Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane ...injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC-random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.
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Understanding of the activation dynamics of C-reactive protein (CRP) on plasma membranes is important in the development of zwitterionic biomaterials for their uses in the tissues of ...inflammation and infection. Previously, the use of a zwitterionic phosphorylcholine group, a biomimetic ligand for CRP in the presence of calcium ions, for binding experiments has revealed that the adsorption dynamics changed by ionic microenvironments. Here we focused on the effect of the ligand density on a surface, a major physicochemical parameter, on the multivalent binding modes. A building block from synthetic origin, a phospholipid analogue with thiol ends, was developed for making a cell membrane-mimicked self-assembled monolayers with tunable lateral ligand density on the molecular basis. The multivalent binding kinetics of CRP, a pentraxin in the original conformation, onto the engineered surface was measured using a surface plasmon resonance technique. The binding experiments revealed that the on-rate and off-rate constants in the first ligand-occupation reaction increased with increasing the ligand density, which resulted in stable values of the dissociation constant. Notably, the binding affinity in the second ligand-occupation reaction showed the optimal value as a function of the ligand density. Moreover, the binding experiments using a monomeric CRP-specific DNA aptamer revealed that pentameric CRP underwent structural transition into the monomers following the adsorption onto the surfaces via multivalent contacts in a pH-dependent manner. The bioengineering-based approach reveals for the first time how the multiple binding reaction is altered by the ligand arrangement at the molecular resolution and how CRP is activated by the conformational transition induced by the multiplex bindings.
C-reactive protein (CRP), a major acute-phase pentraxin, binds to plasma membranes through the multivalent contacts with zwitterionic phosphorylcholine groups. However, details in the molecular dynamics is unknown due to a lack of proper sensing platform. The paper describe the synthesis of thiol-functionalized phosphorylcholine for the development of a robust cell membrane-mimetic surface on a surface plasmon resonance sensor at desired lateral ligand densities. The engineered approach on molecular basis enables a rigorous arrangement of the ligand on the surface, whose tunability and robustness are not achieved using conventional supported lipid layers. The effect of the ligand density on the multivalent binding kinetics provides the understanding of how the multivalent contacts induce conformational transitions of CRP and responses to inflammation.
Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer’s drugs. However, application of enzyme inhibition to odorant sensors is less common ...because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonenethe major odorant of citrus and an AChE inhibitorusing molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 μM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant–enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.
Tracing cell surface sialylation dynamics at a scale of the glycolipoprotein microdomain (lipid rafts) formations remains an intriguing challenge of cellular biology. Here, we demonstrate that this ...goal is accessible, taking advantage of a boronic acid (BA)-based reversible molecular recognition chemistry. A BA-end-functionalized poly(ethylene glycol) was decorated onto an atomic force microscopy (AFM) cantilever, which provided a dynamic and sialic acid (SA)-specific imaging mode. Using this technique, we were able to heat map the SA expression levels not only on protein-decorated substrates but also directly on the cell surfaces, with a submicrometer scale resolution that may be relevant to that of the lipid rafts formation. The SA specificity and the binding reversibility of the probe were confirmed from its pH-dependent characteristics and an inhibition assay using free state SA. This finding may provide a noninvasive means for assessing a variety of SA-involved glycosylation dynamics spanning from physiology to pathology.
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•Synthesis of PEG-polycations with polyether backbone having amine or guanidine groups.•mRNA-loaded micelles were self-assembled by mixing PEG-polycations and mRNA in ...water.•mRNA-loaded micelles from PEG-polycation with guanidine groups show greater stability.•The micelles from PEG-polycation with guanidine enhance intracellular mRNA delivery.•This increased intracellular mRNA delivery promotes high level of protein translation.
Messenger RNA (mRNA) holds great potential as therapeutic agent, though its broad applicability is hampered by its rapid degradation by nuclease attacks and poor access to cells. Polyion complex (PIC) micelles comprising block catiomers are promising mRNA carriers, because of their effective mRNA protection in the PIC core and the capability to promote mRNA cellular uptake. We have recently shown that flexible polycations can promote the interaction between catiomers and mRNA toward improving delivery efficiency, demonstrating that, by engineering the PIC formation, the efficacy of mRNA-loaded PIC micelles (mRNA/m) can be improved. Herein, we focused on the valency between the polycation segment and mRNA, and studied its ability to promote, in collaboration with the flexible catiomers, mRNA stability and delivery efficiency for producing potent nanocarriers. The functionalities of guanidine and primary amine were compared through preparation of two different block copolymers, namely, poly(ethylene glycol)-poly(glycidyl methyl guanidine) (PEG-PGMG) and PEG-poly(glycidyl methyl amine (PEG-PGMA)). mRNA/m from PEG-PGMG (mRNA/mPGMG) showed enhanced stability against biological molecules, including polyanions, urea and nucleases compared to mRNA/m prepared from PEG-PGMA (mRNA/mPGMA), resulting in high efficiency of cellular uptake and protein translation. Our results indicate that multivalent interactions between the guanidine moieties in the polycation segments of block copolymers and the phosphate groups in mRNA are a key factor for stabilizing mRNA/m toward improving mRNA delivery efficiency.
