ACS Applied Bio Materials,
Год журнала:
2024,
Номер
unknown
Опубликована: Дек. 19, 2024
Proteins
are
important
biological
macromolecules
that
perform
a
wide
variety
of
functions
in
the
cell
and
human
body,
can
serve
as
biomarkers
for
early
diagnosis
prognosis
diseases
well
monitoring
effectiveness
disease
treatment.
Hence,
sensitive
accurate
detection
proteins
biospecimens
is
imperative.
However,
at
present,
there
no
ideal
method
available
clinical
samples,
many
which
present
ultralow
(less
than
1
pM)
concentrations
complicated
matrices.
Herein,
we
report
an
ultrasensitive
selective
DNA-assisted
CRISPR-Cas12a
enhanced
fluorescent
assay
(DACEA)
protein
with
limits
reaching
low
attomolar
concentrations.
The
high
sensitivity
was
accomplished
through
combined
DNA
barcode
amplification
(by
using
dual-functionalized
AuNPs)
CRISPR
analysis,
while
selectivity
resistance
to
matrix
effects
our
were
via
formation
protein-antibody
sandwich
structure
specific
recognition
Cas12a
(under
guidance
crRNA)
toward
designed
target
ssDNA.
Given
its
ability
accurately
sensitively
detect
trace
amounts
matrices,
DACEA
platform
pioneered
this
work
has
potential
application
routine
biomarker
testing.
Biosensors and Bioelectronics,
Год журнала:
2025,
Номер
279, С. 117402 - 117402
Опубликована: Март 31, 2025
CRISPR-based
diagnostics
have
gained
increasing
attention
as
biosensing
tools
able
to
address
limitations
in
contemporary
molecular
diagnostic
tests.
To
maximize
the
performance
of
assays,
much
effort
has
focused
on
optimizing
chemistry
and
biology
reaction.
However,
less
been
paid
improving
techniques
used
analyze
data.
date,
decisions
typically
involve
various
forms
slope-based
classification.
Such
methods
are
superior
traditional
based
assessing
absolute
signals,
but
still
limitations.
Herein,
we
establish
benchmarks
(total
accuracy,
sensitivity,
specificity)
using
common
methods.
We
compare
these
benchmark
with
three
different
quadratic
empirical
distribution
function
statistical
tests,
finding
significant
improvements
speed
accuracy
when
applied
a
clinical
data
set.
Two
techniques,
Kolmogorov-Smirnov
Anderson-Darling
report
lowest
time-to-result
highest
total
test
accuracy.
Furthermore,
developed
long
short-term
memory
recurrent
neural
network
classify
CRISPR-biosensing
data,
achieving
100
%
specificity
our
model
Finally,
provide
guidelines
choosing
classification
method
parameters
that
best
suit
assay's
needs.
Nucleic Acids Research,
Год журнала:
2025,
Номер
53(6)
Опубликована: Март 20, 2025
Abstract
Here
we
report
on
the
development
of
a
CRISPR-based
assay
for
sensitive
and
specific
detection
antibodies
antigens
directly
in
complex
sample
matrices.
The
assay,
called
Molecular
Assay
based
antibody-Induced
Guide-RNA
Enzymatic
Transcription
(MAIGRET),
is
use
responsive
synthetic
DNA
template
that
triggers
cell-free
vitro
transcription
guide
RNA
strand
upon
recognition
target
antibody.
Such
transcribed
activates
collateral
activity
Cas12a
enzyme,
leading
to
downstream
cleavage
fluorophore/quencher-labeled
reporter
thus
resulting
an
increase
measured
fluorescence
signal.
We
have
used
MAIGRET
six
different
with
high
sensitivity
(detection
limit
picomolar
range)
specificity
(no
signal
presence
non-target
antibodies).
can
also
be
adapted
competitive
approach
antigens.
With
MAIGRET,
significantly
expand
scope
applicability
sensing
approaches
potentially
enable
measurement
any
molecular
which
antibody
available.
Analytical Chemistry,
Год журнала:
2025,
Номер
unknown
Опубликована: Июнь 5, 2025
Discovery
of
CRISPR/Cas12a
has
revolutionized
broad
fields,
including
gene
editing,
molecular
diagnosis,
and
biosensing.
Flexible
regulation
Cas12a
activity
is
important
for
diverse
applications,
especially
biosensing,
but
it
still
faces
limitations
challenges.
We
find
gold
nanoparticles
(AuNPs)
modified
with
a
single-stranded
DNA
activator
create
huge
steric
barrier
that
strongly
locks
activators
in
one-to-many
manner
inhibits
activity.
This
finding
offers
new
way
to
modulate
the
such
as
designing
versatile
biosensors.
report
spherical
nucleic
acid
(SNA)-modulating
(SNA-Cas)
platform
using
SNAs
signal
translators
target
sensing.
A
stimuli-responsive
SNA
was
constructed
by
modifying
AuNPs
containing
specific
trigger
element,
triggers
reactions
(e.g.,
thiol-exchange
chemical
reaction
RNA-cleaving
DNAzymes)
release
into
solution.
free
initiates
trans-cleavage
CRISPR/Cas12a,
scissoring
fluorescent
reporters
produce
amplified
signals.
To
show
proof
concept,
we
demonstrate
SNA-Cas
sensitively
detect
non-nucleic
targets,
biological
thiol
cysteine,
heavy
metal
Pb2+,
biomarkers
O6-methylguanine-DNA-methyltransferases
(MGMT)
fat
mass
obesity-related
protein
(FTO)
demethylases.
work
opens
one
door
modulating
CRISPR/Cas
shows
great
potential
biosensors
detecting
targets.
ACS Applied Bio Materials,
Год журнала:
2024,
Номер
unknown
Опубликована: Дек. 19, 2024
Proteins
are
important
biological
macromolecules
that
perform
a
wide
variety
of
functions
in
the
cell
and
human
body,
can
serve
as
biomarkers
for
early
diagnosis
prognosis
diseases
well
monitoring
effectiveness
disease
treatment.
Hence,
sensitive
accurate
detection
proteins
biospecimens
is
imperative.
However,
at
present,
there
no
ideal
method
available
clinical
samples,
many
which
present
ultralow
(less
than
1
pM)
concentrations
complicated
matrices.
Herein,
we
report
an
ultrasensitive
selective
DNA-assisted
CRISPR-Cas12a
enhanced
fluorescent
assay
(DACEA)
protein
with
limits
reaching
low
attomolar
concentrations.
The
high
sensitivity
was
accomplished
through
combined
DNA
barcode
amplification
(by
using
dual-functionalized
AuNPs)
CRISPR
analysis,
while
selectivity
resistance
to
matrix
effects
our
were
via
formation
protein-antibody
sandwich
structure
specific
recognition
Cas12a
(under
guidance
crRNA)
toward
designed
target
ssDNA.
Given
its
ability
accurately
sensitively
detect
trace
amounts
matrices,
DACEA
platform
pioneered
this
work
has
potential
application
routine
biomarker
testing.
