Exploring the versatility of dendrimer-stabilized silver nanoparticle platforms: synthesis, characterization, and protein immobilization for enhanced biosensing applications†

Denys R. Oliveira^ab, Aldo J. G. Zarbin*^b and Dênio E. P. Souto*^a
aLaboratório de Espectrometria, Sensores e Biossensores – Department of Chemistry, Federal University of Paraná (UFPR), Curitiba, PR 81530-900, Brazil. E-mail: denio.souto@ufpr.br
^bGrupo de Química de Materiais – Department of Chemistry, Federal University of Paraná (UFPR), Curitiba, PR 81530-900, Brazil. E-mail: aldozarbin@ufpr.br

First published on 8th August 2024

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1 Introduction

Biosensors are detection devices that integrate biologically active elements known as bioreceptors, together with a transducer. The bioreceptors encompass a range of biomaterials such as enzymes, antibodies, antigens, organelles, microorganisms, genetic materials, and biological tissues.^1,2 The role of transducers is to convert the biological response resulting from the interaction between the bioreceptor and the target analyte into a measurable signal. Subsequently, this signal is correlated with the analyte's concentration.^1,2

The surface plasmon resonance (SPR) technique is an optical transducer, which enables several methods to be used in optical sensor platforms, including the variation of refractive index, absorbance, wavelength, and other measurements based on spectroscopy.^1,3 When the optical sensor is based on the surface plasmon excitation, it is referred to as an SPR sensor. SPR has been widely used in biosensing applications because it enables real-time measurement with remarkable sensitivity and is label-free.^1,3 It has stood out in recent decades as a solid and reliable tool in clinical analyses and for the study of biomolecular interactions.^1,3 To ensure the effectiveness of SPR biosensors, the binding of recognition biomolecules and the functionalization of the SPR sensor chip play a crucial role.^4–6 A wide range of materials with different properties can be employed in the functionalization of SPR sensor chips. Self-assembled monolayers of alkanethiols, either combined or not with dendrimers and metallic nanoparticles, are relevant examples of this diversity.^7–9

Dendrimers are monodispersing, highly branched macromolecules with a meticulously designed 3-D architecture. They allow the functionalization of both internal and surface groups without altering the macromolecule structure, thus enabling precise modification of their properties.¹⁰ Poly(amidoamine) (PAMAM) dendrimers were first reported by Tomalia et al.,¹¹ and are nowadays commercially available, contributing significantly to the advancement of knowledge about these macromolecules. They are widely used due to their low toxicity, simplified manufacturing process, and favorable cost-effectiveness.¹² Fig. 1 illustrates the structure of the classic cationic PAMAM dendrimer with amino-terminal groups (–NH₂) of generation 3 (PAMAM-G3).¹³ These molecules have an ethylenediamine core and repeated internal branched arms that are linked to the core.^10,12–16 The generation (G) is determined by the branching layers present in the structure.^10,12–16 In addition to the classic cationic PAMAM, there are two other forms: one terminated with hydroxyl (–OH) groups, which is neutral, and another terminated with carboxyl (–COOH) groups, which is anionic.¹³

Due to its specific structure, PAMAM provides many active sites for the immobilization of biomolecules. Furthermore, given that the use of metallic nanoparticles amplifies and enhances the SPR signal, combining these two materials could potentially lead to significant advancements in the construction of SPR biosensors. It is worth noting that PAMAM has been extensively documented in the literature as a stabilizing agent of metallic nanoparticles.^15,17–19 Crooks et al.²⁰ were the first to report the synthesis, characterization, and applications of dendrimer-encapsulated/stabilized metallic nanoparticles (DENs).²¹ Several alternative routes have been developed afterwards, involving the use of different types of dendrimers, with distinct cores, surface functional groups, and generations, combined with various metals, such as Pt, Pd, Au, Ag, Cu and Co.^{15,17–19,21–33}

The synthesis of PAMAM/metal nanoparticle nanocomposites is typically carried out in water, although organic and biphasic solvents can also be used.^15,20,21 Tertiary amines are the most active components within the PAMAM dendrimer. In favorable cases, metal ions strongly complex with the tertiary amine groups inside the molecule, forming metal-dendrimer complexes that can be reduced by suitable reducing agents.^15,20,21 Sodium borohydride, ascorbic acid, and sodium citrate are the most employed reducing agents.¹⁶ Reduction has also been investigated using UV radiation and X-rays, eliminating the need for chemical reducing agents, which can be advantageous depending on the application.^{15,20,21,25,33}

