupling, so that more SERS hot spots are formed, and detection of hydrogen peroxide with lower concentration is facilitated. Therefore, the method developed by the invention has high sensitivity and strong specificity, can avoid the interference of other components in the sample during detection, does not need pretreatment, and has simpler operation steps. Furthermore, the method is further upgraded and can be applied toDetection of H within a single cell2O2And glucose concentration.
Drawings
FIG. 1 is Fe of the present invention3O4-Au structural nanoparticle pairs H2O2And SERS ultrasensitive detection of glucose concentration.
Nanoparticles, Fe, prepared according to the invention3O4As catalyst, Fe3O4Surface AuseedsThe array may create "hot spots" to strengthen the substrate. With Fe3O4-AuseedsGold particle-based, Au, on the surface of the compositeseedsAnd the nano-particles continue to grow on the ferric oxide cores, so that LSPR peaks of the nano-particles generate red shift, and the Raman excitation is facilitated. The method takes 3,3',5,5' -tetramethylbenzidine molecules as reporter molecules, the reporter molecules are bonded on the surface of gold through Au-S bonds, then hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine undergo redox reaction under the catalysis of ferroferric oxide to generate oxidized 3,3',5,5' -Tetramethylbenzidine (TMB), new Raman characteristic peaks are generated through SERS detection, and the higher the concentration of hydrogen peroxide is, the more 3,3',5,5' -tetramethylbenzidine is oxidized into oxidized 3,3',5,5' -Tetramethylbenzidine (TMB), and the higher the intensity of the generated new Raman characteristic peaks is. Therefore, according to the change of the SERS signal intensity before and after oxidation of the 3,3',5,5' -tetramethylbenzidine molecule, on one hand, H can be detected2O2Sensitive detection of concentration; on the other hand, in the presence of glucose oxidase (GOx), sensitive detection of glucose concentration can be indirectly achieved.
FIG. 2 is Fe of the present invention3O4-aggregation of Au structured nanoparticles under the action of an external magnetic field.
Under the action of no magnetic field, Fe3O4-Au-TMB is a uniformly dispersed colloidal solution; under the action of an external magnetic field, Fe3O4And reversible aggregation is carried out on the Au-TMB nano particles, and local fields among the particles are coupled, so that a 'hot spot effect' is generated, more SERS hot spots are formed, and detection of hydrogen peroxide with lower concentration is facilitated. The method reported herein detects more than previous fluorometryThe sensitivity is improved by about 1 order of magnitude.
FIG. 3 shows Fe synthesized in example 1 of the present invention3O4Au and Fe3O4Au nanoparticle Transmission Electron Microscopy (TEM): (a) fe3O4(M) TEM image of nanoparticles: synthetic Fe3O4The nano-particles have uniform granularity and are Fe3O4The diameter of the nano particles is mostly concentrated between 38nm and 42 nm; (b) TEM image of Au nanoparticles: au coatingseedsThe initial particle size is mostly distributed between 3nm and 4 nm; (c) fe3O4-AuseedsTEM images of nanoparticles: fe3O4After the nano particles are modified by PEI, the nano particles are reacted with AuseedsCoupling reaction is carried out to generate Fe3O4-AuseedsThe surface of the ferroferric oxide particle is small Au as shown in the figureseedsThe particle size is relatively sparse, and the maximum gap between the gold nanoparticles on the surface of the same ferroferric oxide particle is 27 nm; (d) fe3O4TEM images of Au nanoparticles plus 1-fold gold growth solution: after 1 st gold generation, Fe3O4Surface Au of nano-particlesseedsThe size of the gold nanoparticles is increased and the density of the gold nanoparticles is changed, the maximum gap between the gold nanoparticles on the surface of the same ferroferric oxide particle is 10nm, and at the moment, Au is coated on the gold nanoparticlesseedsThe particle sizes of the particles are intensively distributed between 6 nm and 7nm, and the sizes are uniform; (e) fe3O4TEM images of Au nanoparticles plus 2 times gold growth fluid: after the second gold plating, Au is coated on the surface of the ferroferric oxide nano particlesseedsFurther increase of dense, AuseedsThe particle size is intensively distributed between 8.5 nm and 10nm, and the size is uniform; (f) fe3O4TEM images of Au nanoparticles plus 3 times gold growth fluid: au on the surface of ferroferric oxide after 3 rd secondary goldseedsThe particles further grow but now are not uniform in size and the maximum gold particle size has reached about 15 nm.
