The effectiveness of self-nuclei to crystallize the sample is addressed by increasing the cooling rate from sT temperature. The effect of previous standard state on melt memory is analyzed by (a) changing the cooling/heating rate and (b) applying successive self-nucleation and annealing (SSA) technique, observing a strong correlation between melting enthalpy or crystallinity degree and the extent of melt memory. The acquired knowledge can be extended to other semicrystalline polymers to control accurately the melt memory effect and therefore, the time needed to process the material and its final performance.It is widely accepted that melt memory effect on polymer crystallization depends on thermal history of the material, however a systematic study of the different parameters involved in the process has been neglected, so far. In this work, poly(butylene succinate) has been selected to analyze the effect of short times and high cooling/heating rates that are relevant from an industrial point of view by taking advantage of fast scanning calorimetry (FSC). The FSC experiments reveal that the width of melt memory temperature range is reduced with the time spent at the self-nucleation temperature ( The crystallization process of semicrystalline polymers depends on the thermal history of the material. To erase the thermal history, polymers have to be heated to 25–30 °C above their melting temperature. In this way, a homogeneous or isotropic melt is obtained. When the temperatures employed are high enough to produce an isotropic melt, during subsequent cooling in a differential scanning calorimeter (DSC), the material will always crystallize at the same temperature. This temperature is usually denoted as the standard crystallization temperature and it only depends on the cooling rate employed. Nevertheless, if the sample is heated to temperatures that are not high enough to reach an isotropic melt state (or erase melt memory), some self-seeds or self-nuclei survive, which can trigger crystallization at a higher temperature during the subsequent cooling run ,2,3,4.A thermal procedure known as self-nucleation (SN) was developed by Blundell et al.  and was Regarding the theories developed to explain melt memory effects, even though there is no consensus about it, some of them point towards a kinetic nature of melt memory, in the next lines some of these works are summarized. The works of Alamo and Hu ,10 have sT temperature. Despite this, the effect of the time spent at a given sT temperature on self-nucleation Domains, and that of the cooling/heating rates employed, are not known. Considering that in industry short times and high cooling/heating rates are common to process polymers, it is of paramount importance to ascertain the effect of those parameters on self-nucleation.In any case, it is accepted that melt memory depends on the applied thermal history of the sample ,4. HowevAccessing high cooling/heating rates and short times in conventional DSC is not possible, as heating/cooling rates are below 100 K/min. The development of fast scanning chip calorimeter however, allows to study high cooling and heating rates, in the order of several thousands K/min, which are relevant from a processing and an academic point of view. It should be considered that the heating/cooling rate affects the thermal properties of the material and thus, its final performance. From an academic point of view, this technique enables to study the crystallization properties at high undercooling and to investigate the homogeneous nucleation of materials with fast crystallization kinetic, among other topics ,28.Although most of the research has been focused on the basic aspects of melt memory, recently practical applications of this phenomenon have been reported in literature. An interesting application of self-nucleation has been reported by Yuan et al. where they report improvements in the shape memory effect of chemically cross-linked polycaprolactone by taking advantage of a self-nucleation procedure . Zhou etmelt memory Domain [To determine the role of the different parameters involved in melt memory effects, in this work poly(butylene succinate) has been selected since it has a wide y Domain ,4,17,31,Polybutylene succinate was synthetized by melt polycondensation employing tetrabutoxy titane (TBT) as a catalyst . First, the esterification of succinic acid and 1,4-butanediol  was performed followed by the polycondensation under vacuum. The reaction was performed under nitrogen, heating first the flask to 190 °C for 2 h and then to 200 °C for another 2 h under atmospheric pressure. Then, to proceed with polycondensation, the catalyst was added to the flask and stirred for 1 h and 30 min under vacuum, the flask was heated to 230 °C for 1 h and finally to 250 °C for 4 h.In order to purify the polymer, first the obtained material was dissolved in chloroform  and was washed with hydrochloric acid solution (0.1 M) . Afterwards, the catalyst was removed washing the solution twice with demineralized water and precipitated in excess cold heptane . The precipitated polymer was filtered and dried at 40 °C under vacuum until a constant weight was obtained ,32. The The melt memory effect of PBS was studied with a flash DSC 2+  chip calorimeter or differential fast scanning calorimeter. The equipment is connected to a Huber TC-100 intracooler . The sensor was first conditioned and calibrated, then a small sample was placed on the sensor and two heating and cooling steps were conducted to ensure good contact between the polymer and the sensor. The measurements were carried out under nitrogen atmosphere, with a flow rate of 80 mL/min to avoid degradation. The STARe software was used to analyze the data . sT, and was maintained at this temperature for a certain period of time, usually 5 min. After that, the sample was cooled down and heated again. Depending on the conditions employed to perform the self-nucleation procedure and on the sT temperature, the sample can show three different self-nucleation Domains. The temperature region corresponding to each Domain can be determined by analyzing the cooling scan from the sT temperature and the subsequent heating scan. To study melt memory effects, the following self-nucleation procedure see , proposeAs a reference the results obtained applying a heating/cooling rate of 10 K/s were used, this rate was selected considering two factors: to measure the sample at the lowest rate possible without losing accuracy in the flash DSC, to allow the material to fully crystallize. The second factor considered was that no lower rates were employed since otherwise the sample spends longer times at high temperatures (as the measurement takes longer time), which might result in degradation.In this work melting enthalpy values are discussed, which are directly correlated to the crystallinity level, since the mass of the sample employed in the flash DSC was not determined in this case.sT temperatures equal to 118 °C. So, for temperatures equal or above 118 °C, the sample was in Domain I or the melting Domain [sT temperature was reduced to 117 °C or below, an increase in the crystallization temperature with respect to the standard crystallization temperature was observed, which marks the transition to Domain II or the self-nucleation Domain. The enhancement of the crystallization temperature comes from the presence of self-nuclei and self-seeds, which increase the nucleation density.g Domain ,3,4. Howmelt memory Domain or Domain IIa [sT temperature range is called Domain IIb or the self-seeding Domain [PBS crystals were molten at 109 °C according to the FSC results , thus fomain IIa ,24, see Domain III the lowest melting peak was considered . This shift resulted from the crystallization of the material at higher temperatures when cooling from sT, which led to crystals with thicker lamellae and thus, higher melting peaks. Summarizing, at temperatures equal or below 106 °C, the sample is in Domain III or the self-nucleation and annealing Domain [For self-nucleation temperatures equal or lower than 106 °C, if the subsequent heating scan is analyzed b, an addg Domain ,3,4.Domains depend on the conditions employed to perform the experiment [sT temperature [self-nucleation Domains are studied systematically to provide new insights into the control of melt memory effects by taking advantage of its kinetic nature.Melt memory effects are considered to have a kinetic nature, since the temperature regions of the different periment ,3,4. Altperature ,14,15,16Domain III, no effect of time has been observed. For samples in Domain IIb no effect of time or a slight reduction of crystallization temperature has been reported. Finally, for samples in Domain IIa, near Domain I, increasing the time spent at sT temperature, a reduction of crystallization temperature back to the standard crystallization temperature was reported. This means that when the sample is kept for long times at the right sT temperature , it is possible to erase all the self-nuclei, only if the temperature is close to Domain I. All these works have focused on the erasure of melt memory effects by increasing the time spent at sT temperatures and analyzing if the crystallization temperature is reduced due to the “dissolution” of self-nuclei or self-seeds. Concerning the time effect on melt memory, different behaviors have been reported in the literature ,14,15,16sT temperatures that belong to Domain II: the lowest one corresponded to DIIb, and the highest one to DIIa. sT, the crystallization temperature is kept constant and independent of the time spent at sT. For the high sT temperature, there are some small variations, although a specific trend cannot be observed.In this work, we studied the effect of time on crystallization temperature by varying the time from 0.1 to 300 s, for two selected sT, for two different holding times at the different sT values. The results superpose quite well, showing that there was no effect of time, in the range explored in this work, on the crystallization temperature.self-nucleation Domains has not been reported. From a practical point of view, it is more interesting to study how keeping the sample for a very short time at a sT temperature, following the procedures involved in industrial processing, can alter the melt memory effect. It should be highlighted that in literature, only the effect of time on the subsequent crystallization temperature has been studied, but the effect of time on the transition temperature between Domains when the sample was kept at the self-nucleation temperature between 0.1 and 300 s. The transition temperature between Domain I and Domain II, i.e., temperature of the self-nuclei’s ultimate stability, did not change with the time spent at sT temperature for the range of times analyzed in this work. On the other hand, the transition temperature between Domain II and Domain III increased with the time spent at sT. This means that when the sample is kept at sT for short times, lower temperatures are required to produce annealing of the crystal, because the short times spent at this temperature are not enough to anneal the small crystals that are left unmolten at this temperature. m,endT was maintained constant or at least the variation was within 1 °C. For times below 100 s, the transition temperature between Domain II and Domain III was lower than m,endT, which is the usual behavior reported for a wide range of materials and measured with conventional DSC. The light blue area between m,endT and the transition temperature between Domain II and Domain III reflects the temperature range corresponding to Domain IIb, in which there are some crystal fragments  that are the responsible for the increment in nucleation density. However, for the sample that was kept for 300 s at sT, the transition to Domain II occurs at about 1 °C higher than m,endT; this is an unusual behavior that may result from the high heating and cooling rates employed.In Domain II, Domain IIa and Domain IIb vary as a function of the time spent at sT. The width of Domain II reduced with the time spent at sT due to the shift of the transition temperature between Domain II and Domain III to higher temperatures. In this case, the width of Domain II varied from 11 °C for 0.1 s holding time at sT to 7 °C for 300 s. This indicates that it is possible to fine tune the width of the melt memory Domain by varying the time spent at sT. This result implies that it would be possible to take advantage of this effect to reduce the time needed to process a polymer part.Domains is shown as a function of the cooling rate from the applied sT temperature. When self-nuclei and/or self-seeds are produced in the sample, their effectiveness could change depending on the cooling rate employed to generate them. To investigate this effect, the thermal procedure depicted in Domain II and Domain III kept constant for the studied cooling rate range (5–50 K/s) according to DIII) formed at sT were not sensitive to the cooling rates employed.The transition temperature between Domain II (self-nucleation Domain) and Domain I (melting Domain) was reduced by about 3 °C  was the same for all samples since only the cooling from sT was changed. sT, decreased with cooling rate, as expected. However, the transition temperature between out 3 °C b. To undDomain II or self-nucleation Domain. This means that at high cooling rates self-nuclei loose their effectiveness and lower sT temperatures are needed, i.e., a higher number of self-nuclei is required to induce an increase of the crystallization temperature. These results are in line with the ones reported by Jiang et al. [Domain II, which can produce the maximum possible nucleation density without annealing (see ref. 3). Jiang et al. [The results presented in this section show that above a certain cooling rate there is a reduction in the width of g et al. . They peg et al.  showed tg et al.  and Maizg et al. , provingg et al.  the selfDomain I to Domain II, we decided to analyze the subsequent melting step after cooling the sample from the sT temperature. Even if it is not possible to investigate the different Domains, we could consider the melting enthalpy of the final heating scan to ascertain if the self-nuclei left in the sample are effective in crystallizing the sample or if when high cooling rates are used an amorphous sample is obtained, and how this depends on the employed sT temperature.As the crystallization peak broadened when the cooling rate was increased, which hindered an accurate analysis of the transition temperature between sT = 119 °C are shown as a function of the cooling rate from the sT temperature, as an example, in sT for different sT temperatures. For slow cooling rates, independent of the sT to which the sample was heated, all the data show similar melting enthalpy, which means that during cooling from sT the sample was able to develop similar crystallinity. Nevertheless, when the cooling rate increased, significant differences in the melting enthalpy could be observed depending on which Domain the sample was cooled from . Domain I or to high temperatures within Domain II at above 300 K/s, the sample was not able to crystallize, reaching melting enthalpy values below 1 × 10−4 mJ, which was the lowest value obtained for this sample at the highest possible cooling rate, thus it was considered that this value corresponded to an amorphous sample. The presence of some self-nuclei, obtained by heating the sample to the high temperature region within Domain II, were not able to increase the melting enthalpy, and similar values to the sample heated to Domain I were obtained in Domain II, the presence of self-nuclei enhances the melting enthalpy when cooling rate was increased. For example, in the case of the sample heated to 116 °C, which is the lowest temperature within Domain II, an enthalpy of 3 × 10−3 mJ was obtained at 500 K/s cooling rate, whereas the sample heated to higher sT temperatures were completely amorphous or have a negligible enthalpy.When the sample was heated to sT temperatures that correspond to Domain III, in which the crystal fragments left  were able to anneal, it can be observed that for temperatures close to Domain II , the amount of those crystals was really low, since the enthalpy was 5 × 10−4 mJ. However, when lower sT temperatures  were employed, the amount of molten crystals was small, observing only a slight reduction in the melting enthalpy at the highest cooling rate, which was practically negligible.When the sample was heated to Domain II but close to Domain I, the results were the same as the sample cooled down from Domain I. When a higher number of self-nuclei and probably self-seeds were left, at low temperatures within Domain II, the melting enthalpy increased in comparison with the sample cooled from a homogenous melt state. From these results, it could be concluded that to have a significant increase in melting enthalpy, a high density of self-nuclei and self-seeds was required.Overall, the experiments presented in this section show that the effectiveness of self-nuclei depends on the applied cooling rate. When only some self-nuclei were left, at temperatures within Domains should depend on the previously formed semicrystalline standard state. Although in literature, this has been mentioned in several works [Considering the kinetic nature of self-nucleation, the temperature range corresponding to different al works ,3,4, theal works ,36 have al works  or they sT on the self-nucleation Domains, as a different method for the variation of the standard crystalline state. We employed three different rates: 10, 100 and 1000 K/s. The thermal protocol employed is depicted in In this work, we analyzed the effect of the cooling rate from the isotropic melt  and the subsequent heating rate to Domain II as a function of cooling rate. The samples were cooled from a sT = 160 °C (from the isotropic melt or Domain I). It can be observed, that when the heating rate to sT was maintained constant at 10 K/s, the width of Domain II did not change. From these results, we could conclude that the width of Domain II was related to the melting enthalpy of the sample, and thus to the crystallinity level, when it was heated to the sT temperature, which was also constant  was obtained in comparison with the reference or standard conditions that corresponded to 10 K/s cooling/heating rate. If a vertical line is drawn in sT could be observed keeping constant the cooling rate from 160 °C. In all cases, the Domain II width decreased when the heating rate increases, but the decrease was much higher at higher cooling rates.The reduction of Domain II since the melt memory effect could be erased without increasing too much the superheating, as the crystals are more metastable .Although the melting enthalpy was only considered, probably employing high cooling and heating rates the thickness of the crystals could also be reduced, and this should result in narrower Chen et al.  have stuIn this work, a linear homopolymer was analyzed and, thus, the melt memory effect did not come from complex chain topologies but from the presence of intersegmental interactions between the chain segments that were forming the crystal. If the sample contained a higher amount of crystals, the number of interactions between the chain segments previously in the crystals was higher and thus resulted in larger melt memory effects.Domain II is the melting enthalpy  obtained during the subsequent heating to the sT temperature , it was decided to employ the same rates to cool down the sample from 160 °C and to subsequently heat it to the sT temperature for self-nucleation, with the aim of covering a wide range of melting enthalpies. The thermal procedure employed is shown in −2 to 1 × 10−5 mJ. Considering that the key parameter that determines the width of Domains was reduced as the cooling/heating rate was increased, see Domain II, and the sample went directly from Domain I to Domain III. In Domain II and the melting enthalpy, proportional to the crystallinity level, as a function of the heating/cooling rate are depicted. A drastic reduction of both parameters with the increase of cooling/heating rate can be observed. Our results demonstrated the importance of the melting enthalpy  of the sample when it reached the sT temperature on the width of the self-nucleation Domains. The transition temperature between With the aim of creating thicker crystals while increasing the crystallinity degree, the successive self-nucleation and annealing (SSA) technique, designed by Müller et al. ,38 and rSSA is a thermal fractionation technique based on the application of a special thermal protocol that promotes molecular segregation during crystallization and annealing designed to fractionate the material by crystallizable sequence lengths. It is particularly sensitive to defects like branches along the chains, comonomer units, stereo-defects, etc. Once SSA is applied to a polymer sample, the final heating of the material shows several melting peaks, each one corresponding to a different thermal fraction. This distribution of melting points also reflects a distribution of lamellar thickness in the sample provoked by SSA thermal fractionation. This technique has been very useful to characterize ethylene/alfa-olefin copolymers, polypropylenes, block copolymers and more recently, segmented thermoplastic polyurethanes, recycled polyolefin blends, copolyesters and polysulfide based copolymers ,42,43,44sT temperature was reduced by 5 °C in each step, so when sT was reduced, the crystals that were not able to melt underwent annealing. The success of the applied SSA procedure to PBS sample can be observed by analyzing the final heating scan . However, it can be used also to efficiently anneal polymeric crystals, since SSA technique consists of applying successive self-nucleation and annealing steps. The sT temperature and kept at this temperature for 0.1 s. Each time that a new sT was analyzed, first the SSA protocol