Nanotechnology broke into society a few decades ago and is currently present in many aspects of our lives. Its great success is due to its proposal to provide small solutions to big problems. Nanotechnology encompasses several fields of science as chemistry, physics, mathematics, pharmacy, and computer or environmental sciences, being able to create different materials and devices with a huge range of applications. Furthermore, the possibility of combination between a nanosized material into hybrid nanostructures, controlling its size, shape and composition, gives the scientific community a powerful tool to design nanomaterials with multiple functionalities, which has a special importance in biomedicine (1). Nanoparticles are particularly important for biomedical purposes, as their unique properties like high surface-to-volume ratio, similar size of biomolecules, chemically tunable surface or, in many cases, particular physicochemical properties make them unique platforms for certain applications. The huge potential of nanoparticles in the field of nanomedicine has led the scientific community to pay more and more attention to this kind of nanomaterials, which have been very useful in both in vivo and in vitro applications (2). Focusing on the in vitro applications, the use of nanoparticles in diagnosis tends to lead to better assays sensitivity and selectivity, assuming a substantial improvement over conventional diagnostic systems (3). Silica (SiO₂)-composite nanoparticles are undoubtedly the most widely used for a wide variety of applications, being extensively used for diagnostic purposes (4). Their synthesis is cheap and easy, as particle size can be controlled simply, is biocompatible, and allows a versatile surface chemical modification. They also provide a robust solid support for the immobilization of different biomolecules, which makes them ideal platforms in immunoassay applications (5). These highly selective and sensitive assays take the advantage of the specific interaction between an antigen and an antibody, and constitute a powerful tool to determine diverse biological substances.

Herein we focus on the development of nanoparticles for immunoassays applications in the field of the diagnosis of allergic reactions to drugs. Betalactam antibiotics (BLs) are drugs especially important as, due to their extensive use against bacterial infections, they are the most commonly involved in hypersensitivity reactions, becoming a worldwide problem (6). BL allergy can be induced by different immunological mechanisms, being mostly induced by IgE mediated mechanisms (7). Symptoms can range from simple skin problems to anaphylactic shock (6). Their diagnosis is complex, involving the clinical history and in vivo methods (skin test or drug provocation tests) which can be risky. Moreover, in vitro tests consisting on the basophil activation test and the serum specific IgE (sIgE) determination are helpful for diagnosis although with lower sensitivity (8). Based on patient safety, the use of these complementary in vitro methods in combination with skin tests (STs) is preferred for avoiding the performance of the drug provocation tests (9, 10).

Among in vitro tests, serum drug sIgE determination using immunoassays has traditionally been the most common in vitro method for the diagnosis of BL allergy (11). Nowadays the most widely applied method in clinical diagnosis is the commercial ImmunoCAP (Thermo-Fisher), a standardized fluoro-enzyme immunoassay (FEIA). However, when applied to BL allergy, sensitivity is low (0-50%) and variable (10, 12, 13). Moreover, false benzylpenicillin (BP) allergy diagnoses have been reported with ImmunoCAP (14, 15). The previous alternative immunoassay for quantification of BL-sIgE consisted on the conventional radioallergosorbent test (RAST), which is nowadays only performed in specialized laboratories for research purposes (11). These customized immunoassays show higher sensitivity (43-75%) and specificity (68-83%) for BLs (8). The main difference between them refers to the antibody detection technique (fluorescence vs radioactivity), although the cellulose materials in solid phase (sponge matrix contained in a small capsule vs paper discs) are also used for capturing the target sIgE (16). Both solid phases of the commercial ImmunoCAP or the conventional RAST consist on a cellulose polymer activated with cyanogen bromide where the drug-poly-L-lysine (PLL) conjugates are covalently attached. On the other hand, the configuration of the home-made RAST is much more versatile, allowing customizing carriers, solid phases, and activation chemistry (17).

A critical point in this technology is the nature of the drug-carrier conjugate, based on the hapten hypothesis, which states that a drug can only induce an immediate allergic reaction after covalent binding to a carrier protein (18), which is specifically recognized by sIgE antibodies. However, the poor understanding of in vivo drug–protein conjugates could explain the suboptimal diagnostic value of both methodologies. Besides the structure adopted by the drug after covalently binding to the protein and the nature of the carrier molecule, other factors as the solid phase material, its chemical functionalization and sIgE detection techniques can also influence the sensitivity and specificity of immunoassays (8, 11, 17).

