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The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include 5' end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation, and 3' end dephosphorylation, is crucial for regulating numerous cellular processes. Although there are many methods for detecting a single type of DNA phosphorylation, the direct and simultaneous identification of DNA phosphorylation/dephosphorylation on the 5' and/or 3' ends remains a challenge, let alone the unveiling of the heterogeneous catalysis processes of related phosphatases and kinases. Taking advantage of the charge-sensitive aerolysin nanopore interface, herein, an orientation-dependent sensing strategy is developed to enhance phosphorylation-site-dependent interaction with the nanopore sensing interface, enabling the direct and simultaneous electric identification of four heterogeneous phosphorylation statuses of a single DNA. By using this strategy, we can directly evaluate the heterogeneous dephosphorylation process of alkaline phosphatase (ALP) at the single-molecule level. Our results demonstrate that the ALP in fetal bovine serum preferentially catalyzes the 3' phosphate rather than both ends. The quantification of endogenous ALP activity in fetal bovine serum could reach the submilli-IU/L level. Our aerolysin measurements provide a direct look at the heterogeneous phosphorylation status of DNA, allowing the unveiling of the dynamic single-molecule functions of kinase and phosphatase.

With a clear enhancement of the apparent resolution of experimentally determined spectra, excess spectroscopy has been developed as a powerful tool to study solution structures and molecular interactions. In the standard procedure of the method, excess spectra are calculated based on the ideal spectra constructed using two pure compounds. This limits the applications of the method when the pure compounds are unstable or their physical state is different from that of the mixtures. To overcome the problem or to extend the application, we propose generalized excess spectroscopy in this work, where the ideal spectrum is evaluated from the spectra of reference mixtures. After deducing the working equations, we performed digital simulation and then applied the novel approach to a binary system consisting of

Arsenic (As) is a metalloid commonly found worldwide. Environmental As exposure may cause potential health hazards and behavioral changes in humans and animals. However, the effects of environmental As concentrations on social behavior, especially during the juvenile stage, are unclear. In this study, we observed behavioral changes in juvenile zebrafish after 28 days of exposure to inorganic As (NaAsO

During the process of synaptic formation, neurons require not only certain principles for selecting partners to form synapses but also additional mechanisms to avoid undesired cells. However, the means to get around unwanted partners remains largely unknown. In this study, we have identified that the combinatorial expression of clustered protocadherin gammas (γ-PCDHs) is essential in regulating such specificity in the mouse neocortex. Using single-cell sequencing from the 5-prime end, we revealed the combinatorial expression pattern of γ-PCDH variable isoforms in neocortical neurons. Whole-cell patch-clamp recordings demonstrated that increasing the similarity level of this combinatorial pattern in neurons reduced their synaptic connectivity. Our findings reveal a delicate molecular mechanism for assembling the neural network in the mouse neocortex.

DNA lesions such as metholcytosine(mC), 8-OXO-guanine (OG), inosine (I), etc. could cause genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remains a challenge due to massive varieties and less distinguishable readouts for structural variations at the molecular level. Moreover, standard amplification and labeling hardly work in DNA lesion detection. Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor lesion into distinguishable electrochemical readouts. Compared with previous single molecule sensing interfaces, the temporal resolution of the K238Q aerolysin nanopore is enhanced by two orders, which has the best sensing performance in all reported aerolysin nanopores. In this work, the novel K238Q could discriminate directly at least three types of lesions (mC, OG, I) without labeling and quantify modification sites under the mixed heterocomposition conditions of the oligonucleotide. Such a nanopore electrochemistry approach could be further applied to diagnose genetic diseases at high sensitivity., Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor DnA lesion into distinguishable electrochemical readouts.

In this chapter, we introduce the biological membrane protein-based single-biomolecule interface for single molecule sensing, including DNA and protein detection, using synergic effects from steric volume exclusion and weak interactions between the nanopore and the analyte. More attentions will be focused on the state-of-art detection strategies for the measurement of a variety of biomolecules via nanopore electrochemistry by the selected experimental case studies, in particular for tracing short-lived metastable conformation and recognition of multiple targets for the promising clinical diagnosis. This chapter selects experimental cases in an α-hemolysin nanopore and an aerolysin nanopore to demonstrate the sensing ability of biological nanopores in detail.

