Rat Brain Microdialysates Research Articles

Development of a dual-mode Ru-UiO-TPEA MOF probe for highly sensitive and accurate detection of Cu2+ in biological systems

Cu2+ serves as a significant biomarker in various physiological processes. This study presents the development of a novel Ru-UiO-TPEA functionalized metal-organic framework (MOFs) probe for Cu2+ detection, employing both electrochemiluminescence (ECL) and electrochemical (EC) methods. Employing a dual ligand-assisted energy transfer approach, we synthesized Ru-UiO with optimal ECL and EC properties by adjusting the synthesis ratio of biphenyldicarboxylic acid to Ru(dcbpy)32+. This was followed by its conjugation with N-(2-aminoethyl)-N, N′, N′-tris(pyridin-2-ylmethyl) ethane-1,2-diamine (AE-TPEA) to form the highly conjugated Ru-UiO-TPEA probe for Cu2+ detection. As the Cu2+ concentration increased, the probe exhibited a diminished ECL signal and an enhanced differential pulse voltammetry (DPV) signal. The dual-mode detection method enabled cross-validation of the ECL and EC signals, enhancing accuracy and effectively minimizing errors. The ECL and EC methods had detection limits of 0.26 nM (range: 1 nM to 10 μM) and 0.65 nM (range: 2 nM to 80 μM), respectively. Meanwhile, these two methods can effectively detect Cu2+ in rat brain microdialysis fluid. Notably, the measured concentrations correlated well with those determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), highlighting its potential in biological and clinical bioelectronic sensing applications.

Abstract LB174: OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer

Abstract Background: Dysregulation of PI3K signaling is a common driver of solid tumors, with the PI3KαH1047R activating mutation found in 4% of all cancers. Because many cancers, including breast and lung cancers, can metastasize to the CNS, brain penetration should be a key feature of new therapies. The FDA-approved PI3K inhibitor alpelisib lacks CNS activity, and brain-penetrant alternatives such as buparlisib and paxalasib were not approved due to toxicity associated with a lack of mutant selectivity. OKI-219 is a potent, highly selective PI3KαH1047R inhibitor that has shown preclinical anti-tumor activity in multiple cancer models, both as a monotherapy and in combination with HER2- and ER-mediated therapeutics. Here, we demonstrate its brain penetrance across multiple animal species and anti-tumor activity in an intracranial mouse model of PI3KαH1047R mutant breast cancer. Materials and methods: Brain penetrance in mouse, rat, dog, and monkey was quantified by the ratio of unbound drug concentrations in brain to plasma (Kp,uu). Plasma, brain, and cerebrospinal fluid (CSF) concentrations of OKI-219 were measured by LC/MS, and protein binding in plasma, CSF, and brain homogenate were measured by rapid equilibrium dialysis. Additionally, free drug levels in the brain were directly measured by microdialysis in rats. Mice were implanted subcutaneously and intracranially with PI3KαH1047R xxT47D-luc tumors and tumor growth was measured by calipers (subcutaneous tumors) and luminescence (intracranial tumors). Results: At the Cmax of a pharmacologically active 10 mg/kg PO dose, OKI-219 had a Kp,uu of 0.34, 0.61, and 0.58 in rat, dog, and monkey CSF, respectively. Additionally, OKI-219 had an average Kp,uu of 0.67 over 24h post-dose as measured by brain microdialysis in rats at 30 mg/kg, with a dose-proportionate increase in brain free drug levels at 300 mg/kg. After 21 days of treatment, OKI-219 induced a statistically significant dose-dependent anti-tumor effect on both flank and brain xxT47D-luc tumors. Conclusion: Here we show that OKI-219 had consistently good brain penetrance across multiple species by multiple methods of measuring free drug levels in the brain. We further demonstrated that OKI-219 had anti-tumor activity against xxT47D-luc tumors in an intracranial mouse model that is similar to activity in the same tumor when implanted subcutaneously, demonstrating that the brain levels achieved were pharmacologically active. These data indicate the potential for activity of OKI-219 in CNS disease settings, including metastatic brain cancer, which are not currently addressed with existing PI3K pathway inhibitors. OnKure has initiated the PIKture Phase I clinical study (OKI-219-101) to evaluate OKI-219 in patients with PI3KαH1047R-mutated solid tumors. Citation Format: Alexander A. Strait, Molly A. Taylor, Kevin S. Litwiler, Qian Zhao, David A. Mareska, Mark L. Boys, Yevgeniy Izrayelit, Richard Woessner, James D. Winkler. OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB174.