The chemical nature of the hapten(drug)-carrier(protein) conjugate has been extensively studied with the aim to emulate in vitro the in vivo recognition process (18, 19). Moreover, numerous attempts to optimize and increase the sensitivity of the homemade RAST assays have been addressed taking into account the promising benefits of nanomaterials (20, 21). In this context, PAMAM dendrimers have been proposed as perfect substitutes of the carrier protein in the hapten-carrier conjugate. These dendrimer structures provide structural homogeneity, integrity, and controlled multivalence, which allows a high density of haptens in their surface in a reproducible synthetic way (22). These dendrimeric antigen conjugates (DeAns) showed sIgE recognition toward the drug (BP or amoxicillin (AX)) anchored on its periphery (22, 23). Their attachment to cellulose discs allowed the fabrication of discs in which the degree of functionalization, the chemical structure of the conjugate and the linkers were perfectly determined (24). Both the greater degree of functionalization of the cellulose surface disc (24, 25) and the higher generation of dendrimer with which the cellulose was grafted involved an increased density of haptens in the material (26). These factors demonstrated to influence the ability of such materials to capture sIgE from patient sera, showing potential for their use in drug allergy diagnostic tests. These dendrimers, as new carrier molecules, and chemistry linkages, can be combined with other solid supports: nano-sized zeolites (25) and silica particles composites (27) as their high surface area could translate into an improved sensitivity. In vitro evaluation of resulting materials with sera from BL allergic patients led to a successful proof of concept. However, preparation and functionalization of such zeolite turned out to be too expensive to standardize its use for clinical practice, whereas advantages of silica particles justify their use as solid phase for obtaining DeAn-inorganic-organic composites that provide highly specific and sensitive tests for detection of AX-sIgE (27).

Complex and intrinsic issues for diagnosing BL-allergic patients are the fact that drug-sIgE levels are very low (affecting to the low sensibility of immunossays) and the possibility of presenting reactions to more than one kind of BL (affecting to specificity). Based on their sIgE recognition, patients can be classified as allergic to only one kind of penicillin or to several penicillins, or can also present cross-reactivity with other BLs containing similar side chains (28). Diagnosis of such patients implies carrying out the diagnostic algorithm for each suspected penicillin, is time consuming, and requires great efforts in the clinical routine. Therefore, tools for a single screening of BL-IgE antibodies with different specificities could be of interest for a quick diagnosis. In this context, well-characterized bi-epitope DeAns,decorated with both AX and BP allergenic determinants [named amoxicilloyl (AXO) and benzylpenicilloyl (BPO)], showed the ability of specific recognition by IgE of patients allergic to both, AX and BP, with potential in the development for universal in vitro detection of BL allergies (23).

Based on those promising results, herein we present the preparation of new multivalent mono and bi-epitope DeAn@SiO₂ composites, consisting on silica nanoparticles decorated with BL-DeAns. PAMAM-G2 dendrimers were selected as carrier molecule and AX and/or BP as the allergenic determinants. The chemical modification of the particle surface was designed in order to allow checking the quality and composition of the nanocomposites, using standard techniques as zeta potential or IR measurements and NMR spectroscopy. The controlled homogeneous composition of the nano-composites provides the high reproducibility needed for their in vitro applications. The methodology used to anchor the DeAns was optimized in order to minimize the nonspecific interactions and facilitate the effective exposure of the hapten-carrier molecules to specific IgE. This, together with the larger interaction area provided by the nanoparticles, increments the likelihood of capturing sIgE, which is particularly important for overcoming the low sensitivity of current immunoassays. Their ability to quantify AX-sIgE and BP-sIgE was evaluated in sera patients and compared with current immunoassays techniques, showing improvements in terms of sensibility and specificity.

Several aspects have to be taken into account for the clinical application of the nanomaterial. Minimizing non-specific protein adsorption interactions between the nanoparticle surface and the sera to be analyzed is one of the most important factors. A versatile surface chemistry that allows a highly density of allergenic molecules in a non-hydrophobic environment has to be undertaken for a correct application of the nanomaterial in the in vitro test.

An unquestionable property that nanomaterials provide is the intrinsic high surface area to volume ratio of these materials. This promotes a high functionalization ratio of the particles that can be involved in the presence of a large number of recognition sites in a relatively small amount of the material used. This feature is mostly favorable for overcoming sensitivity limitations. Moreover, due to practical aspects in the manipulation of the particles in the day-to-day hospital work, a particle size optimization is needed since particles had to be easily dispersed in the human sera samples for optimizing molecular interactions (IgE-allergenic determinant and IgE-anti IgE) during incubation steps and easily recovered by centrifugation. To ensure agile, operational, and efficient use of the material in clinical diagnosis, a compromise has been reached by preparing nanocomposites of 500 nm in diameter (27).