The combination of PAMAM with metal nanoparticles, such as silver nanoparticles (AgNPs), can enhance the SPR signal and has significant implications for the detectability of biosensors based on this technique.^34–36 The first part of this study involved the synthesis of PAMAN/AgNPs materials (AgDENs) using UV radiation as a reducing agent and investigating the effect of Ag⁺ concentration on the formation of AgDENs. Afterwards, the LbL technique was employed to prepare nanostructured films on the SPR sensor chip, resulting in the development of novel SPR biosensors.

2 Experimental section

The binding of AgDENs to the unmodified gold surface (Au/AgDENs) and their binding to the surface previously modified with CYS-SAM (Au/CYS/AgDENs) were characterized in real-time. Biofunctionalization of the terminal amino groups of AgDENs was achieved through covalent binding with proteins with well-established structures and important functions. As a model system, we initially investigated the binding of CHIP protein to the terminal amino groups of the constructed platforms (Au/AgDENs and Au/CYS/AgDENs) using glutaraldehyde as a binding agent. To characterize each step involved in the construction of the biosensors, morphological and structural analyses were conducted via visible ultraviolet spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), and transmission electron microscopy (TEM). To demonstrate the versatility of the proposed platforms in anchoring different biomolecules, we also explored the immobilization of Hsc70, enzyme Free Candida antarctica lipase B (CALB L) and the C1 protein. To the best of our knowledge, the exploration of some of these proteins as biorecognition materials, through their immobilization on the proposed materials, has not been reported in the literature.

2.1 Reagents

Silver nitrate (AgNO₃, 99.99%), PAMAM dendrimer (ethylenediamine core, generation 3 solution in methanol), cysteamine (CYS, ≥98%), and glutaraldehyde (GLU) (25% aqueous solution, (v/v) were purchased from Aldrich Chemical (St. Louis, MO, USA). Potassium chloride (KCl), disodium hydrogen phosphate (Na₂HPO₄), monopotassium phosphate (KH₂PO₄), potassium hydroxide (KOH), and potassium chloride (KCl) were purchased from LabSynth (Diadema, SP, Brazil). The PBS solution (pH 7.1, 0.1 mol L⁻¹) was prepared by the equimolar addition of 0.1 mol L⁻¹ of KH₂PO₄, Na₂HPO₄, and KCl, and the pH was adjusted by adding KOH. Deionized water was used to prepare the solution after purification using a Milli-Q system.

CHIP³⁷ and Hsc70³⁸ proteins were synthesized by Professor Carlos H.I. Ramos group (Institute of Chemistry, University of Campinas, Campinas, Brazil), and dilutions were made using PBS buffer at pH 7.1. For the CHIP protein and Hsc70, the concentration used was 1 μg mL⁻¹ and 50 μg mL⁻¹, respectively. The enzyme used was the commercial free lipase B from Candida antarctica (Lipozyme CALB L®) by Novozymes Latin America (Araucaria, PR, Brazil). The enzyme was used without additional purification, and the solution was prepared by diluting 0.037 mL of the enzyme in 25 mL of PBS buffer at pH 7.1. The recombinant C1, which is a hypothetical protein from the protozoan Leishmania infantum, it was synthetized by the Leishmaniasis Laboratory of the Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil.

Positive canine serum samples for visceral leishmaniasis were collected from dogs naturally infected with Leishmania infantum antigen of endemic regions of Belo Horizonte, Brazil. For these samples, the diagnoses were performed using IFAT and ELISA serological methods in the Leishmaniasis Laboratory of the UFMG. For negative controls, canine serum samples were obtained from healthy dogs housed in a non-endemic area.

