Research ArticleDevelopment of Antibody-Coated Magnetite Nanoparticles forBiomarker Immobilization
Christian Chapa Gonzalez Carlos A Martiacutenez PeacuterezAlejandro Martiacutenez Martiacutenez Imelda Olivas Armendaacuteriz Oscar Zavala TapiaAdriana Martel-Estrada and Perla E Garciacutea-Casillas
Universidad Autonoma de Ciudad Juarez Instituto de Ingenierıa y Tecnologıa Avenida del Charro 610 Norte Colonia Partido Romero32315 Ciudad Juarez CHIH Mexico
Correspondence should be addressed to Perla E Garcıa-Casillas perlaelviagarciayahoocom
Received 2 September 2013 Accepted 21 January 2014 Published 26 March 2014
Academic Editor Chih-Hung Hsiao
Copyright copy 2014 Christian Chapa Gonzalez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Magnetic nanoparticles (MNPs) have great potential in biomedical applications because of their magnetic response offers thepossibility to direct them to specific areas and target biological entities Magnetic separation of biomolecules is one of the mostimportant applications of MNPs because their versatility in detecting cancer biomarkers However the effectiveness of this methoddepends onmany factors including the type of functionalization ontoMNPsTherefore in this study magnetite nanoparticles havebeen developed in order to separate the 51015840-nucleotidase enzyme (5eNT)The 5eNT is used as a bio-indicator for diagnosing diseasessuch as hepatic ischaemia liver tumor and hepatotoxic drugs damageMagnetic nanoparticles were covered in a coreshell typewithsilica aminosilane and a double shell of silica-aminosilane A ScFv (fragment antibody) and anti-CD73 antibody were attached tothe coated nanoparticles in order to separate the enzymeThemagnetic separation of this enzymewith fragment antibodywas foundto be 28 higher than anti-CD73 antibody and the enzyme adsorption was improved with the double shell due to the increasedlength of the polymeric chainMagnetite nanoparticles with a double shell (silica-aminosilane) were also found to bemore sensitivethan magnetite with a single shell in the detection of biomarkers
1 Introduction
Nanomedicine is generally defined as the biomedical appli-cation of nanotechnology Nanomagnetism is at the forefrontof the nanosciences as magnetic nanomaterials are the mostpromising materials used in the clinical diagnosis and invarious therapeutic applications [1 2] Magnetic particleshave special features that make them viable for biomedicalapplications [3] Their particle size can be controlled inthe nanometric scale and they can be functionalized withbiocompatible molecules to interact with biological entitiesMany researchers have been focusing on the nanoscalebecause magnetic nanoparticles contain a simple magneticdomain and show a superparamagnetic behavior at roomtemperature which means that the magnetization119872 is closeto zero in the absence of a magnetic field but when anexternal magnetic field is applied the magnetic moments are
aligned with the field [4] This kind of magnetic responseis highly desired in biomedical applications because thesematerials offer the possibility of being manipulated to aspecific body area and target biological entities throughan external stimulus This ability of magnetic nanoparticleshas allowed them to be used for labeling and manipulatingbiomolecules as drugs and genes [5ndash8] Drug delivery is themost studied application of the magnetic nanoparticles inorder to develop a new therapeutic method that increasesthe effectiveness of anticancer drug [9] Magnetic drugtargeting (MDT) has also been used to improve localizeddrug delivery to interstitial tumor targets MDT involvesattaching an antibody to the nanoparticles surface in orderto get an antibody-antigen coupling ensuring an efficientand controlled drug release [10] In order to improve theantibody adherence to the nanoparticles they are coated witha biocompatible andor biodegradable material to modify
Hindawi Publishing CorporationJournal of NanomaterialsVolume 2014 Article ID 978284 7 pageshttpdxdoiorg1011552014978284
2 Journal of Nanomaterials
their surface The coated material can be a natural polymersuch as chitosan collagen folic acid or a synthetic materiallike dextran tetraethyl orthosilicate poly-lactic-co-glycolicacid polyethylene glycol and so forth These materialsprovide different functional groups attached to the surfaceof the nanoparticles as aldehyde (ndashCHO) hydroxyl (ndashOH)and amine (ndashNH
2) [9 11ndash14] Biological entities such as
antibodies are attached to the coated nanoparticles throughbonding with these functional groups and the type of liganddefines the effectiveness of these systems
51015840-Nucleotidase (5-ribonucleotide phosphohydrolase5eNT) is an intrinsic membrane glycoprotein present asan ectoenzyme in a wide variety of mammalian cellsAn increased 5eNT concentration is a bioindicator ofhepatobiliary and osseous disease [15 16] In this studymagnetite nanoparticles coated with antibody against5eNT were developed and adherence of antibody against5eNT was investigated with diverse coated materials Theeffectiveness of functional groups such as silanol and aminewas determined A double shell nanoparticle was developedin order to improve the antibody adherence Increase inthe antibody content of the nanoparticles improved theeffectiveness of the MDT systems as a result of betterantibody-antigen coupling
2 Experimental Procedures
21 Synthesis of Magnetite Nanoparticles Magnetic nanopar-ticles (Fe
3O4) were prepared by coprecipitation with a rapid
injection method The starting material was a mixture of13519 g of FeCl
3sdot6H2O (Sigma Aldrich CAS no 10025-77-
1) and 06820 g of FeSO4sdot7H2O in 25mL of distilled water
Subsequently 25mL of NH4OH was added abruptly in a
rapid injection process to increase the pH value to 12 A blackprecipitate was formed it was washed several times until thesupernatant reached a pH value of 65 The precipitate wasdried in a vacuum oven at 30 plusmn 5∘C for 24 h
