No description available.

Two established methods, Electrochemical Impedance Spectroscopy (EIS) and extracellular recording, were implemented into a technology platform for non-invasive whole-cell biosensing. Electrical activity of cardiomyocytes and cell-substrate interaction of human ovarian cancer cells was monitored on electrode array chips. The performance of cells inside a microfluidic or closed low volume environment was investigated. Prior to the development of the entire microfluidic platform the two transducing methods were evaluated in single experiments. Processes as cellular attachment and detachment were monitored using EIS and single frequency impedance sensing. Electrodes of different size and structure were employed and compared for their impedance response. It was shown that small electrodes (A = 9·10-6 cm²) are more sensitive to cell-substrate interaction than larger ones (A = 9·10-5 cm²) and that the frequency used for analysis has a profound influence on the sensitivity. Data were modelled using a common equivalent circuit that represents a cell layer on an electrode resulting in an increase of the impedance magnitude by <170 % due to cell attachment. In order to demonstrate the potential of this method for biomedical applications, experiments related to anti-cancer strategies were performed. Cell detachment was induced by addition of synthetic integrin ligands and by hypericin mediated photodynamic therapy and monitored with impedance-based biosensing. Electrical activity of cardiomyocytes cultured on microelectrode arrays was monitored inside a microfluidic system. The chronotropic drug isoproterenol was applied using a robotic dispensing machine, and the resulting changes in spike rate and duration were compared with results gained by experiments with a large scale MEA chip. The experimental findings inspired the development of a technology platform that was finally evaluated by monitoring extracellular signals from myocytes in response to Isoproterenol. Another topic was the comparison of cell-substrate interaction monitored on various electrode structures.

Class of 2015 Abstract / Objectives: To develop an assay to permit the identification of compounds that can inhibit the interaction between GIT1 and the amino-terminal SH3 domain (SH3-N) of GRB2. Methods: The GIT1 protein was expressed in Sf9 insect cells and purified using Talon resin beads. The SH3-N domain of GRB2 was expressed in the E. coli strain, BL21(DE3)pLysS, and purified using glutathione resin beads. The SH3-N domain was fluorescently tagged on cysteine 32 using Cyanine 3 maleimide. The fluorescence of the assay was measured by using a plate reader with excitation wavelength of 555 nm and emission wavelength of 570 nm. Results: The GIT1 protein was expressed in Sf9 cells and purified using the Talon beads. The SH3-N domain of GRB2 was expressed in BL21 cells and purified from the glutathione resin beads. The SH3-N domain was cleaved from GST by using thrombin, which was engineered into the GST fusion protein and were fluorescently labeled using Cyanine 3 maleimide. Conclusions: The fluorescence polarization assay that will detect the interaction between GIT1 and the SH3-N domain of GRB2 is still under development, but it has progressed towards completion since both components of the assay are in hand.

No description available.

Class of 2009 Abstract / OBJECTIVES: The purpose of this study was to determine the perceptions of the general public in Arizona regarding random drug screening and obtaining and maintaining health care benefits. METHODS: Individuals entering and leaving a grocery store in Chandler, Arizona on one day were invited to voluntarily participate in the study by reviewing a subject disclosure form and completing an 11 item questionnaire. The Likert scale varied from 1 = Strongly Disagree to 6 = Strongly Agree. Individuals were eligible to participate if they were adults and living in Arizona. RESULTS: One hundred adults participated in the study. The results of the study showed general agreement for passing a drug test for illegal substances before being hired (5.0 ± 1.5, mean ± SD), and if testing positive for illegal drugs, being allowed to enroll in a drug treatment program to keep their health care benefits (4.8 ±1.6). Respondents agreed that employers should require random drug testing to maintain employment (4.4 ± 1.8). However, respondents neither agreed or disagreed that people should be required to pass a drug test to be eligible to receive health care benefits (3.9 ± 2.0) or that they should be required to pass a periodic drug test to maintain their health care benefits (3.7 ± 2.0). CONSLUSIONS: Respondents agreed with drug testing to obtain and maintain employment; however, respondents neither agreed or disagreed with the concept of obtaining or maintaining health care benefits with passing a drug test for illegal substances.

