We demonstrate that target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 (MMP-2) activity with zero background. This assay exhibits good specificity and high sensitivity with...
We develop a new fluorescent biosensor for flap endonuclease 1 (FEN1) assay based on CRISPR/Cas12-enhanced single-molecule counting. This biosensor is simple, selective, and sensitive with a detection limit of 2.325...
Alkaline
phosphatase (ALP) is a valuable biomarker and effective
therapeutic target for the diagnosis and treatment of diverse human
diseases, including bone disorder, cardiovascular disease, and cancers.
The reported ALP assays often suffer from laborious procedures, costly
reagents, inadequate sensitivity, and large sample consumption. Herein,
we report a new single-molecule fluorescent biosensor for the simple
and ultrasensitive detection of ALP. In this assay, the ALP-catalyzed
dephosphorylation of detection probe can protect the detection probe
against lambda exonuclease-mediated digestion, and the remaining detection
probes can trigger ceaseless hybridization between two Cy5-labeled
hairpin probes through toehold-mediated DNA strand displacement, generating
a long fluorescent DNA chain, which can be subsequently separated
from unhybridized hairpin probes and disassembled into dispersed Cy5
moieties upon NaOH treatment. The free Cy5 moieties indicate the presence
of ALP and can be directly quantified via single-molecule counting.
This biosensor enables efficient amplification and transduction of
the target ALP signal through enzyme-free assembly and disassembly
processes, significantly simplifying the experimental procedure and
improving the assay accuracy. The proposed biosensor allows specific
and ultrasensitive detection of ALP activity with a detection limit
down to 2.61 × 10–6 U mL–1 and is suitable for ALP inhibition assay and kinetic analysis. Moreover,
this biosensor can be applied for endogenous ALP detection in human
cells and clinical human serum, holding the potential in the ALP biological
function study and clinical diagnosis.
Long
noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic
targets, and they play essential roles in various pathological and
biological processes. So far, the reported lncRNA assays usually suffer
from unsatisfactory sensitivity and time-consuming procedures. Herein,
we develop a mix-and-read assay based on multiple cyclic enzymatic
repairing amplification (ERA) for sensitive and rapid detection of
mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA
MALAT1). In this assay, we design two three-way junction (3WJ) probes
including a 3WJ template and a 3WJ primer to specifically recognize
lncRNA MALAT1, and the formation of a stable 3WJ structure induces
cyclic ERA to generate triggers. The resulting triggers subsequently
hybridize with a free 3WJ template and act as primers to initiate
new rounds of cyclic ERA, generating abundant triggers. The hybridization
of triggers with signal probes forms stable double-stranded DNA duplexes
that can be specifically cleaved by apurinic/apyrimidinic endonuclease
1 to produce a high fluorescence signal. This assay can be carried
out in a mix-and-read manner within 10 min under an isothermal condition
(50 °C), which is the rapidest and simplest method reported so
far for the lncRNA MALAT1 assay. This method can sensitively detect
lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately
measure endogenous lncRNA MALAT1 at the single-cell level. Moreover,
this method can distinguish lncRNA MALAT1 expression in breast cancer
patient tissues and their corresponding healthy adjacent tissues.
Importantly, the extension of this assay to different RNAs detection
can be achieved by simply replacing the corresponding target recognition
sequences.
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