22 Coated Magnetic Nanoparticles To evaluate the enzy-matic adherence to the nanoparticle surface three differentnanoparticles were obtained magnetite with a silica shell(with hydroxyl group) magnetite with aminosilane shell(with amine group) and magnetite with a double shell silica-aminosilane For magnetite with a silica shell (MS samples)1910 g of sodium silicate (Na
2SiO3) was dissolved in 100mL
of deionized water in a flask 020 g of Fe3O4nanoparticles
was added to this solution and sonicated using Ultra-Turraxfor 30 minutes Further the temperature of the mixture wasincreased to 80∘C and hydrochloric acid was added dropwiseuntil pH 60 was attained The material was then washedseveral times with deionized water by magnetic decantationThis procedure was repeated twice in order to ensure that themagnetite was coated with silica
For magnetite with aminosilane shell (MA samples)10mL of N-(2-aminoethyl)-3 aminopropyltrimethoxysilanewas dissolved in a solution containing 250120583LH
2Oand 25mL
of methanol and then 020 g of magnetite nanoparticles wasadded The mixture was exposed to ultrasonic homogenizer
for 30 minutes Subsequently 15mL glycerol was added andthe temperature of the mix was increased to 90∘C with rapidstirring for 6 h The product was washed several times withdeionized water andmethanol and concentrated bymagneticdecantation the material was dried at room temperature Fordouble shell silica-aminosilane nanoparticles (MSA samples)a similar methodology (as mentioned above) was used withthe exception that the starting nanoparticles were precoatedwith silica
23 Antibody Adherence to Coated Nanoparticles Two typesof antibodies against 51015840-nucleotidase were used in orderto compare antigen affinity a commercially purified anti-CD73 monoclonal antibody (Ecto-51015840-nucleotidase 5FB9BD Pharmingen) (namely CD73) and single chain fragmentFv monoclonal antibody obtained by phage display method(namely ScFv) [16] 100 120583L of antibodies solution was incu-bated in a flask containing 002 g of functionalized magneticparticles for 24 h at room temperature with slow agitationThe material was washed several times with 10M pH 74phosphate buffer solution (PBS) Finally the material wassuspended in 100 120583L of PBS and stored at 4∘C The adher-ence of the antibody to functionalized magnetic nanoparti-cles (FMNPs) was determined by measuring absorbance at280 nm in a UV-Vis spectrophotometer (Thermo ScientificNanodrop 2000)
24 51015840-Nucleotidase Enzyme Separation In order to evaluatethe effect of the surface modification on 51015840-nucleotidase(5eNT) separation magnetite with no modification (M)silica-magnetite (MS) silica-aminosilane-magnetite (MSA)silica-amino silane-ScFv antibody-magnetite (MSACF) andsilica-aminosilane-anti-CD73 antibody (MSA-CD73) weretested In the first step an initial solution of 10M of the 5eNT(Sigma-Aldrich CAS no 9027-73-0) in PBS (Sigma-AldrichCAS no 7447-40-7) pH 74 was incubated with 002 g of eachmaterial at room temperature for 24 h in a microcentrifugetube in a rotator with gentle mixing After the immobi-lization step the magnetically loaded nanosorbents carryingprobe enzymes were removed from the medium by applyingan external magnetic field Finally separation efficiencieswere determined by measuring the initial and final enzymeconcentrations before and after the separation step using aBradford colorimetric method at 595 nm wavelengths
25 Characterization of Magnetite Nanoparticles X-raydiffraction (XRD PANalytical XRD84) was used to confirmthe formation of the crystalline phase of magnetite (Fe
3O4)
Field emission scanning electron microscopy (FESEMJEOL 7000F) was used to analyze the morphology andparticle size distribution was determined by using Scandiumsoftware with SEM images The superparamagnetic behaviorwas confirmed by a vibrating sample magnetometer(VSM) Magnetic measurements were performed at roomtemperature in magnetic field up to 20 kOe The Fouriertransform infrared spectra (FTIR Thermo Scientific Nicolet6700) was used to confirm the adherence of the polymericshell to the nanoparticles Enzyme and antibody adsorption
Journal of Nanomaterials 3
20 30 40 50 60 70 80
(220
)
(311
)
(400
)
(422
)(511
) (400
)
(311
)
2120579
Figure 1 X-ray pattern of magnetite nanoparticles
on magnetite nanoparticles was analyzed in a UV-Visspectrophotometer at a wavelength of 280 nm
3 Results and Discussion
The XRD patterns of the nanoparticles obtained (119872) inthis study are shown in Figure 1 The spectra showed sixcharacteristic diffraction peaks of an inverse spinel crystal ofthemagnetite structure Average crystallite sizewas estimatedfrom X-ray pattern by using Scherrerrsquos formula and using theline broadening measurements (FWHM) of the most intensepeak The obtained value was 128 nm which was similarto the particle size values observed by TEM (Figure 2(a))This suggests that themagnetite nanoparticles are monocrys-talline
TEM image shows that spherical particles with a narrowparticle distribution (16 plusmn 4 nm) were obtained and becauseof their size and greater reactivity the nanoparticles formagglomerates between 50 and 80 nm Figures 2(b) and 2(c)show TEM images of the functionalized nanoparticles withsilica shell and double shell silica-aminosilane The nanopar-ticles were obtained with silica shell agglomerated with acoating thickness of 29 plusmn 02 nm however when doubleshell silica-aminosilane was obtained particles distributedbetween 100ndash200 nm with polymagnetic cores and the coat-ing thickness increased to 58 plusmn 08 nm
Hysteresis loops of themagnetite nanoparticles are shownin Figure 3 Magnetite with and without coated materialshows a superparamagnetic behavior that is desirable forbiomedical applications Magnetite has saturation magne-tization (119872
119904) of 559 emug this value decreases by 14
