Congzhou Wang

Conducting polymer-silk biocomposites for flexible and biodegradable electrochemical sensors.

UNLABELLED Approaches to form flexible biosensors require strategies to tune materials for various biomedical applications. We report a facile approach using photolithography to fabricate poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) ( PEDOT PSS) sensors on a fully biodegradable and flexible silk protein fibroin support. A benchtop photolithographic setup is used to fabricate high fidelity and high resolution PEDOT PSS microstructures over a large (cm) area using only water as the solvent. Using the conductive micropatterns as working electrodes, we demonstrate biosensors with excellent electrochemical activity and stability over a number of days. The fabricated biosensors display excellent nonspecific detection of dopamine and ascorbic acid with high sensitivity. These devices are mechanically flexible, optically transparent, electroactive, cytocompatible and biodegradable. The benign fabrication protocol allows the conducting ink to function as a matrix for enzymes as shown by a highly sensitive detection of glucose. These sensors can retain their properties under repeated mechanical deformations, but are completely degradable under enzymatic action. The reported technique is scalable and can be used to develop sensitive, robust, and inexpensive biosensors with controllable biodegradability, leading to applications in transient or implantable bioelectronics and optoelectronics.

Investigating biomolecular recognition at the cell surface using atomic force microscopy.

Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique.

Biopatterning of Silk Proteins for Soft Micro-optics

Silk proteins from spiders and silkworms have been proposed as outstanding candidates for soft micro-optic and photonic applications because of their optical transparency, unique biological properties, and mechanical robustness. Here, we present a method to form microstructures of the two constituent silk proteins, fibroin and sericin for use as an optical biomaterial. Using photolithography, chemically modified silk protein photoresists are patterned in 2D arrays of periodic patterns and Fresnel zone plates. Angle-dependent iridescent colors are produced in these periodic micropatterns because of the Bragg diffraction. Silk protein photolithography can used to form patterns on different substrates including flexible sheets with features of any shape with high fidelity and resolution over large areas. Finally, we show that these mechanically stable and transparent iridescent architectures are also completely biodegradable. This versatile and scalable technique can therefore be used to develop biocompatible, soft micro-optic devices that can be degraded in a controlled manner.

Single cell profiling of surface carbohydrates on Bacillus cereus

Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based ‘recognition force mapping’ as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level.

Real-Time Observation of Antimicrobial Polycation Effects on Escherichia coli: Adapting the Carpet Model for Membrane Disruption to Quaternary Copolyoxetanes.

Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 μg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 μg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 μg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

Morphological and mechanical imaging of Bacillus cereus spore formation at the nanoscale

Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food‐related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterize their surface mechanically. The progression in elasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.

Nanoscale characterization of forensically relevant epithelial cells and surface associated extracellular DNA

Atomic force microscopy provides a novel morphological and physico-chemical perspective to analyze epithelial cell samples in forensic investigations. As a nanoscale, single cell tool, it allows the investigation of scarce samples in a non-destructive fashion. Using chemical force spectroscopy, it permits the identification of specific functional groups or surface molecules. Of specific interest is the presence of extracellular DNA (eDNA) on the surface of epithelial cells that line the exterior skin and interior cavities of human bodies, and can transfer onto surfaces through contact with skin and saliva. To date, this eDNA has only been measured a bulk level. Here, using nanoscale imaging, we first describe the unique differences between keratinized epithelial cells and non-keratinized buccal cells. Then via a force mapping technique, we show how eDNA can be spatially located and quantified on the cell surface. Our results suggest that presence and relative quantity of surface-associated, extracellular DNA signatures can be analyzed on individual epithelial cells from different tissue sources, providing a new tool in the forensic analysis of touch samples.

Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach

In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress-strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Youngs modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials.

Spatial recognition and mapping of proteins using DNA aptamers.

Atomic force microscopy-based adhesion force measurements have emerged as a powerful tool for the biophysical analyses of biological systems. Such measurements can now be extended to detection and mapping of biomolecules on surfaces via integrated imaging and force spectroscopy techniques. Critical to these experiments is the choice of the biomolecular recognition probe. In this study, we demonstrate how oligonucleotide aptamers can be used as versatile probes to simultaneously image and spatially locate targets on surfaces. We focus on two structurally distinct proteins relevant to the clotting cascade - human α-thrombin and vascular endothelial growth factor. Via AFM-recognition mapping using specific DNA aptamers on a commercially available instrument, we show a clear consistency between height and force measurements obtained simultaneously. Importantly, we are able to observe changes in binding due to changes in the external microenvironment, which demonstrate the ability to study fluctuating biological systems in real time. The aptamer specificity and the ability to distinguish their targets are shown through positive and negative controls. It is therefore possible to generate high resolution maps to spatially and temporally identify proteins at the molecular level on complex surfaces.

Nanoscale imaging and hydrophobicity mapping of the antimicrobial effect of copper on bacterial surfaces

Copper has a long historical role in the arena of materials with antimicrobial properties. Various forms of copper ranging from surfaces to impregnation in textiles and particles, have attracted considerable interest owing to their versatility, potency, chemical stability, and low cost. However, the effects and mechanisms of their antimicrobial action is still unclear. In this study, the effect of copper particles on Escherichia coli was studied at the nanoscale using atomic force microscopy (AFM). Time-lapse AFM images at the single cell level show the morphological changes on live E. coli during antimicrobial treatment, in which for the first time, this process was followed in situ on the same cell over time. AFM-based hydrophobicity mapping further showed that incubating cells with Cu decreased the surface hydrophobicity with an increase of incubation time. Specifically, we are able to visualize both morphology and physico-chemical nature of the bacterial cell surface change in response to copper treatment, leading to the membrane damage and cytoplasm leakage. Overall, the time-lapse AFM imaging combined with hydrophobicity mapping approach presented here provides spatio-temporal insight into the antimicrobial mechanisms of copper at the single cell level, and can be applied to design of better metallic antimicrobial materials as well as investigate different microorganisms.