Development
of versatile sensing methods for sensitive and specific
detection of clinically relevant nucleic acids and proteins is of
great value for disease monitoring and diagnosis. In this work, we
propose a novel isothermal Self-primer EXPonential Amplification Reaction
(SPEXPAR) strategy based on a rationally engineered structure-switchable
Metastable Hairpin template (MH-template). The MH-template initially
keeps inactive with its self-primer overhanging a part of target recognition
region to inhibit polymerization. The present targets can specifically
compel the MH-template to transform into an “activate”
conformation that primes a target-recyclable EXPAR. The method is
simple and sensitive, can accurately and facilely detect long-chain
single-stranded nucleic acids or proteins without the need of exogenous
primer probes, and has a high amplification efficiency theoretically
more than 2
n
. For a proof-of-concept demonstration,
the SPEXPAR method was used to sensitively detect the characteristic
sequence of the typical swine fever virus (CSFV) RNA and thrombin,
as nucleic acid and protein models, with limits of detection down
to 43 aM and 39 fM, respectively, and even the CSFV RNA in attenuated
vaccine samples and thrombin in diluted serum samples. The SPEXPAR
method may serve as a powerful technique for the biological research
of single-stranded nucleic acids and proteins.
As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1–S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly–Arg–Pro–Hyp–Gly–Phe–Ser–Pro–Phe–Arg–Ile–Asp–NH2 (VMS2), Ile–Asn–Leu–Lys–Ala–Ile–Ala–Ala–Leu–Ala–Lys–Lys–Leu–Leu–NH2 (VMS3), and Phe–Leu–Pro–Ile–Ile–Gly–Lys–Leu–Leu–Ser–Gly–Leu–Leu–NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.
Sensitive
and specific imaging of microRNA (miRNA) in living cells
is of great value for disease diagnosis and monitoring. Hybridization
chain reaction (HCR) and DNAzyme-based methods have been considered
as powerful tools for miRNA detection, with low efficient intracellular
delivery and limited amplification efficiency. Herein, we propose
a Hairpins@MnO2 nanosystem for intracellular enzyme-free
exponential amplification for miRNA imaging. The enzyme-free exponential
amplification is based on the synergistic cross-activation between
HCR and DNAzymes. The MnO2 nanosheets were employed as
the carrier of three kinds of hairpin DNA probes and further provided
appropriate Mn2+ as DNAzyme cofactors in the living cell.
Upon entering cells and in the presence of highly expressed glutathione
(GSH) in tumors, MnO2 is reduced to release Mn2+ and the three kinds of hairpin DNA probes. In the presence of target
miRNA, the released hairpin DNA H1 and H2 probes self-assemble via
HCR into the wire-shaped active Mn2+-based DNAzymes which
further catalyze the cleavage of H3 to generate numerous new triggers
to reversely stimulate HCR amplifiers, thus offering tremendously
amplified Förster resonance energy transfer readout. The method
has a detection limit of 33 fM, which is 2.4 × 104 times lower than that of the traditional HCR system. The developed
method also has a high specificity; even miRNAs with a single base
difference can be distinguished. Live cell imaging experiments confirmed
that this Hairpins@MnO2 nanosystem allows accurate differentiation
of miRNA expression of cancer cells and normal cells. The method holds
great potential in biological research of nucleic acids.
High sensitivity and specificity imaging of miRNA in living cells plays an important role in understanding miRNA-related regulation and pathological research. Localized DNA circuits have shown good performance in reaction...
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