RESUMO
Mass spectrometry (MS) imaging of lipids in tissues with high structure specificity is challenging in the effective fragmentation of position-selective structures and the sensitive detection of multiple lipid isomers. Herein, we develop an MS3 imaging method for the simultaneous analysis of phospholipid CâC and sn-position isomers by on-tissue photochemical derivatization, nanospray desorption electrospray ionization (nano-DESI), and a dual-linear ion trap MS system. A novel laser-based sensing probe is developed for the real-time adjustment of the probe-to-surface distance for nano-DESI. This method is validated in mouse brain and kidney sections, showing its capability of sensitive resolving and imaging of the fatty acyl chain composition, the sn-position, and the CâC location of phospholipids in an MS3 scan. MS3 imaging of phospholipids has shown the capability of differentiation of cancerous, fibrosis, and adjacent normal regions in liver cancer tissues.
Assuntos
Fosfolipídeos , Espectrometria de Massas por Ionização por Electrospray , Camundongos , Animais , Fosfolipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Isomerismo , Cromatografia Gasosa-Espectrometria de Massas , Diagnóstico por ImagemRESUMO
The measurement of data repeatability in small-molecule metabolites acquired within and among different liquid chromatography-mass spectrometry (LC-MS) platforms is crucial for data sharing or data transfer in natural products research. This work was designed to investigate and evaluate the separation and detection performance of three commercial high-resolution LC-MS platforms (e.g., Agilent 6550 QTOF, Waters Vion IM-QTOF, and Thermo Scientific Orbitrap Exploris 120) using 68 ginsenoside references and the extract of Panax ginseng leaf. The retention time (tR), measured on these three platforms (under the same chromatography condition), showed good stability in different concentration tests, and within/among different instruments for both intra-day and inter-day precision examinations. Correlation in tR of ginsenosides was also highly determined on these three platforms. In spite of the different mass analyzers involved, these three platforms gave the accurate mass determination ability, especially enhanced resolution gained because of the ion mobility (IM) separation facilitated by IM-quadrupole time-of-flight. The current study has systematically evaluated the separation and MS detection performance enabled by three high-resolution LC-MS platforms taking ginsenosides as the template, and the reported findings can benefit the researchers for the selection of analytical platforms and the purpose of data sharing or data transfer.
Assuntos
Ginsenosídeos , Espectrometria de Massas , Panax , Folhas de Planta , Ginsenosídeos/análise , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/química , Panax/química , Folhas de Planta/química , Cromatografia Líquida/métodos , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Early and rapid diagnosis of tumors is essential for clinical treatment or management. In contrast to conventional means, bioimaging has the potential to accurately locate and diagnose tumors at an early stage. Fluorescent probe has been developed as an ideal tool to visualize tumor sites and to detect biological molecules which provides a requirement for noninvasive, real-time, precise, and specific visualization of structures and complex biochemical processes in vivo. Rencently, the development of synthetic organic chemistry and new materials have facilitated the development of near-infrared small molecular sensing platforms and nanoimaging platforms. This provides a competitive tool for various fields of bioimaging such as biological structure and function imaging, disease diagnosis, in situ at the in vivo level, and real-time dynamic imaging. This review systematically focused on the recent progress of small molecular near-infrared fluorescent probes and nano-fluorescent probes as new biomedical imaging tools in the past 3-5 years, and it covers the application of tumor biomarker sensing, tumor microenvironment imaging, and tumor vascular imaging, intraoperative guidance and as an integrated platform for diagnosis, aiming to provide guidance for researchers to design and develop future biomedical diagnostic tools.
Assuntos
Neoplasias , Humanos , Neoplasias/diagnóstico por imagem , Corantes Fluorescentes/química , Imagem Molecular/métodos , Microambiente TumoralRESUMO
Fangwen Jiuwei Decoction is a traditional Chinese medicine preparation for the treatment of pneumonia developed by Shenzhen Bao'an Chinese Medicine Hospital, which shows remarkable clinical responses. Qualitative and quantitative analyses of the main active compounds are crucial for the quality control of traditional Chinese medicine prescription in clinical application. In this study, we identified nine active compounds essential for the pharmacological effects of Fangwen Jiuwei Decoction based on the analysis of the Network Pharmacology and relevant literature. Moreover, these compounds can interact with several crucial drug targets in pneumonia based on molecular docking. We applied high-performance liquid chromatography-tandem mass spectrometry method was established these nine active ingredients' qualitative and quantitative detections. The possible cleavage pathways of nine active components were determined based on secondary ions mass spectrometry. The results of high-performance liquid chromatography-tandem mass spectrometry were further validated, which show a satisfactory correlation coefficient (r > 0.99), recovery rate (≥93.31%), repeatability rate (≤5.62%), stability (≤7.95%), intra-day precision (≤6.68%), and inter-day precision (≤9.78%). The limit of detection was as low as 0.01 ng/ml. In this study, we established a high-performance liquid chromatography-tandem mass spectrometry method to qualitatively and quantitatively analyze the chemical components in the Fangwen Jiuwei Decoction extract.
