Design and fabrication of flexible DNA polymer cocoons to encapsulate live cells.

The capability to encapsulate designated live cells into a biologically and mechanically tunable polymer layer is in high demand. Here, an approach to weave functional DNA polymer cocoons has been proposed as an encapsulation method. By developing in situ DNA-oriented polymerization (isDOP), we demonstrate a localized, programmable, and biocompatible encapsulation approach to graft DNA polymers onto live cells. Further guided by two mutually aided enzymatic reactions, the grafted DNA polymers are assembled into DNA polymer cocoons at the cell surface. Therefore, the coating of bacteria, yeast, and mammalian cells has been achieved. The capabilities of this approach may offer significant opportunities to engineer cell surfaces and enable the precise manipulation of the encapsulated cells, such as encoding, handling, and sorting, for many biomedical applications.

Voltammetric Response of Alizarin Red S-Confined Film-Coated Electrodes to Diol and Polyol Compounds: Use of Phenylboronic Acid-Modified Poly(ethyleneimine) as Film Component.

Alizarin red S (ARS) was confined in layer-by-layer (LbL) films composed of phenylboronic acid-modified poly(ethyleneimine) (PBA-PEI) and carboxymethylcellulose (CMC) to study the voltammetric response to diol and polyol compounds. The LbL film-coated gold (Au) electrode and quartz slide were immersed in an ARS solution to uptake ARS into the film. UV-visible absorption spectra of ARS-confined LbL film suggested that ARS formed boronate ester (ARS-PBS) in the film. The cyclic voltammetry of the ARS-confined LbL film-coated electrodes exhibited oxidation peaks at -0.50 and -0.62 V, which were ascribed to the oxidation reactions of ARS-PBS and free ARS, respectively, in the LbL film. The peak current at -0.62 V increased upon the addition of diol or polyol compounds such as L-dopa, glucose, and sorbitol into the solution, depending on the concentration, whereas the peak current at -0.50 V decreased. The results suggest a possible use of ARS-confined PBA-PEI/CMC LbL film-coated Au electrodes for the construction of voltammetric sensors for diol and polyol compounds.

Host-Guest Chemistry in Layer-by-Layer Assemblies Containing Calix[n]arenes and Cucurbit[n]urils: A Review.

This review provides an overview of the synthesis of layer-by-layer (LbL) assemblies containing calix[n]arene (CA[n]) and cucurbit[n]uril (CB[n]) and their applications. LbL assemblies, such as thin films and microcapsules, containing selective binding sites have attracted considerable attention because of their potential use in separation and purification, sensors for ions and molecules, and controlled release. CA[n]-containing LbL films have been prepared using sulfonated CA[n] and cationic polymers to construct chemical sensors and molecular containers. CA[n]-containing LbL films deposited on the surface of a porous support are useful as ion-selective membranes that exhibit selective permeability to monovalent ions over multivalent ions. CB[n]s have been used as molecular glues for the construction of LbL films and microcapsules by taking advantage of the strong affinity of CB[n]s to aromatic compounds. CB[n]s form a stable 1:1:1 ternary complex with electron-rich and electron-deficient molecules in LbL films to stabilize the assemblies. CB[n]-containing LbL films can also be deposited on the surfaces of micro templates and nanopore membranes to construct microcapsules for controlled release and nanochannels for selective ion transport, respectively.

Lactate-induced decomposition of layer-by-layer films composed of phenylboronic acid-modified poly(allylamine) and poly(vinyl alcohol) under extracellular tumor conditions.

Multilayer films that decompose in the presence of lactate were prepared by depositing phenylboronic acid-modified poly(allylamine) (PBA-PAH) and poly(vinyl alcohol) (PVA) on a lactate oxidase (LOx) layer. The layers adhered through boronate ester bonds. The resulting LOx(PBA-AH/PVA)10 film was stable in pH 7.4 solution but decomposed following the addition of lactate. The carbon-boron bonds in PBA residues were cleaved by oxidative reaction with H2O2 produced by the enzymatic reaction of LOx. Approximately 90% of the film decomposed following exposure for 120 and 30min to 0.05 and 20mM lactate at pH 7.4, respectively. The multilayer film therefore decomposed under conditions comparable to the extracellular environment of tumors (20mM lactate at pH 6.5). Our results show that LOx/(PBA-PAH/PVA)10 multilayer film could be used for cancer drug delivery systems.

