Correlation between the hierarchical structures and nanomechanical properties of amyloid fibrils.

Amyloid fibrils have recently been highlighted due to their excellent mechanical properties, which not only play a role in their biological functions but also imply their applications in biomimetic material design. Despite recent efforts to unveil how the excellent mechanical properties of amyloid fibrils originate, it has remained elusive how the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils are determined. Here, we characterize the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils using atomic force microscopy experiments and atomistic simulations. It is shown that the hierarchical structure of amyloid fibrils plays a crucial role in determining their radial elastic property but does not make any effect on their bending elastic property. This is attributed to the role of intermolecular force acting between the filaments (constituting the fibril) on the radial elastic modulus of amyloid fibrils. Our finding illustrates how the hierarchical structure of amyloid fibrils encodes their anisotropic nanomechanical properties. Our study provides key design principles of amyloid fibrils, which endow valuable insight into the underlying mechanisms of amyloid mechanics.

Electrochemical detection of high-sensitivity CRP inside a microfluidic device by numerical and experimental studies.

The concentration of C-reactive protein (CRP), a classic acute phase plasma protein, increases rapidly in response to tissue infection or inflammation, especially in cases of cardiovascular disease and stroke. Thus, highly sensitive monitoring of the CRP concentration plays a pivotal role in detecting these diseases. Many researchers have studied methods for the detection of CRP concentrations such as optical, mechanical, and electrochemical techniques inside microfluidic devices. While significant progress has been made towards improving the resolution and sensitivity of detection, only a few studies have systematically analyzed the CRP concentration using both numerical and experimental approaches. Specifically, systematic analyses of the electrochemical detection of high-sensitivity CRP (hsCRP) using an enzyme-linked immunosorbant assay (ELISA) inside a microfluidic device have never been conducted. In this paper, we systematically analyzed the electrochemical detection of CRP modified through the attachment of an alkaline phosphatase (ALP-labeled CRP) using ELISA inside a chip. For this analysis, we developed a model based on antigen-antibody binding kinetics theory for the numerical quantification of the CRP concentration. We also experimentally measured the current value corresponding to the ALP-labeled CRP concentration inside the microfluidic chip. The measured value closely matched the calculated value obtained by numerical simulation using the developed model. Through this comparison, we validated the numerical simulation methods, and the calculated and measured values. Lastly, we examined the effects of various microfluidic parameters on electrochemical detection of the ALP-labeled CRP concentration using numerical simulations. The results of these simulations provide insight into the microfluidic electrochemical reactions used for protein detection. Furthermore, the results described in this study should be useful for the design and optimization of electrochemical immunoassay chips for the detection of target proteins.

Single-step electropolymerization patterning of a polypyrrole nanowire by ultra-short pulses via an AFM cantilever.

Conducting polymers (CPs) have attracted a great deal of attention due to their unique properties; these properties are useful in implementing various functional devices, such as memory, and chemical and biological sensors. In particular, the nanopatterning of CPs is a key technology that will accelerate the adoption of CPs in fabricating nanoscaled multifunctional devices. This paper presents an innovative technique for forming polypyrrole nanowire (PPy-NW) patterns, without any additional pretreatment on the gold surface, using atomic force microscopy (AFM) and ultra-short pulse voltage. Applying the ultra-short pulse voltage to the AFM tip has the following advantage: since the electrochemical current is extremely localized around the tip, the successful formation of CP nanowires results. This is because the pulse width is much shorter than the resistor-capacitor (RC) time constant of the equivalent electrochemical circuit of our experimental set-up. This paper provides systematic results regarding the dimensional variation of the PPy-NW patterns produced by varying the electrical conditions of the ultra-short pulse, such as the pulse amplitude, width, and frequency. The results show that use of an ultra-short pulse is essential in fabricating PPy-NW patterns. Additionally, an ultra-short pulse offers excellent pattern controllability for both width (353 nm ∼ 3.37 µm) and height (2.0 ∼ 88.3 nm).

Different Aggregation Pathways and Structures for Aß40 and Aß42 Peptides.

Self-aggregation of amyloid-ß (Aß) peptides has been known to play a vital role in the onset stage of neurodegenerative diseases, indicating the necessity of understanding the aggregation process of Aß peptides. Despite previous studies on the aggregation process of Aß peptides, the aggregation pathways of Aß isoforms (i.e., Aß40 and Aß42) and their related structures have not been fully understood yet. Here, we study the aggregation pathways of Aß40 and Aß42, and the structures of Aß40 and Aß42 aggregates during the process, based on fluorescence and atomic force microscopy (AFM) experiments. It is shown that in the beginning of aggregation process for both Aß40 and Aß42, a number of particles (i.e., spherical oligomers) are formed. These particles are subsequently self-assembled together, resulting in the formation of different shapes of amyloid fibrils. Our finding suggests that the different aggregation pathways of Aß isoforms lead to the amyloid fibrils with contrasting structure.