FIG. 4 shows Fe synthesized in example 1 of the present invention3O4Au and Fe3O4-normalized UV-vis extinction spectrum of Au nanoparticle solution. Wherein M represents Fe3O4A nanoparticle solution; au coatingseedsRefers to Au nanoparticles; m +AuseedsIs Fe3O4-AuseedsA nanoparticle; m + Auseeds+1 means Fe3O4-Au nanoparticles plus 1 gold growth solution; m + Auseeds+2 means Fe3O4-AuseedsAdding 2 times of gold growth solution into the nanoparticles; m + Auseeds+3 means Fe3O4-AuseedsAdding 3 times of gold growth solution into the nanoparticles.
M+AuseedsCurve is Fe3O4Particles and AuseedsLinear summation of particle solution curves. This is because of Fe3O4Au on the surface of nanoparticlesseedsVery sparse distribution, AuseedsThe space between the two plates is large, and the coupling effect cannot be generated. After 1 time of adding the crude gold liquid, the extinction spectrum of the particle solution is obviously red-shifted. This is because of Fe3O4Particle surface AuseedsIncreased diameter, increased particles, AuseedsWith a gradually decreasing distance therebetween, AuseedsAu on the surface of ferroferric oxide by mutual couplingseedsThe array forms a new shell-like structure of surface plasmon modes. After adding the crude gold liquid for another 1 time, the extinction spectrum is further red-shifted. This is because of Fe3O4Particle surface AuseedsFurther increasing the densification, the surface plasma oscillation mode of the shell-like structure is further enhanced. After the 3 rd addition of the crude gold liquid, the extinction spectrum of the solution is red-shifted, but the width of the spectrum peak is increased. This is because Fe is added after the 3 rd addition of the crude gold liquid3O4Particle surface AuseedsThe particle diameters of the particles are uneven, partial particles even form an incomplete shell, the plasma modes on the particle surfaces become complex and various, and the peak width is just the effect of multi-mode superposition.
Detailed Description
The following examples are further detailed descriptions of the present invention in conjunction with the accompanying drawings. The specific measures described are merely illustrative of the invention and are not intended to limit the invention.
FIG. 1 is Fe of the present invention3O4-Au structural nanoparticle pairs H2O2And SERS ultrasensitive detection of glucose concentration.Fe3O4As catalyst, Fe3O4Surface AuseedsThe array may create "hot spots" to strengthen the substrate. With Fe3O4-AuseedsGold particle-based, Au, on the surface of the compositeseedsAnd the nano-particles continue to grow on the ferric oxide cores, so that LSPR peaks of the nano-particles generate red shift, and the Raman excitation is facilitated. 3,3',5,5' -tetramethyl benzidine molecules are used as reporter molecules, hydrogen peroxide and 3,3',5,5' -tetramethyl benzidine are subjected to redox reaction, and detection of hydrogen peroxide concentration is realized according to changes of SERS signal strength before and after oxidation of the 3,3',5,5' -tetramethyl benzidine molecules.
FIG. 2 is Fe of the present invention3O4-aggregation of Au structured nanoparticles under the action of an external magnetic field. Under the action of no magnetic field, Fe3O4-Au-TMB is a uniformly dispersed colloidal solution; under the action of an external magnetic field, Fe3O4And reversible aggregation is carried out on the Au-TMB nano particles, and local fields among the particles are coupled, so that a 'hot spot effect' is generated, more SERS hot spots are formed, and detection of hydrogen peroxide with lower concentration is facilitated. The method reported herein provides an increase in detection sensitivity of about 1 order of magnitude over previous fluorometry methods.