Assuntos
Medicamentos de Ervas Chinesas , Medicamentos de Ervas Chinesas/análise , Espectrometria de Massas em Tandem/métodos , Simulação de Acoplamento Molecular , Medicina Tradicional Chinesa , Cromatografia Líquida de Alta Pressão/métodosRESUMO
A fluorescence aptasensor based on taking the advantage of the combination of magnetic nanoparticles (MNPs), terminal deoxynucleotidyl transferase (TdT), and CRISPR/Cas12a was developed for the determination of nasopharyngeal carcinoma (NPC)-derived exosomes. The MNPs can eliminate background interference due to their magnetic separation capability. TdT can form an ultra-long polynucleotide tail which can bind with multiple crRNA, generating a signal amplification effect. The trans-cleavage activity of CRISPR/Cas12a can be specifically triggered via the crRNA binding with DNA, resulting in the bi-labeled DNA reporter with fluorophore and quencher being cleaved. The excitation wavelength of the fluorescence spectra was 490 nm. Fluorescence spectra with emission wavelengths ranging from 511 to 600 nm were collected. Under the optimization condition, the fabricated fluorescence aptasensor for NPC-derived exosome determination exhibited excellent sensitivity and specificity, with the linear range between 500 to 5 × 104 particles mL-1 and the limit of detection of 100 particles mL-1. It can be used for the determination of NPC-derived exosomes in clinical samples, which has a considerable clinical potential and prospect.
Assuntos
Exossomos , Neoplasias Nasofaríngeas , Humanos , Exossomos/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Sistemas CRISPR-Cas , Carcinoma Nasofaríngeo/diagnóstico , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , DNA/genética , Corantes Fluorescentes/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismoRESUMO
Recently, miRNAs have become a promising biomarker for disease diagnostics. miRNA-145 is closely related to strokes. The accuracy determination of miRNA-145 (miR-145) in stroke patients still remains challenging due to its heterogeneity and low abundance, as well as the complexity of the blood matrix. In this work, we developed a novel electrochemical miRNA-145 biosensor via subtly coupling the cascade strand displacement reaction (CSDR), exonuclease III (Exo III), and magnetic nanoparticles (MNPs). The developed electrochemical biosensor can quantitatively detect miRNA-145 ranging from 1 × 102 to 1 × 106 aM with a detection limit as low down as 100 aM. This biosensor also exhibits excellent specificity to distinguish similar miRNA sequences even with single-base differences. It has been successfully applied to distinguish healthy people from stroke patients. The results of this biosensor are consistent with the results of the reverse transcription quantitative polymerase chain reaction (RT-qPCR). The proposed electrochemical biosensor has great potential applications for biomedical research on and clinical diagnosis of strokes.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Humanos , Eletroquímica , Técnicas Eletroquímicas/métodos , MicroRNAs/genética , Exodesoxirribonucleases , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 105 pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Cocaína , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas , DNA , DNA Nucleotidilexotransferase , DNA Polimerase Dirigida por DNA , Corantes Fluorescentes , HumanosRESUMO
Mahuang Xuanfei Zhike (MXZ) syrup, a Chinese patent medicine, has been widely used in the clinical treatment of cough. However, there is no reported method for the quantitative analysis of the effective components of MXZ syrup in biological samples. In this study, the effective components of MXZ syrup were screened by network pharmacology and molecular docking technology. A sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to test the active components of MXZ syrup in rat plasma and tissue homogenates, including ephedrine, amygdalin, chlorogenic acid, harpagoside, forsythin and forsythoside A. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) and the mass analysis was conducted using a Waters Xevo TQ mass spectrometer using multiple reaction positive and negative ion simultaneous monitoring mode. The results showed that the linearity ranged from 0.3 to 409.4 ng/ml. The extraction recoveries were all <8.33%, and the matrix effects were all <8.45, which met the requirements. The pharmacokinetic and tissue distribution results indicated that the main active components of MXZ syrup were absorbed quickly and eliminated slowly in vivo, and there may be a reabsorption process.
