Immobilization of dengue specific IgM antibodies on magnetite nanoparticles by using facile conjugation strategies

Abstract

IgM antibodies are the first immunoglobulin isotype to appear in several diseases like dengue fever. In this study, five different strategies to conjugate dengue specific IgM antibodies on magnetite nanoparticle surface (Fe₃O₄) were evaluated through commercial IgM antibody-capture ELISA assay for dengue diagnosis. IgM–Fe₃O₄ conjugates were obtained by using physical interactions, covalent bonding and antibody–antigen interactions. In all cases, analytical responses of IgM–Fe₃O₄ conjugates were higher than that of traditional ELISA assay. The most effective conjugation strategy was achieved by antigen–antibody interaction with fluorescence intensity almost 20 times higher than standard ELISA. The results discussed herein illustrate the potential application of magnetite nanoparticles as a new platform for IgM immunoassay.

---

Introduction

In the previous decades the ELISA methodology (enzyme-linked immunosorbent assays) introduced by Engvall and Perlmann in 1971 has been the leading method for quantifying sandwich immune complexes attached to a solid phase using microwell plates.¹ Nevertheless, new commercial ELISA versions are being developed. They employ magnetic microparticles (“beads”)² and nanoparticles (NPs)³ such as magnetite (Fe₃O₄) as solid phase instead of the microwells, in order to improve effectiveness of the antibody (Ab) conjugation for analyte capture,^4,5 purification,⁶ concentration⁷ and signal amplification⁸ along with achieving easy handling.

In this sense, different concepts are used for linking Abs to NPs. The most common strategies for conjugating are summarized in the literature as (a) electrostatic adsorption;⁹ (b) covalent binding via amine groups on the Ab;¹⁰ (c) covalent binding via carbohydrate groups on the Ab;¹¹ (d) ionic adsorption plus covalent binding¹² and (e) use of adapter biomolecules.¹³ The suitable antibodies amount, distribution and orientation are crucial to keep the Ab capability of recognizing its corresponding antigen, once attached onto the NP surface.¹⁴

The most commonly used antibodies for attaching on NPs are monomer classes like IgG.¹⁵ However, to our knowledge there is relative lack of reports on the efficacy of the above-mentioned conjugation strategies for coupling antibody isotype IgM on nanoparticles. Otherwise, numerous IgM antibody-capture ELISA (MAC-ELISA) assays are designed for the diagnosis of illnesses, which have primary specific IgM antibody response, such as brucellosis, syphilis, murine typhus, hepatitis B, yellow fever, leptospirosis, chikungunya, zika and dengue fever.^16–18

Dengue virus is the most common arboviral disease in humans and it is transmitted by Aedes sp. Mosquitoes. Estimates of the global incidence of dengue infections per year have ranged between 50 million and 200 million.¹⁹ During the early stages of the disease, isolated virus, nucleic acid or dengue NS1 antigen can be detected for infection diagnosis.^20,21 More recently, rapid point-of-care (POC) diagnostic devices are being developed.^22–24 However, the highest incidences of dengue are in underdeveloped countries, which cannot afford expensive tests. In a first-time infection, five or more days after the onset of illness, IgM antibodies are the first immunoglobulin isotype to appear so MAC-ELISA is the inexpensive chosen method for diagnosis.²⁵ Therefore, an improved MAC-ELISA analytical performance will be a major benefit.

In the present work, a comparative study among five largely reported and versatile conjugation antibody-nanoparticle strategies is discussed.

The different IgM-dengue immobilization strategies (Scheme 1) were: physical associations through the combined contribution of electrostatic interactions, hydrogen bonding, van der Waals forces and hydrophobic interactions (strategy 1); reaction between IgM-dengue ε-amino groups and carboxylic moieties previously anchored on the magnetite surface via EDC chemistry (strategy 2); formation of Schiff base by reaction of aldehyde-activated (oxidized) sugars moieties attached to IgM-dengue Fc region and hydrazide groups attached on NP surface (strategy 3); reaction via Schiff bases formation and/or Michael addition adducts of IgM-dengue ε-amino groups and quinone groups on polydopamine modified nanoparticles (strategy 4), and IgM-dengue coupling via Fc-binding proteins through antigen–receptor complex formation between IgM-dengue and Anti-Human IgM (μ-chain specific) antibody previously attached on polydopamine modified nanoparticles (strategy 5). In our case, dengue IgM response antibodies (IgM-dengue) are conjugated on magnetite nanoparticles surface in order to improve IgM-dengue recognition effectiveness, likewise, the best strategy was employed for comparing its analytical performance with respect to the conventional ELISA assay.

Scheme 1 Scheme of the different strategies for IgM-dengue coupling on Fe₃O₄ nanoparticles: strategy 1 (Fe₃O₄–IgM), strategy 2 (Fe₃O₄@PEG–COOH–IgM), strategy 3 (Fe₃O₄@PEG–CONHNH₂–IgM), strategy 4 (Fe₃O₄@PDA–IgM) and strategy 5 (Fe₃O₄@PDA–antiIgM-IgM).

Experimental

Materials

All reagents were purchased from Sigma-Aldrich. The antibody Anti-Human IgM (μ-chain specific) produced in rabbit, were purchased from Sigma-Aldrich cod. SAB3701409. UMELISA DENGUE IgM PLUS kit was obtained from Immunoassay Center, Cuba.

