Sensors & Diagnostics,
Год журнала:
2024,
Номер
3(8), С. 1310 - 1318
Опубликована: Янв. 1, 2024
MicroRNAs
(miRNAs)
are
short
(about
18-24
nucleotides)
non-coding
RNAs
and
have
emerged
as
potential
biomarkers
for
various
diseases,
including
cancers.
Due
to
their
lengths,
the
specificity
often
becomes
an
issue
in
conventional
amplification-based
methods.
Next-generation
sequencing
techniques
could
be
alternative,
but
long
analysis
time
expensive
costs
make
them
less
suitable
routine
clinical
diagnosis.
Therefore,
it
is
essential
develop
a
rapid,
selective,
accurate
miRNA
detection
assay
using
simple,
affordable
system.
In
this
work,
we
report
CRISPR/Cas13a-based
biosensing
point-of-care
dark-field
(DF)
imaging.
We
utilized
magnetic-gold
nanoparticle
(MGNPs)
complexes
signal
probes,
which
consist
of
200
nm-sized
magnetic
beads
60
gold
nanoparticles
(AuNPs)
linked
by
DNA
hybridization.
Once
CRISPR/Cas13a
system
recognized
target
miRNAs
(miR-21-5p),
activated
Cas13a
cleaved
bridge
linker
containing
RNA
sequences,
releasing
nm-AuNPs
detected
quantified
portable
DF
imaging
The
combination
CRISPR/Cas13a,
MGNPs,
demonstrated
amplification-free
miR-21-5p
within
30
min
at
limit
500
attomoles
(25
pM)
with
single-base
specificity.
CRISPR/Cas13a-assisted
MGNP-DF
achieved
simple
equipment,
thus
providing
application
cancer
Nucleic Acids Research,
Год журнала:
2025,
Номер
53(2)
Опубликована: Янв. 11, 2025
Abstract
We
present
a
robust
‘splice-at-will’
CRISPR
RNA
(crRNA)
engineering
mechanism
that
overcomes
the
limitations
of
clustered
regularly
interspaced
short
palindromic
repeats
(CRISPR)/Cas
system
in
directly
detecting
ultrashort
RNAs.
In
this
strategy,
an
intact
Cas12a
crRNA
can
be
split
from
almost
any
site
spacer
region
to
obtain
truncated
(tcrRNA)
cannot
activate
even
after
binding
auxiliary
DNA
activator.
While
splicing
tcrRNAs
with
moiety
RNA,
formed
combination
work
together
efficiently,
enabling
engineering.
Importantly,
exhibits
same
trans-cleavage
activation
efficiency
as
conventional
crRNA.
Therefore,
by
rationally
designing
activator
conserved
tcrRNA-complementary
sequence
and
arbitrary
RNA-of-interest
recognition
domain,
general
sensing
is
established
utilizes
traditional
DNA-activated
detect
This
strategy
could
faithfully
sequences
6–8
nt,
which
achieved
Cas13a
systems.
Additionally,
through
flexible
design,
our
method
precisely
distinguish
single-base
differences
microRNA
other
sequences.
has
significantly
expanded
Cas12a-based
diagnostic
toolbox
opened
new
avenues
for
detection.
ACS Nano,
Год журнала:
2022,
Номер
16(12), С. 20693 - 20704
Опубликована: Ноя. 15, 2022
Strategies
utilizing
the
CRISPR/Cas
nucleases
Cas13
and
Cas12
have
shown
great
promise
in
development
of
highly
sensitive
rapid
diagnostic
assays
for
detection
pathogenic
nucleic
acids.
The
most
common
approaches
fluorophore-quencher
molecular
beacons
require
strand
amplification
strategies
or
optical
setups
to
overcome
limitations
readout.
Here,
we
demonstrate
a
flexible
strategy
assembling
luminescent
colorimetric
quantum
dot-nucleic
acid
hairpin
(QD-HP)
use
diagnostics.
