ACS Applied Bio Materials,
Journal Year:
2024,
Volume and Issue:
unknown
Published: Dec. 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.
Journal of Nucleic Acids,
Journal Year:
2024,
Volume and Issue:
2024(1)
Published: Jan. 1, 2024
The
development
of
sensitive
and
specific
diagnostic
tools
for
hepatitis
B
virus
(HBV)
C
(HCV)
remains
crucial
effective
disease
management
control.
In
this
study,
we
utilized
CRISPR‐Cas12
CRISPR‐Cas13
systems
the
detection
HBV
(DNA
virus)
HCV
(RNA
virus),
respectively.
We
designed
tested
multiple
guide
RNAs
(gRNAs)
targeting
both
viruses,
confirming
successful
cleavage
target
sequences
through
gel
electrophoresis
a
fluorescent
reporter
assay.
Using
optimized
gRNAs,
developed
lateral
flow
assay
(LFA)
HCV,
demonstrating
concentration‐dependent
signal
increase.
Importantly,
no
cross‐reactivity
was
observed
with
other
viral
targets.
To
further
enhance
sensitivity,
employed
dual‐enzyme
approach,
combining
Cas12
Cas13
in
single
reaction,
which
significantly
improved
limits
viruses.
Finally,
dual
antigen
LFA
strip
capable
simultaneously
detecting
sample.
This
approach
holds
promise
point‐of‐care
(POC)
diagnostics
where
infection
is
unknown.
study
addresses
current
limitations
CRISPR‐Cas
based
diagnostics,
namely,
need
ultrasensitive
methods
ability
to
detect
antigens
using
test
strip.
Our
findings
demonstrate
feasibility
highly
paving
way
potential
POC
application.
ACS Applied Bio Materials,
Journal Year:
2024,
Volume and Issue:
unknown
Published: Dec. 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.