Synthesis and functionalization of magnetite nanoparticles (Fe₃O₄)

Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method under a N₂ atmosphere.²⁶ FeCl₃·6H₂O (5.4 g) and FeSO₄·7H₂O (2.78 g) were dissolved in 200 mL of 1.2 mM HCl solution. Then, 300 mL aqueous NaOH solution (1.25 M) was slowly added dropwise under vigorous stirring for 30 min, forming a black Fe₃O₄ precipitate. After vigorous stirring for another 30 min, the precipitate was magnetically decanted and washed thoroughly with ultrapure water till the supernatant solution reached neutrality (pH = 7).

Fe₃O₄ nanoparticle surface was functionalized with carboxylic moieties by capping it with polyethyleneglycol dicarboxylic acid (HOOC–PEG–COOH) (M_w 1500 Da) (see the ESI S4 for synthesis details and characterizations (Fig. S4†)). Briefly, 20 mL of magnetite stock solution (20 mg mL⁻¹) were vigorous stirred all night with 0.5 g of polyethyleneglycol dicarboxylic acid. After that, nanoparticles were magnetically decanted and washed thoroughly with ultrapure water and re-dispersed in 20 mL of ultrapure water.

Furthermore, 10 mL of Fe₃O₄@PEG–COOH (20 mg mL⁻¹) were mixed with N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) (0.442 g) and N-hydroxysuccinimide (NHS) (0.053 g) for 1 hour and after that NH₂NH₂·H₂SO₄ (0.2 g) was added by vigorous stirring, overnight. Then, hydrazide modified nanoparticles (Fe₃O₄@PEG–CONHNH₂) were magnetically decanted and washed thoroughly with ultrapure water and re-dispersed in 10 mL of ultrapure water.

Another method used to functionalize Fe₃O₄ was the dopamine self-polymerization onto nanoparticle surface.^27,28 For coating with polydopamine (PDA), 200 mg of Fe₃O₄ were re-dispersed under continuous stirring in 10 mL of 10 mM dopamine solution (PBS, pH = 8.5) at room temperature for 24 hours. The polydopamine-modified nanoparticles (Fe₃O₄@PDA) were carefully magnetically decanted and washed thoroughly with ultrapure water and re-dispersed in 10 mL of ultrapure water (20 mg mL⁻¹).

Characterization

X-ray powder diffraction (XRD) patterns of Fe₃O₄ and Fe₃O₄@PDA were recorded in a D8 Advance (from Bruker) diffractometer in the 10 to 90°/2θ range and CuK_α1 radiation (40 kV, 110 mA). The magnetite nanoparticle size distribution (N = 130), morphologies and the polydopamine film thick on nanoparticles were analyzed by transmission electron microscopy (TEM JEOL JEM 1010) and high resolution TEM (HRTEM JEOL 2010F), using accelerating voltage of 60 kV and 200 kV, respectively. X-ray photoelectron spectroscopy (XPS) of Fe₃O₄ and Fe₃O₄@PDA were carried out on Thermo Scientific K-Alpha spectrometer with monochromatized Al Kα (1487 eV) radiation. All XPS spectra were corrected according to the C 1s line at 284.6 eV. All coated nanoparticles (Fe₃O₄@PEG–COOH, Fe₃O₄@PEG–CONHNH₂ and Fe₃O₄@PDA) were characterized using Fourier transform infrared spectroscopy (FTIR) in Rayleigh WQF-510 spectrometer (in KBr pressed disks). Fluorescence spectra were recorded on a Shimadzu-rf-5301pc spectrofluorometer (Japan). The magnetic behaviour of magnetite nanoparticles was investigated by superconducting interference device magnetometry with MPMS Quantum Design equipment. BET surface area of magnetite nanoparticles was measured in Micromeritics ASAP 2020 V1 device.

Strategies for IgM-dengue coupling on Fe₃O₄ nanoparticles

IgM-dengue antibodies were conjugated on magnetite nanoparticles by coupling 0.5 mg of Fe₃O₄ core (0.05 mL, 10 mg mL⁻¹) with 0.01 mL IgM-dengue negative and positive control (3.4 mg mL⁻¹), from UMELISA DENGUE IgM PLUS kit. The IgM-dengue immobilizations were carried out through five strategies.

Strategy 1 (Fe₃O₄–IgM). IgM-dengue antibodies were incubated with Fe₃O₄ dispersion for 3 hours at 37 °C. The antibody-modified nanoparticles (Fe₃O₄–IgM) were magnetically decanted and washed (4 times) thoroughly with 0.1 mL of washing solution (Tris–NaCl-Tween 20 buffer) to remove the non-reacted IgM, and then, re-dispersed in PBS 1× pH = 7.4 at 10 mg mL⁻¹ of magnetic core. The antibody-modified nanoparticles (Fe₃O₄–IgM) were kept at 4 °C until use.

Strategy 2 (Fe₃O₄@PEG–COOH–IgM). Fe₃O₄@PEG-COOH nanoparticles were dispersed in 0.05 mL of EDC/NHS mixture (50/10 mM) for 1 hour. Then, IgM-dengue antibodies were added at the reaction mixture and incubated for 3 hours at 37 °C. The same procedure of washing and preservation employed in strategy 1 was followed.

