A Hybrid Nanotube Stamp System in Intracellular Protein Delivery for Cancer Treatment and NMR Analytical Techniques.

In contrast to intracellular gene transfer, the direct delivery of expressed proteins is a significantly challenging yet essential technique for elucidating cellular functions, including protein complex structure, liquid-liquid phase separation, therapeutic applications, and reprogramming. In this study, we developed a hybrid nanotube (HyNT) stamp system that physically inserts the HyNTs into adhesive cells, enabling the injection of target molecules through HyNT ducts. This system demonstrates the capability to deliver multiple proteins, such as lactate oxidase (LOx) and ubiquitin (UQ), to approximately 1.8 × 107 adhesive cells with a delivery efficiency of 89.9% and a viability of 97.1%. The delivery of LOx enzyme into HeLa cancer cells induced cell death, while enzyme-delivered healthy cells remained viable. Furthermore, our stamp system can deliver an isotope-labeled UQ into adhesive cells for detection by nuclear magnetic resonance (NMR).

Quantitative analyses of RBC movement in whole blood exposed to DC and ELF electric field.

For the study of biological effects of direct current (DC) and extremely low frequency (ELF) electric fields, we have quantitatively analyzed red blood cell (RBC) movement in whole blood. Considering the inhomogeneous distribution of electric fields in vivo, five different electric field distributions were generated under a microscope. For theoretical analyses, we assumed electrophoresis and dielectrophoresis as basic motive forces and obtained the spatial distribution of blood cell velocity. The RBC velocity was measured using video image analysis. The spatial dependence of the velocity showed good agreement with that predicted by theoretical analysis. This result suggests the validity of the theoretical model based on electrophoresis and dielectrophoresis for the study of ELF electric field exposure to inhomogeneous animal and human bodies. Next, using the same measurement system, we attempted to find the electric field strength at which these effects occur. The threshold values were found to be 0.40 and 1.6 kV/m, respectively, for DC and AC electric field exposures. Furthermore, we investigated the reproducibility of the field effects in more realistic conditions of human exposure. The RBCs in microchannels were exposed to the electric field generated in capacitive coupling using electrodes separated by an air gap. Even in the new condition, similar effects were observed, which also verified the validity of the analysis described above. These results will provide useful information for the safety assessment of field exposure and for the future biomedical applications of electric fields to manipulate RBCs in vivo.

Analysis of Red Blood Cell Movement in Whole Blood Exposed to DC and ELF Electric Fields.

To evaluate hematological effects of direct current (DC) and alternating current (AC) extremely low frequency (ELF) electric field exposure, this study investigated red blood cell (RBC) movement in whole blood. Video images of RBCs were recorded under a microscope using specially designed electrode systems. Video analysis software was then used to measure the RBC velocity. The noise level and measurement system stability were confirmed based on results of a no-field exposure experiment. Using the electrode system to produce a non-homogeneous electric field, different movements were found to occur in DC and AC field exposure. The RBCs moved in the directions of the electric field and the gradient of field distribution, respectively, in the DC and AC fields. Dependences of the RBC velocity on the field strength were, respectively, linear and quadratic in the DC and AC fields. These results suggest that electrophoretic and dielectrophoretic movements were, respectively, dominant in the DC and AC fields. The magnitude of the electric field necessary to cause these effects was found to be 103 -105 times greater than the internationally publicized guideline for human safety. © 2022 Bioelectromagnetics Society.

Surfactant-assisted direct electron transfer between multi-copper oxidases and carbon nanotube-based porous electrodes.

The effects of pre-treatment with surfactants on the electrocatalytic reaction of multi-copper oxidases were quantitatively evaluated using a well-structured carbon nanotube forest electrode. It was found that both the charge polarity of the head group and the aromatics in the tail part of the surfactants affect the efficiency of enzymatic electrocatalysis.

Erratum: Optical parameters estimation in inhomogeneous turbid media using backscattered light: for transcutaneous scattering measurement of intravascular blood: erratum.

[This corrects the article on p. 237 in vol. 15, PMID: 38223194.].

Optical parameters estimation in inhomogeneous turbid media using backscattered light: for transcutaneous scattering measurement of intravascular blood.

