Development of a single nucleotide polymorphism-based strain-identified method for Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 using pan-genomics analysis.

The health benefits conferred by probiotics is specific to individual probiotic strains, highlighting the importance of identifying specific strains for research and production purposes. Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 are exceedingly valuable for commercial use with an excellent mixed-culture fermentation. To differentiate these 2 strains from other S. thermophilus and L. delbrueckii ssp. bulgaricus, a specific, sensitive, accurate, rapid, convenient, and cost-effective method is required. In this study, we conducted a pan-genome analysis of S. thermophilus and L. delbrueckii ssp. bulgaricus to identify species-specific core genes, along with strain-specific SNPs. These genes were used to develop suitable PCR primers, and the conformity of sequence length and unique SNPs was confirmed by sequencing for qualitative identification at the strain level. The results demonstrated that SNPs analysis of PCR products derived from these primers could distinguish CICC 6038 and CICC 6047 accurately and reproducibly from the other strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, respectively. The strain-specific PCR method based on SNPs herein is universally applicable for probiotics identification. It offers valuable insights into identifying probiotics at the strain level that is fit-for-purpose in quality control and compliance assessment of commercial dairy products.

Fermentation characteristics and postacidification of yogurt by Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 at optimal inoculum ratio.

This study aimed to investigate the symbiosis between Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047. In addition, the effect of their different inoculum ratios was determined, and comparison experiments of fermentation characteristics and storage stability of milk fermented by their monocultures and cocultures at optimal inoculum ratio were performed. We found the time to obtain pH 4.6 and ΔpH during storage varied among 6 inoculum ratios (1:1, 2:1, 10:1, 19:1, 50:1, 100:1). By the statistical model to evaluate the optimal ratio, the ratio of 19:1 was selected, which exhibited high acidification rate and low postacidification with pH values remaining between 4.2 and 4.4 after a 50-d storage. Among the 3 groups included in our analyses (i.e., the monocultures of S. thermophilus CICC 6038 [St] and Lb. bulgaricus CICC 6047 [Lb] and their cocultures [St+Lb] at 19:1), the coculture group showed higher acidification activity, improved rheological properties, richer typical volatile compounds, more desirable sensor quality after the fermentation process than the other 2 groups. However, the continuous accumulation of acetic acid during storage showed that acetic acid was more highly correlated with postacidification than d-lactic acid for the Lb group and St+Lb group. Our study emphasized the importance of selecting an appropriate bacterial consortium at the optimal inoculum ratio to achieve favorable fermentation performance and enhanced postacidification stability during storage.

Breathing-Driven Self-Powered Pyroelectric ZnO Integrated Face Mask for Bioprotection.

Rapid spread of infectious diseases is a global threat and has an adverse impact on human health, livelihood, and economic stability, as manifested in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Even though people wear a face mask as protective equipment, direct disinfection of the pathogens is barely feasible, which thereby urges the development of biocidal agents. Meanwhile, repetitive respiration generates temperature variation wherein the heat is regrettably wasted. Herein, a biocidal ZnO nanorod-modified paper (ZNR-paper) composite that is 1) integrated on a face mask, 2) harvests waste breathing-driven thermal energy, 3) facilitates the pyrocatalytic production of reactive oxygen species (ROS), and ultimately 4) exhibits antibacterial and antiviral performance is proposed. Furthermore, in situ generated compressive/tensile strain of the composite by being attached to a curved mask is investigated for high pyroelectricity. The anisotropic ZNR distortion in the bent composite is verified with changes in ZnO bond lengths and OZnO bond angles in a ZnO4 tetrahedron, resulting in an increased polarization state and possibly contributing to the following pyroelectricity. The enhanced pyroelectric behavior is demonstrated by efficient ROS production and notable bioprotection. This study exploring the pre-strain effect on the pyroelectricity of ZNR-paper might provide new insights into the piezo-/pyroelectric material-based applications.

A vertically paired electrode for redox cycling and its application to immunoassays.

An electrochemical immunoassay based on the redox cycling method was presented using vertically paired electrodes (VPEs), which were fabricated using poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) as an electrode material and parylene-C as a dielectric layer. For the application to immunoassays, different electrochemical properties of PEDOT:PSS were analyzed for the redox reaction of 3,3',5,5'-tetramethylbenzidine (TMB, the chromogenic substrate for enzyme-immunoassays) at different pH conditions, including the conductivity (σ), electron transfer rate constant (kapp), and double-layer capacitance (Cdl). The influencing factors on the sensitivity of redox cycling based on VPE based on PEDOT:PSS were analyzed for the redox reaction of TMB, such as the electrode gap and number of electrode pairs. Computer simulation was also performed for the redox cycling results based on VPEs, which had limitations in fabrication, such as VPEs with an electrode gap of less than 100 nm and more than five electrode pairs. Finally, the redox cycling based on VPE was applied to the medical diagnosis of human hepatitis-C virus (hHCV) using a commercial ELISA kit. The sensitivity of the redox cycling method for the medical diagnosis of hHCV was compared with conventional assay methods, such as TMB-based chromogenic detection, luminol-based chemiluminescence assay, and a rapid test kit (lateral flow immunoassay).

Carbon electrode obtained via pyrolysis of plasma-deposited parylene-C for electrochemical immunoassays.

In this study, parylene-C films from plasma deposition as well as thermal deposition were pyrolyzed to prepare a carbon electrode for application in electrochemical immunoassays. Plasma deposition could prepare parylene-C in a faster deposition rate and more precise control over the thickness in comparison with the conventional thermal deposition. To analyze the influence of the deposition method, the crystal and electronic structures of the pyrolyzed parylene-C films obtained via both deposition methods were compared using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. For application as a carbon electrode in immunoassays, the electrochemical properties of the pyrolyzed carbon films from two both deposition methods were analyzed, including the double layer capacitance (2.10 µF cm-2 for plasma deposition and 2.20 µF cm-2 for thermal deposition), the apparent electron transfer rate (approximately 1.1 × 10-3 cm s-1 for both methods), and the electrochemical window (approximately -1.0 ∼ 2.1 V for both methods). Finally, the applicability of the pyrolyzed carbon electrode from parylene-C was demonstrated for the diagnosis of human hepatitis-C using various amperometric methods, such as cyclic voltammetry, chronoamperometry, square-wave voltammetry and differential pulse voltammetry.

Role of hydrogen bond capacity of solvents in reactions of amines with CO2: A computational study.

Various computational methods were employed to investigate the zwitterion formation, a critical step for the reaction of monoethanolamine with CO2, in five solvents (water, monoethanolamine, propylamine, methanol and chloroform) to probe the effect of hydrogen bond capacity of solvents on the reaction of amine with CO2 occurring in the amine-based CO2 capture process. The results indicate that the zwitterion can be formed in all considered solvents except chloroform. For two pairs of solvents (methanol and monoethanolamine, propylamine and chloroform) with similar dielectric constant but different hydrogen bond capacity, the solvents with higher hydrogen bond capacity (monoethanolamine and propylamine) facilitate the zwitterion formation. More importantly, kinetics parameters such as activation free energy for the zwitterion formation are more relevant to the hydrogen bond capacity than to dielectric constant of the considered solvents, clarifying the hydrogen bond capacity could be more important than dielectric constant in determining the kinetics of monoethanolamine with CO2.

Comparative analysis reveals unexpected genome features of newly isolated Thraustochytrids strains: on ecological function and PUFAs biosynthesis.

BACKGROUND: Thraustochytrids are unicellular fungal-like marine protists with ubiquitous existence in marine environments. They are well-known for their ability to produce high-valued omega-3 polyunsaturated fatty acids (ω-3-PUFAs) (e.g., docosahexaenoic acid (DHA)) and hydrolytic enzymes. Thraustochytrid biomass has been estimated to surpass that of bacterioplankton in both coastal and oceanic waters indicating they have an important role in microbial food-web. Nevertheless, the molecular pathway and regulatory network for PUFAs production and the molecular mechanisms underlying ecological functions of thraustochytrids remain largely unknown. RESULTS: The genomes of two thraustochytrids strains (Mn4 and SW8) with ability to produce DHA were sequenced and assembled with a hybrid sequencing approach utilizing Illumina short paired-end reads and Pacific Biosciences long reads to generate a highly accurate genome assembly. Phylogenomic and comparative genomic analyses found that DHA-producing thraustochytrid strains were highly similar and possessed similar gene content. Analysis of the conventional fatty acid synthesis (FAS) and the polyketide synthase (PKS) systems for PUFAs production only detected incomplete and fragmentary pathways in the genome of these two strains. Surprisingly, secreted carbohydrate active enzymes (CAZymes) were found to be significantly depleted in the genomes of these 2 strains as compared to other sequenced relatives. Furthermore, these two strains possess an expanded gene repertoire for signal transduction and self-propelled movement, which could be important for their adaptations to dynamic marine environments. CONCLUSIONS: Our results demonstrate the possibility of a third PUFAs synthesis pathway besides previously described FAS and PKS pathways encoded in the genome of these two thraustochytrid strains. Moreover, lack of a complete set of hydrolytic enzymatic machinery for degrading plant-derived organic materials suggests that these two DHA-producing strains play an important role as a nutritional source rather than a nutrient-producer in marine microbial-food web. Results of this study suggest the existence of two types of saprobic thraustochytrids in the world's ocean. The first group, which does not produce cellulosic enzymes and live as 'left-over' scavenger of bacterioplankton, serves as a dietary source for the plankton of higher trophic levels and the other possesses capacity to live on detrital organic matters in the marine ecosystems.

High phylogenetic diversity and abundance pattern of Labyrinthulomycete protists in the coastal waters of the Bohai Sea.

The unicellular Labyrinthulomycete protists have long been considered to play a significant role in ocean carbon cycling. However, their distribution and biogeochemical function remain poorly understood. We present a large-scale study of their spatiotemporal abundance and diversity in the coastal waters of Bohai Sea using flow cytometry and high-throughput sequencing. These protists display niche preferences and episodic higher biomass than that of bacterioplankton with much phylogenetic diversity (> 4000 OTUs) ever reported. They were ubiquitous with a typical abundance range of 100-1000 cells ml-1 and biomass range of 0.06-574.59 µg C L-1 . The observed spatiotemporal abundance variations support the current 'left-over scavengers' nutritional model and highlight these protists as a significant component of the marine microbial loop. The higher average abundance and phylogenetic diversity in the nearshore compared with those in the offshore reveal their predominant role in the terrigenous matter decomposition. Furthermore, the differential relationship of the protist genera to environmental conditions together with their co-occurrence network suggests their unique substrate preferences and niche partitioning. With few subnetworks and possible keystone species, their network topology indicates community resilience and high connectance level of few operational taxonomic units (OTUs). We demonstrate the significant contribution of these protists to the secondary production and nutrient cycling in the coastal waters. As secondary producers, their role will become more important with increasingly coastal eutrophication.

A High-Resolution Time Series Reveals Distinct Seasonal Patterns of Planktonic Fungi at a Temperate Coastal Ocean Site (Beaufort, North Carolina, USA).

There is a growing awareness of the ecological and biogeochemical importance of fungi in coastal marine systems. While highly diverse fungi have been discovered in these marine systems, still, little is known about their seasonality and associated drivers in coastal waters. Here, we examined fungal communities over 3 years of weekly sampling at a dynamic, temperate coastal site (Pivers Island Coastal Observatory [PICO], Beaufort, NC, USA). Fungal 18S rRNA gene abundance, operational taxonomic unit (OTU) richness, and Shannon's diversity index values exhibited prominent seasonality. Fungal 18S rRNA gene copies peaked in abundance during the summer and fall, with positive correlations with chlorophyll a, SiO4, and oxygen saturation. Diversity (measured using internal transcribed spacer [ITS] libraries) was highest during winter and lowest during summer; it was linked to temperature, pH, chlorophyll a, insolation, salinity, and dissolved inorganic carbon (DIC). Fungal communities derived from ITS libraries were dominated throughout the year by Ascomycota, with contributions from Basidiomycota, Chytridiomycota, and Mucoromycotina, and their seasonal patterns linked to water temperature, light, and the carbonate system. Network analysis revealed that while cooccurrence and exclusion existed within fungus networks, exclusion dominated the fungus-and-phytoplankton network, in contrast with reported pathogenic and nutritional interactions between marine phytoplankton and fungi. Compared with the seasonality of bacterial communities in the same samples, the timing, extent, and associated environmental variables for fungi community are unique. These results highlight the fungal seasonal dynamics in coastal water and improve our understanding of the ecology of planktonic fungi.IMPORTANCE Coastal fungal dynamics were long assumed to be due to terrestrial inputs; here, a high-resolution time series reveals strong, repeating annual patterns linked to in situ environmental conditions, arguing for a resident coastal fungal community shaped by environmental factors. These seasonal patterns do, however, differ from those observed in the bacterioplankton at the same site; e.g., fungal diversity peaks in winter, whereas bacterial diversity maxima occur in the spring and fall. While the dynamics of these communities are linked to water temperature and insolation, fungi are also influenced by the carbonate system (pH and DIC). As both fungi and heterotrophic bacteria are thought to be key organic-material metabolizers, differences in their environmental drivers may offer clues as to which group dominates secondary production at this dynamic site. Overall, this study suggests the unique ecological roles of mycoplankton and their potentially broad niche complementarities to other microbial groups in the coastal ocean.

MLN4924 suppresses the BRCA1 complex and synergizes with PARP inhibition in NSCLC cells.

Like ubiquitination, several studies have demonstrated that neddylation is implicated to be involved in the double strand break repair. BRCA1 is one of the key repair factors in the homologous recombination repair and may play a downstream role of the neddylation. BRCA1 is also a frequently mutated gene in cancers, which serve as the targets for PARP inhibitors. Here we further investigated the correlation between neddylation and BRCA1 complex using neddylation inhibitor MLN4924. MLN4924 efficiently inhibited the recruitment of components of BRCA1 complex to DNA damage sites. Thus MLN4924 may collaborate with PARP inhibitor to suppress tumor. Our results showed that combination MLN4924 and PARP inhibitor Olaparib impaired the DNA repair process in NSCLC cells. Furthermore, MLN4924 and Olaparib significantly inhibited the cancer cell growth. Kaplan-Meier survival analysis from lung cancer patients showed that high expression of NEDD8, BRCA1 and PARPs correlate with worse overall survival. Thus the combination of MLN4924 and PARP inhibitor may serve as a new strategy for NSCLC treatment.

A Method to Generate and Analyze Modified Myristoylated Proteins.

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.

Structure-activity relationship studies of QS11, a small molecule Wnt synergistic agonist.

Both the Wnt/ß-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/ß-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/ß-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11.

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells.

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Wearable fabric-based ZnO nanogenerator for biomechanical and biothermal monitoring.

Wearable devices that can mechanically conform to human skin are a necessity for reliable monitoring and decoding of biomechanical activities through skin. Most inorganic piezoelectrics, however, lack deformability and damage tolerance, impeding stable motion monitoring. Here, we present an air-permeable fabric-based ZnO nanogenerator with mechanical adaptivity to diverse deformations for wearable piezoelectric sensors, collecting biomechanical health data. We fabricate ZnO nanorods incorporated throughout the entire nylon fabric, with a strategically positioned neutral mechanical plane, for bending-sensitive electronics (2.59 µA mm). Its hierarchically interlocked geometry also permits sensitive tactile sensing (0.15 nA kPa-1). Various physiological information about activities, including pulse beating, breathing, saliva swallowing, and coughing, is attained using skin-mounted sensors. Further, the pyroelectric sensing capability of a mask-attached device is demonstrated by identifying specific respiratory patterns. Our wearable healthcare sensors hold great promise for real-time monitoring of health-related vital signs, informing individuals' health status without disrupting their daily lives.

Defect-Passivated Photosensor Based on Cesium Lead Bromide (CsPbBr3) Perovskite Quantum Dots for Microbial Detection.

A defect-passivated photosensor based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QD) was fabricated using parylene films, and the photosensor was applied for the microbial detection. The CsPbBr3 perovskite QDs were synthesized to be homogeneous in size under thermodynamic control, and the perovskite QD-based photosensor was fabricated using MoS2 flakes as the electron transfer layer. In this work, a parylene film with functional groups was deposited on a photosensor for physical protection (waterproof) and defect (halide vacancy) passivation of the perovskite QD. As the first effect of the parylene film, the physical protection of the perovskite QD from water was estimated by comparing the photosensor performance after incubation in water. As the second effect of the parylene, the interaction between the functional groups of the parylene film and the halide vacancies of the perovskite QDs was investigated through the bandgap, crystal structure, and trap-state density analysis. Additionally, density functional theory analysis on Mulliken charges, lattice parameters, and Gibbs free energy demonstrated the effect of the defect passivation by parylene films. Finally, the parylene-passivated QD-based photosensor was applied to the detection of two kinds of food-poisoning and gastroduodenal disease bacteria (Listeria monocytogenes and Helicobacter pylori).

Incorporation of a fluorous diazirine group into phosphatidylinositol 4,5-bisphosphate to illustrate its interaction with ADP-ribosylation factor 1.

Phosphatidylinositides are one family of the most versatile signaling molecules in cells, yet how they interact with different proteins to regulate biological processes is not well understood. Towards a general strategy to identify phosphatidylinositide-protein interactions, a fluorous diazirine group has been incorporated into phosphatidylinositol 4,5-bisphosphate (PIP(2)). The modified PIP(2) was effectively cleaved by phospholipase C, one signaling protein that utilizes PIP(2) as its endogenous substrate. Upon light illumination, the PIP(2) probe effectively crosslinks with small GTPase ADP-ribosylation 1 to form a complex, suggesting that the probe might be suitable to identify PIP(2)-interacting proteins on the proteome level.

MicroRNA-4735-3p Facilitates Ferroptosis in Clear Cell Renal Cell Carcinoma by Targeting SLC40A1.

Objective: Clear cell renal cell carcinoma (ccRCC) is the major histopathological subtype of renal cancer, and ferroptosis is implicated in the pathogenesis of ccRCC. The present study was aimed at investigating the role and underlying mechanisms of microRNA-4735-3p (miR-4735-3p) in ccRCC. Methods: Human ccRCC cell lines were transfected with the miR-4735-3p mimic or inhibitor to manipulate the expression of miR-4735-3p. Cell proliferation, colony formation, cell migration, cell invasion, cell death, oxidative stress, lipid peroxidation, and iron metabolism were determined. To validate the necessity of solute carrier family 40 member 1 (SLC40A1), human ccRCC cell lines were overexpressed with SLC40A1 using adenoviral vectors. Results: miR-4735-3p expression was reduced in human ccRCC tissues and cell lines but elevated upon ferroptotic stimulation. The miR-4735-3p mimic increased, while the miR-4735-3p inhibitor decreased oxidative stress, lipid peroxidation, iron overload, and ferroptosis of human ccRCC cell lines. Mechanistic studies identified SLC40A1 as a direct target of miR-4735-3p, and SLC40A1 overexpression significantly attenuated iron overload and ferroptosis in the miR-4735-3p mimic-treated human ccRCC cell lines. Conclusion: miR-4735-3p facilitates ferroptosis and tumor suppression in ccRCC by targeting SLC40A1.

Capacitive biosensor based on vertically paired electrodes for the detection of SARS-CoV-2.

Vertically paired electrodes (VPEs) with multiple electrode pairs were developed for the enhancement of capacitive measurements by optimizing the electrode gap and number of electrode pairs. The electrode was fabricated using a conductive polymer layer of PEDOT:PSS instead of Ag and Pt metal electrodes to increase the VPE fabrication yield because the PEDOT:PSS layer could be effectively etched using a reactive dry etching process. In this study, sensitivity enhancement was realized by decreasing the electrode gap and increasing the number of VPE electrode pairs. Such an increase in sensitivity according to the electrode gap and the number of electrode pairs was estimated using a model analyte for an immunoassay. Additionally, a computer simulation was performed using VPEs with different electrode gaps and numbers of VPE electrode pairs. Finally, VPEs with multiple electrode pairs were applied for SARS-CoV-2 nucleoprotein (NP) detection. The capacitive biosensor based on the VPE with immobilized anti-SARS-CoV-2 NP was applied for the specific detection of SARS-CoV-2 in viral cultures. Using viral cultures of SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV-strain 229E, the limit of detection (LOD) was estimated to satisfy the cutoff value (dilution factor of 1/800) for the medical diagnosis of COVID-19, and the assay results from the capacitive biosensor were compared with commercial rapid kit based on a lateral flow immunoassay.

Electrochemical One-Step Immunoassay Based on Switching Peptides and Pyrolyzed Carbon Electrodes.

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (Cdl), high electron transfer rate (kapp), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.

Design, synthesis, and incorporation of fluorous 5-methylcytosines into oligonucleotides.

A palladium-catalyzed Negishi coupling reaction has been developed to synthesize fluorous 5-methylcytosines. These fluorous nucleosides are incorporated into the oligonucleotides that correspond to part of the promoter region of Oct4, a master gene that undergoes dynamic DNA demethylation during cellular reprogramming. The separation of the fluorous oligonucleotides from its nonfluorous analogues has been achieved through solid-phase extraction over fluorous silica, suggesting its potential use in probing DNA demethylation.