when magnetite is coated with a silica shell 286 with anaminosilane shell and 23 with a double shell The decreasein the magnetization was due to the presence of the coatingmaterial This property indicates the statistical average of themagnetic moments in the direction of the external magneticfield In this case there are two materials but only magnetitenanoparticles are ferromagnetic material and this propertyis divided by the total mass of the material (magnetitenanoparticles + coated material) [17] According to Das etal 40 emug is enough to easily and quickly manipulatethe nanoparticles in the presence of an external magneticfield in a body [12] The superparamagnetic condition is notinfluenced by agglomeration because each particle has its
own magnetic moment according to Cullity and Grahamthis property depends on the particle size [18]
Functionalized magnetite with silica was confirmed byFTIR analysis Figure 4 shows the characteristic adsorptionband at 586 cmminus1 due to the (FetetrandashO) stretching vibrationsof the magnetite When these particles are coated with silicathe FTIR spectra showed a new band at 1090 cmminus1 due to thepresence of silanol group [13] this bandwas present when theparticles were coated with both silica (MS) and double shellsilica-aminosilane (MSA) In aminosilane coating samplesbands at 3309 and at 1654 cmminus1 were observed which areattributed to the presence of amine group (ndashNH
2) [14 15]
The band observed at 2943 cmminus1 is due to the stretchingof CndashH of the methyl group (ndashCH
2 ndashCH
3) The suggested
mechanism of coated particles is shown in Figure 5 In themagnetite-aminosilane shell the silicon is bonded with theiron through the deprotonation ofmagnetite However whena silica shell is added before the aminosilane groups thesilicon is bonded in the samewaywith themagnetite andwithaminosilane through SndashOndashS bond and the resulting bandappeared at 804 cmminus1 According to these results magnetitenanoparticles coated with silica have silanol functional groupon the surface and magnetite with aminosilane and doubleshell (silica-aminosilane) has the amine group functionalon the nanoparticle surface and the difference betweenthese two samples lies in the length of the polymeric chainThe surface-modified superparamagnetic nanoparticles arebiodegradable and some studies have showed that biodegra-dation time depends on the kind of coating used in the mag-netite nanoparticle In previous studies these nanostructuresshowed 2 of degradation in a physiological fluid at 18 hours[19] However still it is necessary to study the maximumlifetime in a living system
Figure 6 showed the 51015840-nucleotidase immobilizationrates using different functionalized magnetite nanoparticlesClearly it can be observed that the double shell silica-aminosilane magnetite (MSA) has greater enzymatic adher-ence than magnetite (119872) and silica shell magnetite (MS)This characteristic depends on the type of functional groupattached and the length of the polymer chain of the coatingmaterial For this reason two types of antibodies against51015840-nucleotidase were attached to MSA nanoparticles anti-CD73 monoclonal antibody (MSA-CD73 sample) and singlechain fragment Fvmonoclonal antibody (MSA-ScFv sample)When an antibody-coatedmagnetite is used the immobiliza-tion rates were 0365 and 0465 (120583g5eNTmgNPs) with anti-CD73 and ScFv antibody respectively These values werefound to be lower than the polymeric-coated nanoparticlesvalues (MSA 05382 120583g5eNTmgNPs) This can be explaineddue to the underlying differences in the way in whichthe enzymes are bound to the material In the antibody-coated magnetite (MSA-CD73 MSA-ScFv) the immobiliza-tion is given by the antibody-antigen binding The surfaceof the antibody molecule formed by the juxtaposition ofthe complementarity-determining regions or CDRs of theheavy and light chains creates the site to which the antibodybinds to the amino group of the antigen [20] Fragmentsare the minimal portions of an antibody that maintains
4 Journal of Nanomaterials
MM
Freq
uenc
y
80
70
60
50
40
30
20
10
0
Average = 162nmSD = 44nm
Particle size (nm)10 15 20 25 30 35
(a)
MS
(b)
MSA
(c)
Figure 2 TEM image of (a) magnetite nanoparticles (119872) and their particle size distribution (b) magnetite with silica shell (MS) and (c)double shell silica-aminosilane (MSA)
the antigen-combining site Increased immobilization usingfragmentsmay be due to the small size Takashi demonstratedthat antibody fragments had a better distribution in thetumor penetrating in less time than complete antibodies [21]
The nanoparticles containing attached antibodies (MSA-ScFv or MSA-CD73) bind to the enzyme through antibody-antigen coupling Since the antibodies are covalently coupledto antigen the resulting bond is stronger and more selective[22] and hence the antibody-attached magnetite nanoparti-cles showed higher bioselectivity
In the case of polymeric coated magnetite enzyme bindsto the external layer by adsorption mechanism Intermolecu-lar forces surface hydrophobicity and ion electrostatic inter-action alter the protein adsorption [23] In these nanopar-ticles the enzyme is accumulated to the surface forming alayer onto functionalized nanoparticles so differences in thesurface of the nanoparticles make the difference in protein
adsorption Magnetite nanoparticles coated with silica havesilanol functional group on the surface and magnetite withaminosilane and double shell (silica-aminosilane) has theamine group functional on the nanoparticle surface Thedifference in electrostatic interaction makes MSA with anamine groupmore affinity to the protein Another aspect thatincreases the immobilization is the difference in the length ofthe polymeric chain MSA has the largest chain according tothe suggested mechanism of coated magnetite nanoparticles(Figure 5)
Figure 7 shows the ScFv antibody interaction with dif-ferent enzymes bovine serum albumin (BSA) ovalbumin(OVA) and a negative control (CN without enzyme) Theresults showed that there was no interaction in the negativecontrol because of the absence of enzymes The immo-bilization of 51015840-nucleotidase (5eNT) was better than theimmobilization of other enzymes due to the antibody-antigen