2.2 Synthesis of dendrimer-stabilized silver nanoparticles

Dendrimer-stabilized silver nanoparticles (AgDENs) were prepared following previous reports.^27,33,39 Initially, an aqueous solution of AgNO₃ with a concentration of 0.1 mol L⁻¹ at pH 5.7 was prepared and used as a stock solution. Then, the solution was diluted to obtain Ag⁺ concentrations of 0.05, 0.5, and 1 mmol L⁻¹. The diluted AgNO₃ solution was added to a solution of PAMAM dendrimer with an ethylenediamine core, generation 3, at a fixed concentration of 0.05 mmol L⁻¹ in a quartz cuvette. The solutions were stirred for a few minutes and then irradiated with 8 W ultraviolet light (λ = 256 nm) for 1 hour in a dark box. The products were separated by centrifugation for 15 minutes at a speed of 7500 rpm, and the process was repeated.

2.3 Synthesis of citrate-stabilized silver nanoparticles

Citrate-stabilized silver nanoparticles were prepared using Turkevich's method.⁴⁰ The aqueous solution of AgNO₃ (1 mmol L⁻¹) was heated to reach a temperature of about 105 °C. Then sodium citrate solution (1 mmol L⁻¹) at pH 9 was rapidly injected into the AgNO₃ solution. Heating was continued for additional 15 min, and then the solution was cooled in an ice bath. The mixture was then subjected to centrifugation, resulting in the separation of the precipitate, which was subsequently washed with ultrapure water. This washing process was repeated seven times. Finally, the precipitate was dispersed again in ultrapure water for subsequent analysis.

2.4 Materials characterization

UV-Vis spectra were obtained using a Shimadzu UV2450 spectrophotometer, with a 1.0 cm optical path quartz cuvette, and air was used as a reference. FTIR spectra were obtained using an Invenio-R® spectrometer (Bruker) with the total attenuated reflectance (ATR) technique, collecting 256 accumulations at a 2 cm⁻¹ resolution. X-Ray diffraction (XRD) measurements were performed on a Shimadzu XRD-6000 instrument, using CuKα radiation (λ = 1.5418 Å) in a shallow angle mode for thin films with the THA-1101 accessory obtained from Shimadzu. Transmission electron microscopy (TEM) images were acquired in a Jeol JEM 1200EX-II equipment operating at 120 kV by depositing a drop of the sample on copper grids with a thin carbon film, followed by air drying. The ImageJ software⁴¹ was used to measure the diameter of the AgDENs, and a size distribution histogram was constructed based on the analysis of 150 nanoparticles. SPR measurements were conducted on the Autolab Springle instrument (Eco Chemie, The Netherlands) in attenuated total internal reflection mode, employing the Kretschmann configuration.

2.5 SPR sensor chip modification

The SPR sensor chip consists of a glass disc coated with a thin layer of gold, approximately 50 nm thick. For its modifications, two methodologies were employed. In the first methodology, the chip was placed into the equipment, and 50 μL of ultra-pure H₂O was injected to establish a baseline, and the measurement was carried out for 5 minutes. Then, after removing the water, 50 μL of the AgDENs solution was added, and the interaction process was monitored for about 1 hour. Following this, a washing step was carried out by adding approximately 1000 μL of ultra-pure H₂O in flow to remove excess material that did not interact or was weakly bound to the sensor chip. This sequence of procedures, with the same duration, volumes, and use of water to establish the baseline, was applied to silver nanoparticles stabilized by citrate (AgCit) and PAMAM-G3. These materials served as references to evaluate the interaction of AgDENs with the gold surface (SPR sensor chip). In the second methodology, the SPR sensor chip was previously modified with the CYS-SAM. This involved immersing the chip in an ethanolic solution of CYS (1 mmol L⁻¹) for 4 hours to allow the formation of a SAM on the Au surface (Au/CYS). After this period, the chip was washed with ethanol and dried with a pure N₂ flow. Subsequently, the Au/CYS was inserted into the equipment and a baseline was established with H₂O for approximately 5 minutes. Activation of the terminal amino groups of Au/CYS, essential for covalent binding to AgDENs, was then carried out by adding 50 μL of a 1% aqueous solution of glutaraldehyde, monitored for about 10 minutes. After activation, the chip surface was washed under flow with approximately 1000 μL of ultra-pure H₂O. And then, 100 μL of ultra-pure H₂O was added, and the measurement continued for approximately 5 minutes. Following this, 50 μL of the solution containing the AgDENs was injected into the activated Au/CYS, resulting in the formation of the Au/CYS/AgDENs platform. The interaction was monitored for additional 1 hour, followed by surface washing with 1000 μL of ultra-pure water under flow. Finally, 100 μL of ultra-pure H₂O was added, and the measurement continued for approximately 10 minutes.

