Applications of Single-Molecule Vibrational Spectroscopic Techniques for the Structural Investigation of Amyloid Oligomers.

Amyloid oligomeric species, formed during misfolding processes, are believed to play a major role in neurodegenerative and metabolic diseases. Deepening the knowledge about the structure of amyloid intermediates and their aggregation pathways is essential in understanding the underlying mechanisms of misfolding and cytotoxicity. However, structural investigations are challenging due to the low abundance and heterogeneity of those metastable intermediate species. Single-molecule techniques have the potential to overcome these difficulties. This review aims to report some of the recent advances and applications of vibrational spectroscopic techniques for the structural analysis of amyloid oligomers, with special focus on single-molecule studies.

Time-Evolved SERS Signatures of DEP-Trapped Aß and Zn2+Aß Peptides Revealed by a Sub-10 nm Electrode Nanogap.

Alzheimer's disease (AD) has become highly relevant in aging societies, yet the fundamental molecular basis for AD is still poorly understood. New tools to study the undergoing structural conformation changes of amyloid beta (Aß) peptides, the pathogenic hallmark of AD, could play a crucial role in the understanding of the underlying mechanisms of misfolding and cytotoxicity of this peptide. It has been recently reported that Zn2+ interacts with Aß and changes its aggregation pathway away from less harmful fibrillar forms to more toxic species. Here, we present a versatile platform based on a set of sub-10 nm nanogap electrodes for the manipulation and sensing of biomolecules in the physiological condition at a low copy number, where molecules are trapped via dielectrophoresis (DEP) across the nanogap, which also serves as a surface-enhanced Raman spectroscopy hotspot. In this study, we demonstrate that our electrode nanogap platform can be used to study the structural difference between Aß40 and ZnAß40 peptides at different aggregation stages in the physiologically relevant concentration and in solution phase. The Raman spectroscopic signatures of the DEP-captured neuropeptides prove the device to be attractive as a label-free bioanalytical tool.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Frequency modulation of the Min-protein oscillator by nucleoid-associated factors in Escherichia coli.

In E. coli, the Min-protein oscillator, together with the nucleoid occlusion (NO), destabilizes the Z-ring assembly away from the midcell to ensure faithful septation. These two inhibitory pathways are thought to be working independently for division site placement. Even though the Min-protein oscillator has been displayed by synthetic minimal systems, it is unclear the interplays of Min proteins and compartment geometry are sufficient to bolster oscillation stability in vivo. By probing if NO plays a role in the Min oscillation, we study the oscillation frequency in the anucleate and nucleoid-perturbed cells. Surprisingly, we found that the oscillation periods of the Min-protein oscillators were seriously deviated in the anucleate and nucleoid-perturbed cells, but the oscillation frequency either went up in the anucleate or down in the nucleoid-perturbed cells. Intriguingly, enhanced stability and reduced frequency were observed in the cells expressing the NO factor SlmA higher than the native level. Our results reveal an unanticipated role of the nucleoid in modulating the frequency and stability of Min-protein system. SlmA is indicated to facilitate such modulations, potentially via directly interacting with the Min-protein system. A fresh perspective is suggested that frequency modulation of Min-protein oscillator is mediated via the act of nucleoid-associated factors.

Cell Migration in Microfluidic Devices: Invadosomes Formation in Confined Environments.

The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis, allowing the experimentalists to extend their playground to single cells and single molecules, with high throughput and resolution which were inconceivable few decades ago. In particular, microfluidic devices are profoundly changing the conventional way of studying the cell motility and cell migratory dynamics. In this chapter we will furnish a comprehensive view of the advancements made in the research domain of confinement-induced cell migration, thanks to the use of microfluidic devices. The chapter is subdivided in three parts. Each section will be addressing one of the fundamental questions that the microfluidic technology is contributing to unravel: (i) where cell migration takes place, (ii) why cells migrate and, (iii) how the cells migrate. The first introductory part is devoted to a thumbnail, and partially historical, description of microfluidics and its impact in biological sciences. Stress will be put on two aspects of the devices fabrication process, which are crucial for biological applications: materials used and coating methods. The second paragraph concerns the cell migration induced by environmental cues: chemical, leading to chemotaxis, mechanical, at the basis of mechanotaxis, and electrical, which induces electrotaxis. Each of them will be addressed separately, highlighting the fundamental role of microfluidics in providing the well-controlled experimental conditions where cell migration can be induced, investigated and ultimately understood. The third part of the chapter is entirely dedicated to how the cells move in confined environments. Invadosomes (the joint name for podosomes and invadopodia) are cell protrusion that contribute actively to cell migration or invasion. The formation of invadosomes under confinement is a research topic that only recently has caught the attention of the scientific community: microfluidic design is helping shaping the future direction of this emerging field of research.