Characterization of the Neurochemical and Behavioral Effects of the Phenethylamine 2-Cl-4,5-MDMA in Adolescent and Adult Male Rats.

The proliferation of novel psychoactive substances (NPS) in the drug market raises concerns about uncertainty on their pharmacological profile and the health hazard linked to their use. Within the category of synthetic stimulant NPS, the phenethylamine 2-Cl-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA) has been linked to severe intoxication requiring hospitalization. Thereby, the characterization of its pharmacological profile is urgently warranted. By in vivo brain microdialysis in adolescent and adult male rats we investigated the effects of 2-Cl-4,5-MDMA on dopamine (DA) and serotonin (5-HT) neurotransmission in twobrain areas critical for the motivational and rewarding properties of drugs, the nucleus accumbens (NAc) shell and the medial prefrontal cortex (mPFC). Moreover, we evaluated the locomotor and stereotyped activity induced by 2-Cl-4,5-MDMA and the emission of 50-kHz ultrasonic vocalizations (USVs) to characterize its affective properties. 2-Cl-4,5-MDMA increased dialysate DA and 5-HT in a dose-, brain area-, and age-dependent manner. Notably, 2-Cl-4,5-MDMA more markedly increased dialysate DA in the NAc shell and mPFC of adult than adolescent rats, while the opposite was observed on dialysate 5-HT in the NAc shell, with adolescent rats being more responsive. Furthermore, 2-Cl-4,5-MDMA stimulated locomotion and stereotyped activity in both adolescent and adult rats, although to a greater extent in adolescents. Finally, 2-Cl-4,5-MDMA did not stimulate the emission of50-kHz USVs. This is the first pharmacological characterization of 2-Cl-4,5-MDMA demonstrating that its neurochemical and behavioral effects may differ between adolescence and adulthood. These preclinical data could help understanding the central effects of 2-Cl-4,5-MDMA by increasing awareness on possible health damage in users.

Biomimetic mesoporous carbon-silica/AAO asymmetric nanochannel array for electrochemical sensing of K+ in rat brain microdialysates and serum

Acquirement of chemical expression in practical brain system is vital to understand the molecular mechanism involved in physiological and pathological processes in brain. Though nanochannels have been demonstrated to be promising platform for electrochemical sensor, it is a great challenge for nanochannels to be employed in practical brain biofluid. In this work, we rationally designed and created the biomimetic asymmetric nanochannels for sensing of K+ through integrating in situ modification of a two-component mesoporous carbon-silica (MCS) thin film with a pore size of ∼3.6 nm at anodic alumina nanochannel array (AAO) with the ∼40 nm pores (denoted as MCS/AAO). Apparent rectification phenomenon in such functionalized nanochannel array was achieved based on diode-like ion transport. Then, 4′-aminobenzeno-18-crown-6 (SP) was selected to be chemically decorated at MCS/AAO as the specific recognition for K+ (SP/MCS/AAO). The developed SP/MCS/AAO exhibited good selectivity towards K+ detection against the coexisting interferences in brain, and possessed a good linear response to K+ concentration in the range of 0.5–10 mM with a detection limit of 0.1 mM. Combined with microdialysis technique, the variation of K+ was successfully determined in rat brain microdialysates and serums. Compared with normal rats, the concentration of K+ was found to be greatly decreased in the cerebral microdialysates and serum of rats with hypertensive model (SHR). This work unveiled a powerful platform for K+, and promised to be extended to design new strategy for detecting other chemical species, in particular non-electroactive species in biofluid related to physiological and pathological events.

Water-Soluble Photoluminescent Adenosine-Functionalized Gold Nanoclusters as Highly Sensitive and Selective Receptors for Riboflavin Detection in Rat Brain.

Simple, selective, and sensitive detection of cerebral riboflavin is of great significance due to the vital roles of riboflavin in physiological and pathological processes. In the work, water-soluble photoluminescent adenosine-functionalized gold nanoclusters (Ade-AuNCs) are exploited as highly sensitive and selective receptors for cerebral riboflavin detection. The Ade-AuNCs are prepared under aqueous conditions by the one-step "synthesis-functionalization integration" strategy, using chloroauric acid as gold precursors and adenosine as outer-shell ligands. During the Ade-AuNCs synthesis process, adenosine and ascorbic acid are demonstrated to respectively serve as a stabilizer and a reductant, and citrate buffer plays multiple roles including a pH regulator, reductant, and complexing agent. The added riboflavin causes photoluminescence quenching of Ade-AuNCs, and the quenching photoluminescence is applied for well quantifying riboflavin in the range of 0.005-0.1 nM with a detection limit of 0.002 nM. The detailed analytical characterizations reveal that the photoluminescence quenching results from the static photoinduced electron transfer process from the surface functional Ade-AuNCs to riboflavin and the strong affinity between Ade-AuNCs and riboflavin. Moreover, the Ade-AuNC-based sensor exhibits a high selectivity for riboflavin over metal ions, anions, amino acids, and biological substances that possibly exist in the rat brain. Finally, by coupling the microdialysis technique, the proposed sensor is successfully applied to detect riboflavin in living rat brain microdialysates with a basal value of 13.1 ± 2.5 nM (n = 3), and the results are comparable well with those from a reference high-performance liquid chromatography method.

Capillary Electrophoresis-Mass Spectrometry for the Direct Analysis of Metabolites in Highly Saline Samples Using In-Capillary Preconcentration.

Capillary electrophoresis-mass spectrometry (CE-MS) is gaining interest for metabolomics studies because of its high separation efficiency, selectivity, and versatility. The ability to inject nanoliters from only a few microliters of sample in the injection vial makes this approach very suited for volume-limited applications. However, the low injection volumes could compromise the detection sensitivity of CE-MS, thereby potentially limiting its scope in metabolomics. To overcome this issue, online sample preconcentration methods have been developed to increase sample-loading volumes without hampering the intrinsic high separation efficiency of CE. In this protocol, online preconcentration with sample stacking based on pH junction was assessed for the direct profiling of endogenous metabolites in rat brain microdialysates. Sample stacking was realized by a pre-injection of ammonium hydroxide, followed by a large sample injection (i.e., about 17% of the total capillary volume). It is shown that this relatively simple and fast preconcentration procedure is fully compatible with the high-salt concentration in microdialysates and significantly improves the detection sensitivity of the CE-MS method.

Simultaneous determination of 11 neurotransmitters in brain microdialysis samples from rats by UPLC-MS/MS

