Method to Measure Surface Tension of Microdroplets Using Standard AFM Cantilever Tips.

Surface tension is a physical property that is central to our understanding of wetting phenomena. One could easily measure liquid surface tension using commercially available tensiometers (e.g., Wilhelmy plate method) or by optical imaging (e.g., pendant drop method). However, such instruments are designed for bulk liquid volumes on the order of milliliters. In order to perform similar measurements on extremely small sample volumes in the range of femtoliters, atomic force microscope (AFM) is considered as a promising tool. It was previously reported that by fabricating a special "nanoneedle"-shaped cantilever probe, a Wilhelmy-like experiment can be performed with AFM. By measuring the capillary force between such special probes and a liquid surface, surface tension could be calculated. Here, we carried out measurements on microscopic droplets with AFM, but instead, using standard pyramidal cantilever tips. The cantilevers were coated with a hydrophilic polyethylene glycol-based polymer brush in a simple one-step process, which reduced its contact angle hysteresis for most liquids. Numerical simulations of a liquid drop interacting with a pyramidal or conical geometry were used to calculate surface tension from the experimentally measured force. The results on micrometer-sized drops agree well with bulk tensiometer measurement of three test liquids (mineral oil, ionic liquid, and glycerol), within a maximum error of 10%. Our method eliminates the need for specially fabricated "nanoneedle" tips, thus reducing the complexity and cost of measurement.

Ethylene and Jasmonates Signaling Network Mediating Secondary Metabolites under Abiotic Stress.

Plants are sessile organisms that face environmental threats throughout their life cycle, but increasing global warming poses an even more existential threat. Despite these unfavorable circumstances, plants try to adapt by developing a variety of strategies coordinated by plant hormones, resulting in a stress-specific phenotype. In this context, ethylene and jasmonates (JAs) present a fascinating case of synergism and antagonism. Here, Ethylene Insensitive 3/Ethylene Insensitive-Like Protein1 (EIN3/EIL1) and Jasmonate-Zim Domain (JAZs)-MYC2 of the ethylene and JAs signaling pathways, respectively, appear to act as nodes connecting multiple networks to regulate stress responses, including secondary metabolites. Secondary metabolites are multifunctional organic compounds that play crucial roles in stress acclimation of plants. Plants that exhibit high plasticity in their secondary metabolism, which allows them to generate near-infinite chemical diversity through structural and chemical modifications, are likely to have a selective and adaptive advantage, especially in the face of climate change challenges. In contrast, domestication of crop plants has resulted in change or even loss in diversity of phytochemicals, making them significantly more vulnerable to environmental stresses over time. For this reason, there is a need to advance our understanding of the underlying mechanisms by which plant hormones and secondary metabolites respond to abiotic stress. This knowledge may help to improve the adaptability and resilience of plants to changing climatic conditions without compromising yield and productivity. Our aim in this review was to provide a detailed overview of abiotic stress responses mediated by ethylene and JAs and their impact on secondary metabolites.

Do Opposites Attract? Auxin-Abscisic Acid Crosstalk: New Perspectives.

Plants are constantly exposed to a variety of different environmental stresses, including drought, salinity, and elevated temperatures. These stress cues are assumed to intensify in the future driven by the global climate change scenario which we are currently experiencing. These stressors have largely detrimental effects on plant growth and development and, therefore, put global food security in jeopardy. For this reason, it is necessary to expand our understanding of the underlying mechanisms by which plants respond to abiotic stresses. Especially boosting our insight into the ways by which plants balance their growth and their defense programs appear to be of paramount importance, as this may lead to novel perspectives that can pave the way to increase agricultural productivity in a sustainable manner. In this review, our aim was to present a detailed overview of different facets of the crosstalk between the antagonistic plant hormones abscisic acid (ABA) and auxin, two phytohormones that are the main drivers of plant stress responses, on the one hand, and plant growth, on the other.

Using machine learning to link the influence of transferred Agrobacterium rhizogenes genes to the hormone profile and morphological traits in Centella asiatica hairy roots.

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.

