Extraction and Immobilization of SA-α-2,6-Gal Receptors on Magnetic Nanoparticles to Study Receptor...

Extraction and Immobilization of SA-α-2,6-Gal Receptorson Magnetic Nanoparticles to Study Receptor Stabilityand Interaction with Sambucus nigra Lectin

Karla M. Gregorio-Jauregui & Susana A. Carrizalez-Alvarez &

Jorge E. Rivera-Salinas & Hened Saade & José L. Martinez &

Raúl G. López & Elda P. Segura & Anna Ilyina

Received: 11 December 2013 /Accepted: 10 February 2014# Springer Science+Business Media New York 2014

Abstract The interaction between influenza virus hemagglutinins and host cell with terminalsialic acid linked receptors, SA-α-2,6-Gal for human strains is important to obtain insights intothis infectious disease. Sambucus nigra lectin has high affinity for SA-α-2,6-Gal receptors.The goals of this work were: to extract the SA-α-2,6-Gal receptors from porcine airways; toperform receptors immobilization and study their storage stability; and to determine someparameters of interaction between the receptor and S. nigra lectin. The receptor isolation wasmonitored by means of bound sialic acid (BSAc) detection. A major band of protein at66.7 kDa was clearly visible in SDS-PAGE assay. Eighty-one percent of isolated glycoproteinswere immobilized on magnetic nanoparticles. The kinetics of BSAc storage stability at 4 °Cwas approximated as the first order reaction with kinetic constant and half-life estimated as0.062 day−1 and 11.2 days, respectively. The dissociation constant (Kd) calculated fromScatchard's plot was 2.47×10−7 M, and the receptor concentration was equal to 7.92×10−5 M. Procedure for N-SA-α-2,6-Gal -receptors extraction based on their affinity toS. nigra lectin with magnetic nanoparticles, and their immobilization in active form, was notdescribed previously, and may have wide application in designing biosensors or virus removalfrom areas or contaminated samples.

Keywords Sambucus nigra lectin .Magnetic nanoparticles coated with chitosan .

SA-α-2,6-Gal . N-glycans . Immobilization . Kinetic parameters

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0801-x

K. M. Gregorio-Jauregui : S. A. Carrizalez-Alvarez : J. E. Rivera-Salinas : J. L. Martinez : E. P. Segura :A. Ilyina (*)Grupo de Nanobiociencia de la Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila,Boulevard V. Carranza y José Cárdenas Valdés, 25280 Saltillo, Coahuila, Mexicoe-mail: [email protected]

H. Saade : R. G. LópezDepartamento de Procesos de Polimerización, Centro de Investigación en Química Aplicada,Boulevard Enrique Reyna No. 140, 25294 Saltillo, Coahuuila, Mexico

Introduction

Influenza virus is a major cause of respiratory illness and mortality every year, and can infect awide range of animal species including birds, pigs, and humans [1]. Influenza viruses arepolymorphic with a diameter of approximately 80–120 nm and belong to theOrthomyxoviridae family. They are divided into three principal subtypes — A, B, and C —based on the antigenicity of the major surface glycoproteins, the hemagglutinin and neuramin-idase. So far, 16 hemagglutinins and nine neuraminidase subtypes have been identified [2–5].

Influenza virus infection is initiated by the attachment of virus particles to target cellslocated in the upper respiratory tract. This process is facilitated through interaction of viralhemagglutinin, with the host glycans having terminal sialic acid residues [1, 5]. Human andavian influenza viruses are known to have distinct preferences for specifics receptors. Avianinfluenza viruses preferentially bind to sialic acid (SA-α-2,3-Gal) linked receptors, whereashuman strains bind to sialic acid (SA-α-2,6-Gal) linked receptors [6, 7]. Porcine respiratorytract is the main predilection site for influenza infection and the porcine trachea possesses bothSA-α-2,6-Gal and SA-α-2,3-Gal receptors [3, 8].

Plant lectin, such as Sambucus nigra, which is specific to SA-α-2,6-Gal terminated sugars,is usually used to identify this particular type of receptors present in different tissue sections [3,5–9]. Lectins are defined as proteins or glycoproteins that bind reversibly to a specificcarbohydrate [10–12]. They are becoming increasingly valuable as molecular probes, forexample, for labeling of cell-surface components in tissue typing [13–18]. S. nigra lectin isa tetrameric molecule with molecular weight of 126–140 kDa [19, 20] characterized by itsaffinity to SA-α-2,6-Gal receptors [6, 7]. In the present study, the specific interaction betweenS. nigra lectin and N-glycans was used to separate glycoconjugates containing SA-α-2,6-Galreceptors by means of magnetic nanoparticles.

Techniques based on using magnetizable supports have found wide application in biolog-ical fields, for example, in cancer treatment by hyperthermia, magnetic resonance imaging,cellular therapy and labeling, tissue engineering, cell isolation and purification, radio immuneassays, gene and drug delivery [21–27]. Magnetic nanoparticles are also applied inbioseparation process, biosensors, biocatalysis, enzyme and protein immobilization, andbioremediation [16, 28–33]. Magnetic nanoparticles have attracted attention due to theirsuperparamagnetism, absence of coercitivity, small size, low toxicity, biocompatibility, andtheir high specific surface area for the binding [29, 34–37]. In addition, these applicationsrequire special surface coating of the magnetic particles, which has to be not only non-toxicand biocompatible but also allow a targetable delivery with particle localization in a specificarea [27, 31]. Biopolymers such as chitosan can be used for increasing surfacefunctionalization with promising active groups [26, 32, 38]. Recently, we have obtained andcharacterized magnetic nanoparticles coated with chitosan, to be used as carrier of biologicalmolecules, by a one-step co-precipitation method [37, 39].