3 Results

3.1 Structural and morphological characterization

The first indication of the formation of dendrimer-stabilized silver nanoparticles (AgDENs) was the change in the color of the solutions. Initially transparent, the solutions turned light yellow after photoreduction, as shown in Fig. 2a. The entire process of AgNPs production was followed by UV-Vis spectroscopy, as AgNPs exhibit characteristic plasmon bands in the visible region of the electromagnetic spectrum.^42,43 Fig. 2b–d evidence the results obtained for the different proportions of Ag⁺ in the material (AgDENs). For these processes, initially the UV-Vis absorption spectra only showed the characteristic band of the dendrimer at 285 nm, attributed to the π–π* transition of the carbonyl group (CO).³² After photoreduction, a new band appeared at 410 nm, undeniably attributed to the localized surface plasmon resonance (LSPR) originating from collective oscillations of free electrons in the AgNPs.^{22,23,25,32,44–49} The symmetric shapes of these plasmon bands further suggest that they are well-dispersed and spherical structures.^{22,23,25,32,44–50}

A comparative analysis of the 410 nm band in different systems reveals a remarkable increase in its relative intensity. This increase may suggest growth in both the size of the nanoparticles and the amount of metal present. The average size and size distribution, as observed by transmission electron microscopy (TEM) in Fig. 4, are similar for both systems. It is possible to correlate the increase in intensity with the use of a higher concentration of silver during the synthesis process. These results are in line with what was observed by Endo et al.⁵¹ Additionally, the increase in the Ag⁺ concentration ratio in the formation of the nanocomposites did not show a significant influence on the position of this band, which has also been reported by Kéki et al.⁵²

Regarding the 285 nm band, there are still some issues that require clarification despite substantial documentation. The available articles show discrepancies on some aspects, and further investigation is needed to fully understand the appearance and disappearance of this band, along with its intensity and shift.^{22,23,25,32,44–49,52,53} Esumi et al.³³ initially attributed this band to the formation of certain carbonyl compounds, specifically aldehydes, which are formed when the dendrimer is exposed to UV radiation.^27,53 It is known that aliphatic aldehydes, such as propionaldehyde, isobutyraldehyde, and pivalaldehyde, exhibit UV-Vis bands in the 283–285 nm region.^53–55 However, Pande and Crooks⁴⁵ demonstrated that this band arises exclusively from intact or nearly intact dendrimers rather than fragments.⁴⁵ The authors also suggest that this band is conditioned by the protonation and deprotonation of the dendrimer's tertiary amines. In particular, according to the authors, the tertiary amines are protonated at low pH, and the 280–285 nm band is absent.^45,56 However, when these groups are deprotonated at higher pH, this band appears.⁴⁵ These results are in contrast to those reported by Wang et al.,⁵⁷ where the authors showed UV-Vis absorption spectra of PAMAM-G2 at pH levels ranging from 3 to 8. The dendrimer exhibited an absorption band at 285 nm at pH 3. As the pH increased, the absorption intensity decreased. At pH 8, the 285 nm absorption band nearly disappeared. The authors did not provide an explanation for this discrepancy.

The influence of Ag⁺ concentration on the formation of nanocomposites was evaluated. Prior to reduction, we observed that the addition of Ag⁺ ions to the dendrimer solution resulted in a decrease in the intensity of the 285 nm band (Fig. S1, ESI†). This reduction in band intensity is associated with the coordination of Ag⁺ ions with the dendrimer.⁵⁶ After reduction, there is a slight shift of this band towards shorter wavelengths (blue shift). Furthermore, we observed a direct proportionality between the increase in the intensity of this band after reduction and the concentration of Ag⁺. This phenomenon has also been reported by other authors when utilizing Au³⁺ instead of Ag⁺.⁵³

To investigate the impact of radiation on the structure of the dendrimers, we exposed a solution with the same concentration used in the synthesis to UV radiation for 60 minutes. We recorded the spectra before and after exposure (Fig. S2, ESI†). The dendrimer spectra after exposure to radiation revealed an increase in the intensity of the 285 nm band. This increase in intensity is likely due to the concentration increment in the solution, resulting from the evaporation of water during the irradiation time. The observation is that the band remains unchanged, indicating that UV radiation does not degrade the dendrimer structure. This is further confirmed by FTIR-ATR analysis (Fig. S3, ESI†), where no significant change was observed in the aspects obtained before and after radiation exposure.

