The synthetic challenges of radialenes have precluded their practical applications. Here, we report a one-step synthetic protocol of [4]radialene on a copper surface. High-resolution scanning tunneling microscopy measurements reveal that such catalytic reaction proceeds readily with high selectivity at the temperature below 120 K. First-principles calculations show that the reaction pathway is characterized by firstly the cooperative inter-molecular hydrogen tautomerization and then the C–C bond formation. The feasibility of such cyclotetramerization reaction can be interpreted by the surface effect of Cu(100), which firstly plays an important role in directing the molecular assembly and then serves as an active catalyst in the hydrogen tautomerization and C–C coupling processes. This work presents not only a novel strategy to the scant number of synthetic methods to produce [4]radialenes via a novel [1 + 1 + 1 + 1] reaction pathway, but also a successful example of C–C bond coupling reactions guided by the surface-induced C–H/π assembly. Radialenes have distinct structural, electronic and chemical properties from other hydrocarbons, but their synthesis remains a challenge. Here, the authors report a copper catalyzed one-step synthetic protocol of [4]radialene via the cyclotetramerization of phenylacetylene molecules upon thermal activation. Among the family of ring structures, the production of tetramethylenecyclobutane  and their derivatives have attracted extensive theoretical and synthetic interest due to their special cross-conjugated eight-center, eight π-electron systems, which have been reported to show interesting properties such as multiredox potential1. The successful synthesis of [4]radialene was firstly reported by Griffin and Peterson2. Over the past a few decades, the [4]radialenes and their derivatives have been synthesized via two major strategies: (i) the cyclization of precursor olefins  to form the radialene core4 and (ii) instruction of the exocyclic olefin groups after the construction of the four-membered carbon skeleton5. Although tremendous efforts have been devoted to access these compounds, owing to the drastic reaction coordinates, even the most successful synthetic methods are limited to the poor yields and selectivity of the high symmetric cores of [4]radialenes6.The ultimate goal of synthetic chemistry is to obtain the desired molecular structures from readily available precursors with rationally designed step-economic synthetic strategies. In this regard, the constructions of Ideally, the most step-economic and straight forward synthetic strategy to form [4]radialenes is the [1 + 1 + 1 + 1] cyclotetramerization of alkyne groups  and two-dimensional (2D) polymer structures were constructed on metal surfaces30. In these reactions, metal surfaces serve not only as catalysts, but also as 2D templates to selectively tune the in-plane self-assembly of the reactants. For example, Zhang et al. reported that the C–C bond coupling between reaction precursors can be effectively guided by the molecular self-assembly, so that the chirality can be transferred from the assembly structure to the oligomer reaction products31. Our previous studies have shown that acetyls derivatives may self-assemble into magic supra-molecular structures32. In addition, the C–C triple bonds are fairly reactive on metal surfaces36. Thus, correct combinations of surface electronic states, symmetries, and lattices of transition metals may provide possibilities to overcome the above-mentioned three challenges for the proposed [1 + 1 + 1 + 1] synthetic route.Recent developments of “on-surface chemistry” have shown that surfaces may provide effective controlling parameters to the reaction mechanisms of chemical reactionsBy using phenylacetylene as the precursor, here we successfully achieved the selective synthesis of the tetraphenyl[4]radialenes on Cu(100) surfaces. The tetramerized products were visualized by high-resolution scanning tunneling microscopy (STM), and then further evidenced by tip manipulations. Combining with density functional theory (DFT) calculations, we elucidated that the importance of the Cu(100) surfaces is twofold: (i) guiding the self-assembly of phenylacetylene molecules to form a tetramer-oriented adsorption configuration, in which the attractive force between the monomers is the C–H/π interaction and (ii) working as an active catalyst for the cycloaddition reactions. Detailed examinations of the reaction pathway show that such a cyclization of four phenylacetylene molecules to form [4]radialenes was proceeding via an unusual synergic inter-molecular hydrogen tautomerization and inter-molecular C–C bond formation process. This work demonstrates not only a feasible synthetic method to selectively produce [4]radialenes, but also opens a new avenue to C–C coupling guided by the surface-induced C–H/π assembly.32.We firstly dose the phenylacetylene molecules on a Cu(100) substrate with the substrate held at very low temperature (20 K) to identify their pristine adsorption configurations. Figure 36 or the C–Cu bond formation with copper surfaces34. In particular, our previous studies have indicated that depositing phenylacetylene molecules onto the same surface (Cu(100)) held at 120 K leads to the formation of 1D polymers34. Thermodynamically, one should not obtain self-assembly structures after annealing the phenylacetylene decorated Cu(100) surfaces at a temperature much higher than 120 K. Secondly, DFT calculations and STM simulations were carried out to determine the chemical identity of the tetramers. Figure 32, the phenylacetylene molecules were attracted by weak C–H/π interactions . The simulated STM image according to the optimized structure of self-assembly is shown in Fig. 32. However, the simulated images are rather different from that observed on Cu(100) experimentally  calculations indicate that such cyclotetramerization reaction proceeds via an unusual [1 + 1 + 1 + 1] mechanism. Figure Combing the STM observation with the theoretical calculations, the experimental observations can be completely interpreted. At the temperature of 20 K, the adsorbed phenylacetylene molecules were frozen on the copper surface because the thermal activation barrier of molecular diffusion is 0.28 eV. When the surface is heated gradually, the adsorbed molecules start to move around above the surface, and result in the self-assembled configuration as shown in Fig. 34. This controlling experiment has demonstrated a novel example about how to artificially guide the reaction mechanisms by rationally controlling the reaction dynamics.The above analysis reveals that the Cu(100) surface plays a key role in the cyclization reactions, particularly in guiding the formation of the self-assembly intermediate state prior to the tetramerization reaction. This tells why the [1 + 1 + 1 + 1] cyclotetramerization of alkyne groups is hardly achieved in gases or solutions. To further emphasize this point, controlling experiments were performed by dosing phenylacetylene molecules onto the Cu(100) surface at a higher temperature (120 K). At this temperature, the thermal energy is higher than the energy requirement both to break the molecular assembly and to trigger the acetyls reactions on Cu(100). Correspondingly, the molecular-assembled tetramer cannot be formed before the occurrence of the reactions, prohibiting the cooperative inter-molecular hydrogen tautomerization. As a consequence, the C–C coupling reactions occur between phenylacetylene molecules and result in the formation of chain-like polymers37. Figure 32). We choose a region where three tetramers are observed . The different results of tip manipulation analysis for hexamers on Au(111)32 and tetramers on Cu(100) then suggest that the tetramers are not self-assembly structures, but the reaction products of cycloaddition.To further confirm the bonding nature of the experimentally observed tetramers, we carried out delicate tip manipulation experiments, which is a common and effective method to identify the strength of the interactions among the monomers in complex structuresved Fig. . After tved Fig. . FurtherZ/dV  and dI/dV measurements for the clean Cu(100) surface, single PA molecule, and the tetramer  surfaces. Such a tetramerization reaction proceeds via a cooperative [1 + 1 + 1 + 1] cycloaddition mechanism, in which the terminal hydrogen atoms undergo inter-molecular tautomerization and bond to the carbon atoms that directly connect to the phenyl groups. Almost at the same time, the resulted carbon radicals combine with each other to form the core of a tetraphenyl[4]radialene molecule. The feasibility of this reaction pathway is dominated by the lattice symmetry and the electronic structures of Cu(100) surface. To be more specific, prior to the cycloaddition, the Cu(100) surface plays an important role to guide the molecular assembly of four phenylacetylene molecules to form a C–H/π bonded tetramer. Once such self-assembly structure is obtained, the Cu(100) surface serves an active catalyst that allows the cooperative hydrogen tautomerization and C–C bond formation reactions to occur at low temperatures (before the molecular self-assembly is destroyed). In summary, this work presents not only a new and feasible synthetic method to selectively produce [4]radialenes, but also opens a new avenue to the C–C bond coupling reactions guided by the surface-induced assembly.−10 mbar. Phenylacetylene molecules were purified by the standard freeze-pump-thaw process, and the Cu(100) surface was cleaned by standard argon sputtering-annealing cycles. The Cu(100) substrate was kept at around 20 K during the molecular deposition. The tetramers are formed by subsequently annealing the sample to 200 K. A commercial Pt–Ir tip was prepared by gentle field emission at a clean Cu(100) sample. The bias voltage was applied on the sample during the STM observations. All the STM observations were taken at 10 K.Experiments were conducted with a home-built variable temperature STM, whose temperature can vary from 10 to 300 K, and the base pressure is better than 1 × 1045. In all calculations, the electron–ion interactions were described using the projected augmented-wave method46. The exchange-correlation energy was calculated with the general gradient approximation functionals of Perdew–Burke–Ernzerhof47. The plane-wave expansion was stopped at an energy cutoff of 400 eV. The van der waals interactions between the molecules and copper surfaces were corrected with a non-local correlation functional proposed by Dion48. The optimized lattice constant of Cu was 3.65 Å, which is in good agreement with previous theoretical values48. The Cu(100) surface was modeled with a (9 × 9) periodic slab consisting of four atomic layers. A vacuum of 20 Å was adopted to avoid the periodic image interactions normal to the surface. In all cases, the bottom two layers of Cu atoms were fixed at the bulk positions. The transition states were determined with the CI-NEB calculations, in which five structural images were inserted between the initial and the final states49. In all calculations, the reciprocal space was sampled with a 2 × 2 × 1 gamma centered Monkhorst–Pack grid50. The convergence thresholds of atomic forces for structure optimization and CI-NEB calculations were 0.01 eV/Å and 0.03 eV/Å, respectively. The STM simulation was performed with the Tersoff Hamman method using the bSKAN code51.The theoretical study was carried