Comparative Study on the Sensing Kinetics of Carbon and Nitrogen Nutrients in Cancer Tissues and Normal Tissues Based Electrochemical Biosensors.

In this study, an electrochemical sensor was developed by immobilizing colon cancer and the adjacent tissues (peripheral healthy tissues on both sides of the tumor) and was used to investigate the receptor sensing kinetics of glucose, sodium glutamate, disodium inosinate, and sodium lactate. The results showed that the electrical signal triggered by the ligand-receptor interaction presented hyperbolic kinetic characteristics similar to the interaction of an enzyme with its substrate. The results indicated that the activation constant values of the colon cancer tissue and adjacent tissues differed by two orders of magnitude for glucose and sodium glutamate and around one order of magnitude for disodium inosinate. The cancer tissues did not sense sodium lactate, whereas the adjacent tissues could sense sodium lactate. Compared with normal cells, cancer cells have significantly improved nutritional sensing ability, and the improvement of cancer cells' sensing ability mainly depends on the cascade amplification of intracellular signals. However, unlike tumor-adjacent tissues, colon cancer cells lose the ability to sense lactate. This provides key evidence for the Warburg effect of cancer cells. The methods and results in this study are expected to provide a new way for cancer research, treatment, the screening of anticancer drugs, and clinical diagnoses.

Study of the Sensing Kinetics of G Protein-Coupled Estrogen Receptor Sensors for Common Estrogens and Estrogen Analogs.

Endogenous and exogenous estrogens are widely present in food and food packaging, and high levels of natural estrogens and the misuse or illegal use of synthetic estrogens can lead to endocrine disorders and even cancer in humans. Therefore, it is consequently important to accurately evaluate the presence of food-functional ingredients or toxins with estrogen-like effects. In this study, an electrochemical sensor based on G protein-coupled estrogen receptors (GPERs) was fabricated by self-assembly, modified by double-layered gold nanoparticles, and used to measure the sensing kinetics for five GPER ligands. The interconnected allosteric constants (Ka) of the sensor for 17ß-estradiol, resveratrol, G-1, G-15, and bisphenol A were 8.90 × 10-17, 8.35 × 10-16, 8.00 × 10-15, 5.01 × 10-15, and 6.65 × 10-16 mol/L, respectively. The sensitivity of the sensor for the five ligands followed the order of 17ß-estradiol > bisphenol A > resveratrol > G-15 > G-1. The receptor sensor also demonstrated higher sensor sensitivity for natural estrogens than exogenous estrogens. The results of molecular simulation docking showed that the residues Arg, Glu, His, and Asn of GPER mainly formed hydrogen bonds with -OH, C-O-C, or -NH-. In this study, simulating the intracellular receptor signaling cascade with an electrochemical signal amplification system enabled us to directly measure GPER-ligand interactions and explore the kinetics after the self-assembly of GPERs on a biosensor. This study also provides a novel platform for the accurate functional evaluation of food-functional components and toxins.

Development of a sandwich-type rat small intestine tissue sensor for detecting resveratrol and its receptors.

Resveratrol has a variety of biological functions, however, a limited number of studies have assessed its interaction with cell surface receptors. In this study, a sandwich-type rat small intestine tissue sensor (RSIT-sensor) was fabricated to detect the response current from receptor stimulation by different resveratrol concentrations via electrochemical workstation. The results showed that with detection limit of 1 × 10-13 mol/L, the maximum rate of change of the response current was found at the concentration of 8.5 × 10-12 mol/L, indicating that the resveratrol-related receptor was saturated. With comparing the response values of prepared biosensor and bare electrode with resveratrol, it can be concluded that the response value of small intestinal cells to resveratrol has obviously been amplified by the intracellular signal transmission system, and its magnification was about 100 times. In the current research, for the first time, kinetics of the interaction between resveratrol and its receptors and the transmission of signals to the body could be quantitatively measured by a biosensor. Our findings may provide new ideas for resveratrol-related receptor analysis, separation and purification, signal transmission, and evaluation of biological function.

Construction of a Ginseng Root-Meristem Sensor and a Sensing Kinetics Study on the Main Nitrogen Nutrients.

