John D. Olson

A silicon sensor-based filtration immunoassay using biotin-mediated capture

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.

Quantitation of DNA hybridization in a silicon sensor-based system: application to PCR.

Rapid, quantitative hybridization assays with good sensitivity are needed in many applications, for example, determining the amount of specific product from PCR. We have developed an assay which relies on the hybridization of a biotinylated oligomer and a fluoresceinated oligomer to a single-stranded target in solution. The hybridized complex is captured by streptavidin to a biotinylated membrane. After capture, the hybridization complex is detected by an antifluorescein-urease conjugate which binds to the fluoresceinated probe. The membrane-bound urease conjugate is exposed to urea and assayed with a pH-sensitive silicon sensor. The total assay time is less than 2 h and the sensitivity limit is 20 x 10(6) molecules with a coefficient of variation, CV, of less than 10%. The assay was applied to the analysis of a model target using PCR. We were able to measure the amount of specific product and the amplification factor during the exponential phase of PCR. Using extrapolation from the measured amounts of amplified product, the initial amounts of target molecules were calculated to be 1.2 x 10(6) and 4.0 x 10(2) when the added quantities were 3 x 10(6) and 3 x 10(3), as determined by serial dilution.

Immunochemical detection using the light-addressable potentiometric sensor

Rapid, sensitive and flexible assay systems are needed for immunoassays, receptor-ligand binding studies and DNA probe assays. Filtration capture and sensor detection offer several advantages to these areas. Although dependent on the affinity of the specific binders employed, the sensitivity of these techniques can be in the order of 10(-12) M, and total assay time can be less than 15 minutes.

Picogram detection levels of asialofetuin via the carbohydrate moieties using the light addressable potentiometric sensor

Fetal calf serum asialofetuin was assayed in the sandwich format using biotinylated and fluoresceinated ricin toxin (B-RCA and F-RCA). The sandwiched species was captured on a biotin-BSA coated nitrocellulose membrane with streptavidin. Anti-fluorescein antibody-urease conjugate was bound to the complex, and detected and quantitated under microvolume conditions using the light addressable potentiometric sensor. As little as 250 pg of asialofetuin was detectable whereas fetuin gave no response at conditions as high as 32 ng. Using a competitive inhibition assay, we established that the binding constant for the asialofetuin-ricin complex was 3.6×108m−1. This is in good agreement with data published using glycopeptides derived from asialofetuin, and RCA and the ricin agglutinin, RCA120.

DETERMINATION OF DISSOCIATION CONSTANT AND CONCENTRATION OF AN ANTI-DNA ANTIBODY BY USING THE LIGHT-ADDRESSABLE POTENTIOMETRIC SENSOR

The most sensitive assay possible is desired for quantitation of total DNA and DNA probe binding. To reduce background signal, which may reduce assay sensitivity, it is desirable to employ low antibody concentrations. This requires the use of a specific antibody with a high affinity for DNA.