Strategy 3 (Fe₃O₄@PEG–CONHNH₂–IgM). In a previous step, 0.1 mL of IgM-dengue control stock solutions were mixed with 0.02 mL of NaIO₄ solution (100 mM) by continuous stirring for 20 minutes, at room temperature in complete darkness. To verify the oxidation of the carbohydrate, 0.01 mL of antibody solution were added to 0.03 mL of freshly prepared Purpald solution (Sigma, cat. no. 162892). The color of the solution turned purple in a few minutes indicating the presence of aldehydes. The oxidation is stopped by adding PBS 1× pH = 7.4 and filtering the entire volume in a 10k MWCO centrifuge filter (Millipore) at 2000g (6000 rpm) and 4 °C. The pellet was re-suspended in final volume of 0.09 mL of PBS 1× pH = 7.4 to keep the initial concentration. After that, 0.01 mL of oxidized IgM-dengue antibodies solution were incubated with 0.05 mL of Fe₃O₄@PEG–CONHNH₂ for 3 hours at 37 °C. Once the conjugate Fe₃O₄@PEG–CONHNH₂–IgM was obtained, the same procedure of washing and preservation was followed as described in strategy 1.

Strategy 4 (Fe₃O₄@PDA–IgM). IgM-dengue antibodies were incubated with Fe₃O₄@PDA dispersion for 3 hours at 37 °C. The same procedure of washing and preservation was followed as described in strategy 1.

Strategy 5 (Fe₃O₄@PDA–antiIgM–IgM). Fe₃O₄@PDA (10 mg of magnetic core) were resuspended in 1 mL of Anti-Human IgM (μ-chain specific) solution (1 mg mL⁻¹) with moderated stirring for 3 hours at room temperature. Subsequently, the conjugated Fe₃O₄@PDA–antiIgM was magnetically decanted, thoroughly washed (4 times) with 0.1 mL of washing solution (Tris–NaCl-Tween 20 buffer) to remove the non-reacted antiIgM and re-dispersed in PBS (1 mL) at 10 mg mL⁻¹ of magnetic core. After, IgM-dengue antibodies were incubated with Fe₃O₄@PDA–antiIgM dispersion for 3 hours at 37 °C. The same described procedure of washing and preservation was followed as described in strategy 1.

Measuring the amount of IgM-dengue conjugated on Fe₃O₄ by the different conjugation strategies

The amount of IgM-dengue conjugated on Fe₃O₄ surface through the conjugation strategies were analyzed by Bradford assay and SDS gel electrophoresis. For Bradford assay, a reference solution was prepared with exactly the initial antibody concentration and media conditions (pH, ionic strength) and BSA was used as protein standard (successive dilutions 1 : 2 from 0.5 mg mL⁻¹). SDS gel electrophoresis was performed as described by Laemmli²⁹ using a 12.5% polyacrylamide gel (SDS-PAGE) and Coomassie blue staining.

UMELISA dengue IgM plus

Standard. UMELISA Dengue IgM Plus assays were carried out by duplicate and the results were reported as spectra average. In addition, proteins were incubated for 30 minutes at 37 °C in wet atmosphere and all steps were followed by continuous watching (4 times with 0.025 mL of Tris–NaCl-Tween 20 buffer for 30 seconds). Ninety-six-well microtiter plates were coated with 0.02 mL Anti-Human IgM (1 mg mL⁻¹) in coating buffer (pH 9.6) at 4 °C overnight and treated with blocking buffer (0.5% BSA (Sigma) in PBS) for 2 h at 37 °C. Afterward, negative and positive controls of IgM-dengue (0.01 mL, 3.4 mg mL⁻¹) were captured by anti-μ chain specific IgM antibodies anchored onto microplates. After, not linked components were eliminated by washing; 0.01 mL of four serotypes dengue-specific antigens solutions were added to be captured by IgM-dengue antibodies. Once not linked antigens were removed, 0.01 mL of biotinylated anti-dengue antigens monoclonal antibodies solution were added. After another washing step, 0.01 mL of streptavidin-alkaline phosphatase conjugated were added to establish whether immunological chain had taken place. The immune-complex was then revealed by adding 0.02 mL of 4-methylumbeliferil phosphate solution. After 10 min of incubation, 0.01 mL of hydrolyzed substrate was removed from the plate and diluted in 2.5 mL of PBS 1× pH = 7.4. The fluorescence spectra were measured in a spectrofluorometer with the followed conditions: excitation maximum at 360 nm and emission maximum at 450 nm.

Modified with nanoparticles. 0.05 mL of different IgM–Fe₃O₄ conjugates (10 mg mL⁻¹ of magnetic core) identified as Fe₃O₄–IgM, Fe₃O₄@PEG–COOH–IgM, Fe₃O₄@PEG–CONHNH₂–IgM, Fe₃O₄@PDA–IgM and Fe₃O₄@PDA–antiIgM–IgM were incubated with 0.5 mL of BSA (0.5% w/w) for 2 hours at 37 °C in wet atmosphere. Subsequent steps were carried out similarly to the standard procedure. Besides, Fe₃O₄ nanoparticles were employed as solid phase instead of the microwells. Therefore, all ELISA coupling steps were followed by magnetically decanting and washing (4 times) with 0.1 mL of washing solution to remove the non-reacted components.

