Synthesis, Crystallographic, Quantum Chemical, Antitumor, and Molecular Docking/Dynamic Studies of 4-Hydroxycoumarin-Neurotransmitter Derivatives.

In this contribution, four new compounds synthesized from 4-hydroxycoumarin and tyramine/octopamine/norepinephrine/3-methoxytyramine are characterized spectroscopically (IR and NMR), chromatographically (UHPLC-DAD), and structurally at the B3LYP/6-311++G*(d,p) level of theory. The crystal structure of the 4-hydroxycoumarin-octopamine derivative was solved and used as a starting geometry for structural optimization. Along with the previously obtained 4-hydroxycoumarin-dopamine derivative, the intramolecular interactions governing the stability of these compounds were quantified by NBO and QTAIM analyses. Condensed Fukui functions and the HOMO-LUMO gap were calculated and correlated with the number and position of OH groups in the structures. In vitro cytotoxicity experiments were performed to elucidate the possible antitumor activity of the tested substances. For this purpose, four cell lines were selected, namely human colon cancer (HCT-116), human adenocarcinoma (HeLa), human breast cancer (MDA-MB-231), and healthy human lung fibroblast (MRC-5) lines. A significant selectivity towards colorectal carcinoma cells was observed. Molecular docking and molecular dynamics studies with carbonic anhydrase, a prognostic factor in several cancers, complemented the experimental results. The calculated MD binding energies coincided well with the experimental activity, and indicated 4-hydroxycoumarin-dopamine and 4-hydroxycoumarin-3-methoxytyramine as the most active compounds. The ecotoxicology assessment proved that the obtained compounds have a low impact on the daphnia, fish, and green algae population.

N-trans-Feruloyloctopamine Wakes Up BBC3, DDIT3, CDKN1A, and NOXA Signals to Accelerate HCC Cell Apoptosis.

N-trans-Feruloyloctopamine (FO), a natural compound, was reported in our previous study to inhibit a tumor cell malignant phenotype by AKT- and EMT-related signals and might be used as a promising drug for HCC treatment. However, the specific targets and detailed mechanisms still need to be clarified. Screening with RNA-Seq in Huh7 cells treated with FO revealed that 317 genes were modulated, of which 188 genes were upregulated and 129 genes were downregulated. Real-time cell analyzer and flow cytometry data reveal that tumor cell proliferation and apoptosis were impacted by FO. DAVID bioinformatic data showed that most of the biological process GO terms are related to proliferation and apoptosis. KEGG enrichment analysis showed that FO mainly regulates PI3K-AKT- and apoptosis-related signals, in which BBC3, DDIT3, NOXA, and CDKN1A on the surface serve as the novel targets of FO inducing HCC cell apoptosis. The result implied that FO might exacerbate HCC cell apoptosis by regulating BBC3, DDIT3, CDKN1A, and NOXA signals. The obstacle effect of FO can provide new targets and new credibility for the treatment of liver cancer.

Comparative antiradical activity and molecular Docking/Dynamics analysis of octopamine and norepinephrine: the role of OH groups.

Octopamine is a neurotransmitter in invertebrates and a phenol analog of norepinephrine. The crystallographic and spectral (UV-visUV, and NMR) characteristics of octopamine were investigated experimentally and theoretically by applying appropriate level of theory, B3LYP-D3BJ/6-311++G(d,p), which reproduced well the experimental bond lengths and angles. The intramolecular interactions governing the stability of conformers were described by NBO and QTAIM analyses. The antiradical potencies of octopamine and norepinephrine towards DPPH and ABTS+ were examined with special emphasis on the preferred mechanism and effect of catechol moiety. Several techniques were used to distinguish Hydrogen Atom Transfer (HAT) and Proton Coupled Electron Transfer (PCET) mechanisms for reaction with DPPH. The calculated rate constants of the reactions with both radicals showed that Sequential Proton Loss Electron Transfer (SPLET) mechanism was dominant both thermodynamically and kinetically, with values of thermodynamic functions and rate constants clearly proving the importance of the second hydroxyl group in structure. The Molecular Docking and afterward Molecular Dynamics calculations of formed complexes between octopamine/norepinephrine with ß1- and ß2- adrenergic receptors examined in details the interactions that lead to the formation of stable complexes. The number of strong interactions of amino acids with norepinephrine was higher, but the absence of hydroxyl group in octopamine did not lead to a significant change in the type of interactions and stability. The formed complexes showed higher flexibility of amino acids, similar compactness of structure as proteins and increased interatomic distances of the backbone when compared to pure proteins.

Conformational study of octopamine in gas phase and effect of hydrochloride.

This work deals with the molecular modeling and vibrational spectra of all the twenty conformers of an important biomolecule octopamine which have been investigated using the DFT/B3LYP level of theory in combination with the 6-31++g(d,p) as a suitable basis set. The experimental FTIR and FTRaman spectra of octopamine neurotransmitter were recorded in the spectral region 400-4000 cm-1 and 50-4000 cm-1 respectively and correlated with the calculated spectra of the most stable conformer. The effect of hydrochloride on the important geometrical parameters of most stable conformer of octopamine was also studied. The normal coordinate analysis was performed to scale the theoretical frequencies and to calculate potential energy distributions for precise normal mode assignment. Most of the frequencies were in good agreement with experimental one. However, some have been modified. Natural bond orbital analysis was performed in order to confirm the stability of electronic structure of octopamine molecule. HOMO-LUMO analysis for all the twenty conformers was also performed to give the transition profile and to study the chemical reactivity of octopamine.

The solvation properties and effect of d-fructose on the taste behavior of Citrus aurantium active components in aqueous solutions.

The densities and viscosities of synephrine hydrochloride and octopamine hydrochloride aqueous solutions were determined. Apparent molar volumes, apparent molar volume at infinite dilution, viscosity B-coefficients and hydration number were calculated, and it was found that synephrine hydrochloride acts as a better structure maker than octopamine hydrochloride in aqueous solutions. The densities of the investigated salts were measured in aqueous d-fructose solutions, and the corresponding apparent molar volumes of transfer at infinite dilution were determined. Its taste behavior was discussed based on the calculated values for apparent specific volume and intrinsic viscosity. Molecular dynamics simulations and radial distribution functions were applied in order to understand the nature of the interactions and water structuring in the studied systems. The change in taste behavior was observed with increasing concentration of the cosolute, and it was found that the addition of sugar increases the bitterness of the solution. From a molecular docking study on the bitter receptor TAS2R38, the strongest interactions for synephrine-HCl were noted causing the most bitter taste.

Quantification of Biogenic Amines from Individual GFP-Labeled Drosophila Cells by MALDI-TOF Mass Spectrometry.

Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFP-guided single-cell microdissection, on-plate chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) or 2,5-dimethyl-1 H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDI-TOF MS. We were able to determine a limit of detection for OA of 1 fmol/µL, for TA of 2.5 fmol/µL and a lower limit of quantification (LLOQ) of 10 fmol/µL for both substances. SCMS of GFP-labeled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/µL and a TA titer (∼2.5 fmol/µL) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2 > GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.

Octopamine signaling in the metazoan pathogen Schistosoma mansoni: localization, small-molecule screening and opportunities for drug development.

Schistosomiasis is a tropical disease caused by a flatworm trematode parasite that infects over 200 million people worldwide. Treatment and control of the disease rely on just one drug, praziquantel. The possibility of drug resistance coupled with praziquantel's variable efficacy encourages the identification of new drugs and drug targets. Disruption of neuromuscular homeostasis in parasitic worms is a validated strategy for drug development. In schistosomes, however, much remains to be understood about the organization of the nervous system, its component neurotransmitters and potential for drug discovery. Using synapsin as a neuronal marker, we map the central and peripheral nervous systems in the Schistosoma mansoni adult and schistosomulum (post-infective larva). We discover the widespread presence of octopamine (OA), a tyrosine-derived and invertebrate-specific neurotransmitter involved in neuromuscular coordination. OA labeling facilitated the discovery of two pairs of ganglia in the brain of the adult schistosome, rather than the one pair thus far reported for this and other trematodes. In quantitative phenotypic assays, OA and the structurally related tyrosine-derived phenolamine and catecholamine neurotransmitters differentially modulated schistosomulum motility and length. Similarly, from a screen of 28 drug agonists and antagonists of tyrosine-derivative signaling, certain drugs that act on OA and dopamine receptors induced robust and sometimes complex concentration-dependent effects on schistosome motility and length; in some cases, these effects occurred at concentrations achievable in vivo The present data advance our knowledge of the organization of the nervous system in this globally important pathogen and identify a number of drugs that interfere with tyrosine-derivative signaling, one or more of which might provide the basis for a new chemotherapeutic approach to treat schistosomiasis.This article has an associated First Person interview with the first author of the paper.

Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Enhanced intracellular survival (Eis) proteins were found to enhance the intracellular survival of mycobacteria in macrophages by acetylating aminoglycoside antibiotics to confer resistance to these antibiotics and by acetylating DUSP16/MPK-7 to suppress host innate immune defenses. Eis homologs composing of two GCN5 N-acetyltransferase regions and a sterol carrier protein fold are found widely in gram-positive bacteria. In this study, we found that Eis proteins have an unprecedented ability to acetylate many arylalkylamines, are a novel type of arylalkylamine N-acetyltransferase AANAT (EC 2.3.1.87). Sequence alignment and phyletic distribution analysis confirmed Eis belongs to a new aaNAT-like cluster. Among the cluster, we studied three typical Eis proteins: Eis_Mtb from Mycobacterium tuberculosis, Eis_Msm from Mycobacterium smegmatis, and Eis_Sen from Saccharopolyspora erythraea. Eis_Mtb prefers to acetylate histamine and octopamine, while Eis_Msm uses tyramine and octopamine as substrates. Unlike them, Eis_Sen exihibits good catalytic efficiencies for most tested arylalkylamines. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions, future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis, and reveal potential clinical implications for many gram-positive pathogens.

Ligand-binding characterization of simulated ß-adrenergic-like octopamine receptor in Schistocerca gregaria via progressive structure simulation.

It is important to design insecticides having both low drug resistance and less undesirable toxicity for desert locust control. Specific GPCRs of Schistocerca gregaria, especially ß-adrenergic-like octopamine receptor (SgOctßR), can be considered as its potential effective insecticide targets. However, either the unavailability of SgOctßR's structure or the inadequate capability of its sequence lead the development of insecticide for Schistocerca gregaria meets its plateau. To relax this difficulty, this paper develops a promising progressive structure simulation from SgOctßR's sequence, to its predicted structure of SgOctßR in vacuum, to its conformation as well as its complex with endogenous ligand octopamine in a solvent-membrane system. The combined approach of multiple sequence alignment, static structural characterization, and dynamic process of conformational change during binding octopamine reveal three important aspects. The first one is the characterization of SgOctßR's active pocket, including the attending secondary structure elements, its hydrophobic residues and nonpolar surface. The second one is the interaction with octopamine, especially the involved hydrogen bonds and an aromatic stacking of pi-pi interactions. The third one is the potential binding sites, including six highly conserved residues and one highly variable residue for locust insecticide design. This work is definitely helpful for the further structure-based drug design for efficient and eco-friendly insecticides, as well as site-directed mutagenesis biochemical research of SgOctßR.

Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.

p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.

Ancient coexistence of norepinephrine, tyramine, and octopamine signaling in bilaterians.

BACKGROUND: Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are performed by the biogenic amines octopamine and its precursor tyramine. These chemically similar transmitters signal by related families of G-protein-coupled receptors in vertebrates and invertebrates, suggesting that octopamine/tyramine are the invertebrate equivalents of vertebrate norepinephrine. However, the evolutionary relationships and origin of these transmitter systems remain unclear. RESULTS: Using phylogenetic analysis and receptor pharmacology, here we have established that norepinephrine, octopamine, and tyramine receptors coexist in some marine invertebrates. In the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid), we have identified and pharmacologically characterized adrenergic α1 and α2 receptors that coexist with octopamine α, octopamine ß, tyramine type 1, and tyramine type 2 receptors. These receptors represent the first examples of adrenergic receptors in protostomes. In the deuterostome Saccoglossus kowalevskii (a hemichordate), we have identified and characterized octopamine α, octopamine ß, tyramine type 1, and tyramine type 2 receptors, representing the first examples of these receptors in deuterostomes. S. kowalevskii also has adrenergic α1 and α2 receptors, indicating that all three signaling systems coexist in this animal. In phylogenetic analysis, we have also identified adrenergic and tyramine receptor orthologs in xenacoelomorphs. CONCLUSIONS: Our results clarify the history of monoamine signaling in bilaterians. Given that all six receptor families (two each for octopamine, tyramine, and norepinephrine) can be found in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes, all six receptors must have coexisted in the last common ancestor of the protostomes and deuterostomes. Adrenergic receptors were lost from most insects and nematodes, and tyramine and octopamine receptors were lost from most deuterostomes. This complex scenario of differential losses cautions that octopamine signaling in protostomes is not a good model for adrenergic signaling in deuterostomes, and that studies of marine animals where all three transmitter systems coexist will be needed for a better understanding of the origin and ancestral functions of these transmitters.

