Metal Nanoparticles on Board of Low-Cost Devices for Optical Sensing

Abstract

Nowadays nanomaterials (NMs) have become consolidated building blocks for (bio)sensors and (bio)sensing strategies development. In this contest, the metal nanoparticles (MNPs) offer infinite opportunities since their nano-domain provides unique chemical, physical, electrical, and optical features; the most captivating MNPs’ property is the localized surface plasmon resonance (LSPR), which allows them to interact with the electromagnetic radiation, giving rise to typical absorption spectra and naked eye visible colors. Taking advantage of the aforementioned features MNPs have been extensively exploited in the development of optical and colorimetric point-of-need devices.

Herein, device equipped with plasmonic-active thin films of MNPs (gold/Au and silver/Ag nanoparticles/NPs) are presented, suitable for opto-analytical applications. The nanoarchitecture fabrication has been achieved onto ELISA plates. The nano-film was tested as a plasmonic transducer in ELISA plate to evaluate the oxidants capacity of sodium hypochlorite, sodium nitrite, ABTS, H₂O₂, AAPH, DPPH, and ferrous sulfate which are of biological and biochemical interests. Further, life-time studies of the proposed nanoarchitecture were carefully performed with the aim to evaluate the storability of the nano-equipped ELISA plates.

1 Introduction

Oxidants represent a wide class of compounds of chemical, biochemical, biomedical, and biological interest [1, 2]. Oxidants enclose radical species and stronger electron acceptors; among the oxidant agents, hydrogen peroxide (H₂O₂) is the most representative compound, belonging to the reactive oxygen species [1]. However, historically known oxidant species are the ABTS, DPPH and AAPH radical species commonly used for in-vitro study and assays [3,4,5]; further, in the past years, new oxidant species gain increasing interest in the scientific community as the sodium hypochlorite (NaClO) because of its use as disinfectant in the COVID-19 pandemic [2]. Thus, the interest for rapid and easy-to-use methodologies for oxidant species detection is still of primary importance [1, 2]. Nanomaterials (NMs) are widely used to develop analytical strategies to monitor the oxidants species [1, 2]. Several chemiluminescence, fluorescence, and electrochemical-based approaches have been reported in the literature; anyway, the use of biological components (i.e., cells and enzymes) or external probes (i.e., dyes, etc.) are required [1]. Recently, NMs-based optical/colorimetric sensing strategies represent a great alternative to the previous approaches, because of the simple use and easy signal assessment (i.e., visual assessment). In this field, metal nanoparticles (MNPs) are widely used for optical and colorimetric strategies as nano-indicators because of their outstanding optical features; the MNPs are able to interact with the electromagnetic radiation in the Uv-Visible region giving rise to the localized plasmon resonance phenomena (LSPR), resulting in a visible color dependent on the MNPs composition, size, shape, surface doping/functionalization, and interparticle distance [1,2,3]. Further, the MNPs are easy to synthesize by simply employing a metal precursor and a natural or chemical reducing agent, making them appealing for the design of new analytical colorimetric strategies.

Herein, different oxidant agents (i.e., H₂O₂, NaClO, NaNO₂, FeSO₄, ABTS, DPPH, and AAPH) have been evaluated by using a plasmonic-active nanostructured thin-film decorated with gold and silver nanoparticles (AuNPs and AgNPs, respectively), fabricated onto a commonly used optical tool, the ELISA plate. The sensing strategy relies on the etching phenomenon, the selective oxidation of AgNPs driven by the oxidant agents returning in a quantitative LSPR variation of the nanostructured thin film.

In addition, the analyzed oxidant agents have been characterized and classified according to their oxidant capacity/reactivity thanks to metrological indexes herein proposed, and further studies have been conducted to define the life-time of the proposed nanocomposite platform. The experimental theme of this work is part of a broader project of characterization of analytical tools user-friendly and suitable for in-field analysis for which it is necessary to still define the horizons and boundaries.

2 Materials and Methods

2.1 Reagents and Apparatus

Dopamine hydrochloride (DA), hydrogen tetracholoroaurate (Au(III)), silver nitrate (Ag(I)), sodium hydroxide, sodium acetate anhydrous, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, trizma hydrochloride, hydrogen peroxide solution (H₂O₂, 30% v/v), sodium hypochlorite (NaClO), sodium nitrite (NaNO₂), Ferrous Sulfate (FeSO₄), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2-difenil-1-picrilidrazile (DPPH), 2,2 azobis 2 amidinopropane dihydrochloride (AAPH), and methanol were purchased from Sigma-Aldrich (St Louis, MO, USA). Catechin (CT) was purchased from Extrasynthese (Genay, France). Nunc-Immuno MicroWell MaxiSorp (96-well) solid ELISA plates (EPs) were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions of 10 mM CT was prepared in methanol and stored at −20 ℃ in the dark. The absorbance spectra of the modified ELISA plates were recorded using an EnSpire 2300 Multilabel Reader from PerkinElmer (Turku, FI) with a resolution of 5 nm in the λ range 320–800 nm.