out in the framework of DFT using the Vienna ab initio Simulation PackageThe data that support the findings of this study are available from the article and Supplementary Information files, or from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis. The fabrication of DNA microarrays can be accomplished via multiple methods, for example by mechanically spotting purified DNA oligonucleotides onto a solid surface or by in situ DNA synthesis by ink-jet-like printing of activated phosphoramidites14. Photolithography, the original approach to in situ synthesis, allows for the fabrication of high-density microarrays using near ultraviolet light as the 5′-OH deprotection trigger16. Further refinement of the technology led to the Maskless Array Synthesis (MAS) system, which allows for the use of virtual masks in lieu of physical lithography masks. MAS photolithography is a flexible and robust method that can achieve high complexity and high density, not only in DNA synthesis17, but also in RNA synthesis19, peptide synthesis21 and for the synthesis of microarrays of biopolymer mimics with engineered properties23. Like conventional solid phase synthesis of oligonucleotides, microarray synthesis is almost always performed in the 3′ to 5′ direction; however, promising new applications based on enzymatic processing require a free 3′ end and thus need reverse synthesis. Examples of these applications include genotyping by allele-specific primer extension10 and spatial transcriptomics technology, which combines gene expression analysis and visualization of tissue slices while retaining morphological context25. Mechanically spotted DNA arrays are well suited for those experiments, since oligonucleotides are typically attached to the surface using 5′ amino or biotin modifications. Spotting has the additional advantage that synthetic, pre-purified oligonucleotides as well as significantly longer DNA strands, such as PCR products, can be attached to the microarray surface26. While spotting may overcome the length and directionality limitations of in situ array synthesis, spot homogeneity and spot density are severely restricted in comparison with photolithographic synthesis.Since their development, DNA microarrays have had a high impact in biology and medicine and have become widely used, powerful analytical tools in a variety of applications27. Early examples of photoprotected phosphoramidites featured a nitrophenyl core as the photosensitive moiety . Attaching an ethyl group meta to the nitro substituent resulted in a very significant increase in photolysis rate30, leading to the development of NPPOC (2-(2-nitrophenyl)-propoxycarbonyl)29, which Beier and Hoheisel adapted for phosphoramidites and used in the light-directed, in situ fabrication of DNA microarrays30. Recent work on improving the photocleavage efficiency of the NPPOC group culminated with the preparation of benzoyl and thiophenyl-substituted NPPOC , which have photolytic efficiencies up to 12 times higher than standard NPPOC, thereby greatly reducing the overall array synthesis time32.Light-directed MAS requires photolabile protecting groups on nucleoside phosphoramidites in order to carry out the controlled synthesis of oligonucleotides33. While the 5′-hydroxyl group is known to be more reactive than the less nucleophilic 3′-OH group, the stepwise addition of 3′-NPPOC phosphoramidite monomers resulted in high coupling yields34 and thus allowed for their use in the photolithographic in situ synthesis of reverse DNA microarrays. Complex microarrays containing more than 150000 probe sequences were fabricated with reverse, 3′-NPPOC 5′-amidites and the corresponding 5′→3′ oriented strands were found to be accessible substrates for various enzymatic processing reactions34.To broaden the spectrum of applications of high-density photolithographic DNA microarrays by allowing the elongation of oligonucleotides in the 5′→3′ direction, phosphoramidites equipped with an NPPOC group at the 3′-OH position have been synthesized31. Hence, short synthesis times are preferable in order to hinder the degradation of the surface, which in turn poses a limit to oligonucleotide length. Here we report on the use of 3′-benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) as an alternative, high-efficient 3′-photolabile protecting group as well as on improvements in the fabrication process of DNA arrays of reverse orientation --phosphoramidite), 5′-DMTr-dT essentially acts as a capping agent as well since photolithography bypasses the use of an acidic deblocking step. With a high coupling efficiency, 5′-DMTr dT was preferred over standard capping mixtures36. The final step consisted in adding a Cy3 dye at the 3′ end of the oligonucleotide in 50% of the synthesized features. The remaining features were deliberately not labelled and served as a reference for background fluorescence. According to this method, the coupling efficiencies for the stepwise addition of NPPOC and BzNPPOC phosphoramidites were determined for synthesis in the 3′→5′ as well as in the 5′→3′ direction and are shown in Table Commercially available 3′-NPPOC and 3′-BzNPPOC protected phosphoramidites were used to determine the stepwise coupling yield of each monomer in reverse, et al. showed that commercial 3′-DMTr phosphoramidite monomers required increased coupling time compared to their 5′-DMTr variants in order to reach similar coupling efficiencies33. Comparable results were also obtained for the stepwise coupling of 3′-NPPOC monomers34. The lower coupling efficiency of 5′ phosphoramidites may be attributable to the lower nucleophilicity of the 3′-hydroxyl group compared to the more reactive 5′-hydroxyl group. In our hands, we were able to obtain coupling yields equal or superior to 99% for reverse dA, dC and dT monomers and sub 98% for dG, within the same range as those of 5′-NPPOC and 5′-BzNPPOC monomers in standard 3′→5′ oligonucleotide synthesis.Previous coupling experiments performed by Pirrung 32. To determine the optimal light exposure parameters for removing the photolabile protecting groups of the corresponding 3′-OH protected monomers, light exposure gradient experiments were performed. After synthesizing 25mer DNA strands on microarrays using a gradient of UV light exposures, the oligonucleotides were hybridized to their Cy3-labelled complementary strand. The normalized fluorescence intensities for syntheses performed in the forward as well as the reverse direction with either NPPOC or BzNPPOC monomers are shown in Fig. The photolabile NPPOC and BzNPPOC protecting groups show very similar absorbance in the spectral region near 365 nm and are now commonly used in the light-directed maskless synthesis of microarrays. The BzNPPOC group offers a sustainably faster photolysis rate over the standard NPPOC group due to its increased photolytic efficiency, which translates into much faster photocleavage than for the NPPOC group2, for a total radiant exposure of 6 J/cm2, while the equivalent removal of the 5′-BzNPPOC group requires under 30 s, or 3 J/cm2. Comparing the photolysis efficiency of 3′- with 5′-protected amidites with a photosensitive protecting group of the same series reveals a higher photocleavage rate for the 5′-protected monomers than for their 3′-protected counterparts, for both NPPOC and BzNPPOC.The resulting overlapping curves can be understood as photolysis kinetics and thus indicate a photolysis rate for BzNPPOC more than twice as fast as that of NPPOC. Indeed, 95% photocleavage of the 5′-NPPOC group can be achieved with an exposure of 60 s at 100 mW/cmtBPac and iPrPac) at the nucleobase, while phosphoramidites with more stable base protecting groups require an extended treatment in the deprotection solution. In order to define the minimum time period for complete deprotection, mirror-image array pairs originating from the same synthesis were exposed to ethylenediamine/ethanol for either 2 or 12 hours. The optimal settings can be determined by hybridizing Cy3-labelled complementary oligonucleotides to the DNA strands synthesized on the microarray surface. Indeed, partially deprotected nucleobases are expected to reduce hybridization to the complementary strand because the protected exocyclic amines of A, C and G cannot form hydrogen bonds and because the protecting groups may destabilize the base stack via steric hindrance. This effect can be seen in lower Cy3 fluorescence intensity after hybridization following incomplete deprotection. Figure The chemical deprotection at the end of a synthesis removes the protecting groups of the phosphodiester moiety and of the exocyclic amine of the nucleobase. For microarrays, the deprotection usually takes place in a 1:1 (v/v) solution of ethylenediamine and ethanol and is complete within 2 hours for monomers carrying fast-deprotecting groups from the phenoxyacetyl series . In order to take into account the minor degradation of oligonucleotides after repeated exposure to synthesis reagents and solvents, as previously observed31, the four sets of probes were also synthesized in the reverse order in a separate array, starting with 15 seconds of coupling time, then 30, 45 and 60 seconds. The arrays were then hybridized to their fluorescently labelled complement and scanned. The combined average intensity values for each set of microarray syntheses are shown in Fig. The coupling of a phosphoramidite to a growing oligonucleotide chain is made possible after photodeprotection of the 3′-OH, in reverse synthesis. Previously, we set the coupling times to 15 seconds in 3′-to-5′-directed syntheses37. The complementary sequence terminated with Cy3 at the 3′ end had the final cytidine base swapped for a guanine, in order to maintain the identity of the nucleotide directly adjacent to the cyanine dye and prevent any sequence-dependent distortion of the fluorescence intensity39. The recorded fluorescence intensities resulting from hybridization to the DNA strands are shown in Fig. 31. The expected trend is shown in Fig. We wished to combine the multiple optimization phases described above into a new and reliable protocol for the synthesis of reverse oriented DNA strands on microarrays, which could ideally be tested against the protocol for standard, 3′-to-5′-oriented DNA synthesis. In order to be able to make direct comparisons between the quality of the strands synthesized either in the 3′→5′ or 5′→3′ direction, forward and reverse synthesis must be performed on the same substrate to avoid variability inherent in hybridizing and washing separate microarrays. However, to prevent any accidental incorporation of phosphoramidites into a strand of wrong directionality, the microarray was designed with consecutive, rather than parallel, synthesis of forward and reverse sequences. The consecutive synthesis started with the fabrication of a 25mer sequence in the forward direction, followed by the synthesis of the same sequence in the reverse direction; this process order was repeated a second time on the same array. After deprotection, hybridization was performed with an equimolar mix of complementary oligonucleotides with a Cy3 dye either at the 5′ or the 3′ end. The use of an equimolar mix mitigates fluorescence intensity artefacts apparently related to the distance between the dye and the surface2, whereas the other set was used for the forward (3′→5′) synthesis, carried out with 5′-BzNPPOC amidites but otherwise identical synthesis parameters. The gene expression microarray design