Experimental and numerical study of electrochemical nanomachining using an AFM cantilever tip.

We fabricated nanopatterns on Cu thin films via an electrochemical route using an atomic force microscope (AFM). Experimental results were compared with an equivalent electrochemical circuit model representing an electrochemical nanomachining (ECN) technique. In order to precisely construct the nanopatterns, an ultra-short pulse was applied onto the Cu film through the AFM cantilever tip. The line width of the nanopatterns (the lateral dimension) increased with increased pulse amplitude, on-time, and frequency. The tip velocity effect on the nanopattern line width was also investigated. The study described here provides important insight for fabricating nanopatterns precisely using electrochemical methods with an AFM cantilever tip.

Controllable synthesis of single-walled carbon nanotube framework membranes and capsules.

Controlling the morphology of membrane components at the nanometer scale is central to many next-generation technologies in water purification, gas separation, fuel cell, and nanofiltration applications. Toward this end, we report the covalent assembly of single-walled carbon nanotubes (SWNTs) into three-dimensional framework materials with intertube pores controllable by adjusting the size of organic linker molecules. The frameworks are fashioned into multilayer membranes possessing linker spacings from 1.7 to 3.0 nm, and the resulting framework films were characterized, including transport properties. Nanoindentation measurements by atomic force microscopy show that the spring constant of the SWNT framework film (22.6 +/- 1.2 N/m) increased by a factor of 2 from the control value (10.4 +/- 0.1 N/m). The flux ratio comparison in a membrane-permeation experiment showed that larger spacer sizes resulted in larger pore structures. This synthetic method was equally efficient on silica microspheres, which could then be etched to create all-SWNT framework, hollow capsules approximately 5 mum in diameter. These hollow capsules are permeable to organic and inorganic reagents, allowing one to form inorganic nanoparticles, for example, that become entrapped within the capsule. The ability to encapsulate functional nanomaterials inside perm-selective SWNT cages and membranes may find applications in new adsorbents, novel catalysts, and drug delivery vehicles.

Experimental and computational characterization of biological liquid crystals: a review of single-molecule bioassays.

Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.

Atomic force microscopy-based cancer diagnosis by detecting cancer-specific biomolecules and cells.

Atomic force microscopy (AFM) has recently attracted much attention due to its ability to analyze biomolecular interactions and to detect certain biomolecules, which play a crucial role in disease expression. Despite recent studies reporting AFM imaging for the analyses of biomolecules, the application of AFM-based cancer-specific biomolecule/cell detection has remained largely underexplored, especially for the early diagnosis of cancer. In this paper, we review the recent attempts, including our efforts, to analyze and detect cancer-specific biomolecules and cancer cells. We particularly focus on two AFM-based cancer diagnosis techniques: (i) AFM imaging-based biomolecular and cellular detection, (ii) AFM cantilever-based biomolecular sensing and cell analysis. It is shown that AFM-based biomolecular detection has been applied for not only early diagnosing cancer, by measuring the minute amount of cancer-specific proteins, but also monitoring of cancer progression, by correlating the amount of cancer-specific proteins with the progression of cancer. In addition, AFM-based cell imaging and detection have been employed for diagnosing cancer, by detecting cancerous cells in tissue, as well as understanding cancer progression, by characterizing the dynamics of cancer cells. This review, therefore, highlights AFM-based biomolecule/cell detection, which will pave the way for developing a fast and point-of-care diagnostic system for biomedical applications.

Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression.

Matrix metalloproteinase (MMP) is a key marker and target molecule for cancer diagnosis, as MMP is able to cleave peptide chains resulting in degradation of extracellular matrix (ECM), a necessary step for cancer development. In particular, MMP2 has recently been recognized as an important biomarker for lung cancer. Despite the important role of detecting MMP molecules in cancer diagnosis, it is a daunting task to quantitatively understand a correlation between the status of cancer development and the secretion level of MMP in a blood droplet. Here, we demonstrate a nanoscale cancer diagnosis by nanomechanical quantitation of MMP2 molecules under cancer progression with using a blood droplet of lung cancer patients. Specifically, we measured the frequency dynamics of nanomechanical biosensor functionalized with peptide chains mimicking ECM in response to MMP2 secreted from tumors in lung with different metastasis level. It is shown that the frequency shift of the biosensor, which exhibits the detection sensitivity below 1 nM, enables the quantitation of the secretion level of MMP2 molecules during the progression of cancer cells or tumor growth. More importantly, using a blood droplet of lung cancer patients, nanomechanical biosensor is shown to be capable of depicting the correlation between the secretion level of MMP2 molecules and the level of cancer metastasis, which highlights the cantilever-based MMP2 detection for diagnosis of lung cancer. Our finding will broaden the understanding of cancer development activated by MMP and allow for a fast and point-of-care cancer diagnostics.