Results and discussion

Characterization of magnetite nanoparticles

Fe₃O₄ nanoparticles were synthesized by coprecipitation of Fe²⁺/Fe³⁺ ions in alkali media. Fig. 1 summarizes the structural characterization for the nanoparticles to be conjugated. TEM micrographs show magnetite nanoparticles with pseudospherical shapes and average diameter of 8 ± 2 nm (Fig. 1a). The observed size distribution is characteristic for coprecipitation method; due to uncontrollability of the nucleation, crystal growth, coalescence and agglomeration, which simultaneously take place.

Fig. 1 (a) TEM micrographs of Fe₃O₄ nanoparticles with (inside) size distribution (N = 130) and (b) its electron diffraction pattern. (c) HRTEM image of Fe₃O₄ nanoparticles and (inside) graphic representation of 220 and 111 planes. (d) XRD analysis of Fe₃O₄ nanoparticles. (e) High-resolution XPS spectra of Fe₃O₄ nanoparticles and deconvolution for the Fe 2p and O 1s signals.

FFT image (Fig. 1b) exhibits the family of planes corresponding to (111), (220) and (311) of the spinel type structure. The interplanar distance, measured from the adjacent lattice fringes of the nanoparticles, was about 0.414 nm and 0.296 nm corresponding to (111) and (220) planes, respectively, of the Fe₃O₄ single crystal with a cubic spinel structure (Fig. 1c).

XRD pattern (Fig. 1d) confirmed Bragg positions peaks for a cubic spinel structure according to the crystalline nature of Fe₃O₄ (JCPDS 19-629). The pattern showed diffraction peaks at 21.05°, 30.19°, 35.47°, 43.04°, 53.40°, 56.95°, and 62.7° corresponding to the (111), (220), (311), (400), (422), (511) and (440) Bragg reflections with interplanar distances of 0.421, 0.296, 0.252, 0.209, 0.172, 0.161, 0.148 nm, respectively. These patterns suggest the polycrystalline nature of nanoparticles with an inverse spinel structure. The crystallite size was calculated through Debye–Scherrer equation by using the full-width at half diffraction peak (FWHM) for the (331) X-ray diffraction line, resulting a value of 8.6 nm. The unit cell parameter, calculated from the pattern, results 0.8391 nm, which is practically coincident with the reported parameter in the literature (0.8396 nm) for the magnetite phase.³⁰

The XPS spectrum obtained for the synthesized magnetite nanoparticles corresponds with the reported in the literature. In this sense, the deconvolution results of high-resolution spectra for the Fe 2p and O 1s orbitals are presented in Fig. 1e. The XPS peaks of Fe 2p_3/2 (710.7 eV) and Fe 2p_1/2 (724.4 eV) are shown. In the deconvoluted peak of Fe 2p_3/2, the mean relative areas result 0.33 : 0.66 for Fe(II)–O/Fe(III)–O (atomic ratio closer to 0.5), as expected from the Fe₃O₄ stoichiometry, and the slight presence of a satellite peak in approximately 718 eV suggests the presence of magnetite as prevailing phase.³¹ In addition, the small peaks (A) around 714 and 727 eV may be attributed to Fe–OH.³² Deconvolution of the O 1s signal shows the presence of Fe–O (529.8 eV) and Fe–OH (531.4 eV) species.

The magnetic response of magnetite nanoparticles was recorded as a function of applied field at 5 K and 300 K (see Fig. S1(a) in ESI†). At room temperature, nanoparticles exhibited superparamagnetic behavior with the magnetic saturation, 72 emu g⁻¹ lower than the bulk magnetite value; 90 emu g⁻¹. The saturation magnetization can be most likely affected by several features, such as the spin disorder layer, size effect, the incomplete crystallization of magnetite, irregular morphologies and the magnetostatic interaction responsible for the agglomeration of magnetite particles. The coercive field (H_c) was 22,4 Oe and the magnetic diameter calculated by using Chantrell's equations was 4.8 nm. The variation of the magnetization with increasing temperature under a field of 50 Oe was recorded for this sample after cooling with zero field (ZFC) and, after cooling with an applied field of 50 Oe. From these curves the susceptibility (χ_i) versus temperature were obtained (see Fig. 1S(b) in ESI†). According to the behaviour of ZFC/FC curves and the blocking temperature (T_B) near to 300 K, pristine magnetite nanoparticles present magnetic interactions. TEM images reinforce this assumption. The nanoparticles tend to agglomerate in the absence of a capping agent or dispersant media (usually used in magnetic measures to ensure nanoparticles dispersion) provoking the onset of magnetic interactions. Because that reason, T_B (below 20 K) normally determined for superparamagnetic uncovered magnetite nanoparticles spread in solid dispersant media is considerably lower than the real aggregate pristine magnetite nanoparticles (300 K). The extrapolation of the linear part in the graph of the FC inverse susceptibility χ⁻¹ versus temperature, in accordance to the Curie–Weiss law, gives a negative value of the ordering temperature T_C. This suggests a transition from antiferromagnetic or ferrimagnetic ordering to ferromagnetic ordering.

Magnetite nanoparticles were further coated with polyethyleneglycol dicarboxylic acid (Fe₃O₄@PEG–COOH) via the coordination between PEG terminal carboxylic acid moieties on the magnetite surface. On the other hand, magnetite nanoparticles also were capped with hydrazine groups and polydopamine film through the spontaneous oxygen-mediated self-polymerization of dopamine in PBS pH 8.5 (Fe₃O₄@PDA). By naked eyes, all coated nanoparticles improved their colloidal stability in aqueous media during time, suggesting desegregates dispersion.

FT-IR spectra for pristine magnetite nanoparticles and after their functionalization are shown in Fig. 2 and the most relevant vibrations are assigned in Table 1. FT-IR spectra of all capped Fe₃O₄ nanoparticles showed a main absorption band around 572 cm⁻¹ assigned to the Fe–O stretching modes of the magnetite phase. For Fe₃O₄@PEG–COOH, strong absorption bands were obtained at 3250 cm⁻¹ (ν_OH for Fe–OH moieties on nanoparticle surface), 1600 cm⁻¹ and 1400 cm⁻¹ (antisymmetric and symmetric ν_C=O, respectively, for carboxylate group). The absence of ν_C=O signal for carboxylic acid groups suggests the practically completed deprotonation during the coordination between Fe₃O₄ and HOOC–PEG–COOH. In the case of Fe₃O₄@PEG–CONHNH₂, the characteristic bands corresponding to carbonyl and C–N stretching mode of amide moieties are observed. Spectrum of Fe₃O₄@PDA showed aromatic and C–N stretching mode bands of polydopamine shell.

Fig. 2 FT-IR transmittance spectra for (a) Fe₃O₄ nanoparticles and Fe₃O₄ nanoparticles capped with (b) carboxylates, (c) hydrazides and (d) polydopamine.

Table 1 FT-IR spectra assignments for different magnetite nanoparticles

	Value (cm^−1)	Assignment
Fe3O4	572	νFe−O
Fe3O4@PEG–COOH	3250	νOH
	1600
	1400
	572	νFe–O
Fe3O4@PEG–CONHNH2	1615	νC=O (amide)
	1134–975	νC–N
	572	νFe–O
Fe3O4@PDA	1569–1490	νC=C (aromatic)
	1290	νC–N
	890	νC–H (aromatic) or νN–H
	572	νFe–O

---

Polydopamine coated nanoparticles were also characterized by XRD, HRTEM and XPS (Fig. 3).

Fig. 3 (a–c) TEM micrographs of Fe₃O₄@PDA nanoparticles. (d) High-resolution XPS spectra of Fe₃O₄@PDA nanoparticles and deconvolution for the Fe 2p and O 1s signals.

HRTEM analysis revealed that Fe₃O₄ were coated with a polymeric material with thickness ranged from 1 to 1.5 nm (Fig. 3b). Additionally, tubular micrometric assemblies with Fe₃O₄ nanoparticles inside were observed probably due to PDA aggregation processes through non-covalent interactions to build up the supramolecular structure of PDA (Fig. 3c).^33,34 A similar XRD pattern was observed for the core–shell Fe₃O₄@PDA nanoparticles, suggesting that the crystalline structure of the nanomaterial was not affected by coating with the polymer film. The high-resolution XPS spectra of Fe 2p obtained for Fe₃O₄@PDA and Fe₃O₄ were practically equal. The O 1s core-level spectrum could be resolved into three typical peaks at 530.2 eV (Fe–O), 531.4 eV (C=O, quinone) and 533.0 eV (C–OH, catechol). This result suggests the successful deposition of polydopamine film.

Amount of IgM-dengue conjugated on Fe₃O₄ by the different conjugation strategies

The amount of immobilized IgM-dengue on Fe₃O₄ nanoparticle surface is critical for their recognition capability.

SDS-PAGE using Laemmli procedure was employed to determine the relative amount of IgM-dengue conjugated on Fe₃O₄ surface through the four first conjugation strategies (see Fig. S2 in ESI†).

According to the qualitative electrophoresis results, since the amount of positive control of IgM-dengue injected in control lane was 34.21 μg, which is the same original amount used during conjugations, apparently only slight fraction of initial added antibodies was associated on nanoparticles surface. Another possible reasoning is that the antibodies coupled on nanoparticles surfaces are resistant to the reduction with 2-mercaptoethanol used in the reducing SDS-PAGE.

In this regard, another method to quantify IgM coupled on magnetite nanoparticles was necessary employed. In this sense, samples of supernatants after every immobilization process were withdrawn and measured using Bradford assay. A reference solution was prepared with exactly the initial antibody concentration and media conditions (pH, ionic strength) and BSA was used as protein standard. Therefore, the decrease in the supernatant concentration can be directly correlated to the amount of the immobilized antibody.

According to the Bradford results (Table 2), the amounts of conjugated antibodies in strategy 1 (Fe₃O₄–IgM) and 2 (Fe₃O₄@PEG–COOH–IgM) were very similar each other and slightly smaller than that observed for strategy 3 (Fe₃O₄@PEG–CONHNH₂–IgM). The strategy 4 (Fe₃O₄@PDA–IgM) presented higher amount of conjugated antibodies than the strategies from 1 to 3. The strategy 5 (Fe₃O₄@PDA–antiIgM–IgM) had the highest conjugation yield.