In recent years, isothermal nucleic acid amplification techniques have been developed as alternatives to polymerase chain reaction (PCR) requiring thermal cycles. The integration of isothermal ...amplification into electrical or electrochemical devices realizes high throughput nucleic acid-based assays and confers high sensitivity. In this research, we tried to develop electrical/electrochemical biosensors detecting nucleic acids aiming at application to future liquid biopsy. For detection of small DNAs or microRNAs (miRNAs), isothermal amplification methods were employed, and the signal changes based on the extension reaction by polymerases were detected using electrical/electrochemical devices, namely, a chronocoulometric sensor and a micro pH sensor. These small sensors based on electrochemical technique might be a promising tool in on-site detection of nucleic acids related to cancer diagnosis.
Resistive pulse sensing (RPS) is an analytical method that can be used to individually count particles from a small sample. RPS simply monitors the physical characteristics of particles, such as ...size, shape, and charge density, and the integration of RPS with biosensing is an attractive theme to detect biological particles such as virus and bacteria. In this report, a methodology of biosensing on RPS was investigated. Polydopamine (PD), an adhesive component of mussels, was used as the base material to create a sensing surface. PD adheres to most materials, such as noble metals, metal oxides, semiconductors, and polymers; as a result, PD is a versatile intermediate layer for the fabrication of a biosensing surface. As an example of a biological particle, human influenza A virus (H1N1 subtype) was used to monitor translocation of particles through the pore membrane. When virus-specific ligands (6′-sialyllactose) were immobilized on the pore surface, the translocation time of the virus particles was considerably extended. The detailed translocation data suggest that the viral particles were trapped on the sensing surface by specific interactions. In addition, virus translocation processes on different pore surfaces were distinguished using machine learning. The result shows that the simple and versatile PD-based biosensor surface design was effective. This advanced RPS measurement system could be a promising analytical technique.
Chemical and enzymatic modifications of amino acid residues in protein after translation contain rich information about physiological conditions and diseases. Histone acetylation/deacetylation is the ...essential post-translational modification by regulating gene transcription. Such qualitative changes of biomacromolecules need to be detected in point-of-care systems for an early and accurate diagnosis. However, there is no technique to aid this issue. Previously, we have applied an aptamer-functionalized field-effect transistor (FET) to the specific protein biosensing. Quantitative changes of target protein in a physiological solution have been determined by detecting innate charges of captured protein at the gate-solution interface. Moreover, we have succeeded in developing an integrated system of FET and quartz crystal microbalance (QCM) sensors for determining the adsorbed mass and charge, simultaneously or in parallel. Prompted by this, in this study, we developed a new label-free method for detecting histone acetylation using FET and QCM sensors. The loss of positive charge of lysine residue by chemically induced acetylation of histone subunits (H3 and H4) was successfully detected by potentiometric signals using anti-histone aptamer-functionalized FET. The adsorbed mass was determined by the same anti-histone aptamer-functionalized QCM. From these results, the degree of acetylation was correlated to the charge-to-mass ratio of histone subunits. The histone required for the detection was below 100 nM, owing to the high sensitivity of aptamer-functionalized FET and QCM sensors. These findings will guide us to a new way of measuring post-translational modification of protein in a decentralized manner for an early and accurate diagnosis.
The electrochemical behavior of different size gold nanoparticles (AuNPs) was investigated. AuNPs with 2, 5, 10, 15, 20 and 50 nm diameters were immobilized onto a screen printed carbon electrode ...surface by physical adsorption. The impedimetric response was measured for different diameter AuNPs at a fixed value of their surface area, at the same content of gold (Au) and at the same concentration. In a further experiment, the impedimetric response toward AuNP concentration was measured for each diameter. Impedimetric results were compared with results obtained for the detection of Au by stripping voltammetry. Additionally, variability of active surface area and roughness of different electrodes before and after immobilization of AuNPs were carefully evaluated by means of cyclic voltammetry and laser scanning microscopy. Electrochemical impedance spectroscopy (EIS) is a sensitive technique capable of differentiating the signal generated by AuNPs of different sizes, thus providing useful information for the employment of AuNPs in electrochemical biosensors.
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