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
their surface The coated material can be a natural polymersuch as chitosan collagen folic acid or a synthetic materiallike dextran tetraethyl orthosilicate poly-lactic-co-glycolicacid polyethylene glycol and so forth These materialsprovide different functional groups attached to the surfaceof the nanoparticles as aldehyde (ndashCHO) hydroxyl (ndashOH)and amine (ndashNH
2) [9 11ndash14] Biological entities such as
antibodies are attached to the coated nanoparticles throughbonding with these functional groups and the type of liganddefines the effectiveness of these systems
51015840-Nucleotidase (5-ribonucleotide phosphohydrolase5eNT) is an intrinsic membrane glycoprotein present asan ectoenzyme in a wide variety of mammalian cellsAn increased 5eNT concentration is a bioindicator ofhepatobiliary and osseous disease [15 16] In this studymagnetite nanoparticles coated with antibody against5eNT were developed and adherence of antibody against5eNT was investigated with diverse coated materials Theeffectiveness of functional groups such as silanol and aminewas determined A double shell nanoparticle was developedin order to improve the antibody adherence Increase inthe antibody content of the nanoparticles improved theeffectiveness of the MDT systems as a result of betterantibody-antigen coupling
2 Experimental Procedures
21 Synthesis of Magnetite Nanoparticles Magnetic nanopar-ticles (Fe
3O4) were prepared by coprecipitation with a rapid
injection method The starting material was a mixture of13519 g of FeCl
3sdot6H2O (Sigma Aldrich CAS no 10025-77-
1) and 06820 g of FeSO4sdot7H2O in 25mL of distilled water
Subsequently 25mL of NH4OH was added abruptly in a
rapid injection process to increase the pH value to 12 A blackprecipitate was formed it was washed several times until thesupernatant reached a pH value of 65 The precipitate wasdried in a vacuum oven at 30 plusmn 5∘C for 24 h
22 Coated Magnetic Nanoparticles To evaluate the enzy-matic adherence to the nanoparticle surface three differentnanoparticles were obtained magnetite with a silica shell(with hydroxyl group) magnetite with aminosilane shell(with amine group) and magnetite with a double shell silica-aminosilane For magnetite with a silica shell (MS samples)1910 g of sodium silicate (Na
2SiO3) was dissolved in 100mL
of deionized water in a flask 020 g of Fe3O4nanoparticles
was added to this solution and sonicated using Ultra-Turraxfor 30 minutes Further the temperature of the mixture wasincreased to 80∘C and hydrochloric acid was added dropwiseuntil pH 60 was attained The material was then washedseveral times with deionized water by magnetic decantationThis procedure was repeated twice in order to ensure that themagnetite was coated with silica
For magnetite with aminosilane shell (MA samples)10mL of N-(2-aminoethyl)-3 aminopropyltrimethoxysilanewas dissolved in a solution containing 250120583LH
2Oand 25mL
of methanol and then 020 g of magnetite nanoparticles wasadded The mixture was exposed to ultrasonic homogenizer
for 30 minutes Subsequently 15mL glycerol was added andthe temperature of the mix was increased to 90∘C with rapidstirring for 6 h The product was washed several times withdeionized water andmethanol and concentrated bymagneticdecantation the material was dried at room temperature Fordouble shell silica-aminosilane nanoparticles (MSA samples)a similar methodology (as mentioned above) was used withthe exception that the starting nanoparticles were precoatedwith silica
23 Antibody Adherence to Coated Nanoparticles Two typesof antibodies against 51015840-nucleotidase were used in orderto compare antigen affinity a commercially purified anti-CD73 monoclonal antibody (Ecto-51015840-nucleotidase 5FB9BD Pharmingen) (namely CD73) and single chain fragmentFv monoclonal antibody obtained by phage display method(namely ScFv) [16] 100 120583L of antibodies solution was incu-bated in a flask containing 002 g of functionalized magneticparticles for 24 h at room temperature with slow agitationThe material was washed several times with 10M pH 74phosphate buffer solution (PBS) Finally the material wassuspended in 100 120583L of PBS and stored at 4∘C The adher-ence of the antibody to functionalized magnetic nanoparti-cles (FMNPs) was determined by measuring absorbance at280 nm in a UV-Vis spectrophotometer (Thermo ScientificNanodrop 2000)
24 51015840-Nucleotidase Enzyme Separation In order to evaluatethe effect of the surface modification on 51015840-nucleotidase(5eNT) separation magnetite with no modification (M)silica-magnetite (MS) silica-aminosilane-magnetite (MSA)silica-amino silane-ScFv antibody-magnetite (MSACF) andsilica-aminosilane-anti-CD73 antibody (MSA-CD73) weretested In the first step an initial solution of 10M of the 5eNT(Sigma-Aldrich CAS no 9027-73-0) in PBS (Sigma-AldrichCAS no 7447-40-7) pH 74 was incubated with 002 g of eachmaterial at room temperature for 24 h in a microcentrifugetube in a rotator with gentle mixing After the immobi-lization step the magnetically loaded nanosorbents carryingprobe enzymes were removed from the medium by applyingan external magnetic field Finally separation efficiencieswere determined by measuring the initial and final enzymeconcentrations before and after the separation step using aBradford colorimetric method at 595 nm wavelengths
25 Characterization of Magnetite Nanoparticles X-raydiffraction (XRD PANalytical XRD84) was used to confirmthe formation of the crystalline phase of magnetite (Fe
3O4)
Field emission scanning electron microscopy (FESEMJEOL 7000F) was used to analyze the morphology andparticle size distribution was determined by using Scandiumsoftware with SEM images The superparamagnetic behaviorwas confirmed by a vibrating sample magnetometer(VSM) Magnetic measurements were performed at roomtemperature in magnetic field up to 20 kOe The Fouriertransform infrared spectra (FTIR Thermo Scientific Nicolet6700) was used to confirm the adherence of the polymericshell to the nanoparticles Enzyme and antibody adsorption
Journal of Nanomaterials 3
20 30 40 50 60 70 80
(220
)
(311
)
(400
)
(422
)(511
) (400
)
(311
)
2120579
Figure 1 X-ray pattern of magnetite nanoparticles
on magnetite nanoparticles was analyzed in a UV-Visspectrophotometer at a wavelength of 280 nm
3 Results and Discussion
The XRD patterns of the nanoparticles obtained (119872) inthis study are shown in Figure 1 The spectra showed sixcharacteristic diffraction peaks of an inverse spinel crystal ofthemagnetite structure Average crystallite sizewas estimatedfrom X-ray pattern by using Scherrerrsquos formula and using theline broadening measurements (FWHM) of the most intensepeak The obtained value was 128 nm which was similarto the particle size values observed by TEM (Figure 2(a))This suggests that themagnetite nanoparticles are monocrys-talline
TEM image shows that spherical particles with a narrowparticle distribution (16 plusmn 4 nm) were obtained and becauseof their size and greater reactivity the nanoparticles formagglomerates between 50 and 80 nm Figures 2(b) and 2(c)show TEM images of the functionalized nanoparticles withsilica shell and double shell silica-aminosilane The nanopar-ticles were obtained with silica shell agglomerated with acoating thickness of 29 plusmn 02 nm however when doubleshell silica-aminosilane was obtained particles distributedbetween 100ndash200 nm with polymagnetic cores and the coat-ing thickness increased to 58 plusmn 08 nm
Hysteresis loops of themagnetite nanoparticles are shownin Figure 3 Magnetite with and without coated materialshows a superparamagnetic behavior that is desirable forbiomedical applications Magnetite has saturation magne-tization (119872
119904) of 559 emug this value decreases by 14
when magnetite is coated with a silica shell 286 with anaminosilane shell and 23 with a double shell The decreasein the magnetization was due to the presence of the coatingmaterial This property indicates the statistical average of themagnetic moments in the direction of the external magneticfield In this case there are two materials but only magnetitenanoparticles are ferromagnetic material and this propertyis divided by the total mass of the material (magnetitenanoparticles + coated material) [17] According to Das etal 40 emug is enough to easily and quickly manipulatethe nanoparticles in the presence of an external magneticfield in a body [12] The superparamagnetic condition is notinfluenced by agglomeration because each particle has its
own magnetic moment according to Cullity and Grahamthis property depends on the particle size [18]
Functionalized magnetite with silica was confirmed byFTIR analysis Figure 4 shows the characteristic adsorptionband at 586 cmminus1 due to the (FetetrandashO) stretching vibrationsof the magnetite When these particles are coated with silicathe FTIR spectra showed a new band at 1090 cmminus1 due to thepresence of silanol group [13] this bandwas present when theparticles were coated with both silica (MS) and double shellsilica-aminosilane (MSA) In aminosilane coating samplesbands at 3309 and at 1654 cmminus1 were observed which areattributed to the presence of amine group (ndashNH
2) [14 15]
The band observed at 2943 cmminus1 is due to the stretchingof CndashH of the methyl group (ndashCH
2 ndashCH
3) The suggested
mechanism of coated particles is shown in Figure 5 In themagnetite-aminosilane shell the silicon is bonded with theiron through the deprotonation ofmagnetite However whena silica shell is added before the aminosilane groups thesilicon is bonded in the samewaywith themagnetite andwithaminosilane through SndashOndashS bond and the resulting bandappeared at 804 cmminus1 According to these results magnetitenanoparticles coated with silica have silanol functional groupon the surface and magnetite with aminosilane and doubleshell (silica-aminosilane) has the amine group functionalon the nanoparticle surface and the difference betweenthese two samples lies in the length of the polymeric chainThe surface-modified superparamagnetic nanoparticles arebiodegradable and some studies have showed that biodegra-dation time depends on the kind of coating used in the mag-netite nanoparticle In previous studies these nanostructuresshowed 2 of degradation in a physiological fluid at 18 hours[19] However still it is necessary to study the maximumlifetime in a living system
Figure 6 showed the 51015840-nucleotidase immobilizationrates using different functionalized magnetite nanoparticlesClearly it can be observed that the double shell silica-aminosilane magnetite (MSA) has greater enzymatic adher-ence than magnetite (119872) and silica shell magnetite (MS)This characteristic depends on the type of functional groupattached and the length of the polymer chain of the coatingmaterial For this reason two types of antibodies against51015840-nucleotidase were attached to MSA nanoparticles anti-CD73 monoclonal antibody (MSA-CD73 sample) and singlechain fragment Fvmonoclonal antibody (MSA-ScFv sample)When an antibody-coatedmagnetite is used the immobiliza-tion rates were 0365 and 0465 (120583g5eNTmgNPs) with anti-CD73 and ScFv antibody respectively These values werefound to be lower than the polymeric-coated nanoparticlesvalues (MSA 05382 120583g5eNTmgNPs) This can be explaineddue to the underlying differences in the way in whichthe enzymes are bound to the material In the antibody-coated magnetite (MSA-CD73 MSA-ScFv) the immobiliza-tion is given by the antibody-antigen binding The surfaceof the antibody molecule formed by the juxtaposition ofthe complementarity-determining regions or CDRs of theheavy and light chains creates the site to which the antibodybinds to the amino group of the antigen [20] Fragmentsare the minimal portions of an antibody that maintains
4 Journal of Nanomaterials
MM
Freq
uenc
y
80
70