Surface chemical modification is a key point when the particles are intended to be used with human samples. In this sense, a previously studied methodology has been used, in which the reproducibility in the composition of the final composites is guaranteed. However, some of the conditions used during the chemical treatment were slightly different from our previous described procedures in order to increase the degree of amino functionalization. One of the advantages of this synthetic methodology is the possibility of checking all reaction steps, by using well known, cheap, and easy procedures.

Zeta potential (ξ) measurements were used to monitor the derivatization process ( Table 2 and Figure 3 ). Naked particles result negatively charged in PBS (solvent that mimics physiological pH 7.4) due to their pK_a (6.8) (27, 38). The introduction of amino groups implies a change of the charge of the SiO₂-NH₂ particles surfaces, since in PBS conditions they were protonated (27). A variation in both the value and sing of the charge of SiO₂-CO₂H particles surfaces confirms the reaction of a great amount of amino groups, since the introduced carboxylic acid would be in its carboxylate form in PBS (pK _a ~ 4.0 – 5.0), and hence negatively charge. After reaction with PAMAM-G2 dendrimer, a dramatic change in ξ was also detected for SiO₂-De particles. A positive charge that duplicates in value the previous measurement was observed. This is consistent with the introduction of 15 amino groups per dendrimer, since generation 2 PAMAM dendrimer possess 16 amino groups and it can be assumed that only one reacted with a carboxylic unit in the particle surface (25). Consequently 15 primary amines were positively charged per dendrimer, while the internal tertiary amines of the dendrimer structure remain non protonated in PBS solutions (39). Obtaining DeAn@SiO₂ composites involves the reaction of the terminal amino groups of the dendrimers with the carbonyl group on the β–lactam ring of the antibiotics. We can consider that all amino groups on the dendrimer periphery reacted, since we optimized these conditions in our previous work (26). The reaction with the antibiotics caused a shift in the sing of the zeta potential in all cases. For DeAXO@SiO₂ the amoxicilloyl epitope anchored to the dendrimer possesses a phenol group that must be uncharged in PBS solutions since its pK _a = 9.6 (and hence should be in its neutral form) (40). However, the carboxyl group must be in its carboxylate form, meaning negatively charged. On the other hand, the primary amino group of AXO has a pK _a = 7.4, which means that approximately the 50% of these amino groups are protonated at physiological pH (7.4). With this in mind we can assume that 50% of the AXO units anchored to the dendrimers are uncharged (due to the presence of both a negative and a positive charge), while the rest possess a negative charge. In the case of the BPO allergenic determinant, the absence of both the hydroxyl and the amino groups promotes that all BPO units anchored to the dendrimers are negatively charged. This is consistent with the value of ξ observed for DeBPO@SiO₂. As expected, an intermediated value of ξ is detected for DeAXO-BPO@SiO₂.

Infrared spectroscopy (IR) has been also used to characterize the particles ( Figure 3B ). As can be observed, the intense band around 1050 cm^-1, characteristic of Si-O bonds (ν _Si-O), is present in all spectra. Interestingly, two new peaks typical of amide groups (ν _C=O and σ _N-H respectively) appear in all particles where the dendrimer is covalently attached.

Ninhydrin test results indicate the introduction of 113 µmol of terminal primary amino groups per gram of particles ( Figure 3 and Table 1 ). When SiO₂-NH₂ particles were reacted with an excess of succinic anhydride, an important decrease in the number of terminal amino groups was detected (57 µmol per gram of particles). This implies that approximately 50% of the amino groups in the particles surfaces reacted with succinic anhydride and, as consequence, 56 µmol of carboxylic units have been inserted per gram of SiO₂-CO₂H particles ( Figure 3C ). After the covalent coupling with PAMAM-G2 dendrimer, a value of 170 µmol of terminal primary amino groups per gram of SiO₂-De particles was obtained. Keeping in mind that 57 µmol of amino groups were already present per gram of particles, dendrimers contribute with 113 µmol –NH₂/g particles. This implies the introduction of 7.5 µmol of PAMAM-G2/g of SiO₂-De particles (assuming that each PAMAM-G2 possess 15 free amino groups), that is the reaction of approximately 13% of the carboxylic units in the particles ( Figure S4 , ESI). Assuming that particles are prefect spheres of 500 nm of diameter, a density of 2.2 g/cm³ (41), and no aggregation occurs during the modification process ( Figure S3 ), we can estimate that 1 gram of particles possesses a surface area of approximately 6 m² (see ESI) (27). Therefore, we can estimate the presence of approximately one PAMAM-G2 dendrimer per nm² in SiO₂-De particles. Taking into account the previous approximations and assumptions, this value seems to indicate an almost complete coverage of the surface of the particles with the dendrimers.

