Luhang Zhao

A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors

Olfactory receptors, which are responsible for sensing odor molecules, form the largest G protein-coupled receptor (GPCR) family in mammalian animals. These proteins play an important role in the detection of chemical signals and signal transduction to the brain. Currently, only a limited number of olfactory receptors have been characterized, which is mainly due to the lack of sensitive and efficient tools for performing functional assays of these receptors. This paper describes a novel surface acoustic wave (SAW)-based biosensor for highly sensitive functional assays of olfactory receptors. An olfactory receptor of Caenorhabditis elegans, ODR-10, was expressed on the plasma membrane of human breast cancer MCF-7 cells, which was used as a model system for this study. For specific odorant response assays, the membrane fraction of MCF-7 cells containing ODR-10 was extracted and integrated with our SAW sensors. The response of ODR-10 to various odorants was monitored by recording the resonance frequency shifts of SAWs applied to the sensor. Our results show that heterologously expressed ODR-10 receptors can specifically respond to diacetyl, its natural ligand. Dose-dependent responses were obtained by performing measurements using various concentrations of diacetyl. The sensitivity of this biosensor is 2kHz/ng and can detect concentrations as low as 10(-10)mM, which is 10× lower than what has previously been reported. This biosensor can be used to characterize odorant response profiles of olfactory receptors and provide information rich data for functional assays of olfactory receptors. In addition to providing a greater understanding of the biological mechanisms of GPCRs, such data holds great potential in many other fields such as food industry, biomedicine, and environmental protection.

A biomimetic olfactory-based biosensor with high efficiency immobilization of molecular detectors.

The immobilization efficiency of molecular detectors is of great importance with regard to the performances of biosensors such as the sensitivity, stability, and reproducibility. This paper presents a biomimetic olfactory receptor-based biosensor with better performances by improving the immobilization efficiency of molecular detectors for odorant sensing. A mixed self-assembled monolayers (SAMs) functionalized with specific olfactory receptors (ODR-10) was constructed on the sensitive area of surface acoustic wave (SAW) chip. The immobilization of ODR-10 was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The responses of this biosensor to various odorants were recorded by monitoring the resonance frequency shifts of SAW, which is correlated to the mass loading on its sensitive area. All the results demonstrate this biosensor can specifically respond to the natural ligand of ODR-10, diacetyl, with high sensitivity and stability. The sensitivity is 4 kHz/ng, which is 2× higher than that of previous work. The detection limit is 1.2×10(-11) mM. The major advances on immobilization efficiency of molecular detectors presented in this work could substantially promote and accelerate the researches and applications of olfactory receptor-based biosensors with different transducers, such as quartz crystal microbalance (QCM), surface plasma resonance (SPR), and field effect transistors (FET).

An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell

Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

Label-free functional assays of chemical receptors using a bioengineered cell-based biosensor with localized extracellular acidification measurement.

New methods for functional assays of chemical receptors are highly essential for the research of chemical signal transduction mechanisms and for the development of chemical biosensors. This study described a novel bioengineered cell-based biosensor for label-free functional assays of chemical receptors by localized extracellular acidification measurement with a light-addressable potentiometric sensor (LAPS). A human taste receptor, hT2R4, and an olfactory receptor of Caenorhabditis elegans (C. elegans), ODR-10, were selected as models of chemical receptors, which were expressed on the plasma membrane of human embryonic kidney (HEK)-293 cells. The specific ligand binding function of expressed chemical receptors was monitored by localized extracellular acidification measurement using LAPS chip with a movable focused laser illuminating on the desired single cell. The function of expressed olfactory receptors was further validated using MDL12330A, which can specifically inhibit the activity of adenylyl cyclase. The obtained results indicate that both of chemical receptors were successfully expressed in HEK-293 cells and can be functionally assayed by this bioengineered cell-based biosensor that shows dose-dependent responses to the target ligands of chemical receptors. This bioengineered cell-based biosensor exhibits the sensitivity of 1.0 mV/s for hT2R4 assays, and 9.8 mV/s for ODR-10 assays. The negative control cells without any chemical receptor expression show no response to all the chemical stimuli tested. All the results demonstrate this bioengineered cell-based biosensor can be used to detect the interactions between chemical receptors and their ligands. This provides a valuable and promising approach for label-free functional assays of chemical receptors as well as for the research of other GPCRs.

