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In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Survivina/genética , Biomarcadores , Testes ImediatosRESUMO
Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , MicroRNAs , Sistemas CRISPR-Cas/genética , Técnicas Biossensoriais/métodos , Humanos , MicroRNAs/análise , MicroRNAs/metabolismo , Regulação Alostérica , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células HEK293RESUMO
A heteropore covalent organic framework (COF)-based composite membrane material was prepared and proved to have a satisfactory effect on the pretreatment of vegetable samples. The composite membrane was fabricated by in situ growth of a dual-pore COF on the surface of polydopamine (PDA)-aminated non-woven (NW) fabric. Due to the difference in the strength of the interaction between the phytochromes/COF and the pesticides/COF, the removal of phytochromes and the recovery of pesticides can be achieved by adjusting the composition of the solution. Through a simple immersion or filtration operation, NW@PDA@COF composite membrane can quickly and almost completely remove interfering phytochromes in the samples. The recovery of pesticides was determined by HPLC-MS/MS, and the recovery efficiencies were 72.3~101.7% and 67.3~106.7% for immersion and filtration modes of five different vegetable samples, respectively; the RSD is between 1.1 and 19% (n = 3). The limits of detection and quantification for the 13 pesticides investigated were 0.08 µg·L-1 and 0.23 µg·L-1, respectively. A wide linear range of 1~1000 µg·L-1 was observed with R2 values from 0.9774 to 0.9998. The membrane can be repeatedly used for at least 10 times by using a facile elution treatment. Compared to other commonly used sample pretreatment materials, heteropore COF-based composite membrane is superior in terms of sorbent amount, treatment time, operation simplicity, and material reusability.
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BACKGROUND: Hepatocellular carcinoma (HCC), the primary liver cancer, is one of the most malignant human tumors with extremely poor prognosis. The aim of this study was to investigate the anti-cancer effect of berberine in a human hepatocellular carcinoma cell line (HepG2), and to study the underlying mechanisms by focusing on the AMP-activated protein kinase (AMPK) signaling cascade. RESULTS: We found that berberine induced both apoptotic and autophagic death of HepG2 cells, which was associated with a significant activation of AMPK and an increased expression of the inactive form of acetyl-CoA carboxylase (ACC). Inhibition of AMPK by RNA interference (RNAi) or by its inhibitor compound C suppressed berberine-induced caspase-3 cleavage, apoptosis and autophagy in HepG2 cells, while AICAR, the AMPK activator, possessed strong cytotoxic effects. In HepG2 cells, mammalian target of rapamycin complex 1 (mTORC1) activation was important for cell survival, and berberine inhibited mTORC1 via AMPK activation. CONCLUSIONS: Together, these results suggested that berberine-induced both apoptotic and autophagic death requires AMPK activation in HepG2 cells.
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Rapid and highly effective removal of hexavalent chromium (Cr(â ¥)) is extremely vital to water resources restoration and environmental protection. To overcome the pH limitation faced by most ionic absorbents, an always positive covalent organic nanosheet (CON) material was prepared and its Cr(VI) adsorption and removal capability was investigated in detail. As-prepared EB-TFB CON (TFB = 1,3,5-benzaldehyde, EB = ethidium bromide) shows strong electropositivity in the tested pH range of 1 â¼ 10, display a pH-independent Cr(VI) removal ability, and work well for Cr(VI) pollution treatment with good anti-interference capability and reusability in a wide pH range covering almost all Cr(VI)-contaminated real water samples, thus eliminating the requirement for pH adjustment. Moreover, the nanosheet structure, which is obtained by a facile ultrasonic-assisted self-exfoliation, endows EB-TFB CON with fully exposed active sites and shortened mass transfer channels, and the Cr(VI) adsorption equilibrium can be reached within 15 min with a high adsorption capacity of 280.57 mg·g-1. The proposed Cr(VI) removal mechanism, which is attributed to the synergetic contributions of electrostatic adsorption, ion exchange and chemical reduction, is demonstrated by experiments and theoretical calculations. This work not only provides a general Cr(VI) absorbent without pH limitation, but also presents a paradigm to prepare ionic CONs with relatively constant surface charges.
