Haley D. Hill

The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange

The recently developed bio-barcode assay for the detection of nucleic acid and protein targets without PCR has been shown to be extraordinarily sensitive, showing high sensitivity for both nucleic acid and protein targets. Two types of particles are used in the assay: (i) a magnetic microparticle with recognition elements for the target of interest; and (ii) a gold nanoparticle (Au-NP) with a second recognition agent (which can form a sandwich around the target in conjunction with the magnetic particle) and hundreds of thiolated single-strand oligonucleotide barcodes. After reaction with the analyte, a magnetic field is used to localize and collect the sandwich structures, and a DTT solution at elevated temperature is used to release the barcode strands. The barcode strands can be identified on a microarray via scanometric detection or in situ if the barcodes carry with them a detectable marker. The recent modification to the original bio-barcode assay method, utilizing DTT, has streamlined and simplified probe preparation and greatly enhanced the quantitative capabilities of the assay. Here we report the detailed methods for performing the ligand exchange bio-barcode assay for both nucleic acid and protein detection. In total, reagent synthesis, probe preparation and detection require 4 d.*Note: In the version of the article initially published online, incorrect (non-final) versions of the article were posted in both the PDF and HTML formats. The errors have been corrected in all versions of the article.

The role radius of curvature plays in thiolated oligonucleotide loading on gold nanoparticles.

We show that by correlating the radius of curvature of spherical gold nanoparticles of varying sizes with their respective thiol-terminated oligonucleotide loading densities, a mathematical relationship can be derived for predicting the loading of oligonucleotides on anisotropic gold nanomaterials. This mathematical relationship was tested with gold nanorods (radius 17.5 nm, length 475 nm) where the measured number of oligonucleotides per rod (3330 +/- 110) was within experimental error of the predicted loading of 3244 oligonucleotides from the derivation. Additionally, we show that once gold nanoparticles reach a diameter of approximately 60 nm the local surface experienced by the oligonucleotide is highly similar to that of a planar surface.

In vitro high-resolution structural dynamics of single germinating bacterial spores

Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here, we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by NMR. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.

Assembly and organization processes in DNA-directed colloidal crystallization

We present an analysis of the key steps involved in the DNA-directed assembly of nanoparticles into crystallites and polycrystalline aggregates. Additionally, the rate of crystal growth as a function of increased DNA linker length, solution temperature, and self-complementary versus non-self-complementary DNA linker strands (1- versus 2-component systems) has been studied. The data show that the crystals grow via a 3-step process: an initial “random binding” phase resulting in disordered DNA-AuNP aggregates, followed by localized reorganization and subsequent growth of crystalline domain size, where the resulting crystals are well-ordered at all subsequent stages of growth.

Controlling the Lattice Parameters of Gold Nanoparticle FCC Crystals with Duplex DNA Linkers

DNA-functionalized gold nanoparticles can be used to induce the formation and control the unit cell parameters of highly ordered face-centered cubic crystal lattices. Nanoparticle spacing increases linearly with longer DNA interconnect length, yielding maximum unit cell parameters of 77 nm and 0.52% inorganic-filled space for the DNA constructs studied. In general, we show that longer DNA connections result in a decrease in the overall crystallinity and order of the lattice due to greater conformational flexibility.

Three-dimensional hybridization with polyvalent DNA-gold nanoparticle conjugates

We have determined the minimum number of base pairings necessary to stabilize DNA-Au NP aggregates as a function of salt concentration for particles between 15 and 150 nm in diameter. Significantly, we find that sequences containing a single base pair interaction are capable of effecting hybridization between 150 nm DNA-Au NPs. While traditional DNA hybridization involves two strands interacting in one dimension (1D, Z), we propose that hybridization in the context of an aggregate of polyvalent DNA-Au NP conjugates occurs in three dimensions (many oligonucleotides oriented perpendicular to the X, Y plane engage in base pairing), making nanoparticle assembly possible with three or fewer base pairings per DNA strand. These studies enabled us to compare the stability of duplex DNA free in solution and bound to the nanoparticle surface. We estimate that 4-8, 6-19, or 8-33 additional DNA bases must be added to free duplex DNA to achieve melting temperatures equivalent to hybridized systems formed from 15, 60, or 150 nm DNA-Au NPs, respectively. In addition, we estimate that the equilibrium binding constant (K(eq)) for 15 nm DNA-Au NPs (3 base pairs) is approximately 3 orders of magnitude higher than the K(eq) for the corresponding nanoparticle free system.

Chemical tethering of motile bacteria to silicon surfaces.

We chemically immobilized live, motile Escherichia coli on micrometer-scale, photocatalytically patterned silicon surfaces via amine- and carboxylic acid-based chemistries. Immobilization facilitated (i) controlled positioning; (ii) high resolution cell wall imaging via atomic force microscopy (AFM); and (iii) chemical analysis with time-of-flight-secondary ion mass spectrometry (ToF-SIMS). Spinning motion of tethered bacteria, captured with fast-acquisition video, proved microbe viability. We expect our protocols to open new experimental doors for basic and applied studies of microorganisms, from host-pathogen relationships, to microbial forensics and drug discovery, to biosensors and biofuel cell optimization.