Functionalized carbon nanotube nanoelectrodes for biomolecular recognition

The objective of this research is to develop nanoscale ultrasensitive transducers for detection of biological species at molecular level using carbon nanotubes as nanoelectrodes. Rapid detection of ultra low concentration or even single DNA molecules are essential for medical diagnosis and treatment, pharmaceutical applications, gene sequencing as well as forensic analysis. Here the use of functionalized single walled carbon nanotubes (SWNT) as nanoscale detection platform for rapid detection of single DNA molecules is demonstrated. The detection principle is based on obtaining electrical signal from a single amine terminated DNA molecule which is covalently bridged between two ends of an SWNT separated by a nanoscale gap. The synthesis, fabrication, chemical functionalization of nanoelectrodes and DNA attachment were optimized to perform reliable electrical characterization these molecules. Using this detection system fundamental study on charge transport in DNA molecule of both genomic and non genomic sequences is performed. We measured an electrical signal of about 30 pA through a hybridized DNA molecule of 80 base pair in length which encodes a portion of sequence of H5N1 gene of avian Influenza A virus. Due the dynamic nature of the DNA molecules the local environment such as ion concentration, pH and temperature significantly influence its physical properties. We observed a decrease in DNA conductance of about 33% in high vacuum conditions. The counterion variation was analyzed by changing the buffer from sodium acetate to tris(hydroxymethyl) aminomethane, which resulted in a two orders of magnitude increase in the conductivity of the DNA. The fabrication of large array of identical SWNT nanoelectrodes was achieved by using ultralong SWNTs. Using these nanoelectrode array we have investigated the sequence dependent charge transport in DNA. A systematic study performed on PolyG - PolyC sequence with varying number of intervening PolyA - PolyT pairs showed a decrease in electrical signal from 180 pA (PolyG - PolyC) to 30 pA with increasing number of the PolyA - PolyT pairs. This work also led to the development of ultrasensitive nanoelectrodes based on enzyme functionalized vertically aligned high density multiwalled CNTs for electrochemical detection of cholesterol. The nanoelectrodes exhibited selectively detection of cholesterol in the presence of common interferents found in human blood.

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^