10 research outputs found
High Sensitivity Luminescence Nanoparticle Assay for the Detection of Protein Aggregation
Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching
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
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
Intracellular Trafficking of Fluorescent Nanodiamonds and Regulation of Their Cellular Toxicity
In this paper, cellular management of fluorescent
nanodiamonds (FNDs) has been studied for better understanding in the
design for potential applications of FNDs in biomedicine. The FNDs
have shown to be photostable probes for bioimaging and thus are well-suited,
for example, long-term tracking purposes. The FNDs also exhibit good
biocompatibility and, in general, low toxicity for cell labeling.
To demonstrate the underlying mechanism of cells coping the low but
potentially toxic effects by nondegradable FNDs, we have studied their
temporal intracellular trafficking. The FNDs were observed to be localized
as distinct populations inside cells in early endosomes, lysosomes,
and in proximity to the plasma membrane. The localization of FNDs
in early endosomes suggests the internalization of FNDs, and lysosomal
localization, in turn, can be interpreted as a prestate for exocytosis
via lysosomal degradation pathway. The endocytosis and exocytosis
appear to be occurring simultaneously in our observations. The mechanism
of continuous endocytosis and exocytosis of FNDs could be necessary
for cells to maintain normal proliferation. Furthermore, 120 h cell
growth assay was performed to verify the long-term biocompatibility
of FNDs for cellular studies
Advanced Cellulose Fibers for Efficient Immobilization of Enzymes
Biocatalytic
pulp fibers were prepared using surface functionalization
of bleached kraft pulp with amino groups (F) and further immobilization
of a cross-linked glucose oxidase (G*) from Aspergillus
niger. The cross-linked enzymes (G*) were characterized
using X-ray spectroscopy, Fourier transform infrared spectroscopy,
dynamic scanning calorimetry, and dynamic light scattering. According
to standard assays, the G* content on the resulting fibers (FG*) was
of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results
from confocal- and stimulated emission depletion microscopy techniques
demonstrated that glucose oxidase do not penetrate the interlayers
of fibers. The benefit of pulp fiber functionalization was evident
in the present case, as the introduction of amino groups allowed the
immobilization of larger amount of enzymes and rendered more efficient
systems. Using the approach described on this paper, several advanced
materials from wood pulp fibers and new bioprocesses might be developed
by selecting the correct enzyme for the target applications
Advanced Cellulose Fibers for Efficient Immobilization of Enzymes
Biocatalytic
pulp fibers were prepared using surface functionalization
of bleached kraft pulp with amino groups (F) and further immobilization
of a cross-linked glucose oxidase (G*) from Aspergillus
niger. The cross-linked enzymes (G*) were characterized
using X-ray spectroscopy, Fourier transform infrared spectroscopy,
dynamic scanning calorimetry, and dynamic light scattering. According
to standard assays, the G* content on the resulting fibers (FG*) was
of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results
from confocal- and stimulated emission depletion microscopy techniques
demonstrated that glucose oxidase do not penetrate the interlayers
of fibers. The benefit of pulp fiber functionalization was evident
in the present case, as the introduction of amino groups allowed the
immobilization of larger amount of enzymes and rendered more efficient
systems. Using the approach described on this paper, several advanced
materials from wood pulp fibers and new bioprocesses might be developed
by selecting the correct enzyme for the target applications
Protein Quantification Using Resonance Energy Transfer between Donor Nanoparticles and Acceptor Quantum Dots
A homogeneous time-resolved luminescence
resonance energy transfer
(TR-LRET) assay has been developed to quantify proteins. The competitive
assay is based on resonance energy transfer (RET) between two luminescent
nanosized particles. Polystyrene nanoparticles loaded with Eu<sup>3+</sup> chelates (EuNPs) act as donors, while protein-coated quantum
dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS
with emission wavelength at 705 nm (QD705-strep), are acceptors. In
the absence of analyte protein, in our case bovine serum albumin (BSA),
the protein-coated QDs bind nonspecifically to the EuNPs, leading
to RET. In the presence of analyte proteins, the binding of the QDs
to the EuNPs is prevented and the RET signal decreases. RET from the
EuNPs to the QDs was confirmed and characterized with steady-state
and time-resolved luminescence spectroscopy. In accordance with the
Fòˆrster theory, the approximate average donor–acceptor
distance is around 15 nm at RET efficiencies, equal to 15% for QD655
and 13% for QD705 acceptor, respectively. The limits of detection
are below 10 ng of BSA with less than a 10% average coefficient of
variation. The assay sensitivity is improved, when compared to the
most sensitive commercial methods. The presented mix-and-measure method
has potential to be implemented into routine protein quantification
in biological laboratories
Fuzzy Liquid Analysis by an Array of Nonspecifically Interacting Reagents: The Taste of Fluorescence
Complex
or unknown liquid analysis requires extensive instrumentation and
laboratory work; simple field devices usually have serious limitations
in functionality, sensitivity, and applicability. This communication
presents a novel, effective, and simple approach to fingerprinting
liquids. The method is based on nonspecific interactions of the sample
liquid, a long lifetime luminescent europium label, and various surface
modulators in an array form that is readily converted to a field analysis
μTAS system. As compared to existing e-nose or e-tongue techniques,
the method is unique both in terms of sensitivity and usability, mainly
due to the well-known unique properties of the europium label. This
communication demonstrates the use of this new method in distinguishing
different wines, waters, alcohols, and artificially modified berry
juices