The goals of this work were: (1) to extract the SA-α-2,6-Gal receptors of porcine airwaysusing magnetic nanoparticles coated with chitosan and functionalized with S. nigra lectin, 2) toperform receptor immobilization on magnetic nanoparticles coated with chitosan and to studytheir storage stability, and (3) to determine parameters, such as dissociation constant (Kd) oflectin–receptor complex as well as receptor concentration.

Existing methods allow SA-α-2,6-Gal receptors isolation in inactive form, which preventstheir study outside the living organism [5, 7, 8]. The immobilization of receptors, specificallyon magnetic nanoparticles can have significant advantages for the application field [16, 32].The procedure involves covalent bond formation between the chitosan coated magneticnanoparticles and the N-glycan molecules, based on the interaction of the respective functional

Appl Biochem Biotechnol

groups. The complex is magnetically isolated and washed to remove non-specifically boundmolecules. The benefits of these particles for the bioengineering and biotechnological areasinclude their scalability, low-cost, compatibility with complex biological suspensions, speci-ficity, etc. [25].

Materials and Methods

Materials

Porcine trachea was provided by municipal slaughterhouse of Saltillo, Coahuila, Mexico. De-ionized and bi-distilled water was drawn from a Millipore system. All regents were purchasedfrom Sigma-Aldrich (USA).

S. nigra Lectin Immobilization on Magnetic Nanoparticles Coated with Chitosan

Preparation of chitosan-coated magnetic nanoparticles was carried out according the methoddescribed in our previous report [37]. All reactions were performed in quintuplicate. Theprocedure started with mixing in the reactor 50 ml of FeCl3 6H2O (0.32 M) and 50 ml ofFeCl2 4H2O (0.2 M). Then, 0.5 % (w/v) chitosan was added to the reaction mixture, and themixture temperature was raised to 50 °C under mechanical agitation at 400 rpm. Co-precipitation was performed by addition of 20 ml of aqueous ammonia at 0.67 ml/min. Atthe end of the reaction, the particles were recovered by using a permanent magnet, washed 25times with de-ionized water and lyophilized to obtain the final product. Co-precipitationreaction without chitosan was also carried out as a control.

3 g wet nanoparticles coated with chitosan (2.5 g dry basis) were activated by incubation ina solution of 25 % glutaraldehyde for 4 h at 37 °C [40]. After removing of the aldehydesolution and washing with distilled water, nanoparticles were incubated for 12 h at 4 °C on ashaker at 120 rpm with 15 ml of the solution of S. nigra lectin at concentrations of 0.011, 0.024and 0.038 mg/ml. Then, nanoparticles were separated under a magnetic field and washed withTris–HCl buffer 20 mM, pH 8. The degree of lectin immobilization was quantified bymeasuring decrease in protein concentration in the solution by Bradford’s technique [38].Lectin immobilization on magnetic nanoparticles was confirmed by thermogravimetric anal-ysis (TGA).

Extraction of SA-α-2,6-Gal Glycoproteins

Samples of porcine trachea were thoroughly washed with phosphate buffered saline, PBS(0.01 M, pH 7.2), then cut into small pieces and homogenized using a mechanical homoge-nizer followed by a glass-teflon homogenizer in buffer containing 20 mM Tris-sucrose(pH 7.2), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitor. The homog-enate was centrifuged at 1,000×g for 10 min, and the supernatant was incubated with 1 %Triton X-100 for 12 h at 4 °C. The mixture was centrifuged at 5,000×g for 30 min usingconical tubes with 3 kDa filter (AMICON).

Sialidase treatment for negative control sample applied to demonstrate selectiveglycoprotein extraction was performed according to the described technique [42]. Thefraction obtained on 3 kDa filter as described before (0.25 ml), was mixed with 0.25 mlof buffer containing 0.1 M sodium acetate, pH 5.5, with 0.147 mg/ml CaCl2 dihydrateand 5.80 mg/ml NaCl, and then treated with 0.005 ml (50 mU) of sialidase from Vibrio


cholerae (Sigma) at 37 °C for 4 h. The reaction was terminated by increasing of pH to7.0 and boiling for 4 min.

The magnetic nanoparticles functionalized with lectin (prepared with 0.038 mg/ml) wereincubated with both fractions (with and without sialidase treatment) at 37 ° C for 60 min.Receptors were removed from nanocomplex with lectin by washing with the buffer Tris 0.1 M,pH 10. Nanoparticles were recovered by applying magnetic field and introduced into anothercycle of extraction. The samples recovered in all performed cycles were analyzed by SDS-PAGE carried out according to standard methodology [43]. Standard BioRad markers wereused as controls. All assays were performed in triplicate and repeated in two experiments. Thepresence of N-glycans was demonstrated by measuring the concentration of sialic acid [44]and protein [41] quantification.

