3 research outputs found
Improvement of LOD in Fluorescence Detection with Spectrally Nonuniform Background by Optimization of Emission Filtering
The
limit-of-detection (LOD) in analytical instruments with fluorescence
detection can be improved by reducing noise of optical background.
Efficiently reducing optical background noise in systems with spectrally
nonuniform background requires complex optimization of an emission
filter–the main element of spectral filtration. Here, we introduce
a filter-optimization method, which utilizes an expression for the
signal-to-noise ratio (SNR) as a function of (i) all noise components
(dark, shot, and flicker), (ii) emission spectrum of the analyte,
(iii) emission spectrum of the optical background, and (iv) transmittance
spectrum of the emission filter. In essence, the noise components
and the emission spectra are determined experimentally and substituted
into the expression. This leaves a single variable–the transmittance
spectrum of the filter–which is optimized numerically by maximizing
SNR. Maximizing SNR provides an accurate way of filter optimization,
while a previously used approach based on maximizing a signal-to-background
ratio (SBR) is the approximation that can lead to much poorer LOD
specifically in detection of fluorescently labeled biomolecules. The
proposed filter-optimization method will be an indispensable tool
for developing new and improving existing fluorescence-detection systems
aiming at ultimately low LOD
Photosensitized Singlet Oxygen Luminescence from the Protein Matrix of Zn-Substituted Myoglobin
A nanosecond laser near-infrared
spectrometer was used to study
singlet oxygen (<sup>1</sup>O<sub>2</sub>) emission in a protein matrix.
Myoglobin in which the intact heme is substituted by Zn-protoporphyrin
IX (ZnPP) was employed. Every collision of ground state molecular
oxygen with ZnPP in the excited triplet state results in <sup>1</sup>O<sub>2</sub> generation within the protein matrix. The quantum yield
of <sup>1</sup>O<sub>2</sub> generation was found to be equal to 0.9
± 0.1. On the average, six from every 10 <sup>1</sup>O<sub>2</sub> molecules succeed in escaping from the protein matrix into the solvent.
A kinetic model for <sup>1</sup>O<sub>2</sub> generation within the
protein matrix and for a subsequent <sup>1</sup>O<sub>2</sub> deactivation
was introduced and discussed. Rate constants for radiative and nonradiative <sup>1</sup>O<sub>2</sub> deactivation within the protein were determined.
The first-order radiative rate constant for <sup>1</sup>O<sub>2</sub> deactivation within the protein was found to be 8.1 ± 1.3 times
larger than the one in aqueous solutions, indicating the strong influence
of the protein matrix on the radiative <sup>1</sup>O<sub>2</sub> deactivation.
Collisions of singlet oxygen with each protein amino acid and ZnPP
were assumed to contribute independently to the observed radiative
as well as nonradiative rate constants
Accurate MicroRNA Analysis in Crude Cell Lysate by Capillary Electrophoresis-Based Hybridization Assay in Comparison with Quantitative Reverse Transcription-Polymerase Chain Reaction
Accurate
quantitation of microRNA (miRNA) in tissue samples is
required for validation and clinical use of miRNA-based disease biomarkers.
Since sample processing, such as RNA extraction, introduces undesirable
biases, it is advantageous to measure miRNA in a crude cell lysate.
Here, we report on accurate miRNA quantitation in crude cell lysate
by a CE-based hybridization assay termed direct quantitative analysis
of multiple miRNAs (DQAMmiR). Accuracy and precision of miRNA quantitation
were determined for miRNA samples in a crude cell lysate, RNA extract
from the lysate, and a pure buffer. The results showed that the measurements
were matrix-independent with inaccuracies of below 13% from true values
and relative standard deviations of below 11% from the mean values
in a miRNA concentration range of 2 orders of magnitude. We compared
DQAMmiR-derived results with those obtained by a benchmark miRNA-quantitation
method–quantitative reverse transcription-polymerase chain
reaction (qRT-PCR). qRT-PCR-based measurements revealed multifold
inaccuracies and relative standard deviations of up to 70% in crude
cell lysate. Robustness of DQAMmiR to changes in sample matrix makes
it a perfect candidate for validation and clinical use of miRNA-based
disease biomarkers