10 nm deep, sub-nanoliter fluidic nanochannels on germanium for attenuated total reflection infrared (ATR-IR) spectroscopy.

The fabrication of sub-nanoliter fluidic channels is demonstrated, with merely 10 nm depth on germanium, using conventional semiconductor device fabrication methods and a polymer assisted room-temperature sealing method. As a first application, an ultralow volume (650 pL) was studied by ATR-IR spectroscopy. A detection limit of ∼7.9 × 1010 molecules of human serum albumin (HSA) (∼0.2 mM) in D2O was achieved with highly specific ATR-IR spectroscopy.

Microfluidic devices for the study of actin cytoskeleton in constricted environments: Evidence for podosome formation in endothelial cells exposed to a confined slit.

The study of cell behavior in constricted environment is particularly relevant to our understanding of the mechanisms of cell invasion. In this regard, microfluidic systems offer promising platforms as microfabricated fluidic chips provide well-controlled physical, chemical and confined environments to study cell phenotype and behavior. Here, we report a fast and effective manufacturing process of user-friendly microfluidic chips ideally suited for quantitative live cell analysis in combination with immunofluorescence microscopy. The chip body, made of polydimethylsiloxane, is composed of two incubation chambers connected by one rectangular intermediate entry channel which provides access to a series of transversal slits where the observation can be made. The height of the slit is designed to be slightly smaller than that of the cells under study. To validate the chip performance, we analyzed the reorganization of the cytoskeleton of endothelial cells under various degree of spatial confinement. We illustrate how the constricted environment affects endothelial cell behavior in inducing the formation of podosomes. Moreover, the process was stimulated further when the surface of the slit was coated with a thin layer of fibronectin. The study demonstrates the suitability of this technological process for cost-effective fabrication of custom-made single-use chips for biological applications.

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

Low-copy number protein detection by electrode nanogap-enabled dielectrophoretic trapping for surface-enhanced Raman spectroscopy and electronic measurements.

We report a versatile analysis platform, based on a set of nanogap electrodes, for the manipulation and sensing of biomolecules, as demonstrated here for low-copy number protein detection. An array of Ti nanogap electrode with sub-10 nm gap size function as templates for alternating current dielectrophoresis-based molecular trapping, hot spots for surface-enhanced Raman spectroscopy as well as electronic measurements, and fluorescence imaging. During molecular trapping, recorded Raman spectra, conductance measurements across the nanogaps, and fluorescence imaging show unambiguously the presence and characteristics of the trapped proteins. Our platform opens up a simple way for multifunctional low-concentration heterogeneous sample analysis without the need for target preconcentration.

Electrokinetic preconcentration and detection of neuropeptides at patterned graphene-modified electrodes in a nanochannel.

Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions.

Scaling down constriction-based (electrodeless) dielectrophoresis devices for trapping nanoscale bioparticles in physiological media of high-conductivity.

Selective trapping of nanoscale bioparticles (size <100 nm) is significant for the separation and high-sensitivity detection of biomarkers. Dielectrophoresis is capable of highly selective trapping of bioparticles based on their characteristic frequency response. However, the trapping forces fall steeply with particle size, especially within physiological media of high-conductivity where the trapping can be dissipated by electrothermal (ET) flow due to localized Joule heating. Herein, we investigate the influence of device scaling within the electrodeless insulator dielectrophoresis geometry through the application of highly constricted channels of successively smaller channel depth, on the net balance of dielectrophoretic trapping force versus ET drag force on bioparticles. While higher degrees of constriction enable dielectrophoretic trapping of successively smaller bioparticles within a short time, the ETflow due to enhanced Joule heating within media of high conductivity can cause a significant dissipation of bioparticle trapping. This dissipative drag force can be reduced through lowering the depth of the highly constricted channels to submicron sizes, which substantially reduces the degree of Joule heating, thereby enhancing the range of voltages and media conductivities that can be applied toward rapid dielectrophoretic concentration enrichment of silica nanoparticles (∼50 nm) and streptavidin protein biomolecules (∼5 nm). We envision the application of these methodologies toward nanofabrication, optofluidics, biomarker discovery, and early disease diagnostics.

