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This study demonstrates, for the first time, the proof-of-concept of a novel immunosensor, a touchpad-based immunochromatographic strip, that non-invasively extracts and detects skin surface proteins. The strip was composed of a nitrocellulose membrane at the center, where a spot of anti-human IgG capture antibody was physically adsorbed. The capture antibody spot was covered with a glass fiber membrane impregnated with phosphate-buffered saline (PBS) to extract skin surface proteins, avoiding direct contact of the human skin with the capture antibodies. Skin surface IgG was detected in two steps: (1) touching the capture antibody via a glass fiber membrane containing PBS, and (2) dipping the strip into the Au-nanoparticle-labeled secondary antibody to visualize the existence of the captured skin surface IgG on the strip. We qualitatively demonstrated that using a very small amount of PBS while maintaining contact with the skin, skin surface proteins can be concentrated and detected, even with a relatively low-sensitivity immunochromatographic chip. This sensor is expected to be a potential biosensor for the non-invasive diagnosis of the integrity of human skin.
Assuntos
Cromatografia de Afinidade , Pele , Humanos , Pele/química , Cromatografia de Afinidade/métodos , Ouro/química , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Nanopartículas Metálicas/química , Colódio/química , Técnicas Biossensoriais/métodosRESUMO
This study demonstrated for the first time that skin surface pH can be monitored in real-time, using a screen-printed wearable pH sensor, to evaluate the buffering capacity of the human skin. The screen-printed pH sensor was composed of a polyaniline-based pH-sensitive electrode and a nitrocellulose membrane-based liquid junction type of Ag/AgCl reference electrode. This sensor showed a reliable and reversible potentiometric response to pH with long-term potential stability. Intermittent monitoring of the buffering capacity of skin surface pH demonstrated the reliability of the proposed wearable pH sensor, which was comparable to that of a commercially available flat-tip pH sensor. We found that contact of the wearable pH sensor with the subject's skin via aqueous electrolyte solutions was necessary for the sensor to continuously monitor the skin surface pH while sustaining the natural buffer capacity of the human skin surface.
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Dispositivos Eletrônicos Vestíveis , Humanos , Reprodutibilidade dos Testes , Pele , Eletrodos , Concentração de Íons de HidrogênioRESUMO
This is the first report demonstrating proof of concept for the passive, non-invasive extraction and in situ potentiometric detection of human sweat chloride ions (Cl- ions) using a stable printed planar liquid-junction reference electrode-integrated hydrogel-based touch-sensor pad without activities such as exercise to induce perspiration, environmental temperature control, or requiring cholinergic drug administration. The sensor pad was composed entirely of a screen-printed bare Ag/AgCl-based chloride ion-selective electrode and a planar liquid-junction Ag/AgCl reference electrode, which were fully covered by an agarose hydrogel in phosphate-buffered saline (PBS). When human skin contacted the hydrogel pad, sweat Cl- ions were continuously extracted into the gel, followed by in situ potentiometric detection. The planar liquid-junction Ag/AgCl reference electrode had a polymer-based KCl-saturated inner electrolyte layer to stabilize the potential of the Ag/AgCl electrode even with a substantial change in the chloride ion concentration in the hydrogel pad. We expect this fully screen-printed sensor to achieve the low-cost passive and non-invasive daily monitoring of human Cl- ions in sweat in the future.