was applied to the sample: the thermal procedure employed is depicted in sT temperature on top of the melting endotherm is shown in Domains are shown as well. It is interesting to compare the results obtained applying the standard SN procedure (keeping the sample 0.1 s at sT temperature in both cases) and the SN procedure after SSA. Domain III, was observed as well at higher temperatures for samples submitted to SSA before SN, 114 °C, whereas when standard SN was applied Domain III appeared at 106 °C. As expected, thicker crystals (which melt at higher temperatures) could anneal at higher temperatures. After applying SSA, the sample was heated to the selected Ts temperature, the enthalpy was equal to 7.67 × 10−3 mJ, whereas when SSA was applied and then the sample was heated to sT, the melting enthalpy was equal to 1.65 × 10−2 mJ, this means that SSA was very effective in increasing the crystallinity degree. From the final melting endotherms, the melting enthalpies obtained by both protocols were measured. When a standard SN procedure was applied during the heating to mT0 is the equilibrium melting point, σe is the chain folded surface free energy and Domains to higher temperatures for the sample that had undergone SSA procedure before SN corresponded to an increment of lamellar thickness and also to an increase in the degree of crystallinity.From the melting temperature it is possible to estimate the lamellar thickness employing the Gibbs-Thomson equation,ual to 0 ,47. Consual to 0  for the sT did affect the width of Domain II. The effect of the cooling rate from sT temperature on different Domains was investigated, observing a reduction of Domain II with increasing the rate. In addition, these experiments revealed that a high density of self-nuclei and self-seeds are needed to induce the crystallization of the material when high cooling rates are applied. The effect of the previous standard state on melt memory was analyzed, providing a correlation of the width of Domain II and melting enthalpy/crystallinity level of the sample at sT. Moreover, the SSA technique was applied to thicken the crystals and increase the crystallinity degree, which resulted in a shift of transition temperature between Domains to higher temperatures in comparison with the standard state sample.In this work, the parameters affecting the melt memory effect were studied employing PBS and taking advantage of the high cooling/heating rates and short times accessible with flash DSC. For the first time it was proved that although short times spent at self-nucleation temperature did not affect the crystallization temperature (nucleation density), time spent at self-nucleation Domains and provided an example to fine tune the thermal properties of polymers, which can be extended to other semicrystalline material. Considering the short times and high cooling/heating rates involved in industrial processing of materials, this work can help to reduce the time needed to produce plastic parts and modify the thermal properties of polymer ad hoc for each application. Overall, this work shed lights on the kinetic nature of


In the marine environment, the reactive oxygen species (ROS) superoxide is produced through a diverse array of light‐dependent and light‐independent reactions, the latter of which is thought to be primarily controlled by microorganisms. Marine superoxide production influences organic matter remineralization, metal redox cycling, and dissolved oxygen concentrations, yet the relative contributions of different sources to total superoxide production remain poorly constrained. Here we investigate the production, steady‐state concentration, and particle‐associated nature of light‐independent superoxide in productive waters off the northeast coast of North America. We find exceptionally high levels of light‐independent superoxide in the marine water column, with concentrations ranging from 10 pM to in excess of 2,000 pM. The highest superoxide concentrations were particle associated in surface seawater and in aphotic seawater collected meters off the seafloor. Filtration of seawater overlying the continental shelf lowered the light‐independent, steady‐state superoxide concentration by an average of 84%. We identify eukaryotic phytoplankton as the dominant particle‐associated source of superoxide to these coastal waters. We contrast these measurements with those collected at an off‐shelf station, where superoxide concentrations did not exceed 100 pM, and particles account for an average of 40% of the steady‐state superoxide concentration. This study demonstrates the primary role of particles in the production of superoxide in seawater overlying the continental shelf and highlights the importance of light‐independent, dissolved‐phase reactions in marine ROS production. Light‐independent superoxide concentrations exhibit significant variability in coastal waters along the northeast coast of North AmericaParticles account for most light‐independent superoxide production in coastal water, but dissolved‐phase superoxide production also occursChlorophyll and eukaryotes are correlated with superoxide production rate, suggesting that eukaryotic phytoplankton are important ROS producers The formation of ROS within aqueous systems occurs via sequential one‐electron transfer reactions  are short‐lived oxygen‐containing molecules with half‐lives in aquatic systems that range from fractions of seconds to days. The most common forms of ROS in marine systems include hydrogen peroxide  disproportionates superoxide to dioxygen and hydrogen peroxide at or near diffusion‐limited rates in natural waters  has long been appreciated , with typical water depths between 50 and 60 m. The remaining station, chosen as a contrast to the other five shelf sites, was collected off‐shelf in water exceeding 2,000 m depth (Station 4). Samples for ROS measurements were collected as previously described  was added to seawater samples. A small fraction of the superoxide signal is a result of reagent autooxidation; thus, this artifact is removed by taking the difference between two signals as follows. First, the chemiluminescent response, R, in the UFSW, FFSW, and AFSW was quantified relative to the SOD baseline and converted to concentration using the calibration sensitivity, Scalibration . Superoxide detection was based on the reaction between superoxide and a chemiluminescent probe, a methyl cypridina luciferin analog . To prepare the calibration standards, the primary stock solution was further diluted with the calibration matrix to a final superoxide concentration of 5–41 nM. Both primary stock solution and calibration standards were prepared immediately before the analysis. The corresponding chemiluminescent signals were recorded and extrapolated back to the time when the primary standard was quantified, using first‐order decay kinetics. The half‐life of superoxide in AFSW ranged from 0.26 to 0.49 min, and the extrapolation time was 0.5–1 min. Calibration curves were constructed based on the linear regression of the natural logarithm extrapolated chemiluminescent signals versus superoxide concentrations in the calibration standards. Calibrations yielded highly linear curves , with a sensitivity, Scalibration, of 0.16 ± 0.04 (average and standard deviation of different water depths) counts per pM.Calibrations were conducted using potassium dioxide (Sigma) as detailed previously  on a Guava® easyCyte flow cytometer . Three phytoplankton groups were distinguishable based on plots of red fluorescence and forward scatter  and orange fluorescence and forward scatter (phycoerythrin‐containing Synechococcus spp.). Bacteria samples were diluted as needed with filtered (0.2 μm) seawater, stained with SYBR Green I (Invitrogen) according to the manufacturer's instructions, and incubated in the dark at room temperature for at least 30 min before analyzing on the Guava® easyCyte flow cytometer. Bacteria were distinguishable based on green and red fluorescence and green fluorescence and forward scatter. Analyses were performed on the Guava InCyte™ 3.1 software. Detection limits were calculated as 3 times the standard deviation of the filtered (0.2 μm) seawater blank.Flow cytometry samples were filtered (40 μm), preserved in 0.5% glutaraldehyde , flash frozen in liquid nitrogen, and stored at −80°C prior to analysis. Counts (cells per milliliter) were obtained by pipetting 200 μl triplicate samples and filtered seawater blanks (0.2 μm) into 96 well plates and analyzing at a low flow rate  at 16 depths at which all observations were collected and include sample depth, temperature, chlorophyll concentration, PAR, beam transmission, salinity, dissolved oxygen concentration, nitrite concentration, ammonium concentration, nanoeukaryote concentration, picoeukaryote concentration, Synechococcus concentration, bacteria concentration, DOC concentration, superoxide concentration, AFSW decay rate constant, UFSW decay rate constant, fraction of steady‐state superoxide concentration from particles, and superoxide production rate.Measurements collected in this study were analyzed using a combination of principal component analysis (PCA) and pairwise linear least squares regressions. All reported 33.1−2 s−1  and 2 (near New Jersey) exhibited relatively distinct dissolved oxygen and chlorophyll a‐based fluorescence maxima between 10 and 25 m depth, with Station 2 having slightly higher fluorescence at the peak Figure . Station Figures  and S2.Synechococcus) demonstrated that all microbial populations reached a maximum concentration in the top 1–3 sample depths (~3 to ~15 m) and generally decreased with depth. An exception to this was the vertically mixed water column at Georges Bank that showed less structure in vertical profiles  was significantly different from the average concentration at the off‐shelf station .In the absence of light, steady‐state superoxide concentrations within unfiltered waters ranged from approximately 10 pM to as high as 2,400 pM Figures , Table 1At some Stations, such as 1 and 6, light‐independent, steady‐state superoxide concentration profiles were C shaped, with elevated concentrations at the surface and deepest sample and lower concentrations in‐between Figure . At Stat3.3n = 23), which was significantly different from that of the off‐shelf sample location .Across all sample locations and depths, the light‐independent, steady‐state superoxide concentration was lowered by filtration Figures , such thIn the four shelf stations in which particle association of superoxide was investigated , there was no single trend that dominated. At Station 2, superoxide was primarily particle associated at the fluorescence maximum and at the base of the photic zone, but nonparticle‐associated superoxide production contributed significantly to the steady‐state superoxide concentration at the remaining four depths Figure . At all 3.4−1 for UFSW and 0.001 to 0.037 s−1 for aged filtered seawater . Decay rate constants of superoxide in UFSW, where measured (Stations 3–6), were typically the highest at intermediate depths. An exception to this was seen in Station 4 where the highest decay rate constant occurred at 320 m. Decay rate constants in AFSW exhibited a much smaller dynamic range than those seen in UFSW. At Stations 1, 2, and 5, the highest AFSW decay rate constant occurred in the shallowest depth, while the most rapid decay was seen in intermediate depths elsewhere. Average AFSW decay rate constants in the off‐shelf station were approximately fourfold lower than those observed on the shelf.Superoxide pseudo first‐order decay rate constants ranged from 0.021 to 0.125 s3.5The underlying relationships between the set of observations collected in this study were investigated further using PCA. PCA results are shown in the supporting information Figure . In brieR2 < 0.1), with the exception of superoxide production rate  did not correlate well with superoxide production rate  of light‐independent, steady‐state superoxide concentration and superoxide production rate along with several other observations including eukaryote abundance (both picoeukaryote and nanoeukaryote), chlorophyll, dissolved oxygen, superoxide production rate, and both first‐order superoxide decay rate constants Figure . The pai44.1Light‐independent, steady‐state superoxide concentrations along the North Atlantic continental shelf exhibited a range of over 3 orders of magnitude, with the highest values (in excess of 1 nM) observed in waters collected both in and below the photic zone Table  and 3. TR2 = 0.06 and 0.01 respectively, Table R2 = 0.77, Figure Differences between the physical and chemical environments in the shelf stations offer insight into some of the influences on superoxide in coastal seawater. Most of the shelf stations in this study had elevated light‐independent, steady‐state superoxide concentrations in shallow photic water and in aphotic water close to the seafloor Figure . The cleFurther insight into the environmental controls on superoxide production can be gained from contrasting the five on‐shelf sampling locations  with the single off‐shelf sampling location (Station 4). Station 4 had lower chlorophyll and cell counts of all types than the shelf stations Figures . Surface4.2Since superoxide in the ocean has a wide range of potential sources and sinks that vary spatially and temporally, there appears to be no single biological, physical, or chemical influence that is a consistent, robust predictor of superoxide concentrations in seawater. There are, however, some factors that are closely related to superoxide production and steady‐state concentration.For example, particles play a central role in extracellular superoxide production. In this study, particle‐associated production accounted for an average of 73% of the steady‐state superoxide concentration across all stations and depths. Marine particulate matter is a complex mixture of living and nonliving components that can include mineral (of both lithogenic and biogenic origin), whole cells, fecal pellets, and a wide range of adsorbed constituents . On average, the aged, filtered sample had a decay rate constant that was 68% lower than the unfiltered sample. The primary sinks of superoxide in seawater include enzymatic elimination, reactions with dissolved metals, and reactions with dissolved constituents of seawater such as DOC , followed closely by abundances of picoeukaryotes (R2 = 0.74) and nanoeukaryotes . One factor that may underlie this poor correlation is that different bacterial groups may have wildly different production rates  (Wuttig et al., Redox transformations of dissolved metals may also contribute to dissolved‐phase superoxide production (Halliwell & Foyer, It is tempting to categorize several of the dissolved‐phase redox transformations discussed above as incidental or generally outside of the purview of microbial mediation. However, these radical reactions may be part of a larger chemical conversation between microbes and their environment. The production and consumption of ROS coupled with the redox cycling of labile redox‐active compounds in natural waters have been proposed as a mechanism by which microbial communities regulate the redox environment in and immediately surrounding the cell Rose, . While t5Over the last few decades, the paradigm of ROS production in the marine environment has evolved from a model that is primarily driven by photochemistry to one inclusive of photochemistry, abiotic reactions, and microbial production. Indeed, here we show that steady‐state, light‐independent superoxide concentrations in productive waters in the northeast coast of the United States range from 10 to >2,000 pM, with particles as the dominate source along the shelf. Our data also demonstrate the presence of dissolved‐phase superoxide production across a significant range of environmental conditions, most notably in deep waters off the continental shelf. Superoxide production rate is most closely associated with chlorophyll concentration and the abundance of picoeukaryotes and nanoeukaryotes, suggesting that eukaryotic phytoplankton are the main producers of light‐independent, particle‐associated superoxide within our study region.The diversity of extracellular superoxide sources in the ocean has grown increasingly more complex as research over the last several decades has shown that superoxide production, and that of superoxide production in general, varies along dimensions of light, biological composition, and particle association. These biogeochemical pressures conspire to shape the steady‐state concentrations of ROS in the environment and in so doing shape the surrounding redox landscape and dictate the bioavailability of certain nutrients, the flow of electrons through biogeochemical systems, and selective pressures in a given environment.Supporting Information S1Click here for additional data file.


Stretchable electronics promise to extend the application range of conventional electronics by enabling them to keep their electrical functionalities under system deformation. Within this framework, development of printable silver-polymer composite inks is making possible to realize several of the expected applications for stretchable electronics, which range from seamless sensors for human body measurement  to conformable injection moulded structural electronics. However, small rigid electric components are often incorporated in these devices to ensure functionality. Under mechanical loading, these rigid elements cause strain concentrations and a general deterioration of the system’s electrical performance. This work focuses on different strategies to improve electromechanical performance by investigating the deformation behaviour of soft electronic systems comprising rigid devices through Finite Element analyses. Based on the deformation behaviour of a simple stretchable device under tensile loading, three general strategies were proposed: local component encapsulation, direct component shielding, and strain dispersion. The FE behaviour achieved using these strategies was then compared with the experimental results obtained for each design, highlighting the reasons for their different resistance build-up. Furthermore, crack formation in the conductive tracks was analysed under loading to highlight its link with the evolution of the system electrical performance. Devices created with this purpose find application in a wide variety of fields, such as fitness monitoring and health care systems, as well as conformable injection-moulded structural electronics3. The aforementioned applications require the electrical system to deform together with the underlying materials: especially for health-patch applications, this means that such electrical devices have to seamlessly adapt to human skin deformations4, whose deformation levels can be highly heterogeneous5. These reasons make it difficult to restrict the working conditions for this set of systems6, while, on the other hand, they encourage the request for materials that have mechanical properties similar to the human skin, in order to improve comfort to the user and reduce obtrusiveness of such electrical systems8.Soft electronics are designed to overcome the general limitations of conventional electronic systems, that is the possibility to subject these systems to high levels of deformation while maintaining their electrical integrity10 by employing complex geometries for the interconnects such as zigzag, multi-track pattern, helical, and origami-inspired shapes13 and horseshoe designs16. These strategies employ mechanical phenomena such as local buckling and post-buckling behaviour to allow the conductive structure to deform out of the plane defined by the substrate underlying, thus increasing the range of deformability without reaching mechanical failure, thus allowing them to maintain electrical conductivity of bulk metallic conductors even at high overall deformation levels15. However, application of these processes significantly increases the complexity of the fabrication.Stretchable electronic systems generally comprise several components, where each specifically addresses certain functions. These components typically are miniaturized functional modules such as batteries, sensors, radio transmitters and receivers, that are connected by stretchable interconnects. Structures that find vast application as interconnects for stretchable electronics are thin, metallic bi- and tri-dimensional structures, that yield promising results in terms of stability and maximum elongation at electrical failure17.The need to find materials that show conductive properties comparable with metals, while having deformability and stiffness that are closer to polymers and elastomers, resulted in the employment of specific fabrication processes to create micro- and nanostructured elements from metallic materials and the development of new intrinsically conductive materials with better electrical and mechanical properties22. These classes of conductive composites offer high flexibility of properties and many advantages over the thin metallic films that are generally used in this field. Moreover, they can be printed on top of the desired substrate, which can range from thermoplastic polyurethanes (TPUs)23 and polydimethylsiloxanes (PDMS)25 to textiles27. However, conduction in these materials is based on deformation mechanisms that are radically different from the ones available for thin metallic films: in fact, for these materials conduction is carried through the filler particles, that are in contact with each other and act as rigid inclusions, while the polymeric binder typically carries the deformation transferred from the substrate. Because of the heterogeneity of the material and of its peculiar conduction properties and processes, the definition of electrical failure as well as the electrical resistance build-up are properties difficult to extensively characterize and model. Due to the presence of the polymeric binder, the