The ¹H-NMR spectra of DeAXO@SiO₂, correspond to the one previously published (27). For DeBPO@SiO₂ ( Figure S5 ), we used the ¹H-NMR spectra of the butylamine-benzylpenicillin conjugate (Bu-BPO) as a model to study the NMR signals (42). In the spectra of DeBPO@SiO₂ we can clearly observe the presence of the most characteristic peaks of the (3S,5R,6R)-benzylpenicilloate moiety. Signals corresponding to the aromatic ring appear as a multiplet between 7.4 and 7.2 ppm. More interestingly, signals corresponding to H-5 and H-6, bonded to the β–lactam moiety, appear at 4.94 and 4.32 ppm respectively, indicating not only the presence of the BPO unit in the particles, but also the opening of the β–lactam ring of the antibiotics. When this ring is closed, H-5 and H-6 appear closely around 5.5 ppm (33). Additionally, signal corresponding to H-3 appears at 3.50 ppm. All this data indicates the presence of exclusively the open form of the β-lactam ring, which means that all BP present in the particle is covalently attached to the dendrimer, and not adsorbed in the particle surface. In the spectra of DeAXO-BPO@SiO₂, the presence of both AXO and BPO moieties can be observed. In the aromatic region of the spectra, the signals correspond to the aromatic ring of BPO and AXO (two well defined peaks around 7.34, appearing together with the BPO multiplet, and 6.8 ppm) (23). The absence of any signal around 5.5 ppm indicates that no closed β-lactam rings are present, and for instance, no antibiotics are absorbed to the particle surface. The presence of covalently bound antibiotics in DeAXO-BPO@SiO₂ is also confirmed by the signals of H-5, H-6 and H-3 of both AXO and BPO units ( Figure 1 and Figure S6 ). The presence of the PAMAM dendrimer was further confirmed in all the spectra by the signal between 3.7 and 2.0 ppm, typical of this kind of dendrimers (23).

After confirming the high density of allergenic determinants of BP and/or AX over the dendrimer grafted particles, their ability to quantify AX-sIgE and/or BP-sIgE was successfully evaluated in a group of well-diagnosed subjects. Comparison of these results with existing methodologies for BL-sIgE determination indicate the great potential of these developed DeAn@SiO₂ as appropriate platform for capturing antibodies in a specific and selective way.

Nowadays sIgE testing in the context of BL allergy presents relevant limitations in the evaluation of subjects with immediate reactions to penicillins, as the commercial ImmunoCAP method is reported to show low and variable sensitivity (0-50%) (12, 35, 43, 44), although specificity is high (83-100%) (35). The scenario for clinicians is still more complex since not only sIgE detection can fail. Even when the clinical picture is reliable with an immediate drug reaction suggesting IgE-mediated mechanisms, the sensitivity of both ST and serum sIgE detection remains low for BLs (45). Moreover, it is only available for four penicillins (BP, AX, penicillin V, and ampicillin) and one cephalosporin (cefaclor). Therefore, new in vitro tools for consistent diagnosis are needed. Conventional RAST using cellulose discs generally show higher sensitivity than ImmunoCAP (43-75%) (8), though it is still suboptimal. This method is not used in routine clinical diagnosis since it can be performed only in specialized laboratories for preparation of disc solid phases and manipulation of radioactive antibodies. To improve the existing techniques, it is necessary to deepen the causes of their limitations or shortcomings, for whose understanding we focus on the characteristics of the solid phases manufactured for the capture of IgE and the context of the peculiarities of drug allergy.

The reasons for the non-favorable detection in the case of drug allergenic molecules include the following: BLs frequently behave as haptens, which require protein binding to become a complete “allergen” with IgE-binding capability. As a consequence, the structure of the drug after covalent binding to the carrier molecule, their available disposition to sIgE interaction, and the nature of the carrier molecule are relevant (17, 19, 45) for detecting the extremely low concentration of sIgE. Another factor contributing to the diagnosis challenge is that some immediate reactions may not be IgE-mediated; although this is important, in this study we did not address this parameter.

AX-sIgE determination using the different solid phases for individual subjects ( Figure 2A ) of Group A shows values of RAST higher for DeAXO@SiO₂ than for DeAXO-BPO@SiO₂, as the latter solid phase contains half of the AXO groups, and this must be correlated with the binding; even though results of allergic patients are positive, providing the successful detection of sIgE antibodies to both AX and BP. This could present great usefulness to detect such allergen sIgEs in a first screening for BL allergy diagnosis.