Assuntos
Colinérgicos/farmacologia , Eletrólitos , Hidrogéis/química , Íons , Polímeros/química , Potenciometria/instrumentação , Técnicas Biossensoriais , Cloretos/química , Eletroquímica , Eletrodos , Desenho de Equipamento , Humanos , Eletrodos Seletivos de Íons , Potenciometria/métodos , Sefarose/química , Pele/efeitos dos fármacos , Suor , TemperaturaRESUMO
Culturing cell spheroids in microchamber arrays is a widely used method in regenerative medicine and drug discovery while it requires laborious procedures during medium exchange and drug administration. Here, we report a simple method for the medium exchange and drug testing using a hydrogel-based sealed microchamber arrays. Owing to the high molecular permeability of poly(vinyl alcohol) hydrogel, the sealed microchamber allows nutrients and drugs in outer medium to pass through. Thus, automatic medium exchange and drug testing for all the cell spheroids inside the microchamber arrays are achieved by simply transferring the microchamber from old medium to fresh medium. Cell spheroids of human induced pluripotent stem cell-derived cardiomyocytes were cultured inside the sealed microchambers, and it was confirmed that the spheroids were stably positioned inside the microchamber even after transferring 10 times. The cell spheroids showed high viability after culturing for 7 days in the sealed microchamber with the transfer-based medium exchange, which allowed cardiac maturation by simultaneous electrical stimulation. Isoproterenol, a model cardiac drug, was administrated from outside the sealed microchamber to demonstrate the feasibility of drug testing by the rapid transfer method.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Hidrogéis/química , Microtecnologia/instrumentação , Esferoides Celulares/efeitos dos fármacos , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Permeabilidade , Álcool de Polivinil/química , Fatores de TempoRESUMO
An array of porous microneedles was developed for minimally-invasive transdermal electrolytic connection through the human skin barrier, the stratum corneum. The length of microneedle was designed to be 100 µm so that it penetrates into the epidermis layer without pain. Each microneedle was supported by a thicker cylindrical post protruding from a planar substrate to realize its effective penetration even into elastic human skin. Since this support (post and substrate) was equally porous as the needles, the needle chip was entirely permeable for electrolyte. This ion-conductive porous microneedle array was applied to the transdermal conductometry with small direct current for local monitoring of intercellular swelling, edema. The porous needle-based electrode system could be a platform for various transdermal electrical diagnosis and treatments.
Assuntos
Células Epidérmicas , Espaço Extracelular/metabolismo , Microinjeções/instrumentação , Agulhas , Humanos , PorosidadeRESUMO
A commercial painless microneedle was filled with physiological saline agar, and this needle-based salt bridge was inserted into the skin (a piece of porcine skin and a flank skin of a live mouse) to make an electrical contact with its subepidermal region. The transepidermal potential (TEP), the potential difference between the skin surface and the subepidermal region, was measured using this inner electrode and a conventional agar electrode on the surface of the skin. Control of penetration depth of the inner electrode with a spacer and hydrophilic pretreatment with ozone plasma were found to be necessary for stable measurement. The TEP was reduced upon damages on the skin surface by tape stripping and acetone defatting, which indicated the fabricated needle electrode is useful for the minimally-invasive measurement of TEP and evaluation of skin barrier functions. Furthermore, we showed that the device integrating two electrodes into a single compact probe was useful to evaluate the local barrier functions and their mapping on a skin. This device could be a personal diagnostic tool in the fields of medicine and cosmetics in future.
Assuntos
Agulhas , Potenciometria/métodos , Pele/efeitos dos fármacos , Administração Cutânea , Ágar , Animais , Eletrodos , Desenho de Equipamento , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Sais/química , SuínosRESUMO
Non-electroactive bacteria (n-EAB), constituting the majority of known bacteria to date, have been underutilized in electrochemical conversion technologies due to their lack of direct electron transfer to electrodes. In this study, we established an electric wiring between n-EAB (gram-positive Bacillus subtilis and gram-negative Escherichia coli) and an extracellular electrode via a ferrocene-polyethyleneimine-based redox polymer (Fc-PEI). Chronoamperometry recordings indicated that Fc-PEI can transfer intracellular electrons to the extracellular electrode regardless of the molecular organization of PEI (linear or branched) and the membrane structure of bacteria (gram-positive or -negative). As fluorescence staining suggested, Fc-PEI improves the permeability of the bacterial cell membrane, enabling electron carriers in the cell to react with Fc. In addition, experiments with Fc-immobilized electrodes without PEI suggested the existence of an alternative electron transfer pathway from B. subtilis to the extracellular Fc adsorbed onto the cell membrane. Furthermore, we proposed for the first time that the bacteria/Fc-linear PEI modified structure enables selective measurement of immobilized bacterial activity by physically blocking contact between the electrode surface and planktonic cells co-existing in the surrounding media. Such electrodes can be a powerful analytical tool for elucidating the metabolic activities of specific bacteria wired to the electrode even within complex bacterial communities.
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This paper proposes a minimally invasive current-controlled electric stimulation system based on a poly(3,4-ethylenedioxythiophene) (PEDOT)-modified electrode to characterize the dynamics of the membrane potential in Bacillus subtilis. A highly capacitive PEDOT-modified electrode enabled the injection of a large ionic charge to the surface of the cells suppressing cytotoxic pH change in the vicinity of the electrode. The current pulse induced a hyperpolarization response in B. subtilis around the electrode. Using quantitative charge injection through current-controlled electrical stimulation, the threshold charge density to excite B. subtilis was roughly estimated to be 530.8 µC cm-2 (of electrode surface area) for the first time. Our results provide the minimum electrical stimulation conditions necessary to minimal invasively control the bacterial membrane potential.
