L-苯丙氨酸的氨基功能化γ...2O_3磁固相萃取（英文）_秦铭杉.pdf

南 开 大 学 学 报（自然科学版）Acta Scientiarum Naturalium Universitatis NankaiensisVol.561Feb.2023第56卷第1期2023年2月秦铭杉等：L-苯丙氨酸的氨基功能化-Fe2O3磁固相萃取Article ID:0465-7942-(2023)01-0102-10Magnetic Solid Phase Extraction of L-phenylalanineby Amino-functionalized-Fe2O3Qin Mingshan1,Zhu Xinyi1,Wei Dongwei1,Song Qiqi1,Jin Tianming2,Li Cun1,Yang Linyan1(1.Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of AnimalScience and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392,China;2.Tianjin Academy of Agricultural Sciences,Tianjin,300192,China)Abstract:Four aminogenized nanoparticles were prepared as follows:-Fe2O3SiO2-cysteamine,-Fe2O3SiO2-APTMS,-Fe2O3SiO2-PEI,-Fe2O3SiO2-G1.0.Magnetic solid phase extraction experi-ments of L-phenylalanine have been carried out at a concentration of 2.510-6mol/L,different extractiontimes and pH gradients.The lower fluorescence intensity of supernatant indicated the better extract effects.As for-Fe2O3SiO2-G1.0 nanoparticles,the best adsorption was obtained at pH=5,while the adsorptiontime was 8 h.As for-Fe2O3SiO2-PEI nanoparticles,less adsorption than above could be obtained atpH=3 and 0.5 h.-Fe2O3SiO2-PEI nanoparticles took less time to extract L-phenylalanine.Functional-ized-Fe2O3nanoparticles are promising for solid-phase extraction of L-phenylalanine.Keyword:Fe;surface chemistry;IR;TEM;XPS;absorptionCLC number:O658.2Document code:A0IntroductionIn the research and applications of new nanoparticles,magnetic nanoparticles are widely used be-cause of their optical,electrical,magnetic,thermal and catalytic properties.Magnetic nanoparticlesare a kind of nanoparticles which can be manipulated by magnetic fields.This kind of particle usual-ly contains two components.The core is a magnetic material,usually iron,nickel,and cobalt.Thesurface is other materials with functionalities to improve stability,including carbon materials,ionicliquids,Metal-Organic Frameworks,and other polymeric materials.While nanoparticles were lessthan 1m in diameter(typically 5-500 nm)and larger beads were 0.5-500 m.In diameter A groupof magnetic nanoparticles with a diameter of 50-200 nm composed of many individual magneticnanoparticles is called a magnetic nanoparticle cluster,which is the basis for its further magnetic as-Received date：2021-10-25Foundation item：Supported by Outstanding Research Achievement Award Project of Tianjin Agricultural Univer-sity,the Open Topic of Guangxi Key Laboratory for the Chemistry and Molecular Engineer-ing of Medicinal Resources(CMEMR2016-B12),the National Natural Science Foundation ofChina(21875117,31572492),theMunicipal-levelInnovativeandEntrepreneurialTrainingPlan for College Students(202210061109),the Veterinary Bitechnology Scientific Research In-novation Team of Tianjin,China(TD13-5091),the Veterinary“Innovative Talent People ofYoung and Middle Age Key Members Project”of Tianjin,the Natural Science Foundation ofTianjin,China(18JCYBJC30100,19JCQNJC13700)Biography：Qin Mingshan(1995-),male,native place:Heilongjiang Shuangyashan,Master.Corresponding author：Yang Linyan(1982-),female,native place:Hebei Jinzhou,lecturer,direction of re-search:Nanoscale drug carrie.E-mail:y_sembly into a magnetic nano-chain1-3.Magnetic nanoparticles are the core and foundation of magnet-ic solid phase extraction technology.By changing the functional part of the surface,the effective ex-traction of different target analytes can be realized4.Among them,polyamidoamine(PAMAM)dendrimers have the following structural characteristics:accurate molecular structure;high geometric symmetry,a large number of functional groups that canbe modified,and there is a cavity in the molecule.The relative molecular mass is controllable andthe molecule itself is