Example 1
A preparation method of ferroferric oxide/gold structure nanoparticles comprises the following steps:
step one, Fe3O4Preparation of nanoparticles 65mL of water was added to a 250mL three-necked flask and heated to 90 ℃ in a water bath under an oxygen-free atmosphere with nitrogen. 1.280g of ferrous sulfate heptahydrate is weighed and dissolved in 10mL of deionized water to prepare a ferrous sulfate aqueous solution, 5mL of polyethyleneimine (with the relative molecular mass of 10000 and the concentration of 80mg/mL) (the final concentration of the polyethyleneimine is 4g/L), 10mL of the ferrous sulfate aqueous solution, 10mL of a potassium nitrate solution with the concentration of 2.0M and 10mL of a sodium hydroxide solution with the concentration of 1.0M are sequentially added into a three-neck flask, and the mixture is kept for 2 hours. The particles were settled with a magnet and washed 5 times with deionized water. Adding 80mL of deionized water for re-suspension, and then adding the Fe3O4The majority of the nanoparticle diameters are concentrated between 38nm and 42 nm.
Step two, preparing a gold nano seed solution: 1mL of 1% chloroauric acid (25mM) was added to 90mL of water, and after stirring for 1min, 2mL of 38.8mM sodium citrate aqueous solution was added, and after stirring for 1min, 1mL of 38.8mM sodium citrate aqueous solution containing sodium borohydride (0.075%) (75mg) was added. Stirring for 5min, storing at 4 deg.C in dark place, and the particle size of the gold seed particles is distributed between 3-4 nm.
Step three, taking 200. mu.L of the resuspended product of the step one, and adding the product into 9mL of the gold seed solution. Fully shaking and mixing uniformly, then carrying out intermittent ultrasonic treatment for 2min every 10min, and reacting for 2 h. And 5 times of deionized water washing. Then, 9mL of water was added for resuspension, 1mL of 80mg/mL of an aqueous PEI solution was added, and the reaction was carried out at 60 ℃ for 1 hour, during which time aggregation occurred, and dispersion was carried out by sonication. Deionized water was added 2 times and resuspended by adding 2.5mL of deionized water.
Step four, once growing of ferroferric oxide/gold nanoparticles: preparing a gold growth solution: and (3) taking a 5mL test tube, adding 2.5mL of 0.2M hexadecyltrimethylammonium chloride solution with concentration, 125 μ L of 10mM chloroauric acid solution with concentration, 10 μ L of 0.01M potassium bromide solution with degree, 2.265mL of water and 50 μ L of AA (0.04M) solution in sequence, stirring uniformly to prepare a gold growth solution, adding 2.5mL of 0.2M hexadecyltrimethylammonium chloride solution into the product prepared in the step three, adding the gold growth solution, fully shaking uniformly, and reacting for 15 min. At this time, Fe3O4Surface Au of nano-particlesseedsThe particle size of the ferroferric oxide particles is intensively distributed between 6 nm and 7nm, and the maximum gap between the gold nanoparticles on the surface of the same ferroferric oxide particle is 27 nm;
step five, repeatedly regrowing ferroferric oxide/gold nanoparticles: adding 125 μ L of chloroauric acid solution with the concentration of 10mM and 50 μ L of AA solution with the concentration of 0.04M into the product obtained in the fourth step, fully shaking up, performing ultrasonic treatment for 1min, reacting for 15min, and performing iterative growth; repeating the growth step for 1 time again, generating a coupled LSPR peak in the scattering spectrum of the ferroferric oxide/gold nanoparticles, and Au on the surface of the ferroferric oxide nanoparticlesseedsThe particle size is intensively distributed between 8.5-10nm, the size is uniform, the mixture is washed once by deionized water, and 2.5mL of deionized water is added for resuspension.
Seventhly, functionalizing the ferroferric oxide/gold nanoparticle probe: preparing a substance with the mass concentration of 80 mu mol/mL of 3,3',5,5' -tetramethylbenzidine aqueous solution, taking 1mL of ferroferric oxide/gold nano particles iterated twice, adding 10 mu L of 3,3',5,5' -tetramethylbenzidine solution with the mass concentration of 80 mu mol/mL, and reacting at normal temperature for 40 min. Magnet separation, deionized water washing 2 times, adding 1mL deionized water heavy suspension. Taking a small amount of functionalized ferroferric oxide/gold nanoparticles, and testing the Raman spectrum.