To determine the concentration of bound sialic acid (BSAc), the periodate–resorcinolmethod was applied [44]. The assay was performed as follows: a sample of 0.5 ml was addedto 0.1 ml of periodic acid solution. The solution was thoroughly mixed and incubated at 37 °Cfor 100 min. After the addition of the resorcinol reagent (1.25 ml), the solutions were mixed,placed in an ice bath for 5 min, heated at 100 °C for 15 min, cooled in tap water, and 1.25 ml oftert-butyl alcohol were added. Vigorous mixing gave a single phase solution. The tubes wereplaced in 37 °C water bath for 3 min to stabilize the color, cooled to room temperature, and theabsorbance at 630 nm was recorded using a Cary-50 spectrophotometer.

Immobilization of SA-α-2,6-Gal Receptors

Immobilization of SA-α-2,6-Gal receptors was performed according to a technique describedbefore for lectin immobilization. After activation of nanoparticles coated with chitosan byglutaraldehyde reaction, 1.5 g of activated nanoparticles (1.25 g as dry weight) were incubatedin a shaker incubator with 8 ml of N-glycans extract for 10 h at 150 rpm at 0 °C. Immobilizedpreparation was separated from the reaction mixture under a magnetic field and washed severaltimes with deionized water to provide fine powder. The sialic acid and protein concentration ofthe receptor solution before and after immobilization was measured spectrophotometrically[41, 44] to calculate immobilization percentage from the material balance.

Nanocomposites Characterization

The size distribution of the particles was evaluated in a JEOLJSM-7401 F scanning-transmission electron microscope (STEM). Samples were prepared by dispersing the obtainedpowders in water with ultrasonication and then depositing the dispersion on a copper grid.Particle size analysis was carried out for at least 1000 particles using software Image J 1.37c.

Attenuated total reflectance (ATR) Fourier transform infrared spectrometry (FTIR) wascarried out in a Magna IR 550 from Nicolet with germanium crystal to confirm the receptorimmobilization.

The magnetic properties of the nanocomposites were measured on a Physical PropertiesMeasurement System from Quantum Design, model 6000 in vibrating sample magnetometer(VSM) mode with an applied field between −20.0 and 20.0 kOe at room temperature.

Storage Stability of Immobilized System

The storage stability was evaluated by measuring the BSAc in the immobilized preparation ofSA-α-2,6-Gal receptors during storage at 4 °C [44]. Measurements were performed weeklyusing 0.1 g of magnetic nanoparticles functionalized with receptors.


Interaction Between S. nigra Lectin and Magnetic Nanoparticles Functionalizedwith N-Glycans SA-α-2,6-Gal

Lectin-receptor interaction kinetics was evaluated by measurement of lectin concentrationdecrease monitored by Bradford’s method [41]. The reaction was carried out at 37 °C in ashaker incubator at 100 rpm using 3 g of magnetic nanoparticles functionalized with N-glycansand lectin at 0.025 mg/ml.

To calculate the dissociation constant (Kd) and SA-α-2,6-Gal receptors concentration, theadsorption isotherm was obtained using 0.009, 0.013 and 0.017 mg/ml of protein (correspond-ing to lectin concentrations of 61.9, 89.3 and 115.5 nM, respectively) under the sameconditions of the kinetic test. The reaction was performed for 60 min at 37 °C and 100 rpm.Calculation was performed in Scatchard's coordinates [45].

Statistical Analysis

The results are presented as means ± standard deviations. All statistical calculations wereperformed using Microsoft Excel.

Results and Discussion

S. nigra Lectin Immobilization

The results of S. nigra lectin immobilization on magnetic nanoparticles coated with chitosanare shown in Table 1. High percentages of lectin immobilization were obtained using threedifferent lectin concentrations.

Immobilization was carried out using nanoparticles coated with chitosan with an averagediameter of 8–10 nm [3] and surface areas ranging from 314 to 380 nm2. The maximum size ofS. nigra lectin tetramer in solution was estimated by Maveyraud et al. [19] as 9.1±0.1 nm.Considering the spherical model of nanoparticles and circular projection of lectin molecule, aswell as the fact that only 53 % of area is available for intermolecular interactions [39], it can becalculated that approximately two molecules of lectin can be immobilized on one nanoparticle.According to the previous study [39] there are approximately 4×1017 nanoparticles in one g ofdry particles, which gives 8×1017 molecules/g or 1.33×10−6 mol/g of nanoparticles, calculatedby applying Avogadro’s constant. Maveyraud et al. [19] evaluated the molecular weight ofS. nigra lectin tetramer as 126 kDa. Therefore, the theoretically calculated loading of lectin is167 mg/g. Table 1 demonstrates that the experimentally determined quantity of immobilizedlectin was significantly less than theoretically calculated. This also explains why almost 100 %

Table 1 Characterization of Sambucus nigra lectin immobilization on the magnetic nanoparticles coated withchitosan

Lectin concentration 0.011 mg/ml 0.024 mg/ml 0.038 mg/ml

Initial quantity (mg) 0.165±0.005 0.360±0.010 0.570±0.009

Non-immobilized quantity (mg) 0.006±0.002 0.004±0.003 0.006±0.004

% of immobilization 99 99 99

mg of lectin/g of nanoparticles (dry weight) 0.06 0.14 0.22


of immobilization was achieved in the three evaluated cases: the lectin quantities applied inassay were well below the capacity of nanoparticles.

