Sae Ryun Ahn

Ultrasensitive and selective recognition of peptide hormone using close-packed arrays of hPTHR-conjugated polymer nanoparticles.

Recognition of diverse hormones in the human body is a highly significant challenge because numerous diseases can be affected by hormonal imbalances. However, the methodologies reported to date for detecting hormones have exhibited limited performance. Therefore, development of innovative methods is still a major concern in hormone-sensing applications. In this study, we report an immobilization-based approach to facilitate formation of close-packed arrays of carboxylated polypyrrole nanoparticles (CPPyNPs) and their integration with human parathyroid hormone receptor (hPTHR), which is a B-class family of G-protein-coupled receptors (GPCRs). Our devices enabled use of an electrically controllable liquid-ion-gated field-effect transistor by using the surrounding phosphate-buffered saline solution (pH 7.4) as electrolyte solution. Field-induced signals from the peptide hormone sensors were observed and provided highly sensitive and selective recognition of target molecules at unprecedentedly low concentrations (ca. 48 fM). This hormone sensor also showed long-term stability and excellent selectivity in fetal bovine serum. Importantly, the hormone receptor attached on the surface of CPPyNPs enabled GPCR functional studies; synergistic effects corresponding to increased hPTH peptide length were monitored. These results demonstrate that close-packed CPPyNP arrays are a promising approach for high-performance biosensing devices.

Human dopamine receptor nanovesicles for gate-potential modulators in high-performance field-effect transistor biosensors.

The development of molecular detection that allows rapid responses with high sensitivity and selectivity remains challenging. Herein, we demonstrate the strategy of novel bio-nanotechnology to successfully fabricate high-performance dopamine (DA) biosensor using DA Receptor-containing uniform-particle-shaped Nanovesicles-immobilized Carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious diseases, such as Parkinsons and Alzheimers diseases. For the first time, nanovesicles containing a human DA receptor D1 (hDRD1) were successfully constructed from HEK-293 cells, stably expressing hDRD1. The nanovesicles containing hDRD1 as gate-potential modulator on the conducting polymer (CP) nanomaterial transistors provided high-performance responses to DA molecule owing to their uniform, monodispersive morphologies and outstanding discrimination ability. Specifically, the DRNCNs were integrated into a liquid-ion gated field-effect transistor (FET) system via immobilization and attachment processes, leading to high sensitivity and excellent selectivity toward DA in liquid state. Unprecedentedly, the minimum detectable level (MDL) from the field-induced DA responses was as low as 10 pM in real- time, which is 10 times more sensitive than that of previously reported CP based-DA biosensors. Moreover, the FET-type DRNCN biosensor had a rapid response time (<1 s) and showed excellent selectivity in human serum.

Bioelectronic tongue using heterodimeric human taste receptor for the discrimination of sweeteners with human-like performance.

The sense of taste helps humans to obtain information and form a picture of the world by recognizing chemicals in their environments. Over the past decade, large advances have been made in understanding the mechanisms of taste detection and mimicking its capability using artificial sensor devices. However, the detection capability of previous artificial taste sensors has been far inferior to that of animal tongues, in terms of its sensitivity and selectivity. Herein, we developed a bioelectronic tongue using heterodimeric human sweet taste receptors for the detection and discrimination of sweeteners with human-like performance, where single-walled carbon nanotube field-effect transistors were functionalized with nanovesicles containing human sweet taste receptors and used to detect the binding of sweeteners to the taste receptors. The receptors are heterodimeric G-protein-coupled receptors (GPCRs) composed of human taste receptor type 1 member 2 (hTAS1R2) and human taste receptor type 1 member 3 (hTAS1R3), which have multiple binding sites and allow a human tongue-like broad selectivity for the detection of sweeteners. This nanovesicle-based bioelectronic tongue can be a powerful tool for the detection of sweeteners as an alternative to labor-intensive and time-consuming cell-based assays and the sensory evaluation panels used in the food and beverage industry. Furthermore, this study also allows the artificial sensor to exam the functional activity of dimeric GPCRs.

Duplex Bioelectronic Tongue for Sensing Umami and Sweet Tastes Based on Human Taste Receptor Nanovesicles

For several decades, significant efforts have been made in developing artificial taste sensors to recognize the five basic tastes. So far, the well-established taste sensor is an E-tongue, which is constructed with polymer and lipid membranes. However, the previous artificial taste sensors have limitations in various food, beverage, and cosmetic industries because of their failure to mimic human taste reception. There are many interactions between tastants. Therefore, detecting the interactions in a multiplexing system is required. Herein, we developed a duplex bioelectronic tongue (DBT) based on graphene field-effect transistors that were functionalized with heterodimeric human umami taste and sweet taste receptor nanovesicles. Two types of nanovesicles, which have human T1R1/T1R3 for the umami taste and human T1R2/T1R3 for the sweet taste on their membranes, immobilized on micropatterned graphene surfaces were used for the simultaneous detection of the umami and sweet tastants. The DBT platform led to highly sensitive and selective recognition of target tastants at low concentrations (ca. 100 nM). Moreover, our DBT was able to detect the enhancing effect of taste enhancers as in a human taste sensory system. This technique can be a useful tool for the detection of tastes instead of sensory evaluation and development of new artificial tastants in the food and beverage industry.

Purification and functional reconstitution of human olfactory receptor expressed in Escherichia coli

Olfactory receptors (ORs), belonging to the Gprotein coupled receptor (GPCR) family, are very difficult to be overexpressed, purified and reconstituted because of their hydrophobicity and complicated structure. These receptors bind to their specific ligands, thus their specificity is very useful for application as a bioelectronic nose. Furthermore, highly purified and well-reconstituted human olfactory receptor (hOR) can be used in various fields, such as in protein-interaction research, drug screening, and analysis of the hOR structure. In this study, human olfactory receptor, hOR2AG1, was produced with high purity and functionally reconstituted in detergent micelles. The hOR2AG1 was overexpressed in Escherichia coli (E. coli) with glutathione S-transferase (GST) and 6xHis-tag as an inclusion body. The hOR2AG1 fusion protein was solubilized in buffer containing sodium dodecyl sulfate (SDS) and purified using Ni-NTA chromatography. The GST domain was removed using proteolytic cleavage before elution from the column. After purification, the hOR2AG1 was successfully reconstituted using nonionic detergents and methyl-ß-cyclodextrin. Finally highly purified and well-reconstituted hOR was obtained, and its biological characteristics were confirmed by using circular dichroism (CD) spectrum and tryptophan fluorescence assay. These results can be applied to develop protein-based sensing systems including a bioelectronic nose and to analyze the native hOR structure using solid-state NMR, X-ray crystallography, or neutron scattering.

High-performance bioelectronic tongue using ligand binding domain T1R1 VFT for umami taste detection

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. 1 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.