consisted of a total number of 382536 oligonucleotides randomized across a checkerboard-like layout on the microarray surface and composed of human gene probes, reference sequences and quality controls. In detail, two replicates of each of at least 3 unique probes for more than 45000 human genes with a length of 60 nucleotides were synthesized, together with 20–100 replicates of 53mer and 60mer reference sequences and 25mer quality controls. Hybridization experiments with Cy3-labelled cDNA reverse-transcribed from mRNA extracted from a human colon adenocarcinoma cell line (Caco-2) were used to evaluate the quality of the forward and reverse microarrays. For each set of microarrays, one array was hybridized to a Cy3-labelled sample of the cDNA from untreated cells, serving as a control, whereas its mirror-image counterpart was hybridized to the Cy3-labelled cDNA from cells treated with cinnamaldehyde. The quality control metrics from the Cy3-labelled synthetic oligonucleotides  are summarized in Table The next step was to apply our newly established synthesis protocol to the fabrication of highly complex and demanding microarrays. A typical use for high-density arrays is the study of the variation in levels of gene expression between cell populations. Therefore, we synthesized two sets of gene expression microarrays, one set using the newly developed protocol for the reverse (5′→3′) synthesis, employing 3′-BzNPPOC phosphoramidites with a coupling time of 15 seconds and a photodeprotection with a radiant exposure of 3 J/cm2 transformed data are represented in fluorescence intensity scatter plots and shown in Fig. The normalized logIn situ synthesized DNA arrays are almost always synthesized in the traditional 3′→5′ direction, but important new applications of microarrays, such as spatial transcriptomics25 and enzymatic synthesis of RNA40, require a reverse, 5′→3ʹ oriented array synthesis or combined 5′→3′ and 3′→5′ synthesis. Our aim with this project was to adapt the well-established, phosphoramidite and cycle-based techniques for 3′→5′ in situ microarray fabrication to the efficient synthesis of high-density reverse (5′→3′) DNA microarrays of similarly high quality. The robust photolithographic method for high-density DNA array fabrication with up to 786432 individual sequences is being constantly improved and optimized, but those improvements only favoured the classical, 3′→5′ DNA synthesis31. While phosphoramidites with photolabile protecting groups at the 3′-OH position are commercially available, reverse array synthesis has been mostly overlooked. We therefore optimized the photolithographic in situ MAS system to the fabrication of 5′→3′ oligomers.34, we were able to obtain uniformly high coupling yields for 3′-NPPOC as well as for 3′-BzNPPOC amidites and their 5′-equivalents  was found to improve synthesis quality, as indicated by the higher hybridization-based fluorescence intensities. An additional and meaningful reduction of the overall synthesis time is available through the use of 3′-BzNPPOC phosphoramidites. Indeed, further experiments focusing on determining the optimal UV light exposure for complete photolysis revealed that the BzNPPOC group is cleaved more than twice as fast as NPPOC.We first evaluated the coupling efficiency of phosphoramidites carrying BzNPPOC or NPPOC as photolabile protecting groups for the 3′-hydroxyl group. Even though previously published data indicated lower stepwise coupling yields for inverse NPPOC monomers than for their 5′-NPPOC counterpartsts Table . Since aεφ365nm) for amidites carrying the photolabile group at the 3′-OH compared to 5′-OH protected equivalents  solution, while standard protecting groups, e.g. Ac, Bz, iBu, require longer deprotection periods. However, the deprotecting conditions are sufficiently mild to allow overnight deprotection without any degradation of the microarray surface. Microarray deprotection being an important factor in synthesis throughput, the use of faster alternative deprotection conditions, for example aqueous methylamine45, is an alluring prospect but hinges on how the deprotection solutions affect the DNA-glass attachment chemistry, which is beyond the scope of this paper. These improvements now allow for the fabrication of reverse-oriented DNA oligonucleotides by photolithography that rivals the quality of the already optimized, standard 3′→5′ DNA array synthesis. We explored the promising applications of the established reverse synthesis protocol in highly complex and demanding array designs by fabricating a high-density gene expression microarray and hybridizing it to Cy3-labelled genomic cDNA. The comparable fluorescence intensities for 3′→5′ and 5′→3′ oriented gene expression microarrays further reinforce the accuracy and efficiency of light-directed reverse DNA synthesis while the overall distribution of fluorescence values  DNA oligonucleotides on microarrays using commercially available phosphoramidites with 3′-OH photolabile protecting groups as building blocks. Careful optimization of synthesis parameters allowed for the minimization of synthesis time without sacrificing on the quality of the resulting microarrays. Together with our synthesis setup, this method offers a highly flexible design of individual DNA strands, opening up the possibility for combining forward and reverse oriented segments in single strands and the complete in situ synthesis of double-stranded DNA arrays.Here we report on a method for a highly efficient, high-density photolithographic 46. In order to enable access to the synthesis area in-between the two glass substrates , the bottom slide was drilled with two holes of approximately 1 mm diameter. Prior to functionalization, both top and bottom slides were cleaned in an ultrasonic bath and rinsed with purified water. The glass substrates were silanized with N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (Gelest SIT8189.5) in a 95:5 (v/v) solution of ethanol/water containing 0.2% acetic acid for a period of 4 hours at room temperature under gentle agitation47. After two washing steps for 20 minutes each in a 95:5 (v/v) EtOH/H2O + 0.2% AcOH solution, the slides were dried and cured in a vacuum oven overnight at 120 °C. The functionalized slides were stored in a desiccator until use.All microarrays were simultaneously synthesized as mirror image pairs according to a method published earlier46 using a Maskless Array Synthesizer (MAS) instrument which links an optical system with a synchronized chemical delivery system. The computer-controlled optical system uses a digital micromirror device (Texas Instruments 0.7 XGA DMD) consisting of an array of 1024 × 768 individually addressable mirrors, replacing the need for physical photomasks, to pattern UV light onto the synthesis area. A high-power 365 nm UV LED (Nichia NVSU333A U365 surface-mount LED) serves as light source48. The UV light intensity reaching the synthesis area is adjusted using a calibrated intensity meter (SÜSS Micro-Tec 1000). The computer-controlled light exposures which trigger the removal of the photolabile protecting groups are synchronized with a chemical fluidics system (Expedite 8909 nucleic acid synthesizer), which delivers solvents and reagents to the synthesis area. The phosphoramidite chemistry is similar to that of standard solid-phase oligonucleotide synthesis, except for the use of photolabile protecting groups that can be removed upon absorption of a near UV photon in the presence of a weak amine base , leaving either the 5′- or the 3′-hydroxyl group available for the following coupling with an activated monomer. After the synthesis, the phosphodiester and the nucleobase protecting groups are removed by exposing the microarrays to a solution of 1:1 (v/v) ethylenediamine/ethanol for 2 or 12 hours at room temperature. Subsequently, the microarrays are rinsed with deionized water and dried using a microarray centrifuge.The fabrication of microarrays as mirror image pairs was performed as already publishedBzNPPOC and reverse BzNPPOC DNA phosphoramidites were purchased from Orgentis. NPPOC and reverse NPPOC DNA phosphoramidites were obtained from ChemGenes. All phosphoramidites were diluted to 30 mM with acetonitrile . The protecting groups for the exocyclic amines of the nucleobases are shown in Table 35. Microarrays were synthesized with DNA strands of various lengths from 1 to 12mers. To prevent failed sequence additions, each coupling was followed by a capping step with 5′-DMTr-dT (0.03 M) for 60 seconds. Features that were used to determine the background fluorescence remained unlabelled. The other features were terminally labelled by performing two consecutive coupling steps of 300 seconds with Cy3 phosphoramidite (0.05 M). After completion of the synthesis, the microarrays were washed in acetonitrile at room temperature for a period of two hours and dried with a microarray centrifuge. The arrays were scanned as described below and the data was extracted with NimbleScan. SigmaPlot 11.0 (Systat Software) was used to normalize and plot the data. The mathematical model of an exponential decay can be used to describe the decrease in fluorescence intensity as a function of the increasing length of the sequence. The resulting curve was fit to the model curve y = aebx− with y being the fluorescence intensity, a the initial intensity, x the number of couplings and 1 − b the fractional stepwise coupling yield.The yield of each coupling step was determined by the method of terminal labelling as previously describedThe coupling experiments were carried out using the synthesis protocol shown in Table 32. The corresponding microarrays were designed with a light-exposure gradient. In this design, 30 virtual masks are displayed on the DMD within the timeframe of a single, complete exposure step. Each of the virtual masks was displayed for 1/30th of the time required to reach the maximum exposure time necessary for each experiment. The first mask enables the deprotection of phosphoramidites on all features of the microarray where DNA strands are being synthesized. Each of the subsequent masks exposes fewer areas of the microarray in order to obtain oligonucleotide probes which were deprotected with the maximum exposure as well as probes deprotected with successively lower UV light exposure. 