Severe continuous cropping obstacles exist in ginseng cultivation. In order to assess these obstacles, a "sandwich" ginseng root tissue sensor was developed for the kinetic determination of five nitrogen nutrients. The results showed that the sensing parameters of the sensor reached an ultrasensitive level (limit of detection up to 5.451 × 10-24 mol/L) for the five nitrogen nutrients, and exhibited good stability and reproducibility. In the order of two-, four-, and six-year-old ginseng plants, the sensitivity to inorganic nitrogen nutrients (sodium nitrate and urea) showed an upward trend following an initial decline (the interconnected allosteric constant Ka values acted as the parameter). The fluctuations in sensor sensitivity to organic nitrogen nutrients, specifically nucleotides (disodium inosinate and disodium guanylate), were relatively small. The sensor sensitivity of two-, four-, and six-year-old ginseng plants to sodium glutamate was 9.277 × 10-19 mol/L, 6.980 × 10-21 mol/L, and 5.451 × 10-24 mol/L, respectively. Based on the survival rate of the seedlings and mortality rate of the ginseng in each age group, a Hardy-Weinberg equilibrium analysis was carried out. The results showed that the sensing ability of the root system to sodium glutamate may be an important factor affecting its survival under continuous cropping obstacles with increasing age.

Development of a new and simple method for the detection of histidine-tagged proteins based on thionine-chitosan/gold nanoparticles/horseradish peroxidase.

In the current study, an electrochemical biosensing signal amplification system was utilized with thionine-chitosan-gold nanoparticles (Chit-GNPs) that absorbed horseradish peroxidase (HRP) and anti-His tagged protein monoclonal antibody derived from Balb/c mice. In addition, transmission electron microscopy (TEM) was used to characterize the nanogold solution and atomic force microscopy (AFM) was used to characterize the sensor assembly. To evaluate the quality of the immunosensor, the amperometric I-t curve method was applied to determine His-IL23 in PBS. The results indicated that the response current exhibited an optimal linear correlation with the His-IL23 concentration that ranged from 0.01 to 103 ng/ml. The lowest detection limit was noted at 3.3 pg/ml (S/N = 3). The linear equation was deduced as follows: △I = 0.02lgC + 0.037 (R2 = 0.9628). Moreover, it was validated with high sensitivity, reproducibility and rapid response. Apparently, the immunosensor may be a very useful tool for the detection and quantification of His-tagged proteins. In addition, the signal amplification system can be used for the preparation of other immunosensors and to assist in bioassays.

Study on FcγRn Electrochemical Receptor Sensor and Its Kinetics.

Neonatal γ-immunoglobulin (IgG) Fc receptor (FcγRn) is a receptor that transports IgG across the intestinal mucosa, placenta, and mammary gland, ensuring the balance of IgG and albumin in the body. These functions of FcγRn depend on the intracellular signal transduction and activation caused by the combination of its extracellular domain and IgG Fc domain. Nevertheless, there are still no kinetic studies on this interaction. Consequently, in the present study, we successfully constructed the human FcγRn (hFcγRn) electrochemical receptor sensor. The signal amplification system formed by chitosan nanogold-hFcγRn protein and horseradish peroxidase was used to simulate the cell signal amplification system in vivo, and the kinetic effects between seven IgG and hFcγRn receptors from different species were quantitatively measured. The results showed that the interaction of these seven IgGs with hFcγRn was similar to the catalytic kinetics of enzyme and substrate, and there was a ligand-receptor saturation effect. The order of the interconnect allosteric constants (Ka), which is similar to the Michaelis constant (Km), was human IgG < bovine IgG < horse IgG < rabbit IgG < sheep IgG < donkey IgG < quail IgY. The results showed that hFcγRn had the strongest ability to transport human IgG, which was consistent with the evolution of the system. Therefore, our hFcγRn electrochemical receptor sensor can be used to measure and evaluate the interconnected allosteric network. It is also an essential parameter of the interaction between hFcγRn and different IgGs and, thus, provides a new detection and evaluation method for immunoemulsion, therapeutic monoclonal antibody therapy, heteroantibody treatment, and half-life research.