Highly Selective Photothermal Therapy by a Phenoxylated-Dextran-Functionalized Smart Carbon Nanotube Platform.

Near-infrared (NIR) photothermal therapy using biocompatible single-walled carbon nanotubes (SWNTs) is advantageous because as-produced SWNTs, without additional size control, both efficiently absorb NIR light and demonstrate high photothermal conversion efficiency. In addition, covalent attachment of receptor molecules to SWNTs can be used to specifically target infected cells. However, this technique interrupts SWNT optical properties and inevitably lowers photothermal conversion efficiency and thus remains major hurdle for SWNT applications. This paper presents a smart-targeting photothermal therapy platform for inflammatory disease using newly developed phenoxylated-dextran-functionalized SWNTs. Phenoxylated dextran is biocompatible and efficiently suspends SWNTs by noncovalent π-π stacking, thereby minimizing SWNT bundle formations and maintaining original SWNT optical properties. Furthermore, it selectively targets inflammatory macrophages by scavenger-receptor binding without any additional receptor molecules; therefore, its preparation is a simple one-step process. Herein, it is experimentally demonstrated that phenoxylated dextran-SWNTs (pD-SWNTs) are also biocompatible, selectively penetrate inflammatory macrophages over normal cells, and exhibit high photothermal conversion efficiency. Consequently, NIR laser-triggered macrophage treatment can be achieved with high accuracy by pD-SWNT without damaging receptor-free cells. These smart targeting materials can be a novel photothermal agent candidate for inflammatory disease.

Self-assembled amyloid fibrils with controllable conformational heterogeneity.

Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of ß-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled.

Highly efficient exfoliation of individual single-walled carbon nanotubes by biocompatible phenoxylated dextran.

Highly efficient exfoliation of individual single-walled carbon nanotubes (SWNTs) was successfully demonstrated by utilizing biocompatible phenoxylated dextran, a kind of polysaccharide, as a SWNT dispersion agent. Phenoxylated dextran shows greater ability in producing individual SWNTs from raw materials than any other dispersing agent, including anionic surfactants and another polysaccharide. Furthermore, with this novel polymer, SWNT bundles or impurities present in raw materials are removed under much milder processing conditions compared to those of ultra-centrifugation procedures. There exists an optimal composition of phenoxy groups (∼13.6 wt%) that leads to the production of high-quality SWNT suspensions, as confirmed by UV-vis-nIR absorption and nIR fluorescence spectroscopy. Furthermore, phenoxylated dextran strongly adsorbs onto SWNTs, enabling SWNT fluorescence even in solid-state films in which metallic SWNTs co-exist. By bypassing ultra-centrifugation, this low-energy dispersion scheme can potentially be scaled up to industrial production levels.

Carbon Nanotube-Patterned Surface-Based Recognition of Carcinoembryonic Antigens in Tumor Cells for Cancer Diagnosis.

It has been of high significance to devise a biochemical analytical tool kit enabling the detection of few circulating tumor cells (CTCs) for early diagnosis of cancer. Despite recent effort made to detect few CTCs, it is still challenging to sense such cells with their low concentration and/or the minute amount of marker proteins expressed on few CTCs. In this work, we report the label-free recognition of carcinoembryonic antigens (CEAs) expressed on few CTCs by using a carbon nanotube (CNT) sensor coupled with scanning probe microscopy imaging for cancer diagnosis. It is shown that a CNT-patterned surface is able to specifically capture the CEA molecules in the whole cell lysate of CTCs with their concentration even up to 10(-3) cells/mL. Our work sheds light on our bioassay based on a CNT-patterned surface for highly sensitive, label-free detection of marker proteins expressed on few tumor cells, which may open a new avenue in early diagnosis of cancer by providing a novel biochemical analysis tool kit.

Hyaluronic acid receptor-targetable imidazolized nanovectors for induction of gastric cancer cell death by RNA interference.

