Separation detection of different circulating tumor cells in the blood using an electrochemical microfluidic channel modified with a lipid-bonded conducting polymer.

Different circulating tumor cells (CTCs) in blood were separated and detected through the decoration of anti-cancer drug on the target cells, along with chemical modification of the microfluidic channel walls using a lipid attached covalently to the conducting polymer. The working principle of the electrochemical microfluidic device was evaluated with experimental parameters affecting on the separation, in terms of mass and surface charge of target species, fluid flow rate, AC amplitude, and AC frequency. The separated CTCs were selectively detected via the oxidation of daunomycin adsorbed specifically at the cells using an electrochemical sensor installed at the channel end. The fluorescence microscopic examination also confirmed the separation of CTCs in the channel. To evaluate the reliability of the method, blood samples from 37 cancer patients were tested. The device was able to separate the CTCs with 92.0 ±â€¯0.5 % efficiency and 90.9% detection rate.

Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells.

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ±â€¯0.02, 0.76 nM ±â€¯0.02, and 0.48 nM ±â€¯0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated.

Hydrogen peroxide mediates arsenite activation of p70(s6k) and extracellular signal-regulated kinase.

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.

Modulation of constitutive and delayed apoptosis by brefeldin A in human neutrophils.

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by various stimuli. In this study, the effect of brefeldin A (BFA), which affects the biological process of secretion, on constitutive and delayed apoptosis of neutrophils was investigated. Neutrophil apoptosis was determined after culturing for 20 h in vitro by morphological changes, annexin V staining, and DNA electrophoresis. BFA dose-dependently increased the constitutive apoptotic rate of neutrophils. The delay of apoptosis induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) was also blocked by BFA. However, this effect of BFA was less marked when neutrophils were treated with dexamethasone, interleukin-8 (IL-8), or dibutyryl cAMP (dbcAMP). Moreover, the delay of neutrophil apoptosis induced by rottlerin, a specific inhibitor of protein kinase C (PKC)-delta, was significantly abrogated by BFA. Although BFA-induced apoptosis was not blocked by the caspase-3 inhibitor, zDEVD-fmk, myeloid cell leukemia-1 (Mcl-1) expression levels were downregulated by BFA. These results suggest that derangement of vesicular protein transport may be involved in the apoptosis of neutrophils, and that the action of BFA on apoptosis is dependent on changes in the expression of Mcl-1.

Anti-apoptotic role of phospholipase D in spontaneous and delayed apoptosis of human neutrophils.

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by signals induced by various stimuli. We investigated the role of phospholipase D (PLD) in neutrophil apoptosis. The apoptotic rate of neutrophils was found to be increased by 1-butanol and decreased by the exogenous addition of PLD. Moreover, the delay of apoptosis by apoptosis-delaying stimuli such as granulocyte/macrophage colony-stimulating factor or lipopolysaccharide (LPS) was also blocked by 1-butanol. Unstimulated PLD activity in cultured cells for 20 h was higher than that in freshly isolated cells and further increased in cultured cells with LPS. These results suggest that PLD is involved in the up-regulation of neutrophil survival.