Thiol Modification Research Articles

Bioinspired interlaced wetting surfaces for continuous on-demand emulsion separation

Maintaining high separation performance during continuous emulsion separation remains a challenge. Herein, based on biomimetic coupling ideas, hole array interlaced wetting surfaces (HAIWSs) and mastoid array interlaced wetting surfaces (MAIWSs) were prepared by laser processing, electroless silver deposition, thiol modification, and spraying for on-demand emulsion separation. When the separation is going on, randomly moving emulsion droplets are prone to being captured by holes or mastoids due to interlaced wettability. Under this unique interface behavior, the occurrence of filter cake and pore clogging is reduced, thus achieving both high efficiency (∼99.5 and ∼99.3 %). Meanwhile, the high flux can also be maintained (∼3212 and ∼3458 L m−2 h−1). Significantly better than surfaces without pores or mastoid structures. Further, the as-prepared surfaces also exhibit excellent recyclability. After 50 separation cycles, optimized HAIWS and MAIWS still maintained high efficiency (∼96.2 and ∼95.8 %) and high flux (∼3042 and ∼3164 L m−2 h−1), exceeding other surfaces without hole or mastoid structure. Notably, complex physical/chemical cleaning processes are avoided. Besides, even in harsh conditions, HAIWS and MAIWS still maintain excellent stability. The above strategy provides a novel mechanism for effective on-demand emulsion separation and is expected to encourage the creation of new-class separation devices for oily wastewater treatment in industry.

Thiolated dextrin nanoparticles for curcumin delivery: Stability, in vitro release, and binding mechanism

To achieve the effective loading and delivery of curcumin, novel disulfide-crosslinked nanoparticles based on modified dextrin were developed for the encapsulation of curcumin. Thiolated dextrin (Dt-SH) was obtained via sodium periodate oxidation and cysteamine grafting. The Dt-SH exhibited a rough, flake-like morphology, was classified as an amorphous material and demonstrated enhanced enzyme resistance. Subsequently, spherical nanoparticles with sizes ranging from 92.52 to 157.12 nm and zeta potentials between +23.59 and + 29.90 mV were self-assembled in an aqueous solution. Thiol modification promoted interconnection and aggregation of the nanoparticles. These nanoparticles exhibited pH-dependent size variations. Taking curcumin as a hydrophobic model, nanoparticles showed intestinal targeted release in vitro. Fluorescence spectroscopy and thermodynamic analysis indicated that curcumin bound to Dt-SH nanoparticles primarily through hydrogen bonding and van der Waals forces, with hydrophobic interactions contributing. These findings supported the potential of thiolated dextrin nanoparticles in the effective delivery of hydrophobic compounds.

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity

Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.

Developing Field Effect Transistor Sensors Based on DNA Probes to Detect Staphylococcus Lugdunensis Bacteria

The goal of this research is to work with an Electrical Double Layer (EDL)-gated Field Effect Transistor (FET) to detect the bacteria Staphylococcus lugdunensis, which can cause a variety of illnesses, including infections of the skin and soft tissues. In rare cases, Staphylococcus lugdunensis can be successfully identified using the combination of a positive catalase test, a negative tube coagulase test, a positive PYR test, and a positive ornithine decarboxylase test. We are employing an Electrical DoubleLayer (EDL)-gated Field Effect Transistor (FET) to speed up the procedure because this test is time consuming and requires a highly competent individual to perform. An ssDNA probe was developed, and effectively immobilized on a gold surface electrode sensor with a square surface, and due to hybridization with the cDNA at different concentrations, electrical signals were generated in the form of current.Our sensor was fabricated using photolithography where the gold electrode surface having 500 μm x 500 μm gate sensing area consisting of each individually addressable sensor arranged in a 1 x 8 array which is the extent gate was expose for immobilization purpose. The sensor was then subjectedto O2 plasma & HCl solution for surface cleaning and fluorescence image of baregold were taken Figure1(b).The Single-Stranded DNA comprising of fluorescent dye (FAM) at 3’ and thiol modifier at 5’ was drop-casted on the gold surface and was incubated for 24 hours at 24°C. After, it went through a washing process in order to remove the unbound ssDNA and fluorescence images were taken. We observed that intensity was higher than that of baregold which indicates that our probe has successfully immobilized on the gold surface Figure 1(b).Figure 1(a) represents the device that we designed. The system works with N-Channel Enhancement-Mode DMOS FET (VN10LP) in which a continuous drain voltage (3.5 V) was applied and the drain current which is the signal, were measured at two distinct gate bias voltage (2V and 3V). cDNA having quencher (BHQ1) at 5’ was diluted in different concentration namely 1fM, 10fM, 100fM and 1000fM. These cDNA with different concentrations were drop-casted on our sensor with Tris-EDTA (TE) buffer as the baseline and starting with lower concentration to higher concentration every after 12 seconds periodically where we record the change in signal as shown in Figure 1(c).After the electrical measurement, fluorescence image was taken and we can observe that it quenched which indicates that the cDNA and the immobilized ssDNA has successfully hybridized which give the signal change. Figure 1: (a) Surface Immobilization Principal Display in Extended Gate EDL BioFETs. (b)Fluorescence value comparison. (c) Plot showing the change in drain current over time for aptamersensors exposed to various cDNA concentrations. Figure 1

Enhancement of direct electron transfer by aromatic thiol modification with truncated d-fructose dehydrogenase

A heterotrimeric membrane-bound d-fructose dehydrogenase (FDH) from Gluconobacter japonicus, composed of subunits L, C, and S, shows strong direct electron transfer (DET)-type bioelectrocatalytic performance in the two-electron oxidation of d-fructose. The electrode-active site of the enzyme is the heme c moiety in the subunit C. A previous study reported the construction of the subunit L/S subcomplex (ΔC FDH) lacking the subunit C. In this work, we attempted to realize the DET-type reaction of ΔC FDH using porous gold electrodes functionalized with several thiols, and investigated the influence of the thiols on DET. Kinetic analysis of the steady-state catalytic waves revealed that the electrode-active site of ΔC FDH is the [3Fe-4S] iron-sulfur cluster in the L subunit. The experimental results demonstrated that uncharged aromatic thiols on the electrode enhance the DET-type reaction of ΔC FDH. Based on the pH-dependent profile of the DET-type activity and the surface conditions of the modified thiols evaluated by electrochemical reductive desorption, we suggest an enhancement mechanism for DET by aromatic thiols.

A Novel Analog of the Natural Product Fraxinellone Protects against Endogenous and Exogenous Neurotoxicants.

Numerous insults, both endogenous (e.g., glutamate) and exogenous (e.g., pesticides), compromise the function of the nervous system and pose risk factors for damage or later disease. In previous reports, limonoids such as fraxinellone showed significant neuroprotective activity against glutamate (Glu) excitotoxicity and reactive oxygen species (ROS) production in vitro, albeit with minimal mechanistic information provided. Given these findings, a library of novel fraxinellone analogs (including analogs 1 and 2 described here) was synthesized with the goal of identifying compounds exhibiting neuroprotection against insults. Analog 2 was found to be protective against Glu-mediated excitotoxicity with a measured EC50 of 44 and 39 nM for in vitro assays using PC12 and SH-SY5Y cells, respectively. Pretreatment with analog 2 yielded rapid induction of antioxidant genes, namely, Gpx4, Sod1, and Nqo1, as measured via qPCR. Analog 2 mitigated Glu-mediated ROS. Cytoprotection could be replicated using sulforaphane (SFN), a Nrf2 activator, and inhibited via ML-385, which inhibits Nrf2 binding to regulatory DNA sequences, thereby blocking downstream gene expression. Nrf2 DNA-binding activity was demonstrated using a Nrf2 ELISA-based transcription factor assay. In addition, we found that pretreatment with the thiol N-acetyl Cys completely mitigated SFN-mediated induction of antioxidant genes but had no effect on the activity of analog 2, suggesting thiol modification is not critical for its mechanism of action. In summary, our data demonstrate a fraxinellone analog to be a novel, potent, and rapid activator of the Nrf2-mediated antioxidant defense system, providing robust protection against insults.

Completely recyclable and reusable automatic phase-separable catalytic system for the reductive debromination of polybrominated diphenyl ethers

An automatic phase-separable catalytic system was developed for the reductive debromination of polybrominated diphenyl ethers (PBDEs) using thiol-modified Pd nanoparticles (thiol-Pd NPs) as the catalyst, H2 as the reducing agent, and n-butanol (oil phase) and water (water phase) as solvents. This system achieved efficient and complete debromination of various PBDEs with 1–10 bromine atoms and other bromo-organic pollutants at concentrations ranging from 15 to 1500 mg L−1. Typically, this system achieved a debromination efficiency of 100 % within 15 min for all the tested bromo-organic pollutants. Importantly, the debromination reaction was conducted in a microemulsion state under stirring; upon stopping the stirring, the microemulsion automatically separated into an upper oil phase, a down water phase, and a thiol-Pd NP self-assembled membrane phase at the oil/water interface. These three phases could be easily separated from each other, allowing for their reuse across multiple debromination cycles with good reusability. The thiol modifier enhanced the stability and reusability of Pd NPs and improved the adsorption of PBDEs, facilitating their reaction with atomic hydrogen (the dominant reactive species). This method successfully eliminated PBDEs in soils by integrating with a pre-extraction process.

Tetrahydrobiopterin metabolism attenuates ROS generation and radiosensitivity through LDHA S-nitrosylation: novel insight into radiogenic lung injury

Genotoxic therapy triggers reactive oxygen species (ROS) production and oxidative tissue injury. S-nitrosylation is a selective and reversible posttranslational modification of protein thiols by nitric oxide (NO), and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. However, the mechanism by which BH4 affects protein S-nitrosylation and ROS generation has not been determined. Here, we showed that ionizing radiation disrupted the structural integrity of BH4 and downregulated GTP cyclohydrolase I (GCH1), which is the rate-limiting enzyme in BH4 biosynthesis, resulting in deficiency in overall protein S-nitrosylation. GCH1-mediated BH4 synthesis significantly reduced radiation-induced ROS production and fueled the global protein S-nitrosylation that was disrupted by radiation. Likewise, GCH1 overexpression or the administration of exogenous BH4 protected against radiation-induced oxidative injury in vitro and in vivo. Conditional pulmonary Gch1 knockout in mice (Gch1fl/fl; Sftpa1-Cre+/− mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice (Gch1lsl/lsl; Sftpa1-Cre+/− mice) exhibited attenuated radiation-induced pulmonary toxicity. Mechanistically, lactate dehydrogenase (LDHA) mediated ROS generation downstream of the BH4/NO axis, as determined by iodoacetyl tandem mass tag (iodoTMT)-based protein quantification. Notably, S-nitrosylation of LDHA at Cys163 and Cys293 was regulated by BH4 availability and could restrict ROS generation. The loss of S-nitrosylation in LDHA after irradiation increased radiosensitivity. Overall, the results of the present study showed that GCH1-mediated BH4 biosynthesis played a key role in the ROS cascade and radiosensitivity through LDHA S-nitrosylation, identifying novel therapeutic strategies for the treatment of radiation-induced lung injury.

Redox proteomics in melanoma cells: An optimized protocol

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.

A few-minute, simple, and affordable route to functionalized electrodes with DNA

A key challenge in developing DNA-based electrochemical sensors is the rapid and easy immobilization of DNA on different electrode surfaces. The current methods are often laborious and time-consuming. Here, we present a cost-effective and easy-to-perform method for efficiently functionalizing electrode surfaces with DNA in just a few minutes. Our strategy operates in two simple and rapid steps, regardless of the type of electrode, and avoids complicated and time-consuming procedures. The method uses phosphorothioate (PS) modification as an anchoring agent, which is a more cost-effective alternative to thiol (SH) modification, and eliminates the need for complex pretreatment processes. Additionally, a low-pH DNA loading method was used to quickly bind PS DNA to the gold surface without the need for surface-covering agents. We attributed the effect of low pH to the protonation of adenine (A), which leads to the formation of a parallel poly-A duplex (A motif) and facilitates vertical PS-DNA attachment to the electrode surface. Electrochemical techniques were used to confirm the vertical attachment of DNA to the electrode surface, and evaluation of the adsorption stability and hybridization efficiency confirmed the effectiveness of the method. The simplicity and efficiency of this method make it an ideal solution for researchers who require a fast and reliable method for DNA functionalization of electrode surfaces.

Enhancing Cd (II) immobilization with thiol-modified low-temperature pyrolysis biochar: Efficiency, mechanism, and applications

The fixation efficiency of thiol-modified lotus seedpod biochar on Cd (II) was assessed through a comprehensive investigation involving batch experiments and sediment cultivation experiments. The influence of thiol modification on the Cd (II) adsorption performance was investigated under different pyrolysis temperatures (550°C, 700°C, and 850°C). The physicochemical properties, adsorption kinetics, isotherms, and adsorption mechanisms of the modified biochar were studied. Oxidation and thiolation significantly reduced the specific surface area and total pore volume of the biochar, augmenting its hydrophilicity. High-temperature pyrolysis biochar was more suitable for oxidation modification than thiol modification. The abundant oxygen-containing functional groups in low-temperature pyrolysis biochar can serve as active sites for thiol loading. Therefore, it exhibited the highest grafting rate, the highest affinity for Cd (II), and the maximum Cd (II) adsorption capacity of 68 mg/g. Thiol-modified biochar primarily immobilized Cd (II) through chelation. Moreover, it significantly reduced the concentration of bioavailable Cd (II) in the sediment and enhanced enzymatic activity. These findings underscore the potential role of low-temperature pyrolysis thiol-modified biochar in addressing Cd (II) pollution in water and sediments.

The multifunctional flexible conductive viscose fabric prepared by thiol modification followed by copper plating

Based on the current rapid development of electronic products, the development of light-weight, processable, environmentally friendly, long-life, durable, less corrosive, and tunable conductive composite materials with multiple applications may be the development direction of next-generation electronic devices. In this work, for the first time, we employed 3-Mercaptopropyltrimethoxysilane (3-MT) to modify viscose nonwovens and enhance the copper plating process. The prepared samples were characterized by Fourier transform infrared, Wide-angle X-ray diffraction (WAXD), scanning electron microscope + energy dispersive X-ray spectroscopy (SEM + EDS), thermogravimetric analysis (TGA), electrical resistivity, anti-corrosion, Joule heating, and electromagnetic interference (EMI) shielding. Results showed that 3-MT was covalently bound to the viscose surface through hydrolysis and condensation reactions and introduced SH groups. WAXD confirmed that the thiol modification did not change the internal crystal structure of viscose and copper ions. TGA and surface morphology analysis confirmed that the modified viscose promoted the deposition of metal particles in the copper plating process due to the affinity of thiol to metal so that copper particles almost completely wrapped the viscose fibers. In addition, 3MT@Cu@Viscose exhibits extremely low surface and volume resistivity (346.6 and 333.2 mΩ·m), improved corrosion resistance (corrosion rate reduced by 58% compared to the unmodified sample), fast Joule heating response (within 10 s) in low voltage (1 V) and excellent EMI shielding effectiveness (EMI SE > 50 dB). It showed great potential in future multi-functional electronic products such as electric heating sensors, smart clothing, and EMI shielding barrier.

Ageing-dependent thiol oxidation reveals early oxidation of proteins with core proteostasis functions.

Oxidative post-translational modifications of protein thiols are well recognized as a readily occurring alteration of proteins, which can modify their function and thus control cellular processes. The development of techniques enabling the site-specific assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early-oxidation targets in key biological processes governing the de novo production of proteins, protein folding, and degradation, and indicate a hierarchy of cellular responses affected by a reversible redox modification. Comparison with existing datasets in yeast, nematode, fruit fly, and mouse reveals the evolutionary conservation of these oxidation targets. To facilitate accessibility, we integrated the cross-species comparison into the newly developed OxiAge Database.

Dual biomimetic surfaces with anisotropic wettability for multi-scale droplets manipulation

Droplets manipulation on functional biomimetic surfaces have been highly anticipated for their wide range of applications. However, preparing a metal platform for manipulating multi-scale droplets without external energy input remains a challenge. Herein, dual biomimetic surfaces (DBSs) were fabricated by twice laser and thiol modification. The unique interfacial behavior of the droplets can be realized under the synergistic effect of the special “millimeter-micrometer-nanometer” multi-hierarchical composite structure and chemical composition. The as-prepared DBSs can achieve multi-scale droplets manipulation, directional self-cleaning in various media, and efficient directional fog collection. Further, the mechanism of specific interface behavior is analyzed in depth. Nanoparticles endow DBSs with more capture points and condensation cores and micrometer mastoids realized the transmission and aggregation of microscopic droplets through the action of Laplace force. Based on resistance, energy barriers and three-phase contact line, influence of millimeter grooves on anisotropic wettability and directional transportation of the droplets is analyzed. Besides, the multi-hierarchical structure also endows DBSs with good droplets manipulation ability and stability. This work not only provides an advanced biomimetic platform, but also provides an important reference for metal-based lab-on-a-chip for multi-scale droplets manipulation.

Thiolated chitosan encapsulation constituted mucoadhesive nanovaccine confers broad protection against divergent influenza A viruses

Influenza A virus (IAV) poses a significant threat to human and animal health, necessitating the development of universal influenza vaccines that can effectively activate mucosal immunity. Intranasal immunization has attracted significant attention due to its capacity to induce triple immune responses, including mucosal secretory IgA. However, inducing mucosal immunity through vaccination is challenging due to the self-cleansing nature of the mucosal surface. Thiolated chitosan (TCS) were explored for mucosal vaccine delivery, capitalizing on biocompatibility and bioadhesive properties of chitosan, with thiol modification enhancing mucoadhesive capability. The focus was on developing a universal nanovaccine by utilizing TCS-encapsulated virus-like particles displaying conserved B-cell and T-cell epitopes from M2e and NP proteins of IAV. The optimal conditions for nanoparticle formation were investigated by adjusting the thiol groups content of TCS and the amount of sodium tripolyphosphate. The nanovaccine induced robust immune responses and provided complete protection against IAVs from different species following intranasal immunization. The broad protective effect of nanovaccines can be attributed to the synergistic effect of antibodies and T cells. This study developed a universal intranasal nanovaccine and demonstrated the potential of TCS in the development of mucosal vaccines for respiratory infectious diseases.

Synthesis and Characterization of Highly Thiolated Silk Fibroin

AbstractCovalent attachment of thiols to the tyrosine residues of silk fibroin is accomplished with a high degree of functionalization through the reaction of a pyridyldithiol‐containing N‐hydroxysuccinimidal‐ester and an amino‐tyrosine silk intermediate. The extent of thiol modification is characterized by 1H NMR and UV–vis spectroscopy. Further modification of the thiol groups is probed by reacting with an iodoacetamide‐containing small molecule resulting in a novel fluorescent silk derivative. Last, the ability of the thiolated silk to form hydrogels in situ is investigated.

Improvement of Low-Cost Commercial Carbon Screen-Printed Electrodes Conductivities with Controlled Gold Reduction Towards Thiol Modification

Effectively detecting bacteria in the environment is crucial for researchers to make informed decisions about the safety of public areas, such as lakes. This led to an increased need in the development of portable handheld devices, capable of on-the-spot chemical and biological sensing applications. Specific interests lie in electrochemical biosensors and screen-printed electrodes (SPEs) due to the decreased costs, an ability to integrate with handheld devices, and their user-friendly nature. Together, these qualities make the devices more accessible in resource-poor settings. Two of the most common substrates used to fabricate SPEs are carbon and gold. Carbon SPEs are effective in sensing applications yet challenged when attempting to covalently attach biomolecules to the surface. Gold SPEs have higher affinity towards biomolecules and improve the sensitivity, selectivity, and stability of a device; yet they can be costly. A carbon SPE modified with gold may be an ideal candidate to create an efficient low-cost device, using electrochemical gold deposition. In this study, electrochemical gold deposition on SPEs is explored to enhance the surface area and conductivity towards sensing applications. These SPEs were then modified with a thiol-based self-assembled monolayer (SAM) which demonstrates this technique could be used for further modification towards biosensing.

Thermodynamic Overview of Bioconjugation Reactions Pertinent to Lysine and Cysteine Peptide and Protein Residues

Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities.

S-Protected Thiolated Chitosan versus Thiolated Chitosan as Cell Adhesive Biomaterials for Tissue Engineering.

Chitosan (Ch) and different Ch derivatives have been applied in tissue engineering (TE) because of their biocompatibility, favored mechanical properties, and cost-effectiveness. Most of them, however, lack cell adhesive properties that are crucial for TE. In this study, we aimed to design an S-protected thiolated Ch derivative exhibiting high cell adhesive properties serving as a scaffold for TE. 3-((2-Acetamido-3-methoxy-3-oxopropyl)dithio) propanoic acid was covalently attached to Ch via a carbodiimide-mediated reaction. Low-, medium-, and high-modified Chs (Ch-SS-1, Ch-SS-2, and Ch-SS-3) with 54, 107 and 140 μmol of ligand per gram of polymer, respectively, were tested. In parallel, three thiolated Chs, namely Ch-SH-1, Ch-SH-2, and Ch-SH-3, were prepared by conjugating N-acetyl cysteine to Ch at the same degree of modification to compare the effectiveness of disulfide versus thiol modification on cell adhesion. Ch-SS-1 showed better cell adhesion capability than Ch-SS-2 and Ch-SS-3. This can be explained by the more lipophilic surfaces of Ch-SS as a higher modification was made. Although Ch-SH-1, Ch-SH-2, and Ch-SH-3 were shown to be good substrates for cell adhesion, growth, and proliferation, Ch-SS polymers were superior to Ch-SH polymers in the formation of 3D cell cultures. Cryogels structured by Ch-SS-1 (SSg) resulted in homogeneous scaffolds with tunable pore size and mechanical properties by changing the mass ratio between Ch-SS-1 and heparin used as a cross-linker. SSg scaffolds possessing interconnected microporous structures showed good cell migration, adhesion, and proliferation. Therefore, Ch-SS can be used to construct tunable cryogel scaffolds that are suitable for 3D cell culture and TE.

Fabrication of electron tunneling probes for measuring single-protein conductance.

Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein-electrode contact. We use a biotinylated thiol modification, in which a biotin-streptavidin-biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.