Lab-on-a-Tip Based on a Bimetallic Nanoarchitecture Enabling Catalytic 4-Nitrophenol Switch-off

: Mono-and multi-metal nanoparticles (MNPs), thanks to their unique and tunable features, still fascinate the analytical sciences, from their widespread use in sensing and biosensing as nanoplasmonic tags or catalysts up to MNPs-decorated surfaces. Here, a lab µ-Tip decorated with plasmonic-active polymeric films embodying gold/silver nanostructures is presented. The proposed lab-on-a-tip device speed-up the 4-nitrophenol conversion in 4-aminophenol, retaining the performances for more than 10 consecutive measures, acting as an enzyme-like catalyst.


Introduction
Mono-and multi-metal nanoparticles (MNPs) are more and more protagonists of cutting-edge technology, demonstrating unique potentialities in different fields such as medicine, biotechnology, agriculture, etc. [1]. In particular, MNPs, thanks to their unique and tunable optical features, formally named localized surface plasmon resonance, have inspired the development of a new generation of optical (bio)sensing strategies. Among these, colorimetric strategies based on the MNPs formation or aggregation have permitted the selective detection of a plethora of analytes (e.g., ascorbic acid, glucose, polyphenols, pesticides, etc.) giving rise to effective assays [1][2][3][4][5][6]. Furthermore, rationally synthesized MNPs have been also widely employed in bioanalytical applications as nanotags and catalysts [7][8][9][10]. MNPs are often defined as 'nanozyme' due to their ability to catalyze chemical and biochemical reactions with notable effectiveness [7][8][9], allowing both homogeneous and heterogeneous catalysis [7][8][9].
The dyes regeneration aims to convert a dye from a non-active to an active state or from a toxic to a non-toxic form [11]. The 4-nitrophenol (4-NP) is a toxic substance potentially dangerous for the environment widely used in pesticides, drugs, and synthetic dyes production [7][8][9]; besides this, the 4-NP is also the product of several enzymatic reactions (phosphatase, glycosidase, etc.). Fortunately, the 4-NP can be converted into 4-aminophenol (4-AP) resulting in less toxicity; this conversion can be obtained in presence of sodium borohydride, a strong reducing agent, and can be catalyzed throughout different strategies.
Commonly, nanozyme catalyzers reported in literature are used in solution as nanodispersions, and the related drawbacks of colloidal MNPs such as low stability and storability (due to their tendency to aggregate, precipitate, self-oxidize, etc.) need to consider tracing a limit in large-scale application (e.g., wastewater treatment plant). More important, the nature of the nanodispersions makes the reusability of the nano catalyzer not simple, requiring time-spending washing and recovering steps, which can lead to nanodispersion morphology changing with loss of efficiency.
The adhesive nano-films proposed here are composed of AuNPs and AgNPs self-assembled thanks to polydopamine onto the µ-Tips internal surfaces. The µ-Tips modified with the here proposed films result in the ability to catalyze the 4-NP conversion up to 10 reaction cycles, allowing the 4-NP colorimetric decay monitoring. The proposed adhesive nanocomposite represents a springboard for the MNPs-based catalyst, potentially projectable in large-scale production, able to give rise to user-friendly devices as demonstrated by the lab-on-a-tips reported here.
Polystyrene Cuvette and Gilson pipette were purchased from Sigma-Aldrich (St. Louis, MO, USA). Spectrophotometric measurements were carried out using a JENWAY 6400 Spectrophotometer from Barlworld Scientific (Staffordshire, UK).

Metal Nanocomposite Fabrication in Laboratory-Tip
The metal-based nanocomposite formation was performed in 1 mL laboratory µ-Tips. The lab-Tips decoration was performed according to Scroccarello et al. [13,14], with some modifications. A schematization of the whole process is reported in Figure 1 and the main steps are described below.
2.2.1. PDA Film Self-Assembly DA at a concentration of 0.5 g L −1 was prepared in Trizma buffer (10 mM, pH 8.5) and freshly used to fill lab-Tips (800 µL per tip). DA polymerization was carried out in static condition, in a white incubation room, at 25 ± 2 °C, under a light source (20 W, warm light bulb) placed at 50 cm of distance for a total polymerization time of 15 h. Finally, the polydopamine (PDA) formation was blocked, subsequently emptying the tips, which were washed and rinsed with abundant Milli-Q water. The PDA modified laboratory µ-Tips (µ-T@PDA) were let dry at room temperature. The AgNPs decorated µ-T@Au (µ-T@Au@Ag) were left to dry at room temperature.

Gold and Silver Nanodecoration
• Au@AgNPs decoration: For the bimetallic nanocomposite formation, the previous two steps were performed consecutively.