2.2 Nanostructured Plasmonic-Active Film Fabrication

The nanostructured plasmonic-active film synthesis was carried out according to Scroccarello et al. [1] as reported below:

• PDA thin-film formation: 0.5 g L⁻¹ DA was freshly prepared in Trizma buffer (10 mM, pH 8.5) and promptly used to fill EP microwells (200 μL for well). DA polymerization was carried out under static condition at room temperature for 15 h, in a white incubation chamber, using a warm light source (20 W light bulb), placed at 50 cm of distance. DA polymerization was blocked by emptying the microwells and abundantly washing them with Milli-Q water.

• AuSD self-formation: PDA-modified EP wells were filled with 200 μL of 250 μM Au(III) aqueous solution and incubated for 8h in the dark and at room temperature. The reaction was blocked by emptying the microwells and abundantly washing with Milli-Q water.

• AuSD functionalization: PDA@AuSD modified microwells were filled with 150 μL of acetate buffer (10 mM; pH 4.0), 10 μL of 20 mM Au(III) and 40 μL of 1 mM catechin (CT). The reaction was carried out in static condition for 1 h, in the dark and at room temperature. The reaction was blocked by emptying the microwells and abundantly washing them with Milli-Q water.

• AgNPs-nanonetwork formation: The PDA@AuNPs-CT modified microwells were filled with 185 μL of Milli-Q water and 10 μL of 20 mM Ag(I), afterward 5 μL of 4 M NaOH was added. The reaction was carried out under stirring condition by orbitally shaking (SSL1, Stuart equipment, Belfast, UK150) at 150 rpm at room temperature, in the dark, for 6 h. The reaction was blocked by emptying the microwells and abundantly washing them with Milli-Q water.

2.3 Oxidants Capacity Evaluation

The oxidant agents H₂O₂, NaClO, NaNO₂, FeSO₄, ABTS, DPPH, and AAPH were freshly prepared in phosphate buffer (PB, 10 mM, pH 7.0) before use. The PDA@AuNPs-CT@AgNPs modified wells were filled with 180 μL of reaction solvent (Table 1) and 20 μL of oxidant agent standard (diluted in the respective reaction solvent) to reach a final assay volume of 200 μL.

Table 1. Oxidant agents reaction conditions and tested oxidants’ concentration ranges.

The reaction mix was orbitally shaken (60 rpm) for 40 min at controlled temperature (Table 1) in the dark. For FeSO₄ and AAPH different incubation times and temperatures were tested.

The reaction was blocked by emptying the microwells and abundantly washing them with Milli-Q water. The absorbance was recorded at λ = 405 nm in Milli-Q water before (pre-etching) and after (post-etching) the reaction.

2.4 Oxidant Capacity Indexes and Parametrization

The etching phenomenon was monitored by evaluating the absorbance decrease (D%) induced by the etching, mediated by the oxidant agents, according to Eq. 1 (Eq. 1):

$$ D\% = \, \left[ {\left( {Abs_{405pre - etching} - \, Abs_{405post - etching} } \right) \, / \, \left( {Abs_{405pre - etching} } \right)} \right] \, \times \, 100 $$

(1)

According to Della Pelle et al. [3], for each oxidant agents, indexes to study the oxidants reactivity were extrapolated from the dose-response curve (D% vs. Oxidant agent concentration) and reported below:

• m/X_C⁵⁰: ratio between the slope (m) of the linear equation (D% = mX + q) and concentration values (X_C⁵⁰) calculated according to Eq. 2 (Eq. 2) considering the D%⁵⁰ calculated according to Eq. 3 (Eq. 3).

