Taeyun Kwon

Nanomechanical resonators and their applications in biological/chemical detection: Nanomechanics principles

Abstract Recent advances in nanotechnology have led to the development of nano-electro-mechanical systems (NEMS) such as nanomechanical resonators, which have recently received significant attention from the scientific community. This is not only due to their capability of label-free detection of bio/chemical molecules at single-molecule (or atomic) resolution for future applications such as the early diagnosis of diseases like cancer, but also due to their unprecedented ability to detect physical quantities such as molecular weight, elastic stiffness, surface stress, and surface elastic stiffness for adsorbed molecules on the surface. Most experimental works on resonator-based molecular detection have been based on the principle that molecular adsorption onto a resonator surface increases the effective mass, and consequently decreases the resonant frequencies of the nanomechanical resonator. However, this principle is insufficient to provide fundamental insights into resonator-based molecular detection at the nanoscale; this is due to recently proposed novel nanoscale detection principles including various effects such as surface effects, nonlinear oscillations, coupled resonance, and stiffness effects. Furthermore, these effects have only recently been incorporated into existing physical models for resonators, and therefore the universal physical principles governing nanoresonator-based detection have not been completely described. Therefore, our objective in this review is to overview the current attempts to understand the underlying mechanisms in nanoresonator-based detection using physical models coupled to computational simulations and/or experiments. Specifically, we will focus on issues of special relevance to the dynamic behavior of nanoresonators and their applications in biological/chemical detection: the resonance behavior of micro/nanoresonators; resonator-based chemical/biological detection; physical models of various nanoresonators such as nanowires, carbon nanotubes, and graphene. We pay particular attention to experimental and computational approaches that have been useful in elucidating the mechanisms underlying the dynamic behavior of resonators across multiple and disparate spatial/length scales, and the resulting insight into resonator-based detection that has been obtained. We additionally provide extensive discussion regarding potentially fruitful future research directions coupling experiments and simulations in order to develop a fundamental understanding of the basic physical principles that govern NEMS and NEMS-based sensing and detection applications.

Mechanical Properties of Silicon Nanowires

Nanowires have been taken much attention as a nanoscale building block, which can perform the excellent mechanical function as an electromechanical device. Here, we have performed atomic force microscope (AFM)-based nanoindentation experiments of silicon nanowires in order to investigate the mechanical properties of silicon nanowires. It is shown that stiffness of nanowires is well described by Hertz theory and that elastic modulus of silicon nanowires with various diameters from ~100 to ~600 nm is close to that of bulk silicon. This implies that the elastic modulus of silicon nanowires is independent of their diameters if the diameter is larger than 100 nm. This supports that finite size effect (due to surface effect) does not play a role on elastic behavior of silicon nanowires with diameter of >100 nm.

Micromechanical observation of the kinetics of biomolecular interactions

Resonant microcantilevers have recently enabled the label-free detection of biomolecules. Here, we observed the kinetics of biomolecular interactions such as antigen-antibody interactions and/or DNA hybridization based on a resonant frequency shift, obeying Langmuir kinetics, measured in a buffer solution. It is shown that the kinetics of DNA adsorptions on the surface is governed by intermolecular interactions between adsorbed DNA molecules. It is also shown that the kinetics of DNA hybridization is determined by the intermolecular interaction. It is implied that resonant microcantilever in buffer solution may allow for gaining insights into the kinetics of various molecular interactions.

Mapping the surface charge distribution of amyloid fibril

It is of high importance to measure and map the surface charge distribution of amyloids, since electrostatic interaction between amyloidogenic proteins and biomolecules plays a vital role in amyloidogenesis. In this work, we have measured and mapped the surface charge distributions of amyloids (i.e., β-lactoglobulin fibril) using Kelvin probe force microscopy. It is shown that the surface charge distribution is highly dependent on the conformation of amyloids (e.g., the helical pitch of amyloid fibrils) as well as the pH of a solvent.

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 cantilevers 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.

Real‐Time Quantitative Monitoring of Specific Peptide Cleavage by a Proteinase for Cancer Diagnosis

.…(MMPs;greenmissilesinthepicture)expressedonacancercellsurfacecanbesensedby a resonant cantilever device (satellite arm in the picture), as J. Yang, D. S. Yoon, T.Kwon et al. report in their Communication on page 5837 ff. Active MMPs attack thepeptide sequence that is immobilized on the cantilever surface. The peptide cleavageleads to an increase in the resonant frequency of the cantilever, owing to a decrease inthe mass of immobilized peptide.

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.

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.

Aptamer-functionalized nano-pattern based on carbon nanotube for sensitive, selective protein detection

We have developed a horizontally aligned carbon nanotube sensor that enables not only the specific detection of biomolecules with ultra-sensitivity, but also the quantitative characterization of binding affinity between biomolecules and/or interaction between a carbon nanotube and a biomolecule, for future applications in early diagnostics. In particular, we have fabricated horizontally aligned carbon nanotubes, which were functionalized with specific aptamers that are able to specifically bind to biomolecules (i.e. thrombin). Our detection system is based on scanning probe microscopy (SPM) imaging for horizontally aligned aptamer-conjugated carbon nanotubes (ACNTs) that specifically react with target biomolecules at an ultra-low concentration. It is shown that the binding affinity between thrombin molecule and ACNT can be quantitatively characterized using SPM imaging. It is also found that the smart carbon nanotube sensor coupled with SPM imaging permits us to achieve the high detection sensitivity even up to ∼1 pM, which is much higher than that of other bioassay methods. Moreover, we have shown that our method enables a quantitative study on small molecule-mediated inhibition of specific biomolecular interactions. In addition, we have shown that our ACNT-based system allows for the quantitative study of the effect of chemical environment (e.g. pH and ion concentration) on the binding affinity. Our study sheds light on carbon nanotube sensor coupled with SPM imaging, which opens a new avenue to early diagnostics and drug screening with high sensitivity.