This study established a method for simultaneous determination of 11 neurotransmitters, such as acetylcholine, glutamic acid, glycine, and norepinephrine from rat brain microdialysis samples using UPLC-MS/MS. A total of 20 μL of rat brain dialysate was diluted with 60 μL of acetonitrile-water(4∶1) and centrifuged for 10 min at 10 000 r·min~(-1),and 5 μL was injected into UPLC-MS/MS system for assay. Chromatographic separation was performed on a Waters ACQUITY BEH amide column(2.1 mm×100 mm, 1.7 μm) with gradient elution using acetonitrile/0.2% formic acid-water as mobile phases with a flow rate of 0.35 mL·min~(-1) and column temperature of 35 ℃. The eluate was detected by multiple-reaction monitoring(MRM) scanning with an electrospray ionization source operating in the positive ionization mode with an analysis duration of 3.5 min. The relationship between the recovery rate of 11 neurotransmitters and the perfusion rate or the concentration of neurotransmitters was investigated. Furthermore, the effects of puerarin alone or combined with borneol on the content of 11 neurotransmitters in the striatum of rats were investigated. The results showed the calibration curves displayed good linear regression with coefficients all greater than 0.99 and the lower limit of quantification(LLOQ) less than 12.5 nmol·L~(-1) for each analyte. The within-run and between-run precision(RSD) of the 11 neurotransmitters at low, medium, and high levels was less than 9.3%, and the relative error of the accuracy ranged from-8.4% to 9.5%. The stability, recovery, and matrix effects were in line with the biological sample analysis requirements. As revealed by experimental results, the levels of most neurotransmitters in the brain striatum changed significantly after rats were treated with puerarin as compared with the conditions in the blank group. Except for dopamine and norepinephrine, the degree of changes of other neurotransmitters in the combination group(borneol and puerarin) was less than that of the puerarin group. The established UPLC-MS/MS method could be applied to the quantitative determination of 11 neurotransmitters in microdialysis samples, providing an efficient and useful tool to study neurotransmitter changes in animal models of health and diseases.

A Liquid Interfacial SERS Platform on a Nanoparticle Array Stabilized by Rigid Probes for the Quantification of Norepinephrine in Rat Brain Microdialysates.

For the reliable determination of trace chemicals in the brain, we created a SERS platform based on a functionalized AuNPs array formed at a liquid/liquid interface in a uniform fashion over a large substrate area through ternary regulations for real-time quantification of trace norepinephrine (NE). The rigid molecule, 4-(thiophen-3-ylethynyl)-benzaldehyde (RP1) was designed and co-assembled at AuNPs with 4-mercaptophenylboronic acid (MPBA) to chemically define NE via dual recognition. Meanwhile, the rigid structure assembly of RP1 and MPBA efficiently fixed the interparticle gap, guaranteeing reproducible SERS analysis. Furthermore, the Raman peak of C≡C group in the silent region was taken as a response element to further improve the accuracy. Combined with microdialysis, this SERS platform was developed for in-the-field testing of NE in rat brain microdialysates following anxiety.

A Liquid Interfacial SERS Platform on a Nanoparticle Array Stabilized by Rigid Probes for the Quantification of Norepinephrine in Rat Brain Microdialysates

AbstractFor the reliable determination of trace chemicals in the brain, we created a SERS platform based on a functionalized AuNPs array formed at a liquid/liquid interface in a uniform fashion over a large substrate area through ternary regulations for real‐time quantification of trace norepinephrine (NE). The rigid molecule, 4‐(thiophen‐3‐ylethynyl)‐benzaldehyde (RP1) was designed and co‐assembled at AuNPs with 4‐mercaptophenylboronic acid (MPBA) to chemically define NE via dual recognition. Meanwhile, the rigid structure assembly of RP1 and MPBA efficiently fixed the interparticle gap, guaranteeing reproducible SERS analysis. Furthermore, the Raman peak of C≡C group in the silent region was taken as a response element to further improve the accuracy. Combined with microdialysis, this SERS platform was developed for in‐the‐field testing of NE in rat brain microdialysates following anxiety.

Effect of P-glycoprotein Inhibition on the Penetration of Ceftriaxone Across the Blood-Brain Barrier.

Recent studies indicate that inhibition of the efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) may represent a putative strategy to increase the BBB penetration of several antibiotics. Therefore, the present study aimed to investigate the effect of P-gp inhibition on the transport of ceftriaxone (CFX) across the BBB. Blood and brain microdialysis in rats was used to monitor blood and brain unbound CFX concentrations following intravenous administration (50mg/kg), with or without pretreatment with one of the P-gp inhibitors, cyclosporin A (6.25, 12.5, 25mg/kg) or verapamil (5, 10, 20mg/kg). An inhibitory effect was demonstrated by an increase in the ratio of unbound brain to unbound blood concentration (Kp.uu.brain) of CFX. The concentrations of CFX in blood and brain from 0 to 180min after intravenous administration (CFX, 50mg/kg) ranged from 3 to 40μg/ml and 1 to 10μg/ml, respectively. The Kp.uu.brain of CFX was 24.74 ± 1.34%. Pretreatment with cyclosporin A increased the brain concentration and the Kp.uu.brain of CFX in a dose-dependent manner. However, pretreatment with verapamil increased the brain concentration of CFX but not the Kp.uu.brain. The present data shows that CFX might be a substrate of P-gp efflux transporter at the BBB and P-gp inhibition might enhance the brain concentration of CFX. Future studies involving more selective P-gp inhibitors or knockout mouse models should be conducted to specifically elucidate the impact of P-gp inhibition on penetration of CFX across the BBB.