Ultrasensitive Quantitation of Plasma Membrane Proteins via isRTA.

Quantitation of plasma membrane proteins (PMPs) is fundamental and frequently performed daily in the lab. However, challenged by the inherent/interacting heterostructures and complex surroundings of the PMPs in lipid membrane, quantitative techniques for PMP often require complex treatments (e.g., labeling, isolation, purification, and determination), and the sensitivity is usually not satisfactory. To address this problem, we have proposed a novel method that enables quantitation of PMPs with extremely high sensitivity, in an easier-to-manipulate and more streamlined way. This method is based on the design of an in situ rolling cycling replication-templated amplification strategy (isRTA). In fact, two rounds of DNA cascade isothermal amplifications have been conducted. The first round of amplification can provide templates for the second round of amplification; thus, significant enhancement of quantitative signals can be achieved. In this way, PMPs are quantified with ultrahigh sensitivity; as few as 25 copies of PMPs can be detected per cell. Moreover, the advantages of isRTA have been demonstrated by simultaneous identification of several PMP biomarkers (MUC1, EpCAM, and HER2) that are expressed over a wide distribution range on breast cancer cells. The precise typing of breast cancer cell subsets is thus possible because of the "quantitative-to-qualitative" strategy. Therefore, the unprecedented sensitivity and high usability of the isRTA method may present significant prospects for delving into membrane proteins and their related biofunctions in many research fields.

Alizarin Red S-Confined Layer-By-Layer Films as Redox-Active Coatings on Electrodes for the Voltammetric Determination of L-Dopa.

The preparation of redox-active coatings is a key step in fabricating electrochemical biosensors. To this goal, a variety of coating materials have been used in combination with redox-active compounds. In this study, alizarin red S (ARS) was confined in layer-by-layer (LbL) films composed of poly(ethyleneimine) (PEI) and carboxymethylcellulose (CMC) to study the redox properties. A gold (Au) disc electrode coated with PEI/CMC LbL film was immersed in an ARS solution to uptake ARS into the film. ARS was successfully confined in the LbL film through electrostatic interactions. The cyclic voltammogram (CV) of ARS-confined PEI/CMC film-coated electrodes thus prepared exhibited redox waves in the potential range from -0.5 to -0.7 V originating from 9,10-anthraquinone moiety in ARS, demonstrating that ARS preserves its redox activity in the LbL film. An additional oxidation peak appeared around -0.4 V in the CV recorded in the solution containing phenylboronic acid (PBA), due to the formation of a boronate ester of ARS (ARS-PBA) in the film. The oxidation peak current at -0.4 V decreased upon addition of 3,4-dihydroxyphenylalanine (L-dopa) to the solution. Thus, the results suggest a potential use of the ARS-confined PEI/CMC films for constructing voltammetric sensors for L-dopa.

Recent Progress in Nanomaterial-Based Electrochemical Biosensors for Cancer Biomarkers: A Review.

This article reviews recent progress in the development of nanomaterial-based electrochemical biosensors for cancer biomarkers. Because of their high electrical conductivity, high affinity to biomolecules, and high surface area-to-weight ratios, nanomaterials, including metal nanoparticles, carbon nanotubes, and graphene, have been used for fabricating electrochemical biosensors. Electrodes are often coated with nanomaterials to increase the effective surface area of the electrodes and immobilize a large number of biomolecules such as enzymes and antibodies. Alternatively, nanomaterials are used as signaling labels for increasing the output signals of cancer biomarker sensors, in which nanomaterials are conjugated with secondary antibodies and redox compounds. According to this strategy, a variety of biosensors have been developed for detecting cancer biomarkers. Recent studies show that using nanomaterials is highly advantageous in preparing high-performance biosensors for detecting lower levels of cancer biomarkers. This review focuses mainly on the protocols for using nanomaterials to construct cancer biomarker sensors and the performance characteristics of the sensors. Recent trends in the development of cancer biomarker sensors are discussed according to the nanomaterials used.

Photosensitive Layer-by-Layer Assemblies Containing Azobenzene Groups: Synthesis and Biomedical Applications.

This review provides an overview of the syntheses of photosensitive layer-by-layer (LbL) films and microcapsules modified with azobenzene derivatives and their biomedical applications. Photosensitive LbL films and microcapsules can be prepared by alternate deposition of azobenzene-bearing polymers and counter polymers on the surface of flat substrates and microparticles, respectively. Azobenzene residues in the films and microcapsules exhibit trans-to-cis photoisomerization under UV light, which causes changes in the physical or chemical properties of the LbL assemblies. Therefore, azobenzene-functionalized LbL films and microcapsules have been used for the construction of photosensitive biomedical devices. For instance, cell adhesion on the surface of a solid can be controlled by UV light irradiation by coating the surface with azobenzene-containing LbL films. In another example, the ion permeability of porous materials coated with LbL films can be regulated by UV light irradiation. Furthermore, azobenzene-containing LbL films and microcapsules have been used as carriers for drug delivery systems sensitive to light. UV light irradiation triggers permeability changes in the LbL films and/or decomposition of the microcapsules, which results in the release of encapsulated drugs and proteins.

Phenylboronic Acid-Functionalized Layer-by-Layer Assemblies for Biomedical Applications.

Recent progress in the development of phenylboronic acid (PBA)-functionalized layer-by-layer (LbL) assemblies and their biomedical applications was reviewed. Stimuli-sensitive LbL films and microcapsules that exhibit permeability changes or decompose in response to sugars and hydrogen peroxide (H2O2) have been developed using PBA-bearing polymers. The responses of PBA-modified LbL assemblies arise from the competitive binding of sugars to PBA in the films or oxidative decomposition of PBA by H2O2. Electrochemical glucose sensors have been fabricated by coating the surfaces of electrodes by PBA-modified LbL films, while colorimetric and fluorescence sensors can be prepared by modifying LbL films with boronic acid-modified dyes. In addition, PBA-modified LbL films and microcapsules have successfully been used in the construction of drug delivery systems (DDS). Among them, much effort has been devoted to the glucose-triggered insulin delivery systems, which are constructed by encapsulating insulin in PBA-modified LbL films and microcapsules. Insulin is released from the PBA-modified LbL assemblies upon the addition of glucose resulting from changes in the permeability of the films or decomposition of the film entity. Research into insulin DDS is currently focused on the development of high-performance devices that release insulin in response to diabetic levels of glucose (>10 mM) but remain stable at normal levels (~5 mM) under physiological conditions.

Sugar-sensitive dendrimer films as a sacrificial layer for the preparation of freestanding multilayer films.

Multilayer thin films composed of poly(vinyl alcohol) (PVA) and phenylboronic acid-bearing poly(amidoamine) dendrimer (PBA-PAMAM) were used as a sacrificial layer for constructing freestanding polyelectrolyte films consisting of poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). Freestanding (PSS/PAH)15 films were successfully released from substrate by exposing composite (PVA/PBA-PAMAM)n/(PSS/PAH)15 films (n=5 and 10) to sorbitol solutions under mild conditions at pH7.0-9.0. The film release was accelerated in solutions of higher sorbitol concentrations at pH9.0 as well as in solutions with lower concentration of NaCl. The results were rationalized based on the scission of boronate ester bonds between PBA-PAMAM and PVA in the (PVA/PBA-PAMAM)n layer due to a competitive binding of sorbitol to PBA-PAMAM.

Recent Progress in Electrochemical Biosensors for Glycoproteins.

This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

Recent progress in electrochemical biosensors based on phenylboronic acid and derivatives.

This review provides an overview of recent progress made in the development of electrochemical biosensors based on phenylboronic acid (PBA) and its derivatives. PBAs are known to selectively bind 1,2- and 1,3-diols to form negatively charged boronate esters in neutral aqueous media and have been used to construct electrochemical glucose sensors because of this selective binding. PBA-modified metal and carbon electrodes have been widely studied as voltammetric and potentiometric glucose sensors. In some cases, ferroceneboronic acid or ferrocene-modified phenylboronic acids are used as sugar-selective redox compounds. Another option for sensors using PBA-modified electrodes is potentiometric detection, in which the changes in surface potential of the electrodes are detected as an output signal. An ion-sensitive field effect transistor (FET) has been used as a signal transducer in potentiometric sensors. Glycoproteins, such as glycated hemoglobin (HbA1c), avidin, and serum albumin can also be detected by PBA-modified electrodes because they contain hydrocarbon chains on the surface. HbA1c sensors are promising alternatives to enzyme-based glucose sensors for monitoring blood glucose levels over the preceding 2-3months. In addition, PBA-modified electrodes can be used to detect a variety of compounds including hydroxy acids and fluoride (F(-)) ions. PBA-based F(-) ion sensors may be useful if reagentless sensors can be developed.

