Potential-Resolved Electrochemiluminescence for Simultaneous Determination of Triple Latent Tuberculosis Infection Markers

A novel electrochemiluminescence (ECL) immunosensor based on the potential-resolved strategy was first developed for simultaneous determination of triple latent tuberculosis infection (LTBI) markers with high sensitivity, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-2. In this work, luminol and self-prepared carbon quantum dots and CdS quantum dots were integrated onto gold nanoparticles and magnetic beads in sequence to fabricate potential-resolved ECL nanoprobes with signal amplification. IFN-γ-antibody (Ab)­1, TNF-α-Ab1, and IL-2-Ab1 were separately immobilized on three spatially resolved areas of a patterned indium tin oxide electrode to capture the corresponding LTBI markers, which were further recognized by IFN-γ-Ab2, TNF-α-Ab2, and IL-2-Ab2-functionalized ECL nanoprobes. The binding reaction of antibody-functionalized ECL nanoprobes and the captured LTBI markers will generate three sensitive and potential-resolved ECL signals during one potential scanning, and the ECL intensities reflect the concentrations of IFN-γ, TNF-α, and IL-2 in the range of 1.6–200 pg mL^–1. Therefore, the multiplexed ECL immunosensor provided an effective approach for simultaneous detection of triple LTBI markers in human serum, so it will be beneficial to facilitate more accurate and reliable clinical diagnosis for LTBI

Novel Electrochemiluminescence-Sensing Platform for the Precise Analysis of Multiple Latent Tuberculosis Infection Markers

Latent tuberculosis infection (LTBI) is one of the major contributing factors for the high incidence of tuberculosis, and the low contents of LTBI markers in human serum present a great challenge for the diagnosis of LTBI. Here, we reported a novel electrochemiluminescence (ECL)-sensing platform for the precise analysis of multiple LTBI markers, interferon-gamma (IFN-γ) and interleukin (IL)-2. In this approach, self-prepared carbon quantum dots (CQDs) and luminol were integrated onto gold nanoparticles (AuNPs), which were further enriched on the surface of magnetic bead (MB) to create two solid-phase ECL nanoprobes (MB@Au@CQDs and MB@Au@luminol) for improving the detection sensitivity efficiently. Graphene oxide (GO) and AuNPs were electrodeposited onto a patterned indium tin oxide (ITO) electrode with two spatially resolved areas in sequence to form two sensitive and stable sensing areas. IFN-γ-antibody (Ab)₁ and IL-2-Ab₁ were separately immobilized on the two sensing areas to capture the corresponding LTBI markers, which were further recognized by IFN-γ-Ab₂ and IL-2-Ab₂ labeled as MB@Au@CQDs and MB@Au@luminol. The ECL intensity depended linearly on the content of IFN-γ and IL-2 in the range of 0.01–1000 pg mL^–1, with a low detection limit of 10 fg mL^–1. The proposed ECL-sensing platform is simple, sensitive, accurate, reliable, and specific to the detection of rare IFN-γ and IL-2 in human serum and provides a valuable protocol for facilitating fast and precise diagnosis of LTBI

Label-Free Quartz Crystal Microbalance with Dissipation Monitoring of Resveratrol Effect on Mechanical Changes and Folate Receptor Expression Levels of Living MCF‑7 Cells: A Model for Screening of Drugs

Quartz crystal microbalance with dissipation (QCM-D) monitoring was used for real-time and label-free detection of changes and folate receptor (FR) expression levels on living MCF-7 cells for evaluating the anticancer activity of resveratrol. Here, the mechanical changes of cellular responses to resveratrol were tracked by a poly­(l-lysine) (PLL) modified QCM-D sensor, and the inhibition effect of resveratrol on FR expression levels on MCF-7 cells was monitored by chitosan-folic acid (CS-FA) composite membrane functionalized Au substrate for the first time. Changes in morphology and the cellular state of MCF-7 cells stimulated by resveratrol at different concentrations were detected by inverted fluorescence microscopy and flow cytometry. Atomic force microscopy confirmed that resveratrol influenced the cellular mechanical properties. The results indicated that the MCF-7 cells lose their original elasticity and increase their stiffness induced by resveratrol. It was further observed by confocal fluorescence imaging that resveratrol reduced the FR expression levels on the living cells surface. This study established a typical model of the QCM-D biosensor to evaluate the protein biomarker expression levels on the cells surface. QCM-D, which was used to investigate potential targets for an antitumor drug on living cells and realize a better understanding of the drug action mechanism, was expected to be developed into a promising tool for the screening of drugs