The X_C⁵⁰ is the oxidant concentration at which the signal (D%) reached its half-value, named (D%⁵⁰), calculated as the mean value between the highest and lowest signal of the dose-response curve.

$$ X_C^{50} = \, \left( {D\%^{50} - \, q} \right) \, / \, m $$

(2)

$$ {\text{D}}\%^{{5}0} = \, \left( {{\text{D}}\%_{\text{lower point}} + {\text{ D}}\%_{\text{higher point}} } \right) \, /{ 2} $$

(3)

• - Relative Oxidant Capacity, ROC: the m/X_C⁵⁰ ratio of each oxidant normalized against the m/X_C⁵⁰ of H₂O₂ used as a model/reference system and expressed as a percentage according to Eq. 4 (Eq. 4).

  $$ ROC = [\left( {m/X_C^{50} } \right)_{oxidant} /\left( {m/X_C^{50} } \right)_{H_2 O_2 } ]\, \times \,100 $$

  (4)

• - m/ H₂O₂eq: ratio between the slope (m) of H₂O₂ linear equation and the oxidant concentration expressed as H₂O₂ equivalents (H₂O₂eq). The H₂O₂eq of each oxidant agent was calculated from the linear equation of H₂O₂ according to Eq. 5 (Eq. 5):

  $$ H_2 O_2 eq = (D\%^{50}_{oxidant} - q_{{\text{H}}_2 {\text{O}}_2 } )/m_{{\text{H}}_2 {\text{O}}_2 } $$

  (5)

3 Results and Discussion

3.1 Nanostructured Film Assembling

Figure 1 reports the spectrophotometric characterization in the Visible range of the EP modified with polydopamine (PDA) film (Fig. 1A) and nano-decorated with gold (AuNPs, Fig. 1B and 1C) and silver nanoparticles (AgNPs, Fig. 1D). The NPs formation has been confirmed by the localized surface plasmon resonance (LSPR) phenomenon typically exhibited by nanosized Au and Ag at absorption band of 540 ± 10 nm and 410 ± 10 nm, respectively [1,2,3].

Fig. 1.

Visible absorbance spectra of PDA@AuNPs-CT@AgNPs formation steps. (A) PDA thin-film formation: EP well before (black line) and after PDA film formation (orange and grey lines); (B) AuSDs self-formation: PDA film before (black line) and after AuSDs formation(orange and grey lines); (C) AuSDs functionalization: AuSDs before (black line) and after catechin functionalization (yellow and orange lines); (D) AgNPs-nanonetwork formation: Au-nanostructured film before (black line) and after AgNPs nanonetwork formation (orange, blue, and grey lines). The inset schematizes the PDA@AuNPs-CT@AgNPs formation steps.

The PDA nano-decoration with gold occured by a spontaneous self-assembly driven by the reducing free moieties (i.e., catecholic portions) of PDA that are able to reduce the AuSD precursors (Au(III)) from the cationic to the nano-sized and zero-valent state (Au(0)). The AuSDs formation was assessed by the presence of LSPR absorption peak at 550 ± 3 nm (Fig. 1B). Then, the AuSDs were functionalized with catechin (CT) exploiting a seed-growth strategy; the CT acts as a reducing agent inducing an AuSDs growth resulting in an AuSDs enlargement (AuNPs) coupled to the intrinsic surface functionalization [1, 2]. The latter gave rise to an increase in the absorption peak intensity (Fig. 1C). The CT anchoring onto the AuNPs results in a formation of a redox-active corona able to trig the silver nanonetwork formation. In brief, the CT-shell, thanks to the CT-reducing ability, induces the AgNPs formation onto the PDA@AuNPs-CT film resulting in a bimetallic nanostructured film [1, 2]. The AgNPs formation was confirmed by the LSPR profile that exhibits an absorption band at 405 ± 7 nm, proper of silver nanostructures [1, 2]. The LSPR profile of AgNPs hides the AuNPs absorption peak. Each step of the nanostructure fabrication highlighted great reproducibility with RSD values (n = 96 wells) of 6.3% for the PDA film formation, 7.4% and 7.2% for the AuSDs self-formation and CT functionalization, and 8.0% for the AgNPs nanonetwork formation.

3.2 Sensing Strategies, Performances, and Reactivity Indexes

The PDA@AuNPs-Ct@AgNPs thin-film prepared in EP wells is a ready-to-use platform for oxidant agents evaluation without the needing for external reagents [1, 2]. The sensing mechanism relies on the ability of external oxidant agents, the analyte, to etch the MNPs; the etching is the chemical oxidation of MNPs resulting in morphological change until their complete dissolution [1, 2]. Among MNPs, the AgNPs are the most used NPs for etching-based strategies development because more prone to donate electrons compared to other noble metals (i.e., gold, platinum, etc.) [1].

Previous works have demonstrated how oxidant agents such as hydrogen peroxide (H₂O₂), peracetic acid, and sodium hypochlorite (NaClO) can selectively and quantitatively oxidize the AgNPs on similar Au/AgNPs-based nanostructured devices, resulting in a proportional LSPR variation used as analytical signal [1, 2]. Table 2 summarizes all the oxidant agents tested (i.e., H₂O₂, NaClO, NaNO₂, FeSO₄, ABTS, DPPH, and AAPH) by using the PDA@AuNPs-Ct@AgNPs platform and exploiting the etching strategy.

