9 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
Accurate Kd via Transient Incomplete Separation
Current methods for finding the equilibrium dissociation constant, Kd, of protein-small molecule complexes have inherent sources of inaccuracy.We introduce “Accurate Kd via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate Kd value. Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding Kd of protein-small molecule complexes.</div
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
Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR)
Direct quantitative analysis of multiple
miRNAs (DQAMmiR) utilizes
CE with fluorescence detection for fast, accurate, and sensitive quantitation
of multiple miRNAs. Here we report on achieving single-nucleotide
specificity and, thus, overcoming a principle obstacle on the way
of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence
specificity is reached by raising the temperature to the level at
which the probe-miRNA hybrids with mismatches melt while the matches
remain intact. This elevated temperature is used as the hybridization
temperature. Practical implementation of this apparently trivial approach
in DQAMmiR has two major challenges. First, melting temperatures of
all mismatched hybrids should be similar to each other and should
not reach the melting temperature of any of the matched hybrids. Second,
the elevated hybridization temperature should not deteriorate CE separation
of the hybrids from the excess probes and the hybrids from each other.
The second problem is further complicated by the reliance of separation
in DQAMmiR on single-strand DNA binding protein (SSB) whose native
structure and binding properties may be drastically affected by the
elevated temperature. These problems were solved by two approaches.
First, locked nucleic acid (LNA) bases were incorporated into the
probes to normalize the melting temperatures of all target miRNA hybrids
allowing for a single hybridization temperature; binding of SSB was
not affected by LNA bases. Second, a dual-temperature CE was developed
in which separation started with a high capillary temperature required
for proper hybridization and continued at a low capillary temperature
required for quality electrophoretic separation of the hybrids from
excess probes and the hybrids from each other. The developed approach
was sufficiently robust to allow its integration with sample preconcentration
by isotachophoresis to achieve a limit of detection below 10 pM
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