ABSTRACT
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.
Subject(s)
Biosensing Techniques/methods , Gold/chemistry , Immunoassay/methods , Nanoparticles/chemistry , Nanotechnology/methods , Spectrometry, Fluorescence/methods , Troponin T/analysis , Nanoparticles/ultrastructureABSTRACT
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.
Subject(s)
Digoxigenin/analysis , Fluorescent Dyes/chemistry , Gold/chemistry , Immunoassay/methods , Metal Nanoparticles/chemistry , Animals , Antibodies/chemistry , Antibodies/immunology , Cattle , Digoxigenin/immunology , Digoxin/analysis , Digoxin/immunology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Spectrophotometry, UltravioletABSTRACT
In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.