Enzymatically Regulated Peptide Pairing and Catalysis for the Bioanalysis of Extracellular Prometastatic Activities of Functionally Linked Enzymes.

Diseases such as cancer arise from systematical reconfiguration of interactions of exceedingly large numbers of proteins in cell signaling. The study of such complicated molecular mechanisms requires multiplexed detection of the inter-connected activities of several proteins in a disease-associated context. However, the existing methods are generally not well-equipped for this kind of application. Here a method for analyzing functionally linked protein activities is developed based on enzyme controlled pairing between complementary peptide helix strands, which simultaneously enables elaborate regulation of catalytic activity of the paired peptides. This method has been used to detect three different types of protein modification enzymes that participate in the modification of extracellular matrix and the formation of invasion front in tumour. In detecting breast cancer tissue samples using this method, up-regulated activity can be observed for two of the assessed enzymes, while the third enzyme is found to have a subtle fluctuation of activity. These results may point to the application of this method in evaluating prometastatic activities of proteins in tumour.

pH- and sugar-sensitive multilayer films composed of phenylboronic acid (PBA)-modified poly(allylamine hydrochloride) (PBA-PAH) and poly(vinyl alcohol) (PVA): A significant effect of PBA content on the film stability.

Multilayer thin films composed of phenylboronic acid (PBA)-modified poly(allylamine hydrochloride) (PAH), PBA-PAH, with different PBA contents were prepared to study the effect of PBA content on the stability of the films. An alternate deposition of PBA-PAH and poly(vinyl alcohol) (PVA) on the surface of a quartz slide afforded multilayer films through forming boronate ester bonds between PBA-PAH and PVA. The 10-layered (PBA-PAH/PVA)10 films constructed using PBA-PAHs containing 16% and 26% PBA residues were stable in aqueous solutions over the range of pH 4.0-10.0, whereas the multilayer films composed of PBA-PAHs with 5.9% and 8.3% PBA decomposed at pH 8.0 or lower. The pH-sensitive decomposition of the films was rationalized based on the destabilization of the boronate ester bonds in neutral and acidic solutions. In addition, the (PBA-PAH/PVA)10 films decomposed in glucose and fructose solutions as a result of competitive binding of sugars to PBA-PAH in the films. The sugar response of the films depended on the PBA content in PBA-PAH. The (PBA-PAH/PVA)10 films consisting of 16% and 26% PBA-substituted PBA-PAHs are sensitive to physiological relevant level of glucose at pH7.4 while stable in glucose-free solution, suggesting a potential use of the films in constructing glucose-induced delivery systems.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Preparation of Layer-by-Layer Films Composed of Polysaccharides and Poly(Amidoamine) Dendrimer Bearing Phenylboronic Acid and Their pH- and Sugar-Dependent Stability.

Layer-by-layer films composed of polysaccharides and poly(amidoamine) dendrimer bearing phenylboronic acid (PBA-PAMAM) were prepared to study the deposition behavior of the films and their stability in buffer solutions and in sugar solutions. Alginic acid (AGA) and carboxymethylcellulose (CMC) were employed as counter-polymers in constructing LbL films. AGA/PBA-PAMAM films were successfully prepared at pH 6.0-9.0, whereas the preparation of CMC/PBA-PAMAM film was unsuccessful at pH 8.0 and 9.0. The results show that the LbL films formed mainly through electrostatic affinity between PBA-PAMAM and polysaccharides, while, for AGA/PBA-PAMAM films, the participation of boronate ester bonds in the films was suggested. AGA/PBA-PAMAM films were stable in the solutions of pH 6.0-9.0. In contrast, CMC/PBA-PAMAM films decomposed at pH 7.5-9.0. The AGA/PBA-PAMAM films decomposed in response to 5-30 mM fructose at pH 7.5, while the films were stable in glucose solutions. Thus, AGA is useful as a counter-polymer for constructing PBA-PAMAM films that are stable at physiological pH and decompose in response to fructose.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Recent Progress in Electrochemical HbA1c Sensors: A Review.