overall elastic modulus of these conductive inks is several times lower than the one of metallic films, favouring the use of them in applications where unobtrusiveness of the sensing device is important. However, conductive inks have to be strongly bonded to the underlying substrate, since improper adhesion can result in strain concentrations and ink fracture upon applied deformation on the system. This factor heavily limits also the strategies that can be used to improve the overall stretchability of these systems, since they typically involve deformation in a direction normal to the substrate to increase the deformation allowed before rupture.Materials related to this strategy are mainly micro or nanocomposites constituted by different polymers featuring low elastic modulus together with conductive fillers, which increase the electrical conductivity of the medium. Most of these micro and nanocomposites are generally constituted by a conductive material, such as Ag μm- or nm-sized particles or by different carbon-based conductors, such as CNTs and graphene, together with a polymeric matrix33. Recent application of this type of strategy, for example, feature the combination of multiple phenomena, such as prestretching the substrate to induce microbuckling or serpentine structures, together with the use of microislands with specific shapes to enhance the system electrical behaviour under cyclic loading35. However, substrate designs that lead to improvement of the electromechanical behaviour of stretchable systems are also dependent on the complexity on the device itself and on the arrangement of its components, which may strongly influence the local stiffness of the device and its behaviour at high levels of deformation. High levels of prestrain, moreover, may have detrimental effects on the device accurate dimensioning, especially when applied in combination with the creation of stiffer regions on the substrate, which makes the prediction of the device final dimensions far more complex than in the case of a homogeneous system. In order to smoothen the strain transitions in these critical regions, different approaches should be utilised. As seen in a recent review from Xue et al.36, the current methodologies used to increase reliability often relay on the use of shielding frames or stiffening substrates right at the rigid component location to limit the strain fields in the vicinities of rigid components41. However, strain concentration due to the presence of stiffeners and their effect on printed conductive tracks are often taken into account only as a secondary effect of the presence of the stiffener. Moreover, only few of these works focus on strategies to reduce this phenomenon, which promote delamination and premature failure of the system. Other shielding strategies that are currently used rely on open cell substrate to increase breathability and allow to increased deformability of the systems44.The simplest stretchable electronic systems devisable with the materials mentioned above feature a highly deformable substrate, a printed conductive ink such as the ones mentioned above, and surface-mounted devices (SMDs), such as rigid passive components, integrated circuits (ICs), batteries, or connectors. The presence of the rigid components on top of a deformable substrate plays a crucial role in the electromechanical behaviour of these systems, and often there is the need to minimize the dimensions of these ‘rigid islands’ in order to reduce strain concentration happening upon deformation of the system23. In addition, vulnerable regions in the substrate, such as areas with rigid-to-soft connections can be reinforced by local enhancement of the stiffness46. All the cited works, however, mainly refer to increase in system stretchability where the conductive tracks are composed by thin, metallic foils with specific two- or three-dimensional shapes to accommodate high levels of deformation. Currently, very few works focus on the use of printable, electrically conductive inks constituted by conductive Ag-flakes and polymeric matrix, whose mechanical response and electricity conduction diverge strongly from the regular behaviour of pure metallic conductors.As seen in previous publications regarding the presence of interconnects on stretchable electronic systems, it is possible to influence local strain through encapsulation and by modifying the geometry of the conductive ink interconnectlocal component encapsulation; this strategy is very close to the previously proposed framing and micro-island strategies mentioned before.Addition of stiffeners on top of SMD components and conductive tracks, defined as direct component shielding.Addition of stiffeners in regions around the SMDs, defined as strain dispersion.Reduction of stiffness of different regions of the system in order to virtually increase the stiffness around the SMD, denoted as Thus, the aim of this work is to provide and compare suitable strategies to improve the ability of such printed stretchable systems to deform with no destructive effects on the electrical conductivity. In turn, this means to reduce the stress concentrations due to the presence of rigid components, where early failure of the device is often originated. This work presents an approach based on the improvement of electromechanical behaviour of stretchable electronic systems comprising zero-ohm resistors as SMDs through stiffness increase in critical regions. Three fundamental approaches are compared, and critical differences are highlighted:All the strategies above have been thoroughly analysed through FE models and then experimentally tested. The results from the simulations and the experiments were later compared in order to show the details of each strategy and study more in detail the effect of each strategy on the overall behaviour of the stretchable electronic system. Within this context, the reference system will be later mentioned as Reference, while Design 1, 2 and 3 respectively stand for the three strategies mentioned above.The four designs compared for stretchable electronics systems are shown more in detail in Fig. x-direction of Fig. The mechanical behaviour of the different samples was first analysed using ABAQUS/Implicit; after that, samples were prepared and tested. The results comparison allowed to validate the approach used and helped to maximize the information gathered from a single test. Figure entclass1pt{minimax-direction among the different designs. Here it is easier to compare the overall effect of the different shielding methods on the deformation of the conductive track, which results not only in high strain levels in the vicinity of the SMDs, but also strongly depends on the shielding method used. As expected, the highest level of deformation along the track is achieved by the sample without any shielding, while in the region neighbouring the rigid component the other designs show similar levels of deformation for the first 5 mm of the track. However, the deformation levels in Design 1 start to diverge from the other two because of the influence of the stiffener layer, whose edge is located at 11 mm from the beginning of the track. Here it is possible to notice a peak in deformation for Design 1. Deformation levels in Design 2 and 3, instead, increase with a smaller slope in that region. Design 3, in particular, reaches the highest level of deformation along the conductive track direction at roughly 16 mm from the SMD pad, while Design 2 shows a sudden decrease in deformation slightly earlier due to the stiffener on the two sides of the conductive track.Figure This difference in deformation is a direct result of the different shielding systems. While on one hand all three of these designs have the objective of increasing the system stiffness in the vicinity of the SMDs, the final outcome depends strongly on the strategy applied. As already mentioned, both Design 1 and 2 have similar results in the first part of the track because of the similar method of shielding. In fact the addition of the stiffening TPU strips has a similar effect for the first few millimetres of track in both cases. At a distance of about 5 mm the effect of the position of the additional film starts to increase until, at about 10 mm from the beginning of the track, the presence of its edge right under the track and the sudden decrease of stiffness in that region, results in a local strain amplification in the conductive track direction, as can be seen in Fig. It should be noted that in Designs 1 and 2 very similar procedures are applied in order to improve the electromechanical deformation of the system, and the two samples follow the same fabrication process, that involve the use of additional TPU strips placed in the proximities of SMDs followed by hot press binding of the additional strips. However, the results strongly differ and that is mainly due to the location of the additional stiffener on the substrate. In fact, while in one case its presence interferes and creates additional strain concentration regions on the conductive track, in Design 2 the stiffeners location ensures shielding against excessive deformation levels around the SMD. In this design, however, the stiffener does not directly influence the deformation of the conductive track, and thus does not induce relevant deformation peaks around the SMD. Design 3, instead, follows a different strategy to improve the electrical behaviour under a high deformation of the system: shielding of excessive deformation levels around the SMDs is achieved by decreasing the stiffness in regions that are located away from the SMDs, i.e. by removing material from these locations. The effect of material removal is similar to the final outcome achieved in Design 2, but this strategy offers many advantages under a fabrication point of view, since there is no step added to the production process because cutting is already one of the steps needed. Similar FE analyses were also performed on systems featuring serpentine interconnects and are reported in Supplementary Discussion. These analyses yielded similar results than their counterpart featuring straight interconnects, but also highlighted the need to tailor specific strain limiting strategies to the stretchable system’s geometry and mechanical behaviour.The experimental results well matched with the results obtained from the FE modelling. In fact, the lower strain concentration levels of Designs 2 and 3 also result in a lower overall resistance of the systems under deformation, as shown in Fig. Electromechanical failure results for the different Designs are shown more in detail in Fig. Stretched samples were also analysed on a system constituted by movable clamps on a backlit table, in order to better analyse the local evolution of crack density on the conductive tracks under tensile loading. Upon the application of uniaxial tension, the samples and the conductive tracks on them deformed similarly as in the FE analysis, with the main difference coming from