Doppelblinde, randomisierte repetitive In-vivo-Wirksamkeitsstudie zu kommerziellen Hautschutzprodukten gegen Natriumlaurylsulfat (SLS).

HINTERGRUND: In der Primär- und Sekundärprävention von Berufsdermatosen werden Hautschutzprodukte eingesetzt, die eine präexpositionelle Schutzwirkung aufweisen sollen. Insbesondere finden sich auf dem Markt zahlreiche Produkte, die zum Einsatz bei Feuchtarbeit ausgelobt werden. Eine objektive Qualitätsbeurteilung dieser Produkte für Anwender ist auf Grund fehlender vergleichender Wirksamkeitsstudien oft nicht möglich. MATERIAL UND METHODIK: In einer doppelblinden, randomisierten und kontrollierten In-vivo-Studie wurden sechs kommerzielle Hautschutzexterna in einem repetitiven Irritationsmodell untersucht. Sie wurden hinsichtlich ihrer präexpositionellen Schutzwirkung vor dem durch Natriumlaurylsulfat (SLS) induzierten irritativen Kontaktekzem mittels hautphysiologischer Methoden getestet. Es wurde ein Wirkindex anhand des transepidermalen Wasserverlustes (TEWL) gemäß des Protokolls der multizentrischen Studie des Forschungsprojektes FP 275 der Deutschen Gesetzlichen Unfallversicherung DGUV berechnet. ERGEBNISSE: Die untersuchten Externa zeigten eine Schutzwirkung gegen SLS im Vergleich zum unbehandelten Kontrollfeld, wiesen allerdings signifikante Wirkunterschiede auf. Die Auswertung der Nebenparameter Corneometrie und visueller Score erbrachte ebenfalls Unterschiede zwischen den Externa. SCHLUSSFOLGERUNGEN: Die zwischen den untersuchten Externa festgestellten Unterschiede hinsichtlich der Wirkgüte ermöglichten eine Einteilung der Produkte, welche als Grundlage für die Auswahl geeigneter Schutzpräparate dienen kann.

The threshold between life and death in Cistus albidus L. seedlings: mechanisms underlying drought tolerance and resilience.

Drought can lead to important shifts in population dynamics if it occurs during seedling establishment. With the aim of elucidating the underlying mechanisms of drought tolerance and resilience, here we monitored the survival of seedlings of the Mediterranean shrub Cistus albidus L. throughout a year growing in the natural Park of the Montserrat Mountains (Spain) and, additionally, we studied the response to severe drought and subsequent recovery after rewatering of seedlings grown in growth chambers. To find possible mechanisms explaining how seedlings respond to drought, growth and survival together with physiological-related parameters such as chlorophyll contents, vitamin E and stress-related phytohormones were measured. We found that survival decreased by 30% at the end of summer and that the main proxy of seedling survival was total chlorophyll. This proxy was further confirmed in the growth chambers, where we found that seedlings that recovered from drought had higher levels of total chlorophyll compared with the seedlings that did not recover. Furthermore, modulation of vitamin E and jasmonates contents appeared to be crucial in the drought response of C. albidus seedlings. We propose a prediction model of survival that includes total chlorophyll height, leaf mass area and maximum photosystem II efficiency with chlorophyll contents being a good long-term predictor of C. albidus seedling survival under severe stress, which, in turn, could help to better foresee population fluctuations in the field.

Hormonal impact on photosynthesis and photoprotection in plants.

Photosynthesis is not only essential for plants, but it also sustains life on Earth. Phytohormones play crucial roles in developmental processes, from organ initiation to senescence, due to their role as growth and developmental regulators, as well as their central role in the regulation of photosynthesis. Furthermore, phytohormones play a major role in photoprotection of the photosynthetic apparatus under stress conditions. Here, in addition to discussing our current knowledge on the role of the phytohormones auxin, cytokinins, gibberellins, and strigolactones in promoting photosynthesis, we will also highlight the role of abscisic acid beyond stomatal closure in modulating photosynthesis and photoprotection under various stress conditions through crosstalk with ethylene, salicylates, jasmonates, and brassinosteroids. Furthermore, the role of phytohormones in controlling the production and scavenging of photosynthesis-derived reactive oxygen species, the duration and extent of photo-oxidative stress and redox signaling under stress conditions will be discussed in detail. Hormones have a significant impact on the regulation of photosynthetic processes in plants under both optimal and stress conditions, with hormonal interactions, complementation, and crosstalk being important in the spatiotemporal and integrative regulation of photosynthetic processes during organ development at the whole-plant level.

Double blind randomized repetitive efficacy test of various occupational skin protection preparations against sodium lauryl sulphate.

BACKGROUND: Skin protection products, formerly named as barrier creams, are being used as primary and secondary prevention measures against occupational dermatoses. Many of these cosmetic products are claimed by manufacturers to be efficacious against wet work-induced skin barrier impairment if applied prior to exposure. However, results of validated and standardized in vivo efficacy tests allowing the comparison of different products are lacking. MATERIAL AND METHODS: Efficacy of six skin protection products against sodium lauryl sulphate-induced irritant contact dermatitis was investigated in a double blind, randomized and controlled repetitive irritation study in healthy volunteers using skin physiological methods. Transepidermal water loss (TEWL) results were used to calculate an efficacy index of protection by mathematical/statistical means according to a protocol developed in a German Statutory Accident Insurance (DGUV) multicenter study (research project FP 275). RESULTS: All tested products had a demonstrable, protective effect against SLS compared to controls, but their relative efficacy varied significantly. Differences were also seen upon evaluation of other parameters such as corneometry and clinical irritation score (visual score). CONCLUSIONS: Product ranking as a result of the established differences allows improved selection of appropriate skin protection products based on objective parameters, as compared to heterogenous product claims.

Foes or Friends: ABA and Ethylene Interaction under Abiotic Stress.

Due to their sessile nature, plants constantly adapt to their environment by modulating various internal plant hormone signals and distributions, as plants perceive environmental changes. Plant hormones include abscisic acid (ABA), auxins, brassinosteroids, cytokinins, ethylene, gibberellins, jasmonates, salicylic acid, and strigolactones, which collectively regulate plant growth, development, metabolism, and defense. Moreover, plant hormone crosstalk coordinates a sophisticated plant hormone network to achieve specific physiological functions, on both a spatial and temporal level. Thus, the study of hormone-hormone interactions is a competitive field of research for deciphering the underlying regulatory mechanisms. Among plant hormones, ABA and ethylene present a fascinating case of interaction. They are commonly recognized to act antagonistically in the control of plant growth, and development, as well as under stress conditions. However, several studies on ABA and ethylene suggest that they can operate in parallel or even interact positively. Here, an overview is provided of the current knowledge on ABA and ethylene interaction, focusing on abiotic stress conditions and a simplified hypothetical model describing stomatal closure / opening, regulated by ABA and ethylene.

The Arabidopsis thaliana mRNA decay factor PAT1 functions in osmotic stress responses and decaps ABA-responsive genes.

mRNA decapping plays essential roles in regulating gene expression during cellular reprogramming in response to developmental and environmental cues. The evolutionarily conserved PAT1 proteins activate decapping by binding mRNA, recruiting other decapping components, and promoting processing body (PB) assembly. Arabidopsis encodes 3 PAT proteins: PAT1, PATH1, and PATH2. Here, we report that only pat1 mutants exhibit hypersensitivity to ABA and that transcripts of ABA-responsive genes, but not those of ABA biosynthesis genes, persist longer in these mutants. The pat1 mutants also exhibit increased resistance to drought stress and resistance to Pythium irregulare. This is supported by assays showing that PAT1 functions specifically in decapping of the canonical ABA-responsive gene COR15A. In summary, PAT1 protein mediates decay of ABA-responsive genes and, thus, regulates stress responses.

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus.