Assuntos
Medicamentos de Ervas Chinesas , Ephedra sinica , Ratos , Animais , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Distribuição Tecidual , Simulação de Acoplamento Molecular , Medicamentos de Ervas Chinesas/farmacocinéticaRESUMO
Exosomal MicroRNA-21 (miRNA-21, miR-21) is significantly up-regulated in blood samples of patients with lung cancer. Exosomal-derived miR-21 can be used as a promising biomarker for the early diagnosis of lung cancer. This paper develops a fluorescent biosensor based on the combination of magnetic nanoparticles (MNPs), cascade strand displacement reaction (CSDR) and CRISPR/Cas12a to detect the exosomal miR-21 from lung cancer. The powerful separation performance of MNPs can eliminate the potential interference of matrix and reduce the background signal, which is very beneficial for the improvement of specificity and sensitivity. The CSDR can specifically transform one miR-21 into plenty of DNA which can specifically trigger the trans-cleavage nuclease activity of Cas12a, resulting in the cleavage of ssDNA bi-labeled with fluorescent and a quencher. Under the optimized experimental conditions, the developed fluorescence biosensor exhibited high sensitivity and specificity towards the determination of exosomal-derived miR-21 with a linear range from 10 to 1 × 105 fM and a low detection limit of about 0.89 fM. Most importantly, this method can be successfully applied to distinguish the exosomal miR-21 from the lung cancer patients and the healthy people.
Assuntos
Técnicas Biossensoriais , Neoplasias Pulmonares , Nanopartículas de Magnetita , MicroRNAs , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MicroRNAs/genéticaRESUMO
Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography-mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.
Assuntos
Ginsenosídeos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Medicamentos de Ervas Chinesas , Ginsenosídeos/análise , Espectrometria de Massas/métodosRESUMO
MicroRNAs (miRNAs) play a significant role in tumorigenesis and tumor development. Exosomal microRNA-141 (miRNA-141, miR-141) has been reported to be overexpressed in prostate cancer (PCa) and has become a potential biomarker for the diagnosis of PCa. Herein, a novel fluorescent biosensor based on toehold-aided cyclic amplification combined with horseradish peroxidase (HRP) enzyme catalysis and magnetic nanoparticles (MNPs) was designed for determination of the exosomes-derived microRNA-141 (miRNA-141, miR-141). The synergy of HRP enzyme catalysis and toehold mediated strand display reaction (TSDR) increase the sensitivity of the method, and the good separation ability of MNPs ensures the specificity of the method. Therefore, under the optimized experimental conditions, the highly sensitive and specific detection of miRNA-141 can be realized, and the detection limit is as low as 10 fM. More importantly, the biosensor successfully determinates the exosomal miR-141 in the plasma of patients with PCa.
Assuntos
Técnicas Biossensoriais , Exossomos/química , Peroxidase do Rábano Silvestre/metabolismo , Nanopartículas de Magnetita/química , MicroRNAs/sangue , Neoplasias da Próstata/diagnóstico , Biocatálise , Exossomos/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Células PC-3 , Neoplasias da Próstata/sangue , Neoplasias da Próstata/metabolismo , Células Tumorais CultivadasRESUMO
The occurrence and development of cervical cancer threaten women's life and health, HPV-induced cervical cancer is a major health issue among women. We synthesized three Rhopaladins' analogue (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines via a tandem Ugi 4CC/SN cyclization with pyrrolidone as a core structure. In addition, the cytotoxicity of these new compounds in the cervical cancer cell line CaSki was studied by MTT assay. And then we chose one to research the apoptosis and the expression of E6/E7 mRNA in CaSki cells. The results indicated that the new compound can not only inhibited the proliferation of CaSki in dose-dependent and time-dependent manners but also induced the apoptosis, which may be related to the down-regulation of E6/E7 mRNA expression.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Pirrolidinonas/farmacologia , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Ciclização , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Proteínas E7 de Papillomavirus/genética , Pirrolidinonas/síntese químicaRESUMO
A sandwich-type fluorescent biosensor for the determination of tumor-related exosome was designed. It is based on magnetic nanoparticle (MNP) capture and horseradish peroxidase (HRP) catalysis. MNPs were used as the substrate to capture exosomes by modifying the CD63 antibody on MNPs surface. After that, the biotinylated epithelial cell adhesion molecule (EpCAM) antibody was used to capture the tumor-related exosomes, which specifically express EpCAM. A novel method for the fluorescence measurement of tumor-associated exosome was achieved, with a detection limit as low as 200 (± 9) particles mL-1. The analytical range of this method is from 576 (± 15) particles mL-1 to 5.76 × 107 (± 5.1 × 105) particles mL-1. For the fluorescence measurement, the excitation wavelength was set to 320 nm. Fluorescent spectra were collected at emission wavelength in the range 370 to 550 nm; the data shown in the calibration plot were studied by using the fluorescence intensity at 406 nm. This sensor was also able to successfully detect the exosomes from the plasma of patients with hepatocellular carcinoma (HCC) and healthy humans. Graphical abstract Schematic representation of the sensing process of immunoassay-type biosensor based on magnetic nanoparticle capture and the fluorescence signal formed by the horseradish peroxidase (HRP) catalysis for tumor-related exosome determination.
