RESUMO
An integrated aptamer macroarray functionalized with reduced graphene oxide (rGO) to specifically capture and sensitively detect cancer cells is reported. The capture for cancer cells is based on effective recognition of the modified rGO surface through the aptamer against epithelial cell adhesion molecule (EpCAM). The rough structure of rGO enhances morphologic interactions between rGO film interface and the cancer cells, while super-hydrophilicity of modified rGO hinders nonspecific cell capture. The synergistic interactions offer the aptamer macroarray high efficiency of cancer cell capture. By means of a turn-on fluorescence strategy based on the conformation change of the aptamer induced by the target recognition, the enriched cancer cells can be directly read out at excitation/emission wavelengths of 550/680 nm without washing, separation, and dying steps. The working range is 1 × 102 to 2 × 104 cells per mL with a detection limit of 22 cells per mL. The results indicate that the aptamer macroarray has a considerable foreground for early diagnosis, therapy, and monitoring of cancer. Graphical abstract.
Assuntos
Aptâmeros de Nucleotídeos/química , Separação Celular/métodos , Grafite/química , Células Neoplásicas Circulantes , Sequência de Bases , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Pirenos/químicaRESUMO
Biothiols play critical roles in many biological processes and their aberrant is related to a variety of syndromes. A simple and reliable colorimetric method is developed in this work for biothiols detection based on an oxidase mimic, a metal organic framework (MOF) MIL-53(Fe), and a peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB). In this design, MIL-53(Fe) is utilized to catalyze the conversion of TMB to a blue colored 3,3',5,5'-tetramethylbenzidine diimine, which can be read on a spectrophotometer at 652â¯nm. The oxidation-induced blue color generation can be efficiently inhibited by biothiols, thus a colorimetric analytical method is proposed for biothiols detection based on the above system. Under optimal conditions, a linear relationship in a range from 1 to 100⯵M and a limit of detection (LOD) at 120â¯nM are achieved with Cys as a model target. The developed platform is further applied to evaluate cellular biothiols in normal (RWPE-1) and cancer (LNCap) cell lines, revealing that the overall biothiols level in LNCap is much higher than that in RWPE-1. This work renders a powerful tool for identifying cancer cells in a simple manner for biomedical diagnosis associated with biothiols.
Assuntos
Cisteína/análise , Glutationa/análise , Homocisteína/análise , Benzidinas/química , Linhagem Celular , Colorimetria/métodos , Humanos , Estruturas Metalorgânicas/químicaRESUMO
A rolling-mediated cascade (RMC) amplification strategy is described for improved visualization of profiling glycans of mucin 1 (MUC 1) on cell surfaces. CdTe quantum dots (QDs) are used as fluorescent labels. The RMC based amplification allows even distinct glycoforms of MUC1 to be visualized on the surface of MCF-7 cell via an amplified Förster resonance energy transfer (FRET) imaging strategy that works at excitation/emission wavelengths of 345/610 nm. This is achieved by utilizing antibody against MUC1 modified with the fluorescent label 7-amino-4-methylcoumarin-3-acetic acid (AMCA) as the energy donor in FRET. The QDs (used to label surface glycans) act as acceptors. N-Azidoacetylgalactosamine-Acetylated (Ac4GalNAz) as a non-natural azido sugar, can be incorporated into the glycans of the cell surface, which can promote further labeling. The method has the advantage of only requiring a small amount of non-natural sugar to be introduced in metabolic glycan labeling since too much of an artificial sugar will interfere with the physiological functions of cells. Graphical abstract Schematic for the DNA rolling-mediated cascade (RMC)-assisted metabolic labeling of cell surface glycans by using CdTe quantum dots as labels and an intramolecular amplified FRET strategy for imaging glycans on a specific glycosylated protein, MUC1.