Assuntos
Bactérias , Polímeros , Polímeros/química , Estimulação Elétrica , EletrodosRESUMO
This paper reports a new hydrogel extraction technique for detecting blue fluorescent substances in plant leaves. These blue fluorescent substances were extracted by placing a hydrogel film on the leaf of a cherry tomato plant infected with Ralstonia solanacearum; herein, chlorogenic acid was confirmed to be a blue fluorescent substance. The wavelength at the maximum fluorescence intensity of the film after the hydrogel extraction was similar to that of the methanolic extract obtained from the infected cherry tomato leaves. Chlorophyll was not extracted from the hydrogel film because no fluorescence peak was observed at 680 nm. Accordingly, the blue fluorescence of the substances extracted from the hydrogel film was not quenched by the strong absorption of chlorophyll in the blue light region. This hydrogel extraction technique can potentially detect small amounts of blue fluorescent substances and the changes in its amount within the leaves of infected plants. These changes in the amount of blue fluorescent substances in the early stages of infection can be used to detect presymptomatic infections. Therefore, hydrogel extraction is a promising technique for the noninvasive detection of infections before onset.
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Hidrogéis , Solanum lycopersicum , Clorofila , Folhas de Planta , Plantas , Espectrometria de FluorescênciaRESUMO
This is the first report of conducting proof-of-concept study for amperometric acetyltransferase-based L-carnitine sensor by employing the thiol/disulfide exchange reaction. The carnitine acetyltransferase (CrAT) catalyzes the reaction between acetyl-CoA and L-carnitine to produce CoA which is difficult to detect directly by electrochemical methods owing to steric hindrance and electrostatic effect of CoA. The thiol/disulfide exchange reaction between CoA and cystamine was mediated in the enzymatic reaction to produce electrochemically detectable low molecular weight of cationic cysteamine. The sensor exhibited high sensitivity and selectivity for L-carnitine in the concentration range 0.28-50 µM with a limit of detection of 0.28 µM. This is a promising strategy for L-carnitine sensing in point-of-care testing applications.
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Técnicas Biossensoriais , Carnitina , Acetilcoenzima A , Dissulfetos , Compostos de SulfidrilaRESUMO
This report is the first on heat-assisted transferable battery components, enabling manufacturing batteries on non-planer surfaces such as a curved surface and an edge. The transferrable battery components were composed of two layers: a cathode or an anode and a conductive heal-melt adhesive layer on a silicone-based flexible supporting paper. These mechanically-durable, flexible components enabled conformable adhesion even on curved surfaces and substrate edges. As a model battery, the manganese dioxide-zinc system was constructed on a curved surface using transfer techniques and showed a practical capacity of 1.8 mAh cm-2 per unit electrode area. These transferable electrodes allow arbitrary design of batteries according to the power consumption of IoT devices to be fabricated on unreported geometries where has been considered as a dead space.
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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.
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Eletrodos , Hidrogéis , Polímeros , Materiais BiocompatíveisRESUMO
Contractile C(2)C(12) myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C(2)C(12) myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish.
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Fibrina/química , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Técnicas de Cultura de Tecidos/métodos , Alicerces Teciduais/química , Animais , Estimulação Elétrica , Camundongos , Microtecnologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/ultraestruturaRESUMO
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.
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Eletrodos , Sefarose/química , Adsorção , Eletroquímica , Fibronectinas/química , Heparina/química , Microeletrodos , Polietilenoimina/químicaRESUMO
Scanning ion conductance microscopy (SICM) using a nanopipette as a probe and ionic current as a feedback signal was introduced as a novel technique to study live cells in a physiological environment. To avoid contact between the pipette tip and cells during the conventional lateral scanning mode, we adopted a standing approach (STA) mode in which the probe was moved vertically to first approach and then retracted from the cell surface at each measurement point on an XY plane. The STA mode ensured non-contact imaging of the topography of live cells and for a wide range of uneven substrates (500 x 300 microm to 5 x 5 microm). We also used a field-programmable gate array (FPGA) board to enhance feedback distance regulation. FPGA dramatically increased the feedback speed and decreased the imaging time (450 s per image) with enhanced accuracy and quality of live cell images. To evaluate the potential of the STA mode for SICM, we carried out imaging of a convoluted surface of live cell in various scan ranges and estimated the spatial resolutions of these images.