This
utilizes
chimeric
peptide-peptide
(peptide-PNA)
conjugate
fluorescently
labeled
DNA
RNA
hairpins
ZnS-coated
QDs.
QDs
are
particularly
promising
alternatives
due
their
greater
brightness,
strong
UV
absorbance
with
large
emission
offset,
exceptional
photostability,
potential
multiplexing
sharp
peaks.
Using
Förster
resonance
energy
transfer
(FRET),
developed
ratiometric
reporters
capable
pM
target
(without
nucleotide
amplification)
both
RNA,
further
demonstrated
capabilities
camera-phone
detection.
flexibility
this
system
is
imparted
by
dual
functionality
QD
as
FRET
donor
central
nanoscaffold
arranging
acids
fluorescent
acceptors
on
its
surface.
method
also
provides
generalized
approach
that
could
be
applied
other
nuclease
systems.
Lab on a Chip,
Год журнала:
2023,
Номер
23(6), С. 1467 - 1492
Опубликована: Янв. 1, 2023
Critical
development
of
CRISPR-based
diagnostics
coupled
with
nucleic
acid
amplification
and
amplification-free
techniques;
various
purposes
CRISPR
including
determination,
quantification,
multiplexed
point-of-care
diagnostics.
Critical Reviews in Analytical Chemistry,
Год журнала:
2024,
Номер
unknown, С. 1 - 17
Опубликована: Март 15, 2024
MicroRNA
(miRNA)
has
emerged
as
a
promising
biomarker
for
disease
diagnosis
and
potential
therapeutic
targets
drug
development.
The
detection
of
miRNA
can
serve
noninvasive
tool
in
diseases
predicting
prognosis.
CRISPR/Cas12a
system
great
nucleic
acid
due
to
its
high
sensitivity
specificity,
which
been
developed
be
versatile
acid-based
various
fields.
However,
conversion
from
RNA
DNA
with
or
without
amplification
operation
is
necessary
based
on
system,
because
dsDNA
containing
PAM
sequence
ssDNA
traditionally
considered
the
activator
Cas12a.
Until
recently,
direct
by
reported.
In
this
review,
we
provide
an
overview
evolution
biosensors
indirect
direct,
would
beneficial
development
CRISPR/Cas12a-based
sensors
better
performance
miRNA.
ACS Applied Materials & Interfaces,
Год журнала:
2022,
Номер
14(29), С. 32960 - 32969
Опубликована: Июль 15, 2022
In
this
work,
a
CRISPR/Cas12a
initiated
switchable
ternary
electrochemiluminescence
(ECL)
biosensor
combined
with
Co3O4@Au
nanoemitter
is
presented
for
the
in
vitro
monitoring
of
miRNA-141.
Benefiting
from
advantages
high-throughput
cargo
payload
capability
and
superconductivity,
three-dimensional
reduced
graphene
oxide
(3D-rGO)
was
designated
as
an
introductory
conducting
stratum
paper
working
electrode
(PWE).
With
collaborative
participation
NPs,
transmutation
TPrA
Ru(bpy)32+/TPrA
system
can
be
riotously
expedited
into
exorbitant
free
radical
ions
TPrA•,
which
provoked
exaggeration
ECL
signal.
Moreover,
programmable
enzyme-free
hybrid
chain
reaction
(HCR)
amplifier
on
PWE
surface
accurately
anchored
assembly
nucleic
acid
tandem
accomplished
secondary
recursion
Impressively,
multifunctional
nonspecific
cis/trans-splitting
decomposition
manipulated
photoswitch
"on–off"
signal
state
that
avoided
false-positive
diagnosis.
The
multistrategy
cooperative
demonstrated
extraordinary
sensitivity
specificity,
low
detection
limit
3.3
fM
(S/N
=
3)
concentration
scope
10
to
100
nM,
fully
corresponded
expectation.
Overall,
innovative
methodology
paved
generous
avenue
evaluating
multifarious
biotransformations
provided
tremendous
impetus
development
real-time
diagnosis
clinical
other
biomarkers.
