Silver Nanoparticles and Metallic Silver Interfere with the Griess

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Silver nanoparticles and metallic silver interfere with the Griess reaction: reduction of the azo-dye formation via a competing Sandmeyer-like reaction. Angela Kaempfer, Rita La Spina, Douglas Gilliland, Sandro Valzacchi, David Asturiol, Vicki Stone, and Agnieszka Kinsner-Ovaskainen Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00280 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 13, 2017

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Chemical Research in Toxicology

Silver nanoparticles and metallic silver interfere with the Griess reaction: reduction of the azo-dye formation via a competing Sandmeyer-like reaction Angela Kämpfer†, Rita La Spina†, Douglas Gilliland†, Sandro Valzacchi†, David Asturiol†, Vicki Stone§, Agnieszka Kinsner-Ovaskainen†*

†

European Commission Joint Research Centre, Directorate for Health, Consumers and Reference

Materials, Via E. Fermi 2749, TP 125, 21027, Ispra (VA), Italy §

Nanosafety Research Group, School of Life Sciences, Heriot-Watt University, Edinburgh, EH14

4AS, United Kingdom

*Corresponding Author: Agnieszka Kinsner-Ovaskainen European Commission Joint Research Centre, Directorate for Health, Consumers and Reference Materials, Via E. Fermi 2749, TP 127, 21027, Ispra (VA), Italy Tel : +39 0332 78 9246 Email: [email protected]

Keywords: Griess reaction, silver nanoparticles, in vitro assay, interferences, nitric oxide

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Abstract Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g. cytotoxicity, genotoxicity or pro-inflammatory responses. In the case of pro-inflammatory response a frequently used endpoint is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO2-) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO2- in a cell-free environment. Solutions containing a known concentration of NaNO2 were prepared in H2O, PBS or complete cell culture medium (CCM) and analyzed using the Griess reaction in presence of Ag in metallic or ionic state. In MilliQ H2O the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO2-. The effect was more pronounced in PBS and H2O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.



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Introduction Silver is one of the most abundantly used NMs in consumer products. To date, over 400 applications containing nano-sized Ag have been registered, 1 most commonly medical devices2,3 and food contact materials.4 Since consumers can be exposed to Ag NPs leaking or migrating from products5,

6

concerns have been raised regarding potential adverse effects on human health. Whereas the cytotoxic,7,8,9 genotoxic10,11 and reprotoxic12,13 potential of Ag NPs is well documented, their impact on the immune system remains unclear. Some studies observed anti-inflammatory effects both in vitro14,15 and in vivo.16,17 On the other hand, Ag NPs were described to act proinflammatory, e.g. inducing release of pro-inflammatory cytokines,18,19 modulation of immune cell distribution,20 induction of Reactive Oxygen Species (ROS)18,19 and Reactive Nitrogen Species (RNS).21,22 The most inconsistent results regarding Ag NP-induced adverse effects are those documented for the cellular synthesis of the radical ‘nitric oxide’ (·NO). NO can be an important physiological messenger molecule or an indicator for pathophysiological processes depending on its origin.23 Under physiological conditions, the generation of nanomolar-concentrations of NO by constitutively expressed endothelial ((e)NOS) and neuronal ((n)NOS) nitric oxide synthases and is meticulously regulated.24 The generated NO is involved in the regulation of blood pressure25 and neuronal functions.26 In contrast to this, the activation of inducible

(i)NOS

causes the persistent release of

increased concentrations which can reach into the micromolar range.24 The ability to release large quantities of NO has been demonstrated to be an important feature of the innate immune response.27, 28

The prolonged and uncontrolled release of NO can, however, cause damage in neighbouring cells

and is suspected to play a role in various inflammatory and neurodegenerative disease, like Alzheimer’s,29 multiple sclerosis,30 and inflammatory bowel disease.31 The most frequently used assay to indirectly quantify NO is based on the Griess reaction, first described in 1879.32 In this reaction an azo-dye is formed from a nitrosatable compound and a coupling reagent at low pH in the presence of NO2- – a stable breakdown product of NO.33 In 4 ACS Paragon Plus Environment

