When free ferrocene (40 M) was in direct contact with the cells, the cell death was observed in 3 days. polymeric micelles. This resulted in a significant increase in the intensity of the ferrocene signal detected by cyclic voltammetry. cells which were apprehended using conjugated magnetic beads. The beads were then coated on graphite electrodes, and the alkaline phosphatase (AP)-labeled goat anti-antibody catalyzed the production of an electroactive para-aminophenol from its substrate, para-aminophenyl phosphate, which was monitored electrochemically. The obtained signal was directly related to the number of bacterial cells, and the sensor had a sensitivity of 8103 cells/mL. Thus, to overcome such inconveniences, liposomes encapsulated with marker molecules or enzymes have been used.14 However, the main downside of liposomes is their instability throughout their use or storage, 15 which is mainly related to their surface properties. Several solutions were investigated to improve liposome stability. For example, the modification of the composition of the liposome lipids via the incorporation of a bio-adhesive molecule such as chitosan or cholesterol in their membrane significantly improved their stability,16 and also, the cross linking of the hydrophobic core of the liposome membrane via polymerization,17 or through surface-active polymer additives,18 has shown positive effect on their stability. So far, such improvements have been revealed to be very costly and increase the complexity of the fabrication process of these liposome immunoassays which cause them to be less efficient even with enhanced sensitivity range. Another major concern with using conventional liposomes in immunoassay is leakage, especially in the case of low-abundance protein detection. It was found that when small molecules are encapsulated inside liposome as tracers, these molecules have the tendency to leak into the outside medium. Such a problem can greatly affect the performance of the immunoassay.19,20 Therefore, developing a WYE-687 method that does not involve antibody immobilization on an electrode surface or rely on enzymatic reactions to generate the electrochemical signal is highly in demand. Such methods should provide a more robust and stable carrier which will present an important leap toward a more efficient and reliable biosensor for medical and food safety applications. Electrochemical immunoassays based on loaded pH-sensitive polymeric micelles offer an attractive alternative over the existing procedures, especially in applications where high selectivity, sensitivity, and stability are needed. In this work, the MUC1 glycoprotein (protein at the cell surface) was selected as a target for our new biosensor because it is frequently overexpressed in WYE-687 human cancers, particularly in breast cancer.21 In addition, MUC1 has shown a great potential for early detection and grading of tumors, along with the tumor responses to therapy. Furthermore, cell surface-associated epithelial mucin (MUC1) has been tested as a target for gold nanoparticle-mediated cancer diagnosis and therapy.22C24 MUC1 is a high-molecular-weight plasma membrane-bound protein with an obtruding O-glycosylated N-terminal domain that overhangs from the apical surface WYE-687 of glandular epithelial cells in the breast, colon, and lung.25 MUC1 has 72-aa C-terminal domain and a single trans-membrane domain and is regulated and cleaved by tyrosine phosphorylation.26 In this paper, we report the development of an exceedingly sensitive and fast bioassay to probe and quantify biomarkers (breast cancer biomarker). Since these polymeric nanoparticles have been shown to be efficient and stable carriers for hydrophobic molecules, we encapsulated tracer signal generator molecules (ferrocene) inside these polymeric nanoparticles, and upon their release, the ferrocene molecules acted as signal amplifiers for the binding between the antibody and antigen. Experimental section Reagents and materials Ethanol (96%, v/v) from AGA, HPLC-grade tetrahydrofuran (THF) from Sigma-Aldrich, hydrochloric acid (37%, w/w), MTT, Trizol reagent paraformaldehyde, and methoxypolyethylene glycol N-hydroxysuccinimide ester from Sigma-Aldrich Rabbit polyclonal to RPL27A were used. Monoclonal antibodies against the human MUC1 protein, SM3 (Abcam), and C595 (Santa Cruz Biotechnology) were obtained. For cell culture, complete Dulbeccos Modified Eagles Medium or RPMI1640 culture media (DMEM or RPMI1640 basic medium supplemented with 10% fetal bovine serum [FBS; PAA], 2 mM glutamine, 100 units/mL penicillin, and 100 g/mL streptomycin) was prepared. Cell lines were acquired commercially; MCF-7 from ATCC (ATCC HTB-22) and MDA-MB-468.
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