In this study, a novel sandwiched electrochemiluminescence (ECL) immunosensor for the detection of carcinoembryonic antigen (CEA) was developed. The nanocomposite of polydopamine and Ag nanoparticles ...(PDA-AgNPs) was prepared by the redox reaction between Ag+ and dopamine. This nanocomposite not only provided an effective matrix for the immobilization of primary antibody (Ab1) but also enhanced the conductivity of the electrode. Carbon quantum dots (CQDs) were immobilized on the poly(ethylenimine) functionalized graphene oxide (PEI-GO) through amido-bond. Then Au nanoparticles were decorated on the CQDs modified PEI-GO matrix, and the resulted complex AuNPs/CQDs-PEI-GO was introduced to link secondary antibody (Ab2). The CQDs can be connected to the electrode surface through the combination of CEA with Ab1 and Ab2, and then the amplified electrochemiluminescence signal of CQDs was obtained with the synergistic effect of AgNPs, polydopamine, AuNPs and PEI-GO. Under the optimal conditions, the ECL intensity was proportional to the logarithm value of CEA concentration in the linear range from 5pgmL−1 to 500ngmL−1 with a detection limit of 1.67pgmL−1 for CEA detection. The immunosensor was applied for the CEA detection in real samples with satisfactory results. The proposed ECL immunosensor showed good performance with high sensitivity, specificity, reproducibility, stability and will be potential in clinical detection.
•The nanocomposite of polydopamine and Ag nanoparticles (PDA-AgNPs) provided an effective matrix for the immobilization of primary antibody (Ab1).•Carbon quantum dots (CQDs), the electrochemiluminescence material, were immobilized on the poly(ethylenimine) functionalized graphene oxide (PEI-GO) to link secondary antibody (Ab2).•The ECL signals of CQDs were greatly amplified under the synergistic effect of AgNPs, polydopamine, AuNPs and PEI-GO.•The proposed ECL immunosensor can be used for the detection of CEA in real human serum samples.
Ratiometric electrochemiluminescence (ECL) sensors can efficiently remove environmental interference to attain precise detection. Nonetheless, two eligible luminophores or coreactants were usually ...needed, increasing the complexity and restricting their practical application. In this study, a single luminophore of luminol with a single coreactant of H2O2 was employed to construct a dual-potential ratiometric ECL sensor for the detection of carcinoembryonic antigen (CEA). The produced palladium nanoclusters (Pd NCs) employing a DNA duplex as a template could not only stimulate luminol to produce cathodic ECL (I cathodic) but also quench its anodic ECL (I anodic). During the detection process, CEA could damage the double-stranded structure and reduce the Pd NCs’ amount, triggering a significant decrease in the ratio of I cathodic to I anodic (I cathodic /I anodic) and thereby achieving sensitive CEA’s detection. Furthermore, the I cathodic /I anodic was independent of the H2O2 concentration, which avoided a prejudicial effect from H2O2 decomposition and considerably enhanced the detection’s reliability. The developed ratiometric ECL sensor demonstrated a sensitive detection toward CEA with a wide linear range from 100 ag/mL to 10 ng/mL and a detection limit of 87.1 ag/mL (S/N = 3). In conclusion, this study offers a new idea for constructing ratiometric ECL sensors based on a single luminophore and technical support for cancer’s early diagnosis.
Outbreaks of emerging infectious diseases pose a serious threat to public health security, human health and economic development. After an outbreak, an animal model for an emerging infectious disease ...is urgently needed for studying the etiology, host immune mechanisms and pathology of the disease, evaluating the efficiency of vaccines or drugs against infection, and minimizing the time available for animal model development, which is usually hindered by the nonsusceptibility of common laboratory animals to human pathogens. Thus, we summarize the technologies and methods that induce animal susceptibility to human pathogens, which include viral receptor humanization, pathogen-targeted tissue humanization, immunodeficiency induction and screening for naturally susceptible animal species. Furthermore, the advantages and deficiencies of animal models developed using each method were analyzed, and these will guide the selection of susceptible animals and potentially reduce the time needed to develop animal models during epidemics.
The repeated measurements of heart rate variability (HRV) is more relevant than a single HRV measurement in predicting patient prognosis but is less addressed previously. This prospective study aimed ...to investigate the association between repeated measurements of HRV and long-term mortality in chronic hemodialysis patients. The 164 patients (65.0 ± 13.1 years; woman, 57.3%) were enrolled from June 1, 2010, to August 31, 2010, and received four HRV measurements (before and during the index hemodialysis session) after the enrollment. The baseline characteristic and clinical variables, including mortality, were documented. The joint modeling method and Cox regression were used for statistical analyses. After an 8-year follow-up, 79 patients expired, and 85 patients survived. We found that higher normalized high-frequency (nHF) (hazard ratio HR 1.033) as well as lower very-low-frequency (HR 0.990), Variance (HR 0.991), normalized low-frequency (HR 0.999, P = 0.006), and low-frequency/high-frequency ratio (HR 0.796) were independent predictors for cardiovascular mortality. Whereas the independent predictors for infection-associated mortality included higher nHF (HR 1.033) as well as higher age (HR 19.29) and lower serum albumin (HR 0.01, P = 0.001). (all P < 0.001 unless otherwise stated) In conclusion, HRV measurement predicts long-term mortality among hemodialysis patients.
The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer ...and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au–S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 − 4 ×105 U L−1) and a low detection limit (1.5 U L−1) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs.
The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), which is highly expressed in the brain-predominantly in the cerebral cortex-and plays an ...essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of the UNC13B mutation in human disease is not known. In this study, we screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed a favourable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database (gnomAD). Computational modelling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a) knockdown Drosophila. Electrophysiological recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results indicate that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favourable outcome under anti-epileptic therapy in our patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition.
Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD‐(AS‐ODN+p160)) coupled with antisense oligonucleotide (AS‐ODN) and peptide p160 is ...designed for real‐time tracking of targeted delivery of AS‐ODN and regulation of folate receptor‐α (hFR‐α) in MCF‐7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS‐ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD‐(AS‐ODN+p160) is able to specifically target human breast cancer MCF‐7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD‐(AS‐ODN+p160) is energy‐dependent, and the effects of inhibition agents and co‐localization imaging further confirm the endocytic pathway is mainly receptor‐mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC50) value are chosen according to MTT assay. Real‐time PCR shows at these two concentration cases the relative mRNA expression levels of hFR‐α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR‐α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration‐dependent. These results demonstrate that the QD‐(AS‐ODN+p160) probe not only achieves gene silence in a cell‐specific manner but also achieves real‐time tracking during AS‐ODN intracellular delivery.
A new multifunctional quantum dot (QD) probe is prepared by coupled with anti‐hFR‐α antisense oligonucleotide and targeted peptide p160 with QDs through biotin‐streptavidin interaction. Down‐regulation of hFR‐α in a cell‐specific manner is achieved. The probe is effectively transported into the cytoplasm through receptor‐mediated endocytosis, and finally antisense oligonucleotides combine with hFR‐α mRNA and block the protein translation.
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•A ratiometric ECL sensor was constructed using luminol as single luminophor.•The ECLanodic/ECLcathodic was independent with the coreactant H2O2 concentrations.•The ratiometric sensor ...showed superior sensitivity and accuracy than the single-signal sensors.•4. The proposed ECL sensor exhibited high sensitivity for CEA detection with a wide linear range and a low detection limit.
Ratiometric electrochemiluminescence (ECL) assays have attracted widespread attentions in biosensing owing to their precise measurements by eliminating the environmental interferences. However, they mostly needed two eligible luminophors, increasing the complexity of the systems and limiting their practical applications. Herein, using luminol as single luminophor, a dual-potential ratiometric ECL strategy was proposed to detect carcinoembryonic antigen (CEA). The luminol exhibited cathodic and anodic emissions on graphene-ionic liquid-platinum (GR-IL-Pt) composites and Ti3C2 MXenes-Au NPs hybrids, respectively. Then, a sandwich ECL sensor was fabricated using GR-IL-Pt composites as matrix to immobilize the primary antibodies of CEA and Ti3C2 MXenes-Au NPs hybrids as platform to load the secondary antibodies. With the presence of CEA, the ratio of anodic ECL to cathodic ECL (ECLanodic/ECLcathodic) increased obviously, realizing sensitive ratiometric detection of CEA. In addition, the ECLanodic/ECLcathodic was independent with the concentrations of H2O2, greatly improving the test reliability. The developed ECL sensor exhibited a sensitive detection toward CEA, performing a wide linearity in the range of 0.1 pg mL−1 - 10 ng mL−1 with a low detection limit of 34.58 fg mL-1 (S/N = 3). Furthermore, this strategy exhibited a good practicality to detect CEA in human serums, providing a promising strategy in ECL bioanalysis.
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