Table 2 Amount of coupled IgM-dengue antibodies on Fe₃O₄

Strategies	μg (IgM)/mg (Fe3O4)	Conjugation yield (%)
1	12.6	36.8
2	13.2	38.6
3	14.5	42.3
4	15.5	45.3
5	18.8	55.0

---

The fewest number of conjugated IgM-dengue antibodies obtained in strategy 1 is probably due to reversible attachment which is characteristic in physical adsorption. For the strategy 2, the coupling yield depends of the distribution of active amino residues in IgM antibodies and the amount of carboxylic moieties on magnetite surface previously activated it with carbodiimide/N-hydroxysuccinimide. In the case of strategy 3, the steric hindrance that must be generated by the aldehydes moieties in Fc region of IgM pentameric structure can affect the conjugation yield.³⁵

Therefore, the above results suggest that quinone groups on Fe₃O₄@PDA surface are better anchoring points and have larger binding capability than for the others. Finally, despite a drawback of the strategy 5 in which IgM antibodies are not attached on the magnetite surface as strongly as if they were covalently immobilized, this methodology presented the highest yield.

Measurement of efficacies of the IgM–Fe₃O₄ conjugation strategies

UMELISA Dengue IgM Plus assay was carried out for measuring the efficacies of the 5 conjugation strategies, previously mentioned.

The standard ELISA assay was lightly modified by using Fe₃O₄ nanoparticles as solid phase instead of the microwells. It is noticeable to point that the huge weight of IgM antibodies capping magnetite surface, as well as the interacting force lead aggregation of all IgM–Fe₃O₄ conjugates with further nanoparticles precipitation. However, this apparently inconvenient is an advantage considering the practical use of such conjugates as solid ELISA support. Two key issues should be taken in account to achieve suitable results. First, the effectiveness of blocking with BSA solution is a fundamental step. During IgM coupling procedures, the separation of isolated uncovered magnetite nanoparticles from conjugated systems was unpractical. Thus, blockers as BSA are used to avoid unspecific proteins adsorption on naked magnetite nanoparticles allowing low fluorescence background. Secondly, the measured fluorescence intensity is affected by scattering process due to light-nanoparticles interaction. Therefore, hydrolyzed substrate was removed from nanoparticles containing plate's microwells.

The higher the amount of conjugated IgM-dengue, with a suitable spatial conformation for dengue antigen recognition, the higher the fluorescence intensity. Therefore, a high fluorescence intensity will correspond with better efficacy of the employed IgM-dengue conjugation strategies.

Fig. 4 shows fluorescence emission spectra for the different conjugation strategies and the standard UMELISA Dengue IgM Plus assay for 3 hours of IgM-dengue incubation time. Reported emission spectra were obtained by subtracting the negative control spectra to the positive control spectra for each corresponding system (see Fig. S3 in ESI†).

Fig. 4 At the top: fluorescence emission spectra of UMELISA DENGUE IgM PLUS assays for different IgM–Fe₃O₄ conjugates: strategy 1 (Fe₃O₄–IgM), strategy 2 (Fe₃O₄@PEG–COOH–IgM), strategy 3 (Fe₃O₄@PEG–CONHNH₂–IgM), strategy 4 (Fe₃O₄@PDA–IgM) and strategy 5 (Fe₃O₄@PDA–antiIgM–IgM). Bottom: Comparison of the intensity ratio of fluorescence emission at 450 nm of different IgM–Fe₃O₄ conjugates. (F) and the fluorescence emission intensity obtained by using traditional assay (F_{standard ELISA}).

The fluorescence intensities of all conjugates were higher than that of standard ELISA assay. All differences among assays with nanoparticles as solid phase and the traditional ELISA could have explanation taken into account the contact surface area of the different solid supports. The measured BET surface area of magnetite nanoparticles was 92.9 m² g⁻¹ (0.46 m² for 0.5 mg of core Fe₃O₄ used as the solid support) which is much greater than the ELISA plate's microwells (6.4 × 10⁻⁵ m², taking in account geometrical considerations). Moreover, Makky et al.³⁶ reported the apparent lateral size of IgM with pentameric morphology as 38 ± 1 nm (calculated IgM area 1.13 × 10⁻¹⁵ m², considering spherical shape). Therefore, nanoparticles must present higher amount of IgM-dengue antibodies immobilized on their surface (6 × 10⁻¹⁰ mol) than the captured in the traditional ELISA plate (9 × 10⁻¹⁴ mol). Furthermore, the conjugated antibodies retained their recognition capability.

As it is observed in Fig. 4, among different conjugates, strategy 3 (Fe₃O₄@PEG–CONHNH₂–IgM) showed an inconspicuous fluorescence intensity 2.4 times higher than standard ELISA, despite the fact that presents a middle conjugation yield and the covalent attachment of aldehyde-antibodies to hydrazide modified surfaces orients antigen-binding sites in end-on fashion,¹² which could have led a better assay performance. Nevertheless, the chemical modification of sugar moieties also could have affected IgM antigen recognition capability.³⁷ Next, strategy 1 (Fe₃O₄–IgM) and strategy 2 (Fe₃O₄@PEG–COOH–IgM) presented a fluorescent intensity higher than strategy 3 (6 and 5 times higher than standard ELISA, respectively) despite the lower amount of coupled IgM antibodies.