Assuntos
Microscopia/métodos , Imagem Molecular/métodos , Animais , Axônios/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Cricetinae , Dimetilpolisiloxanos/química , Humanos , Fibras Musculares Esqueléticas/citologiaRESUMO
The application of a multienzyme cascade reaction in electrochemical biosensors has the advantage of expanding the target substrates in addition to selectivity combining multiple enzymes on an electrode. However, the multienzyme system has the drawback of inefficient substance conversion because of the time-consuming passing of intermediates between the enzymes and/or diffusional loss of the intermediates. In this study, the optimal construction of a multienzymatic film in an ammonia detection sensor was investigated using a cascade reaction of l-glutamate oxidase and l-glutamate dehydrogenase as a model sensor. Three enzymatic films were prepared: (1) a mixed film designed to have a short diffusional distance between closely located enzymes, (2) a normal-sequential layered film arranged for the correct reaction pathway, and (3) a reverse-sequential layered film as a negative control. This was followed by comparison of the conversion efficiency of ammonia to hydrogen peroxide using time-dependent potentiometric measurements of a Prussian blue electrode determining the hydrogen peroxide amount. The results indicate that the conversion efficiency of the normal-sequential layered film was the highest among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction showed that compared to the mixed film (34%), a higher conversion efficiency of 92% was obtained in the first enzymatic reaction step. These findings will promote the use of multienzymatic cascade reaction systems not only in biosensors and bioreactors but also in various industrial fields.
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This review describes recent advances in biosensors for non-invasive human healthcare applications, especially focusing on sweat analysis, along with approaches for fabricating these biosensors based on printed electronics technology. Human sweat contains various kinds of biomarkers. The relationship between a trace amount of sweat biomarkers partially partitioned from blood and diseases has been investigated by omic analysis. Recent progress in wearable or portable biosensors has enabled periodic or continuous monitoring of some sweat biomarkers while supporting the results of the omic analysis. In this review, we particularly focused on a transistor-based biosensor that is highly sensitive in quantitatively detecting the low level of sweat biomarkers. Furthermore, we showed a new approach of flexible hybrid electronics that has been applied to advanced sweat biosensors to realize fully integrated biosensing systems wirelessly connected to a networked IoT system. These technologies are based on uniquely advanced printing techniques that will facilitate mass fabrication of high-performance biosensors at low cost for future smart healthcare.
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Técnicas Biossensoriais , Impressão Tridimensional , Suor , Humanos , Compostos OrgânicosRESUMO
A highly sensitive and quantitative analysis was performed using a poly(dimethylsiloxane) (PDMS) microwell array in a scanning electrochemical microscopy setup. A microelectrode with a relatively large seal radius was used to cover the top of the cylindrical PDMS microwell (96 pL). The voltammogram for 4 mM ferrocyanide resulted in a charge value of 38 nC, suggesting that almost 100% of the reductant in the microwell was converted to the oxidation current. When genetically modified yeast cells were entrapped in the microwell, the accumulation of p-aminophenol (PAP) produced by expressing beta-galactosidase (betaGAL) was successfully observed.
Assuntos
Saccharomyces cerevisiae/enzimologia , beta-Galactosidase/análise , Difusão , Dimetilpolisiloxanos , Eletroquímica/instrumentação , Eletroquímica/métodos , Microquímica/instrumentação , Microquímica/métodos , MicroeletrodosRESUMO
A hydrogel-based microchamber with organic electrodes for efficient electrical stimulations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is described. The microchamber is made from molecularly permeable, optically transparent, and electrically conductive polyvinyl alcohol (PVA) hydrogel and highly capacitive carbon electrode modified with poly(3,4-ethylenedioxythiophene) (PEDOT). Spheroids of hiPSC-CMs are cultured in microchambers, and electrically stimulated by the electrode for maturation. The large interfacial capacitance of the electrodes enables several days of electrical stimulation without generation of cytotoxic bubbles even when the electrodes are placed near the spheroids. The spheroids can be cultivated in the closed microchambers because of the permeated nutrients through the hydrogel, thus the spheroids are stably addressable and the culture medium around the sealed microchambers can be simply exchanged. Synchronized beating of the spheroids can be optically analyzed in situ, which makes it possible to selectively collect electrically responsive cells for further use. As the hydrogel is electrically conductive, the amount of electrical charge needed for maturing the spheroids can be reduced by configuring electrodes on the top and the bottom of the microchamber. The bioreactor will be useful for efficient production of matured hiPSC-CMs for regenerative medicine and drug screening.