Double-Sided Pressure-Sensitive Adhesive Materials under Human-Centric Extreme Environments.

Maintaining the adhesion strength of flexible pressure-sensitive adhesives (PSAs) is crucial for advanced applications, such as health monitoring. Sustainable mounting is critical for wearable sensor devices, especially under challenging surroundings such as low and high temperatures (e.g., polar regions or deserts), underwater and sweat environments (physical activity), and cyclical shear complex stresses. In this article, we consider the adhesive, mechanical, and optical properties of medical-grade double-sided PSAs by simulating extreme human-centric environments. Diverse temperature conditions, water and humidity exposures, and cyclical loads were selected and tested over long intervals, up to 28 days. We observed that high temperatures increased the shear adhesion strength due to the pore closing and expanding contact area between the adhesive layer and substrate. Conversely, low temperatures caused the adhesive layers to harden and reduce the adhesive strength. Immersion in salty and weakly acidic water and excessive humidity reduced adhesion as water interfered with the interfacial interactions. PSA films showed either adhesive or cohesive failure under extreme mechanical stresses and cyclical loading, which is also affected by the presence of various polar solvents. We demonstrated that the variable adhesive performance, mechanical properties, and optical transparency of pressure-sensitive materials can be directly related to changes in their morphologies, surface roughness, swelling state, and alternation of the mechanical contact area, helping to establish the broader rules of design for wearable human health monitoring sensors for the long-term application of wearable devices, sensors, and electrodes.

Cell surface engineering with edible protein nanoshells.

Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte-based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on-demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.

Orthogonal cell-based biosensing: fluorescent, electrochemical, and colorimetric detection with silica-immobilized cellular communities integrated with an ITO-glass/plastic laminate cartridge.

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

In silico selection of RNA aptamers.

In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer-ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude--significantly accelerating the experimental screening and selection of high-affinity aptamers.

Biofunctionalized zinc oxide field effect transistors for selective sensing of riboflavin with current modulation.

Zinc oxide field effect transistors (ZnO-FET), covalently functionalized with single stranded DNA aptamers, provide a highly selective platform for label-free small molecule sensing. The nanostructured surface morphology of ZnO provides high sensitivity and room temperature deposition allows for a wide array of substrate types. Herein we demonstrate the selective detection of riboflavin down to the pM level in aqueous solution using the negative electrical current response of the ZnO-FET by covalently attaching a riboflavin binding aptamer to the surface. The response of the biofunctionalized ZnO-FET was tuned by attaching a redox tag (ferrocene) to the 3' terminus of the aptamer, resulting in positive current modulation upon exposure to riboflavin down to pM levels.

FRET-based optical assay for monitoring riboswitch activation.

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

Enzyme immobilization in a biomimetic silica support.

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure. Here we show that butyrylcholinesterase entrapped during the precipitation of silica nanospheres retained all of its activity. Ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme. The mechanical properties of silica nanospheres facilitated application in a flow-through reactor. The use of biosilica for enzyme immobilization combines the excellent support properties of a silica matrix with a benign immobilization method that retains enzyme activity.

Cellular internalization and targeting of semiconductor quantum dots.

Peptide-mediated internalization and organelle targeting of quantum dots.

Silk macromolecules with amino acid-poly(ethylene glycol) grafts for controlling layer-by-layer encapsulation and aggregation of recombinant bacterial cells.

This study introduces double-brush designs of functionalized silk polyelectrolytes based upon regenerated silk fibroin (SF), which is modified with poly-L-lysine (SF-PLL), poly-L-glutamic acid (SF-PGA), and poly(ethylene glycol) (PEG) side chains with different grafting architecture and variable amino acid-PEG graft composition for cell encapsulation. The molecular weight of poly amino acids (length of side chains), molecular weight and degree of PEG grafting (D) were varied in order to assess the formation of cytocompatible and robust layer-by-layer (LbL) shells on two types of bacterial cells (Gram-negative and Gram-positive bacteria). We observed that shells assembled with charged polycationic amino acids adversely effected the properties of microbial cells while promoting the formation of large cell aggregates. In contrast, hydrogen-bonded shells with high PEG grafting density were the most cytocompatible, while promoting formation of stable colloidal suspensions of individual cell encapsulates. The stability to degradation of silk shells (under standard cell incubation procedure) was related to the intrinsic properties of thermodynamic bonding forces, with shells based on electrostatic interactions having stronger resistance to deterioration compared to pure hydrogen-bonded silk shells. By optimizing the charge density of silk polyelectrolytes brushes, as well as the length and the degree of PEG side grafts, robust and cytocompatible cell coatings were engineered that can control aggregation of cells for biosensor devices and other potential biomedical applications.

Printed Dual Cell Arrays for Multiplexed Sensing.

We demonstrated inkjet printing of large-scale dual-type encapsulated bacterial cell arrays for prospective multiplexing sensing. The dual cell arrays were constructed on the basis of two types of bioengineered E. coli cells hosting fluorescent reporters (green-GFPa1 and red-turboRFP) capable of detecting different target chemicals. The versatility of inkjet printing allows for the fabrication of uniform multilayered confined structures composed of silk ionomers that served as nests for in-printing different cells. Furthermore, sequential encapsulation of "red" and "green" cells in microscopic silk nest arrays with the preservation of their function allowed for facile confinement of cells into microscopic silk nests, where cells retained dual red-green response to mixed analyte environment. Whole-cell dual arrays immobilized in microscopic biocompatible silk matrices were readily activated after prolonged storage (up to 3 months, ambient conditions), showing red-green pattern and demonstrating an effective prototype of robust and long-living multiplexed biosensors for field applications.

