Fabrication of 3D engineered intestinal tissue producing abundant mucus by air-liquid interface culture using paper-based dual-layer scaffold.

Fabrication of engineered intestinal tissues with the structures and functions as humans is crucial and promising as the tools for developing drugs and functional foods. The aim of this study is to fabricate an engineered intestinal tissue from Caco-2 cells by air-liquid interface culture using a paper-based dual-layer scaffold and analyze its structure and functions. Just by simply placing on a folded paper soaked in the medium, the electrospun gelatin microfiber mesh as the upper cell adhesion layer of the dual-layer scaffold was exposed to the air, while the lower paper layer worked to preserve and supply the cell culture medium to achieve stable culture over several weeks. Unlike the flat tissue produced using the conventional commercial cultureware, Transwell, the engineered intestinal tissue fabricated in this study formed three-dimensional villous architectures. Microvilli and tight junction structures characteristic of epithelial tissue were also formed at the apical side. Furthermore, compared to the tissue prepared by Transwell, mucus production was significantly larger, and the enzymatic activities of drug metabolism and digestion were almost equivalent. In conclusion, the air-liquid interface culture using the paper-based dual-layer scaffold developed in this study was simple but effective in fabricating the engineered intestinal tissue with superior structures and functions.

Aggregation-Based Bacterial Separation with Gram-Positive Selectivity by Using a Benzoxaborole-Modified Dendrimer.

Antimicrobial-resistant (AMR) bacteria have become a critical global issue in recent years. The inefficacy of antimicrobial agents against AMR bacteria has led to increased difficulty in treating many infectious diseases. Analyses of the environmental distribution of bacteria are important for monitoring the AMR problem, and a rapid as well as viable pH- and temperature-independent bacterial separation method is required for collecting and concentrating bacteria from environmental samples. Thus, we aimed to develop a useful and selective bacterial separation method using a chemically synthesized nanoprobe. The metal-free benzoxaborole-based dendrimer probe BenzoB-PAMAM(+), which was synthesized from carboxy-benzoxaborole and a poly(amidoamine) (PAMAM) dendrimer, could help achieve Gram-positive bacterial separation by recognizing Gram-positive bacterial surfaces over a wide pH range, leading to the formation of large aggregations. The recognition site of benzoxaborole has a desirable high acidity and may therefore be responsible for the improved Gram-positive selectivity. The Gram-positive bacterial aggregation was then successfully collected by using a 10 µm membrane filter, with Gram-negative bacteria remaining in the filtrate solution. BenzoB-PAMAM(+) will thus be useful for application in biological analyses and could contribute to further investigations of bacterial distributions in environmental soil or water.

Recognition of d-Glucose in Water with Excellent Sensitivity, Selectivity, and Chiral Selectivity Using γ-Cyclodextrin and Fluorescent Boronic Acid Inclusion Complexes Having a Pseudo-diboronic Acid Moiety.

Fluorescence recognition of d-glucose in water with excellent sensitivity, selectivity, and chiral selectivity is desired because d-glucose is an essential component in biological and pathological processes. We report an innovative approach that exploits the 1:2 stoichiometric inclusion complexes of γ-cyclodextrin (γ-CyD) with two molecules of fluorescent monoboronic acid-based receptors, which form a pseudo-diboronic acid moiety as the recognition site for d-glucose in water. Two monoboronic acids (1F and 2N) were easily synthesized without heating or column purification. The 1:2 stoichiometric inclusion complexes (1F/γ-CyD and 2N/γ-CyD) were prepared in a mixture of dimethyl sulfoxide/water (2/98 in v/v) by mixing γ-CyD and the corresponding monoboronic acids. Both 1F/γ-CyD and 2N/γ-CyD exhibited strong turn-on response to d-glucose with excellent selectivity over nine other saccharides in the water-rich solvent at pH 7.4 owing to the ditopic recognition of d-glucose by the pseudo-diboronic acid moieties. The limits of detection of 1F/γ-CyD and 2N/γ-CyD for d-glucose were 1.1 and 1.8 µM, respectively, indicating the remarkable sensitivity for the detection of d-glucose at µM levels. 1F/γ-CyD and 2N/γ-CyD also demonstrated chiral-selective recognition of d-glucose, which is apparent from the 2.0- and 6.3-fold enhancement of fluorescence by the addition of d-glucose relative to l-glucose addition, owing to the chiral pseudo-diboronic acid moieties produced by the chiral γ-CyD cavity. To the best of our knowledge, 2N/γ-CyD has the highest d/l selectivity among hitherto reported fluorescent diboronic acid-based receptors.

