A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification.

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highly sensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33 pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

Correction: A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

Correction for 'A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs' by Shixin Cai et al., Analyst, 2023, 148, 799-805, https://doi.org/10.1039/D2AN02006H.

A fluorescent aptasensor for ATP based on functional DNAzyme/walker and terminal deoxynucleotidyl transferase-assisted formation of DNA-AgNCs.

The development of sensitive adenosine triphosphate (ATP) sensors is imperative due to the tight relationship between the physiological conditions and ATP levels in vivo. Herein, a fluorescent aptasensor for ATP is presented, which adopts a strategy that combines a split aptamer and a DNAzyme/walker with terminal deoxynucleotidyl transferase (TDT)-assisted formation of DNA-AgNCs to realize fluorescence detection of ATP. A multifunctional oligonucleotide sequence is rationally designed, which integrates a split aptamer, a DNAzyme and a DNA walker. Both multifunctional oligonucleotide and its substrate strand are connected to the surface of Fe3O4@Au nanoparticles via Au-S bonds. The existence of ATP can induce the formation of the complete aptamer, and then activate the DNAzyme to circularly cleave the substrate strand, leaving 2',3'-cyclophosphate at the 3'end of the strand. This blocks the polymerization of dCTP to form poly(C) even in the presence of TDT and dCTP, due to the lack of free 3'-OH. In contrast, when ATP is absent, the DNAzyme/walker cannot work and then TDT catalyzes the formation of poly(C) at the free 3'-OH of the substrate strand, which is subsequently utilized as the template to prepare DNA-AgNCs. The fluorescence response derived from AgNCs thus reflects the ATP concentration. Under the optimum conditions, the aptasensor shows a linear response range from 5 nM to 10 000 nM, with a detection limit of 0.27 nM. The level of ATP in human serum can be effectively measured by the aptasensor with good recovery, indicating its application potential in medical samples.

Development of a Fluorescent Biosensor Based on DNAzyme for Tracing the Release of Zinc in Maize Leaves.

A fluorescent biosensor for real-time monitoring the release of Zn2+ in plants was constructed through immobilization of DNAzyme-containing hairpin DNA on nanofertilizer ZnO@Au nanoparticles (ZnO@Au NPs). A specially designed hairpin DNA containing both DNAzyme and its substrate sequence, which was also labeled with 5'-FAM and 3'-SH groups, was modified on ZnO@Au NPs through the Au-S bond. The fluorescent signal of FAM was initially quenched by AuNPs. When Zn2+ was released from ZnO@Au NPs, DNAzyme was activated and the substrate sequence in hairpin DNA was cleaved. The restored fluorescent signal in Tris-HCl buffer (pH 6.5) was correlated with the concentration of the released Zn2+. The performance of the biosensor was first demonstrated in the solution. The linear detection range was from 50 nM to 1.5 µM, with a detection limit of 30 nM. The biosensor system can penetrate into maize leaves with ZnO@Au NPs. With the release of Zn2+ in leaves, the restored fluorescence can be imaged by a confocal laser scanning microscope and used for monitoring the release and distribution of Zn2+. This work may provide a novel strategy for tracing and understanding the mechanism of nanofertilizers in organisms.

Combined application of biochar and nano-zeolite enhanced cadmium immobilization and promote the growth of Pak Choi in cadmium contaminated soil.

Biochar and zeolite have been demonstrated effective to remove heavy metals in soil; however, the effect of combined application of the both materials on the fraction of Cd and soil-plant system are largely unknown. Cd fractions in soil, growth and Cd uptake of Pak Choi were measured after the combined application of biochar (0, 5, 10 and 20 g·kg-1) and nano-zeolite (0, 5, 10, 20 g·kg-1) by pot experiment. Results showed that both single and combined application reduced the exchangeable Cd in soil and improved the plant growth. However, combined application of 20 g·kg-1 biochar with 10 g·kg-1 nano-zeolite showed the strongest effect, with the residual Cd in soil increased by 214% as compared with control. 20 g·kg-1 biochar with 10 g·kg-1 nano-zeolite Mechanic studies showed that this combination enhanced the antioxidant system, with the SOD, CAT and POD activities enhanced by 56.1%, 133.3% and 235.3%, respectively. The oxidative stress was reduced correspondingly, as shown by the reduced MDA contents (by 46.7%). This combination also showed the best efficiency in regulating soil pH, organic matter and soil enzymes thus improving the plant growth. This study suggests that combined application various materials such as biochar and nano-zeolite may provide new strategies for reducing the bioavailability of Cd in soil and thus the accumulation in edible plants.

[Mechanisms of the mechanically activated ion channel Piezo1 protein in mediating osteogenic differentiation of periodontal ligament stem cells via the Notch signaling pathway].

OBJECTIVE: To explore the mechanism of Piezo1 protein in mediating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) via the Notch signaling pathway. METHODS: In this study, young permanent teeth extracted from impacted teeth of 8-14-year-old children from January 1, 2016 to January 1, 2018 in the Department of Orthodontic, Beijing Children's Hospital were selected as cell sources. hPDLSCs were extracted by enzymatic digestion. Immunohistochemical staining was used to detect the expression of keratin and vimentin, and flow cytometry was used to identify the markers (CD146 and STRO-1) of hPDLSCs. The construction and screening of Piezo1 siRNA gene interference vector and Piezo1 gene overexpression plasmid were completed. Flexcell 4000T mechanical distraction stress instrument was used to construct hPDLSC cell model in vitro. According to the preliminary results, the experiment was divided into five groups: siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of Piezo1, Notch1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and bone sialoprotein (BSP). Western blot was used to detect the expression of ALP and Runx2. Fluo-3 AM probe was used to detect intracellular calcium content. RESULTS: Vimentin staining of hPDLSCs was positive, and keratin staining was negative. Flow cytometry was used to detect the expression of STRO-1 and CD146, markers of hPDLSC. Empty viral vectors, siRNA-Piezo1 interference sequence, and Piezo1 overexpression vector sequence could be transfected into hPDLSC by lentivirus, and the transfection efficiency was high (approximately 90%). The reverse transcription-polymerase chain reaction (RT-PCR) results showed that there were significant differences in Piezo1 gene levels among the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=9.573, P<0.05). The level of Piezo1 in the overexpression group was significantly higher than that in the siRNA interference group (q=3.893, P<0.05). The level of Piezo1 in the stretch stress group was significantly higher than that in the blank control group (q=2.006, P<0.05). The expression of Notch1 and osteogenic genes ALP, Runx2, OCN, and BSP had the same trend. Western blot results showed that there were significant differences in the expression of ALP in the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=11.207, P<0.001). The expression level of ALP in the overexpression group was significantly higher than that in the siRNA interference group (q=2.991, P<0.05). The expression of ALP in the stretch stress group was significantly higher than that in the blank control group (q=3.007, P<0.05). The expression of Runx2 protein showed the same trend. The intracellular calcium fluorescence intensity of the overexpression group was significantly higher than that of the siRNA interference group, and the intracellular calcium fluorescence intensity of the stretch stress group was significantly higher than that of the siRNA interference group. CONCLUSIONS: Mechanical stretch stress can promote the expression of Piezo1 protein. Ca2+ is the second messenger, activates the Notch1 signaling pathway and the expression of ALP, Runx2, OCN, and BSP; and promotes the osteogenic differentiation of hPDLSC. The siRNA-Piezo1 interfering plasmid can block this process. On the contrary, the overexpression plasmid of Piezo1 can promote the osteogenic differentiation of PDLSCs.