A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein.

An electrochemical double-layer Au nanoparticle membrane immunosensor was developed using an electrochemical biosensing signal amplification system with Au nanoparticles, thionine, chitosan, and horseradish peroxidase, which was fabricated using double self-adsorption of Au nanoparticle sol followed by anti-α-fetoprotein Balb/c mouse monoclonal antibody adsorption. The AuNPs sol was characterized by spectrum scanning and transmission electron microscopy. The immunosensor was characterized by atomic force microscopy, cyclic voltammetry, and alternating-current impedance during each stage of adsorption and assembly. The amperometric I-t curve method was used to measure α-fetoprotein (AFP) diluted in phosphate buffered saline. The result indicated a wide linear range, and the change rate of steady-current before and after immune response had linear correlation within the range 0.1-104 pg/mL AFP. The current change rate equation was △I = 5.82334 lgC + 37.01195 (R2 = 0.9922). The lowest limit of detection was 0.03 pg/mL (S/N = 3), and the reproducibility of the sensor was good. Additionally, the sensor could be stably stored above phosphate buffered saline at 4 °C for more than 24 days. More importantly, the sensor is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling.

Design, synthesis and evaluation of novel N-phenylbutanamide derivatives as KCNQ openers for the treatment of epilepsy.

KCNQ (Kv7) has emerged as a validated target for the development of novel anti-epileptic drugs. In this paper, a series of novel N-phenylbutanamide derivatives were designed, synthesized and evaluated as KCNQ openers for the treatment of epilepsy. These compounds were evaluated for their KCNQ opening activity in vitro and in vivo. Several compounds were found to be potent KCNQ openers. Compound 1 with favorable in vitro activity was submitted to evaluation in vivo. Results showed that compound 1 owned significant anti-convulsant activity with no adverse effects. It was also found to posses favorable pharmacokinetic profiles in rat. This research may provide novel potent compounds for the discovery of KCNQ openers in treating epilepsy.

Design, synthesis and evaluation of substituted piperidine based KCNQ openers as novel antiepileptic agents.

Epilepsy is a kind of disease with complicated pathogenesis. KCNQ (Kv7) is a voltage dependent potassium channel that is mostly associated with epilepsy and thus becomes an important target in the treatment of epilepsy. In this paper, a series of substituted piperidine derivatives targeting KCNQ were designed and synthesized by using scaffold hopping and active substructure hybridization. Compounds were evaluated by fluorescence-based thallium influx assay, Rb+ flow assay and electrophysiological patch-clamp assay. Results showed that some compounds possessed more potent potassium channel opening activity than Retigabine. More significantly, compound 11 was found to have good pharmacokinetic profiles in vivo.

R-lipoic acid overdosing affects platelet life span via ROS mediated autophagy.

R-lipoic acid (ALA), a powerful antioxidant valuable for the treatment of diabetes and its complications, has been reported to exhibit an antiplatelet activity in vitro. The aim of this study was to investigate the effect and mechanism of ALA on platelets in vivo. Sprague-Dawley (SD) male rats were intravenously administered with low-dose ALA (20 mg/kg/d), high-dose ALA (80 mg/kg/d) and saline, respectively. Platelets count and bone marrow smear were evaluated and the expressions of markers related to apoptosis and autophagy were measured. Platelet clearance analysis was conducted out on mice. The results showed that high-dose ALA administration could significantly decrease platelet count by 43% compared with control group, whereas, megakaryocytes showed no difference in the number. Moreover, high-dose ALA administration led to significant reduction in half-life of circulating platelets, indicative of enhanced rate of platelet clearance. Interesting, high-dose ALA administration could increase the level of reactive oxygen species (ROS) in platelets and induce autophagy without affecting apoptosis. Our finding also showed that high ALA-induced autophagy in platelets was mediated by class III PtdIns3K activity, which could be reversed by 3-methyladenine (3-MA). Moreover, AKT and MAPK/ERK pathways were also observed to be involved in the regulation of autophagy in platelets. Thus, high-dose ALA could induce autophagy in platelets through modulating the activity of class III PtdIns3K, which was associated with decreased count of circulating platelets and shortened lifespan of platelets.

