3 research outputs found
Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots
In this study, a simple fluorogenic
immunoassay based on in situ
formation of semiconductor quantum dots (QDs) is described. We discovered
that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked
immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent
CdS QDs. ALP-catalyzed hydrolysis of <i>p</i>-nitrophenyl
phosphate (pNPP) leads to the formation of <i>p</i>-nitrophenol
and inorganic phosphate. The latter stabilizes CdS QDs produced in
situ through interaction of Cd<sup>2+</sup> with S<sup>2–</sup> ions. So, the specific interaction of analyte (antibody) with ALP-labeled
antibody can be detected through formation of CdS QDs, monitored by
recording emission spectra at λ<sub>ex</sub> = 290 nm. The fluorescence
intensity showed to be dependent on the concentration of target antibody.
This method allowed us to detect as low as 0.4 ng mL<sup>–1</sup> of analyte antibody with a linear range up to 10 ng mL<sup>–1</sup>. The sensitivity of this novel assay showed to be 1 order of magnitude
better than that of the standard method based on colorimetric <i>p</i>-nitrophenyl phosphate assay
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
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 5′triphosphate (ATP), respectively