Assuntos
Técnicas Biossensoriais , Carcinoma Hepatocelular/sangue , Exossomos/química , Fluorescência , Peroxidase do Rábano Silvestre/metabolismo , Imunoensaio , Neoplasias Hepáticas/sangue , Biocatálise , Calibragem , Carcinoma Hepatocelular/diagnóstico , Exossomos/metabolismo , Células Hep G2 , Peroxidase do Rábano Silvestre/química , Humanos , Neoplasias Hepáticas/diagnóstico , Nanopartículas de Magnetita/química , Tamanho da Partícula , Espectrometria de Fluorescência , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
An electrochemical aptasensing method is described for the determination of the biomarker CA125. It combines aptamer recognition and target-triggered strand displacement amplification. Flower like gold nanostructures were electrodeposited on a screen-printed carbon electrode to increase the sensor surface, to assemble more toehold-containing hairpin probe 1 (Hp1), and to improve the accessibility for DNA strands. Under the optimal conditions, this assay has a linear response in the 0.05 to 50 ngâ¢mL-1 CA125 concentration range, with a low detection limit of 5.0 pgâ¢mL-1. This method is specific and stable. It was successfully applied to the detection of CA125 in spiked biological samples, with recoveries between 82.5% and 104.1%. Graphical abstract.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Antígeno Ca-125/análise , Técnicas Eletroquímicas/métodos , Proteínas de Membrana/análise , Nanopartículas Metálicas/química , Aptâmeros de Nucleotídeos/genética , Antígeno Ca-125/sangue , Antígeno Ca-125/urina , Carbono/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Proteínas de Membrana/sangue , Proteínas de Membrana/urina , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Saliva/químicaRESUMO
A porous polymer coating transfer enrichment method is developed for the direct mass spectrometry (MS) analysis of lipids. The enrichment is fast (ca. 1â min) and enables the profiling and quantitation of lipids in small-volume biofluid samples. Coupled with a photochemical Paternò-Büchi reaction, this method enables the fast determination of lipid structure at the C=C location level and point-of-care lipid biomarker analysis.
Assuntos
Lipídeos/química , Polímeros/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Estrutura MolecularRESUMO
Diacylglycerols (DAGs) are a subclass of neutral lipids actively involved in cell signaling and metabolism. Alteration in DAG metabolism has been associated with onset and progression of several human-related diseases. The structural diversity of DAGs and their low concentrations in biological samples call for the development of methods that are capable of sensitive identification and quantitation of each DAG species as well as rapid profiling when a biochemical pathway is perturbed. In this work, the thiol-ene click chemistry has been employed to introduce a charge-tag, namely, cysteamine (CA), at a carbon-carbon double bond (CâC) of unsaturated DAGs. This one-pot photochemical derivatization is fast (within 1 min), universal (monotagging) for DAGs varying in fatty acyl chain lengths and the number of CâCs, and suitable for small sample volume (e.g., 1-50 µL plasma). Because of the presence of the amine group in CA, tagged DAGs showed at least 10 times increase in response to electrospray ionization as compared to conventional ammonium adduct formation. Low-energy collision-induced dissociation of CA tagged DAGs allowed confident assignment of fatty acyl composition. A neutral loss scan based on characteristic 95 Da loss (a combined loss of CA and H2O) of tagged DAGs has been established as a sensitive means for unsaturated DAG detection (limit of detection = 100 pM) and quantitation from mixtures. The analytical utility of CA tagging was demonstrated by shotgun analysis of unsaturated DAGs in human plasma, including samples from type 2 diabetes mellitus patients.
