12 research outputs found
Sensitive Detection of MicroRNA in Complex Biological Samples via Enzymatic Signal Amplification Using DNA Polymerase Coupled with Nicking Endonuclease
MicroRNA (miRNA) has become an ideal
biomarker candidate for cancer
diagnosis, prognosis, and therapy. In this study, we have developed
a novel one-step method for sensitive and specific miRNA detection
via enzymatic signal amplification and demonstrated its practical
application in biological samples. The proposed signal amplification
strategy is an integrated âbiological circuitâ designed
to initiate a cascade of enzymatic polymerization reactions in order
to detect, amplify, and measure a specific miRNA sequence by using
the isothermal strand-displacement property of a mesophilic DNA polymerase
together with the nicking activity of a restriction endonuclease.
The circuit is composed of two molecular switches operating in series:
the nicking endonuclease-assisted isothermal polymerization reaction
activated by a specific miRNA and the strand-displacement polymerization
reaction designed to initiate molecular beacon-assisted amplification
and signal transduction. The hsa-miR-141 (miR-141) was chosen as a
target miRNA because its level specifically elevates in prostate cancer.
The proposed method allowed quantitative sequence-specific detection
of miR-141 in a dynamic range from 1 fM to 100 nM, with an excellent
ability to discriminate differences among miR-200 family members.
Moreover, the detection assay was applied to quantify miR-141 in cancerous
cell lysates. The results are in excellent agreement with those from
the reverse transcription polymerase chain reaction method. On the
basis of these findings, we believe that this proposed sensitive and
specific assay has great potential as a miRNA quantification method
for use in biomedical research and clinical diagnosis
One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification
Traditional molecular beacons, widely applied for detection
of nucleic acids, have an intrinsic limitation on sensitivity, as
one target molecule converts only one beacon molecule to its fluorescent
form. Herein, we take advantage of the duplex-specific nuclease (DSN)
to create a new signal-amplifying mechanism, duplex-specific nuclease
signal amplification (DSNSA), to increase the detection sensitivity
of molecular beacons (Taqman probes). DSN nuclease is employed to
recycle the process of target-assisted digestion of Taqman probes,
thus, resulting in a significant fluorescence signal amplification
through which one target molecule cleaves thousands of probe molecules.
We further demonstrate the efficiency of this DSNSA strategy for rapid
direct quantification of multiple miRNAs in biological samples. Our
experimental results showed a quantitative measurement of sequence-specific
miRNAs with the detection limit in the femtomolar range, nearly 5
orders of magnitude lower than that of conventional molecular beacons.
This amplification strategy also demonstrated a high selectivity for
discriminating differences between miRNA family members. Considering
the superior sensitivity and specificity, as well as the multiplex
and simple-to-implement features, this method promises a great potential
of becoming a routine tool for simultaneously quantitative analysis
of multiple miRNAs in tissues or cells, and supplies valuable information
for biomedical research and clinical early diagnosis
Overcoming Multidrug Resistance by Base-Editing-Induced Codon Mutation
Multidrug
resistance (MDR) is the main obstacle in cancer chemotherapy.
ATP binding cassette (ABC) transporters on the MDR cell membrane can
transport a wide range of antitumor drugs out of cells, which is one
of the main causes of MDR. Therefore, disturbing ABC transporters
becomes the key to reversing MDR. In this study, we implement a cytosine
base editor (CBE) system to knock out the gene encoding ABC transporters
by base editing. When the CBE system works in MDR cells, the MDR cells
are manipulated, and the genes encoding ABC transporters can be inactivated
by precisely changing single in-frame nucleotides to induce stop (iSTOP)
codons. In this way, the expression of ABC efflux transporters is
reduced and intracellular drug retention is significantly increased
in MDR cells. Ultimately, the drug shows considerable cytotoxicity
to the MDR cancer cells. Moreover, the substantial downregulation
of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)
implies the successful application of the CBE system in the knockout
of different ABC efflux transporters. The recovery of chemosensitivity
of MDR cancer cells to the chemotherapeutic drugs revealed that the
system has a satisfactory universality and applicability. We believe
that the CBE system will provide valuable clues for the use of CRISPR
technology to defeat the MDR of cancer cells
Quantification of Exosome Based on a Copper-Mediated Signal Amplification Strategy
Exosomes, a class
of small extracellular vesicles, play important
roles in various physiological and pathological processes by serving
as vehicles for transferring and delivering membrane and cytosolic
molecules between cells. Since exosomes widely exist in various body
fluids and carry molecular information on their originating cells,
they are being regarded as potential noninvasive biomarkers. Nevertheless,