Fast hepatic biotransformation of p-synephrine and p-octopamine and implications for their oral intake.

Citrus aurantium (bitter orange) extracts have been used in products for weight management and sports performance. These extracts contain large amounts of p-synephrine and much smaller amounts of p-octopamine. Both protoalkaloids exert lipolytic and glycogenolytic activities at similar concentrations. The biotransformation of p-synephrine and p-octopamine is not as well-known as those of other adrenergic amines. For this reason transformation of these amines was investigated in the isolated perfused liver. Special attention was devoted to the single pass extraction of each compound as well as to the kinetics of uptake. The assay of the amines in the outflowing perfusate was done by means of high performance liquid chromatography (HPLC). The single pass extraction of p-synephrine was higher than 90% at a portal concentration of 10 µM. It declined with the concentration, but was still around 30% at the concentration of 500 µM. At low concentrations (10-50 µM) the decreasing sequence of single pass extractions was p-synephrine > p-octopamine ≈ epinephrine > norepinephrine. Rates of uptake versus p-synephrine concentration resulted in a Michaelis-Menten type of relationship, with a KM value of 290.7 ± 32.1 µM and a Vmax of 0.762 ± 0.042 µmol min(-1) g(-1). The rates of uptake of p-octopamine did not present clear saturation and could be approximated by a linear relationship with a first order rate constant of 1.5 min(-1). The rapid hepatic transformation of p-synephrine and p-octopamine means that their concentration in the portal vein exceeds that in the systemic circulation during absorption. Their metabolic effects will, thus, be exerted predominantly in the liver.

Separation of octopamine racemate on (R,S)-2-amino-1-phenylethanol imprinted polymer--Experimental and computational studies.

Ten molecularly imprinted polymers coded as MIP1-MIP10 were prepared by the radical bulk polymerization using (R,S)-(±)-2-amino-1-phenylethanol as the structural analog of the target analyte (R,S)-octopamine. The functional monomers, 4-vinylbenzoic acid (1), methacrylic acid (2), acrylic acid (3), trifluoromethacrylic acid (4), itaconic acid (5), acrylamide (6), isopropenylbenzene (7), 2-hydroxyethyl methacrylate (8), 2-(diethylamino)ethyl methacrylate (9), allylamine (10) were polymerized consecutively with the ethylene glycol dimethacrylate cross-linker in methanol as the porogen. On the basis of the binding capacity of (R,S)-octopamine MIP1 with affinity factor equal to 6.37 was selected for further analysis. The affinity of polymer matrix MIP1 was tested by the non-competitive binding experiments of eight structurally related analytes. Finally, molecularly imprinted solid phase extraction (MISPE) of (R,S)-octopamine from spiked human serum albumin was carried out in order to verify the applicability of novel sorbent. The molecular modeling was employed to rationalize the stereodifferentiation of the analytes by the stereospecific sites formed in the polymer matrix.

Effect of the tyrosinase inhibitor (S)-N-trans-feruloyloctopamine from garlic skin on tyrosinase gene expression and melanine accumulation in melanoma cells.

In our searching for novel tyrosinase inhibitors from natural sources, (S)-N-trans-feruloyloctopamine isolated from garlic skin was found to be a potential mushroom tyrosinase inhibitor. Here, we examined the effects of the potential tyrosinase inhibitor in B16F10 cells on intracellular melanin contents, cytotoxicity, and the signaling mechanism involved in the expression of tyrosinase. The results showed the inhibitor displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin contents in a dose-dependent manner in the α-MSH-stimulated B16F10 cells. Real-time PCR and Western blot analysis showed that it inhibits melanogenesis signaling by down-regulates mRNA and protein expression levels of tyrosinase, which leads to a lower melanin contents. These results suggested that (S)-N-trans-feruloyloctopamine was an ideal tyrosinase inhibitor, and could be used in food and medical industry.

