Polymer coating for improved redox-polymer-mediated enzyme electrodes: A mini-review

Redox polymers have been widely used to facilitate and stabilize bioelectrochemical communication between the active sites of enzymes and electrodes, enabling development of high-performance enzyme electrodes for sensing, provision of power, and in synthesis. This review offers a brief overview of recent efforts in the use of additional polymer layers on top of a redox polymer/enzyme layer, which can both improve the performance and expand the functionality of the resulting bioelectrodes

High-Throughput Counting and Sizing of Therapeutic Protein Aggregates in the Nanometer Size Range by Nano-Flow Cytometry

Protein aggregation is one of the greatest challenges in biopharmaceuticals as it could decrease therapeutic efficacy, induce immunogenicity, and reduce shelf life of protein drugs. However, there lacks high-throughput methods than can count and size protein aggregates in the nanometer size range, especially for those smaller than 100 nm. Employing a laboratory-built nano-flow cytometer (nFCM) that enables light scattering detection of single silica nanoparticles as small as 24 nm with sizing resolution and accuracy comparable to those of electron microscopy, here, we report a new benchmark to analyze single protein aggregates as small as 40 nm. With an analysis rate of up to 10,000 particles/min, the size distribution and particle concentration of nanometer protein aggregates can be acquired in 2–3 min. Employing heat-induced aggregation of bovine serum albumin (BSA) at high concentrations as the model system, effects of different categories of excipients, including sugars, polyols, salts, and amino acids on the inhibition of protein aggregation were investigated. Strikingly enough, as high as 1010 to 1012 particles/mL of protein aggregates were observed in the size range of 40 to 200 nm for therapeutic proteins of human serum albumin injection, reconstituted recombinant human interieukin-2 solution, and human immunoglobulin injection. nFCM opens a new avenue to count and size nanometer protein aggregates, suggesting its future usability in the quality assessment and formulation promotion of therapeutic proteins

Design of a Polyoxometalate-Based Metal–Organic Framework for Photocatalytic C(sp³)–H Oxidation of Toluene

A powerful and promising route for developing novel photocatalysts for light-driven toluene oxidation in water under mild conditions is presented. Herein, a novel polyoxometalate-based metal–organic framework (POMOF), {Co4W22-DPNDI}, is prepared by incorporating the unusual Co4-sandwiched POM anion [Co4(μ-OH)2(SiW11O39)2]10– ({Co4W22}) and the photoactive organic bridging link N,N′-bis­(4-pyridylmethyl)­naphthalene diimide (DPNDI) into a framework. {Co4W22} is a good candidate for photocatalytic water oxidation. DPNDI is easily excited to form the radical species DPNDI* in the presence of an electron donor, which is beneficial for activation of the inert O2. Anion···π interactions and covalent bonds between {Co4W22} and DPNDI facilitate electron–hole separation and electron transfer. {Co4W22-DPNDI} displays high catalytic activity for the activation of the C­(sp3)–H bond of toluene using light as a driving force and inexpensive water as an oxygen source under mild conditions. In particular, the yield and selectivity are improved by replacing oxygen with water, which may be ascribed to the release of protons during the water oxidation process that facilitate the generation of •OH

Quantification of Available Ligand Density on the Surface of Targeted Liposomal Nanomedicines at the Single-Particle Level

Active targeting has been hailed as one of the most promising strategies to further enhance the therapeutic efficacy of liposomal nanomedicines. Owing to the critical role of ligand density in mediating cellular uptake and the intrinsic heterogeneity of liposomal formulations, precise quantification of the surface ligand density on a single-particle basis is of fundamental importance. In this work, we report a method to simultaneously measure the particle size and the number of ligands on the same liposomal nanoparticles by nanoflow cytometry. Then the ligand density for each individual liposome can be determined. With an analysis rate up to 10 000 particles per minute, a statistically representative distribution of ligand density could be determined in minutes. By utilizing fluorescently labeled recombinant receptors as the detection probe against the conjugated ligands, only those available for cell targeting can be exclusively detected. The influence of ligand input, conjugation strategy, and the polyethylene glycol spacer length on the available ligand density of folate-modified liposomes was investigated. The correlation between the available ligand density and cell targeting capability was assessed in a quantitative perspective for liposomes modified with three different targeting moieties. The optimal ligand density was determined to be 0.5–2.0, 0.7, and 0.2 ligand per 100 nm2 for folate-, transferrin-, and HER2-antibody-conjugated liposomes, respectively. These optimal values agreed well with the spike density of the natural counterparts, viruses. The as-developed approach is generally applicable to a wide range of active-targeting nanocarriers

LC-MS based serum metabonomic analysis for renal cell carcinoma diagnosis, staging, and biomarker discovery

A LC−MS based method, which utilizes both reversed-performance (RP) chromatography and hydrophilic interaction chromatography (HILIC) separations, has been carried out in conjunction with multivariate data analysis to discriminate the global serum profiles of renal cell carcinoma (RCC) patients and healthy controls. The HILIC was found necessary for a comprehensive serum metabonomic profiling as well as RP separation. The feasibility of using serum metabonomics for the diagnosis and staging of RCC has been evaluated. One-hundred percent sensitivity in detection has been achieved, and a satisfactory clustering between the early stage and advanced-stage patients is observed. The results suggest that the combination of LC−MS analysis with multivariate statistical analysis can be used for RCC diagnosis and has potential in the staging of RCC. The MS/MS experiments have been carried out to identify the biomarker patterns that made great contribution to the discrimination. As a result, 30 potential biomarkers for RCC are identified. It is possible that the current biomarker patterns are not unique to RCC but just the result of any malignancy disease. To further elucidate the pathophysiology of RCC, related metabolic pathways have been studied. RCC is found to be closely related to disturbed phospholipid catabolism, sphingolipid metabolism, phenylalanine metabolism, tryptophan metabolism, fatty acid beta-oxidation, cholesterol metabolism, and arachidonic acid metabolism

Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein–Protein Interactions via Relative Reporter Protein Expression Measurement

Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein–protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein–protein interaction, and compare interaction strength of different pairs

Rapid, Absolute, and Simultaneous Quantification of Specific Pathogenic Strain and Total Bacterial Cells Using an Ultrasensitive Dual-Color Flow Cytometer

This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli O157:H7 and harmless E. coli DH5α was used as a model system. Pathogenic E. coli O157:H7 cells were selectively labeled by red fluorescent probe via antibody−antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli O157:H7 emits both red and green fluorescence, while E. coli DH5α exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 × 105 cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R2 = 0.9993) and E. coli DH5α (R2 = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5α were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 × 102 cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification

Designing a Polyoxometalate-Incorporated Metal–Organic Framework for Reduction of Nitroarenes to Anilines by Sequential Proton-Coupled Electron Transfers

Developing new photocatalysts for reduction of nitroarenes to anilines under mild conditions is very significant. Herein, a new polyoxometalate-based metal–organic framework (POMOF), {[Co­(H2O)]2[Co2(H2O)6(TPT)]­[Co­(TPT)­PW11O39]}·3H2O·TPT (namely, CoW-TPT, TPT = 2,4,6-tri­(4-pyridyl)-1,3,5-triazine), was prepared by incorporating Co­(II)-substituted Keggin-type anions [PCoW11O39]5– and a photosensitizer (TPT) into a framework. In this structure, the direct coordination bond between [PCoW11O39]5– and TPT ligand and π···π interactions between TPT molecules are beneficial for the separation and migration of photogenerated carriers, thus improving the photocatalytic activity of CoW-TPT. The combination of both photosensitizer TPT and the electron-storable component [PCoW11O39]5– in a cooperative photocatalysis fashion is conducive to the photocatalytic multielectron reduction of nitroarenes. CoW-TPT provided a high conversion of 94.71% in the photocatalytic reduction of nitroarenes to anilines utilizing triethanolamine as the proton source and electron donor by sequential proton-coupled electron transfers

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes