RESUMEN
In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.
Asunto(s)
Hormona Adrenocorticotrópica/aislamiento & purificación , Técnicas Biosensibles , Hormona Liberadora de Corticotropina/aislamiento & purificación , Sistema Hipotálamo-Hipofisario/metabolismo , Enfermedad de Addison/diagnóstico , Enfermedad de Addison/genética , Hormona Adrenocorticotrópica/química , Hormona Liberadora de Corticotropina/química , Síndrome de Cushing/diagnóstico , Síndrome de Cushing/genética , Humanos , Hidrocortisona/química , Hidrocortisona/genética , Sistema Hipófiso-Suprarrenal/metabolismo , Receptor de Melanocortina Tipo 2/química , Receptor de Melanocortina Tipo 2/genética , Receptores de Corticotropina/química , Receptores de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
Field-effect transistor (FET) devices based on conductive nanomaterials have been used to develop biosensors. However, development of FET-based biosensors that allow efficient stability, especially in the gas phase, for obtaining reliable and reproducible responses remains a challenge. In this study, we developed a nanodisc (ND)-functionalized bioelectronic nose (NBN) based on a nickel (Ni)-decorated carboxylated polypyrrole nanoparticle (cPPyNP)-FET that offers the detection of liquid and gaseous cadaverine (CV). The TAAR13c, specifically binding to CV, which is an indicator of food spoilage, was successfully constructed in NDs. The NBN was fabricated by the oriented assembly of TAAR13c-embedded NDs (T13NDs) onto the transistor with Ni/cPPyNPs. The NBN showed high performance in selectivity and sensitivity for the detection of CV, with excellent stability in both aqueous and gas phases. Moreover, the NBN allowed efficient measurement of corrupted real-food samples. It demonstrates the ND-based device can allow the practical biosensor that provides high stability in the gas phase.
Asunto(s)
Cadaverina/análisis , Nariz Electrónica , Nanopartículas/química , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Escherichia coli/genética , Contaminación de Alimentos/análisis , Gases/análisis , Límite de Detección , Níquel/química , Espectroscopía de Fotoelectrones , Polímeros/química , Pirroles/química , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sensibilidad y Especificidad , Transistores Electrónicos , Proteínas de Pez Cebra/genéticaRESUMEN
A new polydiacetylene lipid/human olfactory receptor nano-assembly was fabricated for the visual detection of an odorant for the first time. The assembly consisted of phospholipid-mixed polydiacetylenes (PDAs) and human olfactory receptors (hORs) in detergent micelles. To overcome the limitations of bioelectronic noses, hOR-embedded chromatic complexes (PDA/hORs) were developed, introducing PDAs that showed color and fluorescence transitions against various stimuli. The chromatic nanocomplexes reacted with target molecules, showing a fluorescence intensity increase in a dose-dependent manner and target selectivity among various odorants. As a result, a color transition of the assembly from blue to purple occurred, allowing the visual detection of the odorant geraniol. Through circular dichroism (CD) spectroscopy and a tryptophan fluorescence quenching method, the structural and functional properties of the hORs embedded in the complexes were confirmed. Based on this first work, future array devices, integrating multiple nano-assemblies, can be substantiated and utilized in environmental assessment and analysis of food quality.
Asunto(s)
Lípidos/química , Nanoestructuras/química , Polímero Poliacetilénico/química , Receptores Odorantes/química , Terpenos/análisis , Monoterpenos Acíclicos , Dicroismo Circular , Humanos , Micelas , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Fluorescencia , Terpenos/química , Terpenos/metabolismoRESUMEN
We report a strategy for the human-like smelling of a rose scent utilizing olfactory receptor nanodisc (ND)-based bioelectronic nose devices. In this strategy, a floating electrode (FE)-based carbon nanotube (CNT) field effect transistor (FET) was functionalized with human olfactory receptor 1A2 (hOR1A2)-embedded NDs (hOR1A2NDs). The hOR1A2NDs responded to rose scent molecules specifically, which were monitored electrically using the underlying CNT-FET. This strategy allowed us to quantitatively assess the contents of geraniol and citronellol, the main components of a rose scent, as low as 1 fM and 10 fM, respectively. In addition, it enabled us to selectively discriminate a specific rose odorant from other odorants. Significantly, we also demonstrated that the responses of hOR1A2NDs to a rose scent could be strongly enhanced by enhancer materials like a human nose. Furthermore, the method provided a means to quantitatively evaluate rose scent components in real samples such as rose oil. Since our method allows one to quantitatively evaluate general rose scent ingredients just like a human nose, it could be a powerful strategy for versatile basic research and various applications such as fragrance development.
Asunto(s)
Técnicas Biosensibles/métodos , Nariz/fisiología , Receptores Odorantes/metabolismo , Rosa/metabolismo , Olfato/fisiología , Nariz Electrónica , Humanos , Odorantes , Neuronas Receptoras Olfatorias/metabolismo , Feromonas/metabolismo , Transistores ElectrónicosRESUMEN
Numerous efforts have been made to measure tastes for various purposes. However, most taste information is still obtained by human sensory evaluation. It is difficult to quantify a degree of taste or establish taste standard. Although artificial taste sensors called electronic tongues utilizing synthetic materials such as polymers, semiconductors, or lipid membranes have been developed, they have limited performance due to their low sensitivity and specificity. Recently, bioelectronic tongues fabricated by integrating human taste receptors and nanomaterial-based sensor platforms have been found to have high performance for measuring tastes with human-like taste perception. However, human umami taste receptor is heterodimeric class C GPCR composed of human taste receptor type 1 member 1 (T1R1) and member 3 (T1R3). Such complicated structure makes it difficult to fabricate bioelectronic tongue. The objective of this study was to develop a protein-based bioelectronic tongue for detecting and discriminating umami taste with human-like performance using umami ligand binding domain called venus flytrap (VFT) domain originating from T1R1 instead of using the whole heterodimeric complex of receptors. Such T1R1 VFT was produced from Escherichia coli (E. coli) with purification and refolding process. It was then immobilized onto graphene-based FET. This bioelectronic tongue for umami taste (BTUT) was able to detect monosodium L-glutamate (MSG) with high sensitivity (ca. 1â¯nM) and specificity in real-time. The intensity of umami taste was enhanced by inosine monophosphate (IMP) that is very similar to the human taste system. In addition, BTUT allowed efficient reusable property and storage stability. It maintained 90% of normalized signal intensity for five weeks. To develop bioelectronic tongue, this approach using the ligand binding domain of human taste receptor rather than the whole heterodimeric GPCRs has advantages in mass production, reusability, and stability. It also has great potential for various industrial applications such as food, beverage, and pharmaceutical fields.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Escherichia coli/genética , Humanos , Unión Proteica , Dominios Proteicos , Gusto , LenguaRESUMEN
Olfactory receptors (ORs) are the largest family of the G protein-coupled receptors (GPCRs), which are significantly involved in many human diseases and 40% of all drug targets. A platform containing stable and high-quality OR would be a powerful tool for the development of a practical biosensor that can be applied to various applications, such as the early diagnosis of diseases, assessment of food quality, and drug and fragrance development. Significant efforts have been made to develop the biosensor using GPCRs; nevertheless, they remain a challenge. This chapter describes an attractive methodology for the development of a stable bioelectronic nose using OR-embedded nanodiscs. The ORs were produced in Escherichia coli (E. coli), purified with column chromatography, reconstituted into nanodiscs and applied to a carbon nanotube-field effect transistor (CNT-FET) with floating electrodes. The nanodisc-based bioelectronic nose exhibits high-performance in terms of sensitivity, selectivity and stability. This strategy can be used as a practical method for the receptor-based sensing approach, which represents significant progress in nano-bio technology toward a practical biosensor.
Asunto(s)
Técnicas Biosensibles/métodos , Nariz Electrónica , Nanotubos de Carbono/química , Receptores Odorantes/química , Transistores Electrónicos , Animales , Técnicas Biosensibles/instrumentación , Humanos , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Photosensitive materials contain biologically engineered elements and are constructed using delicate techniques, with special attention devoted to efficiency, stability, and biocompatibility. However, to date, no photosensitive material has been developed to replace damaged visual-systems to detect light and transmit the signal to a neuron in the human body. In the current study, artificial nanovesicle-based photosensitive materials are observed to possess the characteristics of photoreceptors similar to the human eye. The materials exhibit considerably effective spectral characteristics according to each pigment. Four photoreceptors originating from the human eye with color-distinguishability are produced in human embryonic kidney (HEK)-293 cells and partially purified in the form of nanovesicles. Under various wavelengths of visible light, electrochemical measurements are performed to analyze the physiological behavior and kinetics of the photoreceptors, with graphene, performing as an electrode, playing an important role in the lipid bilayer deposition and oxygen reduction processes. Four nanovesicles with different photoreceptors, namely, rhodopsin (Rho), short-, medium-, and longwave sensitive opsin 1 (1SW, 1MW, 1LW), show remarkable color-dependent characteristics, consistent with those of natural human retina. With four different light-emitting diodes for functional verification, the photoreceptors embedded in nanovesicles show remarkably specific color sensitivity. This study demonstrates the potential applications of light-activated platforms in biological optoelectronic industries.
RESUMEN
Cadaverine (CV), a death-associated odor, is an important target molecule for various sensor applications, including the evaluation of food spoilage. In this study, we developed an oriented nanodisc (ND)-functionalized bioelectronic nose (ONBN), based on carbon nanotube transistors and nanodiscs embedded with an olfactory receptor produced in Escherichia coli (E. coli) for detection of CV. To fabricate ONBN devices, a trace-amine-associated receptor 13c (TAAR13c) binding to CV was produced in E. coli, purified, reconstituted into NDs, and assembled, in the desired orientation, onto a carbon- nanotube-based field-effect transistor with floating electrodes. The ONBN showed high performance in terms of sensitivity and selectivity. Moreover, the ONBN was used to measure CV in diverse real-food samples for the determination of food freshness. These results indicate ONBN devices can be utilized to evaluate the quality of food samples quantitatively, which should enable versatile practical applications such as food safety and preservative development. Moreover, the ONBN could provide a useful tool for detection of corpses, which could be practically used in disaster responses.