A complex network of cellular receptors, RNA targeting pathways, and small-molecule signaling provides robust plant immunity and tolerance to viruses. To maximize their fitness, viruses must evolve control mechanisms to balance host immune evasion and plant-damaging effects. The genus Potyvirus comprises plant viruses characterized by RNA genomes that encode large polyproteins led by the P1 protease. A P1 autoinhibitory domain controls polyprotein processing, the release of a downstream functional RNA-silencing suppressor, and viral replication. Here, we show that P1Pro, a plum pox virus clone that lacks the P1 autoinhibitory domain, triggers complex reprogramming of the host transcriptome and high levels of abscisic acid (ABA) accumulation. A meta-analysis highlighted ABA connections with host pathways known to control RNA stability, turnover, maturation, and translation. Transcriptomic changes triggered by P1Pro infection or ABA showed similarities in host RNA abundance and diversity. Genetic and hormone treatment assays showed that ABA promotes plant resistance to potyviral infection. Finally, quantitative mathematical modeling of viral replication in the presence of defense pathways supported self-control of polyprotein processing kinetics as a viral mechanism that attenuates the magnitude of the host antiviral response. Overall, our findings indicate that ABA is an active player in plant antiviral immunity, which is nonetheless evaded by a self-controlled RNA virus.

A defect in BRI1-EMS-SUPPRESSOR 1 (bes1)-mediated brassinosteroid signaling increases photoinhibition and photo-oxidative stress during heat stress in Arabidopsis.

Heat stress has negative effects on plant performance, especially in photosynthesis and photoprotection. To withstand heat stress, plants modulate steroid phytohormones, brassinosteroids (BRs). However, their regulation and functions in heat stress acclimation are still poorly understood. Plant growth, photoinhibition, photo-oxidative stress and endogenous contents of hormones (including not only BRs but also abscisic acid, salicylic acid and jasmonic acid) were evaluated during heat stress in Arabidopsis thaliana wild type plants and loss-of-function mutations in either BR biosynthetic or signaling genes. It was found that a defect in BRI1-EMS-SUPPRESSOR 1 (bes1)-mediated BR signaling showed the most sensitive characteristics to heat stress compared to wild type and other BR mutants. Sensitivity was associated with declined PSII photochemistry efficiency (Fv/Fm) together with increased carotenoid, tocopherol and lipid hydroperoxide contents, which evidences higher photoinhibition and photo-oxidative stress in the bes1 mutant under heat stress. Furthermore, the bes1 mutant showed greater contents of abscisic acid (ABA) after one day of exposure to heat stress. However, all heat stress symptoms in the bes1 mutant could be mitigated by the application of 24-epibrassinolide. Therefore, it is concluded that BES1 transcription factor plays a role in plants responses to heat stress, relieving photoinhibition and photo-oxidative stress, but that alternative BR signaling pathways to BES1 may also be effective in heat stress acclimation. Furthermore, this study emphasizes the complex interplay between BR and ABA in the heat acclimation process.

Interactions between sucrose and jasmonate signalling in the response to cold stress.

BACKGROUND: Jasmonates play an important role in plant stress and defence responses and are also involved in the regulation of anthocyanin synthesis in response to sucrose availability. Here we explore the signalling interactions between sucrose and jasmonates in response to cold stress in Arabidopsis. RESULTS: Sucrose and cold treatments increased anthocyanin content additively. Comprehensive profiling of phytohormone contents demonstrated that jasmonates, salicylic acid and abscisic acid contents increased in response to sucrose treatment in plants grown on agar, but remained considerably lower than in plants grown in compost. The gibberellin GA3 accumulated in response to sucrose treatment but only at warm temperature. The role of jasmonate signalling was explored using the jasmonate response mutants jar1-1 and coi1-16. While the jar1-1 mutant lacked jasmonate-isoleucine and jasmonate-leucine, it accumulated 12-oxo-phytodienoic acid at low temperature on agar medium. Altered patterns of abscisic acid accumulation and higher sugar contents were found in the coi1-16 mutant when grown in compost. Both mutants were able to accumulate anthocyanin and to cold acclimate, but the jar-1-1 mutant showed a larger initial drop in whole-rosette photosystem II efficiency upon transfer to low temperature. CONCLUSIONS: Hormone contents are determined by interactions between temperature and sucrose supply. Some of these effects may be caused indirectly through senescence initiation in response to sucrose availability. During cold stress, the adjustments of hormone contents may compensate for impaired jasmonate signalling, enabling cold acclimation and anthocyanin accumulation in Arabidopsis jasmonate response mutants, e.g. through antagonistic interactions between gibberellin and jasmonate signalling.

