Nanotechnology PAPER Synthesis, characterization and applications of maghemite beads functionalized with rabbit antibodies To cite this article: A F R Rodriguez et al 2018 Nanotechnology 29 365701   View the article online for updates and enhancements. Related content Highly fluorescent and superparamagnetic nanosystem for biomedical applications Mariana P Cabrera, Paulo E Cabral Filho, Camila M C M Silva et al. - Versatile theranostics agents designed by coating ferrite nanoparticles with biocompatible polymers M Zahraei, M Marciello, A Lazaro-Carrillo et al. - Synthesis, characterization and in vitro evaluation of exquisite targeting SPIONs–PEG–HER in HER2+ human breast cancer cells Javad Hamzehalipour Almaki, Rozita Nasiri, Ani Idris et al. - This content was downloaded from IP address 186.217.236.61 on 21/08/2019 at 18:37 https://doi.org/10.1088/1361-6528/aacc21 http://iopscience.iop.org/article/10.1088/1361-6528/aa752a http://iopscience.iop.org/article/10.1088/1361-6528/aa752a http://iopscience.iop.org/article/10.1088/0957-4484/27/25/255702 http://iopscience.iop.org/article/10.1088/0957-4484/27/25/255702 http://iopscience.iop.org/article/10.1088/0957-4484/27/25/255702 http://iopscience.iop.org/article/10.1088/0957-4484/27/10/105601 http://iopscience.iop.org/article/10.1088/0957-4484/27/10/105601 http://iopscience.iop.org/article/10.1088/0957-4484/27/10/105601 http://iopscience.iop.org/article/10.1088/0957-4484/27/10/105601 https://oasc-eu1.247realmedia.com/5c/iopscience.iop.org/226387405/Middle/IOPP/IOPs-Mid-NANO-pdf/IOPs-Mid-NANO-pdf.jpg/1? Synthesis, characterization and applications of maghemite beads functionalized with rabbit antibodies A F R Rodriguez1,6 , C O Rocha5, R D Piazza5, C C dos Santos5, M A Morales2, F S E D V Faria1, M Zubair Iqbal3, L Barbosa4, Y O Chaves4, L A Mariuba4, M Jafelicci Jr5 and R F C Marques5 1 Federal University of Acre, Laboratory of Nanobiotechnology, Rio Branco, 69920-900, AC, Brazil 2 Federal University of Rio Grande do Norte, Department of Theoretical and Experimental Physics, Natal, 59078-970, RN, Brazil 3 Division of Functional Materials and Nano-Devices, Ningbo Institute of Materials Technology & Engineering, Chinese Academy of Sciences, People’s Republic of China 4 Instituto Leonidas e Maria Deane, Fundação Oswaldo Cruz, Adrianópolis, 69057-070, AM, Brazil 5 São Paulo State University—UNESP, Laboratory of Magnetic Materials and Colloids, Institute of Chemistry, Caixa postal 355, Araraquara 14800-900, SP, Brazil E-mail: anselmorodriguez73@gmail.com Received 23 January 2018, revised 4 June 2018 Accepted for publication 12 June 2018 Published 28 June 2018 Abstract Magnetic nanoparticles (NPs) have attracted great attention owing to their applications in the biomedical field. In the present work, maghemite (γFe2O3) NPs of 6.5 nm were prepared using a sonochemical method and used to prepare magnetic beads through silanization with 3-aminopropyltrimethoxysilane (APTS). Subsequently, amino groups in the resulting APTS-γFe2O3 beads were converted to carboxylic acid (CARB-γFe2O3) through the succinic anhydride reaction, as confirmed by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy and dynamic light scattering (DLS) measurements. The size of these beads was measured as 12 nm and their hydrodynamic diameter as 490 nm, using TEM analysis and DLS, respectively. The CARB-γFe2O3 beads were further functionalized by immobilizing rabbit antibodies on their surfaces; the immobilization was confirmed by flow cytometry and ionic strength. The samples were further characterized by Mössbauer spectroscopy and DC magnetization measurements. Studies on magnetic relaxivities showed that magnetic beads present great potential for application in MR imaging. Keywords: magnetic nanobeads, flow cytometry, MRI, mössbauer spectroscopy, XRD, rabbit antibody (Some figures may appear in colour only in the online journal) 1. Introduction During the last few decades, researchers have been develop- ing methods for the immobilization of proteins and enzymes on magnetic nanoparticles (NPs) [1], useful for magnetic resonance imaging (MRI) [2]. Various methods have been employed to synthesize iron oxide magnetic NPs, among the most common being co-precipitation [3], sol–gel [4], solid state [5], sonochemical [6], microwave assisted [7] and thermal decomposition [8]. These solution based methods allow the production of NPs with controllable size, morph- ology and crystallinity, and variable surface chemistry. Their biocompatibility and appropriate functionalization are key conditions for their use in biomedicine [9]. Magnetic iron oxide NPs show superparamagnetic behavior, very low coercivity and high magnetic moment per gram of sample. To Nanotechnology Nanotechnology 29 (2018) 365701 (10pp) https://doi.org/10.1088/1361-6528/aacc21 6 Author to whom any correspondence should be addressed. 0957-4484/18/365701+10$33.00 © 2018 IOP Publishing Ltd Printed in the UK1 https://orcid.org/0000-0002-3034-183X https://orcid.org/0000-0002-3034-183X mailto:anselmorodriguez73@gmail.com https://doi.org/10.1088/1361-6528/aacc21 http://crossmark.crossref.org/dialog/?doi=10.1088/1361-6528/aacc21&domain=pdf&date_stamp=2018-06-28 http://crossmark.crossref.org/dialog/?doi=10.1088/1361-6528/aacc21&domain=pdf&date_stamp=2018-06-28 provide stability in water, iron oxide NPs are usually capped with a variety of functional groups such as amino (-NH2) and carboxylic groups (-COOH). Magnetic beads are small par- ticles consisting of a non-magnetic matrix—e.g. silica or polystyrene—with embedded ferromagnetic NPs [10]. This allows the manipulation of beads through magnetic forces, but avoids agglomeration of the beads since no permanent magnetic moment remains after removing an external magn- etic field. A wide selection of silica-coated magnetic NPs can be used as substrates for the attachment, through polar interac- tions, of a broad range of biomolecules. However, proteins do not normally bind effectively to silica beads; thus, a simple treatment to have more reactive surface groups, such as car- boxylic groups (-COOH) [11] will greatly enhance protein binding. The large surface-to-volume ratio of magnetic beads pro- vides a further use for chemical bonding of target macro- molecules [12]. Magnetic beads are well established for manipulation of biomolecules, such as proteins, cells, or bac- teria [13], and can be applied in magnetic bioseparations [14]. Magnetic beads may also serve as contrast enhancing agents for MRI, for releasing heat upon absorption of radio frequencies, or they may be used for magnetic drug targeting [15, 16]. In this work, we will summarize a chemical route for the synthesis of magnetic beads using APTS and maghemite. Modification of the bead’s surface was performed by succinic anhydride reaction. We have used rabbit antibodies to prove the ability of magnetic beads to couple proteins on their surface. The physical and chemical properties of magnetic beads were studied using Fourier transform IR spectroscopy (FTIR), zeta potential, transmission electron microscopy (TEM), x-ray dif- fraction analysis (XRD), dynamic light scattering (DLS), DC magnetization, Mössbauer spectroscopy and flow cytometry. Finally, we will apply the beads in various aqueous con- centrations for MRI and in high-resolution molecular imaging. 2. Experimental 2.1. Materials All chemicals were used as received from suppliers: Iron (II) chloride tetrahydrate (97%), succinic anhydride (99.0%) and 3-aminopropyltrimethoxysilane (APTS) (98.0%) were purchased from Sigma-Aldrich Brazil. Dimethylforma- mide (DMF, 99.9%) was acquired from Panreac. Sodium hydroxide (NaOH, 97%) and ammonium hydroxide (NH4OH, 30% (v/v)) were purchased from Synth. Iron (III) chloride hexahydrate and sodium chloride (99.0%) were purchased from Mallinckrodt Chemicals. 2.2. Synthesis of magnetic NPs (γFe2O3) and beads with amine groups (APTS- γFe2O3) Magnetite was synthesized by sonoprecipitation method [17]. The NPs were obtained by dissolving 1.019 g of FeCl2·4H2O and 2.773 g of FeCl3·6H2O (in a molar ratio1:2) in 80 ml of distilled water. An aqueous solution at 80 °C was prepared with sodium hydroxide and 1.0 ml aliquots of H2O added until the solution reached pH 12. Then 1.6ml of the iron solution was added to the above alkaline solution. This solution was agitated with an ultrasonic tip probe for one hour, with 3 s of pulse on and 3 s of pulse off. Transformation of magnetite to maghemite occurred after exposing the aqueous suspension of NPs to air at room temperature for one week. To prepare magnetic beads, we added 200 μl of APTS to the above maghemite suspension. The as-produced beads were labeled as APTS-γFe2O3. 2.3. Synthesis of beads with carboxylic groups (CARB- γFe2O3) In a second step, 200 mg of APTS-γFe2O3 beads was dis- persed in 30 ml of dimethylformamide (DMF). A solution of 0.3 g of succinic anhydride and 20 ml of DMF was prepared. These solutions were mixed and placed in a distillation flask with bubbling argon at room temperature and for 8 h [18]. 2.4. Functionalization of magnetic beads with rabbit antibodies The functionalization of magnetic beads with rabbit anti- bodies was done by using the methodology reported by Greg [19]—briefly, 20 mg of maghemite beads were washed three times with phosphate buffer (0.01M) and re-suspended in 1 ml of ultrapure water. Then, 1 ml of rabbit antibody at various concentrations (10, 8, 5, 2.5, 2, 1 and 0.5 μg ml−1), 400 μl (0.54 mgml−1) EDAC (1-ethyl-3-(3-dimethylamino- propyl)carbodiimide, hydrochloride) and 5 mM NHS (N- hydroxysuccinimide) were added to the magnetite dispersion. The beads were washed again, and the blocking buffer (1M glycine at pH 8.0) was added and mixed for 1 h. Then, fluorescent secondary antibody (Anti-rabbit Alexa 488) was added at the dilution of 1/200, incubated for 30 min, and washed twice with PBS. Finally, the magnetic beads were measured by flow cytometry (FACS Canto II). Figure 1. Diffraction patterns of bare iron oxide γFe2O3, APTS-γFe2O3 and CARB-γFe2O3 beads. Numbers are the Miller indices. 2 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al 2.5. XRD measurements Structural characterization of samples was carried out with XRD using a MiniFlexII Rigaku diffractometer equipped with a CuKα radiation source. XRD data were collected in the 2θ range between 10° and 85° with a scan rate of 5°/min and steps of 0.02°. 2.6. TEM A TEM micrograph was obtained on a Philips CM120 microscope. The beads were dispersed in isopropanol and one drop of the dispersion was deposited on a carbon covered copper grid. 2.7. Infrared spectroscopy (FTIR) FTIR spectra were recorded in the reflectance mode on a Perkin−Elmer Frontier spectrometer to identify the compo- sition of the NPs and the lipase content. The samples were milled, dispersed in KBr and pressed into pellets. Spectra were recorded in the range of 4000–400 cm−1 with a nominal resolution of 4.0 cm−1 and through 64 scans. 2.8. Zeta potential and DLS measurements Zeta potential and the particles’ hydrodynamic size were measured using a Zetasizer Nanoseries NanoZS, from Mal- vern Instruments. For Zeta potential measurements, the samples were previously suspended in NaCl 1 mmol L−1 Figure 2. (a) TEM micrograph of bare γFe2O3 NPs, and their size distribution. (b) TEM micrograph of APTS-γFe2O3 beads, and their size distribution. 3 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al solution. The Zeta potential was evaluated in the pH range of 2–10 and the samples were titrated with NaOH and HCL 0.1 mol L−1 solution. 