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nanotoxicological research, the assay is popular because of its straightforward application, simple interpretation, and cost-effectiveness.34 Furthermore, only a few interferences with NMs have been reported to date, e.g. increase of absorbance in the presence of iron oxide and SiO2 NPs,35 or a slight reduction in optical density in the presence of Au NPs. The reported results for Ag NP-induced NO generation vary considerably between studies despite the easy handling and low interference potential of the assay. Whereas some groups quantified significantly increased concentrations of NO2- after exposure to Ag NPs,7,21 other studies did not observe any change.36,37 Yet others noted decreasing concentrations of NO2- after exposure to Ag NPs.38,39 Recently, several groups have started to apply the Griess reaction to demonstrate a NOscavenging effect of Ag NPs.40,41 A possible reason for the large variations between the different studies might be the result of an interference by metallic

(m)Ag

with the Griess reaction, which we have observed that under certain

circumstances can cause false negative results by preventing the azo-dye formation. In this study we will describe the occurrence of this anomalous behaviour of the Griess reaction and propose a possible mechanism by which the silver nanoparticles interfere with the NO quantification.

Experimental procedures Materials for NP-synthesis Silver nitrate (AgNO3) (purity: 99.99% based on Trace Metal Analysis), tannic acid, Gold (III) chloride trihydrate (purity: 99.99% based on Trace Metal Analysis), 3-aminopropyltrimethoxysilane (reagent grade > 98%), ethanol, arginine (reagent grade > 98%), cyclohexane (for HPLC >99.7%), tetraethoxy orthosilane were purchased from Sigma-Aldrich; tri-sodium citrate dehydrate (citrate) (AR grade Cl ≤ 0.001 %), sodium hydroxide (pro-analysis grade; Cl max 0.0005 %) were purchased from Merck. Solutions were prepared in deionized water from Millipore MilliQ water purification system. 5 ACS Paragon Plus Environment


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Materials for cell culture Phosphate Buffered Saline (PBS), cell culture medium (CCM), non-essential amino acids, Sodium Pyruvate, Penicillin/Streptomycin (Pen/Strep), 2-Mercaptoethanol, and L-Glutamine were purchased from Invitrogen. D-Glucose and Phorbol 12-myristate 12-acetate (PMA) were obtained from SigmaAldrich.

Materials for Griess reaction and control experiments The reagents to perform the Griess reaction (Sulfanilamide (SA), N-(1-napthyl) ethylenodiamine hydrochloride (NEDA), and phosphoric acid (H3PO4) as well as sodium nitrite (NaNO2), and all materials used to study the interference with the Griess reaction, such as silver acetate (purity: 99.99% based on Trace Metal Analysis), silver oxide (Ag2O) (purity: 99.99% Based on Trace Metal Analysis), sodium chloride (NaCl) (grade: for molecular biology >98%), and potassium bromide (KBr) were purchased from Sigma-Aldrich. A stock of 30 nm control silver nanoparticles synthesized without tannic acid and stabilized in citrate was obtained from Ted Pella Inc.

Synthesis of NPs Ag NPs: 25 nm Ag NPs were obtained by reduction of AgNO3 with citrate and tannic acid. Full details of Ag NPs synthesis method are given section 1.1 of the Supplementary Information. Au NPs: 30 nm Au NPs were obtained by regrowth method of 15 nm Au NPs by reduction of gold (III) chloride trihydrate with citrate. Details of the Au NPs synthesis are described in section 1.2 of the Supplementary Information. SiO2 NPs: To obtain nominally 100 nm mono-dispersed particles a synthesis method adapted from the procedure in the publication by Hartlen et al.42 was used. Full details of the modified procedure used are described in section 1.3 of the Supplementary information.


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Characterisation of the as-synthesized particles The size and size distribution of the synthesized Ag NPs, Au NPs and SiO2 NPs in dispersion solution were assessed by Centrifugal Liquid Sedimentation (CLS), and Dynamic Light Scattering (DLS) while shape and size were verified by Scanning Electron Microscopy (SEM) imaging. The CLS measurements (instrument model DC24000UHR by CPS Instruments) were performed in an 8 wt %24 wt % sucrose density gradient with a disc speed of 22000 rpm. Each sample injection of 100 µl was preceded by a calibration step using certified polyvinyl chloride (PVC) particle size standards with weight mean size of 280 nm. Measurements of particle size distribution by DLS were done using a Zetasizer Nano-ZS by Malvern Ltd, UK. Each sample was measured in triplicate at 25°C after an equilibration step of 120 sec and using an acquisition time of 80 sec. The hydrodynamic diameter was calculated by the DLS internal software. The size distribution and shape of the particles were also verified using SEM imaging of particles adsorbed from solution onto the polished, chemically modified (functionalized with 3Aminopropyltrimethoxysilane)43 surface of a cleaved silicon wafer. To prepare samples for SEM analysis, small rectangular shards (≈0.5 cm2) of the amino-functionalized silicon wafer were immersed an aqueous dispersion of the particles for 15 min. The silicon shards were then removed from the particle dispersion, gently dipped in MilliQ water to remove excess particles and finally dried in air before insertion in the SEM. SEM analysis was done in secondary electron imaging mode using a FEI NOVA 600, Dual Beam SEM operating with acceleration voltages of 5 KeV. The software ImageJ was used for image analysis with a minimum of 100 particles being counted for size and size distribution calculations. The optical properties of Ag NPs in different matrices were investigated by UV-vis spectroscopy (Thermo Electron Corporation, Nicolett Evolution 300). The results of Ag NPs, Au NPs, and SiO2 NPs are presented in Table S1 and Figure S1.



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Cell culture Monocytic THP-1 cells (ATCC, TIB-202) were differentiated to macrophage-like cells using PMA. A more detailed description of the cell culture is provided in section 1.4 of the Supplementary Information. Subsequently, the cells were exposed to 10 ng/mL lipopolysaccharides (LPS, SigmaAldrich) or varying concentrations of Ag NPs (10-100 µM). To investigate the effect of Ag NPs on the detection of NO2- the supernatants of LPS exposed cells were post-incubated with 10 or 100 µM Ag NPs for 1.5 h before analysis with the Griess reaction.

Nitrite quantification with the Griess reaction The Griess reaction is used for the chemical detection of NO2-, a stable metabolite produced from NO. Briefly, available NO2- reacts with SA in acidic environment to form a diazonium salt as intermediate product. The diazonium intermediate undergoes a coupling reaction with a diamine, in this case NEDA, to form a pink azo-dye. The amount of formed dye is proportional to the amount of NO2- in solution. To each sample (100 µL), SA (stock: 1 mM in MilliQ H2O), H3PO4 (stock: 1.8 M), and NEDA (stock: 1 mM in 95% ethanol) were added to a final concentration of 100 µM, 170 mM, and 100 µM, respectively. The addition of each subsequent reagent was separated by a 30 min incubation time at room temperature. The absorbance of both the background and the dye formed were measured spectrophotometrically (BMG Labtec) at 548 nm. A calibration curve was prepared in each of the matrices using NaNO2 (0-50 µM) dissolved in H2O. The Griess reaction was carried out on the cell supernatant after exposure to LPS (10 ng/mL) or Ag NPs (10 and 100 µM), and on NaNO2 (24,6 µM) spiked samples prepared in ultrapure MilliQ H₂O (hereafter ‘H2O’), PBS, or CCM containing 10% fetal bovine serum. Where applicable, citrate, tannic acid, AgNO3 (at a concentration equivalent to their presence in Ag NPs), silver acetate, Ag2O, Au NPs, SiO2 NPs (at the same concentration as Ag NPs) or varying amounts of silver metal sheet (bulk Ag) were used to substitute Ag NPs. To investigate the influence of salts on the interference with the Griess reaction the reaction mixtures were modified by appropriate additions of NaCl or KBr dissolved in H₂O. 8 ACS Paragon Plus Environment

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Liquid Chromatography-Mass Spectrometry analysis (LC-MS) The removal of the Griess reaction diazonium salt intermediate via a reaction with halogen elements in solution catalyzed by metallic (m)Ag, was proposed as a possible explanation of the interference of Ag NPs on the colorimetric test. For this reason, the formation of 4-Chlorobenzenesulfonamide (4ClBS) and 4-Bromobenzenesulfonamide (4-BrBS) during the Griess reaction was monitored using LC-MS. The analysis was performed with an UPLC Acquity instrument (Waters Corporation, Milford, MA, USA), equipped with an Acquity UPLC BEH C18 analytical column (50 mm x 2.1 mm; 1.7 µm). The UPLC was coupled with a Xevo TQ MS mass spectrometer with electrospray interface (Waters Corporation, Milford, MA, USA). After the development of the Griess reaction, the solutions were centrifuged (10 min at 25.000 rpm) and an aliquot of 5 µL was injected. The chromatographic separation was performed at 40°C, with 5 mM ammonium formate in water (channel A) and 5 mM ammonium formate in 95:5 acetonitrile/water (channel B) as eluent, with the following gradient: 60% A to 10% A in 4.5 min, 10% A for 1 min. The mass spectrometer was operated in negative ionization mode, with 900 L/h desolvation gas flow, 450°C desolvation temperature and 3 KV capillary voltage. The analysis was performed in multiple reaction monitoring (MRM) with 189.9 to 125.9 m/z (Cone voltage 32, collision voltage 30, dwell time 0.1 s) and 189.9 to 79.6 m/z (Cone voltage 32, collision voltage 16, dwell time 0.1 s) as quantifier and qualifier transitions for 4-ClBS and 233.8 to 78.8 m/z (Cone voltage 32, collision voltage 30, dwell time 0.1 s) and 233.8 to 78.8 m/z (Cone voltage 32, collision voltage 16, dwell time 0.1 s) for 4-BrBS. The instrument was calibrated with external standard solutions of the two compounds at five concentration levels in the range 0.1 – 1.5 µg/mL (R=0.998). Calibration standards were prepared in the same matrix as the analytical samples to correct for signal suppression effects.



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Statistical analysis The data were analyzed and the results illustrated using Microsoft Excel. The results are presented as average of three independent experiments. Variations between results are expressed as standard deviation (SD). The data was statistically analyzed by one-way ANOVA using Microsoft Excel. A value of p≤0.05 was accepted as statistically significant.

Results The Griess reaction was used to quantify the presence of NO2- in cell supernatants as indicator for the cellular synthesis of NO after exposure to Ag NPs or LPS. No increase in NO2- was detected in supernatants of PMA-differentiated THP-1 cells exposed to various concentrations of Ag NPs, while treatment with LPS induced a clear increase (Figure 1). To exclude the possibility of false negative results in Ag NPs-treated samples, the supernatants from LPS-treated cells were post-incubated with the two highest Ag NPs-exposure concentrations (10 and 100 µM). When comparing the results of Ag NPs-post-incubated samples with the LPS-treated control a statistically significant (p≤0.001) decrease in dye formation proportional to the Ag NPs concentration was observed, suggesting a reduction in detectable NO2- (Figure 1). This effect was only observed when Ag NPs were present throughout the completion of the Griess reaction. If the particles were eliminated from the test solution by centrifugation before the addition of H3PO4, the detection of NO2- was unimpaired (Figure S2). This suggests that the interference is probably linked to the assay or one of its reagents and is different from the known interaction44 of silver and ·NO.

Interference of reagents, reaction products and NPs with the Griess reaction From the initial observation of the anomalous behaviour of the Griess reaction it was not clear whether the interference was caused by non-metallic reactants, reaction products of the Ag NP synthesis, the Ag NPs themselves, or one of the reagents used in the Griess reaction. To investigate 10 ACS Paragon Plus Environment

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further the origin of the interference, three groups of compounds that are used in the Griess reaction or suspected to interfere with it were tested. The groups were formed by compounds that (i) are needed for the synthesis of Ag NPs (tannic acid, citrate), (ii) contain silver (Ag) in ionic or metallic form (AgNO3, Ag acetate, Ag2O, Ag NPs) or (iii) are nano-sized but have different properties than Ag NPs (SiO2 NPs, Au NPs). No dose-dependent reduction in dye formation was caused by any of the compounds involved in the synthesis of the Ag NPs (Figure 2). Other NMs (Au NPs, SiO2 NPs) (Figure 2) as well as salts containing Ag also failed to inhibit the detection of NO2- (Figure S3). A dose-dependent reduction in dye formation only took place in presence of Ag NPs (Figure 2) and metallic bulk Ag (Figure S4). Interestingly, apart from the dose of Ag NPs also the contact time between Ag NPs and H3PO4 had an influence on the magnitude of the interference (Figure S5). The shorter the contact time between particles and acid was before the addition of NEDA the more dye was formed. Based on these results the observed interference is most likely linked to the presence of Ag NPs, more precisely to (m)Ag. The interference is not attributable to any substances used in the synthesis of the particles, ionic silver, or the reagents of the Griess reaction. The effect cannot be explained with the nano-size of Ag NPs, but may be linked to the surface area or availability of

(m)Ag.

The

concentrations of both SA and NEDA used are 1000 times higher than the minimum quantity needed to entirely cover the surface area of 100 µM Ag NPs (calculations not shown). Hence, the adsorption of the reagents onto Ag NPs can be excluded as main cause for the observed interference.

NP stability in different solvents The degree of aggregation of Ag NPs varies with the matrix in which they are contained. Since the stability of the NPs can affect the way in which they interfere with the Griess reaction, the behaviour of Ag NPs in three different solvents (PBS, H2O, and CCM) was studied. In Figure 3A, the UV-vis spectra of Ag NPs in water show the absorption peak at around 400 nm which is characteristic of the surface plasmon resonance properties of nearly-monodisperse 20-30 nm 11 ACS Paragon Plus Environment


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Ag NPs. When preparing the suspension in PBS, Ag NPs instantly aggregated probably due to the high content of salts in the solution. This aggregation is reflected in a band intensity reduction as measured with UV-vis (Figure S6). In the case of CCM, the presence of serum proteins in the mixture ensured that the Ag NPs would rapidly be coated by a protein corona,45 which effectively stabilizes the particles against the effects of salt induced aggregation. This stabilizing effect of CCM is shown by the persistence of the Ag NP-associated absorbance peak at around 400 nm (Figure S6). The aggregation state of NPs can significantly affect the fate of the particles, especially in the second step of the Griess reaction when the mixtures are acidified by the addition of H3PO4. Figure 3A shows that although Ag NPs diluted in H2O alone are colloidally stable, they dissolved in the strongly acidic (pH95% in presence of 100 µM Ag NPs (Figure 3B). In both CCM and H2O, 100 µM Ag NPs reduced the dye formation significantly, however less dramatically, to 31 and 57% of the control respectively. The fact that the severity of the effect in the three solvents cannot be explained by the colloidal stability of the particles, points out that the effect is probably not attributable to the nano-size of the particles. Instead, we concluded that the interference may be related to the amount (loss by dissolution) and accessibility (protein shielding) of metallic particles following the acidification of the reaction mixture.


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To study whether the interference could be related to an event detectable at the molecular level, samples in H2O, PBS, and CCM were analyzed by LC-MS. The analysis showed that in presence of 100 µM Ag NPs the azo dye was not formed even though SA and NEDA were present. Instead, another compound, 4-ClBS, was formed. Structurally, 4-ClBS is very similar to SA with chlorine replacing nitrogen on the aromatic ring. The formation of 4-ClBS (Table 1) is in line with the results on the influence of the surrounding matrix discussed above: samples prepared in the chloride rich PBS solution yielded the largest quantities of 4-ClBS, whereas H2O-based samples yielded the lowest. In H2O and PBS, increasing the concentrations of NO2- resulted in an increase in 4-ClBS formation. In CCM, the concentration of 4-ClBS remained constant independently of the NO2concentration, which indicates that another species rather than NO2- is the limiting factor of the reaction. In addition, only ~35% of the 4-ClBS that could have been formed based on the available NO2- assuming a 100% conversion rate of SA to the diazonium intermediate was detected in PBS. This suggests that either more than one mechanism is involved in the observed interference, or more than one additional compound is formed which prevented a further dye formation. If the interference depended on the simultaneous presence of Ag NPs and a nucleophile, the addition of chlorine and potentially other halogens to H2O should lead to an increased inhibition in dye formation (Figure 4). In H2O, the azo dye formation was inhibited by ~50% in the presence of 100 µM Ag NPs. The addition of both chlorine- and bromine-containing salts (NaCl and KBr) resulted in a significantly decreased (p≤0.001) dye formation compared to the Ag NPs-containing H2O control (Figure 4). The tendency to inhibit dye formation was greater for KBr than NaCl. The observation that KBr-containing samples contained detectable amounts of the equivalent brominated compound – 4-BrBS – is a clear indication that both halides promote the same reaction. It was also noted that the majority of samples, independently of the deliberate addition of bromide salts, contained detectable amounts of 4-BrBS presumably due to the presence of trace quantities of bromide in one or more of the reagents added in solution (Table S3).



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Discussion Our results show that Ag NPs can significantly interfere with the Griess reaction by reducing the formation of the azo-compound as much as 95% depending on the experimental conditions. Given that this method is frequently used to study the potential of metallic NMs to induce cellular NO synthesis,22, 37, 46 this observation is of utmost importance. This effect may jeopardize the results and interpretation of studies that did not take into account the effects that the presence of Ag NPs in the solution, the type of matrix used, the presence of salts, and the acid contact time can have on the quantification of NO2-. Though initially unexpected, the interference caused by Ag NPs can be explained by assuming that the presence of

(m)Ag

in the Griess reaction triggers a competing process, which most likely

corresponds to the Sandmeyer reaction. In the Griess reaction (Equation 1A), SA (Ar−NH2) reacts with NO2- in acidic conditions to form an aromatic diazonium ion, i.e. Ar−N+≡N.47

NaNO2

A)

Ar−NH2

B)

Ar-N+≡N + NEDA

Ar−N+≡N + H2O

H+

Azo-Dye

Equation 1. Principle mechanism of the Griess reaction: A) Formation of the diazonium ion; B) Dye formation through coupling of diazonium ion and diamine.

When performing the Griess reaction in absence of (m)Ag, the addition of NEDA results in a coupling of the diazonium ion and the diamine to form a pink azo dye (Equation 1B). The amount of dye formed is proportional to the amount of nitrogen in the sample. Thus, by measuring the absorbance of the dye and assuming a yield of 100% the amount of NO2- present in the solution is determined. Our results show that when the Griess reaction is carried out in presence of

(m)Ag

and a suitable

nucleophile, e.g. Cl- or Br-, an alternative competing process that leads to the formation of 4-ClBS or 14 ACS Paragon Plus Environment

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4-BrBS takes place. This competing process is probably analogous to the Sandmeyer reaction. In the conventional Sandmeyer reaction an aryl diazonium salt decomposes in the presence of a copper(I) halide catalyst, to form an aryl halide. Although its mechanism is not fully understood,48 it is generally accepted that it proceeds via an internal electron (e-) transfer reaction49 (Figure 5A). The presence of a catalyst like Cu allows the substitution of an amine group with a nucleophile, e.g. forming Ar-Cl and Ar-Br. Given the chemical similarity of Cu and Ag – both are transition metals of group 11 of the periodic table – and the even more favourable reduction potential of silver with respect to Cu, E0 Cu2+ Cu+ = 0.16 V vs E0 Ag Ag+ = 0.80 V, (m)Ag could be expected to also act as e- donor. This assumed role of

(m)Ag

is especially important in the first part of the Griess reaction

(Equation 1A): we assume that after the addition of SA and H3PO4 the initial amount of NO2- reacts to form the diazonium salt which, in turn, remains available to react with NEDA to form the indicator dye. However, the presence of

(m)Ag

allows for the Sandmeyer reaction to take place and form the

molecules 4-ClBS and 4-BrBS. Thus, in the presence of

(m)Ag

the diamine coupling (Equation 1B)

and the nucleophilic substitution (Figure 5) reactions will compete for the available diazonium ion thereby decreasing the formation of the azo-dye. The suggested mechanism might explain the dependence of the dye formation on the Ag NP concentration, as well as the time-dependence of the interference. As discussed above, this study has identified two products (4-ClBS and 4-BrBS) supporting the idea of this competing reaction. It is possible that other types of nucleophilic substitution occurs50 (e.g. Ar-OH or Ar-H), which has not been investigated further. In the Sandmeyer reaction, the catalytic effect of copper is thought to result from a change of oxidation state of a metal ion Cu(I)=>Cu(II). In the case of Ag, it can exist in several oxidation states such Ag, Ag(I), Ag(II) and Ag(III). However, Ag(II) and Ag(III) are generally unstable in aqueous media and are normally only generated at high temperatures or in the presence of strong oxidizing agents such as persulphate ions or ozone. For this reason, only the Ag(0) to Ag(I) oxidation has been considered as possible explanation for the interference of the Griess reaction. This consideration is