the development of convenient and quantitative exosome analysis methods
is still technically challenging. Here, we present a low-cost assay
for direct capture and rapid detection of exosomes based on a copper-mediated
signal amplification strategy. The assay involves three steps. First,
bulk nanovesicles are magnetically captured by cholesterol-modified
magnetic beads (MB) via hydrophobic interaction between cholesterol
moieties and lipid membranes. Second, bead-binding nanovesicles of
exosomes with a specific membrane protein are anchored with aptamer-modified
copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MBâexosomeâCuO
NP). Third, the resultant sandwich complexes are dissolved by acidolysis
to turn CuO NP into copperÂ(II) ions (Cu<sup>2+</sup>), which can be
reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate
in the presence of polyÂ(thymine). The fluorescence emission of CuNPs
increases with the increase of Cu<sup>2+</sup> concentration, which
is directly proportional to the concentration of exosomes. Our method
allows quantitative analysis of exosomes in the range of 7.5 Ă
10<sup>4</sup> to 1.5 Ă 10<sup>7</sup> particles/ÎźL with
a detection of limit of 4.8 Ă 10<sup>4</sup> particles/ÎźL
in biological sample. The total working time is about 2 h. The assay
has the potential to be a simple and cost-effective method for routine
exosome analysis in biological samples
Simultaneous Surface-Enhanced Raman Spectroscopy Detection of Multiplexed MicroRNA Biomarkers
Simultaneous detection
of cancer biomarkers holds great promise
for the early diagnosis of cancer. In the present work, an ultrasensitive
and reliable surface-enhanced Raman scattering (SERS) sensor has been
developed for simultaneous detection of multiple liver cancer related
microRNA (miRNA) biomarkers. We first proposed a novel strategy for
the synthesis of nanogap-based SERS nanotags by modifying gold nanoparticles
(AuNPs) with thiolated DNA and nonfluorescent small encoding molecules.
We also explored a simple approach to a green synthesis of hollow
silver microspheres (Ag-HMSs) with bacteria as templates. On the basis
of the sandwich hybridization assay, probe DNA-conjugated SERS nanotags
used as SERS nanoprobes and capture DNA-conjugated Ag-HMSs used as
capture substrates were developed for the detection of target miRNA
with a detection limit of 10 fM. Multiplexing capability for simultaneous
detection of the three liver cancer related miRNAs with the high sensitivity
and specificity was demonstrated using the proposed SERS sensor. Furthermore,
the practicability of the SERS sensor was supported by the successful
determination of target miRNA in cancer cells. The experimental results
indicated that the proposed strategy holds significant potential for
multiplex detection of cancer biomarkers and offers the opportunity
for future applications in clinical diagnosis
Copper-Mediated DNA-Scaffolded Silver Nanocluster OnâOff Switch for Detection of Pyrophosphate and Alkaline Phosphatase
We present a new copper-mediated
onâoff switch for detecting
either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on
DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded
sequence containing a 15-nt polythymine spacer between two different
emitters. The switch is based on three favorable properties: the quenching
ability of Cu<sup>2+</sup> for DNA/AgNCs with excitation at 550 nm;
the strong binding capacity of Cu<sup>2+</sup> and PPi; and the ability
of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence
of DNA/AgNCs depends on the concentrations of Cu<sup>2+</sup>, PPi,
and ALP. CopperÂ(II) acts as a mediator to interact specifically with
the Probe, while PPi and ALP convert the signal of the Probe by removing
and recovering Cu<sup>2+</sup>, operating as an onâoff switch.
In the presence of Cu<sup>2+</sup> only, DNA/AgNCs exhibit low fluorescence
because the combination of Cu<sup>2+</sup> and DNA template disturbs
the precise formation of DNA/AgNCs. When PPi is added to the system
containing Cu<sup>2+</sup>, free DNA template is obtained due to the
stronger interaction of PPi and Cu<sup>2+</sup>, leading to a significant
fluorescence increase (ON state) which depends on the concentration
of PPi. Further addition of ALP results in the release of free Cu<sup>2+</sup> via ALP-catalysis of hydrolysis of PPi into Pi, thereby
returning the system to the low fluorescence OFF state. The switch
allows the analysis of either PPi or ALP by observation of the fluorescence
status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi
and ALP, respectively. The AgNCs onâoff switch provides the
advantages of simple design, convenient operation, and low experimental
cost without need of chemical modification, organic dyes, or separation
procedures
Simple and Cost-Effective Glucose Detection Based on Carbon Nanodots Supported on Silver Nanoparticles
We present a new
glucose oxidase (GOx)-mediated strategy for detecting
glucose based on carbon nanodots supported on silver nanoparticles
(C-dots/AgNPs) as nanocomplexes. The strategy involves three processes:
quenching of C-dotsâ fluorescence by AgNPs, production of H<sub>2</sub>O<sub>2</sub> from GOx-catalyzed oxidation of glucose, and
H<sub>2</sub>O<sub>2</sub>-induced etching of AgNPs. In the C-dots/AgNPs
complex, AgNPs act as a ânanoquencherâ to decrease C-dots
fluorescence by surface plasmon-enhanced energy transfer (SPEET) from