In our earlier research, a technique was developed to estimate the effective attenuation coefficient of subcutaneous blood vessels from the skin surface using the spatial distribution of backscattered near-infrared (NIR) light. The scattering effect in surrounding tissues was suppressed through the application of a differential principle, provided that the in vivo structure is known. In this study, a new method is proposed enabling the separate estimation of both scattering and absorption coefficients using NIR light of different wavelengths. The differential technique is newly innovated to make it applicable to the subcutaneous structure without requiring explicit geometrical information. Suppression of the scattering effect from surrounding tissue can be incorporated into the process of estimating the scattering and absorption coefficients. The validity of the proposed technique can be demonstrated through Monte Carlo simulations using both homogeneous and inhomogeneous tissue-simulating models. The estimated results exhibit good coherence with theoretical values (r2 = 0.988-0.999). Moreover, the vulnerability and robustness of the proposed technique against different measurement errors are verified. Optimal conditions for practical measurement are specified under various light-detection conditions. Separate estimation of scattering and absorption coefficients improves the accuracy of turbidity measurements and spectroscopy in biomedical applications considerably, particularly for noninvasive measurements and analysis of blood, lipids, and other components in subcutaneous blood vessels.

A Multienzyme Logic H+ and Na+ Biotransducer.

Sodium ions and protons regulate various fundamental processes at the cell and tissue levels across all biological kingdoms. It is therefore pivotal for bioelectronic devices, such as biosensors and biotransducers, to control the transport of these ions through biological membranes. Our study explores the regulation of proton and sodium concentrations by integrating an Na+-type ATP synthase, a glucose dehydrogenase (GDH), and a urease into a multienzyme logic system. This system is designed to operate using various chemical control input signals, while the output current corresponds to the local change in proton or sodium concentrations. Therein, a H+ and Na+ biotransducer was integrated to fulfill the roles of signal transducers for the monitoring and simultaneous control of Na+ and H+ levels, respectively. To increase the proton concentration at the output, we utilized GDH driven by the inputs of glucose and nicotinamide adenine dinucleotide (NAD+), while recorded the signal change from the biotransducer, together acting as an AND enzyme logic gate. On the contrary, we introduced urease enzyme which hydrolyzed urea to control the decrease in proton concentration, serving as a NOT gate and reset. By integrating these two enzyme logic gates we formed a simple multienzyme logic system for the control of proton concentrations. Furthermore, we also demonstrate a more complex, Na+-type ATP synthase-urease multienzyme logic system, controlled by the two different inputs of ADP and urea. By monitoring the voltage of the peak current as the output signal, this logic system acts as an AND enzyme logic gate. This study explores how multienzyme logic systems can modulate biologically important ion concentrations, opening the door toward advanced biological on-demand control of a variety of bioelectronic enzyme-based devices, such as biosensors and biotransducers.

Proton Logic Gate Based on a Gramicidin-ATP Synthase Integrated Biotransducer.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

pH modulation in adhesive cells with a protonic biotransducer.

Protons (H+) are essential for most physiological functions in organelles and cells. In this study, we have demonstrated a sulfonated polyaniline (SPA) biotransducer that can modulate the intracellular pH in C6 cells with an applied potential, which is directly coupled with H+ to facilitate engineering interactions with physiological processes in the cells. To modulate the pH in the intracellular fluid, we improved the performance of SPA biotransducer by coating of a carbon nanotube (CNT) supportive layer that provides high H+ selectivity in the solution and also high H+ capacity in the hybrid SPA electrode. The intracellular pH modulation was succeeded by applying a potential difference of less than ±0.6 V. pH modulation in the cells is effected by using the biotransducer, which drives the activity of plasma membrane potential and the flow of molecules through the permeable membrane between cells and culture medium, whereas the poly (3,4-ethylenedioxythiophene) (PEDOT)-based biotransducer, which does not have H+ selectivity, was insufficient for modulation. Furthermore, the protonic biotransducer can control the increase/decrease in mitochondria membrane potential, reactive oxygen species and intracellular Ca2+ concentration. Therefore, the protonic biotransducer provides a new perspective to transfer a H+ signal into the cells for modulating the functions.

Self-regulating enzyme-nanotube ensemble films and their application as flexible electrodes for biofuel cells.

Nanostructured carbons have been widely used for fabricating enzyme-modified electrodes due to their large specific surface area. However, because they are random aggregates of particular or tubular nanocarbons, the postmodification of enzymes to their intrananospace is generally hard to control. Here, we describe a free-standing film of carbon nanotube forest (CNTF) that can form a hybrid ensemble with enzymes through liquid-induced shrinkage. This provides in situ regulation of its intrananospace (inter-CNT pitch) to the size of enzymes and eventually serves as a highly active electrode. The CNTF ensemble with fructose dehydrogenase (FDH) showed the oxidation current density of 16 mA cm(-2) in stirred 200 mM fructose solution. The power density of a biofuel cell using the FDH-CNTF anode and the Laccase-CNTF cathode reached 1.8 mW cm(-2) (at 0.45 V) in the stirred oxygenic fructose solution, more than 80% of which could be maintained after continuous operation for 24 h. Application of the free-standing, flexible character of the enzyme-CNTF ensemble electrodes is demonstrated via their use in the patch or wound form.

Fiber-crafted biofuel cell bracelet for wearable electronics.

Wearable devices that generate power using sweat have garnered much attention in the field of skin electronics. These devices require high performance with a small volume and low production rate of sweat by living organisms. Here we demonstrate a high-power biofuel cell bracelet based on the lactate in human sweat. The biofuel cell was developed by using a lactate oxidase/osmium-based mediator/carbon nanotube fiber for lactate oxidation and a bilirubin oxidase/carbon nanotube fiber for oxygen reduction; the fibers were woven into a hydrophilic supportive textile for sweat storage. The storage textile was sandwiched between a hydrophobic textile for sweat absorption from the skin and a hydrophilic textile for water evaporation to improve sweat collection. The performance of the layered cell was 74 µW at 0.39 V in 20 mM artificial sweat lactate, and its performance was maintained at over 80% for 12 h. Furthermore, we demonstrated a series-connection between anode/cathode fibers by tying them up to wrap the bracelet-type biofuel cell on the wrist. The booster six-cell bracelet generated power at 2.0 V that is sufficient for operating digital wrist watches.

Automatic, sequential power generation for prolonging the net lifetime of a miniature biofuel cell stack.

A sequential power generation system for prolonging the net lifetime of a miniature biofuel cell stack has been developed. The system consists of layered chambers of enzyme fuel cells designed to be exposed sequentially to fuel solution by automatically switched fuel flow. The cell chambers were initially separated by magnetized plastic covers sealed with a degradable glue, poly(lactic-co-glycolic acid) (PLGA). The time that the cover was opened by attraction with an external magnet, thereby activating the following cell, was adjustable from a few hours to a few weeks by controlling the weight ratio of Fe(3)O(4) in the covers and the molecular weight of PLGA. By using sequential power generation in this way, the power output of the system was stable for longer periods, and therefore the net lifetime of the stack has been extended as compared with that of a single biofuel cell.

Conducting polymer electrodes printed on hydrogel.

We report herein the micropatterning of poly(3,4-ethylenedioxythiophene) (PEDOT) on a hydrogel, agarose, to provide a fully organic, moist, and flexible electrode. The PEDOT/agarose electrodes were prepared through two electrochemical processes: electropolymerization of PEDOT into the hydrogel and electrochemical-actuation-assisted peeling. We also present a typical application of the PEDOT/agarose electrode to the cultivation of contractile myotubes.

Electrodes combined with an agarose stamp for addressable micropatterning.

We have combined a topographically patterned agarose microstamp with an electrode substrate to develop a novel printing device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. The 10 wt % agarose gel containing 0.1 M PBS + 25 mM KBr showed suitable elasticity for forming stamps and served as the electrolytic medium for the electrochemical oxidation of Br(-) to generate HBrO. The electrode substrate patched with an agarose stamp having 50-microm-high bumps was used for the spatially confined detachment of heparin/polyethyleneimine precoated on glass substrates, followed by micropatterned adsorption of fibronectin. Using the microelectrode array, the addressable micropatterning of protein by the controlled delivery of HBrO to each bump was demonstrated.

A needle-type biofuel cell using enzyme/mediator/carbon nanotube composite fibers for wearable electronics.

To realize direct power generation from biofuels in natural organisms, we demonstrate a needle-type biofuel cell (BFC) using enzyme/mediator/carbon nanotube (CNT) composite fibers with the structure Osmium-based polymer/CNT/glucose oxidase/Os-based polymer/CNT. The composite fibers performed a high current density (10 mA/cm2) in 5 mM artificial blood glucose. Owing to their hydrophilicity, they also provided sufficient ionic conductivity between the needle-type anode and the gas-diffusion cathode. When the tip of the anodic needle was inserted into natural specimens of grape, kiwifruit, and apple, the assembled BFC generated powers of 55, 44, and 33 µW from glucose, respectively. In addition, the power generated from the blood glucose in mouse heart was 16.3 µW at 0.29 V. The lifetime of the BFC was improved by coating an anti-fouling polymer 2-methacryloyloxyethyl phosphorylcholine (MPC) on the anodic electrode, and sealing the cathodic hydrogel chamber with medical tape to minimize the water evaporation without compromising the oxygen permeability.