the ink behaviour. Because of its highly heterogeneous composition, in fact, cracks start to develop in the conductive track as soon as the material is being deformed. This is a typical behaviour for this kind of conductive ink, and occurs because of cracking in the binder matrix, which is the element that actually carries most of the deformation, since the binder has a stiffness that is several times lower the one of the Ag flakes. The development of the cracks has been analysed at different levels of overall deformation of the four designs through photographs of the track with high contrast coming from the presence of the conductive track and of the backlight. Figure The deformation difference between the two regions generates additional cracking in the conductive track right after the additional layer of stiffener, thus generating a high-resistance region in this spot. The cracking density of Design 2, instead, remains constant with a final crack density that is lower than the one seen in the reference sample. Due to the radical differences in design of Design 3, instead, samples from this design tend to wrinkle more under a tensile deformation. This happens because there are no boundary conditions on the side edges of the sample because of the need to load all designs in similar way. However, these wrinkles occur in regions without conductive tracks and do not affect its cracking behaviour.In order to quantitatively compare this behaviour between the different designs, the registered grayscale images of the tracks at a stretch ratio of 1.5 were extracted and processed by averaging the obtained grayscale values over the y-axes shown in Fig. 47. Moreover, the present work also highlights that the mechanical and conductive behaviour of this class of materials should be considered as strongly different from the one of thin metallic foils, because of the several different features seen in terms of cracking behaviour under deformation and general track behaviour in presence of an SMD. Moreover, the different strategies employed in the three designs analysed also show how several aspects related to mechanical behaviour of the components involved and their geometrical features should be considered when choosing a strategy to improve the overall electromechanical behaviour of stretchable electronic devices. In particular, when upscaling the issue taken into account to more complex electrical devices, featuring several components and with a complex general layout, integration of these strategies gets considerably more complex and a coupled mechanical and electrical design is advised to maximize the efficiency and the device electromechanical properties.This behaviour suggests that, for this class of heterogeneous materials, the relationship between resistance and deformation cannot be written in the form that is widely used for thin metal layers, present in the work from Lu et al.The need to limit excessive deformation in stretchable electronic systems arises from the need to overcome the current limitations of electronic devices, and many approaches have been adopted in the past in order to improve their electromechanical performance. This work represents an attempt of improving the behaviour of systems that employ printed Ag flake-based conductive inks under tensile loading through three different strategies, each of which is focused on a separate reasoning or fabrication procedure. This was done by first analysing the mechanical behaviour of a stretchable electronic system including, rigid components, a stretchable substrate, and a conductive paste.Different strategies to improve the overall behaviour of these systems under deformation were then analysed, especially highlighting the behaviour arising from the use of each method. The analysed systems feature printed conductive tracks based on Ag flakes, where stress relieving strategies based on out of plane deformations are not applicable. The approaches represented here show that, even for this kind of stretchable electronic structures it is possible to reduce the resistance change due to the imposed mechanical deformation by stiffening specific regions on the device, thus homogenizing the in-plane strain levels.In particular, the authors showed that stiffening the region right next to rigid electric components tends to create new sudden gradients in material stiffness, thus concluding that it is generally advisable to employ a different strategy to improve the behaviour under deformations of these systems, since this ‘local stiffening’ may lead to formation of strain concentration regions in other, equally critical parts of the sample. Moreover, different outcomes were achieved using different shielding strategies: in particular, stiffening of regions far enough from the SMD to minimize the influence of stiffness gradients proved to be a useful way of improving electromechanical performance of these systems. In Design 2, this was achieved by equipping the samples with additional strips of TPU at a distance of 15 mm from the SMD in order to avoid influences of stiff regions on the conductive track. In Design 3, instead, material was removed to create regions that were more compliant than the region surrounding the SMD in order to disperse the deformation on the whole sample, and thus away from the rigid component. These two strategies yielded similar results in terms of deformation at electrical failure under tensile loading, while fatigue testing revealed important aspects related to delamination under cyclic loading for these samples. The latter strategy revealed to be more efficient in terms of fabrication process, since cutting is already one of the steps included in the fabrication of stretchable electronics systems. Also, more complex systems, e.g. systems featuring smaller SMDs or a higher amount of them, or even different loading conditions, could benefit from similar procedures featuring solutions that arise from a common ground between solid mechanics and electronics, which stands as a fundamental collaboration for novel development in the field of soft and deformable electronics.48 with Finite Element (FE) analyses were used to determine the overall shapes for the samples in Fig. 49. The same kind of potential, with 23. However, Ogden’s model with 50.The parameters used to describe the substrate behaviour were set equal to the values in Supplementary Table 51.Due to the arrangement of its constituents and the type of constituents themselves, the conductive ink is described using a linear elastic to bi-linear perfectly plastic behaviour. The employed zero-ohm resistors are instead handled as rigid components in the model. Since the focus of this work is to find ways to optimize the overall geometrical shape of the system to improve its overall electromechanical performance, perfect bonding is considered between the components. The typical mesh size of the substrate part is 0.3 mm and 0.125 mm for the conductive ink, and the element types used for the two components are C3D8H and C3D8, respectively. The difference in mesh sizes between the two parts is needed to analyse in detail the deformation of the conductive ink, and solution convergence was analysed for similarly sized problems in a separate workThe geometries for the different parts of the models are shown and described more in detail in the supporting information, together with the boundary conditions applied , U4201 provided by Epurex Platilon as the stretchable substrate, which is provided in rolls and shows a high level of deformation at failure and high dielectric properties. The choice of this substrate ensured that, during tensile testing, there would be no failure occurring because of substrate breakage. A slight (3–5%) prestrain was applied to TPU sheets before attaching them to aluminium plate carrier for the printing process. A highly conductive silver flake paste (CI-1036) from Engineered Conductive Materials (ECM) was employed to fabricate the conductive tracks. The curing temperature of the silver paste (125 °C) is lower than the softening temperature of TPU (155–185 °C); this allows to use this paste on the substrate. Deposition of the silver paste on the substrate was done using a semi-automatic screen printer from TIC (SCF-300). Two consecutive printing cycles were used for fabrication of the conductive tracks. After the printing process, samples were annealed in an oven for 30 min at 125 °C. Samples were then removed from the aluminium plate and some of them (Designs 1 and 2) were partially encapsulated using a heat-press machine (Combo heat press) using the same material as the substrate. In order to evenly compare the results and avoid mismatches in properties due to the application of different fabrication processes, however, the other samples were also subjected to the same process without the addition of an additional layer in specific locations. The heat-press process lasted for 1 min at 150 °C. TopLine Corporation supplied the zero ohm resistors (SR2512), whose physical dimensions are 6.3 mm × 3.2 mm. The recommended pad size for this SMD is 1.6 mm × 3.5 mm. Furthermore, a highly flexible isotropic conductive adhesive (ICA) was used to attach the passive components on the top of the interconnects pads. The ICA, supplied by Namics, is the XE184, a thermoset ICA with silver filler that is typically used for bonding of chip components and connectors.Samples were annealed in 100 °C for 30 min for the ICA curing. Before the characterization steps, samples were singulated by a laser cutter (Trotec Speedy 100) into the desired template (140 mm × 38 mm). This ensured high dimensional accuracy and good reproducibility of results.An optical microscope was used to ensure adequate resolution of the conductive tracks and to check for any visible defects. Then, initial resistances of samples were measured with a multimeter. The stretchability of interconnects was evaluated through uniaxial quasi-static tensile loading up to total electrical disconnection with an Instron 4411 tensile tester. The uniaxial deformation level imposed on the samples was monitored as stretch ratio, which is defined as 8. Sets of 10 samples from each of the four groups were analysed. Moreover, the same setup was also used for cyclic loading of the samples under displacement control. During this kind of tests, the samples were cyclically loaded up to a stretch ratio of 1.1 for 1,000 cycles at a 1 Hz rate, while the resistance increase was measured by using the sourcemeter mentioned above at a sampling frequency of 5 Hz.A load sensor of 500 N, pulling speed of 6 mm/min were used for the test. The linking with a Keithley 2425 sourcemeter via a customized software written in LabVIEW for a real-time measurement (sampling frequency of 5 Hz) of the resistance ensured the possibility to monitor the changes in resistance at any moment of the sample stretching under the tensile tester. The interconnects were connected to the Keithley measurement cables using pads of 3M 9703, a pressure-sensitive conductive tape. The schematic of the test setup can be seen from previous worksSamples’ behaviour under deformation was also separately analysed by uniaxially deforming the sample up to a desired deformation level on a backlit table, in order to get information about the cracking behaviour of the conductive tracks upon loading. This behaviour, expected due to the high heterogeneity of the constituents of the conductive ink employed, was captured using a 5 MP industrial camera together with a Schneider Kreuznach Xenoplan 1:4, a telecentric lens. The use of this lens ensured the exclusion of undesired image aberrations due to perspective distortion and ensures a pixel-to-millimetre ratio equal to 0.0137 for any image taken.Supplementary Information


