A reliable method for attaching biological molecules to layer-by-layer self-assemblies.

A method for the stable attachment of biological molecules (oligonucleotides, proteins and haptens) to layer-by-layer self-assemblies is described.

Cloaking cytolytic peptides for liposome-based detection and potential drug delivery.

Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.

Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

A simple method for preparing spectrally encoded magnetic beads for multiplexed detection.

This report describes a simple method for preparing encoded microspheres for use in multiplexed biological detection. In this method, hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) are assembled on polyamine-coated microspheres in chloroform and encapsulated in an outer shell of silica nanoparticles functionalized with a specific recognition surface. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the microspheres are highly luminescent. The amount of QDs assembled depends only on the surface area of the substrate, and therefore, the photoluminescence intensity increased uniformly in proportion to the number of QD layers assembled. The outer shell of silica nanoparticles confers stability on the assembled QDs but has no effect on their photoluminescence because it is transparent to excitatory and emitted light. It was activated with aminosilane and functionalized with a recognition surface of protein antigens using disulfide exchange chemistry. Magnetic beads furnished with spectral codes of up to three colors of QDs matched to specific recognition surfaces were used as multianalyte sensors for serum proteins.

Electrochemiluminescence enzyme immunoassay for TNT.

Details of an electrochemiluminescence (ECL) enzyme immunoassay for TNT (2,4,6-trinitrotoluene) are reported. The design and construction of a computer controlled flow injection electrochemiluminometer in which the immunoassays are carried out is described. This system is used to select and pump solutions through a flow cell, which contains a gold working electrode as part of a three-electrode arrangement. The deposition of a re-usable immunosorbent dextran surface anchored to a gold surface in the flow cell by chemiadsorbed thiol groups is described. Antibodies are labeled with the enzyme glucose oxidase and used in competitive immunoassays in which the separation step is carried out by concentrating unbound antibodies on the immunosorbent surface. Hydrogen peroxide generated by the enzyme label when glucose is pumped through the flow cell is detected using luminol ECL. Light intensity was inversely proportional to the concentration of TNT in the sample in the range 0-100 ppb. The results are compared with colorimetric ELISA's carried out using the same reagents, and potential for developing a portable instrument for use in the field is discussed.

Electrochemiluminescence enzyme immunoassays for TNT and pentaerythritol tetranitrate.

Electrochemiluminescence enzyme immunoassays for 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN) are described. The latter is, to the best of our knowledge, the first report of an immunoassay for PETN. Haptens corresponding to these explosives were covalently attached to high-affinity dextran-coated paramagnetic beads. The beads were mixed with the corresponding Fab fragments and the sample. After adding a second HRP-labeled antispecies-specific antibody, the mixture was pumped into an electrochemiluminometer where beads were concentrated on the working electrode magnetically. The amount of analyte in the sample was determined by measuring light emission when H2O2 was generated electrochemically in the presence of luminol and an enhancer. The detection limits for TNT and PETN were 0.11 and 19.8 ppb, respectively. Details of bead preparation and performance are given. The increase in sensitivity obtained when Fab fragments are used instead of whole antibodies is explained, and the implications of this observation for nanoparticle-based assays are discussed.