RESUMO
We report the first homogeneous sandwich immunoassay with gold nanoparticles (AuNPs) as fluorescence quenchers. The sandwich assay is designed for the detection of the protein cardiac troponin T (cTnT) by its simultaneous interaction with two different antibodies, one attached to AuNPs and the other labeled with fluorescent dyes. We demonstrate the working principle of the assay and using time-resolved fluorescence spectroscopy, we determine the quenching efficiency of the gold nanoparticles. In spite of the relatively large separation distance between dye molecules and AuNPs, ranging from 3 to 22 nm, the AuNPs quench the fluorescence with efficiencies as high as 95%. A limit of detection of 0.02 nM (0.7 ng/mL) was obtained for cTnT, which is the lowest value reported for a homogeneous sandwich assay for cTnT. These results illustrate the use of metallic nanoparticles as fluorescence quenchers in immunoassays where the large biomolecules involved impose distances for which energy transfer between fluorophores would be inefficient.
Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Imunoensaio/métodos , Nanopartículas/química , Nanotecnologia/métodos , Espectrometria de Fluorescência/métodos , Troponina T/análise , Nanopartículas/ultraestruturaRESUMO
We report on a competitive, homogeneous immunoassay for the detection of the hapten digoxigenin. The assay is based on competitive fluorescence quenching by gold nanoparticles. Digoxigenin is indirectly labeled with the fluorophore Cy3B through bovine serum albumin and used as a marker. Gold nanoparticles functionalized with anti-digoxigenin antibodies serve as fluorescence quenchers. Free digoxigenin molecules in the analyte solution compete with the labeled markers for antibodies on the gold nanoparticles. The fluorescence signal depends linearly on the free digoxigenin concentration within a range of concentration from 0.5 to 3 ng mL(-1). The limit of detection is estimated as 0.2 ng mL(-1) and the limit of quantitation is estimated as 0.6 ng mL(-1). The method can be used to detect digoxin, a drug used to cure cardiac arrhythmia.
Assuntos
Digoxigenina/análise , Corantes Fluorescentes/química , Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Animais , Anticorpos/química , Anticorpos/imunologia , Bovinos , Digoxigenina/imunologia , Digoxina/análise , Digoxina/imunologia , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologia , Espectrofotometria UltravioletaRESUMO
We observe an enhancement of fluorescence from a single fluorescent sphere, which is sandwiched between two individual gold nanoparticles, forming a hot spot of strong field enhancement. The fluorescence enhancing hot spot is custom-designed by the deliberate assembly of gold nanoparticles with an atomic force microscope cantilever. The fluorescence intensity is monitored while the separation between the two gold nanoparticles is reduced by gradually pushing the gold nanoparticles closer to the fluorescent sphere. The fluorescence enhancement is maximal when the distance between the two gold nanoparticles is smallest, when the excitation polarization is parallel to the axis of the sandwich, and when the fluorescent sphere is positioned exactly on the axis connecting the two gold nanoparticles.
Assuntos
Cristalização/métodos , Ouro/química , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Espectrometria de Fluorescência/métodos , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Pontos Quânticos , Propriedades de SuperfícieRESUMO
The first application of nanocrystal (NC)-encoded microbeads to clinical proteomics is demonstrated by multiplexed detection of circulating autoantibodies, markers of systemic sclerosis. Two-color complexes, consisting of NC-encoded, antigen-covered beads, anti-antigen antibody or clinical serum samples, and dye-tagged detecting antibodies, were observed using flow cytometry assays and on the surface of single beads. The results of flow cytometry assays correlated with the ELISA technique and provided clear discrimination between the sera samples of healthy donors and patients with autoimmune disease. Microbead fluorescence signals exhibited narrow distribution regardless of their surface antigen staining, without the need of any fluorescence compensation-a parameter determining the limit of sensitivity of flow cytometry assays. In single bead measurements, less than 30 dye-labeled antibodies interacting with the topoI-specific antibodies at the surface of a bead have been detected by the emission of dye excited through the FRET from NCs. In this format, the antibody-bead interaction reaction turns specifically the fluorescence signal from dye label off and on, additionally increasing autoantibody detection sensitivity.