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Non-targeted analysis of chemical hazards in foods plays a crucial role in controlling food safety. However, because it brings forward high demand for sample pretreatment, materials suitable for the pretreatment of foods, especially animal foods, are rare. Herein, covalent organic frameworks (COF)-based monolithic materials were constructed by three successive steps: preparation of polydimethylsiloxane (PDMS) sponge using sugar cube as a sacrificial template, loading of a heteroporous COF on PDMS sponge via ultrasonic or in-situ growth method, coating of the obtained PDMS@COF by polydopamine (PDA) network. As-prepared PDMS@COF@PDA sponges were demonstrated to work well in sample pretreatment of animal foods for non-targeted analysis of chemical hazards. After a simple vortex treatment for about 2 min, more than 98% triglycerides, the main interfering matrix components in animal foods, could be removed from lard and pork samples, accompanied by "full recovery" (recovery efficiencies: ≥63%) of 44 chemical hazards with different physicochemical properties. Besides providing promising sample pretreatment materials for non-targeted food safety analysis, this work also paves a feasible way to improve COF-based monolithic materials and thus promote their practical applications, because we found that the introduction of PDA network on COF-based monolithic material surface could play a role in "killing three birds with one stone": enhancing the stability of the materials by overcoming the detachment of COF during operations; controllably adjusting hydrophobic and hydrogen-bonding interactions on the material surface to promote the removal of triglycerides; weakening the hydrophobic and π-π interactions between COF and chemical hazards to increase the recoveries of chemical hazards.
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Estruturas Metalorgânicas , Animais , Estruturas Metalorgânicas/química , AlimentosRESUMO
A heteropore covalent organic framework incorporated silicone tube (S-tube@PDA@COF) was used as adsorbent to purify the matrices in vegetable extracts. The S-tube@PDA@COF was fabricated by a facile in-situ growth method and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and N2 adsorption-desorption. The as-prepared composite exhibited high removal efficiency of phytochromes and recovery (81.13-116.62%) of 15 chemical hazards from 5 representative vegetable samples. This study opens a promising avenue toward the facile synthesis of covalent organic frameworks (COFs)-derived silicone tubes for streamline operation in food sample pretreatment.
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The successful application of covalent organic frameworks (COFs) depends on not only their unique chemical structures but also their morphology, size, and architecture. Spherical COFs (SCOFs) are attracted special attention due to the superiority of spherical materials in many applications. However, the synthesis of uniform large-sized SCOFs remains a challenge. Herein, by carefully optimizing the synthesis of a heteropore COF, we find that solvent type and catalyst concentration play important roles in determining the morphology and size of COFs, and eventually achieve the controllable synthesis of large SCOFs with uniform sizes ranging from 200 µm to 5 mm. The obtained SCOFs keep the dual-pore feature of the heteropore COF and show good stability and high crystallinity. To exhibit the superior application potential of SCOFs, the SCOFs with a size range of 200-300 µm were demonstrated to be promising solid-phase extraction (SPE) fillers. As-prepared SCOFs-packed SPE column could effectively remove ≥99% phytochrome matrix from 6 different vegetable samples in 10 s, accompanied by 72.56-112.37% recoveries of 33 chemical hazards with different physicochemical properties, thus showing greatly promising application prospects in sample pretreatment of nontargeted food safety analysis. By utilizing acid/base-adjusted reversible color change, millimeter-sized SCOFs were developed as an easy-to-operate and reusable naked-eye indicator of acids.
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Estruturas Metalorgânicas , Catálise , Extração em Fase Sólida , VerdurasRESUMO
A nitrogen (N), oxygen (O)-rich porphyrin-based covalent organic framework (COF), in which interlayer porphyrin molecules are vertically stacked, is prepared and characterized. As-prepared N,O-rich TpTph COF shows a high adsorption capacity for Cd2+ due to the abundant coordination sites. More interesting, it is found that the formation of COF enlarges the porphyrin ring center space, thus facilitating the Cd2+coordination, and the resulting optical signal changes make the ratiometric detection of Cd2+ possible. Furthermore, using carbon fiber (CF) filaments, which are obtained from low cost and easy-to-obtain actived carbon mask, as support, porphyrin COF-based CF@TpTph membrane is prepared through in-situ growth of COF on the support followed by simple mechanical pressing. The CF@TpTph membrane is demonstrated to work well for both Cd2+ removal and enrichment from soil and water samples, and shows the advantages of ease of handling, robust stability, reduced secondary pollution risk to samples, and good reusability. This work provides a powerful tool for Cd2+ removal and enrichment, exhibits that preparing porphyrin-based COFs is a feasible way to promote the interactions between porphyrin ring and Cd2+, and demonstrates that mechanical pressing is a promising strategy for the design of COF-based monolithic materials to promote the practical applications of COFs.