Triticum aestivum vs T. spelta) and production methods  on the nutritional composition of wheat. The retail survey reported here showed that, the use of spelt varieties and organic production results in significantly higher (between 10 and 64%) levels of phenolics and some minerals in wheat flour. However, the relative effect of removing the outer bran and germ during milling was substantially larger; levels of antioxidant activity, and many phenolic compounds and mineral nutrients were 2–5 times higher in whole-grain than white flour. Organic flour contained higher concentrations of the undesirable metals Al and Ni (12% and 81% respectively), and spelt flour had 28% higher concentrations of the toxic metal Cd.There is strong evidence for health benefits from whole-grain wheat consumption and these have been linked to their higher fibre, antioxidant/(poly)phenolic and mineral contents. However, there is still scientific controversy about the relative effect of wheat species ( Wheat flour is the main ingredient in many staple food products such as breakfast cereals, pancakes, pasta, noodles, bread and other bakery products. Most of these foods are made from refined grains, where the outer grain layers (the bran and germ) are removed from the endosperm before further processing (e.g. milling to produce “white” flour)  to around 55% in some developing countries (e.g. India) . In Euro” flour) .However, the use of cereal products made from whole-grain flour (where the whole grain is milled) is increasingly recommended by nutritionists, because there is now substantial evidence for higher whole-grain consumption resulting in a reduced risk of obesity, type 2 diabetes, certain cancers and cardiovascular diseases .The health benefits from whole-grain consumption are thought to be mainly associated with the higher fibre, mineral  and (poly)phenol/antioxidant content (which is mainly in the bran fraction of the grain) of whole-grain flour .A recent meta-analyses of published data found evidence that organic production methods result in significantly higher antioxidant/(poly)phenolic and Zn, but lower protein, nitrate, nitrite and Cd concentrations in cereals . In addiTriticum spelta L.) is a husked-wheat species which in the past was widely grown in Northern Europe but is now considered a minor cereal. Spelt wheat has recently increased its production area and market share in Europe, especially in organic farming  the composition of common and spelt wheat-based food samples that were collected in the same retail outlets are compared and (b) confounding factors such as wheat farming system , grain processing/flour type (e.g. white or whole-grain) were considered in the survey design.overall aim of the study was to obtain a more accurate estimate of the difference in phytochemical and mineral concentrations between spelt and common wheat grain by analysing all accessible retail brands of wheat flour available in Germany and the UK (thereby estimating differences between the common and spelt wheat varieties currently used), while accounting for the confounding effects of farming system and flour type.The specific objectives of this study were therefore to (a) compare antioxidant activity, and protein, (poly)phenolic and mineral concentrations in white and whole-grain common and spelt wheat flour brands/products available in the UK and Germany (b) to study the effect of primary production methods  on antioxidant activity, and protein (poly)phenolic and mineral micronutrient concentrations in wheat flour and (c) identify potential interactions between wheat species, primary production protocols, and post-harvest processing/milling with respect to antioxidant activity, and protein, (poly)phenolic, and mineral concentrations.The The minerals assessed included all plant macro- and micronutrients, the undesirable elements aluminium and nickel and the toxic metal cadmium.The study tested 4 main hypotheses, which were: (a) whole-grain wheat flour has higher antioxidant activity, and protein, phenolic and mineral nutrient concentrations than white flour, (b) organic wheat flour has higher antioxidant activity, and phenolic and mineral nutrient concentrations, but lower protein, Cd and Ni concentrations than conventional wheat flour with the relative differences between organic and conventional brands being greater for whole-grain than white flour and (c) spelt wheat flour has higher antioxidant activity, and protein, phenolic and mineral nutrient concentrations than common wheat flour and (d) the relative impact of processing on antioxidant/phenolic and mineral nutrient concentrations is substantially greater that the impact of farming system and wheat species choice.22.1The retail survey of wheat flour was conducted over two successive years in 2015 and 2016, but no common wheat samples were collected in Germany in 2015. As a result, differences between countries could only be assessed in 2016. In total, 168 samples were purchased from supermarkets in the UK  and Germany  and websites in the UK  in the same period in each year (see T. aestivum or T. spelta), farming system  and flour type (white or whole-grain). Cereal brands were used as replicates, with only one sample per brand (supermarket own or manufacturers brands) being used for each combination of wheat species, farming system, flour type per year. This was primarily done to avoid pseudo-replication, since the use of more than one sample per brand could have resulted in both flour samples originating from the same batch of grain used by the millers; different brands were assumed to have been made by different mills or at least from different grain batches. Samples were transferred from original packages to vacuum food bags and then stored in a −80 °C freezer in containers with silica gel prior to analysis.The experimental design included three factors/variables: wheat species . Around 50 mg of homogenised fresh sample was weighed into a tin foil cup. The cup was carefully folded and squashed into a pellet to expel the air using a tool provided by Elementar. Before each run, a set of control standards were run to ensure that the analyser was working correctly. Results obtained for grain 2.32.3.1Three separate phenolic acid fractions  were extracted from the flour samples using the method described by 2.3.2The total phenolics content in wheat grain was quantified using the Folin-Ciocalteu method  with min2CO3) solution. The microplates were then covered with a plastic cover and incubated in the dark at 40 °C for 30 min. The absorbance of all solutions was measured at 760 nm with a spectrophotometric microplate reader . Final results were presented as µmol gallic acid equivalent (GAE) per g flour (DW).Twenty µL of each sample solution, the serial standard solutions and distilled water as blank were added to wells on a 96-well microplate. Each standard solution and sample solution was tested in duplicate. To each well 130 µL of Folin-Ciocalteu reagent (diluted by distilled water 1:10 (v/v)), was then added, followed after 5 min by 100 µL of 7.5% sodium carbonate . The results were expressed as µmol catechin equivalent (CE) per g flour (DW).Twenty five µL of each sample solution, the serial standard solutions and distilled water as blank were loaded on a 96-well microplate. Each standard solution and sample solution was run in duplicated. 7.5 µL of a 5% NaNO2.3.4Total antioxidant activity of wheat extracts was measured by both the TEAC and FRAP methods . Detaile2.4Extraction for phenolics profile analysis by high-performance liquid chromatography (HPLC) was as described in the phenolic/antioxidant analysis section previously. All chemicals were purchased from Sigma-Aldrich. Phenolic acid standards  were prepared as a stock solution at 0.1 mg/mL in 70:30 methanol:water, and were stored at −20 °C in the dark until HPLC analysis was performed, within three months of extraction.Flour extracts were analysed by HPLC on a Shimadzu Prominence HPLC system equipped with an LC-20AD pump, SIL-20AC antosampler, and SPD-M20A photodiode array detector . Data collection and integration were performed using LabSolution software. Phenolic acids were separated on a reverse-phase Thermo Scientific Hypersil C18 column . The column was heated at 25 °C while the samples tray temperature was set to 4 °C.Mobile phase A was acetonitrile, while mobile phase B was 0.1% acetic acid. The gradient programme for the mobile phase (A:B) was at 0.02 min (5:95), 10 min (20:80), 15 min (25:75), 20 min (35: 65), 25 min (65: 36), 25.01 (100:0), 30 min (100:0), 30.01 (5:95) and 40 (5:95). The flow rate of the mobile phase was 2 mL/m, and the injection volume was 20 µL. Scanning was performed from 190 nm to 800 nm, and phenolic acids were identified by comparing retention times and UV–VIS spectra with those of pure standards. Concentrations, expressed in µg/g DW, were calculated at 230, 270 or 320 nm using calibration curves of phenolic acid standards. The following phenolic acid peaks were identified and quantified according to their spectra and relative retention time: 4-hydroxyvalproic acid, vanillic acid, syringic acid, p-coumaric acid, syringaldeyde, sinapic acid, ferulic acid. The sum of all phenolic acids detected by HPLC was used as an estimate for the total phenolic acid concentration in wheat grain.2.52.5.13) in teflon vessels then digested in a microwave reaction unit  in “vegetable” mode with a four step heating program . After digestion, samples were allowed to cool to room temperature and then were filtered through blue ribbon quantitative filter paper (Whatman Grade 589/3), the filtrates were mixed with distilled water and diluted with ultrapure water in 50 mL flasks. Digested solutions were stored in Sterilin tubes at 4 °C until analysis.Flour (0.25 g) was mixed with 5 mL 69% nitric acid  equipped with a CCD detector . Analytical quality was checked against the certified values of the quality reference material (wheat flour SRM 1567a and apple leaves SRM1515) which were included in every batch of 40 samples.2.6‘nlme’ package in R  derived from Linear mixed-effects (lme) models  was usedage in R . Samplesp < 0.05) interactions between factors, general linear hypothesis tests (Tukey contrasts) were