Session Y40: Bionanotechnology

Probing Single Molecule Capture By a Solid State Nanopore

We investigate the capture of a single molecule of DNA from solution by a solid state nanopore. We model the motion of DNA near the pore as due to a combination of electrophoretic and thermal forces. We test this model by reversing the driving voltage and recapturing individual molecules soon after they translocate through the nanopore. We find that DNA molecules are drawn to the pore by electric forces over micron scale distances, that the probability of capture decreases linearly with distance from the pore over this distance, and that, in our experimental conditions (120 mV applied voltage, $\sim $5 nm dia pore), molecules enter the pore immediately upon arrival.   

Characterization of DNA molecules through solid-state nanopores

We will report the latest results of our ongoing experiment using solid-state nanopores to characterize DNA molecules assisted by optical tweezers.   

Computer simulations of polymer reversal inside a pore

Translocation of biopolymers through pores is implicated in many biological phenomena. Confinement within pores often breaks ergodicity on biological time scales by creating large entropic barriers to rearrangements of the chain. We study one example of such hindered rearrangement, in which the chain reverses its direction inside a long pore. Our goal is two-fold. First, we study the dependence of the polymer reversal timescale on the pore size and on the polymer length. Second, we examine the ability of simple theories, such as transition state theory (TST) and Kramers' theory to quantitatively describe a transition in a system with a complex energy landscape. We find that one-dimensional TST using the polymer extension along the pore axis as the reaction coordinate adequately accounts for the exponentially strong dependence of the reversal rate on the pore radius $r $and the polymer length $N$, while the transmission factor, i.e., the ratio of the exact rate and the TST one, has a much weaker, power law $r$ and $N$ dependence.   

Refoldable Peptide Barrel -- Carbon Nanotube Junctions

We design hybrid bio-nano-junctions formed by cylindrical peptide structures covalently attached to carbon nanotubes. The cylinders are composed of 5 pairs of antiparallel peptide strands that are ``one-to-one'' matched and covalently bonded through ester and amide bonds to the terminal C atoms in two (20,0) carbon nanotubes. The remaining terminal carbons in the CNTs are replaced by nitrogens, forming embedded quinoline-like structures. The used peptide strands are composed of charged amino acids that form cylindrical patterns with preferred stable configurations. By applying a torque to the nanotubes, we can reversibly fold and control the overall structure of the peptide barrels. The junctions might allow the collection and delivery of drugs and activation of biological molecules attached to them.   

In vitro optical measurements of the interaction between human lung cells and single-wall carbon nanotubes

The intrinsic band gap fluorescence of individual semiconducting single-wall carbon nanotubes (SWNTs) stabilized with single-stranded DNA and deoxycholate surfactant is exploited to optically measure the interaction between human lung cells and length-fractionated SWNTs. Using near-infrared (NIR) fluorescence microscopy in microfluidic flow platforms, live human lung fibroblasts (IMR-90) are exposed to controlled quantities of length-sorted single wall nanotubes, and the cellular interaction and uptake of the SWNTs is optically monitored in real space-time. Cell mortality is shown to result from the uptake of shorter nanotubes and is correlated with both SWNT length and concentration. The NIR optical measurements are used to identify potential uptake mechanisms and quantify the kinetics of the interaction.   

AFM and XPS Studies of Oligodeoxyribonucleotides associated with Single Walled Carbon Nanotubes

Oligodeoxyribonucleotides (ODN) disperse single wall carbon nanotubes (SCWNTs) in aqueous solution \textit{via} sonication.$^{...................[1]}$ By developing procedures that produce ODN:SWCNT hybrids uniformly dispersed in an aqueous solution, new biological applications will emerge. We have studied ODN T30:SWCNT hybrids that form after different preparation techniques. Deposition of the resulting ODN T30:SWCNT hybrids onto both insulating and conducting substrates have been studied. AFM under ambient conditions reveals localized features decorating individual SWCNTs having an approximate height consistent with the dimensions of single stranded T30 ODN. XPS confirmed the decorative features are ODN.$^{...........[2]}$ Taking advantage of the ODN negative charge, we studied the deposition of ODN:SWCNT hybrids on Au substrates using electrodeposition techniques. Electrodeposition has advantages since it does not require fuctionalization of ODN or the substrate prior to deposition. Applying a positive potential to the Au substrate can produce a uniform deposit of T30 ODN:SWCNT hybrids. The electrodeposited ODN:SWCNTs were further studied using AFM and XPS. .[1] M. Zheng et al, \textit{Nat Mater} \textbf{2003}, 2, 338. [2] R. R. Lahiji et al, \textit{Small} \textbf{2007}, 3, 1912.   

Atomic Scale Force Spectroscopy of Protein Active Sites

Isotope-specific nuclear resonances allow X-ray photons to monitor local forces at the atomic scale. We use nuclear resonance vibrational spectroscopy (NRVS) to investigate $^{57}$Fe embedded in protein matrices. Access to the complete spectrum of Fe vibrations allows experimental determination of effective local force constants. The\textit{ stiffness }reflects the force required to displace the probe nucleus with respect to its nearest neighbors and provides a direct probe of local structure. In contrast, the \textit{resilience} describes the force required to displace the probe atom with neighboring atoms free to respond, and determines the thermal fluctuations of the Fe. We find that additional covalent links to the protein increase the resilience of the Fe site in cytochrome c as compared to myoglobin   

The Directed Assembly of Conducting Polymer Nanowires

The Directed Electrochemical Nanowire Assembly (DENA) technique is a single-step approach to fabricating metallic nanowires and interconnecting them with external circuitry or other objects. Here we expand this technique to include the growth of non-metallic wires. From aqueous pyrrole solutions, individual wires were grown from the tip of one electrode across a 30 $\mu $m electrode gap and into contact with the tip of the other electrode. Energy dispersive spectroscopy is used to show that the wires are composed of doped polypyrrole. The conductance of these nanowires will be discussed, as well as the interfacing of DENA nanowires with biological cells for cell stimulation studies.   

Directed Electrochemical Nanowire Assembly

\textit{Directed Electrochemical Nanowire Assembly} (DENA) technique is a single-step approach to fabricating metallic nanowires and interconnecting them with external circuitry. We have previously shown that these are near single crystalline metallic nanowires and that they interconnect with on-chip electrodes with very small contact resistances. Here we discuss the user-directed growth of these nanowires up to inter-electrode targets, such as biological cells. Recent results on the delivery of small voltages to these targets will be discussed, as well.   

Electrochemical measurement of DNA in a nanofluidic channel

The elongation of genomic length DNA in confining nanochannels is not only a fascinating exercise in polymer dynamics, but also is of great interest in biotechnology because the elongation of the confined molecule is directly proportional to the actual length of the molecule in basepairs. We will present a way to construct nanochannels using sacrificial PMMA ebeam lithography and to measure non-immobilized DNA molecules inside such a channel electrochemically. This kind of measurements can lead us to fast and precise electronic length measurement, which will open the door to a number of important areas in genomics such as gene exchange and evolution dynamics of single cells.   

A nanofluidic lateral Coulter counter

Optical detection has been the mainstay of detection in many micro-and nanofluidic systems. However, the need for labeling and alignment of detectors or registration of images has slowed transition to application and increased cost. On the other hand, when traditional Coulter counters that detect along the axis of a fluidic channel are integrated with nanofluidics on a chip, leakage and limited resolution often present problems. For that reason, we are developing an electronic detection mechanism that is based on the impedance change of an electrode pair that is laterally integrated with a nanochannel. We will present detection of 50 nm polystyrene beads, and will discuss possible applications in the detection of biological molecules such as DNA.   

On the virus capsid assembly and encapsulation of foreign materials

Icosahedral virus capsids are one of the simplest biological structures, yet poorly understood from a molecular physics point of view, e.g., the paradox between its stability and flexibility, its interaction with the virus genome, and its assembly thermodynamics and kinetics. In the hope of elucidating these problems, we have created a study platform based on virus-like particles (VLPs) -- inorganic nanoparticles encapsulated inside icosahedral virus capsids in place of their genomic cores. These nanoparticles were successfully incorporated when coated with carboxylate-terminated poly-ethyleneglycols, implying a dominating electro-static interaction between the virus capsid and the core. The nano-particle size determines the T number of the icosahedral cage and the efficiency of encapsulation. The current work seeks to understand the mechanism of capsid assembly process using VLP as a model system. Empty capsid assembly kinetics has been also studied for comparison. A simplified kinetically limiting model based on a previously reported master equation model is proposed.   

Assembly and Characterization of NanoComplexes: Quantum Dot Encapsulated Liposomes

Liposome complexes have received significant attention for a variety of biochemical and biomedical applications including drug targeting and drug delivery and tumor imaging and diagnostics. Semiconductor nano-crystals, also known as quantum dots, are now beginning to be used in similar biochemical experiments. Like fluorescent dyes, these quantum dots have the ability to reliably fluoresce at pre-engineered wavelengths. However, these nano-crystals have lifetimes significantly longer comparable to fluorescence dye counterparts. We have successfully encapsulated approximately 10nm CdSe nano-crystals inside approximately 100nm liposomes and studied the resulting complex using fluorescence resonance energy transfer (FRET) microscopy. Further studies were performed using transmission electron microscopy (TEM) showing the details of the encapsulation, and Raman spectroscopy to examine their structural details. Our nano-manufactured quantum dot liposome complexes do not bleach over periods of hours and are general enough to allow the addition of drugs targeted for the vectored cells thus offering the ability to both image and medicate simultaneously over a long period of time.   

Selective Cell Growth on Fibronectin-Carbon Nanotube Hybrid Nanostructures

Carbon nanotubes (CNT) have been considered a promising material for biological applications including biosensors, therapeutic application, and nano-structured scaffolds. However, there are still controversies associated with toxicity and biocompatibility of CNTs on live cells. Here, we report general strategy to functionalize CNTs with cell adhesion molecules (fibronectins) for selective and stable adhesion of cells on CNTs. Interestingly, more fibronectins were adsorbed and activated on CNTs rather than on hydrophobic self assembled monolayers (SAMs) or bare substrates (SiO$_{2})$. We demonstrate the functionality of fibronectins on CNTs with immunofluorescence and molecule-level force measurement study using atomic force microscopy (AFM). These fibronectin-CNT hybrid nanostructures were successfully applied to attract cells selectively onto predefined regions on the substrate. Our strategy was generally available on various cell types including mesenchymal stem cells, KB cells, and NIH3T3 fibroblast cells (\textit{Advanced Materials} \textbf{19,} 2530-2534 (2007)). We will also discuss about its impacts on cell biology combined with CNTs.   

Large-Scale `Linker-Free Assembly' of swCNT-Based Biosensor Arrays

Biosensors based on single-walled carbon nanotubes (swCNTs) have received a great deal of attention due to their potential applications such as genotyping, disease diagnosis, food analysis, etc. However, a lack of reliable mass-production method for such swCNT-based biosensor has been holding back their practical applications. One promising mass-production method for swCNT-based biosensor arrays can be \textit{`linker-free assembly'} process (Nature Nanotechnology 1, 66 (2006)), where non-polar patterns guide the \textit{`selective assembly'} and \textit{`precision alignment'} of carbon nanotubes on bare substrates without using any external forces such as liquid flow, etc. We used this method to fabricate large-scale assembly of swCNT-based integrated devices on virtually general substrates including SiO$_{2}$, Si, Al, Au, etc. To utilize swCNT devices for biosensors, we functionalized swCNT devices on SiO$_{2}$ with receptor biomolecules such as enzyme L-glutamate oxidase or biotin. And then, we could detect the target biomolecules (L-glutamate or streptavidin, respectively) with high sensitivity and selectivity by monitoring the conductance change of swCNT junctions in aqueous environment. These studies provide biological implications on neurotransmitters and proteins onto swCNT patterned surface.