We demonstrate that the hygroscopic and biofluorescent behaviors of living cells can be engineered to design biohybrid wearables, which give multifunctional responsiveness to human sweat. By depositing genetically tractable microbes on a humidity-inert material to form a heterogeneous multilayered structure, we obtained biohybrid films that can reversibly change shape and biofluorescence intensity within a few seconds in response to environmental humidity gradients. Inspired by this hygroscopic information-processing concept , researchers previously developed active materials with unique geometric constraints or structural heterogeneities, including polymers for fabricating hygrovoltaic generators , hydrogels for forming complex micropatterns , spores for harvesting energy , and wood for building innovative pavilions. These previous studies primarily focused on innovative applications by using nonliving materials, the chemical performance and mechanical performance of which are predefined before fabrication. For instance, spores or harvested wood materials are not biologically active and are insusceptible to direct genetic engineering that can introduce new properties or responsiveness to the system. Therefore, the application domains involving customization are limited because of the nature of the raw materials. By contrast, microbes are composed of a portfolio of single-celled living organisms that can be easily genetically modified to acquire new functions and are amenable to production in large-scale bioreactors, but have not yet been fully exploited to create environment-responsive materials. Here, we propose to use genetically tractable microbial cells to create multifunctional, moisture-responsive interfaces. Generally, bio-derived materials, including nucleic acids , proteins , and polysaccharides , can change their structures or volumes when surrounding water activity is altered. Because these three materials are the basic biological components that form cellular structure, we hypothesized that microbial cells can be used as functional building blocks for constructing moisture-responsive materials. We speculated that a bilayer-structured biohybrid film could change shape and functions fluorescence as an example simultaneously with humidity change. The concept of dual responsiveness is illustrated at the macroscopic level , A and B , microscopic level , C and D , cellular level , E and F , and molecular level , G and H. If this hypothesis were true, we could easily embed other functionalities into the existing moisture-responsive cells through modern genetic engineering technologies, such as clustered regularly interspaced short palindromic repeats CRISPR —Cas9 , for instance, to facilely introduce fluorophores , colors , odors , or other metabolic responsiveness induced by environmental change. This will open up a new application space for using hygroscopic transforming materials to tackle real-world problems related to moisture for example, indoor humidity control and warts caused by hyperhidrosis. One intriguing application domain is designing wearable devices to accommodate body sweat for high-intensity working professionals including athletes and soldiers, to prevent frostbite or hypothermia. This research domain for regulating body condition has not been intensively pursued previously because of the lack of multifunctional hygroscopic materials that are facilely accessible, easy to process, and low-cost. A and B The film bends tangibly at low humidity levels A and becomes flat and glows at high humidity levels B. C and D A cross section of the biohybrid film at the microscopic level, where dehydration of the cell layer dark green and light green coated on top of an inert thin film black enables the bending of the film at low humidity levels C , whereas the film becomes flat at high humidity levels D through rehydration. The box on the top left corner indicates the contraction C and expansion D of cells under two conditions. E and F The change of cell size and cellular fluorescence with humidity levels due to moisture desorption E and adsorption F at the cellular level. G and H An example of the conformational change of intracellular eGFP at the molecular level due to water removal G and water binding H at different humidity levels. RESULTS To prove that microbial cells can be used to create a hygroscopic structure that exhibits reversible shape transformation, we used the most common type of Escherichia coli cells to fabricate the biohybrid film. First, the cells were cultured and harvested to prepare a concentrated suspension for printing, before which all the nutrients for example, sugar were removed. Uncoated natural latex sheets cis-1,4-polyisoprene, 150 to 500 μm thick with rough surfaces fig. S1 were selected as the moisture-inert materials, which demonstrated good bacterial biofilm adhesion through hydrophobic interactions in the absence of additional chemical modification. By microprinting these cells thickness, 1 to 5 μm in parallel lines on the latex surface fig. S2 , a multifunctional biohybrid film was produced. This bending behavior movie S1 was reversible over several cycles showing no hysteresis or distortion C , as characterized in a customized humidity-controlled chamber fig. Using atomic force microscopy AFM imaging with humidity control fig. S4 , we observed similar phenomena at the cellular level, where single cells swelled or shrank along all three