David A. LaVan

Label-free immunodetection with CMOS-compatible semiconducting nanowires

Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for the label-free detection of low concentrations of pathogenic microorganisms. Successful solution-phase nanowire sensing has been demonstrated for ions, small molecules, proteins, DNA and viruses; however, ‘bottom-up’ nanowires (or similarly configured carbon nanotubes) used for these demonstrations require hybrid fabrication schemes, which result in severe integration issues that have hindered widespread application. Alternative ‘top-down’ fabrication methods of nanowire-like devices produce disappointing performance because of process-induced material and device degradation. Here we report an approach that uses complementary metal oxide semiconductor (CMOS) field effect transistor compatible technology and hence demonstrate the specific label-free detection of below 100 femtomolar concentrations of antibodies as well as real-time monitoring of the cellular immune response. This approach eliminates the need for hybrid methods and enables system-scale integration of these sensors with signal processing and information systems. Additionally, the ability to monitor antibody binding and sense the cellular immune response in real time with readily available technology should facilitate widespread diagnostic applications.

Nanoparticle-Aptamer Bioconjugates A New Approach for Targeting Prostate Cancer Cells

Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug encapsulated controlled release polymer particles in a cell- or tissue-specific manner. We synthesized a bioconjugate composed of controlled release polymer nanoparticles and aptamers and examined its efficacy for targeted delivery to prostate cancer cells. Specifically, we synthesized poly(lactic acid)-block-polyethylene glycol (PEG) copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH), and encapsulated rhodamine-labeled dextran (as a model drug) within PLA-PEG-COOH nanoparticles. These nanoparticles have the following desirable characteristics: (a) negative surface charge (−50 ± 3 mV, mean ± SD, n = 3), which may minimize nonspecific interaction with the negatively charged nucleic acid aptamers; (b) carboxylic acid groups on the particle surface for potential modification and covalent conjugation to amine-modified aptamers; and (c) presence of PEG on particle surface, which enhances circulating half-life while contributing to decreased uptake in nontargeted cells. Next, we generated nanoparticle-aptamer bioconjugates with RNA aptamers that bind to the prostate-specific membrane antigen, a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. We demonstrated that these bioconjugates can efficiently target and get taken up by the prostate LNCaP epithelial cells, which express the prostate-specific membrane antigen protein (77-fold increase in binding versus control, n = 150 cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced in cells that do not express the prostate-specific membrane antigen protein. To our knowledge, this represents the first report of targeted drug delivery with nanoparticle-aptamer bioconjugates.

Moving smaller in drug discovery and delivery.

Advances in new micro- and nanotechnologies are accelerating the identification and evaluation of drug candidates, and the development of new delivery technologies that are required to transform biological potential into medical reality. This article will highlight the emerging micro- and nanotechnology tools, techniques and devices that are being applied to advance the fields of drug discovery and drug delivery. Many of the promising applications of micro- and nanotechnology are likely to occur at the interfaces between microtechnology, nanotechnology and biochemistry.

Poly(glycerol sebacate) nanofiber scaffolds by core/shell electrospinning.

The novel biomaterial poly(glycerol sebacate) (PGS) holds great promise for tissue engineering and regenerative medicine. PGS is a rubbery, degradable polymer much like elastin; however, it has been limited to cast structures. This work reports on the formation of PGS nanofibers in random non-woven mats for use as tissue engineering scaffolds by coaxial core/shell electrospinning. PGS nanofibers are an inexpensive and synthetic material that mimics the chemical and mechanical environment provided by elastin fibers. Poly(lactide) was used as the shell material to constrain the PGS during the curing process and was removed before cell seeding. Human microvascular endothelial cells from skin (HDMEC) were used to evaluate the in-vitro cellular compatibility of the PGS nanofiber scaffolds. [Figure: see text].

Self-assembled gold nanoparticle molecular probes for detecting proteolytic activity in vivo

Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface-defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5- to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model.