In this work, a facile grinding method for preparation of supramolecular square nanoplates formed by β-cyclodextrin and gallium acetylacetonate was developed. The nanoplates exhibited a highly monodispersed distribution nature at room temperature, and were self-assembled to form supramolecular layer nanosheets at 353 K. Heating to higher temperatures such as 673 K led to the decomposition of the layer nanosheets into γ-Ga2O3 layer nanosheets. The γ-Ga2O3 nanosheets (thickness of about 15 nm) with a very high specific surface area (93.9 m2 g−1) show an improved solar-blind detection performance (e.g., light–dark ratio, 1.64 × 104) compared to devices based on 3D structured γ-Ga2O3. We believe that the grinding-heating-strategy provides a convenient and efficient route to synthesize high-quality supramolecular nanocrystals and inorganic nanomaterials.

RNA sensing is of vital significance to advance our comprehension of gene expression and to further benefit medical diagnostics. Taking advantage of the excellent sensing capability of the aerolysin nanopore as a single-biomolecule interface, we for the first time achieved the direct characterization of single native RNA of Poly(A)4 and Poly(U)4. Poly(A)4 induces ∼10% larger blockade current amplitude than Poly(U)4. The statistical duration of Poly(A)4 is 18.83 ± 1.08 ms, which is 100 times longer than that of Poly(U)4. Our results demonstrated that the capture of RNA homopolymers is restricted by the biased diffusion. The translocation of RNA needs to overcome a lower free-energy barrier than that of DNA. Moreover, the strong RNA-aerolysin interaction is attributed to the hydroxyl in pentose, which prolongs the translocation time. This study opens an avenue for aerolysin nanopores to directly achieve RNA sensing, including discrimination of RNA epigenetic modification and selective detection of miRNA.

Monitoring subtle changes in ionic current flow through a nanopore could be applied to observe single molecule reaction. Here, we introduced cysteine to substitute for lysine at position 238 constructing a mutant aerolysin K238C. It could be regarded as a nanoreactor to efficiently visualize chemical bonds making and breaking. The compound 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was selected as a reactant coming into collisions on the interface of the pore to occur a reversible reaction. Our results showed that the mutant aerolysin could respond to three molecules of DTNB simultaneously and reflect corresponding levels with distinguishable current signals. Therefore, this method constitutes a simple, generic tool for monitoring single molecule reaction, which evokes a guidance for the mutant aerolysin towards the application of tracking other more reactions at single molecule level.

Hexagonal nanoplates of high-quality γ-gallium oxide (γ-Ga2O3) were successfully synthesized by using a competitive and cooperative interaction model based on multiple equilibria, including a precipitation interaction, a coordination interaction and two binding interactions. The synthetic method is straightforward and affords the desired product in very high yield. It is important to note that only a combination of the coordination interaction and binding interactions can contribute to the formation of hexagonal nanoplates by effectively suppressing the generation of gallium oxide hydroxide and directly limiting the assembly of the nanoplates into microflowers. This model is significant because it allows us to understand how the disassembly process of nanostructures is related to the synergistic mechanism of multiple interactions. Furthermore, the γ-Ga2O3 hexagonal nanoplates exhibit good heterogeneous catalytic performance for the oxidation reaction of thiophenes. A possible mechanism was proposed for the catalytic process, and this may open up a new perspective in the study of oxidative desulfurization.

DNA lesion such as metholcytosine(mC), 8-OXO-guanine(OG), inosine(I) etc could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (mC, OG, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

Flavin adenine dinucleotide (FAD) as a cofactor is involved in numerous important metabolic pathways where the biological function is intrinsically related to its transient conformations., Flavin adenine dinucleotide (FAD) as a cofactor is involved in numerous important metabolic pathways where the biological function is intrinsically related to its transient conformations. The confined space of enzymes requires FAD set in its specific intermediate conformation. However, conventional methods only detect stable conformations of FAD molecules, while transient intermediates are hidden in ensemble measurements. There still exists a challenge to uncover the transient conformation of each FAD molecule, which hinders the understanding of the structure–activity relationship of the FAD mechanism. Here, we employ the electrochemically confined space of an aerolysin nanopore to directly characterize a series of transient conformations of every individual FAD. Based on distinguishable current blockages, the “stack”, “open”, and four quasi-stacked FADs are clearly determined in solution, which is further confirmed by temperature-dependent experiments and mutant aerolysin assay. Combined with molecular dynamics simulations, we achieved a direct correlation between the residual current ratio (I/I0) and FAD backbone angle. These results would facilitate further understanding of the structure–activity relationship in the flavoprotein.