Assuntos
Diglicerídeos/sangue , Compostos de Sulfidrila/química , Química Click , Humanos , Lipídeos/química , Lipídeos/isolamento & purificação , Estrutura MolecularRESUMO
Endothelial progenitor cells (EPCs) are a subtype of bone marrow-derived progenitor cells. Stromal cell-derived factor 1 (SDF-1)-mediated EPC mobilization from bone marrow to areas of ischemia plays an important role in angiogenesis. Previous studies have reported that advanced glycation endproducts (AGEs), which are important mediators of diabetes-related vascular pathology, may impair EPC migration and homing, but the mechanism is unclear. Syndecan-4 (synd4) is a ubiquitous heparan sulfate proteoglycan receptor on the cell surface, involved in SDF-1-dependent cell migration. The extracellular domain of synd4 (ext-synd4) is shed in the context of acute inflammation, but the shedding of ext-synd4 in response to AGEs is undefined. Here we investigated changes in ext-synd4 on EPCs in response to AGEs, focusing on the influence of impaired synd4 signaling on EPC migration and homing. We found decreased full length and increased residue of synd4 in cells incubated with AGEs, with concomitant increase in the soluble fragment of ext-synd4 in the cell medium. EPCs from patients with type 2 diabetes expressed less ext-synd4 as assessed by Western blotting. Flow cytometry analysis showed less ext-synd4 on circulating CD34+ peripheral blood mononuclear cells, of which EPCs form a subgroup. We then explored the role of synd4 in EPC migration and homing. Impaired migration of synd4-deficient EPCs was observed by a 2D-chemotaxis slide. Furthermore, poor homing of synd4-/- EPCs was observed in a mouse model of lower limb ischemia. This study demonstrates that the shedding of synd4 from EPCs plays a key role in AGE-mediated dysfunction of EPC migration and homing. Stem Cells 2017;35:522-531.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Produtos Finais de Glicação Avançada/farmacologia , Sindecana-4/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Sindecana-4/química , Sindecana-4/deficiênciaRESUMO
An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5'-thiol group (5'-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL-1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis. Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , DNA/química , Molécula de Adesão da Célula Epitelial/análise , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Eletroquímica , Molécula de Adesão da Célula Epitelial/metabolismo , Estudos de Viabilidade , Ouro/química , HumanosRESUMO
Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R2 of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Microextração em Fase Sólida/métodos , Ioimbina/sangue , Ioimbina/farmacocinética , Administração Oral , Animais , Berberina/sangue , Cromatografia Líquida de Alta Pressão/normas , Estabilidade de Medicamentos , Desenho de Equipamento , Limite de Detecção , Masculino , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Microextração em Fase Sólida/instrumentação , Ioimbina/administração & dosagemRESUMO
Cardiac hypertrophy can be broadly classified as either physiological or pathological. Physiological stimuli such as exercise cause adaptive cardiac hypertrophy and normal heart function. Pathological stimuli including hypertension and aortic valvular stenosis cause maladaptive cardiac remodeling and ultimately heart failure. Syndecan-4 (synd4) is a transmembrane proteoglycan identified as being involved in cardiac adaptation after injury, but whether it takes part in physiological cardiac hypertrophy is unclear. We observed upregulation of synd4 in exercise-induced hypertrophic myocardium. To evaluate the role of synd4 in the physiological form of cardiac hypertrophy, mice lacking synd4 (synd4-/-) were exercised by swimming for 4 wks. Ultrasonic cardiogram (UCG) and histological analysis revealed that swimming induced the hypertrophic phenotype but was blunted in synd4-/- compared with wild-type (WT) mice. The swimming-induced activation of Akt, a key molecule in physiological hypertrophy was also more decreased than in WT controls. In cultured cardiomyocytes, synd4 overexpression could induce cell enlargement, protein synthesis and distinct physiological molecular alternation. Akt activation also was observed in synd4-overexpressed cardiomyocytes. Furthermore, inhibition of protein kinase C (PKC) prevented the synd4-induced hypertrophic phenotype and Akt phosphorylation. This study identified an essential role of synd4 in mediation of physiological cardiac hypertrophy.