Reciprocal inhibition between sensory ASH and ASI neurons modulates nociception and avoidance in Caenorhabditis elegans.

Sensory modulation is essential for animal sensations, behaviours and survival. Peripheral modulations of nociceptive sensations and aversive behaviours are poorly understood. Here we identify a biased cross-inhibitory neural circuit between ASH and ASI sensory neurons. This inhibition is essential to drive normal adaptive avoidance of a CuSO4 (Cu(2+)) challenge in Caenorhabditis elegans. In the circuit, ASHs respond to Cu(2+) robustly and suppress ASIs via electro-synaptically exciting octopaminergic RIC interneurons, which release octopamine (OA), and neuroendocrinally inhibit ASI by acting on the SER-3 receptor. In addition, ASIs sense Cu(2+) and permit a rapid onset of Cu(2+)-evoked responses in Cu(2+)-sensitive ADF neurons via neuropeptides possibly, to inhibit ASHs. ADFs function as interneurons to mediate ASI inhibition of ASHs by releasing serotonin (5-HT) that binds with the SER-5 receptor on ASHs. This elaborate modulation among sensory neurons via reciprocal inhibition fine-tunes the nociception and avoidance behaviour.

Characterization of a prawn OA/TA receptor in Xenopus oocytes suggests functional selectivity between octopamine and tyramine.

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TAMac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TAMac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.

Neurotoxicity and mode of action of N, N-diethyl-meta-toluamide (DEET).

Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD50 ca. 1.5 µg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC50 values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6-12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC50: 120 µM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na(+) and K(+) channels in patch clamped rat cortical neurons, with IC50 values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans.

A separation of tyramine on a 2-(4-methoxyphenyl)ethylamine imprinted polymer: an answer from theoretical and experimental studies.

A 2-(4-methoxyphenyl)ethylamine imprinted polymer (MIP) was successfully applied for the selective separation of tyramine. A computational analysis was used to predict the affinity of the polymer matrix towards tyramine and a preliminary experimental evaluation was made for the target analyte. Then the experimental analysis of polymer towards tyramine was continued. The binding sites were characterized with employment of the Langmuir and Freudlich models. After the optimization of solid phase extraction towards tyramine, the most appropriate systems for the extraction steps were chosen: methanol-water 85:15 v/v for the loading and the washing as well as 0.04 M aq. ammonium acetate-methanol 30:70 v/v for the elution steps. The biogenic compounds as tryptamine, serotonin, octopamine, synephrine, and l-tyrosine were used for the selectivity study on the basis of binding capacities of the analytes on the imprinted and the non-imprinted polymers. The theoretical approach to obtained results allowed to explain the adsorption selectivity of the tested polymer. Finally, the complex matrix of bovine serum albumin was used to show the usefulness of imprinted material for bioanalysis. The obtained recoveries showed the superiority of MIP over the commercial sorbent C18. Total recoveries of tyramine from spiked bovine serum albumin sample were determined as: 95±2%, 14±3%, and 1.9±0.4% for the imprinted, non-imprinted, and commercial C18 sorbents, respectively.

Monoclonal antibody-based immunoassay for analysis of octopamine in housefly.

Octopamine (OA) is one of the biogenic monoamines in the housefly, which acts as an important neurohormone in the physiological process of this pest. In this study, a new hapten of OA was synthesized via aldol condensation. With the hapten, monoclonal antibodies (MAb) were generated and their characterizations were investigated. An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3C11-E3 was established, which required simple sample pre-treatments and had low cross-reactivity with OA structural analogise. The half maximal inhibition concentration (IC50) and the detected range (IC20-IC80) of the icELISA were 128 ng/mL and 12-1438 ng/mL, respectively. Average recoveries of OA ranged from 73 to 129% in the housefly.

3D-printed and CNC milled flow-cells for chemiluminescence detection.

Herein we explore modern fabrication techniques for the development of chemiluminescence detection flow-cells with features not attainable using the traditional coiled tubing approach. This includes the first 3D-printed chemiluminescence flow-cells, and a milled flow-cell designed to split the analyte stream into two separate detection zones within the same polymer chip. The flow-cells are compared to conventional detection systems using flow injection analysis (FIA) and high performance liquid chromatography (HPLC), with the fast chemiluminescence reactions of an acidic potassium permanganate reagent with morphine and a series of adrenergic phenolic amines.