nanometer size.Based on these structural characteristics,dendritic macromole-cules have unique properties:good monodispersity,high stability,large surface area and high activity.Hence,PAMAM dendrimers with magnetic nanoparticles as the core can be prepared as magneticadsorbents5-6.Magnetic solid phase extraction(MSPE)is a solid phase extraction technology with magnetic ormagnetized materials as adsorbent7.During the MSPE process,the magnetic adsorbent is not direct-ly filled into the adsorption column,but is added to the solution or suspension of the sample to ad-sorb the target analyte to the dispersed magnetic adsorbent surface.Under the action of externalmagnetic field,the target analyte migrates with the adsorbent and elutes the measured substancethrough the appropriate solvent to separate from the matrix of the sample.Compared with the con-ventional solid phase extraction materials,the magnetic extraction material has a larger specific sur-face area and does not need a cumbersome column loading process.The sample solution can be add-ed directly for extraction8.In the process of extraction,it can be completely dispersed into the sam-ple solution and in full contact with the measured substance,so only a short equilibrium time and asmall amount of adsorbent are needed in the low concentration trace extraction.Compared with thegeneral solid phase extraction adsorbent,there are super paramagnetic magnetic extraction materials,when there is no external magnetic field,when the adsorption process is completed,The magneticextraction material adsorbed on the surface by external magnetic field is quickly adsorbed to the con-tainer wall to achieve the rapid separation of solid and liquid without other processes.Therefore,thetime required for conventional analysis is greatly shortened,which makes magnetic solid phase ex-traction have higher extraction efficiency and extraction ability than other solid phase extraction.Espe-cially when the sample contains many complex chemical compositions or complex matrix samples inthe form of liquid and internally expanded solid particles,magnetic-solid phase extraction can beused alone or in combination with filtration,precipitation,and solvent exchange for sample pretreat-ment9.Amino acids are the basic building units of many biomolecules,one of the indispensable nutri-tional components in organisms,and play a vital role in maintaining physiological processes.Howev-er,the detection of amino acids poses a great challenge due to their structural similarity,spectral in-ertness,weak carriers,and lack of electrochemical activity.At present,the methods of amino acidanalysis and determination are constantly developing and improving.Many methods have been usedto detect amino acids,including chemical method,spectrophotometry,chromatography,capillary elec-trophoresis and so on.However,most of these methods have the disadvantages of complex instru-ment,high price,single use and long analysis time,so it is still very important to explore newamino acid detection methods which are fast,simple and more sensitive10-14.Phenylalanine is an im-portant amino acid for the human body,which could be transformed into tyrosine and,subsequently,into catecholamine neurotransmitters,while there are individuals born with phenylketonuria.Based on第1期秦铭杉等：L-苯丙氨酸的氨基功能化-Fe2O3磁固相萃取 103 104 南 开 大 学 学 报（自然科学版）第56卷the above reasons,it is very important to determine the concentration of phenylalanine in differentbiologic fluids15.Here,four aminogenized magnetic nanoparticles with -Fe2O3as the nucleus were prepared.Magnetic solid phase extraction(MSPE)experiments of L-phenylalanine(L-Phe)have been carriedout.1ExperimentalSection1.1 Preparation of-Fe2O3SiO2nanoparticles2 g of-Fe2O3nanoparticles and 60 mL of dimethyl sulfoxide(DMSO)were placed in a three-necked flask,magnetically stirred at room temperature for 24 h,sonicated for 1 h.And then 8 mLof tetraethyl orthosilicate(tetraethyl orthosilicate)was added.The mixture was shaken at room tem-perature for 48 h,and the supernatant was removed.The residue was washed three times with etha-nol and ultrapure water,and then vacuum-dried at 60.