65 replicate probes were synthesized at each of the 30 exposure levels with an exposed feature size of 70 × 70 µm, corresponding to a 5 × 5 array of DMD mirrors, and their location randomized across the microarray surface.To examine the photolysis efficiency and determine optimal light exposure parameters for the removal of the photocleavable protecting groups, exposure gradient experiments were performed according to a previously published methodIn order to be able to make direct and reliable comparisons of DNA strands synthesized in different directions or with various coupling times, oligonucleotides need to be synthesized on the same microarray substrate. The desired sequences are randomized across the microarray surface and are fabricated consecutively. Oligonucleotide 25mer probes synthesized early show a ∼6% reduced hybridization intensity, in comparison with the next probe synthesized, due to chemical damage caused by prolonged exposure to solvents and reagents.Forward synthesis describes the fabrication of DNA strands in the 3′ to 5′ direction, whereas the reverse synthesis occurs in the 5′ to 3′ direction. In order to make direct comparisons between forward and reverse syntheses, both were performed on the same substrate. Therefore, the microarray was designed for a consecutive synthesis of the desired sequences, instead of the standard parallel synthesis of the DNA strands. The consecutive synthesis started with the fabrication of a forward sequence, followed by reverse synthesis of the same sequence and a repetition of both syntheses as shown below:3′-TTTTTCTGGTCCCACCAAGTACTACTACTGtttttgtcatcatcatgaaccaccctggtcTTTTTCTGGTCCCACCAAGTAC TACTACTGtttttgtcatcatcatgaaccaccctggtcA, C, G, T: forward, 5′-photoprotected phosphoramiditesa, c, g, t: reverse, 3′-photoprotected phosphoramiditesThe location of the probe replicates of the sequences were randomized across the microarray surface.2 in Dulbecco’s modified Eagle medium (DMEM) with addition of 10% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin49. After having been grown to 80–90% confluence, cells were seeded in 6-well plates at approximately 4 × 105 cells/9.6 cm2 and maintained for 21 days to allow differentiation to an enterocyte-like phenotype. Cultivation medium was changed every second to third day. For the microarray experiments, a previously published protocol was used50. Briefly, cells were treated with DMEM devoid of fetal bovine serum with or without 1000 µM of the test compound, cinnamaldehyde, for 3 hours on day 21. After subsequently washing the Caco-2 cells with ice-cold PBS, RNA isolation was performed using the RNeasy Mini Kit (Qiagen). Concentration and quality of the isolated RNA was determined photometrically using a NanoQuant Plate on an Infinite M200 Tecan reader prior to reverse transcription of a total of 10 µg RNA using Cy3-labelled random nonamer primers (Tebu Bio) as described by Ouellet et al.51. Cy3-labelled cDNA was then purified by means of a Qiagen Qiaquick PCR purification kit.The human colon carcinoma Caco-2 cell line (ATCC) was cultivated under humidified atmosphere at 37 °C and 5% COThe non-genomic microarrays were hybridized to Cy3-labelled complementary oligonucleotides (IDT or Eurogentec). Deprotected microarrays were hybridized in a self-adhesive hybridization chamber (Grace Biolabs SA200). The hybridization mix consists of 150 µL 2x MES hybridization buffer, 110 µL nuclease free water, 13.3 µL acetylated BSA (10 mg/mL) and 26.7 µL of the 100 nM labelled complementary oligonucleotides. The microarrays were incubated with rotation for a period of 2 hours in a hybridization oven (Boekel Scientific) at 42 °C. The hybridization chamber was then removed and the microarrays were washed in non-stringent wash buffer  for 2 minutes, in stringent wash buffer  for 1 minute, followed by a short wash in final wash buffer (0.1x SSC) for a few seconds. A microarray centrifuge was used to dry the microarrays.In most of the performed experiments, the Cy3-labelled complimentary oligonucleotide referred to the following sequence: 5′-GAC CAG GGT GGT TCA TGA TGA TGA C-3′, (QC_25mer). The microarrays used for comparison experiments of forward and reverse synthesized oligonucleotide strands were hybridized to a 1:1 mix of 5′-Cy3 and modified 3′-Cy3 labelled complementary oligonucleotides. The modification of the 3′-Cy3-QC 25mer refers to the presence of a C- instead of a G-base at the 3′-end. Therefore, the sequence of the 3′-Cy3-labelled 25mer oligonucleotide is as follows: 5′-GAC CAG GGT GGT TCA TGA TGA TGA G-3′ (modified QC_25mer).GAC CAG GGT GGT TCA TGA TGA TGA C, QC_25merGAT TTA GGT TTA CAA GTC TAC ACC GAA TTA ACA ACA AAA AAC ACG TTT TGG AG, ECO1BioA1_53merGAA ATG AGG GTG TAA TTG ATT GGG CAA CTG TGC GCC ACG CTA CTT TCT TCT TCG CTT AAC, ECO1BioD2_60merThe hybridization solution used for gene expression experiments consisted of 135 µL 2x MES hybridization buffer, 15 µL acetylated BSA (10 mg/mL), 3 µL herring sperm DNA (10 mg/mL), 10 µL Cy3-labelled QC_25mer (100 nM), 10 µL Cy3-labelled ECO1BioA1 (100 nM), 10 µL Cy3-labelled ECO1BioD2 (100 nM) and the Cy3-labelled cDNA in 85 µL water. The sequences of the 5′-Cy3-labelled oligonucleotides used to evaluate the synthesis and hybridization quality of the microarrays are shown below (5′ to 3′):The gene expression microarray design consisted of 100 probes for QC_25mer, and 140 probes for each of the ECOBio-quality control sequences. The location of each microarray feature was randomized along with all other probe sequences. The gene expression microarrays were incubated with rotation for a period of 20 hours in a hybridization oven (Boekel Scientific) at 42 °C, then washed and dried as described above.A microarray scanner  was used to scan the dried hybridized microarrays at 5 µm resolution with an excitation wavelength of 532 nm. Data extraction of the scanned images was performed with the Software NimbleScan 2.1 (Roche NimbleGen).2 transformed and plotted as treated versus control.A microarray scanner  was used to scan the dried hybridized gene expression microarrays at 2.5 µm resolution with an excitation wavelength of 532 nm. Data extraction of the scanned images was performed with the software NimbleScan 2.1 (Roche NimbleGen). The robust multichip analysis (RMA) function was used to normalize the extracted probe-level data. The normalized intensities of the two biological datasets were log


We discuss how the most extreme δ34S values observed could be related to unique DMSP cycling in the seasonally extreme physiochemical conditions of isolated brine inclusions in winter-spring. Using cell cultures, we show that part of the DMSP δ34S variability could be explained by distinct DMSP metabolism in sea ice microalgae. These findings advance our understanding of the sea ice sulfur cycle and metabolic adaptations of microbes in extreme environments.Sea ice microbial communities produce large amounts of the sulfur metabolite dimethylsulfoniopropionate (DMSP), a precursor of the climate cooling gas dimethylsulfide. Despite their importance to the polar sulfur cycle, drivers and metabolic pathways of sea ice DMSP are uncertain. Here we report the first measurements of sea ice DMSP sulfur isotopic composition ( Gauthier Carnat et al. report the first measurements of the natural sulfur isotopic composition of sea ice from the Antarctic. They find considerable variability between seasons and sea ice horizons, suggesting unique cycling of sulfur metabolites by sea ice microalgae in extreme environments, with important implications for the production of gasses that impact global climate. Multiple physiological roles have been suggested for DMSP to aid in environmental stress adaptation including cryoprotection3, osmoregulation4, and protection against oxidative stress5. DMSP is also a major precursor of the most abundant volatile sulfur compound in oceanic waters, dimethylsulfide (DMS). Oceanic emissions of DMS represent about 25% of the global sulfur flux to the atmosphere6, where it is quickly oxidized to condensable acidic sulfur species7. As a precursor of sunlight-scattering sulfate aerosols and cloud-condensation nuclei, DMS could play a role in climate warming mitigation, although the level of its contribution is still debated9.Dimethylsulfoniopropionate (DMSP) is an important sulfur metabolite synthesized by marine microalgae10. DMS emissions in these regions are strongly influenced by the development of seasonal sea ice. Liquid salty micro-inclusions (brine), which remain trapped between sea ice crystals on freezing, provide a habitat for metabolically active microbial communities (sympagic communities) which can support very high biomass11. These communities are known to periodically produce DMS and DMSP concentrations that are several orders of magnitude greater than global oceanic means13, and release of these sulfur and microbial pools at the ice edge during the spring–summer melt season have been correlated to strong DMS pulses and atmospheric aerosols formation events14.DMS climate-cooling potential may prove to be particularly relevant in the climate-sensitive and aerosol-poor polar regions13 which is currently not well predicted by models. Some of this variability has been correlated with the seasonal evolution of sympagic algal biomass and its vertical zonation in taxonomically distinct assemblages17, DMSP production being known to differ strongly among taxonomic groups18. Sympagic microbial communities also thrive in seasonally and vertically variable physiochemical conditions, mainly driven by changes in sea ice thermodynamics and brine dynamics19. For instance, sympagic microbes must cope with salinities ranging from < 10 g kg−1 in summer melt channels to over 200 g kg−1 in isolated winter brine pockets, and with temperatures ranging from −20 °C in winter surface ice to −1.8 °C at the ice–ocean interface11. High variability in oxygen levels, nutrient supply, light availability, and pH between the different sea ice habitats have also been reported20. As suggested by many22, such variability in physiochemical conditions should strongly influence microbial processes driving the production and degradation of DMS and DMSP1. Unfortunately, only the influence of brine salinity and temperature on microalgal cell physiology and DMSP synthesis was investigated in some details23. Moreover, the exact contribution of sea ice DMS to the annual oceanic DMS flux to the atmosphere remains extremely difficult to quantify. Sea ice may transfer DMS to the polar ocean and ultimately to the atmosphere via multiple pathways such as brine drainage at the ice edge during the entire season24 and melting and ice-breakup in summer14. Direct emissions of DMS from the ice surface and leads have also been observed25.Despite its potential importance for the sulfur cycle in the polar ocean and atmosphere, the biogeochemical cycling of sea ice DMS and DMSP is still poorly understood. Field measurements of sea ice DMS and DMSP concentrations show considerable spatial and temporal variability26. A sensitive method for δ34S analysis in DMS and DMSP in seawater samples was developed in 2013, coupling gas chromatography (GC) and multicollector inductively coupled plasma mass spectrometry (MC-ICPMS)27. Using this method, Amrani et al.28 analysed surface water DMSP samples from six different ocean provinces, revealing a remarkable consistency in δ34S values. Other studies in non-oceanic aquatic environments with different physiochemical conditions such as salt marshes28 and freshwater lakes29 reported very different isotopic values. Until now, the natural sulfur isotopic composition  of DMSP had never been measured in the highly variable sea ice environment.Natural isotopes measurement is a powerful approach to trace sources and transformation processes in complex biogeochemical cycles34S variability in Antarctic sea ice. Considerable variability is revealed across seasons and between sea ice horizons, with multiple values falling out of the range observed in oceanic waters. We show that the highest variability and most extreme DMSP δ34S values are generally found in cold and highly saline isolated brine pockets, while the isotopic signatures in warmer and fresher connected brine channels are more homogeneous. We discuss how the variability could relate to brine inclusions connectivity and its effect on mixing, and to the cycling of DMSP by bacteria and heterotrophs in the isolated brine pockets. Using cell cultures of the polar diatom Fragilariopsis cylindrus in brine conditions, we show that part of the variability could originate from distinct DMSP metabolism in sea ice algae.In this