Smart NIR light-gated CRISPR/Cas12a fluorescent biosensor with boosted biological delivery and trans-cleavage activity for high-performance in vivo operation.

Despite the in vitro usage of CRISPR/Cas12a system in fluorescent biosensors has made remarkable achievements, many challenges such as poor biological delivery, insufficient sensitivity, and uncontrollable initiation compel them hard to conduct in vivo analysis. Thus, we propose here some fruitful sensing concepts. First, the multiple biomolecular components of CRISPR/Cas12a system are collectively carried by MnO2 nanosheets via a simple physical absorption to achieve a highly-efficient biological uptake. Under the reduction of widespread biothiols, not only the sensing frame is easily released but also sufficient Mn2+ is produced to serve as an effective trans-cleavage accelerator. Furthermore, a photocleavge-linker induced smart near-infrared (NIR) light-gated manner is designed to offer a spatiotemporal target recognition, for which a 808 nm NIR light transduced ultraviolet upconversion luminescence with weak thermal effect is employed to completely prevent the sensing flow from pre-initiating during the biological delivery. As a conceptual validation, this biosensor has satisfactory sensitivity and specificity to survivn messenger RNA (a broad-spectrum cancer biomarker). More importantly, it can work as a reliable imaging platform for differentiating cancers in live cellular level and also presents a high-performance operation ability for analyzing live mice, greatly promoting the CRISPR technology in biosensing field.

Genomic analyses of 10,376 individuals in the Westlake BioBank for Chinese (WBBC) pilot project.

We initiate the Westlake BioBank for Chinese (WBBC) pilot project with 4,535 whole-genome sequencing (WGS) individuals and 5,841 high-density genotyping individuals, and identify 81.5 million SNPs and INDELs, of which 38.5% are absent in dbSNP Build 151. We provide a population-specific reference panel and an online imputation server ( https://wbbc.westlake.edu.cn/ ) which could yield substantial improvement of imputation performance in Chinese population, especially for low-frequency and rare variants. By analyzing the singleton density of the WGS data, we find selection signatures in SNX29, DNAH1 and WDR1 genes, and the derived alleles of the alcohol metabolism genes (ADH1A and ADH1B) emerge around 7,000 years ago and tend to be more common from 4,000 years ago in East Asia. Genetic evidence supports the corresponding geographical boundaries of the Qinling-Huaihe Line and Nanling Mountains, which separate the Han Chinese into subgroups, and we reveal that North Han was more homogeneous than South Han.

Light-Activated and Self-Driven Autonomous DNA Nanomachine Enabling Fluorescence Imaging of MicroRNA in Living Cells with Exceptional Precision and Efficiency.

Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.

Photo-gated and self-powered three-dimensional DNA motors with boosted biostability for exceptionally precise and efficient tracing of intracellular survivin mRNA.

Benefiting from the outstanding signal amplification effect and the admirable construction flexibility, the currently proposed DNA motors (particularly DNA walkers) based biosensing concepts have provided a forceful fluorescence imaging tool for intracellular detection. Even so, this promising sensing means is not only subject to poor controllability and prone to produce false signals but also requires exogenous powering forces owing to the common employment of DNAzyme. In response to these challenges, we are herein motivated to present some meaningful solving strategies. For one thing, the surfaces of gold nanoparticles are conducted with a photo-gated walking behavior by introducing a photocleave mode, under which the light-switchable DNA walkers are capable of being selectively activated via an external ultraviolet source to faultlessly prevent the sensing frame from being pre-initiated during cellular uptake and intracellular delivery. For another, the intracellular biothiols are consumed by MnO2 nanosheets to effectively avoid the competitions to Au-S bonds to eliminate potential false outputs and also self-supply sufficient cofactors (Mn2+) to actualize a self-powered operation pattern as well as facilitate the endocytosis process. Following these breakthroughs, a favorable analysis performance towards a model tumor biomarker (survivin mRNA) is endowed with the newly raised biosensor, whose sensitivity is low to pM level with a sound specificity for identifying single base mismatching. Moreover, the significantly improved autonomous three-dimensional DNA walkers can be used to determine and dynamically trace the targets in live cancer cells with an exceptional precise and efficient manner, commendably impelling the sensing ability of DNA motors in biological specimens.

Holographic Optical Tweezers and Boosting Upconversion Luminescent Resonance Energy Transfer Combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a Biosensors.

Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na+). The assay sensitivity is significantly improved by utilizing a bead-supported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.

A boosting upconversion luminescent resonance energy transfer and biomimetic periodic chip integrated CRISPR/Cas12a biosensor for functional DNA regulated transduction of non-nucleic acid targets.

Apart from gene editing capacity, the newly discovered CRISPR/Cas systems offer an exciting option for biosensing field because of their excellent target recognition accuracy. However, the currently constructed sensors are not only limited to nucleic acid analysis but also suffer from poor adaptability in complex samples and unsatisfying sensitivity. We herein introduce some advanced concepts to break through these bottlenecks. First, the sensing targets are extended by skillfully designing a functional DNA such as aptamer (for protein) and DNAzyme (for metal ion) to regulate the transduction of non-nucleic acid species and further activate the trans cleavage of CRISPR/Cas12a. Second, a boosting upconversion luminescent resonance energy is triggered by using a peculiar energy-confining notion, whereby the luminescence domain is intensively restricted in a very narrow space (~2.44 nm) and up to 92.9% of the green emission can be quenched by the approaching BHQ-1 modified reporters. Third, a bio-inspired periodic arrangement biomimetic chip (photonic crystal) is employed to selectively reflect the upconversion luminescence to achieve noteworthy signal enhancement (~35-fold). By utilizing very simple detection devices (a 980 nm portable laser and a smartphone), the CRISPR/Cas12a biosensor shows commendable sensitivity and specificity toward model targets (ATP and Na+, limits of detection are ~ 18 nM and ~0.37 µM, respectively). More importantly, the analysis of real complex samples demonstrate that the as-proposed platform can work as a powerful toolbox for monitoring the ATP fluctuation in single cell and point-of-care testing Na+ in human plasma, enabling a broad application prospect.

Effect of Bias Voltage on Mechanical Properties of HiPIMS/RFMS Cosputtered Zr-Si-N Films.

Zr-Si-N films with atomic ratios of N/(Zr + Si) of 0.54-0.82 were fabricated through high-power impulse magnetron sputtering (HiPIMS)-radio-frequency magnetron sputtering (RFMS) cosputtering by applying an average HiPIMS power of 300 W on the Zr target, various RF power levels on the Si target, and negative bias voltage levels of 0-150 V connected to the substrate holder. Applying a negative bias voltage on substrates enhanced the ion bombardment effect, which affected the chemical compositions, mechanical properties, and residual stress of the Zr-Si-N films. The results indicated that Zr-Si-N films with Si content ranging from 1.4 to 6.3 atom % exhibited a high hardness level of 33.2-34.6 GPa accompanied with a compressive stress of 4.3-6.4 GPa, an H/E* level of 0.080-0.107, an H3/E*2 level of 0.21-0.39 GPa, and an elastic recovery of 62-72%.

[Chemical constituents from roots of Ligularia intermedia].

A new sesquiterpenoid, 8α-hydroxy-6ß-methoxy-1-oxoeremophila-7 (11), 9 (10) -diene-12, 8-olide (1) and five known compounds, petasin (2), caffeic acid (3), hepta-cosanol (4), ß-sitosterol (5) and ß-daucosterol (6) have been isolated from the roots of Ligularia intermedia. The compounds were isolated by column chromatography on silica gel and Sephadex LH-20, and identified based on spectral analyses (MS, 1H-NMR, 13C-NMR).

[A new eremophilane derivative from Ligularia intermedia].

A new eremophilane derivative, (3aR,4R,5S,7S,7aS)-2-acetyl-7,7a-dihydroxy-3a,4-dimethyl-3a,4,5,6,7,7a-hexahydro-3H-inden-5-yl acetate (1) and three known compounds, 10beta-hydroxy-eremophil-7 (11)-en-12,8alpha-olide(2), beta-sitosterol (3) and beta-daucosterol(4) have been isolated from Ligularia intermedia. The compounds were isolated by column chromatography on silica gel and Sephadex LH-20,and identified on the basis of spectral analyses (MS, 1H-NMR, 13C-NMR).

[Origin of and analysis on "wind syndrome" in medicine of Dong nationality].

As a major component of diseases, "wind syndrome" in medicine of Dong nationality is closely related to the way of thinking, ecological surrounding, and culture of Dong ethnic group. This syndrome is very primitive with very extensive inclusion and ethnic uniqueness.