Methodologies for “Wiring” Redox Proteins/Enzymes to Electrode Surfaces

The immobilization of redox proteins or enzymes onto conductive surfaces has application in the analysis of biological processes, the fabrication of biosensors, and in the development of green technologies and biochemical synthetic approaches. This review evaluates the methods through which redox proteins can be attached to electrode surfaces in a “wired” configuration, that is, one that facilitates direct electron transfer. The feasibility of simple electroactive adsorption onto a range of electrode surfaces is illustrated, with a highlight on the recent advances that have been achieved in biotechnological device construction using carbon materials and metal oxides. The covalent crosslinking strategies commonly used for the modification and biofunctionalization of electrode surfaces are also evaluated. Recent innovations in harnessing chemical biology methods for electrically wiring redox biology to surfaces are emphasized

Development of micro immunosensors to study genomic and proteomic biomarkers related to cancer and Alzheimer\u27s disease

A report from the National Institutes of Health defines a disease biomarker as a “characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with biorecognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco

Single Cell Measurements of Genomic and Protein Biomarkers Using Microimmuno Sensors

Abstract not Available.</jats:p

Investigating the affinity of human plasma lipoproteins and albumin to the anti-cancer drug valrubicin

Research Appreciation Day Award Winner - 2017 Institute for Molecular Medicine, Center for Cancer Research - Cancer Biology, Translational Research Poster Presentation AwardBackground: Reconstituted lipoproteins and albumin are promising targeted drug delivery agents. Valrubicin is a hydrophobic anti-cancer drug currently approved for the treatment of in situ BCG –resistant bladder carcinoma through intra-vesical instillation. An injectable formulation with the patients’ own lipoproteins or albumin as carriers would not only make valrubicin an attractive therapy candidate for other types of cancer, but also limit immunological response and off-target effects including cardiotoxicity. Purpose: This study aimed to assess the binding of valrubicin to human plasma lipoproteins and serum albumin (HSA) under different incubation conditions. We hypothesize valrubicin will preferentially associate with high density lipoprotein (HDL) due to its high hydrophobicity index. Materials and Methods: A solution of valrubicin in dimethyl sulfoxide was incubated for up to 24 hours with either pooled whole human plasma or with individual plasma components at room temperature (RT) or 37 degrees Celsius. Plasma component fractions were separated by ultracentrifugation. A 45 mg/mL albumin solution was used for individual incubation. Absorbance at 490 nm was used to estimate valrubicin concentration in low density lipoprotein (LDL), HDL and HSA. Results: Incubation for 2 hours at RT yielded the highest valrubicin concentration in plasma components. Overall, valrubicin association with plasma components moderately increased with the amount of valrubicin added prior to incubation. For instance, 0.185 mg and 1.121 mg of valrubicin was recovered with respectively 1 mg and 5 mg of valrubicin initially added to 1 mL of plasma. About 4%, 10% and 86% of the initial valrubicin added to whole plasma were respectively distributed in the LDL, HDL and the HSA –containing plasma fractions after ultracentrifugation. The incorporation of valrubicin in HDL increased-though not proportionally- with 10 fold concentration of HDL. However, prolonged incubation of valrubicin with 45mg/mL HSA resulted in the dissociation of valrubicin and HSA. Conclusions: Our preliminary data suggests differential interaction of valrubicin with plasma components with preferential binding to HSA. The optimization of the HSA/ valrubicin complex will be required to develop an efficient drug transport formulation. HSA stabilizing agents such as sodium acetyltryptophanate and sodium caprylate could potentially facilitate and stabilize the binding of valrubicin to HSA

Molecular Histopathology Using Gold Nanorods and Optical Coherence Tomography

PURPOSE. To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). METHODS. GNRs were synthesized and covalently attached to anti–glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti–GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. RESULTS. No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. CONCLUSIONS. We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal