Graphene Oxide Signal Reporter Based Multifunctional Immunosensing Platform for Amperometric Profiling of Multiple Cytokines in Serum

Cytokines are small proteins and form complicated cytokine networks to report the status of our health. Thus, accurate profiling and sensitive quantification of multiple cytokines is essential to have a comprehensive and accurate understanding of the complex physiological and pathological conditions in the body. In this study, we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1β, and TNF-α. First, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1β, and TNF-α, respectively, to obtain Ab₂-GO-NB, Ab₂-GO-MB, and Ab₂-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, the capture monoclonal antibody for IL-6, IL-1β, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab₂-GO-NB, Ab₂-GO-MB, and Ab₂-GO-Fc. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1β, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity, and stability, and recovery of 93.6%–105.5% was achieved for determining cytokines spiked in the whole mouse serum

Decoration of Reduced Graphene Oxide Nanosheets with Aryldiazonium Salts and Gold Nanoparticles toward a Label-Free Amperometric Immunosensor for Detecting Cytokine Tumor Necrosis Factor‑α in Live Cells

In this study, a label-free electrochemical immunosensor was developed for detection of cytokine tumor necrosis factor-alpha (TNF-α). First, AuNPs loaded reduced graphene oxides nanocomposites (RGO-ph-AuNP) were prepared, and then, a mixed layer of 4-carbxyphenyl and 4-aminophenyl phosphorylcholine (PPC) was modified to the surface of AuNPs for the subsequent modification of anti-TNF-α capture antibody (Ab₁) to form the capture surface (Au-RGO-ph-AuNP-ph-PPC­(-ph-COOH)) for the analyte TNF-α with the antifouling property. For reporting the presence of analyte, the anti-TNF-α detection antibody (Ab₂) was modified to the graphene oxides which have been modified with the 4-ferrocenylaniline through diazonium chemistry to form Ab₂-GO-ph-Fc. Then, a sandwich assay was formed on gold surfaces for the quantitative detection of TNF-α based on the electrochemical signal of ferrocene. X-ray photoelectron spectra (XPS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), UV–vis, and electrochemistry were used for characterization of the stepwise fabrications on the interface. The prepared electrochemical immunosensor was successfully used for the detection of TNF-α over the range of 0.1–150 pg mL^–1. The lowest detection limit of this immunosensor is 0.1 pg mL^–1 TNF-α in 50 mM phosphate buffer at pH 7.0. The fabricated immunosensor provided high selectivity and stability and can be used to detect TNF-α secreted by live BV-2 cells with comparable accuracy to enzyme-linked immunosorbent assay (ELISA) but with lower limit of detection

Light-Induced Organic Monolayer Modification of Iodinated Carbon Electrodes

We report the modification of carbon electrodes formed from pyrolyzed photoresist films (PPF) via plasma iodination followed by the organic monolayer modification of these surfaces. The iodinated surfaces were characterized using cyclic voltammetry, atomic force microscopy, and X-ray photoelectron spectroscopy to enable the optimization of the iodination while preserving the stability and smoothness of the carbon surface. Subsequently, the C–I surface was further modified with molecules that possess an alkene or alkyne at one end through light activation with low energy (visible range λ 514 nm). The versatility of the modification reaction of the C–I surfaces is shown by reactions with undecylenic acid, 1,8-nonadiyne, and <i>S</i>-undec-10-enyl-2,2,2-trifluoroethanethioate (C₁₁-S-TFA). Modification with 1,8-nonadiyne allows further modification via “click” chemistry with azido-terminated oligo­(ethylene oxide) molecules demonstrated briefly to alter the hydrophilicity of the surface after attachment of ethylene oxide moieties. Furthermore, patterning of C₁₁-S-TFA was demonstrated using a simple photolithography technique. Deprotection of the C₁₁-S-TFA gave a free thiol allowed patterning of gold nanoparticles on the surface as verified using scanning electron microscopy (SEM). These results demonstrate that plasma iodination to form C–I is a versatile, simple, and modular approach to functionalize the carbon surface

The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation

<div><h3>Objective</h3><p>Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx<em>-</em>d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.</p> <h3>Methods</h3><p>We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. <em>In silico</em> analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.</p> <h3>Results</h3><p>HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.</p> <h3>Conclusion</h3><p>miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.</p> </div

miR-338-3p inhibits cell proliferation, especially in LO2/HBx-d382 cells.

<p>(A) A representative cell cycle profile for miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells comparing to their control RNA transfection. (B) Average of G1 phase populations in HBx-expressed LO2 cells after transfected miR-338-3p mimics or inhibitor (*<i>P</i><0.001). (C) Average of S phase populations after transfected miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells (*<i>P</i><0.001). While miR-338-3p overexpresses or suppresses, miR-338-3p induce cell cycle arrest and anti-miR-338-3p increases cell growth, especially in LO2/HBx-d382 cells. Values in (B) and (C) are means ±SD of three separate experiments.</p

miR-338-3p influence the colony formation of transfected LO2 cells.