A new phosphothreonine lyase electrochemical immunosensor for detecting Salmonella based on horseradish peroxidase/GNPs-thionine/chitosan.

In the current study, a novel double-layer gold nanoparticles- electrochemical immunosensor electrode (DGN-EIE) immobilized with Salmonella plasmid virulence C (SpvC) antibody was developed. To increase the fixed quantity of antibodies and electrochemical signal, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, the SpvC monoclonal antibodies (derived from Balb/c mice) were prepared and screened with a high affinity to SpvC. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine Salmonella in PBS. The results showed that the response current had a good linear correlation with the bacterial quantity ranged from 1.0 × 101-5.0 × 104 cfu/mL. The lowest detection limit was found at 5 cfu/mL. Furthermore, the proposed immunosensor has been demonstrated with high sensitivity, good selectivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the bacteria.

A novel glutathione-S transferase immunosensor based on horseradish peroxidase and double-layer gold nanoparticles.

GSTs, a biotransformation enzyme group, can perform metabolism, drug transfer and detoxification functions. Rapid detection of the GSTs with more sensitive approaches is of great importance. In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode (DGN-EIE) immobilized with Glutathione S-Transferase (GST) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signal, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine the GST in PBS. The results showed that the response current had a good linear correlation with the GST concentration ranged from 0.1-10(4) pg/mL. The lowest detection limit was found at 0.03 pg/mL(S/N = 3). The linear equation was deduced as △I/% = 7.386lgC + 22.36 (R(2) = 0.998). Moreover, it was validated with high sensitivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the GST.

A Novel Tetrahydrocannabinol Electrochemical Nano Immunosensor Based on Horseradish Peroxidase and Double-Layer Gold Nanoparticles.

In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode immobilized with tetrahydrocannabinol (THC) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signals, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, a transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of the immunosensor, the amperometric I-t curve method was applied to determine the THC in PBS. The results showed that the response current had a good linear correlation with the THC concentration range from 0.01~10³ ng/mL with a correlation coefficient of 0.9986. The lowest detection limit for THC was 3.3 pg/mL (S/N = 3). Moreover, it was validated with high sensitivity and reproducibility. Apparently, the immunosensor may be a very useful tool for monitoring the THC.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus.

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium, respectively. However, the molecular machinery allowing the taste reaction remains incompletely understood. Through a phosphoproteomic approach, we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades. Ryanodine receptor isoform 1 (RYR1) was further verified by sensory and behavioral assays. We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel, which mediates Ca2+ release from the endoplasmic reticulum, is closed by dephosphorylation in bud tissue after sweet/umami treatment. The alteration in Ca2+ content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction. We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a "metabolic clock" notion based on sweet/umami sensing. Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

Studying allosteric regulation of chemokines and antagonists using a nanoscale hCCR3 receptor sensor.

CC chemokine receptor-3 (hCCR3), a G protein-coupled receptor (GPCR) expressed predominantly on eosinophils, is an important drug target. However, it was unclear how chemokine ligands, activators and antagonists recognize hCCR3, and quantitative measurements of hCCR3 inhibition or activation were rare. This study constructed a nanogold receptor sensor using hCCR3 as the molecular recognition element and horseradish peroxidase as the signal amplifier. We quantified the kinetic antagonism between chemokines and hCCR3 before and after adding hCCR3 antagonists. A molecular docking study was carried out to investigate how hCCR3 and its ligands work. The study results indicate chemokines interact with hCCR3 at low concentrations, and reversible hCCR3 inhibitors solely inhibit hCCR3, not CCLs. Moreover, a quantitative evaluation of hCCR3 chemokine activators and their antagonists was carried out using a directed weighted network. This offers a novel approach to quantitatively evaluate chemokine-receptor activation and antagonism together. This research could potentially offer new insights into the mechanisms of action of chemokines and drug screening.

Isolation of mono-caffeoylquinic acids from tobacco waste using continuous resin-based pre-separation and preparative HPLC.

Three isomers of mono-caffeoylquinic acid, specifically, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid, were successfully isolated from a crude extract of tobacco (Nicotiana tobaccum L.) wastes using continuous resin-based pre-separation and preparative high-performance liquid chromatography (HPLC). The extract of tobacco wastes was continuously pre-separated by resin-based columns packed with D101 and XAD-4, yielding total mono-caffeoylquinic acids with a purity of 67.71% and a recovery rate of 90.06%. Variables affecting resolution and productivity of three mono-caffeoylquinic acid isomers in preparative HPLC (i.e. mobile-phase composition, pH, flow rate and loading amount) were studied. The optimum chromatographic conditions were determined to be a mobile phase consisting of 15% (v/v) methanol and aqueous acetic acid with a pH of 4.5, a flow rate of 4.0 mL/min, a loading amount of 4 mL and a detection wavelength of 360 nm. From 300 mg of loading sample, 56.3 mg of 3-O-caffeoylquinic acid, 92.8 mg of 5-O-caffeoylquinic acid and 73.1 mg of 4-O-caffeoylquinic acid were obtained in a single run, each with a purity of over 98% by HPLC. The structures of the isolated compounds were elucidated by ESI-MS, (1) H-NMR and (13) C-NMR spectral data.

(4R*,5R*)-Diethyl 2-(4-nitro-phen-yl)-1,3-dioxolane-4,5-dicarboxyl-ate.

In the title compound, C(15)H(17)NO(8), the nitro group is essentially coplanar with the aromatic ring [dihedral angle = 6.4 (3) Å]. The five-membered ring has a twist conformation. In the crystal, C-H⋯O inter-actions link the mol-ecules into a helical chain propagating along [010].

(4R*,5R*)-2-(4-Meth-oxy-phen-yl)-1,3-dioxolane-4,5-dicarboxamide.

In the title compound, C(12)H(14)N(2)O(5), the five-membered 1,3-dioxolane ring has a twisted conformation. In the crystal, N-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into a two-dimensional network lying parallel to the ab plane. There are also C-H⋯π inter-actions present in the crystal structure.

Development of a nanozyme-based electrochemical sensor for detection of stringent response.

PpGpp (Guanosine 3',5'-bisdiphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate) are important biological compounds in stringent response of organisms and play a crucial role in their growth and survival. At present, there is no report on the method for comprehensively detection of stringent response. In this study, a nanozyme-based electrochemical sensor was fabricated by self-assembly and was used for detecting stringent response with high sensitivity (Km of ppGpp is 1.498 × 10-12 mol/L and Km of NADPH is 7.489 × 10-13 mol/L) and selectivity. The sensor exhibited advantages of fast response times (∼50 s), high specificity, and simple operation. The sensor was successfully used to detect stringent response in Arabidopsis thaliana leaf extracts, Escherichia coli extracts and serum samples from SD rats. Notably, the method does not require complex sample pretreatment and has high application potential for comprehensively detecting overall nutritional status that is involved in the stringent responses of animals, plants, and microorganisms.

Replacing arginine 99 with leucine to study the kinetics of interconnected allosteric interactions between FFAR4 and naturally occurring fatty acids.

The long-chain fatty acid receptor FFAR4 is the main G-protein-coupled receptor in the body for detecting long-chain fatty acids. It has been shown that Arg99 may be an important residue for fatty acid recognition and for the activation of hFFAR4, though direct evidence is still lacking. In this study, Arg99 on hFFAR4 was substituted with leucine by genetic manipulation, and a double-layer gold nanoparticle biosensor based on hFFAR4 (Arg99 â†’ Leu) was constructed. The interconnected allosteric interaction between 11 naturally occurring fatty acid ligands and the receptor was determined. The results showed that Arg99 is the key residue on hFFAR4 for the recognition of the carboxyl group on fatty acids. This study offered direct quantitative evidence for the role played by different residues in receptor-ligand recognition and interconnected allosterism, providing a new approach for investigating the mechanisms and kinetics of interconnected receptor-ligand allosterism.