Dual Electrochemiluminescence Signal System for <i>In Situ</i> and Simultaneous Evaluation of Multiple Cell-Surface Receptors

A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for <i>in situ</i> and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface. Human mucin1 protein (MUC1) aptamer was immobilized onto AuNPs for capturing MUC1-positive MCF-7 cells. Au@luminol and CdS QDs as ECL nanoprobes were covalently linked with concanavalin A (ConA) and epidermal growth factor (EGF) to label MCF-7 cells on the two areas of the cytosensor separately. Compared to conventional multiplex biosensor, we demonstrated a novel analysis platform for the simultaneous detection of multiple cell-surface receptors; it could provide two sensitive and potential-resolved ECL signals during one potential scanning and avoid cross-reactivity between the two nanoprobes. The quantification of MCF-7 cells on the two spatially resolved areas could be achieved over the linear range from 10² to 1.0 × 10⁶ cells mL^–1 with a detection limit of 20 cells mL^–1. This multiplex cytosensor was further applied for simultaneous quantitative evaluation of the expression levels of mannose and EGFR on MCF-7 cells, revealed that the average numbers of mannose and EGFR per captured MCF-7 cell were 1.2 × 10⁶ and 0.86 × 10⁵ with the relative standard deviation of 5.3% and 4.2%, respectively. The multiplex cytosensor was capable of evaluating multiple cell-surface receptors, which would be beneficial to developing a better diagnostic tool for diseases

Novel Single-Cell Analysis Platform Based on a Solid-State Zinc-Coadsorbed Carbon Quantum Dots Electrochemiluminescence Probe for the Evaluation of CD44 Expression on Breast Cancer Cells

A novel single-cell analysis platform was fabricated using solid-state zinc-coadsorbed carbon quantum dot (ZnCQDs) nanocomposites as an electrochemiluminescence (ECL) probe for the detection of breast cancer cells and evaluation of the CD44 expression level. Solid-state ZnCQDs nanocomposite probes were constructed through the attachment of ZnCQDs to gold nanoparticles and then the loading of magnetic beads to amplify the ECL signal, exhibiting a remarkable 120-fold enhancement of the ECL intensity. Hyaluronic acid (HA)-functionalized solid-state probes were used to label a single breast cancer cell by the specific recognition of HA with CD44 on the cell surface, revealing more stable, sensitive, and effective tagging in comparison with the water-soluble CQDs. This strategy exhibited a good analytical performance for the analysis of MDA-MB-231 and MCF-7 single cells with linear range from 1 to 18 and from 1 to 12 cells, respectively. Furthermore, this single-cell analysis platform was used for evaluation of the CD44 expression level of these two cell lines, in which the MDA-MB-231 cells revealed a 2.8–5.2-fold higher CD44 expression level. A total of 20 single cells were analyzed individually, and the distributions of the ECL intensity revealed larger variations, indicating the high cellular heterogeneity of the CD44 expression level on the same cell line. The as-proposed single-cell analysis platform might provide a novel protocol to effectively study the individual cellular function and cellular heterogeneity

GE11 peptide conjugated selenium nanoparticles for EGFR targeted oridonin delivery to achieve enhanced anticancer efficacy by inhibiting EGFR-mediated PI3K/AKT and Ras/Raf/MEK/ERK pathways

<p>Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.</p

Schematic diagram showing the ROS-mediated oridonin induced KYSE-150 cell apoptosis that can be detected by AFM.

<p>Schematic diagram showing the ROS-mediated oridonin induced KYSE-150 cell apoptosis that can be detected by AFM.</p

ROS scavenger-NAC reversed oridonin induced oesophageal cancer KYSE-150 cell apoptosis.

<p>(A) Annexin V/PI assay of the effects of NAC on oridonin induced KYSE-150 cell apoptosis. (B) Statistical analysis of the effects of NAC on oridonin induced KYSE-150 cell apoptosis, ***p<0.001.</p

Statistical results of NAC reversed oridonin induced oesophageal cancer KYSE-150 cell membrane ultrastructural changes determined by AFM.

<p>(A) Height distribution, (B) root-mean-squared roughness (Rq) and (C) average roughness (Ra) analyzed from 2×2 μm frame ultrastructure images of KYSE-150 cells, n = 30, *p<0.05, ***p<0.001.</p

ROS scavenger-NAC reversed oridonin induced changes of Young’s modulus in oesophageal cancer KYSE-150 cells.

<p>(A) Histogram distribution of Young’s modulus obtained from KYSE-150 cell. (B) Statistical analysis of the effects of NAC on oridonin induced KYSE-150 cell Young’s modulus changes, n>5000, ***p<0.001.</p