<p>(A–B) Gain of miR-338-3p decreases the rate of colony formation of LO2/HBx-d382 and LO2/HBx cells, whereas loss of miR-338-3p enhances the non-anchored growing ability in HBx-expressing cells (*<i>P</i><0.001, <i>**P</i><0.01). (C) The non-anchored growing ability of control LO2/pcDNA3.0 cells are also inhibited by miR-338-3p, although the significant difference is lower than that in HBx-expressing cells (***<i>P</i> = 0.032,#<i>P</i> = 0.025).</p

miR-338-3p inhibits cell proliferation by Edu assay.

<p>(A–B) Representative profiles of Edu cell proliferation after transfection with miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells compared to their negative control transfection (magnification 100×). (C) Rate of Edu positive cells in S phase. Gain of miR-338-3p inhibits the cellular DNA replication in HBx-expressed LO2 cells and control cells, whereas loss of miR-338-3p expression demonstrates an adverse result, especially in LO2/HBx-d382 cells. (*<i>P</i><0.001; **<i>P</i><0.01; ***<i>P</i><0.05).</p

HBx, especially HBx-d382, enhances CyclinD1 expression.

<p>(A) CyclinD1 protein level of the engineered LO2 cells. (B) The histogram of CyclinD1 mRNA and protein level in LO2/HBx and LO2/HBx-d382 cells, compared with LO2/pcDNA3.0 cells (*<i>P</i><0.001,**<i>P</i> = 0.017,***<i>P</i> = 0.029).</p

CyclinD1 is a direct target of and is regulated by miR-338-3p, and the effect of miR-338-3p on CyclinD1 is mainly dependent on the CyclinD1-3′-UTR region (nt 2397–2403).

<p>(A–B) Successfully constructed plasmids of CyclinD1-3′-UTR. (A) Amplification of a DNA fragment containing CyclinD1-3′-UTR region. PCR amplification was performed using specific primers for the types of WT-, Mut-, Mut1-, and Mut2-Cyclin D1-3′-UTR, respectively, and genomic DNA isolated from the genome of LO2/HBx cells was used as PCR template. An approximately 1573 bp band PCR product was detected by agarose gel electrophoresis. (B) Identification of recombinant plasmids. The fragment digested with XhoI and NotI from the recombinant plasmids: pCyclinD1-3′-UTR-WT, pCyclinD1-3′-UTR-Mut, pCyclinD1-3′-UTR-Mut1, and pCyclinD1-3′-UTR-Mut2 were used as a template for confirmation PCR. The PCR product was about 1573 bp, which was finally confirmed by DNA sequencing. miR-338-3p targets CyclinD1. (C) Dual luciferase assay of LO2/HBx cells cotransfected with the Renilla luciferase constructs containing the CyclinD1 WT or Mut 3′-UTR and miR-338-3p mimics or negative RNA (*<i>P</i> = 0.404,**<i>P</i><0.001). (D) The major site of CyclinD1 targeted by miR-338-3p was at position 2397–2403 nt. Effects of miR-338-3p and the negative control on the reporter constructs containing CyclinD1-3′-UTR Mut1 and Mut2 were determined 48 hours after transfection. Renilla luciferase values normalized to Firefly luciferase are presented. (**<i>P</i><0.001,***<i>P</i> = 0.04). (E–G) CyclinD1 protein expression after transfection of miR-338-3p mimics, inhibitor, or negative control (*<i>P</i><0.001,**<i>P</i><0.01). The fold change before and after transfection is more pronounced in LO2/HBx-d382 cells than that in LO2/HBx and control cells. Data are shown as mean ±SD from at least 3 independent experiments. (H) qRT-PCR with the 2^−ΔΔCT method to evaluate the CyclinD1 mRNA expression normalized to GAPDH in LO2/HBx-d382, LO2/HBx, and control cells transfected with miR-338-3p mimics or inhibitor or their respective controls(★<i>P</i> = 0.164,★★ <i>P</i> = 0.438, #<i>P</i> = 0.220,##<i>P</i> = 0.101,* <i>P</i> = 0.254,** <i>P</i> = 0.417).</p

HBx-d382 enhances the non-anchored growing ability of transfected hepatocytes.

<p>(A–B) The identification of stable HBx transfection in LO2 cells. (A) The HBx gene was identified by RT-PCR. (B) Western blotting showed the expression of HBx in LO2 cells. 1: LO2; 2: LO2/pcDNA3.0; 3: LO2/HBx-d382; 4: LO2/HBx; M: marker. (C) Soft agar colony formation assay of transfected LO2 cells. The rate of colony formation in HBx-expressing cells was significantly higher than in control LO2 and LO2/pcDNA3.0 cells (*<i>P</i><0.01). While within the groups of LO2/HBx-d382 LO2/HBx, the former was higher than the latter (#<i>P</i><0.01).</p