ABSTRACT
We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.
Subject(s)
Immunoassay/methods , Immunomagnetic Separation/methods , Magnetics , Nanoparticles/chemistry , Polymerase Chain Reaction/methods , Computer Systems , Nanoparticles/ultrastructureSubject(s)
DNA/chemistry , Nanocomposites/chemistry , Nanoparticles/chemistry , Nucleic Acids/chemistry , Proteins/chemistry , Animals , Antibodies/chemistry , Fluorescent Dyes/pharmacology , Immunoassay , Immunoglobulin G/chemistry , Magnetics , Mice , Microscopy, Electron, Transmission , Nucleic Acid Hybridization , Oligonucleotides/chemistryABSTRACT
The formation and the optical features of two-dimensional aggregates formed by DNA-directed immobilization and cross-linking of bifunctional DNA-gold nanoparticles at flat gold substrates are analyzed. The samples are structurally characterized by atomic force microscopy to evaluate the particle size, the particle densities, and the degree of aggregation. The optical characteristics determined by UV/visible measurements are correlated with the structural features observed.
Subject(s)
DNA/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Base Sequence , Chemistry Techniques, Analytical , Electrophoresis, Agar Gel , Hydrogen-Ion Concentration , Immunoassay/methods , Macromolecular Substances , Nanostructures , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Sensitivity and Specificity , TemperatureABSTRACT
Gold nanoparticles functionalized with thiol-modified single-stranded oligonucleotides are highly useful reagents for a variety of applications, ranging from materials science to bioanalytics. In this chapter, the preparation of citrate stabilized 15-nm Au nanoparticles is described. The nanoparticles are conjugated with 3'-thiol-modified deoxyribonucleic acid oligomers and the resulting conjugates are characterized by determining their shape, size, and surface coverage. The hybridization capabilities are quantified in a microplate assay.
Subject(s)
DNA/chemistry , Gold/chemistry , Nanotechnology/methods , Gold Colloid/chemical synthesis , Microscopy, Electron , Nucleic Acid Hybridization/methods , Oligodeoxyribonucleotides/chemistry , Silver Staining , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , StreptavidinABSTRACT
The DNA-directed self-assembly of surface-bound layers of gold nanoparticles offers a broad range of applications in biomedical analyses as well as in materials science. We here describe a new concept for the assembly of substrate-bound nanoparticle monolayers which employs bifunctional nanoparticles as building blocks, containing two independently addressable DNA oligomer sequences. One of the sequences was utilized for attaching the particle at the solid support, while the other sequence was used to establish cross-links between adjacently immobilized particles. AFM analyses proved the functionality of inter-particle cross-links leading to enhanced surface coverages and the formation of monolayered supramolecular aggregates attached to the substrate. We anticipate that further refinement of this approach will enable applications, for instance, the assembly of ordered layers useful as transducers in biosensing.
Subject(s)
Biosensing Techniques , Coated Materials, Biocompatible/chemistry , DNA/chemistry , Gold/chemistry , Nanotechnology/instrumentation , Nanotechnology/methods , Adsorption , Coated Materials, Biocompatible/chemical synthesis , Macromolecular Substances , Materials Testing , Nucleic Acid Conformation , Particle Size , Surface PropertiesABSTRACT
A new self-assembling peptide-based linker is described. The system comprises three leucine-zipper sequences of de novo design: one peptide, "the belt", templates the co-assembly of the other two-half-sized peptides, "the braces". These basic features were confirmed by circular dichroism spectroscopy and analytical ultracentrifugation: when mixed, the three peptides reversibly formed a predominantly helical and stable 1:1:1 ternary complex. Surface plasmon resonance experiments demonstrated assembly of the complex on gold surfaces, while the ability of the system to bring together peptide-bound cargo was demonstrated using colloidal gold nanoparticles. In the latter experiments, the nanoparticles were derivatized with the brace peptides prior to the addition of the belt. Transmission electron microscopy images of the resulting networks revealed regular approximately 7 nm separations between adjacent particles, consistent with the 42-amino acid helical design of the belt and braces. To our knowledge, belt and braces is a novel concept in leucine-zipper assembly and the first example of employing peptides to guide nanoparticle assembly.