005 Samelisant (SUVN-G3031), a histamine H3 receptor inverse agonist in animal models of sleep disorders

Abstract Introduction Narcolepsy is a chronic sleep disorder characterized by overwhelming daytime drowsiness, sudden attacks of sleep and sometimes accompanied by cataplexy. Although the orexin deficiency is considered to be the primary cause of this disorder, lot of attention has been diverted on targeting histaminergic neurotransmission by blockade of histamine H3 receptor (H3R). Samelisant (SUVN-G3031) is one of the potent and selective H3R inverse agonist currently being evaluated in a Phase 2 study as monotherapy for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). In the current research work, Samelisant was evaluated for neurotransmitter changes in rats and sleep EEG in orexin knockout mice, a reliable proof-of-concept study for treatment of excessive daytime sleepiness and cataplexy in narcolepsy. Methods Binding affinity of Samelisant towards human and rat histamine H3R was evaluated in in-vitro radioligand binding assay and functionality in GTP□S assay. Effect of Samelisant was studied in (R)-α-methyl histamine induced dipsogenia. In rat brain microdialysis, Samelisant was evaluated for its effects on modulation of neurotransmitters like histamine, dopamine and norepinephrine. Male orexin knockout mice were implanted with telemetric device for simultaneous monitoring of electroencephalography (EEG) and electromyography. Effects of Samelisant (3 and 10 mg/kg, p.o.) were evaluated during active period of animals. Results Samelisant is an inverse agonist at histamine H3 receptors with hKi of 8.7 nM and showed minimal binding against over 70 target sites. Samelisant produced significant increase in histamine, dopamine and norepinephrine levels in cortex. Samelisant produced no change in the striatal and accumbal dopamine levels in rats, suggesting no propensity to induce abuse liability. Samelisant blocked R-α-methyl histamine induced water intake and produced dose dependent increase in tele-methylhistamine levels in various brain regions and in cerebrospinal fluid of male Wistar rats. Samelisant produced significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleep in orexin knockout mice. Samelisant also significantly decreased number of cataplectic episodes in orexin knockout mice. Conclusion Samelisant is an inverse agonist at histamine H3 receptor and results from the preclinical studies presented here provide a strong evidence for the potential utility of Samelisant in the treatment of narcolepsy with and without cataplexy. Support (if any):

Brain Microdialysis Coupled to LC-MS/MS Revealed That CVT-10216, a Selective Inhibitor of Aldehyde Dehydrogenase 2, Alters the Neurochemical and Behavioral Effects of Methamphetamine

Methamphetamine (MA), a potent central nervous system stimulant, mainly affects the brain dopaminergic and serotoninergic systems. Monoamine oxidase, catechol-O-methyltransferase, and aldehyde dehydrogenase 2 (ALDH2) are important enzymes in the metabolism of dopamine (DA) and serotonin (5-HT); however, the role of ALDH2 in MA addiction remains unclear. This study focused on the real-time changes in DA, 5-HT, and their metabolites, including 3,4-dihydroxyphenylacetic aldehyde and salsolinol, which are metabolites directly related to ALDH2, to examine the effects of the inhibition of ALDH2 on hyperlocomotion induced by MA. Locomotor activity was evaluated in rats after administration of MA and/or CVT-10216 (a selective ALDH2 inhibitor). Moreover, the simultaneous quantification of DA, 5-HT, and their metabolites in brain microdialysates of the rats was performed using a derivatization-assisted LC-MS/MS method after full validation. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Intraperitoneal (i.p.) administration of 10 or 20 mg/kg of CVT-10216 significantly decreased MA-induced hyperlocomotion (1 mg/kg, i.p.). The analytical results of rat brain microdialysates demonstrated that the administration of CVT-10216 significantly downregulated DA levels, which were increased upon exposure to MA. Moreover, the increase in 3-methoxytyramine levels following coadministration of CVT-10216 and MA could play a potential role in antagonizing the hyperlocomotion induced by MA. All of these findings suggest that the inhibition of ALDH2 protects against MA-induced hyperlocomotion and has therapeutic potential in MA addiction.

Samelisant (SUVN-G3031), a potent, selective and orally active histamine H3 receptor inverse agonist for the potential treatment of narcolepsy: pharmacological and neurochemical characterisation.

Samelisant (SUVN-G3031) is a potent and selective histamine H3 receptor (H3R) inverse agonist with good brain penetration and oral bioavailability. Pharmacological and neurochemical characterisation to support the utility of Samelisant (SUVN-G3031) in the treatment of sleep-related disorders like narcolepsy. Samelisant (SUVN-G3031) was tested in rat brain microdialysis studies for evaluation of modulation in histamine, dopamine and norepinephrine. Sleep EEG studies were carried out in orexin knockout mice to study the effects of Samelisant (SUVN-G3031) on the sleep-wake cycle and cataplexy. Samelisant (SUVN-G3031) has a similar binding affinity towards human (hH3R; Ki = 8.7 nM) and rat (rH3R; Ki = 9.8 nM) H3R indicating no inter-species differences. Samelisant (SUVN-G3031) displays inverse agonist activity and it exhibits very high selectivity towards H3R. Samelisant (SUVN-G3031) treatment in mice produced a dose-dependent increase in tele-methylhistamine levels indicating the activation of histaminergic neurotransmission. Apart from increasing the levels of histamine, Samelisant (SUVN-G3031) also modulates dopamine and norepinephrine levels in the cerebral cortex while it has no effects on dopamine levels in the striatum or nucleus accumbens. Treatment with Samelisant (SUVN-G3031; 10 and 30 mg/kg, p.o.) produced a significant increase in wakefulness with a concomitant decrease in NREM sleep in orexin knockout mice subjected to sleep EEG. Samelisant (SUVN-G3031) also produced a significant decrease in Direct REM sleep onset (DREM) episodes, demonstrating its anticataplectic effects in an animal model relevant to narcolepsy. Modulation in cortical levels of histamine, norepinephrine and dopamine provides the neurochemical basis for wake-promoting and anticataplectic effects observed in orexin knockout mice. Pre-clinical studies of Samelisant (SUVN-G3031) provide a strong support for utility in the treatment of sleep-related disorders related to EDS and is currently being evaluated in a phase 2 proof of concept study in the USA for the treatment of narcolepsy with and without cataplexy.

Quantification of neurotransmitters in microdialysate samples following quetiapine dosing to schizophrenia phenotyped rats using a validated LC-MS/MS method

A versatile method was developed and validated for simultaneous determination of the monoamine neurotransmitters (MNT) dopamine (DA), 3–4-dyhydroxyphenilacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) in rat brain microdialysate samples using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The method allowed for small sample volume, using positive and negative ionization mode in a single run analysis without any derivatization or cleanup steps. Analytes were quantified at concentrations ranging from 100 ng/mL to 0.05, 10, 0.5, 0.1 or 1 ng/mL (lower limit of quantification, LLOQ) of DA, DOPAC, HVA, 5-HT and 5-HIAA, respectively, showing linearity (r > 0.98), accuracy, and precision (R.S.D ± 15%) according to validation limits accepted by international guidelines. The method was successfully applied for monitoring the concentration changes of MNT in microdialysate samples from medium prefrontal cortex of Wistar rats in a neurodevelopmental model of schizophrenia before and after quetiapine 5 mg/kg i.v. bolus dose administration. No alterations in MNTs were observed in schizophrenia phenotyped rats (SPR) in comparison to the baseline shading a light on the limited response rate to antipsychotic drugs observed in chronic schizophrenic patients.