4-Nitrophenol Reduction
The 4-nitrophenol (4-NP) reduction was performed directly in the modified µ-Tips (µ-T@PDA, µ-T@Au, µ-T@Ag, and µ-T@Au@Ag) in presence of NaBH4. The reaction mix was directly prepared in cuvette with 980 µL of 0.01 M NaBH4 and 10 µL of 4-NP at different concentrations (final volume 1 mL). Then, the mix of reaction was sucked in the µ-Tips through Gilson pipette and incubated in the dark. The 4-NP color decay evolution kinetics (conversion at 4-AP, colorless) was monitored in cuvette through absorbance recording every 5 min at 400 nm (tn) against the absorbance at time 0 (t0), according to the following equation: (2)

µ-Tip-Surfaces Nanodecoration
The aim of this work was the development of a reusable and smart nano-catalyst system for the monitoring and degradation of 4-NP. One-milliliter laboratory µ-tips (µ-T) were modified with a PDA film and subsequently decorated with AuNPs, AgNPs, and a bimetallic Au@AgNPs nanocomposite, respectively, and the tips catalytic performance were evaluated against 4-NP reduction ( Figure 1). Dopamine (DA) and metal-precursors concentrations, pH, growth time of both the polymer and the nanoparticles (see Section 2.2) were selected according to our group's previous works [13,14]. In brief, the formation of the PDA thin-film onto the µ-T surface was achieved through a DA polymerization in mild alkaline condition; the resulting polymer, thanks to its redox-active chemistry, allowed the MNPs' (AuNPs and AgNPs) growth and anchoration. The PDA chemistry allows a strong adhesivity permitting an in-situ nanocomposite formation onto different surfaces. Then, exploiting the PDA features, reproducible nanometallic-based films were obtained onto several substrates (Pasteur pipette, Eppendorf, ELISA plate, cuvette, tips, etc.) composed of different materials (glass, polyethylene terephthalate, etc.) (Figure 1).

Reduction of 4-Nitrophenol by Using Nanodecorated µ-Tips
As illustrated in Figure 2, the 4-NP reduction by the nanodecorated µ-Tips occurs in three main steps: (i) Reaction mix absorbance reading at time zero (λ = 400 nm); (ii) reaction mix suctions in the µ-Tips and incubation (15 min), (iii) reacted mix color decay reading (λ = 400 nm). As reported in Figure 3a, it is possible to notice how in the same reaction conditions, the reduction rate of the three different nanodecorated µ-Tips were significantly different. Noteworthily, by using the undecorated polypropylene and PDA decorated µ-Tips, no discoloration occurs, confirming the catalytic role of the nanodecorated surfaces towards the 4-NP reduction (Figure 3a). Among the different nanodecored µ-Tips the µ-T@Au@Ag result the most catalytic, followed by the µ-T@Ag and the µ-T@Au. The µ-T@Au@Ag allows the 4-NP complete reduction (discoloration) in 25 min.
The obtained data result in being totally in accordance with the literature, where it was demonstrated how multi-metallic nanodispersions improves reaction rates compared to single metals. Besides, it is well recognized how the catalytic activity towards the 4-NPs reduction is strictly related to the size, shape, and electron density of the employed nanomaterials [7]. In the case of the µ-T@Au@Ag, it is noticeable that the catalytic performance of the µ-T@Au@Ag is ascribable to the catalyst behavior of the Au and AgNPs (Figure 3a). In our case, for the µ-T@Au@Ag, the superior catalytic performance can be ascribed to the nano Au and Ag catalytic synergistic action; nevertheless, in order to better understand the Au and Ag role, an accurate modeling and parameterization of the kinetic curves is required.
Finally, to study the 4-NP concentration effect on the catalytic reaction, and to obtain quantitative monitoring of the 4-NP decay, dose-response curves in the range of 50-500 µM were built-up with the nanodecorated µ-Tips ( Figure 3b). As expected, the µ-Tips previously observed catalytic behaviors results resumed in the dose-response curves' slopes (see Table 1), where the slope obtained with the µ-T@Au@Ag resulted in being the highest, followed by the µ-T@Ag and the µ-T@Au. As expected, no dose-response curve was obtained with the PDA-modified µ-Tip. The proposed devices result usable up to 10 consecutive 'measures' ensuring the same catalytic activity (RSD ≤ 5%), demonstrating a significant advantage compared to MNPs-based catalysis in colloidal solutions. The devices are storable at room temperature for one month without significant loss in the catalyticity.

Conclusions
Here, a nanodecoration strategy to modify laboratory µ-tips has been proposed for the first time. The proposed procedure allows decorating µ-tips surfaces with AuNPs, AgNPs and their nanocomposites. The superior catalytic activity of the bimetallic films (t@PDA@Au@Ag) for the 4-NP reduction was demonstrated. The here-proposed lab-on-a-tip catalyzer results reusable (up to 10 times), elegantly overcoming the colloidal MNPs limitations, also ensuring a 4-NP quantitatively monitoring. The here-proposed nanodecorated films proved to be versatile and potentially usable on different supports allowing tailored nanodecorations, proposing themselves as new and useful analytical tools potentially suitable for catalysts and biosensors developments.