Thermal degradation curves of magnetic nanomaterials are shown in Fig. 1. The mixturemagnetite–maghemite (without chitosan) lost 2.16 % in weight after being heated from 30 °Cto 800 °C; the nanoparticles coated with chitosan lost 26.9 % of weight, while nanoparticlesfunctionalized with lectin lost 42.3 % of their weight. These results confirm the lectin presenceon nanoparticles due to its immobilization.

To our knowledge there are no reports of S. nigra lectin immobilization on magneticnanoparticles coated with chitosan. Therefore, we compared our results with those obtainedfor enzyme immobilization. Pan et al. [31] immobilized β-galactosidase on nanoparticles (25–30 nm of diameter) coated with chitosan by post-synthesis treatment. The highest degree ofimmobilization was estimated as 92 % that was less than the results reported in Table 1. Thehigh percentage of lectin immobilization achieved in the present study may be related todifferences in magnetic nanoparticles properties. Thus, the smaller diameter leads to highervolume specific surface area (area per volume of material) allowing higher reactivity [27].

Extraction of the N-SA-α-2,6-Gal Glycoproteins

Glycoproteins were extracted from porcine trachea. The extraction of glycoproteins consistedof the incubation at 37 °C for 60 min of nanoparticles functionalized with lectin and thefraction enriched with sialic acid to establish the interaction of lectin with SA-α-2,6-Gal-receptors. Subsequently, receptors were separated by washing with Tris buffer. Table 2 showsthe parameters of extraction process based on bound BSAc quantity as indicator parameters.The extraction was performed four times using repeatedly the same NP-lectin preparation.Only 3.3 % of total BSAc presented in extract was recovered in the first cycle. Theconcentration of specific BSAc was also reduced, thus, the recovery factor was equal to 0.6.

Fig. 1 Thermal degradation curves of nanomaterials before and after lectin (at 0.024 mg/ml) immobilization: ananoparticles without chitosan; b nanoparticles with chitosan; c nanoparticles coated with chitosan after lectinimmobilization


It is common for enzymes that specific activity and subsequently recovery factor increases,while the total activity and yield may decrease substantially due to unavoidable losses [46]. Inthe case of glycoproteins purification, all measured parameters (Table 2) were decreased due tothe extraction based on selective interaction between lectin and SA-α-2,6-Ga that allowed theseparation of other forms of sialic acid. It is also probable that it is due to the limitation in lectinimmobilized on magnetic nanoparticles used for extraction.

The nanocomposite partially lost its ability to interact with SA-α-2,6-Gal-glycans: lesssialic acid was detected after the procedure was repeated several times (Table 2). This may bedue to some alteration of immobilized lectin structure. For example, washing the system withalkaline pH buffer to separate receptors from nanoparticles functionalized with lectin mayaffect its structure, influencing its interaction with N-glycans. Finally, all experimental condi-tions need to be optimized to provide more satisfactory reusing of nanoparticles functionalizedwith lectin.

Electrophoresis was carried out (Fig. 2) to compare extracts and estimate the molecularweight of extracted proteins. Figure 2 shows the SDS-PAGE results obtained using samplesdescribed in Table 2. The protein bands are visible from about 40 to 180 kDa region. A band ofprotein with molecular weight of 66.7 kDa is clearly visible; and so are the bands correspond-ing to proteins with molecular weights of 47.6 and 88.4 kDa. Moreover, three more very faint

Table 2 Purification of SA-α-2,6-receptor from pig trachea fragments using bound sialic acid (BSAc) aspurification criteria

Sample Total BSAc(mg)

Specific BSAc(mg/mg of protein)

% Yield oftotal BSAc

Recoveryfactor

Obtained on 3 kDa filter 0.687 0.782 100 1.0

1. Extraction with NP-lectin 0.023 0.453 3.3 0.6




The data corresponds to extraction performed with NP-lectin obtained using 0.038 mg/ml of lectin

Fig. 2 Left: the electrophoresis of samples extracted from the pig trachea using nanoparticles functionalized withSambucus nigra lectin: no number molecular weight Biorad markers (bands from top to bottom correspond to250, 150, 100, 75, and 50 kDa); 1 fraction obtained from 3 kDa filter applied for the extraction; 2, 3, 4, 5 samplesextracted with the magnetic nanoparticles functionalized with lectin in the first, second, third and fourth repeatedapplications, respectively. Right: calibration curve for the calculation of molecular weight of protein correspond-ing to the electrophoresis samples


bands are present, corresponding to proteins of estimated molecular weight of 127.4, 168.8,and 183.5 kDa. We speculate that the extracted proteins are the N-SA-α-2,6-Gal receptorssince these proteins were isolated based on their ability to interact with S. nigra lectin.

Furthermore, no sialic acid was detected in the negative control sample obtained fromfraction treated with sialidase, and the corresponding bands were not detected in SDS-PAGEgels (not shown), indicating the specificity of glycoproteins isolation.

