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).

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.].

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.

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.

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.

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.

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.

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.

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.

Accelerated Wound Healing on Skin by Electrical Stimulation with a Bioelectric Plaster.

Wound healing on skin involves cell migration and proliferation in response to endogenous electric current. External electrical stimulation by electrical equipment is used to promote these biological processes for the treatment of chronic wounds and ulcers. Miniaturization of the electrical stimulation device for wound healing on skin will make this technology more widely available. Using flexible enzymatic electrodes and stretchable hydrogel, a stretchable bioelectric plaster is fabricated with a built-in enzymatic biofuel cell (EBFC) that fits to skin and generates ionic current along the surface of the skin by enzymatic electrochemical reactions for more than 12 h. To investigate the efficacy of the fabricated bioelectric plaster, an artificial wound is made on the back skin of a live mouse and the wound healing is observed for 7 d in the presence and absence of the ionic current of the bioelectric plaster. The time course of the wound size as well as the hematoxylin and eosin staining of the skin section reveals that the ionic current of the plaster leads to faster and smoother wound healing. The present work demonstrates a proof of concept for the electrical manipulation of biological functions by EBFCs.

Electronic control of H+ current in a bioprotonic device with Gramicidin A and Alamethicin.

In biological systems, intercellular communication is mediated by membrane proteins and ion channels that regulate traffic of ions and small molecules across cell membranes. A bioelectronic device with ion channels that control ionic flow across a supported lipid bilayer (SLB) should therefore be ideal for interfacing with biological systems. Here, we demonstrate a biotic-abiotic bioprotonic device with Pd contacts that regulates proton (H+) flow across an SLB incorporating the ion channels Gramicidin A (gA) and Alamethicin (ALM). We model the device characteristics using the Goldman-Hodgkin-Katz (GHK) solution to the Nernst-Planck equation for transport across the membrane. We derive the permeability for an SLB integrating gA and ALM and demonstrate pH control as a function of applied voltage and membrane permeability. This work opens the door to integrating more complex H+ channels at the Pd contact interface to produce responsive biotic-abiotic devices with increased functionality.

A Disposable paper breathalyzer with an alcohol sensing organic electrochemical transistor.

UNLABELLED: Breathalyzers estimate Blood Alcohol Content (BAC) from the concentration of ethanol in the breath. Breathalyzers are easy to use but are limited either by their high price and by environmental concerns, or by a short lifetime and the need for continuous recalibration. Here, we demonstrate a proof-of-concept disposable breathalyzer using an organic electrochemical transistor (OECT) modified with alcohol dehydrogenase (ADH) as the sensor. The OECT is made with the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) ( PEDOT: PSS), and is printed on paper. ADH and its cofactor nicotinamide adenine dinucleotide (NAD(+)) are immobilized onto the OECT with an electrolyte gel. When the OECT-breathalyzer is exposed to ethanol vapor, the enzymatic reaction of ADH and ethanol transforms NAD(+) into NADH, which causes a decrease in the OECT source drain current. In this fashion, the OECT-breathalyzer easily detects ethanol in the breath equivalent to BAC from 0.01% to 0.2%. The use of a printed OECT may contribute to the development of breathalyzers that are disposable, ecofriendly, and integrated with wearable devices for real-time BAC monitoring.

Proton mediated control of biochemical reactions with bioelectronic pH modulation.

In Nature, protons (H(+)) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H(+) channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H(+) currents and H(+) concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H(+) between PdHx contacts and solution. The present transducer records bistable pH modulation from an "enzymatic flip-flop" circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.

Grotthuss mechanisms: from proton transport in proton wires to bioprotonic devices.

In 1804, Theodore von Grotthuss proposed a mechanism for proton (H(+)) transport between water molecules that involves the exchange of a covalent bond between H and O with a hydrogen bond. This mechanism also supports the transport of OH(-) as a proton hole and is essential in explaining proton transport in intramembrane proton channels. Inspired by the Grotthuss mechanism and its similarity to electron and hole transport in semiconductors, we have developed semiconductor type devices that are able to control and monitor a current of H(+) as well as OH(-) in hydrated biopolymers. In this topical review, we revisit these devices that include protonic diodes, complementary, transistors, memories and transducers as well as a phenomenological description of their behavior that is analogous to electronic semiconductor devices.

Squid beak inspired water processable chitosan composites with tunable mechanical properties.

Natural biological composites often couple light weight with tunable and spatially controlled mechanical properties including stiffness, toughness, and hardness. Examples include the toughness of seashells, the hardness of the chiton tooth, and the stiffness gradient of the squid beak. While seashells and the chiton tooth have a mineralized inorganic component, the squid beak is entirely organic. The squid beak is known as one of the hardest fully organic materials. The hydrated squid beak has a large stiffness gradient from soft, at the interface with the squid mouth, to hard at the tip. This gradient occurs from the spatially controlled cross-linking of chitin nanofibers with a protein matrix aided by catecholamines. Here, we introduce a water processable deacetylated chitin composite with tunable mechanical properties from spatially controlled cross-linking assisted by catecholamines. Given the natural abundance of chitin and the ease of water processing, this composite can find applications for bridging mechanically mismatched materials.

Stretchable biofuel cell with enzyme-modified conductive textiles.

A sheet-type, stretchable biofuel cell was developed by laminating three components: a bioanode textile for fructose oxidation, a hydrogel sheet containing fructose as fuel, and a gas-diffusion biocathode textile for oxygen reduction. The anode and cathode textiles were prepared by modifying carbon nanotube (CNT)-decorated stretchable textiles with fructose dehydrogenase (FDH) and bilirubin oxidase (BOD), respectively. Enzymatic reaction currents of anode and cathode textiles were stable for 30 cycles of 50% stretching, with initial loss of 20-30% in the first few cycles due to the partial breaking of the CNT network at the junction of textile fibers. The assembled laminate biofuel cell showed power of ~0.2 mW/cm(2) with 1.2 kΩ load, which was stable even at stretched, twisted, and wrapped forms.

Organic transdermal iontophoresis patch with built-in biofuel cell.

A completely organic iontophoresis patch is reported. A built-in biofuel cell is mounted on the patch that generates transdermal iontophoretic administration of compounds into the skin. The amplitude of transdermal current is tuned by integrating a conducting polymer-based stretchable resistor of predetermined resistance.

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.