Direct profiling of endogenous metabolites in rat brain microdialysis samples by capillary electrophoresis-mass spectrometry with on-line preconcentration

Metabolic profiling of body fluids from small animal models is often used in (translational) biological studies in order to obtain insight into the underlying molecular mechanisms of (complex) diseases. An example is the use of brain microdialysis samples from rats to study neurological disorders by means of a metabolomics approach. From an analytical point of view, the profiling of (endogenous) metabolites in rat brain microdialysates is challenging because of the limited sample volume for both sample preparation and injection, notably in longitudinal studies. In this work, we have assessed the analytical performance of capillary electrophoresis-mass spectrometry (CE-MS) for the direct profiling of endogenous metabolites in rat brain microdialysates, i.e. without using any sample preparation or derivatization. An on-line preconcentration procedure with sample stacking, which was fully compatible with the high-salt concentration in microdialysates, was used to significantly improve the detection sensitivity of the CE-MS method for metabolic profiling. A response surface methodology, applying a Box-Behnken design, was considered to determine the optimal conditions for preconcentration. A linear response (R2>0.99) for selected metabolites in the concentration range from 0.05 to 10 µM was obtained in perfusate samples. Interday RSD values for peak area and migration time were 2.6-19% and below 3.8%, respectively. Limits of detection ranged from 11 to 284 nM when employing an injection volume of about 291 nL, corresponding to 17% of the total capillary volume. The utility of the CE-MS approach was demonstrated by the direct profiling of endogenous metabolites in rat brain microdialysates. At least 48 compounds could be analyzed of which 25 were provisionally identified and quantified.

The application of bathophenanthroline for the determination of free iron in parallel with hROS in microdialysis samples

BackgroundFree, non-protein bound, Fe(II), which can catalyse the formation of the toxic highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from microdialysis experiments has been limited by the small amounts of sample available. New methodThis work describes the development of a HPLC, with absorbance detection, method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS), which allows a complete extracellular iron analysis with the small sample amounts that are available from in vivo microdialysis in rat brain. ResultsMicrodialysis experiments using 6-hydroxydopamine stimulation, showed that basal-as well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel with measurements of hROS formation. Comparison with existing methodsAlthough a spectrophotometric BS-based assay has been reported for use in microdialysis samples from large animals, the present procedure is applicable to the small sample sizes available from studies in rat brain. It is simpler than the alternative, involving inductively-coupled plasma mass spectrometry. ConclusionsThe procedure described is simple and sensitive, giving a linear response in the Fe(II) concentration range of 50 −2000 nM. A 20 min microdialysis sample (flow-rate 3 μl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II) and total iron whilst leaving sufficient sample volume for determining hROS and amine or amino-acid neurotransmitter release.

Sensitive Fluorescent Sensor for Hydrogen Sulfide in Rat Brain Microdialysis via CsPbBr3 Quantum Dots.

The instability and insolubility of perovskite quantum dots in aqueous solution prohibit applications in polar solvents. As a highly toxic gas pollutant and also an endogenous gaseous signaling molecule existing in a variety of physiological processes, hydrogen sulfide (H2S), with high selectivity and high specificity, detection is of great significance. In this study, a simple device has been designed to separate H2S from aqueous solution and CsPbBr3 quantum dots (CsPbBr3 QDs) have been used as the detection probe to develop a novel fluorescent sensor for rapid H2S detection. The addition of hydrogen sulfide to the phosphoric acid solution results in the escape of H2S from the aqueous sample and hence it passing into the n-hexane solution containing CsPbBr3 QDs, resulting in the quenching of the fluorescence of CsPbBr3 QDs. The fluorescence intensity of the system has a linear relationship with the concentration of H2S in the range of 0-100 μM with the detection limit of 0.18 μM. The proposed system has been applied to detection of H2S in rat brain microdialysate with satisfying results. The potential mechanism regarding the quenching of fluorescence from CsPbBr3 QDs by H2S has been studied as well.