60
50
40
30
20
10
0
Average = 162nmSD = 44nm
Particle size (nm)10 15 20 25 30 35
(a)
MS
(b)
MSA
(c)
Figure 2 TEM image of (a) magnetite nanoparticles (119872) and their particle size distribution (b) magnetite with silica shell (MS) and (c)double shell silica-aminosilane (MSA)
the antigen-combining site Increased immobilization usingfragmentsmay be due to the small size Takashi demonstratedthat antibody fragments had a better distribution in thetumor penetrating in less time than complete antibodies [21]
The nanoparticles containing attached antibodies (MSA-ScFv or MSA-CD73) bind to the enzyme through antibody-antigen coupling Since the antibodies are covalently coupledto antigen the resulting bond is stronger and more selective[22] and hence the antibody-attached magnetite nanoparti-cles showed higher bioselectivity
In the case of polymeric coated magnetite enzyme bindsto the external layer by adsorption mechanism Intermolecu-lar forces surface hydrophobicity and ion electrostatic inter-action alter the protein adsorption [23] In these nanopar-ticles the enzyme is accumulated to the surface forming alayer onto functionalized nanoparticles so differences in thesurface of the nanoparticles make the difference in protein
adsorption Magnetite nanoparticles coated with silica havesilanol functional group on the surface and magnetite withaminosilane and double shell (silica-aminosilane) has theamine group functional on the nanoparticle surface Thedifference in electrostatic interaction makes MSA with anamine groupmore affinity to the protein Another aspect thatincreases the immobilization is the difference in the length ofthe polymeric chain MSA has the largest chain according tothe suggested mechanism of coated magnetite nanoparticles(Figure 5)
Figure 7 shows the ScFv antibody interaction with dif-ferent enzymes bovine serum albumin (BSA) ovalbumin(OVA) and a negative control (CN without enzyme) Theresults showed that there was no interaction in the negativecontrol because of the absence of enzymes The immo-bilization of 51015840-nucleotidase (5eNT) was better than theimmobilization of other enzymes due to the antibody-antigen
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
on magnetite nanoparticles was analyzed in a UV-Visspectrophotometer at a wavelength of 280 nm
3 Results and Discussion
The XRD patterns of the nanoparticles obtained (119872) inthis study are shown in Figure 1 The spectra showed sixcharacteristic diffraction peaks of an inverse spinel crystal ofthemagnetite structure Average crystallite sizewas estimatedfrom X-ray pattern by using Scherrerrsquos formula and using theline broadening measurements (FWHM) of the most intensepeak The obtained value was 128 nm which was similarto the particle size values observed by TEM (Figure 2(a))This suggests that themagnetite nanoparticles are monocrys-talline
TEM image shows that spherical particles with a narrowparticle distribution (16 plusmn 4 nm) were obtained and becauseof their size and greater reactivity the nanoparticles formagglomerates between 50 and 80 nm Figures 2(b) and 2(c)show TEM images of the functionalized nanoparticles withsilica shell and double shell silica-aminosilane The nanopar-ticles were obtained with silica shell agglomerated with acoating thickness of 29 plusmn 02 nm however when doubleshell silica-aminosilane was obtained particles distributedbetween 100ndash200 nm with polymagnetic cores and the coat-ing thickness increased to 58 plusmn 08 nm
Hysteresis loops of themagnetite nanoparticles are shownin Figure 3 Magnetite with and without coated materialshows a superparamagnetic behavior that is desirable forbiomedical applications Magnetite has saturation magne-tization (119872
119904) of 559 emug this value decreases by 14
when magnetite is coated with a silica shell 286 with anaminosilane shell and 23 with a double shell The decreasein the magnetization was due to the presence of the coatingmaterial This property indicates the statistical average of themagnetic moments in the direction of the external magneticfield In this case there are two materials but only magnetitenanoparticles are ferromagnetic material and this propertyis divided by the total mass of the material (magnetitenanoparticles + coated material) [17] According to Das etal 40 emug is enough to easily and quickly manipulatethe nanoparticles in the presence of an external magneticfield in a body [12] The superparamagnetic condition is notinfluenced by agglomeration because each particle has its
own magnetic moment according to Cullity and Grahamthis property depends on the particle size [18]
Functionalized magnetite with silica was confirmed byFTIR analysis Figure 4 shows the characteristic adsorptionband at 586 cmminus1 due to the (FetetrandashO) stretching vibrationsof the magnetite When these particles are coated with silicathe FTIR spectra showed a new band at 1090 cmminus1 due to thepresence of silanol group [13] this bandwas present when theparticles were coated with both silica (MS) and double shellsilica-aminosilane (MSA) In aminosilane coating samplesbands at 3309 and at 1654 cmminus1 were observed which areattributed to the presence of amine group (ndashNH
2) [14 15]
The band observed at 2943 cmminus1 is due to the stretchingof CndashH of the methyl group (ndashCH
2 ndashCH
3) The suggested
mechanism of coated particles is shown in Figure 5 In themagnetite-aminosilane shell the silicon is bonded with theiron through the deprotonation ofmagnetite However whena silica shell is added before the aminosilane groups thesilicon is bonded in the samewaywith themagnetite andwithaminosilane through SndashOndashS bond and the resulting bandappeared at 804 cmminus1 According to these results magnetitenanoparticles coated with silica have silanol functional groupon the surface and magnetite with aminosilane and doubleshell (silica-aminosilane) has the amine group functionalon the nanoparticle surface and the difference betweenthese two samples lies in the length of the polymeric chainThe surface-modified superparamagnetic nanoparticles arebiodegradable and some studies have showed that biodegra-dation time depends on the kind of coating used in the mag-netite nanoparticle In previous studies these nanostructuresshowed 2 of degradation in a physiological fluid at 18 hours[19] However still it is necessary to study the maximumlifetime in a living system