Vitamin E in legume nodules: Occurrence and antioxidant function.

Although the biosynthesis and function of tocochromanols in leaves and seeds have been extensively studied, their occurrence and function in underground tissues, such as roots and nodules, is very poorly understood. Here, we performed a comparative study of the presence of tocochromanols in different plant organs (leaves, roots and nodules) of three legumes (soybean, alfalfa and pea plants). Additionally, we measured variations in tocochromanols as a function of the severity of water stress and evaluated their relationship with the extent of membrane lipid peroxidation and nodule performance (as indicated by thiobarbituric acid-reactive substances assay and 15N isotope labeling, respectively). Results showed the presence of endogenous tocopherols, mainly α-tocopherol, in the three studied organs of the three legumes. Nodules showed higher concentrations of α-tocopherol than roots, but lower than leaves. α-Tocopherol content increased under water shortage in nodules, roots and leaves of soybean as well as in roots of alfalfa, but not in the other plant systems. A strong negative correlation between α-tocopherol and thiobarbituric acid-reactive substances contents was found for roots and especially for nodules. Furthermore, nodule α-tocopherol content positively correlated with nodule N2 fixation (estimated by 15N isotope labeling). We conclude that α-tocopherol is a major antioxidant found in legume nodules.

Contrasting patterns of hormonal and photoprotective isoprenoids in response to stress in Cistus albidus during a Mediterranean winter.

MAIN CONCLUSION: Seasonal accumulation of hormonal and photoprotective isoprenoids, particularly α-tocopherol, carotenoids and abscisic acid, indicate their important role in protecting Cistus albidus plants from environmental stress during a Mediterranean winter. The high diurnal amounts of α-tocopherol and xanthophylls 3 h before maximum light intensity suggest a photoprotective response against the prevailing diurnal changes. The timing to modulate acclimatory/defense responses under changing environmental conditions is one of the most critical points for plant fitness and stress tolerance. Here, we report seasonal and diurnal changes in the contents of isoprenoids originated from the methylerythritol phosphate pathway, including chlorophylls, carotenoids, tocochromanols, and phytohormones (abscisic acid, cytokinins, and gibberellins) in C. albidus during a Mediterranean winter. Plants were subjected not only to typically low winter temperatures but also to drought, as shown by a mean plant water status of 54% during the experimental period. The maximum PSII efficiency, however, remained consistently high (Fv/Fm > 0.8), proving that C. albidus had efficient mechanisms to tolerate combined stress conditions during winter. While seasonal α-tocopherol contents remained high (200-300 µg/g DW) during the experimental period, carotenoid contents increased during winter attaining maximum levels in February (minimum air temperature ≤ 5 °C for 13 days). Following the initial transient increases of bioactive trans-zeatin (about fivefold) during winter, the increased abscisic acid contents proved its important role during abiotic stress tolerance. Diurnal amounts of α-tocopherol and xanthophylls, particularly lutein, zeaxanthin and neoxanthin including the de-epoxidation state, reached maximum levels as early as 2 h after dawn, when solar intensity was 68% lower than the maximum solar radiation at noon. It is concluded that (1) given their proven antioxidant properties, both α-tocopherol and carotenoids seem to play a crucial role protecting the photosynthetic apparatus under severe stress conditions; (2) high seasonal amounts of abscisic acid indicate its important role in abiotic stress tolerance within plant hormones, although under specific environmental conditions, accumulation of bioactive cytokinins appears to be involved to enhance stress tolerance; (3) the concerted diurnal adjustment of α-tocopherol and xanthophylls as early as 3 h before maximum light intensity suggests that plants anticipated the predictable diurnal changes in the environment to protect the photosynthetic apparatus.

Leaf Orientation as Part of the Leaf Developmental Program in the Semi-Deciduous Shrub, Cistus albidus L.: Diurnal, Positional, and Photoprotective Effects During Winter.