New Acid Biosensor for Taste Transduction Based on Extracellular Recording of PKD Channels

This paper describes a polycystic kidney disease-like (PKD) channel-based biosensor for the research of taste transduction by extracellular recording using a micro electrode array (MEA). PKD channels, which were recently proposed as candidates for sour sensation, are heterologously expressed in human embryo kidney-293 cells and coupled with MEA chips to serve as sensitive elements. MEA chips are used as transducers to monitor the responses of PKD channels to sour stimulations by extracellular recording in a noninvasive way for a long term. The results indicate that this biosensor can successfully record the special off-responses of PKD channels to sour stimulations. In addition, this biosensor can be used as a new tool for the functional assays of PKD channels. It is suggested that this biosensor may provide a promising alternative technique for the research of taste transduction, especially for the mechanisms of taste transduction mediated by ion channels. Furthermore, this biosensor also holds the potentials to be developed into electronic tongues for the detection of sour substances.

Biomimetic chemical sensors using bioengineered olfactory and taste cells.

Biological olfactory and taste systems are natural chemical sensing systems with unique performances for the detection of environmental chemical signals. With the advances in olfactory and taste transduction mechanisms, biomimetic chemical sensors have achieved significant progress due to their promising prospects and potential applications. Biomimetic chemical sensors exploit the unique capability of biological functional components for chemical sensing, which are often sourced from sensing units of biological olfactory or taste systems at the tissue level, cellular level, or molecular level. Specifically, at the cellular level, there are mainly two categories of cells have been employed for the development of biomimetic chemical sensors, which are natural cells and bioengineered cells, respectively. Natural cells are directly isolated from biological olfactory and taste systems, which are convenient to achieve. However, natural cells often suffer from the undefined sensing properties and limited amount of identical cells. On the other hand, bioengineered cells have shown decisive advantages to be applied in the development of biomimetic chemical sensors due to the powerful biotechnology for the reconstruction of the cell sensing properties. Here, we briefly summarized the most recent advances of biomimetic chemical sensors using bioengineered olfactory and taste cells. The development challenges and future trends are discussed as well.

Piezoelectric Biosensor Based on Olfactory Receptor Expressed in a Heterologous Cell System for Drug Discovery

Nowadays, there is an urgent need to find novel tools and approaches to improve the overall efficiency of drug discovery. In the present study, we developed a quartz crystal microbalance (QCM)-based biosensor in which olfactory receptor expressed in a heterologous cell system was used as sensitive elements. An olfactory receptor protein of C. elegances, ODR-10, which was serve as a model of G-protein-coupled receptors (GPCRs), was expressed on the plasma membrane of human embryonic kidney (HEK)-293 cells. For targeting the ODR-10 on the surface of cell membrane, the rho-tag import sequence was inserted into the N-terminus of the ODR-10. The membrane expression of ODR-10 was shown by the enhanced green fluorescent protein (EGFP) which sequences was inserted into the C-terminus of the ODR-10. After that, the membrane fraction of HEK-293 cells containing ODR-10 was extracted and then coated on the electrode surface of QCM. The interactions between odorant molecules and olfactory receptors were monitored by QCM. The results indicate that ODR-10 was functionally expressed on the plasma membrane of HEK-293 cells and it can respond to diacetyl with high specificity, which is a natural ligand for ODR-10. All the results suggest one conclusion: this novel QCM-olfactory receptor hybrid biosensor can be used to detect the interactions between GPCRs and their ligands, and suggest it could be a potential tool for drug discovery and even for drug evaluation.

Dual functional extracellular recording using a light-addressable potentiometric sensor for bitter signal transduction

This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells.

Olfactory receptors molecular sensors using surface acoustic wave chip

We present a new surface acoustic wave (SAW) chip for odorant detection by the using of olfactory receptors as molecular sensors. The molecular sensors were originated from the expression of ODR-10 in human breast cancer MCF-7 cells, which is an olfactory receptor of C. elegances. ODR-10 was then combined with SAW chips to serve as molecular sensors for odorant detection. The resonance frequency shifts of SAW can be recorded to characterize the responses of this molecular sensor to various odorants. The recording data show this molecular sensor can recognize its natural ligand of diacetyl with high specificity and sensitivity. It is indicated that this molecular sensor has great potential to be applied in many fields such as biomedicine, environmental monitoring, and food quality control.

A PKD Channel‐based Biosensor for Taste Transduction

This study describes a micro electrode array (MEA)‐based biosensor for taste transduction using heterologous expressed taste polycystic kidney disease‐like (PKD) channels as molecular sensors. Taste PKD1L3/2L1 channels were expressed on the plasma membrane of human embryo kidney (HEK)‐293 cells [1]. Then the cells were cultured on the surface of MEA chip [2] to record the responses of PKD channels to sour stimulations by monitoring membrane potential. The results indicate this MEA‐based biosensor can record the special off‐responses of PKD channels to sour stimulation in a non‐invasive manner for a long term. It may provide an alternative tool for the research of taste transduction, especially for the characterization of taste ion channels.