Boronic Acid-Based Dendrimers with Various Surface Properties for Bacterial Recognition with Adjustable Selectivity.

The need for a selective bacterial recognition method is evident to overcome the global problem of antibiotic resistance. Even though researchers have focused on boronic acid-based nanoprobes that immediately form boronate esters with saccharides at room temperature, the mechanism has not been well studied. We have developed boronic acid-modified poly(amidoamine) (PAMAM) dendrimers with various surface properties to investigate the mechanism of bacterial recognition. The boronic acid-based nanoprobes showed selectivity toward strains, species, or a certain group of bacteria by controlling their surface properties. Our nanoprobes showed selectivity toward Gram-positive bacteria or Escherichia coli K12W3110 without having to modify the boronic acid recognition sites. The results were obtained in 20 min and visible to the naked eye. Selectivity toward Gram-positive bacteria was realized through electrostatic interaction between the bacterial surface and the positively charged nanoprobes. In this case, the recognition target was lipoteichoic acid on the bacterial surface. On the other hand, pseudo-zwitterionic nanoprobes showed selectivity for E. coli K12W3110, indicating that phenylboronic acid did not recognize the outermost O-antigen on the lipopolysaccharide layer. Boronic acid-based nanoprobes with optimized surface properties are expected to be a powerful clinical tool to recognize multidrug-resistant strains or highly pathogenic bacteria.

A simple supramolecular complex of boronic acid-appended ß-cyclodextrin and a fluorescent boronic acid-based probe with excellent selectivity for d-glucose in water.

Various diboronic acid-based chemosensors for d-glucose have been developed for use in diabetes diagnostic systems. However, most of these chemosensors have limitations, such as poor water solubility, difficulties in synthesis, and inability to selectively detect d-glucose from among other saccharides. We report a simple chemosensor based on a supramolecular complex of fluorophenylboronic acid-appended ß-cyclodextrin (FPB-ßCyD) and an anthracene-based probe having a boronic acid moiety (1). Hydrophobic 1 is encapsulated in the cyclodextrin cavity of FPB-ßCyD, making the supramolecular complex (1/FPB-ßCyD) applicable in a water-rich solvent mixture (98% water). Interestingly, 1/FPB-ßCyD showed a strong turn-on response to d-glucose with a 9.6-fold enhancement in fluorescence intensity, and no response to other saccharides. This study uncovers an innovative approach based on the supramolecular assembly of simple components for the development of a water-soluble d-glucose chemosensor with excellent selectivity.

Enzyme-embedded electrospun fiber sensor of hydrophilic polymer for fluorometric ethanol gas imaging in vapor phase.

Non-invasive measurement of volatile organic compounds (VOCs) emitted from living organisms is a powerful technique for diagnosing health conditions or diseases in humans. Bio-based gas sensors are suitable for the sensitive and selective measurement of a target VOC from a complex mixture of VOCs. Conventional bio-based sensors are normally prepared as wet-type probes to maintain proteins such as enzymes in a stable state, resulting in limitations in the commercialization of sensors, their operating environment, and performance. In this study, we present an enzyme-based fluorometric electrospun fiber sensor (eFES) mesh as a gas-phase biosensor in dry form. The eFES mesh targeting ethanol was fabricated by simple one-step electrospinning of polyvinyl alcohol with an alcohol dehydrogenase and an oxidized form of nicotinamide adenine dinucleotide. The enzyme embedded in the eFES mesh worked actively in a dry state without pretreatment. Substrate specificity was also maintained, and the sensor responded well to ethanol with a sufficient dynamic range. Adjustment of the pH and coenzyme quantity in the eFES mesh also affected enzyme activity. The dry-form biosensor-eFES mesh-will open a new direction for gas-phase biosensors because of its remarkable performance and simple fabrication, which is advantageous for commercialization.

Fast and Sensitive Bacteria Detection by Boronic Acid Modified Fluorescent Dendrimer.

This study reports a novel, fast, easy, and sensitive detection method for bacteria which is urgently needed to diagnose infections in their early stages. Our work presents a complex of poly(amidoamine) dendrimer modified by phenylboronic acid and labeled by a fluorescent dansyl group (Dan-B8.5-PAMAM). Our system detects bacteria in 20 min with a sensitivity of approximately 104 colony-forming units (CFU)·mL-1. Moreover, it does not require any peculiar technical skills or expensive materials. The driving force for bacteria recognition is the binding between terminal phenylboronic acids on the probe and bacteria's surface glycolipids, rather than electrostatic interactions. The aggregation caused by such binding reduces fluorescence. Even though our recognition method does not distinguish between live or dead bacteria, it shows selective antibacterial activity towards Gram-negative bacteria. This study may potentially contribute a new method for the convenient detection and killing of bacteria.

Effect of Spacer Length in Pyrene-Modified-Phenylboronic Acid Probe/CyD Complexes on Fluorescence-based Recognition of Monosaccharides in Aqueous Solution.

The chemical sensing of saccharides is of importance for the diagnosis of diabetes. Various enzymatic sensors have been developed, but their heat and pH instability issues need to be resolved. In this regard, the development of artificial saccharide sensors with high stability is attracting attention. We have designed a heat- and pH-stable supramolecular inclusion complex system composed of cyclodextrin (CyD) as a host and a phenylboronic acid (PB) probe possessing pyrene as a fluorescent guest. Several probes possessing alkyl spacers having various lengths between the PB and the pyrene moiety, Cn-APB (n = 1 - 4), were newly synthesized and evaluated with respect to their monosaccharide recognition ability on the basis of the fluorescence response through the cyclic esterification of monosaccharide and PB. These Cn-APB/CyD supramolecular inclusion complexes have exhibited a selective fluorescence response towards fructose in aqueous solution based on the photo-induced electron transfer mechanism. The spacer length of the alkyl group in Cn-APB significantly affects the affinity for saccharides. With respect to the complex between C4-APB and PB-modified CyD (3-PB-γ-CyD), it was found that the supramolecular inclusion complexes had high selectivity for glucose with significant fluorescence enhancement. These results indicate that the lengths of the alkyl spacers in the probe molecules are important to control the recognition of saccharides in aqueous solution.

Rapid Bacterial Recognition over a Wide pH Range by Boronic Acid-Based Ditopic Dendrimer Probes for Gram-Positive Bacteria.

We have developed a convenient and selective method for the detection of Gram-positive bacteria using a ditopic poly(amidoamine) (PAMAM) dendrimer probe. The dendrimer that was modified with dipicolylamine (dpa) and phenylboronic acid groups showed selectivity toward Staphylococcus aureus. The ditopic dendrimer system had higher sensitivity and better pH tolerance than the monotopic PAMAM dendrimer probe. We also investigated the mechanisms of various ditopic PAMAM dendrimer probes and found that the selectivity toward Gram-positive bacteria was dependent on a variety of interactions. Supramolecular interactions, such as electrostatic interaction and hydrophobic interaction, per se, did not contribute to the bacterial recognition ability, nor did they improve the selectivity of the ditopic dendrimer system. In contrast, the ditopic PAMAM dendrimer probe that had a phosphate-sensing dpa group and formed a chelate with metal ions showed improved selectivity toward S. aureus. The results suggested that the targeted ditopic PAMAM dendrimer probe showed selectivity toward Gram-positive bacteria. This study is expected to contribute to the elucidation of the interaction between synthetic molecules and bacterial surface. Moreover, our novel method showed potential for the rapid and species-specific recognition of various bacteria.

Selective Sugar Recognition by Anthracene-Type Boronic Acid Fluorophore/Cyclodextrin Supramolecular Complex Under Physiological pH Condition.

