Submicrometer metallic barcodes.

We synthesized multimetal microrods intrinsically encoded with submicrometer stripes. Complex striping patterns are readily prepared by sequential electrochemical deposition of metal ions into templates with uniformly sized pores. The differential reflectivity of adjacent stripes enables identification of the striping patterns by conventional light microscopy. This readout mechanism does not interfere with the use of fluorescence for detection of analytes bound to particles by affinity capture, as demonstrated by DNA and protein bioassays.

Solution and chip arrays in protein profiling.

The industrialization of proteomics demands reproducible, robust and high-throughput profiling technologies that current two-dimensional gel electrophoresis cannot achieve. New technologies in protein arrays, either on chips or with self-encoded elements in solution, hold much promise for interrogating the diverse and immense proteome.

Surface-enhanced Raman scattering: a structure-specific detection method for capillary electrophoresis.

A new approach to detecting capillary electrophoresis (CE) eluent components by interfacing CE with a surface-enhanced Raman scattering (SERS) system is described. In this approach, CE-based separation of a mixture of trans-1,2-bis(4-pyridyl)ethylene and N,N-dimethyl-4-nitrosoaniline has been detected by SERS in a postcolumn geometry. The retention time obtained from SERS corresponds well with that from conventional UV-visible detection. Meanwhile, CE eluants are identified by their characteristic vibrational spectra, demonstrating the validity of SERS as a structure-specific detection method for CE. In addition, the ability to monitor SERS intensity changes at molecule-specific frequencies makes selective detection of individual analytes possible, even when separation is incomplete. Finally, CE-SERS is evaluated for separation of amino acids (tyrosine and tryptophan) and environmental pollutants (chlorophenol mixtures).

Colloidal Au-enhanced surface plasmon resonance immunosensing.

Surface plasmon resonance (SPR) biosensing using colloidal Au enhancement is reported. Immobilization of approximately 11-nm-diameter colloidal Au to an evaporated Au film results in a large shift in plasmon angle, a broadened plasmon resonance, and an increase in minimum reflectance. The incorporation of colloidal Au into SPR biosensing results in increased SPR sensitivity to protein-protein interactions when a Au film-immobilized antibody and an antigen-colloidal Au conjugate comprise the binding pair. A highly specific particle-enhanced analogue of a sandwich immunoassay is also demonstrated by complexing the Au particle to a secondary antibody. A tremendous signal amplification is observed, as addition of the antibody-Au colloid conjugate results in a 25-fold larger signal than that due to addition of a free antibody solution that is 6 orders of magnitude more concentrated. Picomolar detection of human immunoglobulin G has been realized using particle enhancement, with the theoretical limits for the technique being much lower. Finally, a quasi-linear relationship between particle coverage and plasmon angle shift is presented, thereby providing for a direct correlation between plasmon shift and solution antigen concentration. Together, these results represent significant advances in the generality and sensitivity of SPR as it is applied to biosensing.

Mutagenesis of the Mn2+-binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II.

The present study investigates whether compound I and compound II of manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium utilize the same Mn-binding site for catalysis. Manganese peroxidase was expressed from its cDNA in Escherichia coli and refolded from inclusion bodies to yield fully active enzyme. Three mutants of the enzyme were generated by site-directed mutagenesis. Each of the three amino acid residues proposed to be involved in Mn2+ binding, E35, D179, and E39, was mutated. The acidic side chains of E35 and E39 were shortened by one carbon to the acidic group D, and the acidic side chain of D179 was shortened by one carbon to the alkyl group A. These mutants, E35D, D179A, and E39D, were used to determine whether Mn2+ reacts at the same site with both compound I and compound II of manganese peroxidase and to determine whether phenolic substrates for the enzyme react at this site. Our results conclusively demonstrate that E35 and D179 residues are involved not only in Mn2+ binding but also in electron transfer from Mn2+ to the enzyme for both compound I and compound II. In contrast, E39 is not critically important to either process. None of the three residues is involved in reactions with phenolic substrates or with H2O2.

Nanoscale characterization of gold colloid monolayers: a comparison of four techniques.

Atomic force microscopy (AFM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and near-field scanning optical microscopy (NSOM) have been used to characterize the nanostructure of Au colloid-based surfaces. Because these substrates are composed of particles whose dimensions are known prior to assembly, they are well-suited for a critical comparison of the capabilities and limitations of each nanoscale imaging technique. The three criteria for this comparison, which are relevant to the field of nanoparticle assemblies in general, are (i) accuracy in establishing particle size, particle coverage, and interparticle spacing; (ii) accuracy in delineating surface topography; and (iii) ease of sample preparation, data acquisition, and image analysis. For colloidal Au arrays, TEM gives the most reliable size and spacing information but exhibits the greatest constraints with regard to sample preparation; in contrast, AFM is widely applicable but yields data that are the least straightforward to interpret. For accurate information regarding nanometer-scale architecture of particle-based surfaces, a combination of at least one scanning probe method (AFM, NSOM) and one accelerated-electron method (TEM, FE-SEM) is required.

Self-Assembled Metal Colloid Monolayers: An Approach to SERS Substrates.

The self-assembly of monodisperse gold and silver colloid particles into monolayers on polymer-coated substrates yields macroscopic surfaces that are highly active for surface-enhanced Raman scattering (SERS). Particles are bound to the substrate through multiple bonds between the colloidal metal and functional groups on the polymer such as cyanide (CN), amine (NH(2)), and thiol (SH). Surface evolution, which can be followed in real time by ultraviolet-visible spectroscopy and SERS, can be controlled to yield high reproducibility on both the nanometer and the centimeter scales. On conducting substrates, colloid monolayers are electrochemically addressable and behave like a collection of closely spaced microelectrodes. These favorable properties and the ease of monolayer construction suggest a widespread use for metal colloid-based substrates.

Evidence for retention of biological activity of a non-heme iron enzyme adsorbed on a silver colloid: a surface-enhanced resonance Raman scattering study.

The structure and catalytic properties of the enzyme (E) chlorocatechol dioxygenase (CCD) adsorbed on a citrate-reduced silver colloid are analyzed by surface-enhanced resonance Raman spectroscopy (SERRS). This is the first SERRS study of a non-heme metalloenzyme. It is demonstrated that the native conformation of CCD is retained in the adsorbed state by comparison of resonance Raman scattering (RRS) from CCD in solution with SERRS from CCD adsorbed on the silver colloid. Both spectra show clear evidence of vibrational bands typical of iron-tyrosinate proteins. Furthermore, it is demonstrated that adsorbed CCD retains 60-85% of its enzymatic activity in the reaction of catechol substrate (S) with O2 to give the dioxygenated product (P) cis,cis-muconate. This is accomplished by enzyme assays of Ag-adsorbed CCD and comparison of the SERRS of Ag-adsorbed enzyme-substrate (ES) complex under anaerobic conditions with that of Ag-adsorbed ES in the presence of dioxygen. The SERRS difference spectrum, ES(aerobic)--ES(anaerobic), shows clear evidence for the appearance of the vibrational modes of adsorbed product. The analogous SERR difference spectroscopy experiment is also carried out for the enzyme-inhibitor (EI) complex of CCD with tetrachlorocatechol (TCC). Slow turnover of CCD-TCC is observed by SERRS on exposure to dioxygen which is consistent with the slow rate of turnover of TCC by CCD in solution.