| Abstrakt: |
Nucleic acid diagnostics is a growing sector in life sciences. Numerous applications based on the amplification of nucleic acids are utilized to determine the presence, composition, quality, or quantity of the sequence in question. However, amplification is a time-consuming, laborious, and error-prone process. Therefore, it would be preferable to perform tests with a minimized number of amplification cycles or, alternatively, completely without the amplification. We present two very sensitive sandwich assay concepts for a measurement of nucleic acids. The assays are performed either in standard microtitration wells or on microparticles coated with capture probes. Detection-probe-coated europium(III) nanoparticle labels are used for signal generation. The detection limits of the microtitration well and microparticle applications were 4.0 × 105 and 6.1 × 104 copies of target sequence, respectively. The reference assay, based on the detection of europium(III) chelate-labelled detection probes, had a detection limit of 8.5 × 107 copies. Thus, 1001000-fold improvement in the sensitivity was achieved. The high sensitivity of the designed assays is based on the long-lifetime fluorescence of nanoparticle labels, on time-resolved fluorometry, and on signal rather than target amplification. The presented sandwich assays provide the tool for a development of direct, amplification-free detection of nucleic acids. [ABSTRACT FROM AUTHOR] |