Asunto(s)
Cadaverina/análisis , Escherichia coli/química , Nanopartículas/química , Nanotubos de Carbono/química , Receptores Odorantes/química , Nariz Electrónica , Escherichia coli/metabolismo , Tamaño de la Partícula , Receptores Odorantes/biosíntesis , Propiedades de SuperficieRESUMEN
The field-effect transistor (FET) has been used in the development of diagnostic tools for several decades, leading to high-performance biosensors. Therefore, the FET platform can provide the foundation for the next generation of analytical methods. A major role of G-protein-coupled receptors (GPCRs) is in the transfer of external signals into the cell and promoting human body functions; thus, their principle application is in the screening of new drugs. The research community uses efficient systems to screen potential GPCR drugs; nevertheless, the need to develop GPCR-conjugated analytical devices remains for next-generation new drug screening. In this study, we proposed an approach for studying receptor agonism and antagonism by combining the roles of FETs and GPCRs in a dopamine receptor D1 (DRD1)-conjugated FET system, which is a suitable substitute for conventional cell-based receptor assays. DRD1 was reconstituted and purified to mimic native binding pockets that have highly discriminative interactions with DRD1 agonists/antagonists. The real-time responses from the DRD1-nanohybrid FET were highly sensitive and selective for dopamine agonists/antagonists, and their maximal response levels were clearly different depending on their DRD1 affinities. Moreover, the equilibrium constants (K) were estimated by fitting the response levels. Each K value indicates the variation in the affinity between DRD1 and the agonists/antagonists; a greater K value corresponds to a stronger DRD1 affinity in agonism, whereas a lower K value in antagonism indicates a stronger dopamine-blocking effect.
Asunto(s)
Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/instrumentación , Diseño de Equipo , Humanos , Proteínas Inmovilizadas/agonistas , Proteínas Inmovilizadas/antagonistas & inhibidores , Proteínas Inmovilizadas/metabolismo , Receptores de Dopamina D1/metabolismo , Transistores ElectrónicosRESUMEN
In the brain and central nervous system, dopamine plays a crucial role as a neurotransmitter or a local chemical messenger for interneuronal communication. Dopamine is associated with renal, hormonal, and cardiovascular systems. Additionally, dopamine dysfunction is known to cause serious illnesses, such as Parkinson's disease and Alzheimer's disease. Therefore, dopamine detection is essential for medical diagnosis and disease prevention and requires a novel strategy with high sensitivity and selectivity and a rapid response. Herein, we present a novel human dopamine receptor (hDRD1)-conjugated multidimensional conducting polymer nanofiber (NF) membrane for the selective and sensitive detection of dopamine. The membrane, which consists of multidimensional carboxylated poly(3,4-ethylenedioxythiophene) (MCPEDOT) NFs with nanorods, is used as a transistor in a liquid-ion gated field-effect transistor (FET)-based biosensor. Interestingly, hDRD1 is first expressed in Escherichia coli before it is immobilized onto the MCPEDOT NF. The hDRD1-MCPEDOT NF-based FET exhibits a rapid real-time response (<2 s) with high dopamine selectivity and sensitivity performance (approximately 100 fM). Furthermore, this FET device can be integrated into a poly(dimethylsiloxane)-based microfluidic system and also can retain its high performance in the integrated system, which results in the generation of large-scale dopamine biosensors with a novel geometry.
Asunto(s)
Nanofibras , Técnicas Biosensibles , Dopamina , Humanos , Polímeros , Receptores DopaminérgicosRESUMEN
Human sensory-mimicking systems, such as electronic brains, tongues, skin, and ears, have been promoted for use in improving social welfare. However, no significant achievements have been made in mimicking the human nose due to the complexity of olfactory sensory neurons. Combinational coding of human olfactory receptors (hORs) is essential for odorant discrimination in mixtures, and the development of hOR-combined multiplexed systems has progressed slowly. Here, we report the first demonstration of an artificial multiplexed superbioelectronic nose (MSB-nose) that mimics the human olfactory sensory system, leading to high-performance odorant discriminatory ability in mixtures. Specifically, portable MSB-noses were constructed using highly uniform graphene micropatterns (GMs) that were conjugated with two different hORs, which were employed as transducers in a liquid-ion gated field-effect transistor (FET). Field-induced signals from the MSB-nose were monitored and provided high sensitivity and selectivity toward target odorants (minimum detectable level: 0.1 fM). More importantly, the potential of the MSB-nose as a tool to encode hOR combinations was demonstrated using principal component analysis.