High-quality γ-Ga2O3 nanospheres (diameter, 130 nm) were successfully synthesized by direct conversion of a precursor complex of Ga3+ ions and tartrate ions (L2−) in water. The addition of even a small amount of L2− ions can efficiently suppress the hydrolysis of Ga3+ ions to GaOOH. This synthetic method is new and has great advantages of simplicity, organic solvent-free processing and high efficiency. The γ-Ga2O3 nanospheres which consist of primary nanoparticles with a diameter of about 10 nm show an extremely high specific surface area (83.7 m2 g−1), thereby possessing improved solar-blind detection performance (e.g., light current, 1.83 μA; light–dark ratio, 2.29 × 103 and photocurrent fluctuation

Aerolysin has been used as a biological nanopore for studying peptides, proteins, and oligosaccharides in the past two decades. Here, we report that wild-type aerolysin could be utilized for polynucleotide analysis. Driven a short polynucleotide of four nucleotides length through aerolysin occludes nearly 50% amplitude of the open pore current. Furthermore, the result of total internal reflection fluorescence measurement provides direct evidence for the driven translocation of single polynucleotide through aerolysin.

Wind-turbine main bearing has to withstand dynamic loads with different directions and different magnitudes in complex environments, and its stable operation has a vital impact on the performance of the entire wind turbine. Therefore, the fatigue strength test of wind turbine bearing is helpful to ensure the normal operation of the whole wind turbine. According to the actual force of main bearing in natural environment, this paper designed a large-scale wind-turbine bearing test bench to detect the deformation performance of the bearing. Through the establishment of the mechanical loading model, the simulation of the actual working conditions of main bearing is realized by the loading of the eight hydraulic cylinders of the test bench, and the radial and axial displacement of the test bearing under different wind conditions are recorded by displacement sensors. A number of temperature sensors are used to monitor the real-time temperature change of the bearing inner ring. The test results show that the loading effect of eight hydraulic cylinders can realize the force of wind turbine bearing under wind load, and the test bench can effectively detect wind-turbine bearings with a diameter of 2.5 m. The mechanical loading method and test results can provide guidance for further inspection of the wind-turbine bearing.

Nanopore analysis is a powerful technique for single molecule analysis by virtue of its electrochemically confined effects. As a single molecule translocates through the nanopore, the featured ionic current pattern on the time scale contains single molecule characteristics including volume, charge, and conformational properties. Although the characteristics of a single molecule in a nanopore have been written to the featured ionic current, extracting the dynamic information from a complex current trace is still a big challenge. Here, we present an applicable nanopore analysis method employing the Hilbert-Huang Transform (HHT) to study the vibrational features and interactions of a single molecule during the dynamic translocation process through the confined space of a nanopore. The HHT method is specially developed for analyzing nonlinear and non-stationary data that is highly compatible with nanopore data with a high frequency resolution. To provide proof-of-concept, we applied HHT to measure the frequency response for the wild-type (WT) aerolysin and mutant K238E aerolysin nanopores with and without the presence of poly(dA)4, respectively. The energy-frequency-time distribution spectra demonstrate that the biological nanopore contributes greatly to the characteristics of the high frequency component (>2 kHz) in the current recording. Our results suggest that poly(dA)4 undergoes relatively more consistent and confined interactions with K238E than WT, leading to a prolonging of the duration time. Therefore, the characteristics in frequency analysis could be regarded as an "single-molecule ionic spectrum" inside the nanopore, which encodes the detailed behaviours of single-molecule weak interactions.

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.

Aiming at the difficulty and low automation in installing slabstone under dry-hanging technology which is heavy and large in size, a slabstone dry-hanging installation robot system was developed. According to the working requirements such as load, installing precision and process, we researched a 6-DOF of series-parallel manipulator, analyzed its structural and completed the inverse solution. The inverse position equation of the robot was derived based on the kinematical theory. Mathematical relation between the Position-Orientation parameters and the direction cosine matrix was established. Experiments show that the robot can achieve slabstone auto- installing, and solve the problems in the dry-hanging installing.

Direct, low-cost, label-free, and enzyme-free identification of single nucleobase is a great challenge for genomic studies. Here, this study reports that wild-type aerolysin can directly identify the difference of four types of single nucleobase (adenine, thymine, cytosine, and guanine) in a free DNA oligomer while avoiding the operations of additional DNA immobilization, adapter incorporation, and the use of the processing enzyme. The nanoconfined space of aerolysin enables DNA molecules to be limited in the narrow pore. Moreover, aerolysin exhibits an unexpected capability of detecting DNA oligomers at the femtomolar concentration. In the future, by virtue of the high sensitivity of aerolysin and its high capture ability for DNA oligomers, aerolysin will play an important role in the studies of single nucleobase variations and open up new avenues for a broad range of nucleic-acid-based sensing and disease diagnosis.