-Fe2O3SiO2nanoparticles could be ob-tained16.2.2 Preparation of cysteamine modified-Fe2O3SiO2nanoparticles100 mg of -Fe2O3SiO2,2g of LT-570,and 20 mL of 0.4%acetic acid solution weremixed and stirred for 3 h.The mixture was washed with the ultrapure water five times,4 mL ofcysteamine solution(50 mg/mL)was added,and the obtained mixture was irradiated under 254 nmUV for 18 h.After washing with ethanol and ultrapure water for twice,the residue was treatedwith triethylamine solution(pH=9)for 5 min,washed with ethanol for twice and ultrapure water forthree times,and then vacuum-dried at 60.-Fe2O3SiO2-cysteamine nanoparticles could be ob-tained17.2.3 Preparation ofAPTMS modified-Fe2O3SiO2nanoparticles1 g of-Fe2O3SiO2nanoparticles was dispersed in 50 mL ethanol and 2 mL of(3-aminopro-pyl)trimethoxysilane(APTMS)was added.The obtained mixture was stirred at 80 for 8 h andwashed with ultrapure water for five times,and then vacuum-dried at 60 ,to obtain -Fe2O3SiO2-APTMS nanoparticles16.2.4 Preparation of PEI modified-Fe2O3SiO2nanoparticles10 mL of polyethyleneimine(PEI)aqueous solution(2 mg/mL)and 2 g of -Fe2O3SiO2nanoparticles were mixed with 30 mL of ultrapure water.The mixture was shaken at room tempera-ture for 18 h,and the supernatant was removed.The residue was washed with ethanol and ultrapurewater three times,and then vacuum-dried at 60.-Fe2O3SiO2-PEI nanoparticles could be ob-tained.2.5 Preparation of-Fe2O3SiO2&PAMAM nanoparticles2.5.1 Preparation of G0.5-G1.0:Under nitrogen atmosphere,17.5 mL of APTMS and 20 mL ofmethanol were mixed.The methanol solution(20 mL)of methyl acrylate(MA,19 mL)was addeddropwise within 1 h at 0.The mixture was stirred for 30 min,heated to 25 and stirred for24 h.G0.5 could be obtained by vacuum evaporation.10 g of G0.5 and 15 mL of methanol were mixed.The mixture of diethylenetriamine(DETA,17 mL)and methanol(14 mL)was added dropwise within 2 h at 0.The mixture was stirred for30 min,heated to 25 and stirred for 72 h.After vacuum evaporation,G1.0 could be obtained18.2.5.2 Preparation of -Fe2O3SiO2-G1.0 Nanoparticles:Under nitrogen atmosphere,5 g of -Fe2O3SiO2nanoparticles was dispersed into 150 mL of toluene,and 10 g of G1.0 were added.Af-ter stirring for 24 h at 70,the mixture was washed with ethanol three times,washed with ultra-pure water twice,and then vacuum-dried at 60.-Fe2O3SiO2-G1.0 could be obtained19.2.6 MSPE of amino acidsThe L-phenylalanine(L-Phe)solutions were prepared at a concentration of 2.510-6mol/L andset pH gradients to 3,5,7,9,and 11.20 mg of the aminated nanoparticles was dispersed in 4 mL solution of L-Phe.The extractiontime was set to 8,4,2,1,and 0.5 h.3 mL of the supernatant was taken,and the supernatantwas used for fluorescence detection.2.7 Fluorescence detection of amino acidsPreparation of 0.1%o-phthalaldehyde derivative:50 mg of o-phthalaldehyde(OPA)was addedinto 1 mL of ethanol,and then 19 mL of boric acid buffer(pH=9.5)and 200 L of-mercaptoeth-anol were added.The mixture was diluted to 50 mL with boric acid buffer,and stored in refrigera-tor at 4.Preparation of 0.1 mol/L phosphate buffer:disodium hydrogen phosphate solution(0.1 mol/L)and sodium dihydrogen phosphate solution(0.1 mol/L)were prepared respectively.And then 94.7mL of disodium hydrogen phosphate solution and 5.3 mL of sodium dihydrogen phosphate solutionwere mixed.1 mL of