axes in response to changes in humidity , D to F. This indicated that the bending effect of the tangible film was induced by the net force generated through the volume change of each individual cell movie S2. A and B Shape transformation of a biohybrid film 1. The top layer is composed of E. It bends at 15% RH A and becomes flat at 95% RH B. C The bending curvature of this biohybrid film at different RH levels. D and E Topological images of a cell at 15% RH C and 95% RH D obtained from AFM. F The cell volume change at different RH levels scanned by AFM. G and H Fluorescence images of a biohybrid film coated with E. It shows little fluorescence at 15% RH G compared with that at 95% RH H. I Fluorescence intensity varies along with the bending curvature of the film when exposed to humid air in a dry environment. RFU, relative fluorescence units. J Bending angle for different types of cells. K Bending angle for major cellular biological components. L and M Simulation symbol S and theoretical model symbol T are consistent with experimentally measured bending symbol E for varying numbers of cell layer symbol L thickness L or latex substrate thickness M. Beyond mechanical response to moisture, we sought to introduce additional functionalities through genetic engineering of the E. Here, we used enhanced green fluorescent protein eGFP to demonstrate this concept. It is known that GFP will lose all of its fluoresce ability in ethanol because of dehydration , G and H. In our experiment, we found that the fluorescence intensity of biohybrid films using GFP-expressing E. S5 and table S1 was correlated with humidity , G to I. The fluorescence intensity of the cell significantly decreased under dry conditions, consistent with dehydration-induced conformational changes of eGFP at low levels of water activity. When environmental humidity increased, eGFP renatured and the film re-exhibited fluorescence movie S3. We further validated that eGFP itself changed fluorescence intensity under different conditions by imaging the cell lysate through confocal microscopy fig. These sets of experiments verified our hypothesis that E. To generalize this concept to other types of microbial cells to suit the needs of different application domains, we measured the bending angles of a biohybrid film consisting of two Gram-positive bacteria Bacillus subtilis and Rhodococcus erythropolis , two Gram-negative bacteria E. The results showed that different types of cells have similar moisture responsiveness, which indicates that this property may arise from macromolecular building blocks nucleic acids, proteins, carbohydrates, and lipids that are common to the cells. To examine this hypothesis, we investigated the bending behavior of the biohybrid films prepared by printing each of the four major intracellular macromolecules on latex K. The results showed that with the same dry weight, proteins induced a higher contractile force than nucleic acids and polysaccharides. As expected, lipids did not change the bending angles because the hydrophobicity of lipids prevented water absorption. This provides us with a pragmatic approach to further improve the bending performance by overexpressing proteins intracellularly using genetic engineering. The enlarged pool of hygroscopic biological materials including generally regarded as safe GRAS materials will definitely facilitate material selection in application and function customization. To quantitatively understand the mechanical performance of these hygroscopic materials, we measured the bending angles of the bilayer-structured biohybrid films geometry in note S1 with varied cell layer and substrate thicknesses under different humidity conditions. The experimental data of the bending angle were then compared with a theoretical formula based on the classical Stoney formula and the results from a three-dimensional finite-element model fig. AFM revealed that the volume depends linearly on humidity at the single-cell level F , which can be described by the empirical relation in terms of the normalized volume. The volume of the fully wet state is based on the measurement at 95% RH, the maximum value possible while avoiding the formation of water droplets. The total volume change for the cell layer should also follow this relation, because the cells are densely packed to form a continuous film. Although the single cell exhibits anisotropic contraction, the average effect over a large amount of cells results in the cell layer deforming isotopically in each dimension. The contraction strain of the cell layer can thus be expressed as. The fitted elastic modulus of the cell layer is 70 MPa, consistent with previous reports ; the stress and strain of the cell layer in the experiments were also estimated fig. Furthermore, the bending of the biohybrid films is well described by numerical simulation movie S4 and note S3. After characterizing the system, we turn to application of the biohybrid films for making wearables that respond to body sweat during motion. Because the bilayer structure curls naturally under ambient conditions 30 to 70% RH , which makes it hard to handle for example, cutting, shaping, and assembling , we developed a sandwich structure where cell layers are coated on both sides of a moisture-inert material. This structure allows the film to respond only to a localized moisture gradient across the film while ensuring the flatness of the film with balanced contractile forces on b