2.9. Flow cytometry The samples were analyzed by FSC (Forware Scatter) size, SSC (Side Scatter) granularity and fluorescence in a flow cytometer (FACS Canto®II), and the results were analyzed using FlowJo® software version 9.3.2 of the Tree Star © with the flow cytometry platform at Fiocruz-ILMD. 2.10. Mössbauer spectroscopy 57Fe Mossbauer spectra were recorded in the transmission mode at 300 and 12 K by using a spectrometer from SEECo equipped with a helium closed cycle cryostat from Janis; the gamma ray source had an activity of 25 μCi. The spectra were fitted by using Normos90 software; the reported isomer shifts are related to α-Fe metal at room temperature. 2.11. Magnetic characterization DC magnetization measurements were made in a physical properties measurement system, (PPMS-Dynacool from Quantum Design) equipped with a vibrating sample mag- netometer (VSM). The magnetization as a function of temp- erature was determined in the zero field cooled (Mzfc) and field cooled (Mfc) modes under a magnetic field of 70 Oe; both measurements were made in the warming mode. 2.12. MRI The transverse (T2) and longitudinal (T1) relaxation times and relaxivities (r2, r1) of as-prepared NPs were performed on a 0.55 T MRI instrument (Shanghai Niumai Corporation Ration NM120-Analyst). Dilutions of iron oxide NPs samples in water with various concentrations (0.08, 0.18, 0.36, 0.72 and 1.43mM) were used for transverse and longitudinal relaxation times and T2 imaging with additional concentrations (2.84 and 5.68mM) as compared to water. Relaxation times T2 and T1 measurements were performed using TE (echo time)=20ms and TR (repetition time)=4000ms. T2-weighted MR images were performed on running a SE (spin-echo) sequence with TR=1600 ms and TE=20ms. 3. Results and discussion 3.1. Structural characterization 3.1.1. XRD. The patterns of bare maghemite particles and beads are shown in figure 1. The diffractograms show peaks relative to the inverse spinel structure due to maghemite, no second phase like hematite or ferrihydrite was found. By using the Scherrer equation and the full width half maximum of the peak [311], [220], [511] and [440] we have determined the γFe2O3 particle size of (6.9±0.9) nm, the samples APTS-γFe2O3 and CARB-γFe2O3 presented similar crystallite sizes to the bare γFe2O3 NPs, revealing that the procedure of coating with succinic anhydride and 3-aminopropyltrimethoxysilane do not affect the magnetic core. In the APTS-γFe2O3 and CARB-γFe2O3 patterns, the broad peak at about 22° is related to the amorphous SiO2. The diffraction peaks in figure 1 are consistent with the standard structure of maghemite (JCPDS card No. 39-1346). 3.1.2. TEM analyses. A TEM image of bare γFe2O3 NPs is presented in figure 2(a) along with their size distribution; the NPs have a spherical morphology and are not aggregated. The size distribution has a mean diameter of 6.5 nm. This result is Figure 3. Infrared spectroscopy of γFe2O3, APTS- γFe2O3 and CARB- γFe2O3 samples. Figure 4. DLS of APTS-γFe2O3 NPs dispersed in water. Figure 5. pH dependence of Zeta potential of γFe2O3, APTS- γFe2O3 and CARB- γFe2O3 samples. 4 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al in close agreement with the crystallite size obtained in the XRD analysis. A TEM image of APTS-γFe2O3 beads is presented in figure 2(b) along with their size distribution; the beads have a rounded morphology and form small agglomerates. The size distribution of the sample has a mean diameter of 12 nm. This result confirms the SiO2 coating of the γFe2O3 NPs. 3.2. Vibrational characterization of magnetic beads 3.2.1. Infrared spectroscopy. The FTIR spectrum presented in figure 3 shows bands at 628 and 574 cm−1 which are assigned to the Fe−O stretching vibration in γFe2O3, these bands appear slightly shifted in the spectra of the APTS-γFe2O3 and CARB-γFe2O3 samples. The bands at 3400 and 1600 cm−1 correspond to the O−H stretching and bending vibrations of adsorbed water, respectively [20]. The spectrum of the APTS-γFe2O3 sample has absorption bands at 1034 cm−1 and 1110 cm−1 which correspond to Si−O stretchings, confirming the silanization reaction. The amine group shows N−H out-of- plane bending at 870 cm−1 and C−N stretchings at 1352 cm−1. The asymmetric and symmetric absorption bands at 1648 and 1425 cm−1 correspond to carboxylate stretching vibrations, and confirm the functionalization with succinic anhydride. The bands located in between 1425–1380 cm−1 are a combination of C−O stretchings and O−H deformation vibration. The band at Figure 6. Overlap of APTS-γFe2O3 and CARB-γFe2O3 beads by FSC size and complexity SSC (A), surface chart of the correlation between FSC, SSC and the amount of NPs demonstrated by the APTS-γFe2O3 beads (B) and CARB-γFe2O3 beads (C). Figure 7. Evaluation of the uniformity of the samples correlating the size of the FSC-A area by the FSC-H height. 5 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al 1264 cm−1 indicates the presence of carboxylic acid dimer [20]. Furthermore, the signals at 2923 cm−1 and 2852 cm−1, correspond to asymmetric and symmetric C−H vibrations, these bands are also observed in CARB-γFe2O3 and APTS-γFe2O3 samples. 3.2.2. DLS and zeta potential. Figure 4 shows the DLS measurement of APTS-γFe2O3 particles in water. It shows beads with hydrodynamic mean diameter of 490 nm. Comparison of bead size from DLS and TEM techniques reveal aggregation of this sample. In addition, the magnetic beads were evaluated by Zeta potential measurements. The isoelectric point (IEP) corre- sponds to the regime where the net charge on the NPs is zero. The zeta potentials as a function of pH curves are shown in figure 5. The IEP for γFe2O3 NPs is located at pH=3.22. It can be seen that the polycondensation with APTS shifted the IEP to 6.41, which corresponds to the APTS pKa value. Therefore, the APTS-γFe2O3 beads have a positive electrical potential at low pH (<6.41) and negative potential at high pH [21, 22]. Similar behavior is observed after functionaliza- tion of APTS-γFe2O3 beads with succinic anhydride. The pKa value of succinic acid is 4.2, while the IEP for CARB-γFe2O3 is at 3.76; this result confirms the functiona- lization of APTS-γFe2O3 with carboxylic groups. 3.3. Morphometric analysis of beads (size and complexity) The morphometric characteristics of the NPs were analyzed by flow cytometry via the fluorescence detector parameters that measure the degree of direct scattering (FSC, Y-axis), and the degree of lateral dispersion or complexity—lateral dis- persion (SSC, X-axis), and plotted on a logarithmic scale (figure 6(A)). The results show that the APTS-γFe2O3 beads were smaller in size than the CARB-γFe2O3 beads observed in 3D-size plots by complexity (figures 6(B), (C)). 3.3.1. Morphometric analysis of beads (uniformity). Although the initial morphometric characteristics showed that the particles had different sizes, it was verified that there was aggregation between the particles during the treatment; the singlet was analyzed by checking the particle uniformity in the parameters of lateral dispersion intensity (SSC-A) and uniformity (FSC-A) and (FSC-H). The results show that there is no difference between mean lateral dispersion intensity in both particle types compared to the untreated control (figure 7(B)) and that the particles follow a uniformity pattern (figure 7(A)) of 98.7% for APTS-γFe2O3 and 97.4% for CARB-γFe2O3. Thus, we have performed several experiments to characterize the morphometric parameters of the beads essential to applications in biotechnology; the purity analysis can facilitate the identification of beads by flow cytometry. 3.3.2. Evaluation of fluorescence coupling capacity in beads. The uniformity of the samples correlating the size of the FSC- A area by the FSC-H height (see figure 8) was also evaluated, showing that both samples have approximately uniform sizes comprising 92.2% and 92.9% within the analysis range, respectively. Two subpopulations of coupled NPs were analyzed, in order to determine the percentage of fluorescence coupling according to the types of pretreatment; these results are presented in table 1. The percentage of coupling is calculated by the initial analysis of the percentage of events within the gate determined by size and complexity (figure 6); after determining the area of analysis of the beads, the percentage of fluorescence is determined in relation to the beads without coupling or cut off. The fluorescence above the cut line determines the percentage of fluorescence (figure 8) calculated through the Flowjo program or by multiplying the fluorescence gate (fluorescence) mean by the gate (count) of size and complexity. In the fluorescence analysis, the histograms show that both samples have about 95% coupling; this is confirmed in figure 8 by the overlapping histogram graph, that shows two populations of magnetic NPs: low coupling and high coupling (see table 1). ATPS-γFe2O3 magnetic beads exhibited larger populations with high coupling percentage (93.3%) when compared to CARB-γFe2O3 magnetic beads (85%) at either of the 10 μg or 0.5 μg of rabbit antibody concentrations shown in table 1. However, no significant variation of fluorescence between dilutions of the antibody in the coupling was observed, indicating that a test with smaller antibody values would be required in order to determine the best concentration for coupling and the limiting dilution. 3.4. Magnetic studies Figure 9 shows the magnetization as a function of magnetic field of CARB-γFe2O3 and ATPS-γFe2O3 beads. At 300K, the CARB-γFe2O3 and ATPS-γFe2O3 samples showed saturation magnetizations of 27.4 emu g−1 and 26.4 emu g−1, respectively. At 5 K, their saturation magnetizations were of 35.5 emu g−1 and 33.9 emu g−1 for CARB-γFe2O3 and ATPS-γFe2O3 beads, respectively. The low saturation magnetization is due to the SiO2 contribution to the sample mass—as shown in figure 2, there is a thick layer of SiO2 on the surfaces of -γFe2O3 NPs—and to the Figure 8.Overlays of the histograms showing the fluorescence of the secondary Alexa 488 Anti-Rabbit in the APTS-γFe2O3 and CARB-γFe2O3 beads conjugated in 10 μg concentrations of primary rabbit. 6 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al reduced γFe2O3 particle size, which enhances the total surface of the sample; in this case, the outer Fe layers have broken bonds and their Fe moments may experience a canting effect and therefore show a lower saturation magnetization [23]. At room temperature the coercivity field for CARB-γFe2O3 and ATPS-γFe2O3 samples were smaller than 50 Oe. Although the NPs are below the critical size to be superparamagnetic at room temperature, the low coercivity field suggest that there is some agglomeration of NPs and there are dipolar interactions between them. Figure 10 shows the Mzfc and Mfc measurements per- formed for the ATPS-γFe2O3 sample. The cusp observed at TB VSM=129 K indicates the thermal blocking temperature for the ATPS-γFe2O3 NPs, this temperature may be larger than expected for a system of NPs without interaction [24]. The magnetic dipolar interaction between NPs can be studied through long-range dipolar forces in terms of the interacting superparamagnetic model and the Vogel−Fulcher law [24]. Since the CARB-γFe2O3 sample was obtained by functiona- lization of the ATPS-γFe2O3 sample with succinic acid, it is reasonable to conclude that both samples should have similar blocking temperatures. The 57Fe Mössbauer spectra for the CARB-γFe2O3 sample is presented in figure 11. The spectrum recorded at room temperature, figure 11(a), is analyzed by taking into account a broad spectrum (S1) due to slow relaxing Fe magnetic moments, and a doublet (S2) related to fast relaxing Fe magnetic moments. The doublet has isomer shift and Table 1. Percentage of APTS-γFe2O3 and CARB-γFe2O3 beads coupled with antibodies separated into high coupling and low coupling. Sample High % Low % APTS-γFe2O3 CARB-γFe2O3 APTS-γFe2O3 CARB-γFe2O3 CN 0 0.052 0.37 2.57 10 μg 95.2 88.1 4.62 10.4 8 μg 88.9 76.2 11 2.7 5 μg 93.1 87 6.8 10.6 2.5 μg 95.9 82.4 3.92 15.9 2 μg 90.9 89 8.94 9.32 1 μg 96.6 81.1 3.29 14.2 0.5 μg 94.8 91 4.89 7.86 Mean 93.3 85 6.21 12.7 SD 2.83 5.24 2.85 4.49 Figure 9. M−H curves at 5 K and 300 K for (a) Carboxylic acid (CARB-γFe2O3) beads and (b) ATPS (ATPS-γFe2O3) beads. Figure 10. Mzfc and Mfc measurement under a field of 70 Oe for ATPS-γFe2O3 beads. Figure 11. Mössbauer spectroscopy measurements performed at (a) 300 K and (b) 12 K for CARB-γFe2O3 beads. 