Mediterranean ecosystems harbor a very important part of Earth's biodiversity, and they are a conservation priority due to the effects of global change. Here, we examined the performance of the semi-deciduous shrub Cistus albidus under Mediterranean conditions during winter, including changes in leaf angle governed by diurnal, seasonal, and positional effects and their relationship with winter photoinhibition and photoprotection. We found marked diurnal variations in leaf angle during the day in autumn, which disappeared as the photoperiod shortened during winter due to a progressive decrease in the predawn leaf angle from November to January. During this period, auxin contents decreased, while those of melatonin increased, and the F v/F m ratio, chlorophyll, and tocopherol contents kept unaltered, thus indicating the absence of photoinhibitory damage. A marked decrease in the leaf angle toward the shoot apex occurred during winter, which was associated with slightly higher F v/F m ratios. An analysis of the inter-individual variability and sun orientation effects on leaf movements in a population growing in the Montserrat mountains revealed a very marked variability of 46.8% in the leaf angle, while F v/F m ratio showed a variation of 7.5% only. West orientation, which was associated with reduced leaf temperatures, but with no differences in the photosynthetic photon flux density, led to enhanced tocopherol contents, while leaf angle, F v/F m ratio, chlorophyll, auxin, and melatonin contents kept unaltered. It is concluded that (1) C. albidus has very effective and fine-regulated photoprotection mechanisms, including an adequate orientation of decussate leaves as part of the developmental program, (2) leaf angle is modulated on a diurnal and seasonal basis, thus contributing to prevent photoinhibition as a first line of defense, and (3) enhanced tocopherol contents help withstand combined high light with low temperature stress in C. albidus growing at high elevation.

Biosynthesis, Metabolism and Function of Auxin, Salicylic Acid and Melatonin in Climacteric and Non-climacteric Fruits.

Climacteric and non-climacteric fruits are differentiated by the ripening process, in particular by the involvement of ethylene, high respiration rates and the nature of the process, being autocatalytic or not, respectively. Here, we focus on the biosynthesis, metabolism and function of three compounds (auxin, salicylic acid and melatonin) sharing not only a common precursor (chorismate), but also regulatory functions in plants, and therefore in fruits. Aside from describing their biosynthesis in plants, with a particular emphasis on common precursors and points of metabolic diversion, we will discuss recent advances on their role in fruit ripening and the regulation of bioactive compounds accumulation, both in climacteric and non-climacteric fruits.

Engulfment control of platinum nanoparticles into oxidized silicon substrates for fabrication of dense solid-state nanopore arrays.

We found that platinum (Pt) nanoparticles, upon annealing at high temperature of 1000 °C, are engulfed into amorphous fused-silica or thermal oxide silicon substrates. The same phenomenon was previously published for gold (Au) nanoparticles. Similar to the Au nanoparticles, the engulfed Pt nanoparticles connect to the surface of the substrates through conical nanopores, and the size of the Pt nanoparticles decreases with increasing depth of the nanopores. We explain the phenomena as driven by the formation of platinum oxide by reaction of the platinum with atmospheric oxygen, with platinum oxide evaporating to the environment. We found that the use of Pt provides much better controllability than the use of Au. Due to the high vapor pressure of platinum oxide, the engulfment of the Pt nanoparticles into oxidized silicon (SiO2) substrates is faster than of Au nanoparticles. At high temperature annealing we also find that the aggregation of Pt nanoparticles on the substrate surface is insignificant. As a result, the Pt nanoparticles are uniformly engulfed into the substrates, leading to an opportunity for patterning dense nanopore arrays. Moreover, the use of oxidized Si substrates enables us to precisely control the depth of the nanopores since the engulfment of Pt nanoparticles stops at a short distance above the SiO x /Si interface. After subsequent etching steps, a membrane with dense nanopore through-holes with diameters down to sub-30 nm is obtained. With its simple operation and high controllability, this fabrication method provides an alternative for rapid patterning of dense arrays of solid-state nanopores at low-cost.