FIG. 3 (a) is Fe prepared in the first step of example 13O4(M) TEM image of nanoparticles, Fe3O4The nanometer particles have uniform granularity, and most of the diameters are concentrated between 38nm and 42 nm; FIG. 3 (b) shows Au prepared in step two of example 1seedsTEM image of nanoparticles, AuseedsThe particle size of the nano particles is uniform, and the particle size is mostly distributed between 3nm and 4 nm; FIG. 3 (c) shows Fe prepared in step three of example 13O4-AuseedsTEM image of nanoparticles with small Au on the surface of large ferroferric oxide particlesseedsThe particles are distributed sparsely, and the maximum gap between the gold nanoparticles on the surface of the same ferroferric oxide particle is 27 nm; FIG. 3 (d) is Fe prepared in step four of example 13O4TEM image of Au nanoparticles plus 1 XAu growth fluid, Fe3O4Surface Au of nano-particlesseedsThe size of the gold nanoparticles is increased and the density of the gold nanoparticles is changed, the maximum gap between the gold nanoparticles on the surface of the same ferroferric oxide particle is 10nm, and at the moment, Au is coated on the gold nanoparticlesseedsThe particle sizes of the particles are intensively distributed between 6 nm and 7nm, and the sizes are uniform; FIG. 3 (e) is Fe prepared in step five of example 13O4TEM image of Au nanoparticles plus 2 gold growth liquids, Au on the surface of ferroferric oxide nanoparticlesseedsFurther increase of dense, AuseedsThe particle size is intensively distributed between 8.5 nm and 10nm, and the size is uniform; FIG. 3 (f) is Fe prepared in step five of example 13O4TEM image of Au nanoparticles plus 3 gold growth liquids, Au on ferroferric oxide surfaceseedsThe particles further grow but now are not uniform in size and the maximum gold particle size has reached about 15 nm.
FIG. 4 is the synthesis of Fe in example 13O4Au and Fe3O4-normalized UV-vis extinction spectrum of Au nanoparticle solution. Wherein M represents Fe3O4UV-vis extinction spectrogram of the nanoparticle solution; au coatingseedsRefers to the UV-vis extinction spectrogram of the Au nanoparticle; m + AuseedsIs Fe3O4-AuseedsNanoparticle UV-vis extinction spectrum, Fe3O4Au on the surface of nanoparticlesseedsVery sparse distribution, AuseedsThe spacing is large, and the coupling effect cannot be generated; m + Auseeds+1 means Fe3O4The UV-vis extinction spectrum of the-Au nano-particles and the 1-time gold growth solution obviously generates red shift, which indicates that Fe3O4Particle surface AuseedsIncreased diameter, increased particles, AuseedsWith a gradually decreasing distance therebetween, AuseedsAu on the surface of ferroferric oxide by mutual couplingseedsThe array forms a new shell-like structure surface plasma oscillation mode; m + Auseeds+2 means Fe3O4-AuseedsAnd (3) adding 2 times of UV-vis extinction spectrums of the gold growth solution to the nanoparticles, and further performing red shift on the extinction spectrums. This is because of Fe3O4Particle surface AuseedsThe densification is further increased, and the surface plasma oscillation mode of the shell-like structure is further enhanced; m + Auseeds+3 means Fe3O4-AuseedsAnd (3) adding the UV-vis extinction spectrum of the gold growth liquid to the nano particles, wherein the extinction spectrum is subjected to red shift, but the spectrum peak width is increased. This is because Fe is added after the 3 rd addition of the crude gold liquid3O4Particle surface AuseedsThe particle diameters of the particles are uneven, partial particles even form an incomplete shell, the plasma modes on the particle surfaces become complex and various, and the peak width is just the effect of multi-mode superposition.
Example 2
H is detected by using the ferroferric oxide/gold nanoparticles prepared in example 1 as an SERS probe and 3,3',5,5' -tetramethylbenzidine as a reporter molecule2O2The concentration comprises the following steps:
step one, preparing a hydrogen peroxide solution with a certain concentration gradient: a certain amount of 30% aqueous hydrogen peroxide solution is taken and prepared into 1mL of aqueous hydrogen peroxide solution with certain gradient concentration.
Step two, SERS detection of hydrogen peroxide solutions with different concentrations: and adding 100 mu L of the functionalized probe into hydrogen peroxide solutions with different concentrations, reacting for 15min, magnetically absorbing the probe, and dropwise adding the probe onto an analytically pure aluminum sheet for Raman test, wherein the wavelength of Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
Example 3
The method for detecting the glucose concentration by using the ferroferric oxide/gold nanoparticles prepared in the embodiment 1 as an SERS probe and using 3,3',5,5' -tetramethylbenzidine as a signal molecule comprises the following steps:
step one, enzyme treatment of a glucose solution with a certain concentration gradient: a certain amount of glucose is weighed and prepared into 1mL of glucose aqueous solution with a certain concentration gradient. Adding 20 mu L of 20mg/mL glucose oxidase GOx, and carrying out water bath reaction at 37.5 ℃ for 30 min;
step two, SERS detection of glucose solutions with different concentrations: for a certain concentration gradient (1-10)-9mol/L) glucose solution, adding 100 mu L of the functionalized probe, reacting for 15min, magnetically absorbing the probe, and dripping the probe on an analytically pure aluminum sheet for Raman test. Wherein the wavelength of the Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A preparation method of ferroferric oxide/gold structure nano particles is characterized by comprising the following steps: the method comprises the following specific steps:
(1) preparation of Fe3O4 nanoparticle solution:
under the environment of nitrogen and water bath, sequentially adding a polyethyleneimine solution, a ferrous sulfate solution, a potassium nitrate solution and a sodium hydroxide solution, reacting for 1-3h, settling particles by using a magnet, and washing by using deionized water to obtain a Fe3O4 nano-particle solution, wherein the particle size of the Fe3O4 nano-particles is 30-60 nm;
(2) preparing a gold nano seed solution:
preparing a mixed solution A of sodium borohydride and sodium citrate; preparing a chloroauric acid solution with a certain concentration, adding a sodium citrate solution under magnetic stirring, adding a mixed solution A, reacting for a period of time to obtain a gold nano-seed solution, wherein the particle size of the gold nano-seeds is 3-5 nm;
(3) preparing ferroferric oxide/gold nanoparticles:
mixing the Fe3O4 nano-particle solution prepared in the step (1) with the gold nano-seed solution prepared in the step (2), fully stirring, reacting for 1-3h, performing magnetic separation, washing with deionized water to remove excessive gold nano-seeds, adding a polyethyleneimine solution, reacting for 0.5-2h at 55-65 ℃ to prepare ferroferric oxide/gold nano-particles, and washing with deionized water for 1-2 times, wherein the maximum gap between the gold nano-particles on the surface of the same ferroferric oxide nano-particle is 27 nm;
(4) once growing of ferroferric oxide/gold nanoparticles:
preparing a gold growth solution according to the mass ratio of the hexadecyl trimethyl ammonium chloride to the chloroauric acid to the potassium bromide to the AA solution of 5000:1.25:1: 20; adding a hexadecyl trimethyl ammonium chloride solution with the same amount of substances into the ferroferric oxide/gold nano particle solution prepared in the step (3), uniformly stirring, adding a gold growth solution, and reacting for 15-20 min;
(5) and (2) repeatedly regrowing ferroferric oxide/gold nanoparticles:
adding a chloroauric acid solution with the same volume as the chloroauric acid solution in the step (4) into the product obtained in the step (4), adding an AA solution with the same volume as the AA solution in the step (4), reacting for 15-20min, and growing gold particles again; the step of growing again is generally repeated for 2 to 5 times until a coupled LSPR peak is generated in the scattering spectrum of the ferroferric oxide/gold nanoparticles, and the coupled LSPR peak is washed for 1 to 2 times by deionized water for standby; the particle size of the ferroferric oxide/gold nanoparticles regrown for many times is 40nm-75 nm.