Entropy-driven single molecule tug-of-war of DNA at micro-nanofluidic interfaces.

Entropy-driven polymer dynamics at the nanoscale is fundamentally important in biological systems but the dependence of the entropic force on the nanoconfinement remains elusive. Here, we established an entropy-driven single molecule tug-of-war (TOW) at two micro-nanofluidic interfaces bridged by a nanoslit, performed the force analysis from a modified wormlike chain in the TOW scenario and the entropic recoiling process, and determined the associated scalings on the nanoconfinement. Our results provide a direct experimental evidence that the entropic forces in these two regimes, though unequal, are essentially constant at defined slit heights, irrespective of the slit lengths and the DNA segments within. Our findings have the implications to polymer transport at the nanoscale, device design for single molecule analysis, and biotechnological applications.

Nanoscale molecular traps and dams for ultrafast protein enrichment in high-conductivity buffers.

We report a new approach, molecular dam, to enhance mass transport for protein enrichment in nanofluidic channels by nanoscale electrodeless dielectrophoresis under physiological buffer conditions. Dielectric nanoconstrictions down to 30 nm embedded in nanofluidic devices serve as field-focusing lenses capable of magnifying the applied field to 10(5)-fold when combined with a micro- to nanofluidic step interface. With this strong field and the associated field gradient at the nanoconstrictions, proteins are enriched by the molecular damming effect faster than the trapping effect, to >10(5)-fold in 20 s, orders of magnitude faster than most reported methods. Our study opens further possibilities of using nanoscale molecular dams in miniaturized sensing platforms for rapid and sensitive protein analysis and biomarker discovery, with potential applications in precipitation studies and protein crystallization and possible extensions to small-molecules enrichment or screening.

Nano-constriction device for rapid protein preconcentration in physiological media through a balance of electrokinetic forces.

We describe a methodology to steeply enhance streptavidin protein preconcentration within physiological media over that achieved by negative dielectrophoresis (NDEP) through utilizing a DC offset to the AC field at nanoscale constriction gap devices. Within devices containing approximately 50-nm constriction gaps, we find that the addition of a critical DC field offset (1.5 V/cm) to the NDEP condition (∼200 V(pp) /cm at 1 MHz) results in an exponentially enhanced extent of protein depletion across the device to cause a rapid and steeply rising degree of protein preconcentration. Under these conditions, an elliptical-shaped protein depletion zone that is extended along the device centerline axis forms instantaneously around the constrictions to result in protein preconcentration along the constriction sidewall direction. Through a potential energy diagram to describe the electrokinetic force balance across the device, we find that the potential energy barrier due to NDEP is gradually tilted upon addition of DC fields, to cause successively steeper potential wells along the sidewall direction for devices containing smaller constriction gaps. Hence, for approximately 50-nm constriction gaps at a critical DC field, the ensuing narrow and deep potential energy wells enable steep protein preconcentration, due to depletion over an exponentially enhanced extent across the device.

Self-Searching Writing of Human-Organ-Scale Three-Dimensional Topographic Scaffolds with Shape Memory by Silkworm-like Electrospun Autopilot Jet.

Bioengineered scaffolds satisfying both the physiological and anatomical considerations could potentially repair partially damaged tissues to whole organs. Although three-dimensional (3D) printing has become a popular approach in making 3D topographic scaffolds, electrospinning stands out from all other techniques for fabricating extracellular matrix mimicking fibrous scaffolds. However, its complex charge-influenced jet-field interactions and the associated random motion were hardly overcome for almost a century, thus preventing it from being a viable technique for 3D topographic scaffold construction. Herein, we constructed, for the first time, geometrically challenging 3D fibrous scaffolds using biodegradable poly(ε-caprolactone), mimicking human-organ-scale face, female breast, nipple, and vascular graft, with exceptional shape memory and free-standing features by a novel field self-searching process of autopilot polymer jet, essentially resembling the silkworm-like cocoon spinning. With a simple electrospinning setup and innovative writing strategies supported by simulation, we successfully overcame the intricate jet-field interactions while preserving high-fidelity template topographies, via excellent target recognition, with pattern features ranging from 100's µm to 10's cm. A 3D cell culture study ensured the anatomical compatibility of the so-made 3D scaffolds. Our approach brings the century-old electrospinning to the new list of viable 3D scaffold constructing techniques, which goes beyond applications in tissue engineering.