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Estruturas Metalorgânicas , Porfirinas , Adsorção , Cádmio , Fibra de Carbono , Estruturas Metalorgânicas/química , Porfirinas/químicaRESUMO
Nontargeted analysis of food safety requires selective removal of interference matrices and highly efficient recovery of chemical hazards. Porous materials such as covalent organic frameworks (COFs) show great promise in selective adsorption of matrix molecules via size selectivity. Considering the complexity of interference matrices, we prepared crystalline heteropore COFs whose two kinds of pores have comparable sizes to those of several common phytochromes, main interference matrices in vegetable sample analysis. By controlling the growth of COFs on the surface of Fe3O4 nanoparticles or by utilizing a facile co-electrospinning method, heteropore COF-based magnetic nanospheres or electrospun nanofiber films were prepared, respectively. Both the nanospheres and the films maintain the dual-pore structures of COFs and show good stability and excellent reusability. Via simple magnetic separation or immersion operation, respectively, they were successfully used for the complete removal of phytochromes and highly efficient recovery of 15 pesticides from the extracts of four vegetable samples, and the recoveries are in the range of 83.10-114.00 and 60.52-107.35%, respectively. Film-based immersion operation gives better sample pretreatment performance than the film-based filtration one. This work highlights the great application potentials of heteropore COFs in sample pretreatment for nontargeted analysis, thus opening up a new way to achieve high-performance sample preparation in many fields such as food safety analysis, environment monitoring, and so on.
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Nanopartículas de Magnetita/química , Estruturas Metalorgânicas/química , Nanosferas/química , Resíduos de Praguicidas/isolamento & purificação , Fitocromo/isolamento & purificação , Adsorção , Brassica napus/química , Capsicum/química , Contaminação de Alimentos/análise , Kelp/química , Fenômenos Magnéticos , Nanofibras/química , Resíduos de Praguicidas/química , Fitocromo/química , Extração em Fase Sólida/métodos , Spinacia oleracea/química , Verduras/químicaRESUMO
The development of multimodal imaging probes carrying more than one modifiable site is very important in medical diagnosis. Herein, we demonstrate that amino acids, including acidic, neutral and basic amino acids, can be used as stabilizers and functional agents for the simple, one-step hydrothermal synthesis of hydrophilic upconversion nanoparticles (UCNPs) with a pure hexagonal phase and strong upconversion luminescence (UCL). The surface of the as-prepared UCNPs was capped with both carboxyl and amino groups, which not only provided the NPs with good dispersity in water, but also made further conjugation with two different biomolecules (e.g. targeted molecules and functional agents) possible. By co-doping different lanthanide ions, amino acid-functionalized UCNPs with different-colored UCL and different functions were obtained. For example, aspartate (Asp)-functionalized NaLuF4 co-doped with Tm3+ and Gd3+ not only emitted strong UCL in the range of the biological transparent window, but also has great potential as a T1-weighted magnetic resonance (MR) imaging contrast agent. The as-prepared Asp-NaLuF4:Gd/Yb/Tm UCNPs were successfully used in the UCL/MR bimodal in vivo imaging of nude mice.
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Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.
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Corantes Fluorescentes/química , Quadruplex G , MicroRNAs/análise , Humanos , Limite de DetecçãoRESUMO
As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1 × 10(-4)-1 U/mL with a detection limit of 1 × 10(-4)U/mL.
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DNA Catalítico/química , Quadruplex G , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Espectrofotometria Ultravioleta/instrumentação , Uracila-DNA Glicosidase/análise , Uracila-DNA Glicosidase/química , Quebras de DNA de Cadeia Simples , DNA Catalítico/genética , Ativação Enzimática , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report that QATPE, an aggregation-induced emission-active tetraphenylethene dye, can be used as a non-sequence-specific ssDNA probe for real-time monitoring of all rolling circle amplification (RCA) reactions, thus making RCA more suitable for biosensing applications.
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Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , Técnicas de Amplificação de Ácido NucleicoRESUMO
G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na(+), K(+), Mg(2+) and Ca(2+), promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca(2+) has the strongest stabilizing effect, followed by K(+), Mg(2+), and Na(+) in a decreasing order. The binding of K(+) to G-triplexes is accompanied by exothermic heats, and the binding of Ca(2+) with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K(+) or Ca(2+). These observations imply that stable G-triplexes may be formed under physiological conditions.
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Cátions Bivalentes/química , Guanina/química , Oligonucleotídeos/química , Sequência de Bases , Soluções Tampão , Calorimetria , Dicroísmo Circular , Quadruplex G , Conformação de Ácido Nucleico , Pinças Ópticas , Transição de Fase , Termodinâmica , Temperatura de TransiçãoRESUMO
As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.