Wearable high-powered biofuel cells using enzyme/carbon nanotube composite fibers on textile cloth.

Wearable biofuel cells with flexible enzyme/carbon nanotube (CNT) fibers were designed on a cotton textile cloth by integrating two components: bioanode fibers for glucose oxidation and O2-diffusion biocathode fibers for oxygen reduction. The anode and cathode fibers were prepared through modification with glucose dehydrogenase and bilirubin oxidase, respectively, on multi-walled carbon nanotube-coated carbon fibers. Both biofibers woven on the cloth generated a power density of 48 µW/cm2 at 0.24 V from 0.1 mM glucose (human sweat amount), and of 216 µW/cm2 at 0.36 V, when glucose was supplied from a hydrogel tank containing 200 mM glucose. Our fiber-based biofuel cell deformed to an S-shape without a significant loss in cell performance. Furthermore, we demonstrated a series-connection involving the tying of biofibers on a cloth with batik-based ionic isolation. The booster four cells generate power at 1.9 V that illuminated an LED on the cloth.

Nanostraw membrane stamping for direct delivery of molecules into adhesive cells.

Delivering ions and molecules into living cells has become an important challenge in medical and biological fields. Conventional molecular delivery, however, has several issues such as physical and chemical damage to biological cells. Here, we present a method of directly delivering molecules into adhesive cells with an Au-based nanostraw membrane stamp that can physically inject a target molecule into the cytoplasm through a nanostraw duct. We successfully delivered calcein target molecules into adhesive cells with high efficiency (85%) and viability (90%). Furthermore, we modeled the molecular flow through Au nanostraws and then demonstrated the control of calcein flow by changing the concentration and geometry of Au nanostraws. Our Au membrane stamping provides a new way of accessing the cytoplasm to modulate cellular functions via injected molecules.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 microM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.

Smoking trends before, during, and after pregnancy among women and their spouses.

BACKGROUND: Pregnancy and childbirth can act as strong factors motivating parents to spontaneously quit smoking. The aim of the present survey was to establish smoking cessation guidelines for this group. The objectives were to clarify the smoking status of parents before, during and after pregnancy, as well as the factors associated with continuous smoking during pregnancy and post-partum smoking relapse among women who had stopped smoking during pregnancy. METHODS: A cross-sectional survey with self-administered questionnaires was conducted for the parents of the 908 infants who participated in the official medical and dental examination for 18-month-old infants in Itabashi ward, Tokyo, Japan. RESULTS: The prevalence of smoking among women before, during pregnancy, and at post-partum 18 months at 95% confidence intervals was 29.3% (23.3-35.3%), 9.8% (3.0-16.6%), and 23.1 (16.8%-29.4%), and among their spouses it was 64.3% (60.0-68.6%), 58.1% (53.4-62.7%), and 58.2% (53.5-62.8%), respectively. The positive factors associated with smoking among pregnant women were marriage to a smoker, less education, under stress, and part-time employment rather than unemployment. The positive factors associated with smoking relapse after childbirth among women were breast-feeding <6 months and age under 30 years. CONCLUSION: Compared with Western countries, the rate of smoking cessation during pregnancy was relatively high in Japan. It is necessary that smoking cessation intervention and support for men be initiated at the early stages of their spouses' pregnancies. These observations could influence the targeting and design of maternal smoking intervention.

Use of alcohol and hypnotic medication as aids to sleep among the Japanese general population.

OBJECTIVE: The present study was conducted to clarify the prevalence of the use of alcohol and hypnotic medication as sleep aids, and associated factors, in the general population in Japan. METHODS: The survey was conducted in June 2000, using self-administered questionnaires, targeting a population that was selected randomly from among 300 communities throughout Japan. A total of 18,205 responses indicating alcohol use and 16,804 responses indicating hypnotic medication use were analyzed. RESULTS: The prevalence of alcohol use as a sleep aid one or more times per week was 48.3% among men and 18.3% among women. The prevalence of the use of hypnotic medication one or more times per week was 4.3% among men and 5.9% among women. The prevalence of alcohol used as a sleep aid increased gradually for men and women up to age 55-59 years and 40-44 years, respectively, and then declined with increasing age thereafter. The prevalence of the use of hypnotic medication among both men and women showed a trend toward a gradual increase with age. The use of alcohol as a sleep aid was associated with "difficulty maintaining sleep," but no such problem was associated with the use of hypnotic medication. CONCLUSIONS: Alcohol is a more popular sleep aid than hypnotic medication. The factors associated with the use of alcohol and of hypnotic medication are different.