The presence of several bands (Fig. 2) suggests that there are several polymorphic proteinsthat can serve as lectin (or influenza virus) binding receptors. These results are in agreementwith the reports by Sriwilaijaroen et al. [5], on partial characterization of N-glycans from theporcine respiratory tract, in which these proteins were denatured and extracted from porcineairways in organic solvents. Elimination of many proteins present in the initial extract wasconfirmed by the presence of fewer bands in isolates. In the present study SA-α-2,6-Galreceptors were extracted by means of a rapid technique thereby retaining their activity tointeract with lectin outside of organism. This provides novel opportunities for studyinginfluenza virus infection.

Immobilization of SA-α-2,6-Gal Receptors

Analysis of the results of immobilization shows that 81 % of glycosylated proteins and ~99 %of BSAc were captured on magnetic nanoparticles (Table 3). The ratio between theimmobilized protein and BSAc was 0.56 mg of BSAc/mg of protein.

Even though the amount of protein applied for immobilization was not high, the degree ofimmobilization has not reached 100 % of the calculated maximal binding. This suggests thatcertain factors limit the immobilization process, such as difficulties related to three-dimensional structures of support and proteins, cross-linking of amino groups of chitosanwith glutaraldehyde, etc. [47–50]. However, the immobilized proteins contained high level ofBSAc (99 %).

STEM analysis showed that nanoparticles functionalized with receptors have mostlyspherical shape (Fig. 3). Measurements of the size of more than 1000 nanoparticles werecarried out using an image analysis program (ImageJ 1.37c). These results are presented inFig. 4. Using these date the number-average diameter (Dn) was calculated by the followingequation:

Dn ¼X

niDiXni

;

where ni is the number of particles of diameter Di. The obtained values were 8.32 and 8.20 nmbefore and after receptor immobilization, respectively. Both systems were characterized by

Table 3 Characterization of SA-α-2,6-Gal receptors immobilization on magnetic nanoparticles coated withchitosan

Parameter Protein BSAc

Initial quantity 0.22±0.01 mg 0.101±0.11 mg

Non-immobilized quantity 0.04±0.01 mg 0.0007±0.09 mg

Immobilized quantity 0.18±0.01 mg 0.10±0.05 mg

% of immobilization 81 % 99 %

mg/g of nanoparticles (dry weight) 0.144 0.08


mean diameter of less than 10 nm. No significant size variation was found for the particlescontaining bound receptor. It is noteworthy that the measured diameter of the particle is lowerthan that reported in other studies: Bai et al. [29] reported average diameter of particles of20 nm through the immobilization of lipase on magnetic nanoparticles and Zhu et al. [36]reported 100 nm with 5-fluorouracil immobilization.

FT-IR measurements were carried out in order to confirm the receptors coupling reaction tothe surface of the iron oxide nanoparticles coated with chitosan. Figure 5 shows the FT-IRspectra of the magnetic nanoparticles after the modification. The comparison with extract ofSA-α-2,6-Gal receptors before immobilization was also performed.

As seen in Fig. 5, SA-α-2, 6-Gal receptors showed characteristic bands at 3,344 cm−1,corresponding to stretching vibrations of valence with hydrogen OH, NH; 2,929 cm−1,stretching vibrations CH; 1,646 cm−1, corresponding to NH2 groups; 1,418 cm−1,CCstretching and 1,045 cm−1, which belongs to the COC bond [36]. The spectrum of ananocomposite obtained by receptors immobilization showed the five bands similar to char-acteristic for receptors. According to the above, the magnetic nanoparticles have immobilizedSA-α-2,6-Gal receptors.

Fig. 3 STEM micrograph of nanocomposites prepared by co-precipitation method at 50°C with chitosan at0.5 w/v % before (a) and after receptor immobilization (b)

Fig. 4 Particle diameter histogram of nanocomposites prepared by co-precipitation method at 50°C withchitosan at 0.5 w/v % before (right) and after receptors immobilization (left)


The magnetic properties of the nanocomposite also reveal the presence of receptors.Figure 6 shows the magnetization curves obtained for three sequentially modifiednanopreparations. The higher magnetization at 20 kOe of the nanoparticles functionalizedwith receptors was about 44 emu/g, while for magnetic nanoparticles with and withoutchitosan are 53 and 77 emu/g, respectively.

From magnetization curves remnant magnetization and coercivity values were defined. Themagnetic particles showed remnant magnetization of 2.05 emu/g and coercivity of 26.86 Oe,while for magnetic nanoparticles with chitosan and with chitosan-receptors, these parameterswere decreased to 24.2 and 19.49 Oe, and 1.59 and 1.27 emu/g, respectively. These values arevery small and indicate a superparamagnetic behavior, which is characteristic of magnetite andmaghemite nanoparticles with diameters smaller than 10–15 nm [29, 34]. Superparamagnetismarises from thermal energy overcoming the magnetic anisotropy energy barriers of single-domain particles; indicating that nanoparticles can be easily redispersed after the removal ofthe external field. One of the most attractive features of superparamagnetic nanoparticles istheir demagnetization once the applied magnetic field is removed, which allows their reuse.

The decrease in magnetization was most likely attributed to the existence of coatedmaterials on the surface of magnetic nanoparticles that reflect less magnetic mass, which isconsistent with literature [26, 51]. The consistency in shapes of the curves further confirms thatthe decrease in magnetization is primarily due to lower concentration of magnetic material inthe preparation.