Ultrasensitive and Low-Cost Paper-Based Graphene Oxide Nanobiosensor for Monitoring Water-Borne Bacterial Contamination.

Water-borne pathogens are mostly generated due to poor sanitation, industrial effluents, and sewage sludge, leading to a significant increase in mortality rate. To prevent this, we need a simple, user-friendly, and rapid on-site detection tool of pathogens, i.e., a biosensor. As contaminated water mainly contains (80%) coliform bacteria, of which Escherichia coli is the major species, we have developed a screen-printed paper-based, label-free biosensor for the detection of E. coli in water. A nanoarchitectured graphene oxide (GO), as a fast electron-transfer flatland, was deposited on the screen-printed graphene (G) on a hydrophobic paper, followed by the immobilization of lectin Concanavalin A (ConA) as a biorecognition element for a GGO_ConA-biosensing electrode. The electrochemical characterization of GGO_ConA shows fast electron transfer with a calculated electroactive surface area of 0.16 cm2. The biosensor performance was tested in the sludge water and beach water (real sample) as an analyte using the electrochemical impedance spectroscopy (EIS) technique. The charge-transfer resistance (Rct) of GGO_ConA increases linearly with the bacterial concentration in the range of 10-108 CFU mL-1 with an estimated limit of detection (LOD) of 10 CFU mL-1, which indicates the ultrasensitivity of our biosensor, with 100 times more sensitivity than previous studies. Our reported biosensor, being cost-effective, eco-friendly, and ultrasensitive, may serve greatly as a portable monitoring kit for checking water-borne bacterial contamination.

Gelatin-polycaprolactone-nanohydroxyapatite electrospun nanocomposite scaffold for bone tissue engineering.

Bone injuries and fractures generally take a long period to heal itself. To address this problem, bone tissue engineering (BTE) has gained significant research impetus. Among the several techniques used for scaffold fabrication, electrospinning ought to be the most promising technique for the development of the nanostructured scaffolds. The present study was carried out to fabricate an electrospun nanocomposite scaffold for BTE by using gelatin, polycaprolactone (PCL), and nanohydroxyapatite (nHAp). To prepare Gelatin-PCL-nHAp nanocomposite scaffold: Gelatin-PCL blend was electrospun and then treated with nHAp (1 wt%) for different time periods. The fabricated nanocomposite scaffold was analysed by field emission scanning electron microscopy (FESEM) to determine the fiber diameter and evaluate the fiber morphology. The Gelatin-PCL-nHAp nanocomposite scaffold-20 min exhibited the average fiber diameter of 615±269 nm and average pore size 4.7±1.04 µm, and also revealed the presence of nHAp particles over the Gelatin-PCL scaffold surface. Further, X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy and thermogravimetric (TG) analysis also indicated the deposition of nHAp over the Gelatin-PCL scaffold surface. MTT assay and DNA quantification showed good viability and significant proliferation of human osteoblasts on Gelatin-PCL-nHAp nanocomposite scaffold. Moreover, cell-scaffold constructs illustrated efficient cellular attachment and adequately spread cells, and it also depicts characteristic polygonal morphology of osteoblasts over the Gelatin-PCL-nHAp nanocomposite scaffold. Thus, the results of in-vitro analysis of electrospun nanocomposite scaffold suggest that the Gelatin-PCL-nHAp scaffold can be a potential candidate for BTE applications.

Novel Self-Directing Single-Polymer Jet Developing Layered-Like 3D Buckled Microfibrous Scaffolds for Tissue Engineering Applications.