We synthesized novel PET (photoinduced electron transfer)-type fluorescence glucose probe 1 [(4-(anthracen-2-yl-carbamoyl)-3-fluorophenyl)boronic acid], which has a phenylboronic acid (PBA) moiety as the recognition site and anthracene as the fluorescent part. Although the PBA derivatives dissociate and bind with sugar in the basic condition, our new fluorescent probe can recognize sugars in the physiological pH by introducing an electron-withdrawing fluorine group into the PBA moiety. As a result, the pK a value of this fluorescent probe was lowered and the probe was able to recognize sugars at the physiological pH of 7.4. The sensor was found to produce two types of fluorescent signals, monomer fluorescence and dimer fluorescence, by forming a supramolecular 2:1 complex of 1 with glucose inside a γ-cyclodextrin (γ-CyD) cavity. Selective ratiometric sensing of glucose by the 1/γ-CyD complex was achieved in water at physiological pH.

Rapid and Selective Discrimination of Gram-Positive and Gram-Negative Bacteria by Boronic Acid-Modified Poly(amidoamine) Dendrimer.

There is an urgent need to develop a rapid and selective method for the detection of bacteria because delayed diagnosis and the overuse of antibiotics have triggered drug resistance in bacteria. To this end, we prepared boronic acid-modified poly(amidoamine) generation 4 (B-PAMAM(G4)) dendrimer as cross-linking molecules that form aggregates with bacteria. Within 5 min of adding B-PAMAM(G4) dendrimer solution to a bacterial suspension, large aggregates were observed. Interestingly, the aggregate formation with various bacteria was pH-dependent. In basic pH, both Gram-positive and Gram-negative bacteria formed aggregates, but in neutral pH, only Gram-positive bacteria formed aggregates. We revealed that this bacteria-selective aggregation involved the bacterial surface recognition of the phenylboronic acid moiety of B-PAMAM(G4) dendrimer. In addition, we demonstrated that the spherical structure of B-PAMAM(G4) was one of the important factors for the formation of large aggregates. The aggregation was also observed in the presence of ≤10 mM fructose. B-PAMAM(G4) dendrimer is expected to be a powerful tool for the rapid and selective discrimination between Gram-positive and Gram-negative bacteria.

Nanogelation and Thermal Stabilization of Enzyme by Vitamin B6-Bearing Polysaccharide as Biocrosslinker.

Nanogels containing a protein (carbonic anhydrase, CA) were prepared by cross-linking CA and pyridoxal (vitamin B6)-bearing pullulan (PLPP) as a biocrosslinker via Schiff base formation. UV titration and high-performance liquid chromatography confirmed that CA was quantitatively complexed with PLPP in the presence of zinc ions. Dynamic light scattering and transmission electron microscopy showed that the nanogel diameter was about 20 nm. CA retained 90% of its native activity after complexation with PLPP. Moreover, the residual enzymatic activity of CA after heating and its long-term storage stability at room temperature were improved by complexation with PLPP. Enzyme nanogelation with PLPP is an efficient method for enzyme stabilization.

Metal and Phosphate Ion Recognition Using Dipicolylamine-modified Fluorescent Silica Nanoparticles.

Dipicolylamine-modified fluorescent silica nanoparticles were prepared by introducing dipicolylamine to the surface of silica nanoparticles possessing terminal amines. We examined the selectivities of dipicolylamine-hydroxycoumarin carbonate (dpa-HCC) and dpa-HCC/fluorescent silica nanoparticles (FSiNP) for metal ions and phosphate anions. The dipicolylamine-modified silica nanoparticles responded to PPi, Tri and Pb2+, indicating novel selectivity derived from the assembly effect of dpa-HCC on the silica nanoparticle surface. Surface-modified fluorescent silica nanoparticles are expected to be used as a sensor for environmental and biological applications.

Development of Dipicolylamine-Modified Cyclodextrins for the Design of Selective Guest-Responsive Receptors for ATP.