Localized surface plasmon resonances in metallic nanoparticles have garnered significant interest, making them relevant for a wide range of applications, primarily due to the significant enhancement of the electromagnetic field near the metal surface.⁵⁸ These resonances are also highly dependent on the size, shape, and local dielectric environment of the nanoparticle.^22,59,60 Thus, we attribute the increase in intensity in this band at 280 nm, mainly to the change in the local dielectric environment of the silver nanoparticles after the formation of the nanocomposite.²² The observations reported above, based on UV-Vis absorption spectra, regarding size and size distribution, were confirmed by XRD and TEM techniques, as shown in Fig. 3 and 4, respectively.

The crystalline nature of the nanoparticles was confirmed through XRD analysis, as demonstrated in Fig. 3. The XRD diffractogram for both systems exhibited a peak at 2θ = 38.14°, which could be attributed to the (111) plane of the face-centered cubic (fcc) phase of Ag (JCPDS 04-0783).^61–65 As the silver concentration decreased, it was not possible to observe the peak using the same sample volume. The average size of the AgNps was estimated for the AgDEN system (20:1) and is approximately 3.5 nm.

As can be observed from the images obtained by TEM of the AgDENs (Fig. 4), the nanoparticles predominantly exhibit a spherical shape with diameters ranging from 3.7 to 16.2 nm. Furthermore, it is important to note that the nanoparticles were well-separated, indicating the absence of any signs of aggregation. Upon conducting a comparative analysis among different systems, we did not detect significant variations in the size and shape of the particles.

Taking into consideration that the ideal diameter of the PAMAM-G3 dendrimer sphere is 3.6 nm,²¹ the analysis of the images shows that the average sizes of the AgDENs are more than twice the hydrodynamic diameter of the dendrimer. It is not expected that the dendrimer core is larger than its hydrodynamic diameter.⁶⁶ In the images, it is possible that there are clusters of dendrimers covering and stabilizing the silver nanoparticles. Based on these results, it can be concluded that the nanoparticles are primarily stabilized by the forces between molecules. PAMAM-G3 dendrimers have a lower density of terminal groups compared to higher-generation dendrimers. Consequently, they possess a more open structure, which allows for the complexation of metal ions both on their periphery and internally.^39,67

The FTIR-ATR analysis was performed to understand and monitor the coordination of metal ions with PAMAM-G3. Additionally, the presence of various functional groups on the surface of the synthesized nanoparticles was confirmed.^31,68 The spectra of PAMAM-G3 and AgDENs before and after photoreduction are illustrated in Fig. 5a and b, respectively. The spectrum of PAMAM-G3 reveals characteristic bands, extensively described in the literature.^{26,31,32,66,68–77} The bands at 3270 cm⁻¹ and 3078 cm⁻¹ are associated with the symmetric and asymmetric stretching of N–H in primary amines, while the bands at 2936 cm⁻¹ and 2840 cm⁻¹ are attributed to the asymmetric and symmetric stretching vibrations of CH₂, respectively.^{26,32,66,68–72,74,77} The absorption band at 1634 cm⁻¹ is assigned to the CO stretching vibrations (amide I), while the band centered at 1555 cm⁻¹ is attributed to the C–N stretching/N–H bending vibrations (amide II).^26,32,78,79 This latter band corresponds to the amide of dendrimer branches, serving as the main characteristic of the branch conformation.²⁶ Stretching vibrations of C–N can be observed at 1325 cm⁻¹ and 1467 cm⁻¹,⁷⁴ and finally, the CH₂ scissoring mode can be observed at 1436 cm⁻¹.⁶⁶

The analysis of the spectra before reduction shows that the bands originally present in PAMAM-G3 exhibit a significant reduction in intensity and eventually disappear from the spectrum (Fig. 5a). PAMAM dendrimers have a high availability of functional groups that can participate in reactions with Ag⁺ ions.⁵³ Therefore, this observation was attributed to the coordination of Ag⁺ ions with all the available groups within the dendrimer's structure. The literature reports that initially Ag⁺ ions coordinate with the external primary amines present in the dendrimer's structure, and after reduction, they penetrate the interior of the macromolecule.^39,67

The FTIR-ATR spectra of dendrimer-stabilized silver nanoparticle solutions (see Fig. 5b) are slightly different when compared to pure dendrimer solutions. For both studied systems, there was a shift in the bands attributed to amide I and C–N stretching after reduction. No significant changes were observed in the remaining bands after reduction. These alterations suggest the involvement of the amide group in the interaction with silver nanoparticles, as reported by other authors.^{31,66,73–76}

3.2 In situ evaluation via SPR of the interaction of PAMAM-G3, AgNPs and AgDENs on the metallic surface (gold) for biosensing applications

Sensorgrams (Δθ_SPR vs. time) were obtained (Fig. 6) for the binding process involving different proportions of AgDENs (1:1, 10:1, and 20:1), PAMAM-G3 (DEN), and citrate-stabilized silver nanoparticles (AgCIT).

For the PAMAM-G3 dendrimer and for the conjugated material (AgDENs), even after the washing step, only a slight decrease in variations of the angle of resonance (Δθ_SPR) is observed. On the other hand, it is observed that for silver nanoparticles protected by citrate (AgCIT), there is a significant decrease in Δθ_SPR(m°), dropping below the pre-established baseline. This demonstrates that there was no significant interaction between the Au surface and AgCIT. The greater stability observed for PAMAM-G3 and AgDENs can be explained by two factors, as demonstrated by Tokuhisa et al.⁸⁰ First, the large surface area of PAMAM-G3 and its dense exterior result in a strong van der Waals interaction with the Au surface.⁸⁰ Second, the large number of amine groups in PAMAM-G3 chemically adsorb onto the Au surface and, analogous to polydentate metal-ion ligand chemistry, stabilize the amine/Au interaction.⁸⁰

To achieve greater chemical and mechanical stability for the bioreceptor binding on the sensor chip, a layer-by-layer (LbL) system was also explored. The use of self-assembled monolayers (SAMs) is one of the most widely employed strategies in the construction of SPR biosensors, as mentioned earlier. Cysteamine (CYS), a low-cost short chain alkanethiol, was used to modify the SPR sensor chip. Its structure contains a thiol group (–SH) at one end, which forms gold-thiol bonds, and an amino-terminal functional group (–NH₂) at the other.⁹ The strategy adopted for immobilizing AgDENs on the SPR sensor chip modified with CYS-SAM, as well as the immobilization of different biomolecules on the constructed platforms, involved the use of glutaraldehyde as a crosslinking agent. Glutaraldehyde (GLU) is a bifunctional reagent commonly used for immobilizing biomolecules on metallic surfaces. It possesses highly reactive aldehyde groups, allowing it to interact with different proteins via lysine ε-amino and N-terminal groups, although it may occasionally react with other groups such as thiol, phenol, and imidazole.^81–83 For better understanding, the immobilization and activation processes are schematically represented in Fig. 7. The immobilization of AgDENs on the unmodified SPR sensor chip (route 1, Fig. 7) and on the CYS-SAM modified chip (route 2, Fig. 7) was evaluated in real time.

To compare the binding process for the different routes, sensorgrams (Δθ_SPR vs. time) and SPR reflectance curves were included (Fig. S4, ESI†), illustrating the interaction of AgDENs directly on the gold surface (Au/AgDENs) and their interaction on the gold surface previously modified with the CYS-SAM (Au/CYS/AgDENs). SPR experiments revealed that the most significant changes in Δθ_SPR(m°) occurred when AgDENs were immobilized directly on the Au surface. It was observed that the interaction process between AgDENs and CYS-SAM was less favored. This is evident when observing the full width at half maximum; the use of the CYS-SAM led to an increase in this width. Furthermore, with the exception of the 10:1 system, there was an increase in the minimum value of the SPR reflectance curve (increase in the percentage of reflected light), for the other systems studied. This effect is likely caused by the increased distance between AgNPs and the planar Au surface after the formation of the intermediate CYS-SAM layer. These results suggest a slightly more pronounced plasmonic effect for the direct binding of AgDENs to the Au surface.

3.3 Immobilization of the CHIP protein on the platforms Au/AgDENs and Au/CYS/AgDENs for biosensing applications

In the biosensor project, the immobilization of biomolecules, also known as bioreceptors, is a fundamental step. This phase is crucial to confer and determine the selectivity and specificity of the biosensor. The efficiency of the developed platforms in terms of anchoring capacity for different biomolecules was evaluated through tests using proteins with distinct physico-chemical properties, each with important and known biological functions, such as CHIP, Hsc70, and CALB L proteins. Through a precise analysis of the binding processes involving each protein on the proposed platforms, it was possible to infer the versatility and efficiency of the proposed material (AgDENs) in biosensor construction.

Initially, we examined the binding of the CHIP protein on the SPR sensor chip previously functionalized and non-functionalized with the CYS-SAM, as a proof of concept (Fig. 8).

The CHIP protein is mainly composed of two domains: one is the amino-terminal TPR domain, responsible for binding with Hsp70 to fulfill its role as a molecular chaperone; the other is the carboxyl-terminal U-box domain, which mainly plays a role in ubiquitin ligase E3 activity.⁸⁴ These domains confer CHIP a crucial role in protein content balance.⁸⁴

CHIP is an essential player in several fundamental cellular processes related to the pathogenesis of neurological diseases, including intracerebral hemorrhage, ischemic stroke, Alzheimer's disease, Parkinson's disease, and other conditions.^85,86 Understanding the role of the CHIP protein in the development of these diseases may be crucial for the development of new potential therapies and diagnostic biomarkers.⁸⁷

The CHIP immobilization process was carried out by SPR (Fig. 8). The covalent binding of this protein on Au/AgDENs and Au/CYS/AgDENs platforms was evaluated, and the results obtained show that the binding of CHIP on both platforms produced a significant change in the Δθ_SPR(m°) response. Even after successive washes with PBS buffer at pH 7.1, only a slight decrease in Δθ_SPR(m°) is observed, demonstrating that CHIP was effectively immobilized on the different platforms. Comparing the changes in the resonance angle (θ_SPR), it is observed that for AgDENs (1:1) and AgDENs (20:1), there was an increased variation in the resonance angle (Δθ_SPR) when AgDENs were immobilized directly on the gold surface. This reflects the sensitivity of the proposed SPR biosensor.⁸⁸ For AgDENs (10:1), no significant alteration was observed with and without the use of the CYS-SAM. This confirms that the direct application of AgDENs on the SPR sensor chip is more advantageous, considering the time and reagent savings for biosensor fabrication.

3.4 Immobilization of Hsc70 and CALB L on the platform Au/AgDENs for biosensing applications

Hsc70 is a constituent member of the 70 kDa heat shock protein family, widely present in the adult central nervous system.^89–91 It plays crucial roles that include a variety of physiological processes, such as ATP metabolism, assisting in the folding and formation of functional structural domains of client proteins, endocytosis, and serving as a key component of chaperone-mediated autophagy, which might remove damaged cellular components in anoxically stressed cells and restrict neuroinflammation after stroke.^89–91 Hsc70 also participates in multiple non-communicable diseases and some pathogen-caused infectious disease.⁹¹ In general, the presence of heat shock proteins in the blood has been considered a biomarker of damage, stress or inflammation. High cellular or circulating levels of Hsp70 have been reported to be prognostic in multiple cancers.⁹² On the other hand, enzyme-catalyzed reactions have emerged as a more sustainable alternative compared to chemical-catalyzed reactions due to their low energy consumption and reduced operational costs.⁹³ An effective technique for improving enzymatic catalytic performance and reusability is their immobilization on solid supports.⁹⁴ In particular, Candida antarctica lipase B (CALB L) is widely recognized in various scientific studies and industrial applications as an efficient biocatalyst.⁹⁴ CALB L is a typical lipase and a member of the α/β-hydrolase fold family. It is incorporated into a conserved catalytic triad consisting of serine (Ser105), aspartate (Asp187), and histidine (His224), which forms a substrate binding site for biocatalytic reactions.^93,95