Electromagnetic needles with submicron pole tip radii for nanomanipulation of biomolecules and living cells

We describe the design and fabrication of a temperature-controlled electromagnetic microneedle (EMN) to generate custom magnetic field gradients for biomedical and biophysical applications. An electropolishing technique was developed to sharpen the EMN pole tip to any desired radius between 100 nm and 20 μm. The EMN can be used to apply strong static or dynamic forces (>50nN) to micrometer- or nanometer-sized magnetic beads without producing significant heating or needle movement. Large tip radii (20 μm) allow magnetic force application to multiple magnetic beads over a large area, while small radii (0.1–6 μm) can be used to selectively pull or capture single magnetic beads from within a large population of similar particles. The customizable EMN is thus well suited for micro- and nanomanipulation of magnetic particles linked to biomolecules or living cells.

Three-dimensional conductive constructs for nerve regeneration.

The unique electrochemical properties of conductive polymers can be utilized to form stand-alone polymeric tubes and arrays of tubes that are suitable for guides to promote peripheral nerve regeneration. Noncomposite, polypyrrole (PPy) tubes ranging in inner diameter from 25 microm to 1.6 mm as well as multichannel tubes were fabricated by electrodeposition. While oxidation of the pyrrole monomer causes growth of the film, brief subsequent reduction allowed mechanical dissociation from the electrode mold, creating a stand-alone, conductive PPy tube. Conductive polymer nerve guides made in this manner were placed in transected rat sciatic nerves and shown to support nerve regeneration over an 8-week time period.

Analysis of soft cantilevers as force transducers

High density arrays of polydimethylsiloxane (PDMS) cantilevers to measure basolateral cell forces offer new insight into cell mechanics and motility, but the analysis of the force from displacement data often violates simplifications in the deformation theories. Theoretical and numerical solutions for PDMS cantilever deflection are presented, incorporating both shear strain and large-deflection theory. Classical large-deflection theory was found to be appropriate for PDMS cantilevers with L∕D of 10 or higher, but numerical solutions of large deformation that also incorporate shear strain were necessary when L∕D was smaller than 10.

Structural and biomechanical characteristics of the diaphragmatic tendon in infancy and childhood: an initial analysis

BACKGROUND Engineered tendon grafts have been shown, experimentally, to be promising alternatives for partial diaphragmatic replacement. This study was aimed at determining the cellularity, extracellular matrix composition, and biomechanical characteristics of the diaphragmatic tendon in infants and children to be used as a reference for proper diaphragmatic graft engineering. METHODS The left diaphragmatic tendon was procured at autopsy from 13 patients divided into 2 groups. Group I (n = 9) consisted of newborns and infants. Group II (n = 4) consisted of children and adolescents. Samples underwent quantitative assays for total DNA, glycosaminoglycans, collagen, and elastin contents. Biomechanical measurements included modular and ultimate tensile strength analyses. Statistical comparisons were by the 2-sample Students t test. RESULTS Group I showed significantly higher levels of total DNA, glycosaminoglycans, collagen, and elastin than group II. Conversely, group II tended to have higher modular and ultimate tensile strengths. CONCLUSIONS In neonates and infants, the diaphragmatic tendon has increased cell density and higher levels of major extracellular matrix components than in older children, in whom the diaphragmatic tendon tends to have higher tensile strength. Engineered diaphragmatic constructs should be tailored to the distinct anatomical, functional, and biomechanical characteristics of the diaphragmatic tendon at different age groups.

Optimization of force produced by electromagnet needles acting on superparamagnetic microparticles

The design of an electromagnet needle (EMN) has been numerically optimized based on finite element analysis of the detailed interaction between an EMN and a typical superparamagnetic microparticle. The nonlinear magnetization curves of the core materials and particles are considered; the model calculates the force acting on the particle without far-field simplifications. The EMN optimized for maximum force per unit of heating power generates over 40 000 times the force per unit of heating power of typical optical tweezers. The EMN optimized for a fixed high power level produced 56.7nN, a ten-fold improvement over prior EMNs.