All characteristics defined for nanocomposite with receptors indicate that the magneticnanoparticles are a good carrier for receptor immobilization, which after modification con-serves its nanostructure and superparamagnetic properties. The important properties of thepreparation are its stability and the capacity of immobilized SA-α-2,6-Gal receptors to interactwith S. nigra lectin, which may be considered as a model for affinity to virus hemagglutinin.

Fig. 5 FTIR of SA-α-2,6-Gal receptors (a), and nanocomposites obtained after receptor immobilization (b)


Stability of Immobilized Biological System

The level of the BSAc can be used for evaluating the stability of the immobilized receptors.Sialic acid interacts with the influenza virus and the detachment or chemical modification ofthe sialic acid indicates inactivation of the receptor. Immobilization often leads to increasedstability of proteins including enzymes, receptors, antibodies, etc. [29, 32, 35].

The receptors of the porcine airways are not stable once removed from the tissue. Themeasurement of the levels of BSAc demonstrated that it was reduced to zero after 2 weeks ofstorage at 4 °C in the case of receptors suspension, while the immobilized receptors conserved60 % of the initial concentration (Fig. 7). The results obtained with the immobilized prepara-tion (Fig. 7) demonstrated the increase in storage stability through their immobilization, whichhelped to maintain detectable levels of BSAc for more than 40 days. Figure 7 shows that thekinetics of BSAc level variation is linearized at semi-logarithmic coordinates. This could beinterpreted as evidence that the inactivation process can be considered as a first order reaction.From the tangent the inactivation constant was calculated as 0.062 day−1. From this parameterthe half-life of immobilized receptors was estimated at 11.2 days. Thus, the immobilization onmagnetic nanoparticles coated with chitosan preserved the viability of receptors.

Interaction Between Magnetic Nanoparticles Functionalized with SA-α-2,6-Gal Receptorsand S. nigra Lectin

Kinetics of interaction between S. nigra lectin and immobilized N-glycans is presented inFig. 8. The rapid decrease of S. nigra lectin concentration was detected during the first 10 minof interaction. This signifies that the higher amount of lectin is bound to the receptors. After

Fig. 6 Magnetization curves determined at room temperature for nanoparticles: without chitosan (filled square);with chitosan (filled circle); nanoparticles functionalized with receptors (filled triangle)


30 min of reaction, the lectin concentration approached a stationary level. According to thebackground of the interaction between human respiratory tissue receptors and S. nigra lectin,labeled with fluorescent groups, 30 min was enough for equilibrium to be established [52].These reported data are consistent with those observed for the kinetic study (Fig. 8).

Fig. 7 Bound sialic acid (BSAc) concentration during storage of immobilized SA-α-2,6-Gal receptors at 4 °C,and kinetics linearization in semi-logarithmic coordinates

Fig. 8 Kinetics of Sambucus nigra lectin interaction with the receptors immobilized on magnetic nanoparticles


Calculation from the material balance of the amount of lectin applied in the assay and theprotein level recovered upon completion of the reaction shows that 48 % of lectin interactswith the receptor, which corresponds to 0.04 mg of protein per gram of nanoparticles. Theseresults confirm the ability of purified and immobilized SA-α-2,6-Gal receptors to interact withS. nigra lectin.

Interaction between receptors and lectin is described by classic equation of reversiblereaction: L+R ↔ LR, which is characterized by dissociation constant Kd=[L][R]/[LR].

Applying the balance equation for receptor concentration [R0]=[LR]+[R] and simplemathematic transformation, the Scatchard's equation is obtained:

1=Kd ¼ LR½ �= R0½ �− LR½ �ð Þð L½ �Þand LR½ �= L½ � ¼ 1=Kdx R0½ �−1=Kdx LR½ �

Figure 9 demonstrates the isotherm and its presentation in Scatchard's coordinates: [LR]/[L]versus [LR]. From the linear function the parameter characterizing the ligand–receptor inter-action may be defined: the slope is equal to affinity constant: Ka=4.04×10

6 M and dissociationconstant, Kd=1/Ka=2.47×10

−7 M, as well as abscise in the point of line intersection with axisis equal to [R0]=7.92×10

−5 M.Varfolomeev and Gurevich [45] described that values of dissociation constant for ligand–

receptor interaction generally fall within the range from 10−6 to 10−11 M. The value ofdissociation constant for interaction between SA-α-2,6-Gal receptors and S. nigra lectinestimated in the present study lies in the same range. The high value of affinity constant (orlow value of dissociation constant) signifies the high specificity and recognition betweenligand and receptor. Frequently the systems with high affinity, such as antibody to antigen, arecharacterized by dissociation constant from 10−9 to 10−11 M [45]. In the case of interactionbetween SA-α-2,6-Gal receptors and S. nigra lectin the dissociation constant is not as low, thatsignifies less affinity between this ligand and receptors. The receptor concentration ([R0]) isrelatively high, but not having any data for comparison is difficult to discuss this result.

The obtained results demonstrate that the system of SA-α-2,6-Gal receptors immobilizedon magnetic nanoparticles coated with chitosan maintains their affinity to ligand, for exampleS. nigra lectin, after immobilization.

Fig. 9 Isotherm of the interaction between Sambucus nigra lectin and the receptors immobilized on magneticnanoparticles: left: lectin concentration; right: the same curve presented on Scatchard’s coordinates


Conclusion

In this study, the SA-α-2,6-Gal receptors isolation, their immobilization on a magnetic support,and the characterization of obtained system were performed. This method provides for a rapidand simple way for the isolation and immobilization of specific receptors. These results arepromising for future studies that focus on hemagglutinin and virus interaction with receptors,design of biosensors for virus detection, and, possibly, for virus elimination from contaminatedareas by applying magnetic field. The extraction of N-SA-α-2,6-Gal receptors based on theiraffinity to S. nigra lectin, and their immobilization on magnetic nanoparticles in active formhas not been reported previously.

Acknowledgements The authors are grateful to Julieta Sánchez (CIQA) for her technical assistance in assayrelated to nanoparticles characterization. We thank CONACYT for Ph.D. thesis scholarship.

References

1. Eijk, M. V., White, M. R., Batenburg, J. J., Vaandrager, A. B., Golde, L. M. G. V., & Haagsman, H. P.(2004). American Journal of Respiratory Cell and Molecular Biology, 30, 871–879.

2. Ibricevic, A., Pekosz, A., Walter, M. J., Newby, C., Battaile, J. T., Brown, E. G., Holtzman, M. J., & Brody,S. L. (2006). Journal of Virology, 80, 7469–7480.

3. Nelli, R. K., Kuchipudi, S. V., White, G. A., Baquero-Perez, B., Dunham, S. P., & Chang, K. C. (2010).BMC Veterinary Research, 6, 2–9.

4. Yassine, H. M., Lee, C. W., Gourapura, R., & Saif, Y. M. (2010). Animal Health Research Reviews, 11, 53–72.

5. Sriwilaijaroen, N., Kondo, S., Yagi, H., Takemae, N., Saito, T., Hiramatsu, H., Kato, K., & Suzuki, Y. (2011).pLoS ONE, 6, 1–8.

6. Ito, T., Couceiro, J. N., Kelm, S., Baum, L. G., Krauss, S., Castrucci, M. R., Donatelli, I., Kida, H., Paulson,J. C., Webster, R. G., & Kawaoka, Y. (1998). Journal of Virology, 72, 7367–7373.

7. Poucke, S. G. M., Nicholls, J. M., Nauwynck, H. J., & Reeth, K. V. (2010). Virology Journal, 7, 1–14.8. Thongratsakul, S., Susuki, Y., Hiramatsu, H., Sakpuaram, T., Sirinarumitr, T., Poolkhet, C., Moonjit, P.,

Yodsheewan, R., & Songserm, T. (2010). Asian Pacific Journal of Allergy and Immunology, 28, 294–301.9. Kirkeby, S., Martel, C. J. M., & Aasted, B. (2009). Virus Research, 144, 225–232.10. da Cardoso Silva, C. D., Cavalcanti Coriolano, M., da Silva Lino, M. A., de Lagos Melo, C. M., de Souza

Bezerra, R., de Matoso Maciel Carvalho, E. V., Guerra dos Santos, A. J., Alves Pereira, V. R., & BreitenbachBarroso Coelho, L. C. (2012). Applied Biochemistry and Biotechnology, 166, 424–435.

11. Sureshkumar, T., & Priya, S. (2012). Applied Biochemistry and Biotechnology, 168, 2257–2267.12. Sucupira Maciel, M. I., de Mendonça Cavalcanti, M. S., Napoleão, T. H., Guedes Paiva, P. M., de Jansem

Almeida Catanho, M. T., & Breitenbach Barroso Coelho, L. C. (2012). Applied Biochemistry andBiotechnology, 168, 580–591.

13. Wang, W. C., & Cummings, R. D. (1988). Journal of Biological Chemistry, 263, 4576–4585.14. Yamamoto, K., Konami, Y., & Irimura, T. (1997). Journal of Biochemistry, 121, 756–761.15. Potts, S. J., Slaughter, D. C., & Thompson, J. F. (2000). Journal of Food Science, 65, 346–350.16. Heeboll-Nielsen, A., Dalki r, M., Hubbuch, J. J., & Thomas, O. R. T. (2004). Biotechnology and

Bioengineering, 87, 311–323.17. Komath, S. S., Kavitha, M., & Swamy, M. J. (2006). Organic and Biomolecular Chemistry, 4, 973–988.18. Sharma, A., Ng, T. B., Wong, J. H., & Lin, P. (2009). Journal of Biomedicine Biotechnology, 2009, 1–9.19. Maveyraud, L., Niwa, H., Guillet, V., Svergun, D. I., Konarev, P. V., Palmer, R. A., Peumans, W. J., Rougé,

P., Van-Damme, E. J., Reynolds, C. D., & Mourey, L. (2009). Proteins, 75, 89–103.20. Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B., & Peumans, W. J. (1984). Biochemical Journal, 221, 163–

169.21. Enpuku, K., Inoue, K., & Soejima, K. (2005). Japanese Journal of Applied Physics, 44, 149–155.22. Kubik, T., Bogunia-Kubik, K., & Sugisaka, M. (2005). Current Pharmaceutical Biotechnology, 6, 17–33.23. Osaka, T., Matsunaga, T., Nakanishi, T., Arakaki, A., Niwa, D., & Iida, H. (2006). Analytical and

Bioanalytical Chemistry, 384, 593–600.


24. Naka, K., Narita, A., Tanaka, H., Chujo, Y., Morita, M., Inubushi, T., Nishimura, I., Hiruta, J., Shibayama,H., Koga, M., Ishibashi, S., Seki, J., Kizaka-Kondoh, S., & Hiraoka, M. (2008). Polymers for AdvancedTechnologies, 19, 1421–1429.

25. Saiyed, Z. M., Ramchand, C. M., & Telang, S. D. (2008). Journal of Physics: Condensed Matter, 20, 1–5.26. Ge, Y., Zhang, Y., He, S., Nie, F., Teng, G., & Gu, N. (2009). Nanoscale Research Letters, 4, 287–295.27. Pankhurst, Q. A., Thanh, N. K. T., Jones, S. K., & Dobson, J. (2009). Journal of Physics D: Applied Physics,

42, 167–181.28. Amagliani, G., Omiccioli, E., Del-Campo, A., Bruce, I. J., Brandi, G., & Magnani, M. (2006). Journal of

Applied Microbiology, 100, 375–383.29. Bai, S., Guo, Z., Liu, W., & Sun, Y. (2006). Food Chemistry, 96, 1–7.30. Jaffrezic-Renault, N., Martelet, C., Chevolot, Y., & Cloarec, J. P. (2007). Sensors, 7, 589–614.31. Kekkonen, V., Lafreniere, N., Ebara, M., & Saito, A. (2009). Journal of Magnetism and Magnetic Materials,

321, 1393–1396.32. Pan, C., Hu, B., Li, W., Sun, Y., Ye, H., & Zeng, X. (2009). Journal of Molecular Catalysis B: Enzymatic,

61, 208–215.33. Kuo, C., Liu, Y., Liu, C., Chang, C., Chen, J., Chang, C., & Shieh, C. (2012). Carbohydrate Polymers, 87,

2538–2545.34. Wan, S., Huang, J., Yan, H., & Liu, K. (2006). Journal of Materials Chemistry, 16, 298–303.35. Yang, S. Y., Jian, Z. F., Horng, H. E., Hong, C. Y., Yang, H. C., Wu, C. C., & Lee, Y. H. (2008). Journal of

Magnetism and Magnetic Materials, 320, 2688–2691.36. Zhuo, Y., Yuan, P., Yuan, R., Chai, Y., & Hong, C. (2009). Biomaterials, 30, 2284–2290.37. Gregorio-Jauregui, K. M., Pineda, M. G., Rivera-Salinas, J. E., Hurtado, G., Saade, H., Martínez, J. L.,

Ilyina, A., & López, R. G. (2012). Journal of Nanomaterials, 2012, 1–8.38. Dung, D. T. K., Hai, T. H., Phuc, L. H., Long, B. D., Vihn, L. K., & Truc, P. N. (2009). Journal of Physics:

Conference, 187, 1–5.39. Osuna, Y., Gregorio-Jáuregui, K. M., Gaona-Lozano, J. G., Garza-Garcia, I. M., Ilyina, A., Barriga-Castro,

E. D., Saade, H., & López, R. G. (2012). Journal of Nanomaterials, 2012, 1–7.40. Segura-Ceniceros, E. P., Dabek-Klapko, R., & Ilyina, A. (2006). Vestnik Moskovskogo Universiteta, Khimiya,

47, 143–148.41. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.42. Varki, A., & Diaz, S. (1984). Analytical Biochemistry, 137, 236–247.43. Hames, B. D. (1998). Gel electrophoresis of protein. A practical approach (3rd ed., pp. 13–33). New York:

Oxford University Press.44. Jourdian, G. W., Lawrence, D., & Roseman, S. (1971). Journal of Biological Chemistry, 246, 430–435.45. Varfolomeev, S. D., & Gurevich, K. G. (1999). Biokinetika Prakticheskii Kurs (Biokinetics practical course).

Moscow: Fair-press.46. Ward, W. W., & Swiatek, G. (2009). Current Analytical Chemistry, 5, 1–21.47. Lu, S. Y., Qian, J. Q., Wu, Z. G., Ye, W. D., Wu, G. F., Pan, Y. B., & Zhang, K. Y. (2009). Journal of

Biochemical Technology, 1, 79–84.48. Tischer, W., Wedekind, F., & Wedekind, F. (1999). Topics in current chemistry, Immobilized enzymes:

methods and applications (pp. 96–123). Berlin: Springer.49. Metin, A. U. (2013). Macromolecular Research, 21, 1145–1152.50. Cao, L. (2006). Carrier-bound immobilized enzymes: principles, application and design (pp. 1–293).

Germany: Wiley-VCH.51. Mohapatra, S., Pal, D., Ghosh, S. K., & Pramanik, P. (2007). Journal of Nanoscience and Nanotechnology, 7,

3193–3199.52. Nicholls, J. M., Bourne, A. J., Chen, H., Guan, Y., & Peiris, M. J. S. (2007). Respiratory Research, 8, 73–77.


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