C-dots (donor) to AgNPs (acceptor). The H<sub>2</sub>O<sub>2</sub> formed by GOx-catalyzed oxidation of glucose etches the AgNPs to
silver ions, thus freeing the C-dots from the AgNPs surfaces and restoring
the C-dotsâ fluorescence. Therefore, the increase in fluorescence
depends directly on the concentration of H<sub>2</sub>O<sub>2</sub>, which, in turn, depends on the concentration of glucose. The strategy
allows the quantitative analysis of glucose with a detection limit
of 1.39 ÎźM. The method based on C-dots/AgNPs offers the following
advantages: simplicity of design and facile preparation of nanomaterials,
as well as low experimental cost, because chemical modification and
separation procedures are not needed
Attomolar Ultrasensitive MicroRNA Detection by DNA-Scaffolded Silver-Nanocluster Probe Based on Isothermal Amplification
MicroRNAs (miRNAs) play vital roles in a plethora of
biological and cellular processes. The levels of miRNAs can be useful
biomarkers for cellular events or disease diagnosis, thus the method
for sensitive and selective detection of miRNAs is imperative to miRNA
discovery, study, and clinical diagnosis. Here we develop a novel
method to quantify miRNA expression levels as low as attomolar sensitivity
by target-assisted isothermal exponential amplification coupled with
fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility
in the application of detecting miRNA in real samples. The method
reveals superior sensitivity with a detection limit of miRNA of 2
aM synthetic spike-in target miRNA under pure conditions (approximately
15 copies of a miRNA molecule in a volume of 10 ÎźL) and can
detect at least a 10 aM spike-in target miRNA in cell lysates. The
method also shows the high selectivity for discriminating differences
between miRNA family members, thus providing a promising alternative
to standard approaches for quantitative detection of miRNA. This simple
and cost-effective strategy has a potential of becoming the major
tool for simultaneous quantitative analysis of multiple miRNAs (biomarkers)
in tissues or cells and supplies valuable information for biomedical
research and clinical early diagnosis
Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer
We have developed a label-free method
for sequence-specific DNA detection based on surface plasmon enhanced
energy transfer (SPEET) process between fluorescent DNA/AgNC string
and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded
DNA template encoded two emitter-nucleation sequences at its termini
and an oligo spacer in the middle, was rationally designed to produce
bright fluorescence emission. The proposed method takes advantage
of two strategies. The first one is the difference in binding properties
of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward
AuNPs. The second one is SPEET process between fluorescent DNA/AgNC
string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously
adsorbed onto the surface of AuNPs and correspondingly AuNPs serve
as ânanoquencherâ to quench the fluorescence of DNA/AgNC
string. In the presence of target DNA, the sensing probe hybridized
with target DNA to form duplex DNA, leading to a salt-induced AuNP
aggregation and subsequently weakened SPEET process between fluorescent
DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and
a concomitant fluorescence increase were clearly observed in the sensing
system, which had a concentration dependent manner with specific DNA.
The proposed method achieved a detection limit of âź2.5 nM,
offering the following merits of simple design, convenient operation,
and low experimental cost because of no chemical modification, organic
dye, enzymatic reaction, or separation procedure involved
Direct Exosome Quantification via Bivalent-Cholesterol-Labeled DNA Anchor for Signal Amplification
Exosomes, as an important
subpopulation of extracellular vesicles
(EVs), play an important role in intercellular communications in various
important pathophysiological processes, especially cancer-related.
However, reliable and convenient quantitative methods for their determination
are still technically challenging. In this study, we developed an
efficient and direct method by combining immunoaffinity and lipid
membrane surface modification into a single platform for specific
isolation and accurate quantification of exosomes. Exosomes are specifically
captured by immunomagnetic beads, and then a bivalent-cholesterol
(B-Chol)-labeled DNA anchor with high affinity is spontaneously inserted
into the exosome membrane. The rationally designed sticky end of the
anchor acts as the initiator for the subsequent horseradish peroxidase
(HRP)-linked hybridization chain reaction (HCR) for signal amplification.
Detection is based on the color change of HRP-catalyzed H<sub>2</sub>O<sub>2</sub>-mediated oxidation of 3,3â˛,5,5â˛- tetramethyl
benzidine (TMB), which can be conveniently observed by the naked eye
and monitored by UVâvis spectrometry. This proposed method
enables sensitive detection of 2.2 Ă 10<sup>3</sup> exosomes
per microliter with a relative standard deviation of <5.6%, with
100-fold higher sensitivity compared to conventional ELISA. We believe
that our assay has considerable potential as a routine bioassay (cost-efficient,
reliable, and easy to operate) for the accurate quantification of
exosomes in clinical samples