7 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al quadrupole splitting in agreement with Fe3+. The relative absorption spectral areas (RAA) are of 63% and 37% for the S1 and S2 Fe components. Since RAA(S1)/RAA(S2)>1, we can conclude that this sample is thermally blocked at 300 K. This result does not contradict the M−H analyses because the Mössbauer spectroscopy measuring time (of∼10−7 s) is smaller than the magnetometry measuring time (∼10 s); this fact will lead to a higher Mössbauer blocking temperature. 57Fe Mössbauer spectroscopy is an element-specific technique which provides local information on the spin relaxation and charge of iron ions, and allows an easy iden- tification of iron oxides through their hyperfine magnetic field, isomer shift and quadrupole splitting [25]. The measurement recorded at 12 K is shown in figure 11(b). This spectrum exhibits a single sextet with hyperfine magnetic field of 51 T and isomer shift of 0.38 mm s−1; both parameters are in close agreement with the formation of maghemite [25]. No other second phase con- taining Fe was observed. 3.5. Magnetic resonance studies Interest in the use of nanometric particles has increased over recent years, and has been applied in several studies. Many studies have sought to characterize and investigate applica- tions, even proposing the use of the tool as a diagnostic test material due to its easy applicability. Flow cytometry has been used as a tool to evaluate the morphometric character- istics [26] and coupling capacity [27] of these compounds, as well as the interaction which these NPs can have with human cells, increasing the applicability of these compounds in the field of immunology [27–30]. In our study, beads prepared with APTS-γFe2O3 and CARB-γFe2O3 showed similar morphologies (figure 6) as well as sample uniformity (figure 7). The ability to investigate these morphological parameters and the coupling capacity of these NPs analyzed by flow cytometry may be fundamental for the development of a future tool for applications in nanobiotechnology [31–33]. In order to examine the features of as-synthesized beads CARB-γFe2O3 as an MR contrast agent, magnetic relaxation times and T2 magnetic resonance images were measured on 0.55 T scanner. The longitudinal and transverse relaxation times (r2, r1) of CARB-γFe2O3 beads are shown in figures 12(a), (b). The r1 value is 2.5 s −1mM−1 and the r2 value 60 s −1mM−1, giving an r2/r1 ratio of 24— demonstrating that the fabricated beads are an efficient T2 weighted contrast agent. Furthermore, to prove the relation time in the favor of T2 contrast agent, MR imaging was acquired. Figure 13 shows the T2-weighted MRI of beads and water. It is clearly seen that the MRI signals of NPs becomes brighter at initial low concentration because of the size of the NPs and then become darkened at high concentration as compared to the MRI image of pure water. The MRI shows that imaging intensity increases with the increase of Fe concentration. The MR relaxation data and MRI results showed the potential of as- synthesized NPs being used as T2 MRI contrast agent. Figure 12.MRI relaxation time of the as-prepared beads CARB-γFe2O3 at various concentrations of Fe (0.08, 0.18, 0.36, 0.72 and 1.43 mM). Figure 13. T2 MR imaging of pure water and CARB-γFe2O3 beads at various concentrations of Fe (0.08, 0.18, 0.36, 0.72, 1.43, 2.14 and 2.85 mM). 