Biophysical characterisation of protein – ligand interactions can provide vital information to dissect complex biochemical binding mechanisms. C3d has been shown to interact with a number of different protein ligands, namely CD21 SCR1-2, S. aureus Efb-C, S. aureus Ehp, S. aureus Sbi and complement regulatory protein factor H. Although much is known about the relationship of C3d and CD21 in the induction of humoral immunity, the structural aspects of this interaction remained controversial until very recently. The aim of this thesis was to gain a detailed understanding of the C3d/CD21 SCR1-2 interaction using different biophysical methods and to identify potential low molecular weight inhibitors of the interaction. A crystal structure of the C3d/CD21 complex solved by Szakonyi et al. in 2001 indicated the C3d binding site on CD21 was in the SCR2 domain. It did not agree with mutagenesis studies and recent NMR titration experiments show that only residues from the SCR1 domain of CD21 appear to mediate binding under physiologically relevant ionic strength. In the current work, NMR was employed to monitor ligand binding to C3d and to provide residue specific information that reflects a physiologically relevant binding mode to complement the crystallographic model solved by van den Elsen and Isenman in 2011. Microcalorimetric analysis on the site-directed mutagenesis studies also supported a model of hydrophobically- and electrostatically-driven protein-protein interaction for C3d and CD21 SCR1-2. Complement C3d forms a non-specific thioester linkage with antigen, which then binds to CD21 SCR1-2 and coligates with membrane immunoglobulin of the B cell receptor. While the interactions triggers B cell activation and hence the production of antibody under normal circumstances, it has been demonstrated that the interactions also lead to undue B cell activation and auto-antibody production. There is a well established collagen-induced arthritis (CIA) mouse model to support the significance of C3d and CD21 in disease susceptibility. To this end, a high-throughput SPR-based screening platform was set up to screen a fragment library against C3d, so as to identify low molecular weight compounds that could serve as a starting point for drug development programme. Unfortunately, the work did not yield C3d-binding inhibitors and future work could include screening large fragment libraries that are designed to target protein-protein interfaces.

Firstly, this work demonstrates that contactless dielectrophoresis (cDEP) was useful to detect a reversal in the electrical phenotype of late-stage ovarian cancer cells to a profile similar to that of slow-growing early-stage ovarian epithelial cells after treatment with a non-toxic bioactive metabolite, sphingosine. Current chemotherapeutics are highly toxic to patients and can cause severe adverse side effects, so non-toxic treatments that could slow or reverse cancer growth would be advantageous. This is the first instance of cDEP for detecting induced changes in cell structure, showing its potential as a rapid, non-biomarker-based drug screening platform. Specifically, low frequency contactless dielectrophoresis devices previously designed by Sano et al were used to extract the crossover frequency and specific membrane capacitance of early and late stage mouse ovarian surface epithelial (MOSE-E and MOSE-L) cells when untreated, treated with the anti-cancer sphingosine (So) metabolite and with a generally cancer-supporting sphingosine-1-phosphate (S1P) metabolite. The specific membrane capacitance of MOSE-L cells treated with So decreased and the normalized crossover frequency increased to levels matching MOSE-E cells. Secondly, a new multilayer cDEP device featuring curved interdigitated electrode channels overlaying a straight sample channel for the purpose of cell sorting was designed, computationally modeled, fabricated, and tested. The goal of this design was to achieve continuous multi-stream sorting of cells, and preliminary testing demonstrated that prostate cancer PC3 cells were continuously deflected toward the top of the channel under an electric field, as predicted by the numerical model. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Paper spray mass spectrometry is an alternative technique for toxicological screening that is able to quickly and adequately screen for compounds encountered in postmortem investigations with little sample handling and no sample preparation. For analysis of dried blood spots using a triple quadrupole mass spectrometer, detection criteria were defined to align with relevant regulatory guidelines while considering how fragment ion selection, method sensitivity, and fragment ion ratio tolerances are best utilized in paper spray mass spectrometry. For analysis, drugs and drug metabolites relevant to postmortem investigations were spiked into drug-free blood, and by monitoring two fragment ion channels in selected reaction monitoring mode, as well as the ratio between the two fragment ions, a method was developed capable of detecting over 120 drug and drug metabolites at concentrations relevant to postmortem drug screening. Total analysis time for the developed method is less than 8 minutes, and less than 50µL of sample and 5mL of solvent are consumed during analysis.

This research developed a biosensor for kinase drug discovery applications. In particular it combined electronic techniques with optical techniques to understand the phosphorylation of proteins. There are two major electronic characteristics of phosphorylation that aid in its detection and subsequently biosensor development: first is the release of a proton upon phosphorylation of a protein (change in pH) and second is the addition of negative charge to the protein upon its phosphorylation. The work in this thesis reports an electrolyte–insulator–semiconductor sensing structures to detect the pH changes associated with phosphorylation and metal–insulator–semiconductor structures to detect the charge change upon phosphorylation of proteins. Major application of the developed devices would be to screen inhibitors of kinase that mediate phosphorylation of proteins. Inhibitors of kinase act as drugs to prevent or cure diseases due to the phosphorylation of proteins. With the advancements in VLSI and microfluidics technology this method can be extended into arrays for high throughput screening for discovering drugs.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.