Universal Method For Fabrication Research Articles

Exploration of DNA Binding Proteins As a Versatile Tool for Fabrication of DNA-Protein Complexes and Its Application to Biosensing System

Introduction DNA-protein complexes composed of a DNA aptamer and an enzyme are useful to realize innovative biosensing systems; however, the stoichiometric and efficient fabrication method for DNA-protein complexes is yet to be developed. Chemical modification using amino acids on the surface of the protein is known as one irreversible conjugation way to fabricate DNA-protein complexes, but stoichiometric conjugation and retention of protein function to ensure high detection sensitivity are still a challenge. Therefore, in recent years, DNA binding proteins that covalently bind to specific DNA sequences have been utilized to fabricate DNA-protein complexes and applied as sensing elements. However, the binding efficiency to DNA of the DNA binding protein was low, which caused a decrease in detection sensitivity.Here, we introduce our challenge to utilize DNA binding proteins with high DNA binding ability as a versatile tool suitable for the fabrication of DNA-protein complexes. By fusing such DNA binding proteins to protein of interest, can be covalently linked to the fusion protein, leading to the preparation of stoichiometric DNA-protein complexes universally and conveniently (Fig. 1). We demonstrated the feasibility to construct a biosensing system of hemoglobin (Hb), composed of glucose oxidase (GOx) and anti-Hb aptamer as a representative example. Results & Discussion We explored the DNA binding proteins that can covalently bind to specific DNA sequences using the database to fabricate the stoichiometric and stable DNA-protein complex. Seventeen candidates were selected and classified into two families: HUH-endonuclease and uracil DNA glycosylase. We then selected three kinds of HUH-endonucleases: Rep domain of tomato yellow leaf curl virus (TYLCV), replication protein B (RepB), and conjugation transfer relaxase/helicase TraI (TraI). We expected them to have high recombinant production in the E. coli expression system because TYLCV and RepB have a relatively low molecular weight and TraI is derived from E. coli. We also selected UdgX because it is the only uracil DNA glycosylase known to form covalent bonds with DNA after removing uracil that aberrantly exists on DNA1). Thus, four DNA binding proteins with the ability to covalently bind to DNA were selected for further characterization.The binding ability of the four DNA binding proteins was compared. Each DNA binding protein was reacted with single stranded DNA (ssDNA) containing the corresponding recognition sequence and subjected to SDS-PAGE analysis. An ssDNA-dependent concentration band shift to higher molecular weight was observed for all DNA binding proteins upon covalent binding to each ssDNA. Based on the band quantification, we confirmed that UdgX had the highest binding efficiency and thus we used UdgX in following experiments.We attempted to detect Hb utilizing a biosensing element composed of GOx and anti-Hb aptamer fabricated using UdgX. We recently successfully integrated a protein coupling module, SpyCatcher/Tag system, into the universal fabrication method of sensing elements2-4). GOx and UdgX were also conjugated using SpyCatcher/SpyTag system, and then reacted with anti-Hb aptamer to fabricate aptamer-GOx complex. Hb has peroxidase activity and catalyzes chemiluminescence of luminol. Therefore, by using the aptamer-GOx complex, hydrogen peroxide produced by GOx is supplied to the Hb efficiently, and chemiluminescence of luminol is observed (Fig. 2).We confirmed homogeneous preparation of the aptamer-GOx complex by SDS-PAGE, and also the binding ability of aptamer and enzymatic activity of GOx. Using thus prepared DNA-protein complex, Hb was successfully detected with a high linearity in buffer and also in human serum without any washing procedures. The detection range was 4.0×10-3 to 32×10-3 g/L, this can meet the clinically required one (70 to 200 g/L) by diluting an actual sample. Because this system uses redox enzyme, it can be applied into the electrochemical measurement to achieve more rapid Hb detection. In this system, owing to integration of DNA binding protein and SpyCatcher/SpyTag system into sensing elements, a set of DNA aptamer and enzyme can be also easily exchanged, thus which has potential of various applications. References 1) P. B. Sang et al., Nucleic Acids Res., 43, 8452–8463 (2015).2) H. Kimura et al., Anal. Chem., 90, 14500-14506 (2018).3) H. Kimura et al., Biosens. Bioelectron., 175, 112885 (2021).4) D. Miura et al., Talanta, 234, 122638 (2021). Figure 1

Fabrication of free-standing flexible and highly efficient carbon nanotube film/PEDOT: PSS thermoelectric composites

Carbon nanotube film (CNTF) with two-dimensional CNT network structure is adopted to prepare CNTF/poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) thermoelectric composites, which overcomes the disadvantages of low content, easy aggregation, and random orientation of CNTs when dispersed in polymer. A vacuum-assisted filtration method was proposed, which can uniformly and sufficiently penetrate the polymer into CNTF along thickness direction for fabrication of CNTF/PEDOT:PSS composites. A highest electrical conductivity of 806.2 S/cm at 300 K was achieved for the composites with 60 wt% PEDOT:PSS loading, which was 51.0% higher than that of the original CNTF (534.1 S/cm). A maximum power factor of 339.6 μW·m−1·K−2 at 320 K was achieved with a corresponding Seebeck coefficient of 67.7 μV/K. This study provides a universal method for fabrication of other kinds of CNTF/conductive polymer thermoelectric composites.

A universal method to fabricating porous carbon for Li-O2 battery

The main bottleneck in the application of biomass-based carbon material in energy field is the development of an economical, nanoscale controllable and universal fabrication method. Herein, we demonstrate that a biomass-derived carbon consisting of loose carbon nanosphere clusters could serve as an ideal cathode material for high performance Li-O2 batteries. The carbon nanomaterial can be prepared from both biomass derivatives and crude biomass by a low cost, facile and nanoscale controllable method. The open slit-shaped hierarchical pore structure endows the electrode sufficient active surface sites and mass transport channels that are not easily asphyxiated by the discharge products. The concentration of oxygenic groups and structure defects on the surface of obtained carbon materials is very low, which minimizes the side reactions associated with carbon decomposition. The unique pore structure and surface properties of the carbon electrode give rise to a superhigh specific capacity of 20,300 mA h g−1 and an extremely long cycle life which a cyclic capacity retention of 100% is maintained for 543 cycles with current density of 0.2 mA cm−2 at controlled discharge-charge depths of 500 mA h g−1.

Convenient and Universal Fabrication Method for Antibody-Enzyme Complexes as Sensing Elements Using the SpyCatcher/SpyTag System.

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.

Facile synthesis of magnetic metal organic frameworks for highly efficient proteolytic digestion used in mass spectrometry-based proteomics

Mass spectrometry (MS) based bottom-up strategy is now the first choice for proteomics analysis. In this process, highly efficient and complete enzymatic degradation of protein samples is extremely important to achieve in-depth protein coverage and high-throughput protein profiling. However, conventional in-solution digestion suffer from long digestion time and enzyme autolysis that limit the protein sample processing throughput and identification accuracy. Here, we developed a novel type of magnetic metal organic frameworks (MOFs)-based immobilized enzyme reactor, Fe3O4@DOTA-ZIF-90-trypsin. By introducing a stable chelator, 1,4,7,10-tetraazacyclododecane N,N′,N″,N‴-tetraacetic acid (DOTA), onto the magnetic cores, the hybrid supporting matrix of the immobilized enzyme reactor Fe3O4@DOTA-ZIF-90 has novel characteristics that include: i) favourable magnetic response (1.01 emu g−1) that makes the operation easy and convenient, ii) ultrahigh surface area (565.21 m2 g-11) and active sites that ensure high loading amounts and covalent linkage of enzyme, and iii) excellent structural and thermal stability that endows the immobilized enzyme reactor a prolonged lifespan. The performance of the magnetic MOFs-immobilized trypsin is first investigated using the standard protein, BSA, and the results showed that the immobilized enzyme reactor exhibits satisfactory digestion efficiency within only 1 min with the sequence coverage (80%) that is comparable or even better than that (70%) of the traditional 12 h-free trypsin digestion. To test the applicability of the magnetic MOFs-based immobilized enzyme reactor, protein samples extracted from 400 oocytes in mice were digested through the new immobilized enzyme reactor. In total, 8957 peptides corresponding to 1843 protein groups are identified, which are nearly of 40% and 67% increases in the number of identified proteins and peptides compared to using in-solution digestion with free proteases. Specifically, the identification of oocyte-specific proteins was critical in the discovery of and understanding the regulation mechanism of oocyte maturation. Thus, this synthetic procedure of Fe3O4@DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of Fe3O4@DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.

Mussel-Inspired Polyglycerol Coatings with Controlled Wettability: From Superhydrophilic to Superhydrophobic Surface Coatings.

Facile approaches to substrate-independent surface coatings with special wettability properties, such as superhydrophobicity, superhydrophilicity, and superamphiphobicity, have been limited. To address this problem, we combined two separate biomimetic concepts of mussel-inspired adhesion and highly hierarchical lotuslike surface structures to develop a universal fabrication method for various superwetting systems on any kind of material. In this feature article, we summarize our work on mussel-inspired polyglycerol (MI-dPG) and its application in the area of superwetting interfacial materials. MI-dPG mimics not only the functional groups of mfp-5 but also their molecular weight and molecular structure, which results in strong and rapid adhesion to the substrate. Furthermore, the MI-dPG coating process provides precise roughness control. The construction of highly hierarchical and superhydrophilic structures was achieved either directly by pH-controlled aggregation or in combination with nanoparticles. Subsequent postmodification of these highly hierarchical structures with different fluorinated or nonfluorinated hydrophobic molecules yielded a surface with superhydrophobic and even superamphiphobic properties.

Microfabricated cells for chip-scale atomic clock based on coherent population trapping: Fabrication and investigation

A universal method for fabrication of miniature cells for frequency standards and quantum magnetometers containing 87Rb atoms in the atmosphere of inert gas neon based on integrated technologies is considered. The results of experimental studies of coherent population trapping signals observed for a series of cells which provided recovery of vapors of an alkali metal from the rubidium dichromate salt with the help of laser radiation are presented. The coherent population trapping signals with a typical linewidth of 2–3 kHz and a signal-to-noise ratio of 1500 in the 1-Hz bandwidth were observed, which allows one to provide a relative frequency stability of atomic clock of 10−11 at 100 s.

Fabrication of holographic blazed grating with Fourier synthesis exposure

A simple, direct, and universal fabrication method for holographic blazed gratings is proposed and a 79 lines/mm holographic echelle grating with 4 μm depth quasi-triangular grooves experimentally demonstrated as an example of a blazed grating fabricated directly by Fourier synthesis exposure. With the method, we converted a periodic grating groove profile into the exposure energy spatial distribution on the surface of a photoresist with the response characteristics of the particular photoresist. Then the exposure energy could be decomposed into a series of sinusoidal functions using Fourier series, and realized by superposing a series of two-beam interference patterns. In contrast with mechanically ruled gratings, the fabrication process of holographic gratings is quite quick and low cost, and especially no Rowland ghosts appear in their dispersion spectrum. The reported work will offer a new approach for blazed grating fabrication with a holographic process.

English

Introduction The fabrication methods and functionalization of lipid tubes are described. The fabrication methods of lipid tubes were classified into three categories, i.e. self-assembly of lipid molecules, microfluidic way to form tubes and stretching of giant v esicles using an electric field. The functionalization of lipid tubes was summarized as metallization and deposition of inor ganic silica onto tube surface, encapsulation of nanoparticles into hollow cylindrical tube of tubular microstructures, and embedding nanoparticles into the wall of lipid tubes to form functional nanocomposites or nanostructures. The applications of these nano- and microstructures are also given. This review gives a brief overview of the interesting field of lipid tubes. Conclusion The fabrication of tubular microstructures demonstrated the precise self-assembly property of lipid molecules. To explore a new universal fabrication method for lipid tubes is still a direction of this field. Templating technology using lipid tubular microstructures is a promising route to make diverse nanostructures. Duo to the high biocompatibility of lipid tubes, they can be designed as drug carriers to encapsulate hydrophilic molecules inside the tube or hydrophobic molecules within the lipid bilay er for drug delivery in biomedical research.