Regarding BP-sIgE determination ( Figure 2B ), the percentage of positive cases was 25% for Group A corresponding to AX selective patients (false-positive results) using ImmunoCAP, in agreement with reported cases of false positive results (15). This could be explained by the presence of a not clinically relevant sIgE to phenylethylamine, a structure that can be present in the ImmunoCAP^® and that shares benzyl allergenic epitopes with BP (14). False positive results were also found with BPO-PLL-discs in 45% of patients from Group A, showing suboptimal selectivity. It is interesting that three of the cases of false positive diagnosis to BP (patients 4A, 5A and 6A) coincide with these both methods ( Table 3 ). A common characteristic of these patients is the high values of total IgE, in agreement with previous observations indicating a negative influence of levels higher than 200 kU_A/L in the serum on the detection of BL-sIgE (46). Great results were obtained with DeBPO@SiO₂ for which all the patients of Group A and Group B were correctly classified ( Figure 2B ).

Although further research is still needed in a higher number of patients for clinical validation, the innovative approach described in this study provides more sensitive and reliable in vitro tests with the possibility of testing different BLs, potentially simultaneously. Improvements can be explained by the nano precision, including the following: Monodisperse PAMAM dendrimers offer advantages over PLL polydisperse polymers, displaying controlled and exact number of haptens, resulting in high reproducible materials. The coupling of DeAns on NPs provides solid phases with higher surface area compared to cellulose polymers, which increment the sensibility of the immunoassays given the low concentration of BL-sIgE.

Nanoscaled DeAn@SiO₂ materials are the solution for the structural precision that are translated into accurate immunological tests. Mono and bi-epitope DeAn@SiO₂ composites have been successfully prepared and their ability to recognize sIgE from patient sera has been confirmed. Both, the particle synthesis and its chemical modification protocols have been well established to ensure reproducibility in composition, which is critical for its use in in vitro diagnostic applications. The synthetic methodology has been designed in such a way that it allows an exhaustive monitoring of the chemical reactions taking place in the particle surfaces using standard techniques. The control of the degree of functionalization allows the amount of dendrimers covalently attached to be maxima in the prepared nano-composites, ensuring the effective exposure of the hapten-carrier conjugate to specific IgE, and decreasing the uncovered particle surface that could lead to non-specific interactions with sIgE. The three configurations of DeAn@SiO₂ composites, including the mono-allergenic derivatives and the bi-allergenic DeAXO-BPO@SiO₂ demonstrated their ability to quantify AX-sIgE and/or BP-sIgE in patients’ sera. These solid phases allowed an excellent distinction between allergic patients and tolerant controls in the reduced selected group of studied subjects, improving the results of existing immunoassays techniques in terms of sensitivity and specificity. Remarkably, these new nano-based platforms provide a suitable tool for accurate sIgE testing in patients to more than one BL simultaneously, representing the starting point for an in vitro screening assay that can simplify future diagnostic approaches. Moreover, the described design of DeAn@SiO₂ can be extrapolated to other penicillins, providing a wider range of culprit BLs to evaluate. All these observations suggest that DeAn@SiO₂ are potential solid phases for developing diagnostic tools that can overcome limitations of available techniques for BL-sIgE detection.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Provincial Ethics Committee of Malaga. The patients/participants provided their written informed consent to participate in this study.

EP-I, CM, MT, MM, and YV conceived and designed the study. VG-O performed the Nps synthesis and chemical characterization as well as RAST with particles. CM supervised all in vitro tests and analyzed immunoassays data. IJ and MM radiolabeled anti-IgE. IJ and JC performed ImmunoCAP and RAST with discs. ID and MT evaluated and selected patients and controls. YV and MM analyzed all the data, prepared figures, and wrote the paper with input from EP-I, CM, and MT. 3All authors contributed to the article and approved the submitted version.

This work was supported by the Spanish Ministerio de Ciencia e Innovación (Proyectos de I+D+I «Programación Conjunta Internacional», EuroNanoMed 2019 (PCI2019-111825-2), Ministerio de Ciencia y Educación (PID2019-104293GB-I00), Instituto de Salud Carlos III (ISCIII) of MINECO (grants cofunded by ERDF: ‘‘Una manera de hacer Europa’’ (Euronanomed Program AC19/00082, PI20/01734, PI18/00095, RETIC ARADYAL, RD16/0006/0001 and RD16/0006/0012, and Miguel Servet II program (CPII20/00028)), Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007), Andalusian Regional Ministry of Health (PE-0172-2018) and Nicolas Monardes Program (RC-0004-2021).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.