Development of Nanosensors and Bioprobes

We describe the development and application of nanosensors having bioreceptor probes for bioanalysis. The nanoprobes were fabricated with optical fibers pulled down to tips having distal end sizes of approximately 30–60nm. The use of two different types of receptors was investigated. Fiberoptic nanoprobes were covalently bound either with bioreceptors, such as antibodies, or with other receptors, such as cyclodextrins that are selective for the size and chemical structure of the analyte molecules. Theoretical calculations were performed to model the binding of beta-cyclodextrin with pyrene and 5,6-benzoquinoline, and to illustrate the possibility of comparing experimental data with theoretical data. The antibody-based nanoprobe was used for in situ measurements of benzopyrene tetrol in single cells. The performance of the nanosensor is illustrated by intracellular measurements performed on a rat liver epithelial cell line (Clone 9) used as the model cell system. The usefulness and potential of these nanotechnology-based biosensors in biological research and applications are discussed.     

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.     

A beta-cyclodextrin based fiber-optic chemical sensor: a fractal analysis

A fractal analysis is presented for the binding of pyrene in solution to beta-cyclodextrin attached to a fiber-optic chemical sensor. The specific (k(l)) and non-specific binding rate coefficients and the fractal dimension (D(f)) (specific binding case only) both tend to increase as the pyrene concentration in solution increases from 12.4 to 124 ng ml(-1). Predictive relations for the binding rate coefficient (specific as well as non-specific binding) and for D(f) (specific binding case only) as a function of pyrene concentration are provided. These relations fit the calculated k(l) and D(f) values in the pyrene concentration range reasonably well. Fractal analysis data seem to indicate that an increase in the pyrene concentration in solution increases the "ruggedness" or inhomogeneity on the fiber-optic biosensor surface. The fractal analysis provides novel physical insights into the reactions occuring on the fiber-optic chemical surface and should assist in the design of fiber-optic chemical sensors.     

Concavity cuts for disjoint bilinear programming

We pursue the study of concavity cuts for the disjoint bilinear programming problem. This optimization problem has two equivalent symmetric linear maxmin reformulations, leading to two sets of concavity cuts. We first examine the depth of these cuts by considering the assumptions on the boundedness of the feasible regions of both maxmin and bilinear formulations. We next propose a branch and bound algorithm which make use of concavity cuts. We also present a procedure that eliminates degenerate solutions. Extensive computational experiences are reported. Sparse problems with up to 500 variables in each disjoint sets and 100 constraints, and dense problems with up to 60 variables again in each sets and 60 constraints are solved in reasonable computing times. Received: October 1999 / Accepted: January 2001?Published online March 22, 2001     

Fluorescence monitoring of a benzo[a]pyrene metabolite using a regenerable immunochemical-based fiber-optic sensor

Microscale regenerable biosensors are described and utilized to measure the natural fluorophor benzo[a]pyrene tetraol (BPT). The sensors combine laser-excited/fiber-optic remote sensing principles with a unique capillary tube delivery system to make repetitive, heterogeneous fluoroimmunoassay measurements. Two sensor configurations and modes of operation are described. Concentrations of BPT in the nanomolar range are easily measured with a reproducibility of 10% or better, depending on the sensor design, selective measurements can be made in ca. 20 min, then the sensor can be regenerated by delivering new reagents to the sensing chamber, without removing the sensor from the sample.     

Electrophoretic injection bias in a microchip valving scheme

The pinched injection strategy, implemented on microfabricated fluidic devices (microchips), was investigated for an electrophoretic injection bias. Both the sample loading and dispensing steps were found to contribute to the injection bias whereby neutral species were injected preferentially to anionic species. In the sample loading step, neutral species filled a larger volume in the cross intersection than anionic species. Similarly, in the dispensing step, a larger volume of neutral analyte was injected than anionic analyte. Up to a 27% difference in injected volumes was observed. Fluorescently labeled amino acids were used as model analytes.     

Evaluation of antibody immobilization technqiues for fiber optic-based fluoroimmunosensing

Fiber optic chemical sensors have been developed that employ immunochemicals to perform fluoroimmunoassays. A regenerable fiber optic sensor was developed with a capillary delivery system to perform repetitive assays using antibodies immobilized to beads, “immunobeads”. The sensitivity of these sensors is directly proportional to the amount of antibody present. For the immunobeads, the amount immobilized (loaded) and the ability of the immobilized antibody to maintain antibody recognition (activity) are two criteria which will affect the sensitivity of the sensors. Four reagents, [1,1′-carbonyldiimidazole (CDI), Protein A, glycidoxypropyltrimethoxysilane (GOPS) and 2-fluoro-1-methylpyridium toluene-4-sulfonate (FMP)], were evaluated using these two criteria. Two antibody—antigen systems were employed to investigate the four procedures. The first combination is a polyclonal rabbit anti-human IgG with a F(ab)^′₂ fragment human IgG as the antigen. The second combination is a monoclonal mouse anti-benzo[a]pyrene tetraol (BPT) IgG with BPT as the hapten. In the case of the BPT, CDI demonstrated superior performance, combining high loading and high retention of antibody activity. In the case of the large antigen, CDI again immobilized the most antibody but suffered activity losses of about 40%. The large amount of inactive antibody may make this procedure less attractive than the Protein A beads, which maintained the activity of the anti-human IgG but exhibited less loading than the CDI. However, in terms of active antibody there are similar amounts. FMP yielded similar results to CDI for the large antigen case but the sample preparation is more labor intensive. GOPS yielded losses of up to 70% of the antibody activity, which makes it unattractive as a reagent. The two systems tested give an indication of antibody—antigen interactions but may not be representative of all cases. The ideal immobilization reagent and conditions will probably change from system to system.