The construction of supramolecular recognition systems based on specific host-guest interactions has been studied in order to design selective chemical sensors. In this study, guest-responsive receptors for ATP have been designed with cyclodextrins (CyDs) as a basic prototype of the turn-on type fluorescent indicator. We synthesized dipicolylamine (DPA)-modified CyD-Cu2+ complexes (Cu·1α, Cu·1ß, and Cu·1γ), and evaluated their recognition capabilities toward phosphoric acid derivatives in water. The UV-Vis absorption and fluorescence spectra revealed that Cu·1ß selectively recognized ATP over other organic and inorganic phosphates, and that ß-CyD had the most suitable cavity size for complexation with ATP. The 1D and 2D NMR analyses suggested that the ATP recognition was based on the host-guest interaction between the adenine moiety of ATP and the CyD cavity, as well as the recognition of phosphoric moieties by the Cu2+-DPA complex site. The specific interactions between the CyD cavity and the nucleobases enabled us to distinguish ATP from other nucleoside triphosphates, such as guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). This study clarified the basic mechanisms of molecular recognition by modified CyDs, and suggested the potential for further application of CyDs in the design of highly selective supramolecular recognition systems for certain molecular targets in water.

Development of Supramolecular Saccharide Sensors Based on Cyclodextrin Complexes and Self-assembling Systems.

Cyclodextrins (CDs) are water-soluble host compounds having nano-size hydrophobic cavities that enable them to incorporate organic molecules in water. Optically inert CDs can be efficiently combined with various types of chromoionophores and fluoroionophores. In this study, using diverse combinations of phenylboronic acid fluorescent sensors and azoprobes with CDs, the unique saccharide recognition functions of CD, chemically modified CD, and CD gel complexes based on their synergistic function are clarified, thereby confirming their use as supramolecular saccharide sensors. To realize novel supramolecular chirality, the twisted structure of two ditopic azoprobes inside the γ-CD chiral cavity is controlled by multi-point recognition of guest ions in water. As different types of supramolecular saccharide sensors, phenylboronic acid-based self-assembling systems are also reviewed.

Design and Function of Supramolecular Recognition Systems Based on Guest-Targeting Probe-Modified Cyclodextrin Receptors for ATP.

In this study, we have developed a rational design strategy to obtain highly selective supramolecular recognition systems of cyclodextrins (CyDs) on the basis of the lock and key principle. We designed and synthesized dipicolylamine (dpa)-modified γ-CyD-Cu2+ complexes possessing an azobenzene unit (Cu·1-γ-CyD) and examined how they recognized phosphoric acid derivatives in water. The results revealed that Cu·1-γ-CyD recognized ATP with high selectivity over other phosphoric acid derivatives. The significant blue shift in the UV-vis spectra and 1H NMR analysis suggested that the selective ATP recognition was based on the multipoint interactions between the adenine moiety of ATP and both the CyD cavity and the azobenzene unit in addition to the recognition of phosphoric moieties by the Cu-dpa complex site. Our unique receptor made it capable of distinguishing ATP from AMP and ADP, revealing the discrimination of even a length of one phosphoric group. This study demonstrates that, compared to conventional recognition systems of CyDs, this multipoint recognition system confers a higher degree of selectivity for certain organic molecules, such as ATP, over their similar derivatives.

Saccharide Recognition Based on Self-Assembly of Amphiphilic Phenylboronic Acid Azoprobes.

We designed amphiphilic phenylboronic acid azoprobes (B-Azo-Cn) and evaluated their saccharide recognition function in relation to the micelle formation changes of the self-assembled B-Azo-Cn. First, we evaluated B-Azo-C8 in a 1% methanol-99% water solution under basic conditions. The wavelength of maximum absorption in the ultraviolet-visible (UV-vis) spectra of B-Azo-C8 was shifted, and the solution showed a color change with the addition of saccharides. The morphology of B-Azo-C8 was evaluated using dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) observations. B-Azo-C8 formed aggregates in the absence of saccharides and in the presence of glucose. In the presence of fructose, micelle-formed B-Azo-C8 was dispersed, indicating that B-Azo-C8 changed its dispersion state by recognizing fructose. The effect of alkyl chain length on the saccharide recognition ability was examined as well. B-Azo-C4 and B-Azo-C12 did not recognize saccharides in a 1% methanol-99% water solution under basic conditions, indicating that an appropriate alkyl chain length was required for recognizing saccharides. The control of the hydrophilic-lipophilic balance (HLB) was a key factor for saccharide recognition.