In chemico methodology for engineered nanomaterial categorization according to number, nature and oxidative potential of reactive surface sites

Methanol probe chemisorption quantifies the number of reactive sites at the surface of engineered nanomaterials, enabling normalization per reactive site in reactivity and toxicity tests, rather than per mass or physical surface area. Subsequent temperature-programmed surface reaction (TPSR) of chemisorbed methanol identifies the reactive nature of surface sites (acidic, basic, redox or combination thereof) and their reactivity. Complementary to the methanol assay, a dithiothreitol (DTT) probe oxidation reaction is used to evaluate the oxidation capacity. These acellular approaches to quantify the number, nature, and reactivity of surface sites constitute a new approach methodology (NAM) for site-specific classification of nanomaterials. As a proof of concept, CuO, CeO2, ZnO, Fe3O4, CuFe2O4, Co3O4 and two TiO2 nanomaterials were probed. A harmonized reactive descriptor for ENMs was obtained: the DTT oxidation rate per reactive surface site, or oxidative turnover frequency (OxTOF). CuO and CuFe2O4 ENMs exhibit the largest reactive site surface density and possess the highest oxidizing ability in the series, as estimated by the DTT probe reaction, followed by CeO2 NM-211 and then titania nanomaterials (DT-51 and NM-101) and Fe3O4. DTT depletion for ZnO NM-110 was associated with dissolved zinc ions rather than the ZnO particles; however, the basic characteristics of the ZnO NM-110 particles were evidenced by methanol TPSR. These acellular assays allow ranking the eight nanomaterials into three categories with statistically different oxidative potentials: CuO, CuFe2O4 and Co3O4 are the most reactive; ceria exhibits a moderate reactivity; and iron oxide and the titanias possess a low oxidative potential.

for it is chemisorbing.As the surface reaches its maximum capacity of methanol chemisorption, there is a decrease in the online H 2 O signal, and once the surface is saturated with methanol, new methanol molecules that enter the system do not have a binding site and thus leave the reactor, as observed by online methanol m/z 31 signal increase.When the sample is saturated with methoxy, the feed is switched to 100 mL/min Ar to first purge residual methanol vapor, and then run the TPSR of the chemisorbed methoxy species by linearly heating the sample from 100 to 450 ⁰C at 10 ⁰C/min.Thise signal must be integrated during the chemisorption step to quantify the moles or molecules of methanol retained by the material on the surface until saturation (MeOH s ), and during the TPSR step to calculate the fraction of methanol that is released unreacted (MeOH u ), as follows: When methanol is fed to the reactor (t=0) for chemisorption the signal in the reactor outlet is initially low (or even 0), because methanol is mainly (or even totally) adsorbed on the nanomaterial.Outlet concentration of methanol progressively increases as the surface sites are filled.When the surface is saturated (t=s), methanol signal remains constant and equal to the by-pass value, corresponding to 2000 ppm.MeOH s is determined by calculating the total amount of methanol fed to the reactor from t=0 to t=s and subtracting the integrated signal of methanol in the same period, corresponding to the released methanol that was not adsorbed.Methanol molar flow is readily calculated from its concentration and controlled flow rate (100 mL/min).
Upon heating, the fraction of methanol that is released unreacted is calculated by integrating the methanol signal.The reacted methanol (MeOH r ) is then obtained by subtracting both values, and is equal to the number of surface sites.The specific number of active sites is calculated by dividing this number by the mass of NM used for the experiment.The specific number of surface sites divided by the NM BET area delivers the active sites surface density.

Methanol-TPSR (Temperature-Programmed Surface Reaction) reaction equations
The sample saturated with methoxy and purged in Ar is linearly heated from 100 to 450 ⁰C at 10 ⁰C/min for TPSR.The m/z values followed in the residual gas analyzer were: CH 3 OH (methanol) The type and amount of products and the temperature at which they are detected correlate with the nature, amount and reactivity of the surface sites, respectively (Figure S 1A): oxidation of methoxy species to formaldehyde (HCHO) is indicative of redox sites (Eq.S1); dehydration of two adjacent methoxy to dimethyl ether (CH 3 OCH 3 ), of acidic sites (Eq.S2); and formation of carbon dioxide (CO 2 ), of basic sites, which strongly bind methanol that is typically burnt and desorbed at high temperatures (Eq.S3); highly reactive redox sites will also produce CO 2 due to fast formaldehyde combustion (over-oxidation reaction), but at significantly lower temperature. 1Bifunctional sites may be present at the surface of NMs if two kind of reactive sites are in close vicinity, generating different products: methyl formate (HCOOCH 3 ) originates from basic-redox sites (Eq.S4) and dimethoxymethane ((CH 3 O) 2 CH 2 ) from acid-redox sites (Eq.S5).Water and CO are secondary products formed in several reactions and may not be correlated with a specific site.On occasions, less reactive sites release unreacted methoxy species as methanol, which typically occurs at low temperatures in the TPSR profile.

3
Figure S 1. Probe reactions for quantitative reactive characterization of NMs: A) Methanol chemisorption on the surface of a NMs with formation of a methoxy group per active site, followed by surface reaction and products desorption; redox sites lead to formaldehyde formation, basic sites produce carbon dioxide, and two nearby acid sites generate dimethyl ether.B) Oxidation of dithiothreitol (DTT) catalysed by a nanoparticle (NP) and quantification of non-oxidized DTT with Ellman's reagent (DTNB) by UV-Vis spectrophotometry detection at 412 nm of the coloured product.
Figure S1.Probe reactions for quantitative reactive characterization of NMs: A) Methanol chemisorption on the surface of a NMs with formation of a methoxy group per active site, followed by surface reaction and products desorption; redox sites lead to formaldehyde formation, basic sites produce carbon dioxide, and two nearby acid sites generate dimethyl ether.B) Oxidation of dithiothreitol (DTT) catalysed by a nanoparticle (NP) and quantification of non-oxidized DTT with Ellman's reagent (DTNB) by UV-Vis spectrophotometry detection at 412 nm of the coloured product.

Table S 2
. In vitro toxicity data reported in the literature for TiO 2 NM-101, CeO 2 NM-211, ZnO NM-110 and CuO indicating for a given exposure time the concentration at which a NM induces effects significantly different to control (p<0.05,0.01 or 0.001) or the half-maximal effective concentration (EC 50 )

Table S3 .
Data of methanol chemisorption

Table S2 .
In vitro toxicity data reported in the literature for TiO 2 NM-101, CeO 2 NM-211, ZnO NM-110 and CuO

Table S3 .
Data of methanol chemisorption Wachs IE, Patience GS, Dai YM.Experimental methods in chemical engineering: Temperature programmed surface reaction spectroscopy-TPSR.