Figure 6 showed the 51015840-nucleotidase immobilizationrates using different functionalized magnetite nanoparticlesClearly it can be observed that the double shell silica-aminosilane magnetite (MSA) has greater enzymatic adher-ence than magnetite (119872) and silica shell magnetite (MS)This characteristic depends on the type of functional groupattached and the length of the polymer chain of the coatingmaterial For this reason two types of antibodies against51015840-nucleotidase were attached to MSA nanoparticles anti-CD73 monoclonal antibody (MSA-CD73 sample) and singlechain fragment Fvmonoclonal antibody (MSA-ScFv sample)When an antibody-coatedmagnetite is used the immobiliza-tion rates were 0365 and 0465 (120583g5eNTmgNPs) with anti-CD73 and ScFv antibody respectively These values werefound to be lower than the polymeric-coated nanoparticlesvalues (MSA 05382 120583g5eNTmgNPs) This can be explaineddue to the underlying differences in the way in whichthe enzymes are bound to the material In the antibody-coated magnetite (MSA-CD73 MSA-ScFv) the immobiliza-tion is given by the antibody-antigen binding The surfaceof the antibody molecule formed by the juxtaposition ofthe complementarity-determining regions or CDRs of theheavy and light chains creates the site to which the antibodybinds to the amino group of the antigen [20] Fragmentsare the minimal portions of an antibody that maintains
4 Journal of Nanomaterials
MM
Freq
uenc
y
80
70
60
50
40
30
20
10
0
Average = 162nmSD = 44nm
Particle size (nm)10 15 20 25 30 35
(a)
MS
(b)
MSA
(c)
Figure 2 TEM image of (a) magnetite nanoparticles (119872) and their particle size distribution (b) magnetite with silica shell (MS) and (c)double shell silica-aminosilane (MSA)
the antigen-combining site Increased immobilization usingfragmentsmay be due to the small size Takashi demonstratedthat antibody fragments had a better distribution in thetumor penetrating in less time than complete antibodies [21]
The nanoparticles containing attached antibodies (MSA-ScFv or MSA-CD73) bind to the enzyme through antibody-antigen coupling Since the antibodies are covalently coupledto antigen the resulting bond is stronger and more selective[22] and hence the antibody-attached magnetite nanoparti-cles showed higher bioselectivity
In the case of polymeric coated magnetite enzyme bindsto the external layer by adsorption mechanism Intermolecu-lar forces surface hydrophobicity and ion electrostatic inter-action alter the protein adsorption [23] In these nanopar-ticles the enzyme is accumulated to the surface forming alayer onto functionalized nanoparticles so differences in thesurface of the nanoparticles make the difference in protein
adsorption Magnetite nanoparticles coated with silica havesilanol functional group on the surface and magnetite withaminosilane and double shell (silica-aminosilane) has theamine group functional on the nanoparticle surface Thedifference in electrostatic interaction makes MSA with anamine groupmore affinity to the protein Another aspect thatincreases the immobilization is the difference in the length ofthe polymeric chain MSA has the largest chain according tothe suggested mechanism of coated magnetite nanoparticles(Figure 5)
Figure 7 shows the ScFv antibody interaction with dif-ferent enzymes bovine serum albumin (BSA) ovalbumin(OVA) and a negative control (CN without enzyme) Theresults showed that there was no interaction in the negativecontrol because of the absence of enzymes The immo-bilization of 51015840-nucleotidase (5eNT) was better than theimmobilization of other enzymes due to the antibody-antigen
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
Figure 2 TEM image of (a) magnetite nanoparticles (119872) and their particle size distribution (b) magnetite with silica shell (MS) and (c)double shell silica-aminosilane (MSA)
the antigen-combining site Increased immobilization usingfragmentsmay be due to the small size Takashi demonstratedthat antibody fragments had a better distribution in thetumor penetrating in less time than complete antibodies [21]
The nanoparticles containing attached antibodies (MSA-ScFv or MSA-CD73) bind to the enzyme through antibody-antigen coupling Since the antibodies are covalently coupledto antigen the resulting bond is stronger and more selective[22] and hence the antibody-attached magnetite nanoparti-cles showed higher bioselectivity
In the case of polymeric coated magnetite enzyme bindsto the external layer by adsorption mechanism Intermolecu-lar forces surface hydrophobicity and ion electrostatic inter-action alter the protein adsorption [23] In these nanopar-ticles the enzyme is accumulated to the surface forming alayer onto functionalized nanoparticles so differences in thesurface of the nanoparticles make the difference in protein
adsorption Magnetite nanoparticles coated with silica havesilanol functional group on the surface and magnetite withaminosilane and double shell (silica-aminosilane) has theamine group functional on the nanoparticle surface Thedifference in electrostatic interaction makes MSA with anamine groupmore affinity to the protein Another aspect thatincreases the immobilization is the difference in the length ofthe polymeric chain MSA has the largest chain according tothe suggested mechanism of coated magnetite nanoparticles(Figure 5)
Figure 7 shows the ScFv antibody interaction with dif-ferent enzymes bovine serum albumin (BSA) ovalbumin(OVA) and a negative control (CN without enzyme) Theresults showed that there was no interaction in the negativecontrol because of the absence of enzymes The immo-bilization of 51015840-nucleotidase (5eNT) was better than theimmobilization of other enzymes due to the antibody-antigen