In the case of strategy 4 (Fe₃O₄@PDA–IgM) the fluorescence was significantly increased despite the probably randomly spatial orientation of antibodies on the nanoparticles surface. In this case, the fluorescence intensity was 12 times higher than standard ELISA. These results suggest that polydopamine coating provides a versatile platform for the antibodies immobilization with high conjugation yield and without losing their recognition capabilities.

The highest intensity of light emitted by fluorescence was obtained in strategy 5 (Fe₃O₄@PDA–antiIgM–IgM), which was 18 times more effective than standard ELISA. In this methodology, besides presenting the highest IgM conjugation yield, the Anti-Human IgM (μ-chain specific) antibodies anchored on Fe₃O₄ surface ensure site-specific conjugation of IgM-dengue through their Fc region and free Fab regions far away from the NP surface which allows a better dengue's antigens recognition.

In order to demonstrate the sensitivity improvement of Fe₃O₄@PDA–antiIgM conjugate as a new platform for dengue recognition, a sandwich immunoassay for measuring different IgM-dengue antibody concentrations was carried out and compared with the standard assay. The corresponding calibration curves are presented in Fig. 5. Good linearity of the calibration curve was obtained in the concentration range from 0 to 100 μg mL⁻¹ (y = 3.0x + 91, r² = 0.974 for Fe₃O₄@PDA–antiIgM and y = 0.31x + 25.7, r² = 0.995 for standard ELISA). These results reveal that Fe₃O₄@PDA–antiIgM is almost 10 times more sensitive than standard ELISA (interpreted as the calibration curve slope). Additionally, Fe₃O₄@PDA–antiIgM conjugate showed 6 times lower limit of detection (31.1 ng, S/N = 3) than the standard ELISA (193 ng).

Fig. 5 At the top: fluorescence spectra for Fe₃O₄@PDA–antiIgM with increasing concentrations of IgM-dengue. Bottom: Calibration curves for Fe₃O₄@PDA–antiIgM and standard UMELISA DENGUE IgM PLUS assay.

These results are consequence of a combination of factors such as: (i) the very high aspect ratio (surface area/volume) of the Fe₃O₄ nanoparticles which permits an increase of the number of antibodies on nanoparticle surface; (ii) the proved efficacy of polydopamine scaffold for biomolecules coupling and (iii) the anchoring of antiIgM receptors on Fe₃O₄ surface allows specificity and effective IgM-dengue antibodies capture.

Conclusions

In summary, five different strategies to conjugate IgM-dengue antibodies on magnetite nanoparticles were evaluated and compared with traditional ELISA assay for dengue diagnostic. All IgM–Fe₃O₄ conjugates had better analytical response signals (fluorescence) than the traditional ELISA. The most effective strategy was IgM-dengue coupling through antigen–receptor complex formation with Anti-Human IgM (μ-chain specific) antibody previously attached on core–shell Fe₃O₄@PDA nanoparticles. The analytical performance of Fe₃O₄@PDA–antiIgM approach was almost 10 times more sensitive than traditional UMELISA DENGUE IgM PLUS assays. These results suggest that this system could be an effective and versatile platform for dengue IgM detection.

Acknowledgements

The authors acknowledge the Center of Protein Studies, Faculty of Biology, University of Havana, Cuba; the Immunoassay Center, Cuba; the Center of Molecular Immunology, Cuba and the Center for Applied Science and Advanced Technology of IPN, Legaria Unit, Mexico for access to their experimental facilities. This study was partially supported by the CONACyT (Mexico) project CB-2014-01-235840. G. O. thanks the support provided by SIP-IPN through an innovation project for student.