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supported by the fact that the interference only occurred when metallic Ag was available and it was not observed when only ionic Ag was present. In this context, also the influence of the surrounding matrix on the magnitude of the interference can be explained as follows. In the case of PBS the particles rapidly aggregated but the Ag is still present in its metallic state. The aggregation of Ag NP reduces the surface area exposed to acid (H3PO4) and thus slows the dissolution of the metallic silver, thereby permitting the competing reaction to occur to some extent. Additionally, the presence of the salt also provides a large excess of the nucleophile (Cl), which favours the formation of the products, i.e. the displacement of the (-N+≡N) group of the diazonium salt. In pure H2O, however, the well-dispersed Ag NPs dissolve almost immediately to Ag+ after addition of H3PO4 eliminating the source of (m)Ag. In PBS, the dissolution proceeds over a prolonged period of time, most likely under generation of e- from

(m)Ag.

Furthermore, when

conducting the reaction in pure H2O the lack of nucleophiles will not displace the equilibrium towards the products and the formation of 4-ClBS or 4-BrBS will not occur. When H2O is spiked with salts, however, Ag NPs behave as in PBS: the particles aggregate before the addition of H3PO4, resulting in decreased surface area but, nonetheless, leaving silver in the metallic state. In this case, the addition of H3PO4 likely induces again a formation of e- from

(m)Ag

leading to the generation of

gaseous N2 and subsequent substitution with available nucleophiles. When the reaction is performed in CCM, the protein corona reduces accessibility of H3PO4 or the diazonium salt to the particle surface. As H3PO4 cannot attack the particles’ surface, the availability of e- will be reduced. Hence, the inhibition of dye formation is less pronounced in CCM compared to PBS or H2O + salts, even though both (m)Ag and nucleophiles are available. We do not think that the observation of this effect is entirely new, however, its interpretation and application is novel. The same mechanism was exploited by Qian et al.46 who described the ability of Palladium-covered Au NPs to effectively reduce NO2- as environmental contaminant. In this case, Palladium acted as catalyst. In the case of silver metal, a number of studies have reported on its e-donating properties. Whereas both Furuya and Ritter51 and Wang et al.52 have investigated the 16 ACS Paragon Plus Environment

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fluorination of diazonium salts using Ag-Triflate complexes, Ramamurthy and colleagues40 probably observed the same interference we described. However, in this cases the group attributed the reduced dye formation to the NO-scavenging effect of (m)Ag as described previously by Kim et al.44 and not to an interference with the assay. However, since the group did not state whether the Ag NPs were still present in the sample when the Griess reaction was performed this remains an assumption. It might come as a surprise that another group recently did not find any interference of Ag NPs with the Griess reaction.53 The group’s conclusion is not in contradiction with our findings, though, if the differences in the experimental set-up are taken into account: Instead of using the reagents separately as done in our group, Liang et al. performed the assay with a mixture of the reagents. By adding all three reagents simultaneously, reaction 1B and the nucleophilic substitution reaction (Figure 5) immediately compete with each other. In this case, NEDA has the higher affinity for the diazonium ion. Therefore, the majority of the available diazonium ion reacts with the diamine to form the azodye instead of undergoing the nucleophilic substitution. In conclusion, in the presence of (m)Ag the accuracy of the Griess reaction in quantifying NO2- can be strongly impaired and thus may produce false negative results. Instead of coupling SA with NEDA to form an azo-dye a nucleophilic substitution via a Sandmeyer reaction may occur. Most reports on NO-induction by Ag or other metallic NPs do not mention explicitly whether the particles were eliminated before performing the assay. Hence, it is not clear whether the results of these reports may have underestimated the NO2- measures. Despite the demonstrated interference, the Griess reaction remains a valid and useful assay for in vitro research provided that the following is considered: Performing the assay in presence of e- donors should be avoided, especially if the Griess reagents are added separately with increased incubation times (>10 min). In case experiments are performed using metallic NMs, we recommend to eliminate the particles from the supernatant before the assay, e.g. by centrifugation. Alternatively, the reaction can be run by adding a complete mixture of the reagents including H3PO4. The assay’s suitability to determine the radical-scavenging potential of Ag NPs as



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suggested by40 or any other metal-based NMs requires the inclusion of extensive control experiments to ensure that the measured values are not affected by competing reactions.