Entrapment of enzymes and nanoparticles using biomimetically synthesized silica.

Entrapment of enzymes and nanoparticles using biosilicification reactions.

Identification of peptides that promote the rapid precipitation of germania nanoparticle networks via use of a peptide display library.

Peptides that promote the rapid, room-temperature precipitation of amorphous germania nanoparticle networks from solution have been identified via use of a combinatorial peptide display library.

Controlled formation of biosilica structures in vitro.

Herein we describe the controlled formation of biosilica structures by manipulation of the physical reaction environment; we were able to synthesize arched and elongated silica structures using a synthetic peptide; the results presented here are evidence that in vitro biocatalysis may be controlled in order to form desired silica structures.

Biological infrared imaging and sensing.

A variety of thermoreceptors are present in animals and insects, which aid them in hunting, feeding and survival. Infrared (IR) imaging pit organs in Crotaline and Boid snakes enable them to detect, locate and apprehend their prey by detecting the IR radiation they emit. IR pit organs of common vampire bats (Desmodus rotundus) enable them to detect IR radiation emitted by blood-rich locations on homeothermic prey. The beetle Melanophila acuminata locates forest fires by IR-detecting pit organs in order to lay their eggs in freshly killed conifers. Thermoreceptors located in the wings and antennae of darkly pigmented butterflies (Pachliopta aristolochiae and Troides rhadamathus plateni) protect them from heat damage while sun basking. Blood-sucking bugs (Triatoma infestans) are speculated to possess thermoreceptors, which enable them to perceive the radiant heat emitted by homeothermic prey and estimate its temperature at a distance. This is a review of the diverse types of biological thermoreceptors, their structure and function, and how electron microscopy has been instrumental in determining their ultrastructure.

Silica-precipitating peptides isolated from a combinatorial phage display peptide library.

Many biological organisms contain specialized structures composed of inorganic materials. Cellular processes in vivo facilitate the organized assembly of mineral building blocks into complex structures. The structural hierarchy and complexity across a range of length scales are providing new ideas and concepts for materials chemistry. Proteins that direct biomineralization can be used to control the production of nanostructured materials and facilitate the fabrication of new structures. Here, we demonstrate that some of the silica-binding peptides isolated from a combinatorial phage peptide display library can be used in precipitating silica from a solution of silicic acid. The results described in this report demonstrate that peptides displayed by phages act as templates in inorganic material synthesis and provide a means of understanding how some of the biological systems may be carrying out materials chemistry in vivo.

Computational design of RNA libraries for in vitro selection of aptamers.

Selection of aptamers that bind a specific ligand usually begins with a random library of RNA sequences, and many aptamers selected from such random pools have a simple stem-loop structure. We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and designed a protocol for RNA 3D structure prediction. The proposed approach significantly reduces the RNA sequence search space, thus accelerating the experimental screening and selection of high-affinity aptamers.

FRET-based optical assay for selection of artificial riboswitches.

Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E. coli cells that is based on TEV protease-FRET substrate reporter system.

Detection of orexin A neuropeptide in biological fluids using a zinc oxide field effect transistor.

Biomarkers which are indicative of acute physiological and emotional states are studied in a number of different areas in cognitive neuroscience. Currently, many cognitive studies are conducted based on programmed tasks followed by timed biofluid sampling, central laboratory processing, and followed by data analysis. In this work, we present a sensor platform capable of rapid biomarker detection specific for detecting neuropeptide orexin A, found in blood and saliva and known as an indicator of fatigue and cognitive performance. A peptide recognition element that selectively binds to orexin A was designed, characterized, and functionalized onto a zinc oxide field effect transistor to enable rapid detection. The detection limit using the sensor platform was sub-picomolar in water, and picomolar to nanomolar levels in saliva and serum. The transistor and recognition element sensor platform can be easily expanded, allowing for multiple biomarkers to be detected simultaneously, lending itself to complex biomarker analysis applicable to rapid feedback for neuroscience research and physiological monitoring.

Development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in E. coli cells.

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.

pH-responsive layer-by-layer nanoshells for direct regulation of cell activity.

Saccharomyces cerevisiae yeast cells encapsulated with pH-responsive synthetic nanoshells from lightly cross-linked polymethacrylic acid showed a high viability rate of around 90%, an indication of high biocompatibility of synthetic pH-responsive shells. We demonstrated that increasing pH above the isoelectric point of the polymer shell leads to a delay in growth rate; however, it does not affect the expression of enhanced green fluorescent protein. We suggest that progressive ionization and charge accumulation within the synthetic shells evoke a structural change in the outer shells which affect the membrane transport. This change facilitates the ability to manipulate growth kinetics and functionality of the cells with the surrounding environment. We observed that hollow layer-by layer nanoshells showed a remarkable degree of reversible swelling/deswelling over a narrow pH range (pH 5.0-6.0), but their assembly directly on the cell surface resulted in the suppression of large dimensional changes. We suggest that the variation in surface charges caused by deprotonation/protonation of carboxylic groups in the nanoshells controlled cell growth and cell function, which can be utilized for external chemical control of cell-based biosensors.