Table 2. Oxidant agents dose-response curve parameters and respective oxidant capacity indexes.

According to previous studies, the AgNPs LSPR peak intensity decrease (D%) induced by the analyte-mediated etching was used as analytical signal. To this aim, the H₂O₂ was employed as a probe to explore and study the AgNPs oxidation progress during the time by monitoring the LSPR absorbance spectra evolution (Fig. 2A). The LSPR absorbance spectra variation along the reaction progress confirms the H₂O₂-mediated AgNPs etching by peak intensity decreases at 405 nm, which is the typical absorption band of AgNPs [1, 2]. Further, the selective etching of AgNPs was proved by the LSPR absorbance spectrum recorded after 40 min of reaction (Fig. 2A, bottom spectrum). Indeed, the LSPR peak of AgNPs completely disappeared due to the full oxidation of AgNPs leading to the AuNPs LSPR profile occurrence with absorption band at 520 nm (Fig. 2A, bottom spectrum).

Fig. 2.

(A) Absorbance spectra evolution before (brown line) and during the PDA@AuNPs-CT@AgNPs etching mediated by 200 µM H₂O₂ along time; every 10 min for a total reaction time of 40 min. The inset reports the PDA@AuNPs-CT@AgNPs colorimetric progress, the reaction time increase from top (t₀) to bottom (t₄₀). (B) Dose-response curves of H₂O₂ in the range of 1–200 µM, the inset reports the kinetic curves of etching mediated by H₂O₂. Dose-response curves of (C) NaClO in the range of 25–900 µM; and (D) ABTS•⁺ in the range of 20–1000 µM.

Among the evaluated oxidant only the H₂O₂, NaClO, and ABTS were able to drive the AgNPs etching (Table 2). H₂O₂ and NaClO are oxidizers, and their mechanism of action relies on the formation of radical species (hydroxyl radical, OH•; active chlorine species OCl⁻, HOCl, Cl⁻, and Cl₂) responsible for the oxidative processes [1, 2], meanwhile, the ABTS radical activity was confirmed. The ABTS is a radical cation commonly used in a photometric assay for the antioxidant capacity evaluation based ABTS scavenging mediated by the analyte [3].

Dose-response curves, for the quantitative determination of H₂O₂, NaClO, and ABTS, were built using increasing amounts of H₂O₂ (Fig. 2B), NaClO (Fig. 2C), and ABTS (Fig. 2D) by plotting the signal (D%) against concentration values (µM). Linear ranges from 1 to 200 µM for H₂O₂, from 25 to 900 µM for NaClO, and from 20 to 1000 µM for ABTS were reported; Table 2 reports parameters of the dose-response curves. No LSPR variation was observed for respective blanks of reaction (reaction medium without analyte). Good determination coefficient (R² ≥ 0.9929) and reproducible signals (RSD ≤ 9.6%, n = 5) were obtained for the analytes. Remarkable limit of detection (LOD) 0f 0.3 for H₂O₂, 2.9 for NaClO, and 6.3 for ABTS were obtained, the LOD was calculated according to the following equation: LOD = [(3 x σ_blank)/linear regression slope] [1].

Despite the NaNO₂, FeSO₄, DPPH, and AAPH are oxidant agents (i.e., NaNO₂, and FeSO₄,) and radical species (i.e., DPPH, and AAPH) no AgNPs etching was reported. The ineffectiveness of these species could be attributed to the low oxidation capacity in the experimental reaction conditions; indeed, NaNO₂ exhibits oxidant capacity at acidic pH [6], the FeSO₄ is used as an oxidant stressor for in-vitro studies then long reaction times (t > 48 h) and physiological conditions are required [7], and DPPH principally retains its radical behaviour in ethanolic/methanolic solution [4]. The AAPH is a strong radical generator, known for its ability to initiate/trigger oxidation processes in living organisms; although the potential oxidant ability of AAPH, no etching phenomenon was observed because of the higher instability of AAPH radicals resulting in short half-life [5].

Eventually, the H₂O₂, NaClO, and ABTS reactivity were evaluated and compared. To this aim, m/X_C⁵⁰, ROC, and m/H₂O₂eq parameters with metrological characteristics have been extrapolated by the linear equation (see Sect. 2.4). These parameters allow comparing different oxidants overcoming the partial information given using the single parameters as the m or linear range in terms of concentrations and signal reached. In detail, the m/X_C⁵⁰ encloses the dose-response curve slopes (change in response per unit of concentration) and the mean oxidant concentration of the linearity range, allowing to directly assess and compare the oxidant capacity and effectiveness. The higher the value of m/X_C⁵⁰, the greater the oxidizing activity [3].