Bionanosensor based on N-doped graphene quantum dots coupled with CoOOH nanosheets and their application for in vivo analysis of ascorbic acid

Herein, we employ 3D nitrogen-doped porous graphene frameworks (NPG) as raw material to prepare emissive nitrogen doped graphene quantum dots (r-NGQDs) via chemical oxidation method. The as-prepared fluorescent r-NGQDs was integrated with CoOOH nanosheets to construct a sensing platform for in vivo ascorbic acid (AA) analysis. Initially, the fluorescence emission intensity of r-NGQDs was quenched by CoOOH nanosheets based on the inner filter effect (IFE). Then the quenched intensity of r-NGQDs and CoOOH nanosheets system was enlightened by addition of AA, since AA could consume CoOOH nanosheets through redox reaction, leading to the release of r-NGQDs and fluorescence restoration. Moreover, the restored fluorescence intensity of r-NGQDs is highly dependent on the concentration of AA which endows them as a quantitative analysis of AA with a limit of detection (LOD) reach up to1.85 μM (n = 3) in aqueous solution. Finally, the as constructed bionanosensor was further employed for in vivo analysis of AA in living rat brain microdialysate with basal value up to 9.4 ± 1.4 μM (n = 3).

Emissive carbon dots derived from natural liquid fuels and its biological sensing for copper ions

Liquid fuels rich in aliphatic and aromatic carbon compounds are utilized as the carbon precursors for the production of carbon dots (CDs) for the first time. Herein we demonstrate the preparation of liquid fuels derived CDs (d-CDs and f-CDs) via chemical oxidation approach and the subsequent application for cerebral copper ions (Cu2+) sensing in rat brain microdialysate. The as-prepared CDs exhibits different morphology and photoluminescence properties depending on the different component of precursors. The luminescence of the as-prepared d-CDs can be significantly quenched upon addition of Cu2+, in which Cu2+ are trapped by the oxygen and nitrogen functional groups surrounding the emissive d-CDs. Moreover, the fluorescence intensity of d-CDs is sensitive to the concentration of Cu2+ with a linear relationship in the range of 0–4 μM, and a detection limit was estimated to be 0.039 μM. Simultaneously, we found out that the luminescent d-CDs manifests an extraordinarily high selectivity which can clearly discriminate Cu2+ from other interference species in aqueous solution and therefore substantially endows d-CDs as a fluorescent sensing platform for Cu2+. According to this nature, we employed d-CDs for the in vivo analysis of Cu2+ in the rat brain microdialysate and the measured basal level of Cu2+ was estimated to be 2.82 ± 0.16 μM (n = 3) even in the presence of other metal ions, biological substances and amino acids that commonly existing in the rat brain.

Turn-on fluorescent assay for antioxidants based on their inhibiting polymerization of dopamine on graphene quantum dots.

We describe a sensitive turn-on fluorescent assay for antioxidants by using fluorescence-tunable graphene quantum dots (GQDs). GQDs exhibited strong fluorescence without dopamine (DA). DA could self-polymerize to a thin polydopamine (PDA) film on the surface of GQDs under alkaline environment, resulting in the fluorescence quenching of GQDs via fluorescence resonance energy transfer (FRET). However, the self-polymerization of DA could be effectively inhibited in the presence of antioxidants including glutathione (GSH), ascorbic acid (AA), cysteine (Cys), and homocysteine (Hcys). Thus, the fluorescence of GQDs restored. The "turn-on" sensing of antioxidants could be achieved with high sensitivity. The detection limit for GSH, AA, Cys, and Hcys could be achieved as low as 2.4 nM, 1.5 nM, 4.2 nM, and 4.4 nM, respectively. Finally, the GQDs@PDA system was applied for monitoring cerebral antioxidants in rat brain microdialysates. This work promises new opportunities to evaluate antioxidant capacity in physiological and pathological fields.