Assuntos
Corantes Fluorescentes/química , Mucina-1/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Polissacarídeos/análise , Pontos Quânticos/química , Anticorpos/imunologia , Compostos de Cádmio/química , Compostos de Cádmio/toxicidade , Cumarínicos/química , DNA/química , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Células MCF-7 , Mucina-1/imunologia , Polissacarídeos/química , Pontos Quânticos/toxicidade , Telúrio/química , Telúrio/toxicidadeRESUMO
To explore the effects of different electron acceptors on soil methane emission and responses of soil microorganisms to different light conditions, a strict anaerobic 20-day incubation experiment was conducted with eight treatments: darkness + Fe3+ (DF); darkness + NO3- (DN); darkness +SO42- (DS); darkness + distilled water (DCK); light + Fe3+ (LF); light + NO3- (LN); light +SO42- (LS); light + distilled water (LCK). The changes of methane concentration in the anaerobic incubation flask and the variation of the abundance of bacteria, archaea, fungi and six soil functional genes were analyzed. Results showed that soil methane emission under NO3-, SO42- addition and control (CK) was significantly lower under light conditions than dark, except the Fe3+ treatment. DN, DCK and LF treatments had the highest abundance of bacteria, fungi and archaea genes, respectively. The gene abundance of methanogenic mcrA, sulfate-reducing bacteria Dsr, and carbon-fixing CbbL were significantly up-regulated in the LF, while that of methanotrophs pmoA, iron-reducing bacteria Geo, and denitrifying bacteria nosZ were significantly up-regulated in the LN, DCK and LCK, respectively. Results of Pearson correlation and RDA analysis showed that CH4 emission was significantly positively correlated with CO2 concentration, pH, ammonium-nitrogen, and total N contents, and negatively correlated with N2O concentration, Eh, nitrate, and total C contents. Under dark condition, methane emission was positively correlated with archaea and pmoA genes abundance, and negatively correlated with other genes abundance. Under light condition, methane emission was negatively correlated with the abundance of soil microbe and functional genes. In general, methane emission under light condition was significantly lower than that under dark condition (except for the Fe3+ treatment). These results showed that it was helpful to reduce methane emission under light condition, but the increase or decrease of methane emission was closely related to the type of electron acceptors and the functional responses of soil micro-organisms.
Assuntos
Elétrons , Solo , Archaea/genética , Metano/análise , Solo/química , Microbiologia do SoloRESUMO
The capacity to diagnose cancer with the existing endogenous biomarkers remains limited because biomarkers usually act at the tumor site and are thus challenging to be detected directly from body fluids with high sensitivity and specificity, especially in the early stage of tumorigenesis. Here, we demonstrate an exogenous tumor-penetrating nanomarker composed of fluorescent nanoparticles conjugated with specific fluorescein-labeled peptides. The injectable nanomarkers perform four functions: they penetrate the tumor, target sites of cancer, cleave specific peptides by on-target protease, and drop off the labeled peptide into host urine for fluorescent detection. Sensitive in vivo tracking and monitoring of the cyclic process of the nanomarker was also accomplished. The nanomarker can noninvasively diagnose and monitor tumors with a volume of about 17 mm3 without invasive core biopsies. Enhanced capacity of early point-of-care detection for cancer is accomplished by receptor-dependent specificity of the signal generation in the urine compared with clinically used blood biomarkers.
Assuntos
Nanopartículas , Neoplasias , Biomarcadores , Diagnóstico por Imagem , Humanos , Neoplasias/diagnóstico , PeptídeosRESUMO
OBJECTIVE: To study the effects of calponin-1 expression inhibition on the proliferation , invasiveness, apoptosis and cytoskeleton of uterine smooth muscle cells, and explore the molecular mechanism of calponin-1 in the uterine smooth muscle cells for labor onset. METHODS: siRNA-calponin-1 adenovirus plasmid was constructed and transfected into primarily cultured uterine smooth muscle cells. The proliferation, invasiveness and apoptosis of the cells were determined by MTT assay, matrigel invasion assays and flow cytometry, respectively. Rhodamine-Phalloidin was used for labeling filamentous actin (F-actin), and the morphology and the distribution of F-actin was observed under fluorescence microscopy and analyzed quantitatively. RESULTS: The motor ability of uterine smooth muscle cells decreased significantly after transfection with siRNA-calponin-1 adenovirus plasmid (P<0.05). The transfected cells showed thinner, loosened and irregular F-actin microfibers, and the cells in the empty vector and blank control groups showed thicker and longer F-actin microfibers. CONCLUSION: Inhibition of calponin-1 expression can inhibit uterine smooth muscle cell migration and cause the morphological change and rearrangement of F-actin without affecting its proliferation and apoptosis in vitro, suggesting that the morphological change and rearrangement of F-actin of uterine smooth muscle cell may be one of the important mechanisms in the labor onset.