8 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al 4. Conclusions Magnetic γFe2O3 NPs with mean size 6.5 nm were successfully synthesized by a sonochemical method. Polycondensation of APTS on γFe2O3 NPs led to the formation of APTS-γFe2O3 beads. These beads were modified with succinic anhydride to obtain CARB-γFe2O3 beads. The size of the beads was eval- uated by TEM, which returned a mean size of 12 nm. The DLS results showed that the beads were aggregated and have a hydrodynamic size of 490 nm. The CARB-γFe2O3 and APTS-γFe2O3 beads had isoelectric points at pH of 3.76 and 6.41, respectively. The FTIR studies showed characteristic bands which demonstrated the presence of the amino and silane groups, at 870 cm−1 and 1352 cm−1, respectively, as well as carboxylate groups, at 1648 cm−1 and 1425 cm−1. To prove the ability of magnetic beads to couple proteins, rabbit antibody was covalently immobilized on these chemically modified beads. Mössbauer spectroscopy and VSM measurements showed that a large fraction of the beads have superparamagnetic behavior within the VSM measuring time. The XRD and Mössbauer results showed that capping with APTS did not induce any phase change in the maghemite phase. Immobilization time of rabbit antibodies was 4 h at a concentration of 120mg per gram of NPs. The MRI results suggest that magnetic beads indeed constitute an excellent T2 contrast agent candidate. Acknowledgments Authors thank the financial support of the Brazilian agencies MCT/CNPq, CAPES and INCT in Nanobiotechnology. Morales M thanks CNPq for his fellowship. ORCID iDs A F R Rodriguez https://orcid.org/0000-0002-3034-183X References [1] Dyal A, Loos K, Noto M, Chang S W, Spagnoli C, Shafi K V P M, Ulman A, Cowman M and Gross R A 2003 Activity of candida rugosa lipase immobilized on γ-Fe2O3 magnetic nanoparticles J. Am. Chem. Soc. 125 1684–5 [2] Yang H, Zhang S, Chen X, Zhuang Z, Xu J and Wang X 2004 Magnetite-containing spherical silica nanoparticles for biocatalysis and bioseparations Anal. Chem. 76 1316–21 [3] Lee Y, Lee H, Kim Y B, Kim J, Hyeon T, Park H W, Messersmith P B and Park T G 2008 Bioinspired surface immobilization of hyaluronic acid on monodisperse magnetite nanocrystals for targeted cancer imaging Adv. 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Protocols 7 1311–26 10 Nanotechnology 29 (2018) 365701 A F R Rodriguez et al https://doi.org/10.1007/10_2007_072 https://doi.org/10.1007/10_2007_072 https://doi.org/10.1007/10_2007_072 https://doi.org/10.1007/978-94-007-2555-3_3 https://doi.org/10.1007/978-94-007-2555-3_3 https://doi.org/10.1007/978-94-007-2555-3_3 https://doi.org/10.1007/s10895-009-0546-z https://doi.org/10.1007/s10895-009-0546-z https://doi.org/10.1007/s10895-009-0546-z https://doi.org/10.1088/0957-4484/19/34/345102 https://doi.org/10.1039/C6PY00251J https://doi.org/10.1039/C6PY00251J https://doi.org/10.1039/C6PY00251J https://doi.org/10.1093/nar/gng054 https://doi.org/10.1038/nprot.2012.065 https://doi.org/10.1038/nprot.2012.065 https://doi.org/10.1038/nprot.2012.065 1. Introduction 2. Experimental 2.1. Materials 2.2. Synthesis of magnetic NPs (γFe2O3) and beads with amine groups (APTS- γFe2O3) 2.3. Synthesis of beads with carboxylic groups (CARB- γFe2O3) 2.4. Functionalization of magnetic beads with rabbit antibodies 2.5. XRD measurements 2.6. TEM 2.7. Infrared spectroscopy (FTIR) 2.8. Zeta potential and DLS measurements 2.9. Flow cytometry 2.10. Mössbauer spectroscopy 2.11. Magnetic characterization 2.12. MRI 3. Results and discussion 3.1. Structural characterization 3.1.1. XRD 3.1.2. TEM analyses 3.2. Vibrational characterization of magnetic beads 3.2.1. Infrared spectroscopy 3.2.2. DLS and zeta potential 3.3. Morphometric analysis of beads (size and complexity) 3.3.1. Morphometric analysis of beads (uniformity) 3.3.2. Evaluation of fluorescence coupling capacity in beads 3.4. Magnetic studies 3.5. Magnetic resonance studies 4. Conclusions Acknowledgments References