Funding Sources The work was funded by the institutional budget of the European Commission`s Joint Research Centre. Angela Kämpfer’s work was supported by a PhD grant from the European Commissions’ Joint Research Centre (contract number: 2012-IPR-I-20-00646).

Declaration of interest statement The authors have no relevant affiliation or financial involvement with any organization or entity with a financial interest or conflict concerning the information presented in this manuscript.

Abbreviations: 4-Bromobenzenesulfonamide (4-BrBS); 4-Chlorobenzenesulfonamide (4-ClBS); Ag NPs: Silver Nanoparticles; Au NPs: Gold Nanoparticles; CCM: Cell Culture Medium; CLS: Centrifugal Liquid Sedimentation; DLS: Dynamic Light Scattering; LPS: Lipopolysaccharide; NEDA: N-(1-napthyl) ethylenodiamine hydrochloride; NM: nanomaterial; NO: Nitric Oxide; NOS: Nitric Oxide Synthase; ROS: Reactive Oxygen Species; RNS: Reactive Nitrogen Species; SA: Sulfanilamide; SEM: Scanning Electron Microscope; SiO2 NPs: silicon dioxide nanoparticles.

Supporting Information Details of synthesis of Ag NPs, Au NPs and SiO2 NPs; Details of THP-1 cell culture; Results of Ag NPs, Au NPs and SiO2 NPs nanoparticle characterisation; Additional supplementary figures related to assay interferences [Influence of Ag NPs concentration and incubation time after addition of H3PO4 on the detection of nitrite; Influence of non-metallic silver compounds on the detection of nitrite in PBS; Influence of metallic silver on the detection of nitrite – nano-sized vs. bulk material; Stability of 18 ACS Paragon Plus Environment

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Ag NPs in PBS, complete cell culture medium and water after the addition of Griess Reagents without addition of H3PO4]; Analysis of halogenated compounds formed during Griess reaction. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table 1

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Concentration of 4-ClBS (in µg/mL) after performing the Griess reaction in presence of 100 µM Ag NPs (NO2¯ concentrations of ≥ 3 µM can be reliably detected with the Griess reaction54).

NO2- (µM)

H2 O

CCM

PBS

0

N/A

N/A

< detection limit

40

0.149

0.711

0.801

80

0.965

0.617

1.477


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Figure legends Figure 1. Nitrite detection in presence or absence of Ag NPs ; LPS exposure: 100 ng/mL.

Figure 2. Detection of NO2- in presence of Ag NPs (10 or 100 µM), their by-products (i.e. citrate, tannic acid, AgNO3), and other NMs (Au NPs, SiO2 NPs); Nota bene: concentrations for tannic acid (TA), citrate and AgNO3 are equivalent to their concentration in 10 or 100 µM Ag NPs (*p≤0.05 compared to control; #p≤0.05 compared to 10 µM Ag NPs).

Figure 3. Influence of the surrounding matrix on (A) the integrity of Ag NPs after adding H3PO4; (B) the detection of NaNO2 in presence of 0, 10, or 100 µM Ag NPs (*p≤0.05 compared to 0 µM Ag NPs in PBS, #p≤0.05 compared to 0 µM Ag NPs in H2O; +p≤0.05 compared to 0 µM Ag NPs in CCM).

Figure 4. Detection of NaNO2 in the presence of 1.5M NaCl and 0.5 M KBr (****p≤0.005 compared to 100 µM Ag NPs in H2O without added salts).

Figure 5. The Sandmeyer reaction: (A) suggested working principle of the Sandmeyer reaction ; (B) examples of reaction products formed by means of the Sandmeyer reaction.50



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Figure 1


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Figure 2



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Figure 3

A

B


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Figure 4



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Figure 5

A

B


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Silver Nanoparticles and Metallic Silver Interfere with the Griess

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