Electrospinning is a promising technique for the fabrication of bioscaffolds in tissue engineering applications. Pertaining issues of multiple polymer jets and bending instabilities result in random paths which lend poor controllability over scaffolds morphology for affecting the porosity and mechanical stability. The present study alleviates these challenges by demonstrating a novel self-directing single jet taking a specifically patterned path to deposit fibers into circular and uniform scaffolds without tuning any externally controlled parameters. High-speed camera observation revealed that the charge retention and dissipation on the collected fibers caused rapid autojet switching between the two jetting modes, namely, a microcantilever-like armed jet motion and a whipping motion, which sequentially expand the area and thickness of the scaffolds, respectively, in a layered-like fashion. The physical properties showed that the self-switching dual-jet modes generated multilayered microfibrous scaffolds (MFSs) with dual morphologies and varied fiber packing density, thereby establishing the gradient porosity and mechanical strength (through buckled fibers) in the scaffolds. In vitro studies showed that as-spun scaffolds are cell-permeable hierarchical 3D microporous structures enabling lateral cell seeding into multiple layers. The cell proliferation on days 6 and 9 increased 21% and 38% correspondingly on MFSs than on nanofibrous scaffolds (NFSs) done by conventional multijets electrospinning. Remarkably, this novel and single-step process is highly reproducible and tunable for developing fibrous scaffolds for tissue engineering applications.

Interplay of electrical forces for alignment of sub-100 nm electrospun nanofibers on insulator gap collectors.

We present a quantitative design methodology for optimizing insulator gap width, gap resistivity, and collector to needle height for the alignment of sub-100 nm electrospun nanofibers at insulator gaps of metal collectors. Enhancement of the spatial extent of alignment forces at insulator gaps, due to the concerted action of attractive stretching forces from the modified electric fields and repulsive forces from residual charges on undischarged fibers in the gap, is studied. At gap widths considerably smaller than the collector to needle height (<2%), the spatial extent of stretching forces is large as evidenced by successive reduction in nanofiber size with gap width; however, the low magnitude of repulsive forces limits the degree of nanofiber alignment. At successively larger gap widths less than the needle height, the spatial extent of the stretching forces is gradually restricted toward the metal-insulator edges, while the influence of repulsive forces is gradually extended across the rest of the spatial extent of the gap, to cause enhanced nanofiber alignment through the concerted action of these forces. At gap widths greater than the needle height, the limited spatial extent and lowered maximum value of the stretching forces at the metal-insulator edge reduces their influence on fiber stretching and alignment. The collection of sub-100 nm electrospun poly(lactic acid-co-glycolic acid) nanofibers with a good degree of alignment (≤10° deviation) is found to require intermediate size gaps (∼2% of needle height) of high resistivity (≥10(12) ohm-cm), to enhance the spatial extent of stretching forces while maintaining the dominance of repulsive forces due to residual charge across a majority of the spatial extent of the gap.

Gelatin-alginate-cerium oxide nanocomposite scaffold for bone regeneration.

Worldwide the number of bone damage/fracture, due to traumatic and accidental injuries, has been growing exponentially. Currently available treatments for bone repairing are slow, and often full functional recovery is not achieved. During slow healing process, free radicals are generated at fractured site, which causes further delay in healing process. To overcome these problems, bone tissue engineering (BTE) based approaches, i.e., polymeric scaffolds loaded with free radical scavenging capabilities, seem to be a potential alternative. Cerium oxide nanoparticles (nanoceria, NC) show very good free radical scavenging capabilities. In this study, NC was incorporated in gelatin-alginate (GA) scaffolds to obtain nanocomposite scaffolds (GA-NCs) by freeze drying. Further, the effect of varying nanoceria concentration on the physicochemical and biological properties of the nanocomposite scaffolds has been evaluated. Field emission scanning electron microscopy (FESEM) images of the scaffolds revealed presence of interconnected pores. Furthermore, incorporation of NC has increased the mechanical properties, bio-mineralization, and decreased the swelling and in-vitro weight loss of the scaffolds. Additionally, GA-NCs depicts competent cell attachment, proliferation and viability. The results for osteogenic differentiation studies (i.e. ALP activity, RunX2 and osteocalcin expression) have indicated that GA-NCs scaffolds hold potential to assist differentiation of mesenchymal stem cells (MSCs) to osteoblast. Finally, the results for free radical scavenging functionality demonstrate that GA-NCs are capable of reducing free radicals. Thus, it could be stated that NC incorporated GA nanocomposite scaffold has vital importance for applications in bone tissue-engineering in future regenerative therapies.