the supernatant was taken and reacted with 1 mL water,2 mL o-phthalaldehyde de-rivative(0.1%)and 2 mL phosphoric acid buffer(0.1 mol/L,pH=8)in the dark for 5 min.Theproducts were analyzed using fluorescence spectrophotometer(ex=360 nm,slit=5 nm)based on ami-no acid.2Resultsanddiscussion2.1 IRAnalysisA series of amino-based magnetic nanoparticleswere prepared of-Fe2O3.Infrared Spectroscopy(IR)wasusedforcharacterizationoftheobtainednanoparticles.As shown in Fig.1,the IR spectra of-Fe2O3SiO2nanoparticles,-Fe2O3SiO2-LT-570nanoparticles,-Fe2O3SiO2-cysteamine nanoparti-cles,-Fe2O3SiO2-APTMSnanoparticles,-Fe2O3SiO2-PEInanoparticles,-Fe2O3SiO2-G1.0 nanoparticles could be obtained.For -Fe2O3SiO2nanoparticles,the peak of440-650 cm-1was caused by the stretching vibra-tion of Fe-O bond,the peak of 1 000-1 100cm-1was caused by the stretching vibration of Si-O-Sibond,andthepeakof3440cm-1wascaused by the stretching vibration of Si-OH bond.Fig.1 IR spectra of-Fe2O3SiO2nanoparticles,-Fe2O3SiO2-LT-570 nanoparticles,-Fe2O3SiO2-cysteamine nanoparticles,-Fe2O3SiO2-APTMSnanoparticles,-Fe2O3SiO2-PEI nanoparticles,-Fe2O3SiO2-G1.0nanoparticles4 5003 5002 5001 500500wavenumbers/cm-1Fe2O3Fe2O3SiO2Fe2O3SiO2-LT-570Fe2O3SiO2-cysteamineFe2O3SiO2-APTMSFe2O3SiO2-PEIFe2O3SiO2-G1.03 4402 9001 6401 100440-650第1期秦铭杉等：L-苯丙氨酸的氨基功能化-Fe2O3磁固相萃取 105 106 南 开 大 学 学 报（自然科学版）第56卷The results showed that-Fe2O3was successfully coated with SiO219-21.For other nanoparticles,because of the newly introduced nitrogen-containing components afteramination,a characteristic peak at about 2 900 cm-1could be assigned to C-H stretching vibrationpeak and the peak of 1 550-1 640 cm-1could be representing N-H bending vibrations22-23.2.2 TEM analysisThe TEM image of -Fe2O3SiO2-G1.0 was shown in Fig.2.The -Fe2O3SiO2-G1.0nanoparticleswerecluster-shaped,withasingle cluster size of approximately 60-100nm.Itcouldbeseenthatthemagneticcompositeof-Fe2O3SiO2-G1.0shouldbe a secondary particle formed by the re-union of the-Fe2O3SiO2-G1.0 nanopar-ticles.It could be seen from Fig.2 that themagnetic composite displays core-shell mor-phology with dark inner-Fe2O3core andlight contrast outer silica shell,which illus-trated the successful decoration of-Fe2O3with silica gel and functional PAMAM dendrimer24-25.2.3 XPS analysisFig.3(A)shows the low-resolution XPS survey spectra of -Fe2O3SiO2nanoparticles,-Fe2O3SiO2-LT-570 nanoparticles,-Fe2O3SiO2-cysteamine nanoparticles,-Fe2O3SiO2-APT-MS nanoparticles,-Fe2O3SiO2-PEI nanoparticles,-Fe2O3SiO2-G1.0 nanoparticles.The peaksA:XPS spectra of-Fe2O3SiO2nanoparticles,-Fe2O3SiO2-LT-570 nanoparticles,-Fe2O3SiO2-cysteamine nanoparticles,-Fe2O3SiO2-PEI nanoparticles,-Fe2O3SiO2-G1.0 nanoparticles,B:high-resolution C1s spectrum of-Fe2O3SiO2nanoparticles,C:high-resolution C1s spectrum of-Fe2O3SiO2-LT-570nanoparticles,D:high-resolutionC1sspectrumof-Fe2O3SiO2-cysteaminenanoparticles,E:high-resolution C1s spectrum of-Fe2O3SiO2-PEI nanoparticles,F:high-resolutionC1s spectrum of-Fe2O3SiO2-G1.0 nanoparticles,G:high-resolution O1s spectrum of-Fe2O3SiO2nanoparticles,H:high-resolution O1s spectrum of-Fe2O3SiO2-LT-570 nanoparticles,I:high-resolu-tion O1s spectrum of-Fe2O3SiO2-cysteamine nanoparticles,J:high-resolution O1s spectrum of-Fe2O3SiO2-PEI nanoparticles,K:high-resolution O1s spectrum of-Fe2O3SiO2-G1.0 nanoparticlesFig.3 XPS spectraFe2O3SiO2Fe2O3SiO2-LT-570Fe2O3SiO2-cysteamineFe2O3SiO2-APTMSFe2O3SiO2-PEIFe2O3SiO2-G1.0Fe2pO1sN1sC1sSi2p1 4001 000600200-200binding energy/eV(A)C-C/C-HC=OC-O/C-N300295290285280275binding energy/eV(B)Fig.2 TEM image of-Fe2O3SiO2-G1.0加速电压200 kV放大倍率40 000相机长度-200 nmof Fe2p,Si2p,C1s,O1s and N1s appeared in the XPS spectrum.According to these XPS spectra,it could be seen that the appearance of Si2p peak in Fig