5 research outputs found
Assays for Methionine Ī³āLyase and <i>S</i>āAdenosylālāhomocysteine Hydrolase Based on Enzymatic Formation of CdS Quantum Dots <i>in Situ</i>
<i>S</i>-Adenosyl-l-homocysteine hydrolase
(AHCY)
hydrolyzes its substrate <i>S</i>-adenosyl-l-homocysteine
(AdoHcy) to l-homocysteine (Hcy). Methionine Ī³-lyase
(MGL) catalyzes the decomposition of Hcy to hydrogen sulfide which
forms fluorescent CdS nanoparticles in the presence of CdĀ(NO<sub>3</sub>)<sub>2</sub>. On the basis of these enzymatic reactions, two new
simple and robust fluorogenic enzymatic assays for MGL and AHCY were
developed and applied to detection of AHCY inhibitors
Enzymatic Product-Mediated Stabilization of CdS Quantum Dots Produced <i>In Situ</i>: Application for Detection of Reduced Glutathione, NADPH, and Glutathione Reductase Activity
Glutathione is the most abundant
nonprotein molecule in the cell
and plays an important role in many biological processes, including
the maintenance of intracellular redox states, detoxification, and
metabolism. Furthermore, glutathione levels have been linked to several
human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease,
cardiovascular disease, diabetes mellitus, and cancer. A novel concept
in bioanalysis is introduced and applied to the highly sensitive and
inexpensive detection of reduced glutathione (GSH), over its oxidized
form (GSSG), and glutathione reductase (GR) in human serum. This new
fluorogenic bioanalytical system is based on the GSH-mediated stabilization
of growing CdS nanoparticles. The sensitivity of this new assay is
5 pM of GR, which is 3 orders of magnitude better than other fluorogenic
methods previously reported
Microbead QD-ELISA: Microbead ELISA Using Biocatalytic Formation of Quantum Dots for Ultra High Sensitive Optical and Electrochemical Detection
Electrochemical
detection strategies employing semiconductor quantum
dots (QDs) open up new opportunities for highly sensitive detection
of biological targets. We designed a new assay based on microbead
linked enzymatic generation of CdS QDs (Microbead QD-ELISA) and employed
it in optical and electrochemical affinity assays for the cancer biomarker
superoxide dismutase 2 (SOD2). Biotinylated antibodies against SOD2
were immobilized on the surface of polyvinyl chloride microbeads bearing
streptavidin. In order to prevent any non-specific adsorption the
microbeads were further blocked with bovine serum albumin. The analyte,
SOD2 was captured on microbeads and labeled with alkaline phosphatase-conjugated
antibody linked with mouse antibody against SOD2. Hydrolysis of <i>para</i>-nitrophenylphosphate by immobilized alkaline phosphatase
triggered the rapid formation of phosphate-stabilized CdS QDs on the
surface of microbeads. The resulting semiconductor nanoparticles were
detected by fluorescence spectroscopy, microscopy, and square-wave
voltammetry (SWV). The electrochemical assay based on the detection
with square-wave voltammograms of Cd<sup>2+</sup> ions originating
from immobilized CdS QDs showed linearity up to 45 ng mL<sup>ā1</sup>, and the limit of SOD2 detection equal to 0.44 ng mL<sup>ā1</sup> (1.96 Ć 10<sup>ā11</sup> M). This detection limit is
lower by 2 orders of magnitude in comparison with that of other previously
published assays for superoxide dismutase. The electrochemical assay
was validated with HepG2 (Human hepatocellular carcinoma) cell lysate
containing SOD2
Peroxidase-Mimicking DNAzyme Modulated Growth of CdS Nanocrystalline Structures in Situ through Redox Reaction: Application to Development of Genosensors and Aptasensors
This work demonstrates the use of
the peroxidase-mimicking DNAzyme
(peroxidase-DNAzyme) as general and inexpensive platform for development
of fluorogenic assays that do not require organic fluorophores. The
system is based on the affinity interaction between the peroxidase-DNAzyme
bearing hairpin sequence and the analyte (DNA or low molecular weight
molecule), which changes the folding of the hairpin structure and
consequently the activity of peroxidase-DNAzyme. Hence, in the presence
of the analyte the peroxidase-DNAzyme structure is disrupted and does
not catalyze the aerobic oxidation of l-cysteine to cystine.
Thus, l-cysteine is not removed from the system and the fluorescence
of the assay increases due to the in situ formation of fluorescent
CdS nanocrystals. The capability of the system as a platform for fluorogenic
assays was demonstrated through designing model geno- and aptasensor
for the detection of a tumor marker DNA and a low molecular weight
analyte, adenosine 5ā²triphosphate (ATP), respectively
Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors
The anisotropic morphology
of gold nanorods (AuNRs) has been shown
to lead to nonuniform ligand distribution and preferential etching
through their tips. We have recently demonstrated that this effect
can be achieved by biocatalytic oxidation with hydrogen peroxide,
catalyzed by the enzyme horseradish peroxidase (HRP). We report here
that modification of AuNRs with thiol-containing organic molecules
such as glutathione and thiocholine hinders enzymatic AuNR etching.
Higher concentrations of thiol-containing molecules in the reaction
mixture gradually decrease the rate of enzymatic etching, which can
be monitored by UVāvis spectroscopy through changes in the
AuNR longitudinal plasmon band. This effect can be applied to develop
novel optical assays for acetylcholinesterase (AChE) activity. The
biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine,
which prevents enzymatic AuNR etching in the presence of HRP. Additionally,
the same bioassay can be used for the detection of nanomolar concentrations
of AChE inhibitors such as paraoxon and galanthamine