The process of covalent immobilization of Hsc70 and CALB L on Au/AgDENs was monitored in real-time by SPR, and the reflectance curves obtained are shown in Fig. S5 and S6, respectively (ESI†). As shown in the figures, the binding between the proteins and the detection surface (AgDENs) resulted in an obvious shift in the SPR resonance angle. Both studied biomolecules exhibited similar behavior. It was observed that the use of the AgDEN system (10:1) caused a greater shift in the resonance angle of SPR compared to other proportions, reflecting the sensitivity of the SPR biosensor.⁸⁸ It was also noted that the minimum reflectance value decreased following the immobilization of biomolecules in both systems, indicating maximum coupling of incident light with the surface plasmon wave (SPW).⁸⁸

The study highlighted the sensitive application of the SPR technique to investigate real-time interactions of nanocomposites with CYS-SAM and the sensor chip, as well as the interaction of the different proteins with the prepared nanocomposites. Comparative analysis reveals a better stability of AgDENs, attributed to van der Waals interaction and chemical adsorption. Comparison with the use of the layer-by-layer (LbL) system suggests a preference for the direct immobilization of AgDENs on the SPR sensor chip. Tests with different biomolecules, such as CHIP, Hsc70, and CALB L, demonstrate the versatility and efficiency of the proposed material in SPR biosensors.

3.5 Application of the proposed platform (AgDEN) for the construction of an immunosensor for the detection of Leishmania infantum antibodies

The use of the dendrimer-stabilized silver nanoparticles as platforms for the construction of an immunosensor was explored using a recombinant protein (C1 protein) from the protozoan Leishmania infantum. Fig. 9a shows the SPR analysis obtained during the activation of the amino-terminal groups of the dendrimer by the addition of 1% (w/v) glutaraldehyde and during the immobilization of the C1 protein (200 μg mL⁻¹ = 4.90 μmol L⁻¹) on Au/AgDEN (10:1). It is possible to observe a characteristic profile for both steps, with a higher θ_SPR value, even after successive washes with PBS buffer at pH 7. The values of the effective responses can also be seen in Fig. 9b.

The proposed SPR biosensor based on C1 protein/AgDEN was applied to detect antibodies against Leishmania infantum in canine serum samples from the positive and negative groups for visceral leishmaniasis. Fig. 9c shows the association step obtained by adding positive and negative canine sera, followed by the dissociation step. As can be seen, there is a much higher effective Δθ_SPR value when the positive sample is added (Fig. 9d), which demonstrates excellent selectivity of the proposed SPR immunosensor.

4 Conclusions

In this study, we highlight the immense potential of dendrimer-stabilized silver nanoparticles for SPR biosensing applications. Structural and morphological characterization revealed that AgNPs are stabilized both at the periphery and internally in PAMAM-G3. FTIR-ATR analysis before reduction showed that silver binds to all groups present in the dendrimer, and after reduction, there was a shift in bands attributed to amide I and CN stretching; these changes suggest the involvement of the amide group in the interaction with silver nanoparticles. UV-VIS analysis revealed characteristic bands of both dendrimer and silver nanoparticles, and it was demonstrated that the intensity of the band at 410 nm depends on the silver concentration used in the synthesis. Additionally, it was confirmed that radiation does not alter the dendrimer's structure. From TEM images of AgDENs, it was observed that the nanoparticles predominantly exhibit a spherical shape, with diameters ranging from 3.7 to 16.2 nm. The use of AgDENs proved to be a highly attractive strategy for the development of SPR biosensors, primarily due to their high surface area and excellent chemical stability. The formed film exhibited exceptional anchoring capacity for different biomolecules studied. There was a significant increase in the sensitivity of the proposed platform, likely due to the interaction between silver nanoparticles (AgNPs) and the surface plasmonic wave on the gold planar surface. To demonstrate a specific application, the proposed platform was successfully explored in the construction of an immunosensor for the detection of antibodies against Leishmania infantum, obtaining excellent selectivity to discriminate canine serum samples from the positive and negative groups for visceral leishmaniasis. These results open new possibilities to enhance the efficiency of biosensors for a variety of analytical and biomolecule detection applications.

Data availability

Data reported in this work will be made available on request.

Conflicts of interest

This manuscript has not been previously published and is not being considered for publication elsewhere, and all the authors have no conflict of interest to declare.

Acknowledgements

The authors acknowledge the National Institute of Science and Technology of Nanomaterials for Life (INCT NanoLife – Process 406079/2022-6) and the Brazilian Federal Foundation for Support and Evaluation of Graduate Education – CAPES.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nj02593h

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