2. The method for preparing ferroferric oxide/gold structured nanoparticles according to claim 1, wherein the method comprises the following steps: in the step (1), the water bath heating temperature is 80-100 ℃, and the molar ratio of the polyethyleneimine, the ferrous sulfate heptahydrate, the potassium nitrate and the sodium hydroxide is 1:115:500: 250.
3. The method for preparing ferroferric oxide/gold structured nanoparticles according to claim 1, wherein the method comprises the following steps: in the step (2), preparing a mixed solution A containing 0.075% by mass of sodium borohydride and sodium citrate; and (3) mixing the chloroauric acid aqueous solution and the sodium citrate solution according to the mass concentration of 1:2 under magnetic stirring, and adding the solution A to prepare the gold nano seed solution.
4. The method for preparing ferroferric oxide/gold structured nanoparticles according to claim 1, wherein the method comprises the following steps: in the step (3), mixing the ferroferric oxide nano particle solution and the gold nano seed solution according to the volume ratio of 1:45, fully shaking and uniformly mixing, reacting for 2 hours, washing for 3-5 times by using deionized water, adding deionized water with the same volume as the gold nano seed solution, fully shaking and mixing, adding 0-4g/L polyethyleneimine solution, reacting for 1 hour at 55-65 ℃, and if aggregation occurs during the reaction, performing ultrasonic dispersion, and washing for 3-5 times by using deionized water to obtain the ferroferric oxide/gold nano particles.
5. The application of the ferroferric oxide/gold structure nanoparticles prepared by the method according to any one of claims 1 to 4 in SERS (surface enhanced Raman scattering) ultra-sensitive detection of H2O2 concentration.
6. The use of claim 5, wherein: the application uses ferroferric oxide/gold structure nano particles as probes and 3,3',5,5' -tetramethyl benzidine as signal molecules, and the specific method is as follows:
(1) functionalization of ferroferric oxide/gold nanoparticles:
preparing 3,3',5,5' -tetramethylbenzidine aqueous solution with the substance quantity concentration of 80 mu mol/mL, adding ferroferric oxide/gold structure nanoparticle solution into the solution, wherein the volume ratio of the ferroferric oxide/gold structure nanoparticle solution to the 3,3',5,5' -tetramethylbenzidine solution is 100:1, reacting at normal temperature for 40-60min, separating by using a magnet, washing twice by using deionized water, and adding deionized water for heavy suspension to obtain the SERS probe;
(2) SERS probes detect H2O2 solutions at different concentrations:
preparing a certain concentration gradient (1-10-9 mol/L) H2O2 solution, adding 100 mu L of the SERS probe prepared in the step (1), reacting for 5-20min, absorbing the probe by magnetic force, and dropwise adding the probe to an analytical pure aluminum sheet for Raman testing; wherein the wavelength of the Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
7. The application of the ferroferric oxide/gold structure nano-particles prepared by the method according to any one of claims 1 to 4 in SERS (surface enhanced Raman scattering) ultra-sensitive detection of glucose concentration.
8. The use of claim 7, wherein: the application uses ferroferric oxide/gold structure nano particles as probes and 3,3',5,5' -tetramethyl benzidine as signal molecules, and the specific method is as follows:
(1) the method (1) according to claim 6, wherein the SERS probe is prepared;
(2) enzyme treatment of glucose solutions of different concentrations:
preparing a glucose aqueous solution with a certain concentration gradient (1-10-9 mol/L), adding 20 mu L of 20mg/mL glucose oxidase GOx solution into 1mL of glucose solution with different concentrations, and carrying out water bath reaction at 37.5 ℃ for 20-40 min;
(3) SERS detects glucose solutions of different concentrations:
adding 100 mu L of SERS probe into glucose solution with different concentrations, reacting for 5-20min, magnetically absorbing the probe, and dropwise adding the probe onto an analytically pure aluminum sheet for Raman testing; wherein the wavelength of the Raman exciting light is 633nm, the power is 10%, a static mode is adopted, the exposure time is 1s, and the integration frequency is 2 times.
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