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
Figure 5 Suggested mechanism of coated magnetite nanoparticles
M MS MSA MSA-CD73 MSA-ScFv00
02
04
(120583g
of5
eNT
mg
NPS
)100
Figure 6 51015840-Nucleotidase immobilization rates in different func-tionalized magnetite nanoparticles
interaction Therefore the use of antibodies onto functional-ized nanoparticles resulted in the greater selectivity duringthe immobilization of enzymes
4 Conclusions
Antibody-coatedmagnetite nanoparticles for immobilizationof 51015840-nucleotidase were developed The enzyme immobiliza-tion grade depends on the type of functional group used andthe length of the polymer chain of the coatingmaterial In thisstudy amine functional group showed higher affinity than
Test 1 Test 2 Test 30000
0005
0010
0015
0020
0025
0030
0035
BSAOVA CN
120583g
of en
zym
e120583
L so
lutio
n
5998400NT
Figure 7 Affinity of the ScFv antibody with the 51015840-nucleotida
silanol functional group The adherence of 51015840-nucleotidaseenzyme improved on increasing the polymeric chain lengthof the coating material This shows that the magnetitenanoparticles with a double shell (silica-aminosilane) aremore sensitive thanmagnetite with a single shell in the detec-tion of biomarkers but when antibody-coated nanoparticlesare used the enzyme immobilization becomesmore selective
Journal of Nanomaterials 7
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] VVaradan L Chen and J SieNanomedicine Design andAppli-cations of Magnetic Nanomaterials Nanosensors and Nanosys-tems Wiley-VCH Weinheim Germany 2008
[2] G A O Jinhao G U Hongwei and X U Bing ldquoMultifunc-tional magnetic nanoparticles design synthesis and biomedi-cal applicationsrdquo Accounts of Chemical Research vol 42 no 8pp 1097ndash1107 2009
[3] Q A Pankhurst J Connolly S K Jones and J Dobson ldquoAppli-cations of magnetic nanoparticles in biomedicinerdquo Journal ofPhysics D vol 36 no 13 pp R167ndashR181 2003
[4] R Cornell and U Schwertmann The Iron Oxides StructureProperties Reactions Occurrences and Uses Wiley-VCHWein-heim Germany 2003
[5] D-M Fan H-R Shi Z-M Chen Q-HWu H-N Liu and R-T Zhang ldquoEarly detection of ovarian carcinoma by proteomerofiling based on magnetic bead separation and atrix-assistedlaser desorptionionization time of flight mass spectrometryrdquoAfrican Journal of Microbiology Research vol 4 no 10 pp 940ndash951 2010
[6] A Figuerola R Di Corato L Manna and T Pellegrino ldquoFromiron oxide nanoparticles towards advanced iron-based inor-ganic materials designed for biomedical applicationsrdquo Pharma-cological Research vol 62 no 2 pp 126ndash143 2010
[7] A Jordan R Scholz P Wust H Fahling and R Felix ldquoMag-netic fluid hyperthermia (MFH) cancer treatment with ACmagnetic field induced excitation of biocompatible superpara-magnetic nanoparticlesrdquo Journal of Magnetism and MagneticMaterials vol 201 no 1ndash3 pp 413ndash419 1999
[8] K Eunkyung L Sanghwa J Aram et al ldquoA single-molecule dis-section of ligand binding to a protein with intrinsic dynamicsrdquoNature Chemical Biology vol 9 pp 313ndash318 2013
[9] J-H Park K-H Im S-H Lee et al ldquoPreparation and char-acterization of magnetic chitosan particles for hyperthermiaapplicationrdquo Journal of Magnetism and Magnetic Materials vol293 no 1 pp 328ndash333 2005
[10] S Dandamudi and R B Campbell ldquoThe drug loading cytotoxi-cty and tumor vascular targeting characteristics of magnetite inmagnetic drug targetingrdquo Biomaterials vol 28 no 31 pp 4673ndash4683 2007
[11] R T Reza C M Perez A M Martınez D B Baques and PGarcıa-Casillas ldquoStudy of the particle size effect on themagneticseparation of bovine serum albumin (BSA)rdquo Sensor Letters vol8 no 3 pp 476ndash481 2010
[12] M Das D Mishra T K Maiti A Basak and P PramanikldquoBio-functionalization of magnetite nanoparticles using anaminophosphonic acid coupling agent new ultradispersediron-oxide folate nanoconjugates for cancer-specific targetingrdquoNanotechnology vol 41 no 19 Article ID 415101 2008
[13] A del Campo T Sena J Lellouchec and J Bruce ldquoMultifunc-tional magnetite and silica-magnetite nanoparticles synthesissurface activation and applications in life sciencesrdquo Journal ofMagnetism and Magnetic Materials vol 293 no 1 pp 33ndash402005
[14] H Zimmermann ldquo5rsquo-Nucleotidase molecular structure andfunctional aspectsrdquo Biochemical Journal vol 285 part 2 pp345ndash365 1992
[15] F Pagani and M Panteghini ldquo51015840-Nucleotidase in the detectionof increased activity of the liver form of alkaline phosphatase inserumrdquo Clinical Chemistry vol 47 no 11 pp 2046ndash2048 2001
[16] A Martinez Martinez C Flores-Flores F J Campo E Munoz-Delgado C Fini and C J Vidadl ldquoBiochemical propertiesof 51015840-nucleotidase from mouse skeletal musclerdquo Biochimica etBiophysica Acta Protein Structure and Molecular Enzymologyvol 1386 no 1 pp 16ndash28 1998
[17] B Buschow Physics of Magnetism and Magnetic MaterialsKluwer Academic Germany 2004
[18] B D Cullity and C D Graham Introduction to MagneticMaterials John Wiley amp Sons New York NY USA 2009
[19] K I Baca Ramos C A Martinez Perez C A R Gonzalez etal Stability of Functionalized Magnetic Particles in a PhysiologicFluid vol 1 of NSTI-Nanotech 2012
[20] C A Janeway P Travers and M Walport Immunobiology TheImmune System in Health and Disease Part II The Recognitionof Antigen Garland Science New York NY USA 2001
[21] T Yokota D E Milenic M Whitlow and J Schlom ldquoRapidtumor penetration of a single-chain Fv and comparison withother immunoglobulin formsrdquo Cancer Research vol 52 no 12pp 3402ndash3408 1992
[22] S Nandimi Immunology Introductory Textbook New AgeInternational USA 2005
[23] I Lundstrom ldquoModels of protein adsorption on solid surfacesrdquoin Surfactants Adsorption Surface Spectroscopy and DisperseSystems pp 76ndash82 1985