The m/X_C⁵⁰ values of H₂O₂, NaClO, and ABTS are reported in Table 2. As expected, H₂O₂ (m/X_C⁵⁰ = 1.5 x 10^–3) is the most reactive chemical species respect with NaClO (m/X_C⁵⁰ = 1.5 × 10^–4), and ABTS (m/X_C⁵⁰ = 6.0 x 10^–5); for this reason, the H₂O₂ was used as a reference/model system for the calculation of the ROC and m/ H₂O₂eq indexes. The ROC and m/H₂O₂eq are relative indexes allowing to relate the oxidant reactivity to the reference standard resulting useful for samples analysis.

As evinced in Table 2, according to m/X_C⁵⁰, ROC, and m/ H₂O₂eq parameters the reactivity order is H₂O₂ > NaClO > ABTS.

3.3 Platform Storability

Eventually, the storability test of PDA@AuNPs-Ct@AgNPs platform to study the life-time of the proposed platform has been performed. Different storing conditions have been tested: environmental atmosphere/air and nitrogen (N₂)-modified atmosphere, different buffer/solvents (water/H₂O, methanol/MeOH, methanolic solution in water, phosphate buffer/PB, saline solution), and antioxidant/reducing compounds (ascorbic acid/AA, sodium citrate). Figure 3 report the signal evolution (D%) the PDA@AuNPs-Ct@AgNPs platform stored during time. In all the conditions tested the platform resulted storable for one month, and after 16 days no further signal variation was reported. The most promising results have been obtained with methanolic solution in water (80, 60 and 40% of MeOH) and 10 mM sodium citrate aqueous solution retaining more than the 89% of the starting signal (Fig. 3). In all the storing conditions reproducible signals were highlighted (RSD ≤ 2.9%, n = 5) proving the stability of the nanostructured film during the time.

Fig. 3.

Solvents screening for PDA@AuNPs-CT@AgNPs storabililty assessment during time. (A) Solvents and gaseous environments that act better than water (blue line): 10 mM sodium citrate aqueous solution (yellow line), nitrogen (green line), 40% methanolic solution in water (violet line), 60% methanolic solution in water (brown line), 80% methanolic solution in water (red line), 100% methanol (olive line). (B) Solvents and gaseous environments that act worse than water (blue line): 20% methanolic solution in water (brown line), environmental atmosphere/ air (orange line), 10 mM ascorbic acid aqueous solution (green line),10 mM phosphate buffer (pH 7.0) (yellow line), physiological solution/0.9% saline solution (grey line). The signal was expressed as Storability/% vs. time monitoring the Absorbance intensity retention (Abs%, λ = 405 nm) during time. The absorbance intensity retention has been reported as % and calculated according to the following equation: Abs% = (Abs_t / Abs_t0) x 100, where Abs_t0 is the absorbance value before the storability test and Abs_tx in the absorbance value recorded after different times of storage.

For the better storing conditions, no aggregation or collapse phenomena are observed, indicating that Ag is well anchored and embedded in the film. Whereas for the worse storing condition the absorbance intensity decrease could be attributed to (i) a slight peak decrease due to AgNPs oxidation driven by the solvents (i.e., MeOH of air), (ii) a shift and/or broadening of the AgNPs LSPR peak induced by AgNPs morphology changes or interaction of solvents with the AgNPs surface (i.e., saline solution).

4 Conclusions

In this study, a nanostructured thin film using polydopamine self-decorated with Au and AgNPs were developed. The nanostructure fabrication can be performed onto any substrate thanks to the use of polydopamine which is able to form ubiquitously adhesive redox-active polymer. Herein, the nanostructured thin film was realized onto ELISA plate to provide easy-to-use systems for the evaluation of oxidant species by using the etching strategy. The developed platform allowed the monitoring and quantification of the H₂O₂, NaClO, and ABTS which are analytes of and biological interest.

Useful indexes (m/X_C⁵⁰, ROC, and m/ H₂O₂eq) were extrapolated from the oxidants dose-response curves which enable the direct comparison of the oxidant capacity of oxidant species as well as the evaluation of their intrinsic reactivity. The indexes herein proposed are universally exploitable for the comparison of molecules with the same class of reactivity (i.e., oxidant agents, antioxidant agents, etc.).

Finally, the storability of the proposed nanostructured platform in several storing conditions (solvents, modified atmosphere, etc.) has been assessed. Definitely, herein is proposed the realization of ready-to-use kits, potentially useful in different bioanalytical industrial/environmental applications.