14 research outputs found
Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods
New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.Siirretty Doriast
Rapid detection of trace amounts of surfactants using nanoparticles in fluorometric assays
High Sensitivity Luminescence Nanoparticle Assay for the Detection of Protein Aggregation
Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching
Lanthanide Label Array Method for Identification and Adulteration of Honey and Cacao
A generic,
cost-effective, and simple method has been developed
to fingerprint liquids to differentiate food brands and ingredients.
The method is based on a label array using nonspecific long lifetime
unstable luminescent lanthanide labels. The interaction between the
liquid sample and the label is typically detrimental to the luminescence
of the unstable chelate leading to a sample-dependent luminescence-intensity
array. The label-array method is a unique approach as the array of
unstable chelates is extremely inexpensive to produce and possesses
high sensitivity due to spectral as well as unstable structural properties
of the lanthanide label. The global method has been applied to distinguish
commercial honey and cacao brands to demonstrate its feasibility as
honey and cacao are among the most adulterated food products
Method for Estimation of Protein Isoelectric Point
Adsorption of sample protein to Eu<sup>3+</sup> chelate-labeled
nanoparticles is the basis of the developed noncompetitive and homogeneous
method for the estimation of the protein isoelectric point (pI). The
lanthanide ion of the nanoparticle surface-conjugated Eu<sup>3+</sup> chelate is dissociated at a low pH, therefore decreasing the luminescence
signal. A nanoparticle-adsorbed sample protein prevents the dissociation
of the chelate, leading to a high luminescence signal. The adsorption
efficiency of the sample protein is reduced above the isoelectric
point due to the decreased electrostatic attraction between the negatively
charged protein and the negatively charged particle. Four proteins
with isoelectric points ranging from ∼5 to 9 were tested to
show the performance of the method. These pI values measured with
the developed method were close to the theoretical and experimental
literature values. The method is sensitive and requires a low analyte
concentration of submilligrams per liter, which is nearly 10000 times
lower than the concentration required for the traditional isoelectric
focusing. Moreover, the method is significantly faster and simpler
than the existing methods, as a ready-to-go assay was prepared for
the microtiter plate format. This mix-and-measure concept is a highly
attractive alternative for routine laboratory work
Luminometric Label Array for Counting and Differentiation of Bacteria
Methods for simple and fast detection and differentiation of bacterial species are required, for instance, in medicine, water quality monitoring, and the food industry. Here, we have developed a novel label array method for the counting and differentiation of bacterial species. This method is based on the nonspecific interactions of multiple unstable lanthanide chelates and selected chemicals within the sample leading to a luminescence signal profile that is unique to the bacterial species. It is simple, cost-effective, and/or user-friendly compared to many existing methods, such as plate counts on selective media, automatic (hemocytometer-based) cell counters, flow cytometry, and polymerase chain reaction (PCR)-based methods for identification. The performance of the method was demonstrated with nine single strains of bacteria in pure culture. The limit of detection for counting was below 1000 bacteria per mL, with an average coefficient of variation of 10% achieved with the developed label array. A predictive model was trained with the measured luminescence signals and its ability to differentiate all tested bacterial species from each other, including members of the same genus Bacillus licheniformis and Bacillus subtilis, was confirmed via leave-one-out cross-validation. The suitability of the method for analysis of mixtures of bacterial species was shown with ternary mixtures of Bacillus licheniformis, Escherichia coli JM109, and Lactobacillus reuteri ATCC PTA 4659. The potential future application of the method could be monitoring for contamination in pure cultures; analysis of mixed bacterial cultures, where examining one species in the presence of another could inform industrial microbial processes; and the analysis of bacterial biofilms, where nonspecific methods based on physical and chemical characteristics are required instead of methods specific to individual bacterial species
Rapid detection of trace amounts of surfactants using nanoparticles in fluorometric assays
Rapid microtiter assays that utilize the time-resolved fluorescenceresonance energy transfer or quenching of dye-labeled proteinsadsorbed onto the surfaces of polystyrene or maghemite nanoparticleshave been developed for the detection and quantification oftrace amounts of surfactants at concentrations down to 10 nM.authorCount :7</p
Simple Nanoparticle-Based Luminometric Method for Molecular Weight Determination of Polymeric Compounds
A nanoparticle-based method utilizing
time-resolved luminescence
resonance energy transfer (TR-LRET) was developed for molecular weight
determination. This mix-and-measure nanoparticle method is based on
the competitive adsorption between the analyte and the acceptor-labeled
protein to donor EuÂ(III) nanoparticles. The size-dependent adsorption
of molecules enables the molecular weight determination of differently
sized polymeric compounds down to a concentration level of micrograms
per liter. The molecular weight determination from 1 to 10 kDa for
polyamino acids and from 0.3 to 70 kDa for polyethylene imines is
demonstrated. The simple and cost-effective nanoparticle method as
microtiter plate assay format shows great potential for the detection
of the changes in molecular weight or for quantification of differently
sized molecules in biochemical laboratories and in industrial polymeric
processes
Sensitive Luminometric Method for Protein Quantification in Bacterial Cell Lysate Based on Particle Adsorption and Dissociation of Chelated Europium
A sensitive and rapid assay for the quantification of
proteins,
based on sample protein adsorption to Eu<sup>3+</sup>-chelate-labeled
nanoparticles, was developed. The lanthanide ion of the surface-conjugated
Eu<sup>3+</sup> chelate is dissociated at a low pH, decreasing the
luminescence signal. The increased concentration of the sample protein
prevents dissociation of the chelate, leading to a high luminescence
signal due to the nanoparticle-bound protein. The assay sensitivity
for the quantification of proteins was 130 pg for bovine serum albumin
(BSA), which is an improvement of nearly 100-fold from the most sensitive
commercial methods. The average coefficient of variation for the assay
of BSA was 8%. The protein-to-protein variability was sufficiently
low; the signal values varied within a 28% coefficient of variation
for nine different proteins. The developed method is relatively insensitive
to the presence of contaminants, such as nonionic detergents commonly
found in biological samples. The existing methods tested for the total
protein quantification failed to measure protein concentration in
the presence of bacterial cell lysate. The developed method quantified
protein also in samples containing insoluble cell components reducing
the need for additional centrifugal assay steps and making the concept
highly attractive for routine laboratory work