References

1. R. M. Lequin, Clin. Chem., 2005, 51, 2415–2418.
2. A. D. Stefano, G. Volpe, G. Adornetto, S. Bernardini, M. Nuccetelli, G. Gallucci, L. D. Ruvo and D. Moscone, Electroanalysis, 2014, 26, 1382–1389.
3. M. Muluneh and D. Issadore, Adv. Drug Delivery Rev., 2014, 66, 101–109.
4. J. He, M. Huang, D. Wang, Z. Zhang and G. Li, J. Pharm. Biomed. Anal., 2014, 101, 84–101.
5. X. Zhou, W. Xu, Y. Wang, Q. Kuang, Y. Shi, L. Zhong and Q. Zhang, J. Phys. Chem. C, 2010, 114, 19607–19613.
6. S. D. Soelberg, R. C. Stevens, A. P. Limaye and C. E. Furlong, Anal. Chem., 2009, 81, 2357–2363.
7. B. A. Otieno, C. E. Krause, A. Latus, B. V. Chikkaveeraiah, R. C. Faria and J. F. Rusling, Biosens. Bioelectron., 2014, 53, 268–274.
8. S. Krishnan, V. Mani, D. Wasalathanthri, C. V. Kumar and J. F. Rusling, Angew. Chem., 2011, 123, 1207–1210.
9. M. S. Szymanski and R. A. Porter, J. Immunol. Methods, 2013, 387, 262–269.
10. H. Tumturk, F. Sahin and E. Turan, Analyst, 2013, 139, 1093–1100.
11. S. Kumar, J. Aaron and K. Sokolov, Nat. Protoc., 2008, 3, 314–320.
12. J. Montenegro, V. Grazu, A. Sukhanova, S. Agarwal, J. M. D. L. Fuente, I. Nabiev, A. Greiner and W. J. Parak, Adv. Drug Delivery Rev., 2013, 65, 677–688.
13. Y. Xing, Q. Chaudry, C. Shen, K. Y. Kong, H. E. Zhau, L. W. Chung, J. A. Petros, R. M. O'Regan, M. V. Yezhelyev, J. W. Simons, M. D. Wang and S. Nie, Nat. Protoc., 2007, 2, 1152–1165.
14. S. Puertas, P. Batalla, M. Moros, E. Polo, P. D. Pino, J. M. Guisán, V. Grazú and J. M. D. L. Fuente, ACS Nano, 2011, 5, 4521–4528.
15. D. C. Andrade, I. C. Borges, H. Laitinen, N. Ekström, P. V. Adrian, A. Meinke, A. Barral, C. M. Nascimento-Carvalho and H. Käyhty, J. Immunol. Methods, 2014, 405, 130–143.
16. D. Saxena, M. Parida, P. V. L. Rao and J. S. Kumar, Diagn. Microbiol. Infect. Dis., 2013, 75, 396–401.
17. R. Priya, M. Khan, M. Kameswara-Rao and M. Parida, J. Virol. Methods, 2014, 203, 15–22.
18. W. Li, L. Mao, L. Yang, B. Zhou and J. Jiang, J. Virol. Methods, 2013, 191, 63–68.
19. N. Evelyn, A. Murray, M. B. Quam and A. Wilder-Smith, Clinical Epidemiology, 2013, 5, 299–309.
20. S. Patramoola, R. H. E. Bernard, L. Natthanej, N. Chazal, P. Surasombatpattanaa, P. Ekchariyawat, S. Daoust, S. Thongrungkiat, F. Thomas, L. Briant and D. Misséa, J. Virol. Methods, 2013, 193, 55–61.
21. N. T. Darwish, Y. B. Alias and S. M. Khor, TrAC, Trends Anal. Chem., 2015, 67, 45–55.
22. C.-W. Yen, H. de Puig, J. O. Tam, J. Gomez-Marquez, I. Bosch, K. Hamad-Schifferli and L. Gehrke, Lab Chip, 2015, 15, 1638–1641.
23. Y. Zhang, J. Bai and J. Y. Ying, Lab Chip, 2015, 15, 1465–1471.
24. P. D. Sinawang, V. Rai, R. E. Ionescu and R. S. Marks, Biosens. Bioelectron., 2016, 77, 400–408.
25. Dengue guidelines for diagnostic, treatment, prevention and control, World Health Organization (WHO) and the Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, 2nd edn, 2009, ISBN 978 92 4 154787 1.
26. W. Sacchi-Peternele, V. Monge-Fuentes, M. L. Fascineli, J. R. D. Silva, R. Carvalho-Silva, C. Madeira-Lucci and R. B. D. Azevedo, J. Nanomater., 2014, 1–10.
27. M. Martín, A. G. Orive, P. Lorenzo-Luis, A. H. Creus, J. L. González-Mora and P. Salazar, ChemPhysChem, 2014, 15, 3742–3752.
28. M. Martin, P. Salazar, R. Villalonga, S. Campuzano, J. M. Pingarron and J. L. Gonzalez-Moraa, J. Mater. Chem. B, 2014, 2, 739–746.
29. U. K. Laemmli, Nature, 1970, 227, 680–685.
30. W. Kima, C. Y. Suha, S. W. Choa, K. M. Roha, H. Kwona, K. Songa and I. J. Shonb, Talanta, 2012, 94, 348–352.
31. T. Yamashita and P. Hayes, Appl. Surf. Sci., 2008, 254, 2441–2449.
32. O. F. Odio, L. Lartundo-Rojas, P. Santiago-Jacinto, R. Martínez and E. Reguera, J. Phys. Chem. C, 2014, 118, 2776–2791.
33. Y. Ding, L. Weng, M. Yang, Z. Yang, X. Lu, N. Huang and Y. Leng, Langmuir, 2014, 30, 12258–12269.
34. J. Zhou, C. Wang, P. Wang, P. B. Messersmith and H. Duan, Chem. Mater., 2015, 27, 3071–3076.
35. J. N. Arnold, M. R. Wormald, R. B. Sim, P. M. Rudd and R. A. Dwek, Annu. Rev. Immunol., 2007, 25, 21–50.
36. A. Makky, T. Berthelot, C. Feraudet-Tarisse, H. Volland, P. Viel and J. Polesel-Maris, Sens. Actuators, B, 2012, 162, 269–277.
37. V. B. Klimovich, Biochemistry, 2011, 76, 534–549.

---

Footnote

† Electronic supplementary information (ESI) available: Details of mass magnetization versus applied field and ZFC/FC curves for magnetite nanoparticles, SDS-page analysis of different IgM–Fe₃O₄ conjugates, fluorescence spectra for ELISA assays for IgM-dengue coupled on magnetite nanoparticles by the different strategies and the synthesis and characterization of polyethyleneglycol dicarboxylic acid. See DOI: 10.1039/c6ra23260d

---