This article reviews recent progress made in the development of electrochemical glycated hemoglobin (HbA1c) sensors for the diagnosis and management of diabetes mellitus. Electrochemical HbA1c sensors are divided into two categories based on the detection protocol of the sensors. The first type of sensor directly detects HbA1c by binding HbA1c on the surface of an electrode through bio-affinity of antibody and boronic acids, followed by an appropriate mode of signal transduction. In the second type of sensor, HbA1c is indirectly determined by detecting a digestion product of HbA1c, fructosyl valine (FV). Thus, the former sensors rely on the selective binding of HbA1c to the surface of the electrodes followed by electrochemical signaling in amperometric, voltammetric, impedometric, or potentiometric mode. Redox active markers, such as ferrocene derivatives and ferricyanide/ferrocyanide ions, are often used for electrochemical signaling. For the latter sensors, HbA1c must be digested in advance by proteolytic enzymes to produce the FV fragment. FV is electrochemically detected through catalytic oxidation by fructosyl amine oxidase or by selective binding to imprinted polymers. The performance characteristics of HbA1c sensors are discussed in relation to their use in the diagnosis and control of diabetic mellitus.

Recent Progress in Lectin-Based Biosensors.

This article reviews recent progress in the development of lectin-based biosensors used for the determination of glucose, pathogenic bacteria and toxins, cancer cells, and lectins. Lectin proteins have been widely used for the construction of optical and electrochemical biosensors by exploiting the specific binding affinity to carbohydrates. Among lectin proteins, concanavalin A (Con A) is most frequently used for this purpose as glucose- and mannose-selective lectin. Con A is useful for immobilizing enzymes including glucose oxidase (GOx) and horseradish peroxidase (HRP) on the surface of a solid support to construct glucose and hydrogen peroxide sensors, because these enzymes are covered with intrinsic hydrocarbon chains. Con A-modified electrodes can be used as biosensors sensitive to glucose, cancer cells, and pathogenic bacteria covered with hydrocarbon chains. The target substrates are selectively adsorbed to the surface of Con A-modified electrodes through strong affinity of Con A to hydrocarbon chains. A recent topic in the development of lectin-based biosensors is a successful use of nanomaterials, such as metal nanoparticles and carbon nanotubes, for amplifying output signals of the sensors. In addition, lectin-based biosensors are useful for studying glycan expression on living cells.

Glucose-induced decomposition of layer-by-layer films composed of phenylboronic acid-bearing poly(allylamine) and poly(vinyl alcohol) under physiological conditions.

Multilayer thin films prepared via layer-by-layer (LbL) deposition of phenylboronic acid-bearing poly(allylamine hydrochloride) (PBA-PAH) and poly(vinyl alcohol) (PVA) were combined with glucose oxidase (GOx) to develop LbL films that can be decomposed in response to glucose. LbL deposition of PBA-PAH and PVA on a GOx-modified quartz slide afforded GOx/(PBA-PAH/PVA)n films, in which PBA-PAH and PVA layers were connected to each other through boronate ester bonds, while GOx was immobilized electrostatically. The GOx/(PBA-PAH/PVA)n films were stable in glucose-free solutions under physiological conditions. In contrast, films decomposed in the presence of glucose, depending on the concentration of glucose and solution pH. The GOx/(PBA-PAH/PVA)10 film decomposed almost completely in 60 min in the presence of 10 mM glucose at pH 7.4, while it took 120 min in 0.1 mM glucose solution. Film decomposition was accelerated at pH 5.0-7.4, whereas it was suppressed in acidic and basic media (pH 3.0 and 9.0, respectively) due to the lower catalytic activity of GOx. Glucose-induced decomposition of the film can be rationalized based on the scission of the carbon-boron bonds of PBA in the film by hydrogen peroxide (H2O2), which is produced through a GOx-catalyzed oxidation reaction of glucose.