study, we present the first assessment of DMSP δ34S values in sea ice was assessed from a set of Antarctic sea ice cores collected in two different regions of the Southern Ocean , and at four different stations in the central Ross Sea and Ross Sea marginal ice zone (PIPERS field study31). One land-fast ice station was visited seven times during a year-round time series study in the McMurdo Sound, Ross Sea (YROSIAE field study13). At each station, DMSP δ34S were determined as a function of depth, targeting distinct sea ice horizons  and their associated microalgal assemblages.The variability of DMSP δ34S showed very high variability, with values ranging between +10.6 and +23.6‰ (n = 65)  waters)  δ34S values. Such light DMSP δ34S have been recorded in non-oceanic aquatic environments such as salt marshes (+11.3‰)28 and freshwater lakes (+11.4‰)29, where the source sulfate for DMSP metabolism was much lighter than seawater sulfate. Closer inspection of sea ice DMSP δ34S values reveals substantial variability between the three sets of ice cores collected  was also much lighter than in the two other sets. No correlation was found between DMSP δ34S and DMSP concentrations in any of the ice core sets  Fig. . To put rs) Fig. . The majted Fig. . Pack ic13. This set of ice cores was therefore selected to study the temporal/seasonal variability in sea ice DMSP δ34S, since the two other sets integrated spatial variability and were sampled over a much shorter time frame31. Temporal trends during the YROSIAE study are described here in four distinct sea ice horizons corresponding to distinct microalgal assemblages 13  as described in ref. 13. While the mean DMSP δ34S of each station were relatively similar , sharp contrasts could be observed between the shape and range of the depth profiles , and a strong vertical variability between the surface/interior ice horizons and bottom/ice–ocean interface horizons. This variability observed in <2 m of sea ice was higher than the overall variability observed in surface oceanic waters, and in 120 m depth seawater profiles from the Red Sea28. Interestingly, the late-winter core (YRS4) also had a heavy δ34S value in its interior ice horizon. On the other hand, the spring–summer transition cores showed more homogeneity. A trend of 34S depletion in surface ice compared to sub-surface ice was observed in all cores, and bottom/ice–ocean interface δ34S values were always very close to the typical oceanic waters range.The YROSIAE study in the McMurdo Sound provided an opportunity to sample the same fast-ice site from the winter–spring transition (mid-September) to the spring–summer transition (late-November), covering the full seasonal cycle (growth and decay) of a sympagic bloom)13 Fig. . Two conles Fig. . The win34S values sampled in six different ocean provinces encompass a wide range of hydrological, meteorological, and biological conditions32 , which fall within the range of near-surface oceanic waters signatures , taken up through assimilatory sulfate reduction34. The concentration of SO42− in sea ice is regulated by a complex interplay between the concentration/dilution of salts as sea ice forms/melts, the temperature-driven sequential precipitation/dissolution of sulfate minerals (gypsum and mirabilite), and brine exchanges with seawater in the case of an open permeable brine system37. As a result, the bioavailability of sulfate is much more variable in the brine habitat than in oceanic waters which show a stable concentration of 28 mM34. Evaluating the SO42− concentration and δ34S signature in our sea ice sample was therefore of specific interest. Sulfate concentrations were determined in bulk ice from the YROSIAE study and weighted by the brine volume fraction to estimate the amount of sulfate available in brine 39. Sulfur isotope measurements of bulk ice sulfate in the ice core set from the McMurdo Sound . These results are consistent with those reported in frost flowers and sea ice in the Canadian High Arctic, and support the assumption of no isotopic fractionation during mirabilite formation40.The S source for microalgal DMSP synthesis in oxygenated parts of the oceanic water column is widely accepted to be dissolved sulfate  and temperature (T) shifts typically encountered by these organisms in the brine habitat across seasons20. F. cylindrus is an ice-associated diatom widespread in both polar regions whose cell physiology and DMSP metabolism in sea ice physiochemical conditions have been studied in some details23. Temperature and salinity are known not only to control DMSP production in ice-associated algae23 but also to control the abundance and activity of enzymes involved in microalgal sulfate assimilation41 and DMSP synthesis42. F. cylindrus cultures were brought from cold oceanic conditions  to high salinity conditions , and to combined high salinity–low temperature conditions   was measured in both experiments  at the start of the experiment and after 48 h were relatively similar, averaging +3.1‰ ± 0.2‰ (n = 4). Considering that the source sulfate in the culture water  had a δ34S of +6.5‰ (± 0.1‰), the corresponding apparent fractionation factor from sulfate to DMSP was on average −3.4‰ ± 0.06‰ (n = 4)  after 24 h, 48 h, and 1 week were also relatively similar  but ±1‰ lighter than in the control culture, yielding an apparent fractionation factor of −4.3‰ ± 0.09‰ (n = 6) on average  were slightly more variable  and 1 week (+2.1‰) were comparable to those of the high salinity–low temperature culture, but heavier after 48 h (+2.9‰), yielding an apparent fractionation factor of −4.2‰ ± 0.4‰ on average (n = 6)  Fig. . This vaage Fig. . DMSP δ3 6) Fig. .Fig. 3Ch34S in all the ice core sets suggests that more than one process drive the isotopic signature of DMSP in sea ice. The results of the present study point toward an important role of sea ice thermodynamic growth and the vertical/seasonal development of the brine inclusions network. In particular, the stark contrast between small, cold, and highly saline isolated brine pockets that develop in cold ice, and larger, warmer, and fresher connected brine channels that develop in warmer ice warrants further consideration  than in cultures in cold oceanic conditions (−3.4‰). The exact mechanism driving this difference in fractionation remains uncertain. Enzymatic biotransformation of assimilated sulfate to methionine followed by methionine transaminase are the common metabolic pathways of DMSP in most oceanic taxonomic groups46. Several steps in these pathways remain poorly understood42. Depending on physiochemical conditions, reactions in these pathways could be catalysed by different enzymes isoforms, with different kinetic properties and different cellular compartments localisations41. Looking at the proteome response to increased brine salinity in F. cylindrus, Lyon et al.23 suggested that degradation of light harvesting proteins could be an alternative source of methionine for DMSP synthesis. They also identified key enzymes that could catalyse the conversion of methionine to DMSP, enzymes that were not identified in oceanic diatoms42.Results of our controlled experiments with 34S observed in our sea ice samples  on the RV-Polarstern in June–August 2013. A general overview of the AWECS sampling strategy and area is given in ref. 30. Three pack ice stations of AWECS were analysed in this work . The last set of ice cores was collected during the PIPERS (Polynyas and Ice Production in the Ross Sea) study on the RV Nathaniel B. Palmer in the Ross Sea in April–June 2017. Two stations were visited in the marginal ice zone (PIP1 and 4), and two in the central Ross Sea ice pack (PIP29 and 23).Three sets of ice cores were collected for this work. The first set was sampled in the McMurdo Sound during the YROSIAE (Year Round Survey of Ocean-Sea Ice-Air Exchanges in Antarctica) time series study in 2011–2012. One fast ice station located at Cape Evans was sampled seven times throughout the seasons  as described in ref. 13 was followed during the three studies. Briefly, an electropolished stainless-steel (SS) ice corer with an internal diameter of 14 cm was used to retrieve ice cores. Cores were immediately wrapped in polyethylene (PE) bags and kept at <−30 °C horizontally in the dark to prevent brine drainage from the cores and limit the physiological activity of ice algae47. One core per sampling event was fully dedicated to all the S analysis . Two other cores  were used to determine ancillary parameters as described below. The distance between the three cores collected was always <20 cm to limit spatial variability.The same ice coring procedure48.Liquid samples (sea water and brine) were collected during the PIPERS study. Sea water was sampled at three different SOCCOM float locations in the Ross Sea sector of the Southern Ocean using a CTD-Rosette sampler (5 m depth). Liquid samples were transferred to pre-acidified no headspace glass vials for long-term conservation at 4 °C in the dark47. This technique was developed to prevent artificial conversion of DMSP to DMS on melting. One of the ice cuboids was inserted with two grade-316 SS marbles into an air-tight grade-316 SS container. The container was mechanically shaken by a fast up and down motion of the crushing device, reducing the cuboid into a fine ice powder by repeated impacts with the marbles. The ice powder resulting from the crushing step was retrieved and the DMSP content was then quantified as DMS after cold alkali cleavage of DMSP into DMS. The ice powder was left to melt at 4 °C overnight with NaOH pellets in several sealed sparging vials equipped with a Teflon septum. One vial was then connected to a traditional purge-and-trap apparatus, and DMSP quantified as DMS with an Agilent 7890A GC-dual FPD system (DB1-Sulfur Specific column)13. The system was calibrated with pure (>99%) DMS (Merck®) dilutions in Milli-Q water. Triplicate measurements of DMSP in ice samples gave relative standard deviations <12%.The S dedicated ice core was cut into 5 or 10 cm vertical sections. Several smaller ice cuboids were then cut in the centre part of these sections for the different S analysis. Ice cuboids for DMSP concentrations were cut and processed in the field within 48 h of sampling. DMS was first extracted from the ice matrix using the dry crushing technique47. Storage times were 4 years, 3 years, and 5 months for the YROSIAE, AWECS, and PIPERS ice cores sets, respectively. Total DMSP concentrations are usually well preserved after long-term storage in ice cores as shown by the storage tests (>2 years) of Stefels et al.47.Ice cuboids for sulfate concentrations and sulfur isotope measurements were cut in the S dedicated ice core sections in the home laboratory. Ice core sections were kept at all time wrapped in PE bags in the dark and at −30 °C between sampling and analysis. This storing procedure has been proven effective in limiting brine drainage from the samples and physiological activity of microalgae34S) and reported relative to the international reference standard Vienna Cañon Diablo Troilite (V-CDT) in units of permil (‰) following equation:34R is the integrated 34S/32S ion-current ratio of the sample and standard peaks. A sparging vial was connected to a purge-and-trap apparatus (see ref. 27 for a detailed description), and DMS resulting from the base-cleavage of DMSP subsequently transferred via a six-way valve  into a Trace GC  for separation to individual compounds using a Agilent J&W capillary column . Separated DMS was then transferred via a heated (200 °C) transfer line to a multi-collector inductively coupled plasma mass spectrometer  to determine its sulfur isotopic composition. Standard DMS and DMSP solutions with known δ34S values  were injected for calibration every four samples, and a bracketing technique was applied to correct for instrumental mass bias49. Analytical precision and accuracy of DMS and DMSP analysis of standards were usually better than 0.2‰ (1σ standard deviation). The precision of sulfur isotope analysis for duplicate or triplicate samples of seawater or ice averaged 0.3‰ but in few extreme cases reached up to 1‰.Sulfur isotope measurements are presented in this study using the standard delta (δ) notation (δ42−]) precision of these analyses were around 20 nM. Split of this solution was treated with 10% BaCl2 solution and the BaSO4 analysed for its sulfur isotope composition by a conventional elemental analyser (EA) coupled to isotope ratio mass spectrometer (IRMS) method50 using Delta Plus, Thermo. The sulfur isotope reference materials NBS-127 , IAEA-S-1 , and IAEA-SO-6  were purchased from the National Institute of Standards and Technology (NIST) and used for calibration. Precision of this method for duplicates/triplicates was usually better than 0.3‰.Quantification of sulfate was determined from a melted ice cuboid. The melt aliquot was filtered with polycarbonate filters (0.45 μm) to remove particulate organic matter. Aliquot (50 μL) of the filtered solution was diluted 400× and introduced in a Dionex-ICS5000 liquid chromatograph for ion analysis  was always measured in situ with a fast-response handheld portable digital thermometer equipped with a calibrated probe (TESTO®720)51. The probe was inserted in 4 mm diameter holes drilled to the centre of the physical core at 5 cm intervals. The precision of the probe was ± 0.1 °C with an accuracy of ± 0.2 °C. Brine salinity (Sb) was directly computed from sea ice temperature (T) assuming thermodynamic equilibrium of the brine with surrounding ice52:Vb) (brine volume/bulk ice volume) was computed from sea ice temperature (T) and bulk ice salinity (S) using the equations of Cox and Weeks52 for ice temperature <−2 °C and of Lepparänta and Manninen for ice temperature ≥−2 °C53.Sea ice temperature 54. Then, the settled cells were visualized (measured and enumerated) using a Leica Leitz DM IL inverted light microscope equipped with 10× and 40× objectives and 10× and 12.5× oculars (magnification of 500×) with an attached digital camera (LeicaDC300F) for documentation. Species identification was based on Medlin and Priddle55, Thomas56, and Scott and Marchant57.Microalgal taxonomy was determined on the biology core through light microscopic cell enumeration in 200 mL aliquots of melted sea ice stored in brown glass bottles and preserved with acid Lugol’s solution. The bottles were stored in the dark until the cells were settled from a 50, 10, 2, or 1 mL volume for up to 24 hF. cylindrus were maintained in exponential growth at 4 °C and a salinity of 34 g kg−1 under a 16:8 light:dark cycle (75 μE m−2 s−1) in a refrigerated incubator (RUMED® Rubarth Apparate GmbH). F. cylindrus was grown in artificial seawater, created by mixing filtered (0.2 μm) Antarctic seawater from the McMurdo Sound area with artificial sea salts (Instant Ocean®) to a salinity of 100 g kg−1 , and diluting it with mQ water to a salinity of 34 g kg−1. This mixing was done because the volume of Antarctic seawater available was relatively limited. A standard f/2 medium was also added to the water58. These culture conditions are referred in the study as cold oceanic conditions.Cultures of F. cylindrus was initially grown under cold oceanic conditions  in 1 L Nalgene® bottles (inoculation: 5·106 cells mL−1). After 21 days, this control culture was sampled to determine the cell density, the carbon biomass, and the concentration in chlorophyll a, and DMSP (following the same purge and trap-GC procedure detailed above). The remaining volume of the 1 L culture was divided into sub-cultures (cultures in brine conditions). The salinity of both sub-cultures was modified by three additions  of the initial salt mixture  to reach a salinity of 75 g kg−1 . The temperature of the second sub-culture was simultaneously decreased to −4.4 °C by inserting the culture bottles in a cooling alcohol bath (PolyScience®). The cultures were then sampled after 24 h, 48 h, 72 h, and 1 week following the final salt addition and analysed for the same parameters as the control culture. Each culture (control and brine conditions) was treated with a set of antibiotics (penicillin-G and streptomycin)59 to limit bacterial contamination.In the experiment, 34S value in all the culture samples (control and brine conditions) as verified by EA-IRMS analysis of each solution (+6.5‰ ± 0.1‰). This allowed the determination of difference fractionation factors between sulfate and DMSP.The sulfur isotopic composition of DMSP in each culture (control and brine conditions) was determined by GC-MC-ICPMS as described above. Artificial sea salts additions to the cultures logically modified the isotopic signature of the source sulfate for DMSP synthesis. Since all culture waters  were prepared from the same initial salt mixture , this source sulfate had the same δSupplementary Information


O-ethoxyzorb[4]arene (ZB4). Similar to per-O-ethoxyoxatub[4]arene, ZB4 is capable of accommodating a wide range of organic cations. However, ZB4 does not show large amplitude conformational responses to the electronic substituents on the guests. Instead of a linear free-energy relationship, ZB4 follows a parabolic free-energy relationship. This is explained by invoking the influence of secondary C–H···O hydrogen bonds on the primary cation···π interactions based on the information obtained from four representative crystal structures. In addition, heat capacity changes (ΔCp) and enthalpy–entropy compensation phenomena both indicate that solvent reorganization is also involved during the binding. This research further deepens our understanding on the binding behavior of ZB4 and lays the basis for the construction of stimuli-responsive materials with ZB4 as a major component.Large amplitude conformational change is one of the features of biomolecular recognition and is also the basis for allosteric effects and signal transduction in functional biological systems. However, synthetic receptors with controllable conformational changes are rare. In this article, we present a thorough study on the host–guest chemistry of a conformationally adaptive macrocycle, namely per- Macrocyclic receptors are the principal workhorses used in supramolecular chemistry . A myriaendo-functionalized molecular tubes [n]arenes [n]arenes [n]arenes and found that the macrocycles have many unique properties. For example, oxatub[4]arene has a wide guest scope and can bind almost all of the common organic cations [O-methoxy- and per-O-ethoxyzorb[4]arene were shown to be effective tetramethylammonium ion receptors [O-n-butoxyzorb[4]arene .During the last five years, we have developed two classes of macrocyclic receptors with biomimetic structures : endo-fuar tubes –30 and car tubes –37. Amonn]arenes –36 and z cations . It also cations  and remo cations . In addieceptors . The perene ZB4,  was onlyZB4 is also a conformationally adaptive macrocycle. We wondered whether ZB4 has a wide guest binding scope. It was reported that quaternary ammonium-based organic cations (1+–3+) can be hosted by zorb[4]arenes [4+ and 5+ and other types of organic cations hosted by TA4 (6+–10+) were tested with ZB4. Most of these guests can indeed be complexed. But there are some exceptions. Changing the core quaternary ammonium structure of 3+ completely shuts down the binding, because no obvious complexation-induced shifts were detected in the 1:1 mixture of ZB4 with 4+ or 5+  were then performed to obtain the association constants. For small guests such as ZB4 shows it to exist as a self-inclusion conformation in the solid state  [ZB4 shows no response to the electronic substituents on the guests. This is quite different from TA4.e guests . We wondymmetry)  to be thTA4, indicating that the binding affinities are affected by substituents through a field/inductive effect [ZB4. The association constants of ZB4 to these guests were determined by ITC titrations  [3, OMe, Me, SMe, F, Cl, Br, and I, the binding affinities increase with increasing σp. However, the binding affinities decrease with further increasing σp . The guest with iodo substituent (18+) is the best, with an association constant of 4.9 × 105 M−1 at 25 °C. It is interesting to note that although the substituents of guests 12+ and 19+ are quite different in view of their electronic properties, they share very similar binding affinities.In addition, there has been a linear free energy relationship between electronic properties of substituents present in the guests and their binding affinities with e effect . Howeverter (σp)   of the substituents as shown in ZB4 to adapt according to the need of the guests. Simultaneously, the guest may also conformationally adapt to better interact with ZB4. As shown in 18+ in 18+@ZB4-III is slightly different in shape from free cation 18+.What is the underlying reason for the response of binding affinities to the electronic substituent effect on the guests? Luckily, single crystals of complexes ZB4 and 18+ were determined by ITC experiments  for the formation of 18+@ZB4 is −0.13 kJ mol−1 K−1 as determined from the slope of the linear fitting in plots of ΔH versus temperature from 283 to 313 K , while the changes in ΔH and −TΔS are much larger (ca. 4–4.3 kJ mol−1). The changes in ΔH and −TΔS are opposite in signs and perfectly compensate each other. The enthalpy–entropy compensation phenomenon may be explained by invoking a solvent reorganization during the formation of 18+@ZB4 complex, which is common for reactions taking place in aqueous solution [In addition, thermodynamic parameters at different temperatures for the complex between to 313 K . The relsolution .ZB4 using NMR, ITC titration and X-ray crystallography. Similar to TA4, ZB4 is able to host a wide range of organic cations. However, in contrast to TA4, ZB4 shows no large amplitude of conformational response to the electronic nature of substituents on the guests, and its binding affinities follow a parabolic rather than a linear free energy relationship. A closer look at four representative crystal structures suggested that the parabolic free energy relationship may be caused through influencing the major interactions in the host–guest complexes by tuning the weak C–H∙∙∙O hydrogen bonds. Heat capacity changes and enthalpy–entropy compensation indicate that solvent reorganization is also involved during the host–guest binding. Generally, ZB4 is quite different from TA4, and further enriches the arsenal of conformationally adaptive macrocycles. With these model systems, we may further understand the importance of conformational adaptivity in biomolecular recognition and even design stimuli-responsive materials by harnessing this large amplitude of conformational changes.In summary, we systematically studied the guest binding scope, electronic substituent effects and thermodynamic origin on the molecular recognition of File 1Experimental procedures, NMR spectra, mass spectra, determination of association constants and X-ray single crystal data.


These features allow nanoelectrodes based on honeycomb alumina nanoscaffold synergizing both effective ion migration and ample electroactive surface area within limited footprint. A micro-supercapacitor is finally constructed and exhibits record high performance, suggesting the feasibility of the current design for energy storage devices.Downsizing the cell size of honeycomb monoliths to nanoscale would offer high freedom of nanostructure design beyond their capability for broad applications in different fields. However, the microminiaturization of honeycomb monoliths remains a challenge. Here, we report the fabrication of microminiaturized honeycomb monoliths—honeycomb alumina nanoscaffold—and thus as a robust nanostructuring platform to assemble active materials for micro-supercapacitors. The representative honeycomb alumina nanoscaffold with hexagonal cell arrangement and 400 nm inter-cell spacing has an ultrathin but stiff nanoscaffold with only 16 ± 2 nm cell-wall-thickness, resulting in a cell density of 4.65 × 10 Micro-supercapacitors are promising energy storage systems to power the future electronic devices. Here, the authors utilize honeycomb alumina nanoscaffold as a nanostructuring platform to design nanoelectrodes and construct micro-supercapacitors with impressive performance. HMs have been widely exploited as catalyst supporters in different gaseous reactor applications such as chemical and refining processes, catalytic combustion, ozone abatement, and photocatalytic air purification7. As catalyst supporters, HMs have higher surface-to-volume ratio comparing to that of planar substrates for providing high contact efficiencies between the supported catalysts and the gaseous reactants, meanwhile their parallel and straight channels facilitate mass transport of gas with low diffusion resistance. Besides these gas-phase catalysis applications, the cellular and homogeneous structure of the HM presents potential opportunities for other functional applications. For example, it could be a desirable platform to assemble an electroactive electrode for electrochemical devices , in which an efficient electrochemical reaction requires both high specific surface area of the electrode and favorable ionic transport within the electrode11. Despite these merits, so far HMs have not been widely utilized for electrochemical applications mainly due to the bulky structure of the existing HMs. The present electrode design based on HMs makes electrochemical devices cumbersome , and meanwhile the macrostructural channels of HMs require an impractically large amount of electrolytes to fill up all the channels in order to ensure a sufficient contact between the supported electroactive materials and the electrolyte, which is different from gas-phase catalysis applications. Moreover, the specific surface area of the conventional HMs, although it is higher than that of planar substrate counterparts, is far from sufficient to satisfy the requirements of electrochemical applications. Currently, the best reported HM can only attain 1.6 × 104 cells per square inch (cpsi) and about 3 μm in cell wall thickness12, with a specific surface area not being comparable with that of widely used nanoelectrodes . However, most of those nanoelectrodes suffer from the limit of aspect ratio to maintain their highly oriented nature by avoiding the agglomeration since nanowires and nanotubes with high aspect ratio would prefer to form into many dense clusters, and consequently are difficult to simultaneously satisfy both high specific surface area and low ion transport resistance. To this end, downsizing the cell size  of the conventional HMs to nanoscale shall be a solution to efficiently address the challenges for extending the application potentials of HMs. Nevertheless, the microminiaturization of the HMs encounters technological difficulties in creating exactly parallel and straight nanoscale channels over a large area by all reported HM fabrication approaches whatever in industry or in laboratory, and meanwhile faces the challenge to guarantee that the microminiaturized HM would hold the similar excellent mechanical stabilities as the conventional HM.Honeycomb monoliths (HMs) are a kind of cellular materials with two-dimensional cell configuration consisting of parallel and straight channels extended throughout the body9 cpsi that is five orders of magnitude higher than that of the reported highest value. Meanwhile, the HAN with such ultrathin cell wall is stiff and can sustain surprisingly high mechanical stability identified by an experimental nanoindentation. Such robust HAN offers a stable nanostructuring platform to assemble electroactive materials for micro-supercapacitors (MSCs). The insulating HAN is retained in the nanoelectrodes and thus endows the nanoelectrodes with vertically aligned and robustly stable nanoporous structure to simultaneously achieve both effective ion migration and ample electroactive surface area within limited footprint. As a result, a MSC constructed with the HAN-based nanoelectrodes exhibits record high-energy performance among the reported micro-supercapacitors. The maximum capacitance of the MSC reaches 128 mF cm−2 at a current density of 0.5 mA cm−2, and the peak energy and power densities are 160 μWh cm−2 and 40 mW cm−2, respectively.In this article, microminiaturized HMs with ultrathin and stiff nanoscaffold—honeycomb alumina nanoscaffold (HAN)—is realized for the first time. Specifically, a large-scale HAN is fabricated by using a nanoindentation-anodization-etching process with a high-purity aluminum foil. A representative HAN is actually a microminiaturized HM with hexagonal cell arrangement, 400 nm inter-cell spacing and only 16 ± 2 nm cell wall thickness. Impressively, the cell density of the HAN reaches 4.65 × 102O3) with a hexagonal cell arrangement and a cell periodicity of 400 nm. Notablely, the as-prepared nanoporous alumina has a double-layer cell structure  adjacent to the cell center and a relatively pure Al2O3 thin layer (in light-gray color with the thickness of 21 ± 2 nm) remote from the cell center12. By applying a precisely controlled two-step etching in aqueous H3PO4 solutions, the acid anion-contaminated Al2O3 thick layer are easy to be totally dissolved and meanwhile the pure Al2O3 thin layer is partially etched. Finally, a honeycomb nanoscaffold consisting of only an ultrathin layer of pure Al2O3 is left with the cell wall thickness of 16 ± 2 nm  in electrodes/devices will be simultaneously satisfied, benefiting to accomplishing high device performance. Not limited to hexagonal cell arrangement with 400 nm inter-cell spacing, two other HANs also have been produced through tuning the nanopatterns on the surface of aluminum foils generated by nanoindentation, one with hexagonal cell arrangement but 800 nm inter-cell spacing  images, are shown in Fig. ure Fig. , with an nm Fig. , which iHAN Fig. . Noting ing Fig.  and the ing Fig. . Note thing Fig. , which i14. Obviously, such high extrusion pressure would bring forward the critical challenges and requirements to the mechanical stabilities of the nanoelectrodes for supercapacitors and batteries. As known, the cellular solids with honeycombs are much stiffer and stronger to manifest high compressive strength when loaded at the out-of-plane direction  than at the in-plane direction16, thus nanoindentation experiments have been further conducted for characterizing the nanomechanical properties of the HAN. The Young’s modulus of the HAN with hexagonal cell arrangement, 400 nm inter-cell spacing and only 16 nm cell wall thickness are obtained in the range of 1.41–3.22 GPa measured at five different positions  and supercapacitors for the purpose of maintaining intimate contact between the electrodes and the current collectors as well as preventing active materials delamination and deformation in the electrodes during operation2) layer was conformally coated onto the HAN with hexagonal cell arrangement and 400 nm inter-cell spacing  preserves the original honeycomb features with the cell wall thickness increasing from 16 ± 2 to 40 ± 2 nm. To verify the possibility of HAN@SnO2 as nanostructured current collectors, a symmetric supercapacitor based on two HAN@SnO2 electrodes, HAN@SnO2//HAN@SnO2, were assembled and characterized since SnO2 is also one kind of electroactive materials for supercapacitors. Rate performance of supercapacitors, especially under high scan rate, could indirectly reflect the ionic and electronic transport behavior of electrodes. The cyclic voltammetry (CV) curves of the device at different scan rates are shown in Fig. −1, which indicates the typical double-layer capacitive behavior of the device. Figure −1, suggesting the high instantaneous power characteristics of the device. The representative Nyquist plot of the device, and the corresponding expanded view at the high-frequency region, again reveals the capacitive behavior even at high frequencies that are attributed to the fully accessible surface area of the electrodes for electrolyte ions adsorption/desorption  of only 1.8 Ω in HAN@SnO2//HAN@SnO2 device, also contributing to realize high-rate performance. Therefore, high-rate performance should be attributed not only to the rapid mass transport of ions but also to the efficient charge transport and collection in HAN@SnO2 nanoelectrodes during the ultrafast charge-discharge process21.As aforementioned, the HAN is a kind of microminiaturized HMs with much higher cell density than the conventional HMs, and the cellular and homogeneous structure of the HAN presents opportunities as nanostructuring platform to assemble electroactive materials for electrochemical devices. Given the insulating nature of the HAN, the first step to design nanoelectrodes with the HAN for electrochemical applications is to convert the insulating HAN to be a nanostructured current collector. As shown in Fig. ing Fig.  through −1  coated HAN@SnO2 (HAN@SnO2@MnO2) and polypyrrole (PPy) coated HAN@SnO2 (HAN@SnO2@PPy) nanoelectrodes, respectively. The pore depth of HAN in both two nanoelectrodes was 25 μm, and the mass loading of MnO2 and PPy was 0.65 and 1.32 mg cm−2, respectively. The detailed cross-sectional SEM images of different sections  curves of