A simple Clark-type online electrochemical cell design, consisting of a platinum gauze working electrode and incorporating ionic liquids (IL) as electrolytes, has been successfully applied for the amperometric sensing of oxygen. Studying ILs comprising the bis(trifluoromethylsulfonyl)imide anion, the obtained analytical parameters were found to be strongly dependent on the choice of cation. Compared with a conventional Clark cell design based on an aqueous supporting electrolyte, the modified oxygen sensor achieves substantial improvements in performance and stability. A limit of detection for oxygen as low as 0.05 vol %, linearity over an oxygen partial pressure between 0% and 20%, and a steady-state response time of 2 min was demonstrated, with a stable analytical response shown over the examined period of 90 days with no obvious fouling of the electrode surface. Based on the attractive physical attributes of ionic liquids (e.g., thermal stability beyond 150 °C), one can envision intriguing utility in nonstandard conditions and long-term online applications, as well as extension to the determination of other gases, such as methane and nitric oxide.

CD20, expressed on greater than 90% of B-lymphocytic lymphomas, is an attractive target for antibody therapy. Rituximab is a chimeric murine/human-engineered monoclonal antibody and can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. The immobilization of B-lymphoblast-like Burkitt's lymphoma Raji cells on the quartz crystal microbalance (QCM) gold electrode surface using RGD tripeptide was electrochemically confirmed. The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (Δf0) in the studied antibody concentration range from 5 to 250 µg mL−1 following the Langmuir adsorption model. From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6×106 M−1. Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved the specific interaction between Rituximab and B cells. The effects of Ca2+ and Mn2+ ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular, further indicating that CD20 is a calcium ion channel that can transport these metal ions into the cells and accelerate the cell lysis induced by Rituximab. Thus the real time capability of QCM and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms.

Endothelial cell (EC) activation and their subsequent binding with different cells have various mechanical consequences that, if monitored real time, can serve as a functional biomarker of many pathophysiological response mechanisms. This work presents an innovative and facile strategy to conduct such monitoring using quartz crystal microbalance (QCM), thereby relating the shifts in its frequency and motional resistance to morphological changes upon cell-cell and cell-substrate interactions. By activating ECs with TNF-α and then characterizing their binding with HL-60 and KG-1 leukemia cells, we are able to induce the mechanical changes in ECs especially in the region of cell-substrate contact which resulted in dynamically coupled mass and viscoelastic changes representing the extent of both activation and binding. The activated ECs suffered a decrease of cellular contact area, leading to positive frequency shift and decreased motional resistance. The binding of leukemia cells onto pre-activated ECs exerted a mechanical force to regain the cell surface contact which resulted in the obvious QCM responses opposite to that of activation, and proportional to the number of cells added, in spite of the fact that these added cells are extremely outside the extinction depth of the shear wave generated by QCM. Different cell lines demonstrate different attachment behavior, which was detected by the QCM. Despite these variations are quite subtle, yet the sensitivity of the technique for dynamic changes at the interface makes them detectable. Moreover, the reproducibility of the generated data determined at each step by deviation measurements (<10%) in response plot was very high despite the high possible heterogeneity in cell populations. The results are explained on the basis of simple theoretical and physical models, although, the development of a more quantitative and precise model is underway in our laboratory.

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Background: Antigens like CD20 antigen, are established targets for antibody therapy with monoclonal antibodies (mAb) like Rituximab. Mixed clinico-pathological responses to mAb have been reported either due to presence of antibodies, rapid clearance or low density of target receptor/antigens. This demands need for an assay to monitor serum mAb therapeutic levels to ensure appropriate dosage. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used technique to detect mAb level in human serum which is expensive and time consuming. Understanding properties and interactions of antigens is quintessential for developing better targeted agents and overcoming resistance. Flow cytometry is still the most widely used technique to detect CD20 level in human serum which is expensive, time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to not only monitor levels but also study real time interaction of antigens with antibodies using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance to therapy. Method:Mimotope was used as a substitute for the antigen like CD20 and HER2 receptor protein in QCM assays to detect mAb level. The validation samples were prepared from the standard T solution in 10% human serum at three concentrations (10, 20 and 40 ug/ml). The changes in frequencies (ΔF) of sera from 3 female patients were obtained by calculating the differences between frequency shifts in pre and post mAb infusion. mAb level was calculated by equation, (ΔF +1.0022) ÷ 0.9997 μg / ml. The real-time processes of attachment of Cells like Raji cells on the gold electrode and the subsequent binding of antibody like Rituximab to the cells were studied using QCM biosensor. The interaction between Antigen and Antibody led to the increased resonant frequency shifts (df0) in the studied antibody concentration range from 5 to 250 μg mL-1 . Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved very specific interaction between Rituximab and CD20 antigens on B cells Results: Antigen and Antibody binding was very specific. This binding decreased the electrochemical activity and stability of the cells, supporting the cell lysis mechanisms of action of Rituximab. We showed that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used. QCM assay was capable of detecting mAb like Trastuzumab serum level as low as 0.038 nM (linear operating range of 0.038-0.859 nM). The time frame of assay was 20-30 minutes. These results were in concordance with previously published results using ELISA. We have shown a systematic approach for using QCM technique to quantify the apparent binding constant between antigen and antibody can reveal antigen density. Conclusion: We have established a low cost, highly sensitive, fast, synthetic peptide based QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays to monitor mAb serum levels like Rituximab, Trastuzumab and other monoclonal antibodies, helping physicians to determine the clinical efficacy of these drugs and ensuring appropriate dosages. Moreover antigen density and interactions of antigens with respective monoclonal antibodies like CD20 with Rituximab will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like Rituximab and Ofatumumab. This could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the peptide mimotopes could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization. Disclosures No relevant conflicts of interest to declare.