Hsu-Kun Wang

Femtomolar sensitivity using a channel-etched thin film waveguide fluoroimmunosensor

A dual channel, evanescent fluoroimmunoassay format is used to detect femtomolar analyte concentrations (i.e. less than 1 part per trillion [w/w]) on an etched channel siliconoxynitride thin film integrated optical waveguide. Two assays are used to demonstrate the dose-response behaviour of the sensor: (1) a direct assay of a fluorescently-labeled protein ligand binding to an immobilized protein receptor, and (2) an indirect sandwich assay of a non-fluorescent protein ligand binding to an immobilized protein receptor, as detected by the binding of a fluorescently-labeled secondary receptor protein. A red-emitting cyanine dye (Cy-5), which minimized background fluorescence and scatter losses of the waveguide, was used in both assays. To our knowledge, this is the first report of femtomolar sensitivity in an immunosensing instrument.

Planar Waveguide Biosensors for Nucleic Acid Hybridization Reactions

Oligonucleotide probes derived from (1) the T3 RNA polymerase promoter sequence (T3) and (2) prostate-specific antigen messenger RNA (PSA) were prepared and labeled with a red-emitting fluorescent dye (Cy5). The complimentary oligonucleotides were prepared and labeled with biotin. Initially, a feasibility study was performed in which the hybridization rate of the T3/anti T3 Oligonucleotide pair was examined. Specifically, biotinylated anti T3 was immobilized to a neutravidin-coated waveguide and solutions containing increasing concentrations of Cy5-labeled T3 were injected into the biosensor. Fluorescence emission was detected with an evanescent wave imaging fluorometer. The hybridization reaction proceeded rapidly with a significant amount of binding occurring during the first 5 minutes. A Michaelis-Menton kinetics model was used to analyze hybridization rate data and gave values of 78 nanomolar for the apparent affinity of the hybridization reaction and 1.4 picomolar for the analytical sensitivity of the hybridization assay. In subsequent studies the hybridization rate of the PSA/anti PSA oligonucleotide pair was examined. Biotinylated anti PSA was immobilized to the waveguide and solutions containing increasing concentration of Cy5-labeled PSA were injected into the biosensor. The hybridization rate observed for formation of the PSA/anti PSA pari was comparable to the high rates observed for the T3/anti T3 pari. Lastly, the selectivity of the biosensor was examined using an Oligonucleotide probe derived from human glandular kallikrein, which exhibits a high degree of homology to PSA. The two Oligonucleotide probes only differed in 7 out of 20 positions. Interestingly, the hybridization rate observed for Cy5-labeled hGK was very low - not statistically different from the non-specific binding rate of the hybridization assay.

Rapid clinical diagnostics assays using injection-molded planar waveguides

The goal of our research program is to develop an evanescent wave immunoassay system that can be used in point-of-care and critical care settings. Several key attributes are required to accomplish this goal: (1) the assay system should be at least as sensitive as present day immunoassays; (2) assay time should be 5 minutes or less; (3) the assay protocol should be relatively simple; (4) the sensor should be capable of performing more than one assay on a single specimen; (5) the assay system should be able to accommodate specimens such as serum, plasma and whole blood; and (6) the sensor should be an inexpensive, disposable cartridge. Our laboratory has developed an injection-molded planar waveguide sensor that meets most, if not all, of these attributes. This sensor has been evaluated in a number of different immunoassays for analytes such as bovine serum albumin, human chorionic gonadotrophin, creatine phosphokinase MB and cardiac troponin I.

How well can a T-cell epitope replace its parent carrier protein? A dose-response study.

AbstractPurpose. This work examines the effectiveness of synthetic peptide immunogens derived from immunodominant T-cell epitopes as replacements for their intact parent protein in vaccines. Methods. Fluorescein was conjugated to hen egg lysozyme (FL-HEL, positive control) and three synthetic peptide immunogens: (a) murine B10.A (H-2a) immunodominant T-cell epitope of HEL [FL-(T-cell epitope)]; (b) multiple antigenic peptide (MAP) multimer of this epitope {[FL-(T epitope)]n-MAP, n = 2-4}; and (c) negative control MAP with T-cell epitope residues replaced with glycine [(FL-Gly18)4-MAP]. The dose response of each immunogen was examined over a 300-fold range in B10.A mice. The immune response was monitored using antifluorescein ELISA assays. Results. FL-(T epitope)s immune response correlated positively with dose, with maximum response comparable to that of [FL-(T epitope)]n-MAP, or FL-HEL. This trend was consistent across 1°, 2°, and 3° responses, although interanimal variability was higher in the latter two because of an all-or-none response in mice immunized with this peptide. [FL-(T epitope)]n-MAPs immune response was consistently high and nearly dose independent, a trend observed across 1°, 2°, and 3° responses. FL-HELs immune response correlated negatively to dose in the 1° response but was nearly dose independent in the 2° and 3° responses. The magnitude of these latter responses was comparable to that observed for [FL-(T epitope)]n-MAP. (FL-Gly18)4-MAP did not elicit an immune response except at the highest dose. This trend was consistent across 1°, 2°, and 3° responses. Conclusions. The monomeric epitope was 300-fold less potent than its parent carrier protein, but increasing immunogen valency using MAP technology compensated totally for reduced potency. (FL-Gly18)4-MAPs lack of response at all but the highest dose strongly suggests that a specific immunodominant T-cell epitope sequence for HEL is necessary for successful peptide mimicry of HEL. This work also demonstrates the importance of quality assessment of commercial MAP core resins.