performed using the ‘glht’ function of the ‘multcomp’ package  and antioxidant activity (TEAC), with spelt having an 11% higher phenolic content and 15% higher antioxidant activity (TEAC) . SignifiSignificant 2-way interactions between wheat species and flour type were detected for concentrations of all phytochemicals (except flavonoids) and antioxidant activity (FRAP and TEAC) . When whA significant 2-way interaction between farming system and flour type was only detected for antioxidant activity (FRAP) . AntioxiSignificant 3-way interactions were detected for total flavonoids, ferulic acid concentrations and total phenolic compounds concentration detected by HPLC . SignifiWhen concentrations of free, bound and conjugated phenolic, flavonoid, concentrations and antioxidant activity in these fractions were compared, overall trends were broadly similar to those found for total concentrations and activity .3.23.2.1N-content (Protein concentrations were estimated based on nitrogen (N) concentrations in grains and ANOVA results were therefore identical for protein and -content . Signifi-content .Table 5M3.2.1.1Significant main effects of wheat species and flour type were detected for Na concentrations, which were significantly higher in common wheat than spelt flour, and whole-grain than white flour .There was a 2-way interaction between wheat species and flour type. Significantly higher sodium concentration in common wheat than spelt wheat were only detected in whole-grain flour but not in white flour samples .3.2.1.2Significant main effects of all 3 experimental factors were detected for P, K and Mg concentrations, which were significantly higher in spelt compared with common wheat flour , organic compared with conventional flour  and whole-grain compared with white flour  . SimilarFor P, K and Mg, there were significant 2-way interactions between farming system and flour type , with siFor S there were significant 2-way interactions between (a) wheat species and flour type and (b) wheat species and farming system . Signifi3.2.1.3For Ca a significant interaction between wheat species and flour type was detected . White c3.3Significant main effects of all 3 experimental factors were detected for manganese (Mn), zinc (Zn), copper (Cu) and Molybdenum (Mo) concentrations, which were significantly higher in spelt compared with common wheat flour , organic compared with conventional flour  and whole-grain compared with white flour  . SimilarFor Mo a significant 2-way interaction between wheat species and flour type was detected , with coFor Mn, Zn and Cu significant 2-way interactions were detected between farming systems and flour type , with coFor Cu a significant 2-way interaction was also detected between wheat species and farming systems . Cu conc3.4A significant main effect of wheat species (but not farming system and flour type) was detected for the toxic metal cadmium (Cd), with concentrations found to be significantly higher (28%) in spelt than common wheat flour . SignifiFor Al there were significant 2-way interactions between (a) farming system and flour type and wheat species and farming system . Al conc4In the wheat flour survey reported here, all accessible brands of white and wholegrain flour available in the UK and Germany were analysed. Results therefore reflect the (a) variability associated with the currently used spelt and common wheat varieties, agronomic practices and milling protocols used by commercial farmers and processors in these countries and (b) range of wheat flour quality available to consumers. However, since information on location of farms, crop varieties and specific agronomic practices used by organic and conventional farmers and post-harvest drying, storage, cleaning, and milling protocols used for organic and conventional grains were not recorded, it was not possible to assess to what extent these parameters affected the flour composition parameters measured.Overall, results from this study suggest that there are significant differences in antioxidant and mineral composition between (a) spelt and common wheat and (b) organic and conventional flour and that refining of grains  during the production of white flour removes a large proportion of these nutrients. The relative impacts and interactions between wheat genetics, agronomic practices and grain processing on the nutritional quality and associated potential health impacts are discussed in separate sections below.4.1T. aestivum vs T. spelta) and production protocols , (b) diminishes the differences in antioxidant activity, and phenolic and mineral concentrations in wheat flour produced with grain from contrasting farming systems and wheat species.The results of this study which showed that whole-grain flour has a substantially higher nutritional value than white flour is consistent with previous studies, which reported that whole-grain flour and cereal products have a substantially higher protein, fibre, phenolic and mineral macro-, and micronutrient content than white flour and cereal products . ResultsSimilar to several previous studies  the survThe finding that phenolic, ferrulic acid, sinapic acid and antioxidant activity (FRAP and TEAC) were higher in common than spelt whole-grain samples, but higher in spelt than common wheat white flour is reported for the first time here. It suggests that the negative impact of grain refining on antioxidant levels is greater in common than spelt wheat. This view is supported by the finding that the difference in N/protein concentrations between whole-grain and white flour was also greater in common (11%) than spelt (3%) wheat. This could be due to differences in (a) grain physiology/morphology and/or (b) the amount of bran and germ being removed in the refining process used for spelt and common wheat grain as suggested in previous reports .It is well known that changes to the refining process affect the percentage of bran and germ loss and thus nutritional quality and it has been recommended that studies focused on comparing the nutritional composition of white flour use standardised extraction and analytical methods  to allow4.2Results from this study suggest that spelt flour had slightly, but significantly higher phenolic concentrations and antioxidant activity than common wheat, and that the differences were greater in whole-grain than white flour. Concentrations of protein/N, S, most mineral micro-nutrients analysed and cd were also significantly higher in spelt than common wheat. The results reported here are consistent with some but not all previous studies that compared the composition of spelt and common wheat flour. For example, T. aestivum and T. spelta cultivars/genotypes and it is likely that results were affected by within species variation, which can be considerable  common compared to spelt wheat flour, may indicate that only white common wheat flour is fortified with Ca in the UK and Germany.4.3meta-analysis of data from 343 peer-reviewed publications, which reported that antioxidant/(poly-phenolic) concentrations were higher in organic than conventional crops , Mg and Zn concentrations, and/or antioxidant activity in organic compared with conventional wheat flour, is consistent with the results of a al crops . Howeverperiment  also sugperiment .N-fertiliser, herbicides and modern short-straw varieties can all have a negative effect on antioxidant/(poly)-phenolic concentrations in wheat. Mineral N-fertiliser use was reported as a major driver for the lower concentrations of (poly)phenolics/antioxidants found in both grains and leaves of conventional cereal crops  drivers on the wheat flour composition detected in this study could not be determined.4.4The most recent review of the role of phenolics/antioxidants in modern nutrition by There is now substantial epidemiological evidence that a diet high in phenolics/antioxidants-rich food protects against developing cardiovascular disease and type 2 diabetes . HoweverCereals and cereal-based foods are important sources of minerals in the diet . HoweverThe higher phenolic and antioxidant activity in organic wheat flour may, at least partially explain the results of recent cohort studies which compared health outcomes in individuals with low and high levels of organic food consumption. These studies reported significant positive associations between high levels of organic food consumption and lower risks of obesity, metabolic syndrome, pre-eclampsia and eczema, hypospadias and cancer . However5The finding of substantially higher antioxidant concentrations and essential mineral micronutrients such as Mg, Fe, Zn, Cu in whole-grain flour supports hypothesis 1 and lends further support to current dietary recommendations to switch to whole-grain cereal product consumption.The finding of higher antioxidant activity and/or phenolic and mineral nutrient concentrations in organic compared with conventional, and spelt compared with common wheat flour supports hypotheses 2 and 3. Since the flour survey reported here was based on all spelt and common wheat flour brands that could be found on the shelves of major UK and German retailers, the results can be assumed to accurately reflect the nutritional composition of different wheat flour products available in these countries. The study therefore also suggests that organic and spelt flour consumption would result in higher antioxidant and mineral micronutrient intakes and associated potential health benefits.meta-analysis which reported that Cd and/or Ni concentrations are higher in conventional cereal (see Introduction).The finding of similar Cd and higher Ni concentrations in organic compared with conventional flour does not support hypothesis 2 and contradicts the findings of a recent systematic review/The finding that grain processing  has a substantially larger impact on nutritionally desirable antioxidant activity, and phenolic and mineral nutrient concentrations than primary production protocols and wheat species choice supports hypothesis 4.Juan Wang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Eleni Chatzidimitriou: Investigation, Methodology, Supervision. Liza Wood: Investigation. Gultakin Hasanalieva: Investigation, Methodology. Emilia Markelou: Conceptualization, Writing - review & editing. Per Ole Iversen: Validation, Writing - review & editing. Chris Seal: Methodology, Project administration, Resources, Supervision, Validation, Writing - review & editing. Marcin Baranski: Data curation, Formal analysis, Validation. Vanessa Vigar: Conceptualization, Writing - review & editing. Laura Ernst: Writing - review & editing. Adam Willson: Writing - review & editing. Manisha Thapa: Writing - review & editing. Bronwyn J. Barkla: Validation, Writing - review & editing. Carlo Leifert: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - original draft, Writing - review & editing. Leonidas Rempelos: Data curation, Formal analysis, Investigation, Supervision, Visualization, Writing - original draft, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.