We have developed a nanovector consisting of hyaluronic acid (HA) and poly-L-lysine-graft-imidazole (PLI)-based polyplexes containing Bcl-xL-specific shRNA-encoding plasmid DNA (HA/PLI/pDNA) for CD44 targeted gastric cancer therapy. The prepared ternary polyplexes have a negative surface charge of -24 mV and a size of approximately 100 nm at an N/P ratio of 5 with HA/PLI molar ratio of 0.03. Gel electrophoresis and cell viability experiments demonstrated that the ternary polyplexes showed high stability and no cytotoxicity due to the anchored HA molecules on the surface of PLI/pDNA binary polyplexes. Selective cancer cell death was achieved by CD44-mediated gene delivery and the internalized gene was effectively escaped from endosomes due to the buffering capacity of imidazole groups in an acidic environment. These nanovectors may be highly efficient gene delivery tools that allow the selective destruction of metastatic gastric cancer cells.

Nanomechanical actuation driven by light-induced DNA fuel.

We report the reversible nanomechanical actuation of a microcantilever driven by the light irradiation-induced conformational changes of i-motif DNA chains, which are functionalized on the cantilever's surface. It is shown that light irradiation-driven nanomechanical actuation can be manipulated using DNA hybridization and/or ionic concentrations.

Nanomechanical characterization of chemical interaction between gold nanoparticles and chemical functional groups.

We report on how to quantify the binding affinity between a nanoparticle and chemical functional group using various experimental methods such as cantilever assay, PeakForce quantitative nanomechanical property mapping, and lateral force microscopy. For the immobilization of Au nanoparticles (AuNPs) onto a microscale silicon substrate, we have considered two different chemical functional molecules of amine and catecholamine (here, dopamine was used). It is found that catecholamine-modified surface is more effective for the functionalization of AuNPs onto the surface than the amine-modified surface, which has been shown from our various experiments. The dimensionless parameter (i.e., ratio of binding affinity) introduced in this work from such experiments is useful in quantitatively depicting such binding affinity, indicating that the binding affinity and stability between AuNPs and catecholamine is approximately 1.5 times stronger than that between amine and AuNPs. Our study sheds light on the experiment-based quantitative characterization of the binding affinity between nanomaterial and chemical groups, which will eventually provide an insight into how to effectively design the functional material using chemical groups.

Microfluidic multifunctional probe array dielectrophoretic force spectroscopy with wide loading rates.

The simultaneous investigation of a large number of events with different types of intermolecular interactions, from nonequilibrium high-force pulling assays to quasi-equilibrium unbinding events in the same environment, can be very important for fully understanding intermolecular bond-rupture mechanisms. Here, we describe a novel dielectrophoretic force spectroscopy technique that utilizes microsized beads as multifunctional probes for parallel measurement of intermolecular forces with an extremely wide range of force rate (10(-4) to 10(4) pN/s) inside a microfluidic device. In our experiments, various forces, which broadly form the basis of all molecular interactions, were measured across a range of force loading rates by multifunctional probes of various diameters with a throughput of over 600 events per mm(2), simultaneously and in the same environment. Furthermore, the individual bond-rupture forces, the parameters for the characterization of entire energy landscapes, and the effective stiffness of the force spectroscopy were determined on the basis of the measured results. This method of determining intermolecular forces could be very useful for the precise and simultaneous examination of various molecular interactions, as it can be easily and cost-effectively implemented within a microfluidic device for a range of applications including immunoassays, molecular mechanics, chemical and biological screening, and mechanobiology.

Single-molecule recognition of biomolecular interaction via Kelvin probe force microscopy.

We report the scanning probe microscope (SPM)-based single-molecule recognition of biomolecular interactions between protein kinase and small ligands (i.e., ATP and Imatinib). In general, it is difficult to sense and detect the small ligands bound to protein kinase (at single-molecule resolution) using a conventional atomic force microscope (AFM) due to the limited resolution of conventional AFM for detecting the miniscule changes in molecular size driven by ligand binding. In this study, we have demonstrated that Kelvin probe force microscopy (KPFM) is able to articulate the surface potential of biomolecules interacting with ligands (i.e., the protein kinase-ATP interactions and inhibition phenomena induced by antagonistic molecules) in a label-free manner. Furthermore, measured surface potentials for biomolecular interactions enable quantitative descriptions on the ability of protein kinase to interact with small ligands such as ATP or antagonistic molecules. Our study sheds light on KPFM that allows the precise recognition of single-molecule interactions, which opens a new avenue for the design and development of novel molecular therapeutics.

Nanomechanical in situ monitoring of proteolysis of peptide by Cathepsin B.

Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever's overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics.