Bionanoscience at LLNL

Science in Support of National Objectives

PLS conducts bionanocience research projects that apply nanoscience and nanotechnology to cutting-edge problems in biophysics, life, and materials science. We focus on developing novel detection methods and platforms for a variety of national security interests ranging from nuclear nonproliferation to biosecurity applications. Our research is focused on developing:

  1. Probe microscopy techniques for biosciences and biosecurity applications
  2. Functional self-assembly in one-dimensional bionanosystems
  3. Carbon nanotube-based membranes for molecular-scale filtration and separation applications
  4. Ultrafast microfluidic mixing devices for protein-folding studies
  5. Ultrasensitive optical spectroscopy and microscopy

1. Probe Microscopy Techniques for Biosciences and Biosecurity Applications

PLS is developing probe microscopy techniques to assemble a nanotechnology toolbox for biosciences and biosecurity applications. We are using probe microscopy techniques to better understand structure-function relationships and the life cycle of microbial and cellular systems. We are also studying the mechanism of biominerization and biologically-inspired fabrication of nanostructures and nanodevices. Our work also entails probing and measuring chemical and biological interactions on a single molecule level with chemical force microscopy.

Structure-Function Relationships and the Life Cycle of Microbial and Cellular Systems

Elucidating the molecular structure and architecture of human pathogen surfaces is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, and environmental resistance so that we can develop countermeasures against bioterrorist agents. We are investigating the architecture, proteomic structure, and function of pathogens through a combination of high-resolution in vitro atomic force microscopy (AFM) and AFM-based immuno-labeling with threat-specific antibodies. This work provides a foundation for identifying structures of pathogens that could lead to the development of vaccines, detection and attribution technologies and improved decontamination systems.

We have demonstrated, using various species of bacterial spores, strikingly different species- and formulation- dependent crystalline structures of the spore coat appear to be a consequence of crystallization mechanisms that regulate the assembly of the spore coat. We also mapped the proteomic structures of cell surfaces and revealed molecular-scale structural dynamics of single germination spores and a cell outgrowth during the germination process. These results could enable the development of targeted pathogen-specific therapeutic countermeasures, diagnostics, bioforensics, and vaccines for pathogen biodefense.

AFM images of spores
Left: High-resolution atomic force microscopy image of the rodlet layer covering the outer coat of Bacillus atrophaeus spore. The scale bar is 50 nm. Right: The development of a dormant Bacillus atrophaeus spore  into a live vegetative cell (grey) was captured with in vitro AFM.


Plomp, M., T. J. Leighton, K. E. Wheeler, H. D. Hill, and A. J. Malkin, “In Vitro High-Resolution Structural Dynamics of Single Germinating Bacterial Spores,” Proc. Natl. Acad. Sci. 104: 9644-9649 (2007).

Plomp, M., T. J. Leighton, K. E. Wheeler, and A. J. Malkin, “The High-Resolution Architecture and Structural Dynamics of Bacillus spores,” Biophys. J. 88: 603-608 (2005).


Mechanism of Biominerization

Understanding of the physical mechanisms by which biological systems use small molecules and macromolecules to control crystallization can provide insights into methods of synthesizing crystalline structures for applications across a wide range of technologies. Moreover, developing this understanding also presents a potential opportunity for creating new strategies towards synthesis of novel therapeutic agents for controlling pathogenic crystallization. For the past decade, we have been combining in situ AFM and molecular modeling to reveal the underlying principles, energetic factors, and stereochemical relationships that enable the biological control of inorganic molecular assembly of various model systems including calcium oxalate monohydrate (COM), a main constituent of human kidney stones. We obtained the first molecular-scale views of COM modification by two urinary constituents—citrate (figure below) and osteopontin—and found that, while both molecules inhibit the growth kinetics and modify growth shape, they do so by attacking different faces on the COM crystals. The results have significant implications for kidney stone disease therapy.

AFM images of spores
Molecular-scale views of calcium oxalate monohydrate (COM) modification by citrate (image size = 6 micrometers). Left: Atomic force microscopy (AFM) image showing COM grows on dislocation hillocks. Center: Molecular modeling reveals that citrate interacts strongly with specific steps on existing crystal face by stereochemical match. Right: AFM image displaying altered morphology due to strong interaction between citrate and COM steps. The growth hillock has been changed from triangular to disc-like shape.


Qiu, S. R., A. Wierzbicki, C. A. Orme, A. M. Cody, J. R. Hoyer, G. H. Nancollas, S. Zepeda, and J. J. De Yoreo, “Molecular Modulation of Calcium Oxalate Crystallization by Osteopontin and Citrate,” Proc. Natl. Acad. Sci. 101, 1811-1815 (2004). (Cover Article)

Qiu, S. R., A. Wierzbicki, E. A. Salter, S. Zepeda, C. A. Orme, J. R. Hoyer, G. H. Nancollas, A. M. Cody, and J. J. De Yoreo, “Modulation of Calcium Oxalate Monohydrate Crystallization by Citrate through Selective Binding to Atomic Steps,” J. Am. Chem. Soc. 127, 9036-9044 (2005).


Biologically-Inspired Fabrication of Nanostructures and Nanodevices

The use of macromolecular scaffolds for hierarchical organization of molecules and materials is a common strategy in living systems. For example, in proteins complexes, micrometer-scale structures are generated from nanometer-scale building blocks possessing high-density functionality. We are mimicking this strategy by creating nanoscale chemical templates to direct the organization of engineered macromolecules and complexes, such as DNA, RNA, proteins, and viruses. These building blocks then serve as scaffolds for the assembly of materials and hierarchical organization of macromolecules such as metallic and semiconductor nanocrystals or artificial-light harvesting complexes. These efforts not only provide well-controlled systems for developing a fundamental understanding of the physical principles governing the macromolecular assembly processes—they also offer exploratory routes to define a new technology for device fabrication of ultradense multicomponent architectures, such as signature-based, chemical and biological sensors that are effective against a wide range of known targets.

AFM images of spores Atomic force microscopy images of biologically driven fabrication on nanostructures on chemical templates.
A. Functionalized alkyl thiol molecules—i.e., maleimide terminated (left) and nitrotriacetic acid (NTA) terminated (right) alkyl thiol—line and dot patterns (line width = ca. 25 nanometers). They are fabricated via nanografting on atomically flat Au substrates.
B. 2D assembly of Cowpea Mosaic Virus (CPMV) on atomically flat mica surfaces.
C. 1D CPMV assembly fully covered on Ni-NTA line patterns fabricated via a route similar to A. The figure shows the single line of CPMV particles.
D. Assembled RNA aptamer catalyzed by hexagonal Pd nanoplates assemble on 2D chemical templates where RNA catalysts are covalently immobilized. TEM inset image shows single hexagonal Pd nanoplate.


Huang, Y., C. Y. Chiang, S. K. Lee, Y. Gao, E. L. Hu, J. J. De Yoreo, and A. M. Belcher, “Programmable Assembly of Nanoarchitectures Using Genetically Engineered Viruses,” Nano Lett. 5, 1429-1434 (2005).

Cheung, C. L., S.-W. Chung, A. Chatterji, T. Lin, J. E. Johnson, S. Hok, J. Perkins, and J. J. De Yoreo, “Directed Self-Assembly of Virus Particles at Chemical Templates,” J.A.C.S. 128, 10801-1807 (2006).


Chemical and Biological Interactions on a Single Molecule Level

We are exploiting the nanoscale precision and manipulation capabilities of atomic force microscopes to measure, characterize, and map nanoscale interactions with chemical force microscopy (CFM). CFM is a scanning probe microscopy technique that uses a tip of a scanning probe microscope modified with a specific chemical functionality to detect and probe specific interactions with surface chemical groups. We are using CFM on a variety of systems ranging from probing interactions of chemical functional groups with single carbon nanotubes to measuring interactions between biological molecules, as well as between biological molecules and cell surfaces. Recent highlights include using CFM to quantify the strength of single and multiple bonds for interactions of multivalent cancer drugs with their targets, and measurement of interactions of a single functional group with a carbon nanotube surface.

AFM images of spores

Left: Chemical force microscopy measurement of the affinity of a multivalent antibody construct to the surface-immobilized targets (MUC1 peptides). Polymer tethers link individual antibody fragments to the AFM tip surface.
Right, top: A representative force vs distance trace showing different parts of the measurement: Cantilever touches the sample surface in region I, pulls away from the surface at region II, ruptures the antibody–protein bond at III, and returns to the undeflected state at IV.
Right, bottom: Dynamic force spectra measured for the rupture of one-, two-, and three-peptide-antibody bonds. These measurements provided the first-ever experimental proof for the prediction of Markovian model of multivalent bond strength (solid lines).


Sulchek, T. A., R. W. Friddle, and A. Noy, “Strength of Multiple Biological Bonds,” Biophys. J. 90, 4686-4691 (2006).

Sulchek, T. A., R. W. Friddle, K. Langry, E. Lau, H. Albrecht, T. V. Ratto, S.J. DeNardo, M. Colvin, and A. Noy, “Dynamic Force Spectroscopy of Parallel Individual M ucin1­Antibody Bonds,” Proc. Natl. Acad. Sci. USA,102, 16638-16643 (2005).


2. Functional Self-Assembly in One Dimensional Bionanosystems

One-dimensional nanoscale materials have unique properties that we can use to create functional devices and nanostructures. These nanostructures could combine material and electronic properties of nanotubes and nanowires with the sophisticated functionality of biological machines. We are concentrating on using carbon nanotubes and silicon nanowires as one-dimensional self-assembly scaffolds to create biomimetic supramolecular structures for potential use as advanced embedded nanoscale sensors and as a broad platform for detection and translation of biological signals.

We have recently created a new bionano architecture, i.e., a one-dimensional lipid bilayer that consists of a functional continuous lipid membrane wrapped around an inorganic nanowire. Our current efforts are focused in the following areas: (1) we are continuing to study the fundamental processes that govern self-assembly in one-dimensional systems, specifically the role of substrate curvature in determining the fundamental properties of the self-assembled lipid and polymer layers; (2) we are working on integrating biological channels in nanotube and nanowire devices with the goal of creating a new generation of biomimetic interfaces for advanced detection technologies.

1D bilayer Left: A scanning confocal microscopy image of a 1D bilayer assembled on a single carbon nanotube.
Right: A schematic representation of a 1D bilayer structure.


Artyukhin, A. B., M. Stadermann, R. W. Friddle, P. Stroeve, O. Bakajin, and A. Noy, “Controlled Electrostatic Gating of Carbon Nanotube FET Devices,” Nano Lett. 6, 2080-2085 (2006).

Huang, S.-C., A. B. Artyukhin, Y. Wang, J.-W. Ju, P. Stroeve, and A. Noy, “Persistence Length Control of the Polyelectrolyte Layer-by-Layer Self-Assembly on Carbon Nanotubes.” J. Am. Chem. Soc. 127, 14176-14177 (2005).


3. Development of Carbon Nanotube-Based Membrane for Filtration and Separation Applications

Molecules traveling through a carbon nanotube Artist's vision of methane molecules traveling through a carbon nanotube.

Carbon nanotubes are an excellent platform for the fundamental studies of transport through channels commensurate with molecular size. Water transport through carbon nanotubes is also believed to be similar to transport in biological channels such as aquaporins.

We have developed a process to microfabricate a membrane with sub-2-nanometer, aligned carbon nanotubes as ideal atomically-smooth pores. The measured gas flow through carbon nanotubes in this membrane exceeds predictions of the Knudsen diffusion model by more than an order of magnitude. The measured water flow exceeded values calculated from continuum hydrodynamics models by more than three orders of magnitude and is comparable to flow rates extrapolated from molecular dynamics simulations and measured for aquaporins.

We are currently investigating the fundamentals of mass transport through carbon nanotubes and exploring applications that exploit these unique nanofluidic phenomena. The extremely high permeabilities of these membranes, combined with their small pore size, may enable energy efficient filtration and eventually decrease the cost of water desalination and of separations of industrial gases and biomolecules. 


Holt, J. K., H. G. Park, Y. Wang, M. Stadermann, A. B. Artyukhin, C. P. Grigoropoulos, A. Noy, and O. Bakajin, “Fast Mass Transport through Sub-2nm Carbon Nanotubes, Science 312, 1034-1037 (2006). (Cover Article)


4. Development of Ultrafast Microfluidic Mixing Devices for Protein Folding Studies

We are developing microfluidic mixers for use in studying protein folding. These mixers allow us to measure protein-folding kinetics at fast timescales using a range of spectroscopic techniques: fluorescence resonance energy transfer (FRET), tryptophan fluorescence, and circular dichroism. By piecing together the complementary information that these techniques provide, we are trying to understand the conformational changes that occur during the first milliseconds of folding.

Using mixers compatible with synchrotron radiation circular dichroism spectroscopy, we studied transiently populated collapsed unfolded proteins. The results indicate a β-structure content of the collapsed unfolded state of about 20% compared to the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, a situation previously found only for highly expanded unfolded proteins.

Schematic of the ultrafast mixer Schematic of the ultrafast mixer

Using mixers made out of fused silica, we demonstrated that the submillisecond protein-folding process referred to as “collapse” actually consists of at least two separate processes. We observed the ultraviolet fluorescence spectrum from naturally occurring tryptophans in three well-studied proteins—cytochrome c, apomyoglobin, and lysozyme—as a function of time in a microfluidic mixer with a dead time of ~20 microseconds. We attributed the first process to hydrophobic collapse and the second process to the formation of the first native tertiary contacts.

Recently designed mixers with a mixing time of 1 ± 1 µs with sample consumption on the order of femtomoles are currently being used for FRET and tryptophan fluorescence studies.


Lapidus, L. J., S. Yao, K. S. McGarrity, D. E. Hertzog, E. Tubman, and O. Bakajin, “Protein Hydrophobic Collapse and Early Folding Steps Observed in a Microfluidic Mixer,” Biophysical Journal 99, 218-224 (2007).

Hoffmann, A., A. Kane, D. Nettels, D. E. Hertzog, P. Baumgärtel, J. Lengefeld , G. Reichardt, D. A. Horsley, R. Seckler, O. Bakajin, and B. Schuler, “Mapping Protein Collapse with Single Molecule Fluorescence and Kinetic Synchrotron Radiation Circular Dichroism Spectroscopy,” Proc. Nat. Acad. Sci. 104,105-110 (2007).


5. Development of Ultrasensitive Optical Spectroscopy and Microscopy

We are developing ultrasensitive optical spectroscopy and microscopy—including single molecule fluorescence spectroscopy, surface-enhanced Raman spectroscopy, and micro-Raman spectroscopy of molecules, biological cells, and crystals—to enable the development of detailed molecular descriptions of cellular processes. There are three theme areas in our research. First, we are studying the structure, function, interactions, and dynamics of the multiprotein machines involved in DNA replication and repair. Using solution-based, single-molecule spectroscopy, we have studied the motion of the polIII β-subunit DNA sliding clamp (“β-clamp”) on DNA and demonstrated that the clamp not only acts as a tether, but also a placeholder.

Second, our goal is to obtain a quantitative description of entire biological networks of interacting molecules and to describe emergent properties of the systems. We are developing the capability to obtain quantitative information on the interactions and dynamics of proteins and study the pathogenicity of selected pathogens in real time and at the single cell level.

Third, we are developing methods for measuring intracellular concentrations of a wide variety of analytes using surface-enhanced Raman scattering from functionalized metallic nanoparticles. Surface-enhanced Raman spectroscopy (SERS) allows sensitive detection of changes in the state of chemical groups attached to single nanoparticles. We have tested a nanoscale pH meter in a cell-free medium, measuring the pH of the solution immediately surrounding the nanoparticles.


 Miller, A. E., A. J. Fischer, T. Laurence, C. W. Hollars, R. J. Saykally, J. C. Lagarias, and T. Huser, “Single-Molecule Dynamics of Phytochrome-Bound Fluorophores Probed by Fluorescence Correlation Spectroscopy,” Proc. Natl. Acad. Sci. USA 103, 11136-11141 (2006).

Contact: Alex Malkin [bio], 925-423-7817,


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Alex Noy

Olgica Bakajin and Alex Noy

New Frontiers

Availability of safe, clean, and inexpensive water has emerged as one of the defining global problems for the 21st century. Water shortages are one of the main obstacles for the continuation of the economic growth, as well as one of the root causes for the societal disruptions such as epidemics, environmental disasters, tribal and ethnic conflicts, political destabilization, and wars. One of the more appealing approaches to mitigating water supply problems could be based on a widespread use of new technological solutions that enable energy-efficient conversion of seawater, brackish water, or polluted water into clean water. Such solutions ultimately must rely on qualitative improvements to the existing membrane technologies. Our group is working on developing such technologies based on our recent discovery of ultrafast water flow in sub-2-nanometer diameter carbon nanotube membranes. Transport measurements in these membranes show that water flows through these pores at least three orders of magnitude faster than the predictions of a classical Hagen-Poiselle slip flow model. Thus, small-pore carbon nanotube membranes are much more efficient water transporters than conventional purification membranes that have much larger pores! We are currently exploring how these unique characteristics of carbon nanotube membranes could be used to develop the next generation of membrane-based solutions for efficient water desalination and purification.

Don Sirbuly

Don Sirbuly

We are developing novel photonic materials capable of multiple complementary sensing modalities for advanced optical components that can be used in portable biological diagnostics and for chemical sensing. Subwavelength semiconductor nanowires transport light at or below the diffraction limit for compact optical detection systems. Since these thin waveguides can carry a substantial amount of the guided optical power in the evanescent field (up to 30%), it is possible to perform absorbance, fluorescence, and surface-enhanced Raman spectroscopy at the sub-10 femtoliter probe volume level. To produce biocompatible optical surfaces for biochemical sensing, we have developed a rapid means of coating the waveguides with functional phospholipid bilayer membranes. These biomimetic membranes allow the nanophotonic materials to be encoded with chemical specificity and lay the groundwork for all-optical in vitro biological monitoring. To promote chemical vapor detection with these fibers, chemo-responsive nanoparticles are submersed within the evanescent field to alter the optical performance of the waveguide upon adsorption to vapor molecules.