2 research outputs found
Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection
G-triplex has recently been identified
as a new secondary structure
in G-rich sequences. However, its functions and biological roles remain
largely unknown. This study first developed two kinds of Amplex Red
oxidases, which were based on relatively new G-triplex structure and
a common G-quadruplex one. A collection of DNA binding assays including
circular dichroism (CD) spectroscopy, a CD melting assay, and a UV
titration study were used to determine the G-triplex structure of
G3 oligomer. The low intrinsic oxidative activity of hemin was significantly
enhanced using G-triplex or G-quadruplex. Only one key guanine deletion
from the G3 oligomer or G4 one could result in a much decreased Amplex
Red oxidation activity. To the best of our knowledge, this is the
first case reporting direct use of air as the oxidant for fluorescence
generation based on DNAzyme strategies. Further mechanism studies
demonstrated an involvement of on-site H<sub>2</sub>O<sub>2</sub> generation
from O<sub>2</sub> and water and a following oxidation of Amplex Red
to resorufin, causing a fluorescence enhancement. Furthermore, the
newly developed oxidases have been effectively used in microRNA detection,
using only one biotin-labeled probe and one small-molecule substrate.
The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming
sequence enabled one to produce G-triplex or G-quadruplex by endonuclease
in the presence of a slight amount of miRNA and amplify the signal
of fluorescence from the oxidation of Amplex Red. Our findings of
novel Amplex Red oxidases could potentially be used in a wide range
of applications
An Ultrasensitive Diagnostic Biochip Based on Biomimetic Periodic Nanostructure-Assisted Rolling Circle Amplification
Developing
portable and sensitive devices for point of care detection
of low abundance bioactive molecules is highly valuable in early diagnosis
of disease. Herein, an ultrasensitive photonic crystals-assisted rolling
circle amplification (PCs-RCA) biochip was constructed and further
applied to circulating microRNAs (miRNAs) detection in serum. The
biochip integrated the optical signal enhancement capability of biomimetic
PCs surface with the thousand-fold signal amplification feature of
RCA. The biomimetic PCs displayed periodic dielectric nanostructure
and significantly enhanced the signal intensity of RCA reaction, leading
to efficient improvement of detection sensitivity. A limit of detection
(LOD) as low as 0.7 aM was obtained on the PCs-RCA biochip, and the
LOD was 7 orders of magnitude lower than that of standard RCA. Moreover,
the PCs-RCA biochip could discriminate a single base variation in
miRNAs. Accurate quantification of ultralow-abundance circulating
miRNAs in clinical serum samples was further achieved with the PCs-RCA
biochip, and patients with the nonsmall cell lung carcinoma were successfully
distinguished from healthy donors. The PCs-RCA biochip can detect
bioactive molecules with ultrahigh sensitivity and good specificity,
making it valuable in clinical disease diagnosis and health assessment