HAN@SnO2@MnO2//HAN@SnO2@MnO2 MSCs at different current densities are shown in Fig. 2, the layer thickness of MnO2 was kept the same in all HAN@SnO2@MnO2 electrodes, thus electrodes with deeper HAN have higher MnO2 mass loading. Figure 2 mass loading as a function of scan rates. The device capacitance depends strongly on the pore depth of original HAN and the subsequent changes of MnO2 mass loading of HAN@SnO2@MnO2 electrodes. For HAN@SnO2@MnO2 electrodes consisting of 25-μm-pore-deep HAN, the MnO2 mass loading was 0.65 mg cm−2, consequently resulting in highest device capacitance of 137 mF cm−2 at a scan rate of 2 mV s−1  were also assembled and characterized to further understand the role of SnO2 layer in HAN@SnO2 based pseudocapacitive electrodes. It can be concluded from Fig. −2) of HAN@SnO2//HAN@SnO2 MSCs, in which the pore depth of HAN in all electrodes was 25 μm, suggests the function of SnO2 layer in HAN@SnO2 based pseudocapacitive electrodes mostly for charge transport rather than charge storage. Furthermore, electrochemical impendence spectroscopy (EIS) was performed in order to clearly understand the role of HAN@SnO2 as nanostructured current collectors. Figure 2//HAN@SnO2 MSCs with different pore depth of original HAN . All MSCs have very similar equivalent series resistances (ESR), however, there is a significant raise in Rct. The gradually increased Rct should be the inevitable result arising from the limited electrical conductivity of SnO2, which is much lower than that of metals. The intrinsic electrical resistance of SnO2 leads the total resistance of HAN@SnO2//HAN@SnO2 MSCs to be progressively increased accompanying with HAN pore depth increase, and then consequently results in an increase in Rct. With respect to HAN@SnO2@MnO2//HAN@SnO2@MnO2 and HAN@SnO2@PPy//HAN@SnO2@PPy MSCs . When the current density is increased from 0.5 to 5 mA cm−2, the capacitance drops to 93 mF cm−2 (72% retention) and it recovers to 123 mF cm−2 (96% retention) with the current density again being decreased to 0.5 mA cm−2  based stacked MSCs (~40 μWh cm−2) but with a similar peak power density24. Additionally, the areal energy density of the asymmetric MSCs with EMIM-TFSI electrolyte is even comparable with that of some state-of-the-art 3D micro-batteries but with much higher areal power density36.When further replacing aqueous electrolyte with the ionic liquid electrolyte (1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, EMIM-TFSI), the asymmetric MSCs can work efficiently in an enlarged potential range of 0–3.0 V Fig. . The dism−2 Fig. , revealiSCs Fig. 21,24–34.The insufficient energy of MSCs is their crucial flaw compared with micro-batteries and remains a major challenge to overcome. This work signifies the capability of the HAN as a promising nanostructuring platform to assemble pseudocapacitive materials toward rationally designing nanoelectrodes for MSCs with high-energy performance. In comparison with the conventional HMs, the HAN as a kind of microminiaturized HMs has the highest cell density, lower relative density, higher surface area enhancement factor, and excellent compressive performance. With such robust HAN as the keel, the as-prepared nanoelectrodes could possess vertically aligned and robustly stable nanoporous structure. And in contrast to nanoelectrodes design based on nanowires and nanotubes, there is no limit about the aspect ratio to sustain such vertically aligned nanoporous structure. Besides high surface area to provide abundant electroactive sites for surface Faradaic reactions, the vertically aligned nanoporous structure affords the smooth channels to favor the permeability for electrolyte infiltration and ion diffusion to access the surface of pseudocapacitive materials, guaranteeing a high utilization efficiency of the pseudocapacitive materials. Taken together, the HAN-based nanoelectrodes design synergizes the effects of both effective ion migration and high electroactive surface area, thus enabling high and reversible capacitive behavior even at high charge–discharge rates. In the current HAN-based nanoelectrodes design, four strategies have been combined together into single MSCs aiming to achieve high-energy storage capability: (i) the utilization of pseudocapacitive materials with high specific capacitance in both positive and negative electrodes; (ii) the adoption of asymmetric device configuration to extend the operating potential window of MSCs; (iii) the application of ionic liquid electrolyte to further widen the operating potential window of MSCs; and more importantly, (iv) the prominent nanoelectrode architecture design based on HAN for both positive and negative electrodes. The combination of pseudocapacitive materials with unique nanoelectrodes architecture results in the increased areal capacitance with reduced footprint, meanwhile the adoption of asymmetric device configuration coupled with ionic liquid electrolyte helps to overcome the narrow operating potential window of pseudocapacitive materials. As a result, the asymmetric MSCs constructed with the HAN-based nanoelectrodes exhibit one of the most remarkable comprehensive areal device performance metrics.We emphasize that nanoelectrode design with the robust HAN as the keel to assemble electroactive materials inherits the unique structural characteristics of the HMs and can be manipulated to achieve desired application requirements for electrochemical devices beyond MSCs. Our work opens up the ample opportunities for further expanding the application range of the HMs and also provides a paradigm about rationally designing nanostructures for various functional devices with new features and high performance.3PO4) aqueous solution at 160 V for different time. The temperature of the solution and the aluminium foil was kept at 10 °C during the anodization process. After anodization, the unanodized aluminum was etched by a mixture aqueous solution of CuCl2 (85 wt%) and HCl (15 wt%). Thereafter a two-step chemical etching process was applied to obtain the HAN: (1) etching with a 5 wt% H3PO4 solution at 60 °C to achieve an open-end membrane; (2) further etching with a 5 wt% H3PO4 solution at 30 °C to finally obtain HAN. The surface area enhancement factor (A) for HAN was calculated by Eq. d and h are corresponding to the cell diameter and depth of the cell, respectively, s is the intercell distances, measured between the centers of the cells, and θ is the angle of the pattern in which the cells are positioned. For the typical HAN structure with cell depth of 25 μm, the specific values are d = 384 nm, h = 25 μm, s = 400 nm and θ = 60°, respectively. Accordingly, the A is calculated to be 240.The HAN was fabricated through a nanoindentation-anodization process, followed by a precisely controlled two-step chemical etching process. Firstly, a hexagonal patterns was generated onto the surface of aluminum foil by nanoindentation, then the aluminium foil was anodized in a 0.4 M phosphoric acid . The precursors were tin (IV) chloride (SnCl4) for tin and ultrapure water for oxygen, respectively, and high-purity nitrogen was the carrier and purging gas. Specifically, each ALD cycle consisted of a 0.2 s pulse of SnCl4 and a 4 s purge of N2, followed by a 1 s pulse of H2O and an 8 s purge of N2, and this procedure was repeated 1500 times. The growth rate of SnO2 is estimated to be about 0.16 Å per cycle. With the as-obtained HAN@SnO2 as the working electrodes, the HAN-based nanoelectrodes were fabricated through electrochemical deposition method. The HAN@SnO2@PPy electrode was prepared by electrochemical polymerization of PPy onto HAN@SnO2 substrate. The plating solution consisted of 0.1 M pyrrole monomer (98%) and 0.2 M oxalic acid, and the applied potential was 0.8 V (vs. Ag/AgCl)37. Likewise, the HAN@SnO2@MnO2 electrode was fabricated by electrochemical deposition of MnO2 onto the HAN@SnO2 substrate by using an electrolyte consisting of 50 mM manganese acetate and 100 mM sodium acetate with a constant current density of 1 mA cm−2. The active mass of the electrodes were determined according to Faraday’s law and 100% charge efficiency was assumed, according to Eq. Q is the charge passed during the electrochemical deposition process of active materials . And it is worth noting that the charge is precisely adjust here in order to achieve the same layer thickness of active materials for HAN@SnO2 with different cell depth. M is the molar mass of the active electrode material , F the Faraday constant and z the number of transferred electrons per active electrode atom (z = 2 for MnO2 and PPy deposition).The HAN@SnOThe surface morphologies and microstructures of the electrodes were characterized using scanning electron microscopy (ZEISS AURIGA) equipped with an EDX detector. Cyclic voltammetry (CV), galvanostatic charge–discharge (GCD), electrochemical impedance spectroscopy (EIS) measurements, and cycling performance measurements were conducted with a Potentiostat . Nanoindentation was conducted with the Triboindenter (Hysitron) equipped with a Berkovich diamond indenter with an approximately 600-nm-radius.2SO4 aqueous electrolyte and a glass microfiber filter  as separator as well as 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM-TFSI) ionic liquid electrolyte (1 M in acetonitrile). The CV curves were collected with various scan rates of 5 to 2000 mV s−1, and the GCD curves were obtained at current densities of 0.2, 0.5, 1, 2, 5, 10, 15, 20 and 25 mA cm−2. The EIS measurement was performed with a frequency range from 100 MHz to 10 mHz and a 5 mV AC amplitude.All the electrochemical measurements were carried out in a two-electrode configuration with 1.0 M NaCGCD (mF cm−2) is determined from the GCD profiles based on Eq. I (mA) is the discharge current, A (cm2) is total footprint area of device, and ΔV (V) is the voltage window.The capacitance of the device CCV (mF cm−2) can also be calculated from CV curves based on Eq. I (A) is the response current and ν (V s−1) is the potential scan rate.The device capacitance E, unit: μWh cm−2) and power density  of the device were calculated by Eqs. C (mF cm−2) is the areal capacitance of the device, ∆t (s) is the discharge time.With GCD plots, the energy density (Supplementary InformationPeer Review File